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https://openalex.org/W2739747493	https://repository.ubn.ru.nl/bitstream/2066/181882/1/181882.pdf	English	null	Cognitive rehabilitation and mindfulness in multiple sclerosis (REMIND-MS): a study protocol for a randomised controlled trial	BMC neurology	2,017	cc-by	7,968	Cognitive rehabilitation and mindfulness in multiple sclerosis (REMIND-MS): A study protocol for a randomised controlled trial Nauta, I.M.; Speckens, A.E.M.; Kessels, R.P.C.; Geurts, J.J.G.; Groot, V. de; Uitdehaag, B.M.J.; Fasotti, L.; Jong, B.A. de 2017, Article / Letter to editor (BMC Neurology, 17, (2017), article 201) Doi link to publisher: https://doi.org/10.1186/s12883-017-0979-y Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/181882 Download date: 2024-10-24 * Correspondence: i.nauta1@vumc.nl 1Department of Neurology, Amsterdam Neuroscience, MS Center Amsterdam, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, the Netherlands Full list of author information is available at the end of the article Note: Note: To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Nauta et al. BMC Neurology (2017) 17:201 DOI 10.1186/s12883-017-0979-y Open Access Cognitive rehabilitation and mindfulness in multiple sclerosis (REMIND-MS): a study protocol for a randomised controlled trial Ilse M. Nauta1*, Anne E. M. Speckens2, Roy P. C. Kessels3,4, Jeroen J. G. Geurts5, Vincent de Groot6, Bernard M. J. Uitdehaag1, Luciano Fasotti3,7 and Brigit A. de Jong1 © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background treatment option is cognitive rehabilitation therapy (CRT) [10]. CRT entails the learning of new cognitive strategies aimed at compensating for cognitive problems. The use of these strategies shows positive effects on cognitive function among stroke and brain injury patients [11]. There is also some evidence for positive effects of CRT on cognitive func- tion among MS patients. However, no final conclusion on the effectiveness of CRT can be established due to contra- dictory findings [12, 13]. These contradictory findings may be explained by small sample sizes, heterogeneous interven- tions across studies and methodological limitations (e.g. biased selection) [12, 13]. Multiple sclerosis (MS) is a chronic disease of the central nervous system, which leads to physical, neuropsychiatric and cognitive problems. Cognitive problems are commonly reported by MS patients, with prevalence rates of objective cognitive deficits varying between 43 and 70% [1]. The most frequently affected cognitive domains are information processing speed, memory, attention, visuospatial process- ing and executive function. These objective cognitive defi- cits (i.e. assessed with cognitive tests) only show a weak relation with the cognitive complaints reported by MS patients themselves [2, 3]. Despite this weak relation, sub- jectively experienced cognitive problems are arguably as important as objective cognitive deficits, since they may re- flect the burden of cognitive problems in daily life. A second promising non-pharmacological treatment option is mindfulness-based cognitive therapy (MBCT) [14]. MBCT entails mindfulness training combined with elements of cognitive behavioural therapy. There is prelim- inary evidence that mindfulness-based interventions posi- tively affect cognitive function in healthy individuals [14, 15] and they may even influence brain structures and functions that are involved in cognitive function [14, 16, 17]. In MS patients, positive effects of mind- fulness-based interventions on psychological symptoms have been found [18–20], and a recent pilot study re- ported some positive effects of mindfulness on objective cognitive function [19]. To our knowledge, no other stud- ies have investigated the effect of mindfulness-based inter- ventions on cognitive function among MS patients. In summary, well-designed studies are necessary to investi- gate the effect of MBCT and CRT on cognitive function among patients with MS. The impact of cognitive problems on daily life can be extensive given the relatively young age of disease onset. Problems in social relations and work participation are likely to occur, consequently negatively affecting the quality of life of MS patients [1, 4]. Background This highlights the need for effective cognitive treatment options for MS pa- tients. To develop and guide effective cognitive treat- ments, knowledge about the aetiology of objective and subjective cognitive problems is essential. Objectives This study primarily aims to examine the effectiveness of CRT and MBCT on subjectively experienced cognitive problems among MS patients. We hypothesise that both CRT and MBCT positively affect subjective cognitive function compared to enhanced treatment as usual (ETAU). We also expect positive effects on the second- ary outcome measures objective cognitive function, functional brain network measures, psychological symp- toms, well-being, quality of life and daily life functioning. Additionally, we will evaluate in an exploratory way The REMIND-MS study The REMIND-MS study is a randomised controlled trial (RCT) that investigates the effect of CRT and MBCT on subjective and objective cognitive function in MS pa- tients. Additionally, resting-state magnetoencephalogra- phy (MEG) data will be obtained to gain additional knowledge about the aetiology of subjective and object- ive cognitive problems with respect to functional brain networks, and to unravel if cognitive improvements after both interventions are associated with functional brain network changes. Whereas the aetiology of objective cognitive deficits is widely studied, studies focusing on the aetiology of subjective cognitive complaints are rare. Since subjective and objective cognitive problems correlate weakly [2, 3], their aetiology might be different [9]. One recent study found that subject- ively experienced cognitive problems could not be explained by brain pathology, but no measures of brain networks were included [9]. To date, the study of brain networks and their relation to objective and subjective cognitive function among MS patients is still in its infancy. Additional well-designed studies are needed to unravel the aetiology of objective and subjective cognitive problems in MS. Abstract Background: Cognitive problems frequently occur in patients with multiple sclerosis (MS) and profoundly affect their quality of life. So far, the best cognitive treatment options for MS patients are a matter of debate. Therefore, this study aims to investigate the effectiveness of two promising non-pharmacological treatments: cognitive rehabilitation therapy (CRT) and mindfulness-based cognitive therapy (MBCT). Furthermore, this study aims to gain additional knowledge about the aetiology of cognitive problems among MS patients, since this may help to develop and guide effective cognitive treatments. Methods/design: In a dual-centre, single-blind randomised controlled trial (RCT), 120 MS patients will be randomised into one of three parallel groups: CRT, MBCT or enhanced treatment as usual (ETAU). Both CRT and MBCT consist of a structured 9-week program. ETAU consists of one appointment with an MS specialist nurse. Measurements will be performed at baseline, post-intervention and 6 months after the interventions. The primary outcome measure is the level of subjective cognitive complaints. Secondary outcome measures are objective cognitive function, functional brain network measures (using magnetoencephalography), psychological symptoms, well-being, quality of life and daily life functioning. Discussion: To our knowledge, this will be the first RCT that investigates the effect of MBCT on cognitive function among MS patients. In addition, studying the effect of CRT on cognitive function may provide direction to the contradictory evidence that is currently available. This study will also provide information on changes in functional brain networks in relation to cognitive function. To conclude, this study may help to understand and treat cognitive problems among MS patients. Trial registration: This trial was prospectively registered at the Dutch Trial Registration (number NTR6459, registered on 31 May 2017). Keywords: Multiple sclerosis, Cognition, Cognitive rehabilitation therapy, Mindfulness-based cognitive therapy, Brain networks, Randomised controlled trial © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nauta et al. BMC Neurology (2017) 17:201 Page 2 of 10 Page 2 of 10 Aetiology of cognitive problems The aetiology of objective and subjective cognitive prob- lems in MS is complex and not completely understood. Objective cognitive deficits in MS patients have been linked to cortical, deep grey matter and white matter damage [5, 6]. Researchers have argued that this wide- spread pathology may result in a disruption of the con- nectivity between brain regions, which in turn may result in cognitive decline [7]. Changes in brain net- works are indeed present in MS patients: studies have reported changes in functional connectivity [7] and a loss of hierarchal structure [8], which both related to re- duced objective cognitive performance in MS patients. Participants Mixed model analyses will be applied with three mea- surements comparing two groups (MBCT vs. ETAU, CRT vs. ETAU). There are no previous studies with MS patients that investigated the effect of CRT or MBCT on the primary outcome measure, the Cognitive Failure Questionnaire (CFQ) [23]. Based on a previous RCT using a comparable outcome measure, a medium effect size can be expected [24, 25]. With an alpha of .05, a power of .80, an intra-class correlation of .06, and 33 participants per group, a minimal difference of 0.62 SD can be detected between two groups. Taking into ac- count drop-out and loss to follow-up, we intend to re- cruit 40 MS patients per group. Exclusion criteria Participants who meet any of the following criteria are excluded from participation: (1) psychosis, (2) suicidal ideation, (3) an inability to speak or read Dutch, (4) pre- vious experience with a similar intervention (e.g. a com- parable cognitive rehabilitation training or mindfulness training), (5) physical or cognitive disabilities, comorbid- ities or treatments that would interfere too much with the interventions to enrol in this study (to be evaluated on an individual level). The reasons for excluding partic- ipants who express interest in the study will be accur- ately documented. Treatment of cognitive problems The best cognitive treatment options for MS patients are still a matter of debate [10]. A promising non-pharmacological Nauta et al. BMC Neurology (2017) 17:201 Page 3 of 10 Page 3 of 10 whether there are differences in intervention effects be- tween CRT and MBCT. participants who are still interested to participate are in- vited by the trial coordinator to sign the informed consent form. After signing the informed consent form, it will be checked whether the participants are fully eligible. Secondary study objectives are: 1) to explore the role of functional brain network measures (using MEG) in subjective and objective cognitive problems, and to evaluate whether there are differences in functional brain network measures between these types of cognitive problems; On the informed consent form, participants have the op- tion to give permission for an informant to be contacted. If permission is given, an informant of the participants will also receive written information and an informed consent form, as informants will be asked to complete one ques- tionnaire at three time-points (see outcome measures). On the informed consent form, participants and their infor- mants also have the option to give permission for using their data for other research, for sharing their data with re- searchers outside of the European Union and to be con- tacted again for follow-up research. 2) to explore the role of functional brain network measures as possible mediators in the effect of the interventions; 3) to evaluate whether alterations in objective cognitive function, functional brain network measures, psychological symptoms, well-being, quality of life and daily life functioning are mediating factors that determine subjective cognitive function; Methods-design The REMIND-MS study is a dual-centre, single-blind RCT with three parallel groups: CRT, MBCT and ETAU. All interventions last nine weeks in total. Measurements take place at baseline, post-intervention and after a 6- month follow-up period. The full trial design is sum- marised in Fig. 1. Setting Selection and measurements take place at the VU Univer- sity Medical Center in Amsterdam, the Netherlands. Part of the measurements, that is, the self-report questionnaires, can be completed by the participants at home. The inter- ventions take place at two centres in the Netherlands: VU University Medical Center in Amsterdam and Klimmendaal Rehabilitation Center in Arnhem. Inclusion criteria P j g 4) to evaluate which factors determine whether a patient is likely to benefit from one of the therapies, such disease severity, severity of cognitive problems and mood at baseline, or gender. Participants are eligible to participate if they meet the following criteria: (1) between 18 and 65 years of age, (2) confirmed diagnosis of MS according to the McDonald 2010 criteria [21], (3) a minimum score of 23 on the Multiple Sclerosis Neuropsychological Questionnaire – Patient version (MSNQ-P), which measures subjective cognitive complaints [22]. Recruitment and consent Participants are recruited through the participating cen- tres (VU University Medical Center and Klimmendaal Re- habilitation Center), the ‘VUmc MS Center Amsterdam’ website and MS patient associations. All potentially eli- gible participants who express interest in the study are provided with written trial information, which contains information about the rationale, purpose and personal implications of the study. The information sheet also includes contact details of the trial coordinator and of an independent medical doctor who is not part of the research team, who can both be contacted for additional in- formation. After sufficient time for consideration, potential Nauta et al. BMC Neurology (2017) 17:201 Page 4 of 10 VUmc: eligibility check Decision which location: VUmc or KR VUmc: randomisation t = 9 weeks MBCT CRT ETAU VUmc: Baseline assessment KR: randomisation MBCT CRT ETAU VUmc: Post-intervention assessment VUmc: 6-month follow-up assessment Lost to follow-up or discontinued Informed consent Information provided Excluded: no longer willing to participate Excluded: based on in- and exclusion criteria Confirm willingness to participate Excluded: no informed consent Fig. 1 Flowchart of the trial design. KR = Klimmendaal Rehabilitation Center; VUmc = VU University Medical Center Cognitive rehabilitation therapy (CRT) Interventions Interventions All interventions last nine weeks. The CRT consists of nine 2.5-h group sessions, MBCT of eight 2.5-h group sessions and one ‘silent day’, and enhanced treatment as usual (ETAU) of one individual appointment within the 9-week period. For CRT and MBCT, the optimal group size was determined based on previous experiences. Groups will consist of a maximum of 6 people in the CRT group and a maximum of 10 people in the MBCT group. Professionally trained psychologists will guide the CRT sessions, certified mindfulness trainers will teach the MBCT sessions and MS specialist nurses will have appointments with participants from the ETAU group. All trainers will be instructed and supervised by the same specialists. The interventions will be provided in a standardised manner using a written protocol. The trainers are instructed not to disclose treatment infor- mation to trainers from another treatment arm. All par- ticipants will receive an information brochure on MS and cognition. The CRT protocol focuses on the following cognitive do- mains: speed of information processing, memory, execu- tive function and mental fatigue. Cognitive impairments will be treated by a combination of compensatory strategy training and psycho-education. The proposed strategies are based on MS-tailored variants of evidence-based treat- ments that have been developed in CRT research with brain-injured subjects. Treatment of problems in informa- tion processing speed will be based on ‘Time Pressure Management’ [26, 27], memory on ‘Training Memory strategies’ [28], executive function on a ‘Multifaceted Treatment for Executive Dysfunction’ [29, 30] and mental fatigue on ‘Cognitive and Graded Activity Training’ [31, 32]. These four treatments are incorporated in the protocol as described by Geusgens, Baars-Elsinga, Visser- Meily and van Heugten [33]. In addition to cognitive strat- egy training, CRT focuses on emotional and behavioural changes, and grief resolution. Grief resolution will be in- cluded by explaining the stages of bereavement and by Page 5 of 10 Nauta et al. BMC Neurology (2017) 17:201 Page 5 of 10 discussing the loss of physical independence, mobility, cog- nitive ability and emotional control on self-esteem and fu- ture perspective. The participants will receive homework assignments aimed at identifying their own cognitive prob- lems and at applying the learned strategies in daily life situ- ation. These homework assignments will take 30 to 45 min a day, 6 days per week. assignments will be checked and evaluated during each visit of the treatment period. Outcome measures Outcome measures All outcome measures will be administered at each as- sessment: at baseline, post-intervention and 6-months follow-up (see Table 1). Mindfulness based cognitive therapy (MBCT) Mindfulness based cognitive therapy (MBCT) The MBCT protocol is primarily based on the MBCT program by Segal, Williams and Teasdale [34]. MBCT is an intervention in which aspects of mindfulness meditation are combined with aspects of cognitive behavioural therapy. MBCT focuses on increasing awareness of the present mo- ment. To achieve this, participants will be trained in both self-regulation of attention and non-judgmental awareness of moment-to-moment experience. Patients will become more aware of their emotions, thoughts and behaviours and will learn to use more adaptive behaviour to respond to their symptoms. The program will be adapted to the MS patients in terms of tailoring psycho-educative elements to themes relevant to the MS patient (e.g. cognitive problems) and modified movement exercises (for patients suffering from physical impairments). Participants will receive guided mindfulness meditation exercises of 30 to 45 min, 6 days per week, for home practice and a reader with home prac- tice instructions and background information. All thera- pists will fulfil the advanced criteria of the Association of Mindfulness Based Teachers in the Netherlands and Flanders, which are in concordance with those of the UK Mindfulness-Based Teacher Trainer Network [35]. Replacement and follow-up of withdrawn participants Participants can leave the study at any time for any reason without any consequences. Follow-up measurements will still be scheduled if the participant is willing and able to participate in follow-up measurements. There will be no replacement of individual participants after withdrawal. If a high rate of participants drops out during the study, more participants will be included in the study. These partici- pants will be randomly allocated to one of three parallel groups using the randomisation and minimisation proced- ure as described under ‘randomisation and blinding’. Relevant concomitant care and interventions During the intervention period and the 6-months follow- up period, patients are asked not to follow an intervention outside this study that focuses on mindfulness or cogni- tion, and to keep their level of care constant during this period when possible. Naturally, usual care should con- tinue, as do new treatment options when the health situ- ation of the patient changes. Enhanced treatment as usual (ETAU) ( ) Enhanced treatment as usual (ETAU) entails an appoint- ment with an MS specialist nurse in addition to usual care. The appointment will focus on psycho-education. More specifically, the MS specialist nurse will provide the participants with information on the frequently affected cognitive domains in MS and their relation to brain path- ology. This will occur in a standardised manner. Interventions For the MBCT group, ad- herence will be assessed during the entire treatment period with a calendar on which participants fill out whether they adhere to both formal (e.g. the sitting meditation) and informal (e.g. 3-min breathing space) mindfulness exercises. Demographic and patient characteristics g p p At baseline, the following demographic characteristics are collected: age, gender, work status and education. In addition, the following clinical characteristics will be noted: comorbid condition as defined by the Cumulative Illness Rating Scale (CIRS) [37], subtype of MS, year of diagnosis, disease duration and MS disability as defined by the Expanded Disability Status Scale (EDSS) [38]. If an EDSS score is not available, or if this score has been determined more than three months ago, a new EDSS score will be gathered at baseline. The use of medication will be noted at each assessment. Health care consump- tion will be measured with a questionnaire on healthcare utilisation and productivity losses in patients with a psy- chiatric disorder (TIC-P) [39] and will be administered at each measurement. Table 1 presents an overview of the demographic and patient characteristics that will be collected at each assessment. Teacher ratings CRT and MBCT sessions will be recorded on video to evaluate teacher competence and protocol adherence. These video recordings will solely be used for the pur- pose of trainer evaluation, and the camera will be di- rected at the trainer. For the CRT sessions, adherence to the protocol will be checked using a checklist. For the MBCT sessions, the Mindfulness-Based Interventions - Teachers Assessment Criteria [36] will be used. Primary outcome measure The primary outcome measure is the level of subjective cognitive complaints and is measured with the CFQ [23]. Subjective cognitive complaints in terms of execu- tive function will be measured with the Behaviour Rating Inventory of Executive Function – Adult Version (BRIEF-A) [40]. The BRIEF-A consists of a self- and an informant report version. Adherence For all groups, attendance to the sessions will be docu- mented. For the CRT group, adherence to homework Page 6 of 10 Nauta et al. BMC Neurology (2017) 17:201 Table 1 Overview of outcome measures per assessment Assessment Baseline Post-intervention Follow-up Demographic characteristics X Medical history (e.g. MS subtype) X Use of medication X X X Expanded Disability Status Scale (EDSS) X Health care consumption X X X Questionnaires measuring subjective cognitive complaints, psychological symptoms, quality of life, well-being and daily life functioning X X X Neuropsychological assessment X X X Magnetoencephalography (MEG) X X X Qualitative data to improve the interventions X Qualitative data to improve the interventions with available scripts [49]. The MEG data will be used to determine resting-state functional connectivity and brain network organisation. To study functional connectivity, synchronisation measures will be computed, such as the phase-lag index [7, 50]. To study brain network organ- isation, tools from modern network theory will be ap- plied to the entire network and a subset of the network (i.e. the minimum spanning tree (MST)) [8, 50]. Mea- sures such as degree, clustering coefficient and path length will be computed, as well as MST-network de- rived measures, such as betweenness centrality, tree hierarchy and eccentricity. There will be an emphasis on node centrality measures to identify the ‘hubs’ (i.e. highly connected nodes) of the network [51]. Since the field of modern network science is constantly developing, the best methods and measures will be selected once the study is completed. Secondary outcome measures Cognitive function A test battery based on the Minimal Assessment of Cognitive Function in MS (MACFIMS) will be used [41]. Verbal learning and memory is assessed with the Dutch version of the California Verbal learning Test (CVLT) [42]. Spatial learning and memory are measured with the Brief Visuospatial Memory Test-Revised (BVMT- R) [43]. Visual-spatial abilities are measured with the Benton Judgment of Line Orientation Test (JLO) [44]. Visual processing speed and working memory are mea- sured with the Symbol Digit Modalities Test (SDMT) [45]. Verbal fluency and memory retrieval are assessed with the Controlled Oral Word Association Test (COWAT) [46]. Higher executive function is measured with the Delis- Kaplan Executive Function System sorting test (D-KEFS) free sorting condition [47]. Selective attention and response inhibition are measured with the Stroop Colour-Word Test [48]. When available, parallel versions of tests will be administered for repeated assessment to ac- count for material-specific learning effects. Psychological symptoms Depression and anxiety are measured with the Hospital Anxiety and Depression Scale (HADS) [52]. The level of fatigue is measured with the Checklist Individual Strength-20-r (CIS-20-r) [53]. The tendency to ruminate when being sad or depressed is measured with the subscale ‘brooding’ of the Dutch Ruminative Response Scale (RRS-NL) [54]. Quality of life Health-related quality of life is measured with the Multiple Sclerosis Quality of Life Questionnaire (MSQoL-54) [55]. Well-being Emotional, psychological and social well- being is measured with the Mental Health Continuum- Short Form (MHC-SF) [56]. The ability to be mindful, that is, non-judgmental awareness of moment-to-moment experience, is measured with the Five Facets of the Mind- fulness Questionnaire short form (FFMQ-SF) [57]. Self- compassion, that is, the ability to act with compassion towards oneself in difficult times, is measured with the short form of the Self-Compassion Scale (SCS-SF) [58]. Functional brain networks Resting-state MEG data will be recorded using a 306-channel whole-head MEG system (Elekta Neuromag Inc., Helsinki, Finland) in a magnetically shielded room (Vacuumschmelze GmbH, Hanau, Germany) at the VU University Medical Center. Magnetic fields will be recorded during resting state (i.e. a no-task, eyes-closed condition). Pre-processing of data and removal of noise will be done on Linux computers Page 7 of 10 Page 7 of 10 Nauta et al. BMC Neurology (2017) 17:201 Daily life functioning Participation in society is mea- sured with the Utrecht Scale for Evaluation of Rehabili- tation – Participation (USER-P) [59]. Monitoring and harms A i d d i An independent monitor, the Clinical Research Bureau (CRB) of the VU University Medical Center, will monitor the data of this study according to GCP. The CRB will check the following aspects of the participants: (1) in- formed consents, (2) source data verification, (3) the re- ported (serious) adverse events ((S)AEs). Considering the nature of this study, SAEs are not expected. All AEs that are reported spontaneously by the participant or observed by the research staff or therapists will be recorded. All SAEs will be reported by the investigator to the sponsor, and the sponsor will inform the accredited Medical Ethics Committee (MEC). Secondary outcome measures Goal Attainment Scaling (GAS) is used to determine the effect of the treatment on personalised goals in daily situations [60]. ETAU, MBCT vs. ETAU, CRT vs. MBCT, drop-outs vs. treatment completers) in demographic and clinical charac- teristics and outcome measurements at baseline are analysed using independent samples t-tests (normally dis- tributed continuous outcome variables), Mann-Whitney U tests (skewed continuous outcome variables) and Pearson’s chi-square tests (categorical outcome variables). Primary and secondary objectives Primary and secondary objectives To evaluate the effectiveness of the interventions, mixed-model analyses will be performed for the primary and secondary outcome measurements with time (base- line, post-intervention, follow-up) as a within subjects fac- tor and condition (CRT vs. ETAU, MBCT vs. ETAU, and exploratory: CRT vs. MBCT) as a between-subjects factor. These analyses will be performed using an intention-to- treat approach, including all randomised participants re- gardless of adherence and measurement completion. Secondarily, per-protocol analyses will be performed for further exploration of the intervention effects. To evaluate the secondary study aims, mediation and moderation analyses [61] will be performed to evaluate whether alterations in functional brain networks play a role in the effect of the interventions. In addition, cross- sectional associations between functional brain networks and cognitive function (subjective and objective) will be analysed using Pearson’s correlation and linear regression analyses. To evaluate whether alterations in secondary study parameters are mediating factors that determine subjective cognitive function, mediation analyses [61] and linear regression analyses will be performed. Fi- nally, logistic and linear regression models will be per- formed to evaluate which factors determine whether a patient is likely to benefit from one of the therapies. Data management The collected data will be labelled with a participant identification code. The name and other identifiers of the participant will be removed from the data. The link between the participant identification code and the names of the participants will be kept separately. An electronic case report form is developed according to the guidelines of Good Clinical Practice (GCP) to docu- ment the data collected in the study. This case report form will include demographic and clinical characteris- tics, and all outcomes of the study parameters. The data will be treated confidentially and will only be available to the trial coordinator and principal investigator. Other in- vestigators can only get access to the data for the pur- pose of research and with permission of the principal investigator. The data gathered in this study will be pro- tected in accordance with the Dutch Personal Data Pro- tection Act and the Medical Treatment Contracts Act. For all analyses, confounding variables will be inserted, such as age and education. Bonferroni corrections will be applied to correct for multiple comparisons within each objective. Randomisation and blinding Following baseline assessment, participants will be ran- domly allocated to one of three treatment arms (MBCT, CRT or ETAU). First, the location of the intervention will be determined based on the patient’s living location and preference. For each location, randomisation will be performed in variable blocks of 6 and 9, and with an 1:1:1 allocation ratio. A minimisation program will be used to ensure balance between all groups. Minimisation will be performed on three factors: (1) subjective cogni- tive function, (2) age and (3) gender. Weighting is equal for each factor. The minimisation program will be con- structed before the start of the study by an independent scientific programmer. The randomisation procedure will be performed by a researcher who is not involved in administering any outcome measure. Outcome measure- ments will be administered by assessors who are blind to treatment assignment, but this blinding is not feasible with regard to participants and therapists. Prior to each post-measurement, participants will be reminded not to disclose their group allocation to the assessor. Authorship Th i i The principal investigator will justify the names for authorship. Individuals who fulfil authorship criteria will be an author on the manuscripts. An important strength of our study is that we use functional brain network measures, such as functional connectivity and nodal centrality, as an outcome vari- able. These measures may help to explain treatment ef- fects and may provide information on whether network deterioration can be halted. Additionally, if functional brain network measures at baseline predict treatment outcomes, network analyses can be used as a prognostic factor. We will also relate functional brain network mea- sures to objective and subjective cognitive problems in MS, which may help to understand the overlap and dis- tinctiveness between these types of cognitive problems. Dissemination policy When the data collection is completed, the total data set will be analysed and the results will be published in scientific journals and presented at (inter)national scientific meetings. A summary of the results will be released to the participants of this study. The identity of the participants will not be disclosed in any of these publication forms. The researchers of this study will attempt to reduce the time between the completion of data collection and the release of the study results. Statistical analysis Descriptive statistics Data on demographic and clinical characteristics will be summarised in a table. For adherence and other feasibility indicators, frequencies and percentages will be calculated. Satisfaction with the program will be summarised in quali- tative descriptions. Differences between groups (CRT vs. Nauta et al. BMC Neurology (2017) 17:201 Nauta et al. BMC Neurology (2017) 17:201 Page 8 of 10 Page 8 of 10 Ancillary and post-trial care At the end of the study, participants can take part in one of the other interventions of the study. The REMIND-MS study does not provide additional care outside these interventions. Author details 1 1Department of Neurology, Amsterdam Neuroscience, MS Center Amsterdam, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, the Netherlands. 2Department of Psychiatry, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, the Netherlands. 3Donders Institute for Brain, Cognition and Behaviour, Radboud University, PO Box 9101, 6500 HB Nijmegen, the Netherlands. 4Department of Medical Psychology, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, the Netherlands. 5Department of Anatomy and Neurosciences, Amsterdam Neuroscience, MS Center Amsterdam, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, the Netherlands. 6Department of Rehabilitation Medicine, MS Center Amsterdam, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, the Netherlands. 7Klimmendaal Rehabilitation Center, PO Box 9044, 6800 CG Arnhem, the Netherlands. USER-P: Utrecht Scale for Evaluation of Rehabilitation – Participation; VUmc: VU University Medical Center Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Authors’ contributions BAJ initiated the study, applied for funding and is the principal investigator. IMN is the study coordinator, drafted this manuscript and will perform the data collection. BAJ, LF, AEMS, RPCK, JJGG, VG and BMJU were involved in the conception and design of the study. LF contributed his CRT expertise to the study protocol development and the CRT intervention protocol, and he will train and supervise the CRT therapists. AEMS contributed her mindfulness expertise to the study protocol development and the MBCT intervention protocol, and she will train and supervise the MBCT therapists. RPCK made important statistical contributions. All authors provided feedback on drafts of this paper and read and approved the final manuscript. In summary, this study may help to unravel and treat cognitive problems among MS patients. Availability of data and materials h d h ll b ll d h The data that will be collected in this study will be available from the principal investigator on reasonable request. Acknowledgements ld l k h We would like to thank the Dutch MS Research Foundation for funding our project. Discussion the protocol, and will not have any impact on data generation, statistical analyses or writing of the final manuscript. The best treatment options for cognitive problems in MS patients are still a matter of debate. This study will therefore investigate the effect of two promising non- pharmacological treatments: MBCT and CRT. To our knowledge, this will be the first RCT that investigates the effect of MBCT on cognitive function among MS patients. In addition, studying the effect of CRT on cog- nitive function may provide direction to the contradict- ory evidence that is currently available [12, 13]. If these treatments appear to be effective, we will investigate which factors predict this beneficial effect. These prog- nostic factors may lead towards tailored treatments for MS patients who suffer from cognitive problems. Abbreviations AE Ad This study has been reviewed and approved by the MEC of the VU University Medical Center (METC 2017.009, protocol version 5, 19 May 2017) and the Scientific Research Committee of Amsterdam Neuroscience (CWO nr.16–14). All substantial protocol amendments undergo review and approval by the MEC of the VU University Medical Center. All participants will provide written informed consent prior to study participation. The study is registered in Dutch Trial registry (NTR6459). Abbreviations AE: Adverse event; BRIEF-A: Behaviour Rating Inventory of Executive Function – Adult Version; BVMT-R: Brief visuospatial memory test-revised; CFQ: Cognitive failure questionnaire; CIRS: Cumulative illness rating scale; CIS-20-r: Checklist individual strength-20-r; COWAT: Controlled oral word association test; CRB: Clinical Research Bureau; CRT: Cognitive rehabilitation therapy; CVLT: California verbal learning test; CWO: Scientific Research Committee; D-KEFS: Delis-Kaplan Executive Function System sorting test; EDSS: Expanded disability status scale; ETAU: Enhanced treatment as usual; FFMQ-SF: Five facets of mindfulness questionnaire short form; GAS: Goal attainment scale; GCP: Good clinical practice; HADS: Hospital anxiety and depression scale; JLO: Judgment of line orientation test; KR: Klimmendaal Rehabilitation Center; MBCT: Mindfulness-based cognitive therapy; MEC: Medical Ethics Committee; MEG: Magnetoencephalography; MHC-SF: Mental Health Continuum-Short Form; MS: Multiple sclerosis; MSNQ-P: Multiple Sclerosis Neuropsychological Questionnaire – Patient version; MSQoL-54: Multiple sclerosis quality of life questionnaire; MST: Minimum spanning tree; RCT: Randomised controlled trial; REMIND-MS: Cognitive Rehabilitation and Mindfulness in Multiple Sclerosis; RRS-NL: Dutch Ruminative Response Scale; SAE: Serious adverse event; SCS-SF: Short form of the self-compassion scale; SDMT: Symbol digit modalities test; TIC-P: Questionnaire on healthcare utilisation and productivity losses in patients with a psychiatric disorder; USER-P: Utrecht Scale for Evaluation of Rehabilitation – Participation; VUmc: VU University Medical Center Consent for publication Not applicable. Competing interests The authors declare to have no competing interests. Competing interests The authors declare to have no competing interests. References 38. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology. 1983;33(11):1444–52. 13. Rosti-Otajarvi EM, Hamalainen PI. Neuropsychological rehabilitation for multiple sclerosis. Cochrane Database Syst Rev. 2014;2:CD009131. 14. Tang YY, Holzel BK, Posner MI. The neuroscience of mindfulness meditation. Nat Rev Neurosci. 2015;16(4):213–25. 39. Bouwmans C, Jong KD, Timman R, Zijlstra-Vlasveld M, Van d, Feltz-Cornelis C, Tan SS, et al. Feasibility, reliability and validity of a questionnaire on healthcare consumption and productivity loss in patients with a psychiatric disorder (TiC-P). BMC Health Serv Res. 2013;13 15. Chiesa A, Calati R, Serretti A. Does mindfulness training improve cognitive abilities? A systematic review of neuropsychological findings. Clin Psychol Rev. 2011;31(3):449–64. 40. Roth RM, Isquith PK, Gioia GA, Widows M. Development of the Behavior Rating Inventory of Executive Function-Adult version. Arch Clin Neuropsych. 2005;20(7):906. 16. Fox KC, Nijeboer S, Dixon ML, Floman JL, Ellamil M, Rumak SP, et al. Is meditation associated with altered brain structure? A systematic review and meta-analysis of morphometric neuroimaging in meditation practitioners. Neurosci Biobehav Rev. 2014;43:48–73. 41. Benedict RH, Cookfair D, Gavett R, Gunther M, Munschauer F, Garg N, et al. Validity of the minimal assessment of cognitive function in multiple sclerosis (MACFIMS). J Int Neuropsychol Soc. 2006;12(4):549–58. 17. Tomasino B, Chiesa A, Fabbro F. Disentangling the neural mechanisms involved in Hinduism- and Buddhism-related meditations. Brain Cogn. 2014;90:32–40. 42. Mulder JL, Dekker R, Dekker DH. Verbale Leer- & Geheugen test: Handleiding [Verbal Learning & Memory Test: manual]. Lisse, the Netherlands: Swets & Zeitlinger; 1996. 18. Bogosian A, Chadwick P, Windgassen S, Norton S, McCrone P, Mosweu I, et al. Distress improves after mindfulness training for progressive MS: a pilot randomised trial. Mult Scler. 2015;21(9):1184–94. 43. Benedict RHB, Schretlen D, Groninger L, Dobraski M, Shpritz B. Revision of the brief visuospatial memory test: studies of normal performance, reliability, and validity. Psychol Assessment. 1996;8(2):145–53. 19. Blankespoor RJ, Schellekens MPJ, Vos SH, Speckens AEM, de Jong BA. The effectiveness of mindfulness-based stress reduction on psychological distress and cognitive functioning in patients with multiple sclerosis: a pilot study. Mindfulness. 2017; doi:10.1007/s12671-017-0701-6. 44. Benton AL, Hamsher KD, Varney NR, Spreen O. Judgment of line orientation. New York: Oxford University Press; 1983. 45. Smith A. Symbol digit modality test (SDMT): manual (revised). Los Angeles. Psychol Serv. 1982; 20. Grossman P, Kappos L, Gensicke H, D'Souza M, Mohr DC, Penner IK, et al. Funding Thi i This investigator-initiated trial is funded by the Dutch MS Research Founda- tion (project number 15–911). The funder had no influence on the design of Page 9 of 10 Page 9 of 10 Nauta et al. BMC Neurology (2017) 17:201 Nauta et al. BMC Neurology (2017) 17:201 Nauta et al. BMC Neurology (2017) 17:201 Received: 13 July 2017 Accepted: 12 November 2017 References Rocca MA, Amato MP, De Stefano N, Enzinger C, Geurts JJ, Penner IK, et al. Clinical and imaging assessment of cognitive dysfunction in multiple sclerosis. Lancet Neurol. 2015;14(3):302–17. 31. Zedlitz AM, Fasotti L, Geurts AC. Post-stroke fatigue: a treatment protocol that is being evaluated. Clin Rehabil. 2011;25(6):487–500. 32. Zedlitz AM, Rietveld TC, Geurts AC, Fasotti L. Cognitive and graded activity training can alleviate persistent fatigue after stroke: a randomized, controlled trial. Stroke. 2012;43(4):1046–51. 7. Tewarie P, Schoonheim MM, Stam CJ, van der Meer ML, van Dijk BW, Barkhof F, et al. Cognitive and clinical dysfunction, altered MEG resting-state networks and thalamic atrophy in multiple sclerosis. PLoS One. 2013;8(7):e69318. 33. Geusgens C, Baars-Elsinga A, Visser-Meily A, van Heugten C. Niet Rennen maar Plannen: Trends Service in kommunikatie B.V.; 2012. 8. Tewarie P, Hillebrand A, Schoonheim MM, van Dijk BW, Geurts JJ, Barkhof F, et al. Functional brain network analysis using minimum spanning trees in multiple sclerosis: an MEG source-space study. NeuroImage. 2014;88:308–18. 34. Segal ZV, Williams JMG, Teasdale JD. Mindfulness-based cognitive therapy for depression. second ed. New York: The Guilford Press; 2013. 9. Hulst HE, Gehring K, Uitdehaag BM, Visser LH, Polman CH, Barkhof F, et al. Indicators for cognitive performance and subjective cognitive complaints in multiple sclerosis: a role for advanced MRI? Mult Scler. 2014;20(8):1131–4. 35. Good practice guidelines for teaching mindfulness-based courses. UK Mindfulness-Based Teacher Trainer Network. 2011. http://mindfulnessteachersuk. org.uk/pdf/teacher-guidelines.pdf. Accessed 1 June 2017. 10. Lovera J, Kovner B. Cognitive impairment in multiple sclerosis. Curr Neurol Neurosci Rep. 2012;12(5):618–27. 36. Crane RS, Kuyken W, Williams JM, Hastings RP, Cooper L, Fennell MJ. Competence in teaching mindfulness-based courses: concepts, development and assessment. Mindfulness (N Y). 2012;3(1):76–84. 11. Cicerone KD, Langenbahn DM, Braden C, Malec JF, Kalmar K, Fraas M, et al. Evidence-based cognitive rehabilitation: updated review of the literature from 2003 through 2008. Arch Phys Med Rehabil. 2011;92(4):519–30. 37. Miller MD, Paradis CF, Houck PR, Mazumdar S, Stack JA, Rifai AH, et al. Rating chronic medical illness burden in Geropsychiatric practice and research - application of the cumulative illness rating-scale. Psychiatry Res. 1992;41(3):237–48. 12. Mitolo M, Venneri A, Wilkinson ID, Sharrack B. Cognitive rehabilitation in multiple sclerosis: a systematic review. J Neurol Sci. 2015;354(1–2):1–9. 12. Mitolo M, Venneri A, Wilkinson ID, Sharrack B. Cognitive rehabilitation in multiple sclerosis: a systematic review. J Neurol Sci. 2015;354(1–2):1–9. 13. Rosti-Otajarvi EM, Hamalainen PI. Neuropsychological rehabilitation for multiple sclerosis. Cochrane Database Syst Rev. 2014;2:CD009131. References 1. Chiaravalloti ND, DeLuca J. Cognitive impairment in multiple sclerosis. Lancet Neurol. 2008;7(12):1139–51. 1. Chiaravalloti ND, DeLuca J. Cognitive impairment in multiple sclerosis. Lancet Neurol. 2008;7(12):1139–51. 26. Fasotti L, Kovacs F, Eling PATM, Brouwer WH. Time pressure management as a compensatory strategy training after closed head injury. Neuropsychol Rehabil. 2000;10(1):47–65. 2. Benedict RH, Munschauer F, Linn R, Miller C, Murphy E, Foley F, et al. Screening for multiple sclerosis cognitive impairment using a self- administered 15-item questionnaire. Mult Scler. 2003;9(1):95–101. 2. Benedict RH, Munschauer F, Linn R, Miller C, Murphy E, Foley F, et al. Screening for multiple sclerosis cognitive impairment using a self- administered 15-item questionnaire. Mult Scler. 2003;9(1):95–101. 27. Winkens I, Van Heugten CM, Wade DT, Fasotti L. Training patients in time pressure management, a cognitive strategy for mental slowness. Clin Rehabil. 2009;23(1):79–90. 3. Kinsinger SW, Lattie E, Mohr DC. Relationship between depression, fatigue, subjective cognitive impairment, and objective neuropsychological functioning in patients with multiple sclerosis. Neuropsychology. 2010;24(5):573–80. 3. Kinsinger SW, Lattie E, Mohr DC. Relationship between depression, fatigue, subjective cognitive impairment, and objective neuropsychological functioning in patients with multiple sclerosis. Neuropsychology. 2010;24(5):573–80. 28. van Kessel M, Fasotti L, Berg I, van Hout M, Wekking E. Training geheugenstrategieën. Amsterdam: Boom Uitgevers; 2010. 4. Wynia K, Middel B, van Dijk JP, De Keyser JH, Reijneveld SA. The impact of disabilities on quality of life in people with multiple sclerosis. Mult Scler. 2008;14(7):972–80. 4. Wynia K, Middel B, van Dijk JP, De Keyser JH, Reijneveld SA. The impact of disabilities on quality of life in people with multiple sclerosis. Mult Scler. 2008;14(7):972–80. 29. Boelen DH, Spikman JM, Fasotti L. Rehabilitation of executive disorders after brain injury: are interventions effective? J Neuropsychol. 2011;5(Pt 1):73–113. 30. Spikman JM, Boelen DH, Lamberts KF, Brouwer WH, Fasotti L. Effects of a multifaceted treatment program for executive dysfunction after acquired brain injury on indications of executive functioning in daily life. J Int Neuropsychol Soc. 2010;16(1):118–29. 5. DeLuca GC, Yates RL, Beale H, Morrow SA. Cognitive impairment in multiple sclerosis: clinical, radiologic and pathologic insights. Brain Pathol. 2015; 25(1):79–98. 5. DeLuca GC, Yates RL, Beale H, Morrow SA. Cognitive impairment in multiple sclerosis: clinical, radiologic and pathologic insights. Brain Pathol. 2015; 25(1):79–98. 6. Rocca MA, Amato MP, De Stefano N, Enzinger C, Geurts JJ, Penner IK, et al. Clinical and imaging assessment of cognitive dysfunction in multiple sclerosis. Lancet Neurol. 2015;14(3):302–17. 6. Received: 13 July 2017 Accepted: 12 November 2017 but reduces perceived cognitive deficits in patients with multiple sclerosis: a randomised, controlled, multi-centre trial. Mult Scler. 2014;20(1):99–107. but reduces perceived cognitive deficits in patients with multiple sclerosis: a randomised, controlled, multi-centre trial. Mult Scler. 2014;20(1):99–107. 25. Rosti-Otajarvi E, Mantynen A, Koivisto K, Huhtala H, Hamalainen P. Neuropsychological rehabilitation has beneficial effects on perceived cognitive deficits in multiple sclerosis during nine-month follow-up. J Neurol Sci. 2013;334(1–2):154–60. References MS quality of life, depression, and fatigue improve after mindfulness training: a randomized trial. Neurology. 2010;75(13):1141–9. 46. Benton LA, Hamsher KD, Sivan AB. Controlled oral word association test. Multilingual aphasia examination. 3 ed. Iowa City, IA: AJA; 1994. 21. Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol. 2011;69(2):292–302. 47. Delis DC, Kaplan E, Kramer JH. The delis-Kaplan executive function system: Examiner's manual. San Antonio: The Psychological Corporation; 2001. 22. Sonder JM, Mokkink LB, van der Linden FA, Polman CH, Uitdehaag BM. Validation and interpretation of the Dutch version of the multiple sclerosis neuropsychological screening questionnaire. J Neurol Sci. 2012;320(1–2):91–6. 48. Hammes JGW. The STROOP color-word test: manual. Amsterdam: Swets and Zeitlinger; 1973. 49. Hillebrand A, Barnes GR, Bosboom JL, Berendse HW, Stam CJ. Frequency- dependent functional connectivity within resting-state networks: an atlas- based MEG beamformer solution. NeuroImage. 2012;59(4):3909–21. 23. Merckelbach H, Muris P, Nijman H, de Jong PJ. Self-reported cognitive failures and neurotic symptomatology. Pers Indiv Differ. 1996;20(6):715–24. 24. Mantynen A, Rosti-Otajarvi E, Koivisto K, Lilja A, Huhtala H, Hamalainen P. Neuropsychological rehabilitation does not improve cognitive performance 50. Stam CJ, van Straaten ECW. The organization of physiological brain networks. Clin Neurophysiol. 2012;123(6):1067–87. Page 10 of 10 Nauta et al. BMC Neurology (2017) 17:201 Nauta et al. BMC Neurology (2017) 17:201 51. Stam CJ. Modern network science of neurological disorders. Nat Rev Neurosci. 2014;15(10):683–95. 52. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiat. Scandinavica. 1983;67(6):361–70. Psychiat. Scandinavica. 1983;67(6):361–70. 53. Vercoulen JHMM, Alberts M, Bleijenberg G. De checkl spankracht (CIS). Gedragstherapie. 1999;32:131–6. 54. Raes F, Hermans D, Eelen P. Kort instrumenteel De Nederlandstalige versie van de Ruminative Response Scale (RRS-NL) en de Rumination on Sadness Scale (RSS-NL). Gedragstherapie. 2003;36(2):97–104. 55. Vickrey BG, Hays RD, Harooni R, Myers LW, Ellison GWA. Health-related quality-of-life measure for multiple-sclerosis. Qual Life Res. 1995;4(3):187–206. quality-of-life measure for multiple-sclerosis. Qual Life Res. 1995;4(3):187 56. Lamers SMA, Westerhof GJ, Bohlmeijer ET, ten Klooster PM, Keyes CLM. Evaluating the psychometric properties of the mental health continuum- short form (MHC-SF). J Clin Psychol. 2011;67(1):99–110. 57. Bohlmeijer E, ten Klooster PM, Fledderus M, Veehof M, Baer R. Psychometric properties of the five facet mindfulness questionnaire in depressed adults and development of a short form. Assessment 2011;18(3):308–320. 58. Raes F, Pommier E, Neff KD, Van Gucht D. Nauta et al. BMC Neurology (2017) 17:201 References Construction and factorial validation of a short form of the self-compassion scale. Clin Psychol Psychot 2011;18(3):250–255. 59. Post MWM, van der Zee CH, Hennink J, Schafrat CG, Visser-Meily JMA, van Berlekom SB. Validity of the Utrecht scale for evaluation of rehabilitation- participation. Disabil Rehabil. 2012;34(6):478–85. 60. Kiresuk TJ, Sherman RE. Goal attainment scaling - general method for evaluating comprehensive community mental health programs. Community Ment Hlt J. 1968;4(6):443–53. 61. Holmbeck GN. Toward terminological, conceptual, and statistical clarity in the study of mediators and moderators: examples from the child-clinical and pediatric psychology literatures. J Consult Clin Psychol. 1997;65(4):599–610. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W3022286624	https://europepmc.org/articles/pmc7845155?pdf=render	English	null	An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action	F1000Research	2,021	cc-by	26,758	F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Abstract Research software has become a central asset in academic research. It optimizes existing and enables new research methods, implements and embeds research knowledge, and constitutes an essential research product in itself. Research software must be sustainable in order to understand, replicate, reproduce, and build upon existing research or conduct new research effectively. In other words, software must be available, discoverable, usable, and adaptable to new needs, both now and in the future. Research software therefore requires an environment that supports sustainability. Willi Hasselbring , Kiel University, Kiel, Germany 1. Hence, a change is needed in the way research software development and maintenance are currently motivated, incentivized, funded, structurally and infrastructurally supported, and legally treated. Failing to do so will threaten the quality and validity of research. In this paper, we identify challenges for research software sustainability in Germany and beyond, in terms of motivation, selection, research software engineering personnel, funding, infrastructure, and legal aspects. Besides researchers, we specifically address political and academic decision-makers to increase awareness of the importance and needs of sustainable research software practices. In particular, we recommend strategies and measures to create an environment for sustainable research software, with the ultimate goal to ensure that software-driven research is valid, reproducible and sustainable, and that software is recognized as a first class citizen in research. This paper is the outcome of two workshops run in Germany in 2019, at deRSE19 - the first International Conference of Research Software Engineers in Germany - and a dedicated DFG-supported follow-up workshop in Berlin. F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 27FIZ Karlsruhe - Leibniz Institute for Information Infrastructure, Karlsruhe, Germany 28University of Goettingen, Göttingen, Germany 29University of Münster, Münster, Germany 30Institute for Advanced Sustainability Studies, Potsdam, Germany 31Ulm University, Ulm, Germany 32Linköping University, Linköping, Sweden 33Ludwig Maximilian University of Munich, München, Germany 34Leibniz University Hannover, Hannover, Germany 35Eindhoven University of Technology, Eindhoven, The Netherlands 36Julius Kühn-Institut (JKI), Quedlinburg, Germany * Equal contributors First published: 27 Apr 2020, 9:295 https://doi.org/10.12688/f1000research.23224.1 Latest published: 26 Jan 2021, 9:295 https://doi.org/10.12688/f1000research.23224.2 v2 OPINION ARTICLE An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action [version 2; peer review: 2 approved] Hartwig Anzt1,2, Felix Bach 1, Stephan Druskat 3-5, Frank Löffler3,6, Axel Loewe 1, Bernhard Y. Renard7, Gunnar Seemann 8, Alexander Struck 5, Elke Achhammer9, Piush Aggarwal 10, Franziska Appel11, Michael Bader9, Lutz Brusch 12, Christian Busse 13, Gerasimos Chourdakis 9, Piotr Wojciech Dabrowski 14, Peter Ebert15, Bernd Flemisch16, Sven Friedl 17, Bernadette Fritzsch18, Maximilian D. Funk19, Volker Gast3, Florian Goth20, Jean-Noël Grad 16, Jan Hegewald 18, Sibylle Hermann16, Florian Hohmann21, Stephan Janosch22, Dominik Kutra 23, Jan Linxweiler 24, Thilo Muth 25, Wolfgang Peters-Kottig 26, Fabian Rack27, Fabian H.C. Raters 28, Stephan Rave 29, Guido Reina 16, Malte Reißig 30, Timo Ropinski31,32, Joerg Schaarschmidt1, Heidi Seibold 33, Jan P. Thiele 34, Benjamin Uekermann 35, Stefan Unger36, Rudolf Weeber16 1Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany 2University of Tennessee, Knoxville, TN, USA 3Friedrich Schiller University, Jena, Germany 4German Aerospace Center (DLR), Berlin, Germany 5Humboldt-Universität zu Berlin, Berlin, Germany 6Louisiana State University, Baton Rouge, LA, USA 7Hasso Plattner Institute, Digital Engineering Faculty, University of Potsdam, Potsdam, Germany 8University Heart Centre Freiburg Bad Krozingen, Freiburg, Germany 9Technische Universität München, München, Germany 10Universität Duisburg-Essen, Duisburg, Germany 11Leibniz Institute of Agricultural Development in Transition Economies (IAMO), Halle (Saale), Germany 12Technische Universität Dresden, Dresden, Germany 13Deutsches Krebsforschungszentrum, Heidelberg, Germany 14Hochschule für Technik und Wirtschaft Berlin, Berlin, Germany 15Saarland Informatics Campus, Saarbrücken, Germany 16University of Stuttgart, Stuttgart, Germany 17Berlin Institute of Health, Berlin, Germany 18Alfred Wegener Institute, Bremerhaven, Germany 19Max-Planck-Gesellschaft e.V., München, Germany 20Universität Würzburg, Würzburg, Germany 21Universität Bremen, Bremen, Germany 22Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 23European Molecular Biology Laboratory, Heidelberg, Germany 24Technische Universität Braunschweig, Braunschweig, Germany 25Federal Institute for Materials Research and Testing, Berlin, Germany 26Konrad-Zuse-Zentrum für Informationstechnik Berlin (ZIB), Berlin, Germany 16University of Stuttgart, Stuttgart, Germany 17Berlin Institute of Health, Berlin, Germany 18Alfred Wegener Institute, Bremerhaven, Germany Page 1 of 34 This article is included in the Science Policy Research gateway. This article is included in the Science Policy Research gateway. This article is included in the Science Policy Research gateway. This article is included in the Science Policy Research gateway. ng authors: Axel Loewe (axel.loewe@kit.edu), Gunnar Seemann (gunnar.seemann@universitaets-herzzentrum.de Author roles: Anzt H: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing; Bach F: Conceptualization, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing; Druskat S: Conceptualization, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing; Löffler F: Conceptualization, Investigation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Loewe A: Conceptualization, Funding Acquisition, Investigation, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing; Renard BY: Conceptualization, Investigation, Writing – Original Draft Preparation, Writing – Review & Editing; Seemann G: Conceptualization, Funding Acquisition, Investigation, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing; Struck A: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing; Achhammer E: Writing – Original Draft Preparation; Aggarwal P: Writing – Original Draft Preparation; Appel F: Writing – Original Draft Preparation, Writing – Review & Editing; Bader M: Writing – Original Draft Preparation, Writing – Review & Editing; Brusch L: Writing – Original Draft Preparation, Writing – Review & Editing; Busse C: Writing – Review & Editing; Chourdakis G: Writing – Review & Editing; Dabrowski PW: Writing – Review & Editing; Ebert P: Writing – Original Draft Preparation; Flemisch B: Writing – Original Draft Preparation; Friedl S: Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Fritzsch B: Writing – Review & Editing; Funk MD: Writing – Original Draft Preparation, Writing – Review & Editing; Gast V: Writing – Review & Editing; Goth F: Writing – Original Draft Preparation; Grad JN: Writing – Original Draft Preparation; Hegewald J: Writing – Review & Editing; Hermann S: Writing – Original Draft Preparation, Writing – Review & Editing; Hohmann F: Writing – Original Draft Preparation; Janosch S: Writing – Review & Editing; Kutra D: Writing – Original Draft Preparation; Linxweiler J: Writing – Original Draft Preparation; Muth T: Writing – Original Draft Preparation; Peters-Kottig W: Writing – Original Draft Preparation; Rack F: Writing – Original Draft Preparation, Writing – Review & Editing; Raters FHC: Writing – Original Draft Preparation; Rave S: Writing – Original Draft Preparation; Reina G: Writing – Original Draft Preparation, Writing – Review & Editing; Reißig M: Writing – Review & Editing; Ropinski T: Writing – Original Draft Preparation; Schaarschmidt J: Writing – Original Draft Preparation; Seibold H: Writing – Review & Editing; Thiele JP: Writing – Original Draft Preparation, Writing – Review & Editing; Uekermann B: Writing – Original Draft Preparation, Writing – Review & Editing; Unger S: Visualization, Writing – Original Draft Preparation; Weeber R: Writing – Original Draft Preparation Competing interests: No competing interests were disclosed. Keywords Sustainable Software Development, Academic Software, Software Infrastructure, Software Training, Software Licensing, Research Software Page 2 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 This article is included in the Science Policy Research gateway. Grant information: The authors thank the Deutsche Forschungsgemeinschaft (DFG) for funding a meeting (Rundgespräch, grants LO 2093/3-1 and SE 1758/6-1) during which the initial draft of this paper has been created. We are particularly grateful for the support from Dr. Matthias Katerbow (DFG). This work was additionally supported by Research Software Sustainability grants funded by the DFG: Aggarwal: 390886566; PI: Zesch. Appel: 391099391; PI: Balmann. Bach & Loewe & Seemann: 391128822; PIs: Loewe/Scholze/Seemann/Selzer/Streit/Upmeier.Bader: 391134334; PIs: Bader/Gabriel/Frank. Brusch: 391070520; PI: Brusch. Druskat & Gast: 391160252; PI: Gast/Lüdeling. Ebert: 391137747; PI: Marschall.Flemisch & Hermann: 391049448; PIs: Boehringer/Flemisch/Hermann.Hohmann: 391054082; PI: Hepp. Goth: 390966303; PI: Assaad. Grad & Weeber: 391126171; PI: Holm. Kutra: 391125810; PI: Kreshuk.Mehl & Uekermann: 391150578; PIs: Bungartz/Mehl/Uekermann. Peters-Kottig: 391087700; PIs: Gleixner/Peters-Kottig/Shinano/Sperber. Raters: 39099699; PI:Herwartz. Reina: 391302154; PIs: Ertl/Reina. Muth & Renard: 391179955; PIs Renard/Fuchs. Ropinski:391107954; PI: Ropinski. Alexander Struck acknowledges the support of the Cluster of Excellence Matters of Activity. Image Space Material funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – EXC 2025. We acknowledge support by the KIT-Publication Fund of the Karlsruhe Institute of Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Grant information: The authors thank the Deutsche Forschungsgemeinschaft (DFG) for funding a meeting (Rundgespräch, grants LO 2093/3-1 and SE 1758/6-1) during which the initial draft of this paper has been created. We are particularly grateful for the support from Dr. Matthias Katerbow (DFG). This work was additionally supported by Research Software Sustainability grants funded by the DFG: Aggarwal: 390886566; PI: Zesch. Appel: 391099391; PI: Balmann. Bach & Loewe & Seemann: 391128822; PIs: Copyright: © 2021 Anzt H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Anzt H, Bach F, Druskat S et al. An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action [version 2; peer review: 2 approved] F1000Research 2021, 9:295 https://doi.org/10.12688/f1000research.23224.2 How to cite this article: Anzt H, Bach F, Druskat S et al. This article is included in the Science Policy Research gateway. An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action [version 2; peer review: 2 approved] F1000Research 2021, 9:295 https://doi.org/10.12688/f1000research.23224.2 First published: 27 Apr 2020, 9:295 https://doi.org/10.12688/f1000research.23224.1 Page 3 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 In combination with the predominance of temporary positions in research, this results in a highly inefficient system where millions of lines of code are generated every year that will not be re-used after the termination of the developer’s position. Part of the problem is the reluctance to accept research software engineering as an academic profession that results in a lack of incentives to produce high-quality software: producing high software quality needs sufficient resources, and although the San Francisco Declaration on Research Assessment (DORA) demands a change in the academic credit system, many institutions base promotion and appointments on traditional metrics like the Hirsch index4. It is obvious that an extraordi- nary amount of idealism is required to write sustainable code, including documentation and installation routines, as well as running infrastructure and giving support to others when resources can be used more profitably in writing scientific publications based on fragile prototype software5,6. In combination with the predominance of temporary positions in research, this results in a highly inefficient system where millions of lines of code are generated every year that will not be re-used after the termination of the developer’s position. Part of the problem is the reluctance to accept research software engineering as an academic profession that results in a lack of incentives to produce high-quality software: producing high software quality needs sufficient resources, and although the San Francisco Declaration on Research Assessment Background Similarly, it is research software that allows us to get a glimpse of the consequences our actions today have on the climate of tomorrow. However, an implication of computer-based research is that findings and data can only be reproduced, understood, and validated if the software that was used in the research process is sustained and their functionality maintained. We mainly focus on the situation of research software and RSEs in Germany, where funding bodies increasingly acknowl- edge the importance and value of sustainable research software and related infrastructures. The DFG, the largest funding body for fundamental research in Germany, for example, opened a call for sustainable research software development at the end of 2016 and a second call for quality management in research software in June 2019. The first call was oversub- scribed by a factor of 10-15, a strong indicator of unmet demand. As another example, the 2019 “Guidelines for Safeguarding Good Research Practice” codex of the DFG now explicitly lists software side-by-side with other research results and data. The FAIR principles for research data10 provide guidelines for data archiving, but enabling full reproducibility and traceability of research software requires additional steps11. In consequence, there are ongoing discussions on whether software should be considered as a specific kind of research data or as a separate entity12. At the same time, sustaining research software, and in particu- lar open research software, comes with a number of challenges. Commercial research software often has revenue flows that can facilitate sustainable software development, mainte- nance, and documentation as well as the operation of adequate infrastructure. However, a large share of researchers base their research on software that was developed in-house or as a community effort. Many of these software stacks can not be sustained – often because research software was not a first class deliverable in a research project and hence remained in a prototype state, or because of missing incentives and resources to maintain the software after project funding ended. Another fundamental difference to industrial software devel- opment is that most developers of academic research software (often doctoral students or postdoctoral researchers) never receive training in sustainable software development3. In particular, as they see themselves usually as the primary user of a software product, there are virtually no incentives to invest in sustainability measures such as code documentation or portability. Amendments from Version 1 REVISED Besides fixing some typographic errors and adding references as suggested by the reviewers, We separated the legal decision tress from this manuscript. As they were not the focus of this position paper and diluted its key messages, they were published separately: https://doi. org/10.5281/zenodo.4327147 Other aspects that were elaborated on include: testing, infrastructure for cross-institutional use, sustainable funding, the relation between software quality and transparency, a clear statement pro open source, the potential role of legal help desks. (DORA) demands a change in the academic credit system, many institutions base promotion and appointments on traditional metrics like the Hirsch index4. It is obvious that an extraordi- nary amount of idealism is required to write sustainable code, including documentation and installation routines, as well as running infrastructure and giving support to others when resources can be used more profitably in writing scientific publications based on fragile prototype software5,6. Jan Hegewald was added to the list of authors. He already contributed to initial submission but unfortunately his name was missing in the list. Any further responses from the reviewers can be found at the end of the article Thus, one main factor for the poor sustainability of research software is the lack of long-term funding for research software engineers (RSEs)7,8 who take care of the appropriate architecture, organization, implementation, documentation, and community interaction for the software, paired with the implementation of measures towards making the software sustainable during and beyond the development process9. Background Meet Kim, who is currently a post-grad PhD student in researchonomy at the University of Arcadia (UofA). We will follow Kim’s fictional career in order to understand different aspects of research software sustainability. Note that in Kim’s world, many of the changes this paper calls for have already been implemented. (In our example, Kim is a female person. Of course, research software engineers (RSEs) can be of any gender.) In this paper, we describe the state of the practice and current challenges for research software sustainability and suggest measures towards improvements that can solve these challenges. The paper is the result of a community effort, with work under- taken during two workshops and subsequent collaborative work across the larger RSE community in Germany. It has been initiated during a half-day workshop at first International Confer- ence for Research Software Engineers in Germany (deRSE19) in Potsdam, Germany on June 5th, 2019, and continued during a dedicated two-day workshop in Berlin, Germany on November 7th and 8th, 2019, which was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG). Subse- quently, the draft produced during the latter event was opened up for collaborative discussion by the German RSE community through de-RSE e.V. - Society for Research Software. Computational analysis of large data sets, computer-based simu- lations, and software technology in general play a central role for virtually all scientific breakthroughs of at least the 21st century. The first image of a black hole may be the most promi- nent recent example where astrophysical experiments and the collection and processing of data had to be complemented with sophisticated algorithms and software to enable research excellence1,2. Similarly, it is research software that allows us to get a glimpse of the consequences our actions today have on the climate of tomorrow. However, an implication of computer-based research is that findings and data can only be reproduced, understood, and validated if the software that was used in the research process is sustained and their functionality maintained. Computational analysis of large data sets, computer-based simu- lations, and software technology in general play a central role for virtually all scientific breakthroughs of at least the 21st century. The first image of a black hole may be the most promi- nent recent example where astrophysical experiments and the collection and processing of data had to be complemented with sophisticated algorithms and software to enable research excellence1,2. Why sustainable research software in the first place? In order to support research, a sustainable software must ideally be correct15–17, and validatable. Due to the experimental nature of some research software, this may not be possible in all cases, e.g., due to lack, or infeasible implementation, of a test oracle18, vast configuration spaces, or large and heterogeneous data inputs19. While it must be accepted that precise, oracle-based testing may not be possible here, alternative solutions should be implemented, such as metamorphic testing, runtime assertions, test input sampling and generation (e.g. via machine learning), and input data modeling. Sustainable software must also be understandable, documented, publicly released, adequately published (i.e. in persistently identifiable form as software source code20, and potentially in an addi- tional paper which describes the software concept, design decisions, and development rationale), actively maintained, and (re-)usable21–23. We also argue that truly sustainable research software should ideally be published under a Free/Libre Open Source Software (FLOSS) license, and follow an open develop- ment model, to (1) enable the validation of research results that have been produced using the software, (2) enable the repro- ducibility of software-based research, (3) enable improvement and (re-) use of the software to support more and better research, and reduce resources to be spent on software devel- opment, (4) reduce legal issues (see section below), (5) meet ethical obligations from public funding, and (6) open research software to the general public, i.e., the stakeholder group with arguably the greatest interest in furthering research knowledge and improving research for the benefit of all. After graduation, Kim joins a fixed-term researchonomical research project. For her PhD thesis, she wants to crunch some data. Her colleague recommends learning some Boa, which is an all-purpose programming language often used in researchonomy. Luckily, the UofA runs regular Software Plumbery courses for researchers, including a Boa course. Kim takes the course and gains a solid understanding of the basics of the Hash shell, version control with Tig, and the basics of Boa. She starts writing scripts, which help her a lot with the data processing. Unfortunately, Kim’s scripts are quite slow and actually break after she installs a newer version of Boa. She visits the weekly Code Café organized by her university’s central RSE team. The RSEs not only help her update her scripts but also suggest some changes which speed up the computation by a factor of 25. Background These positive developments notwithstanding, guidelines and policies for sustainable research software development in Page 4 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Germany are unfortunately still lacking, and long-term funding strategies are missing. This all leads to unmet requirements and unsolved challenges that we want to highlight in this paper by elaborating on (1) why research software engineering needs to be considered an integral part of academic research; (2) how to decide which software to sustain; (3) who sustains research software; (4) how software can be funded sustainably; (5) what infrastructure is needed for sustainable software develop- ment; and (6) legal aspects of research software development in academia. While we specifically focus on the research soft- ware landscape in Germany, we are convinced that many of the analyses, findings, and recommendations may carry beyond. We want to address RSEs who are experiencing simi- lar challenges and newcomers to the field of research software development, but first and foremost political and academic decision makers to raise awareness of the importance of and requirements for sustainable software development. As a community, we work hard on overcoming the challenges of software development in an academic setting, but we need support – and reliable funding options and institutional recognition in particular – for the sake of better research. Our credibility as researchers in society hinges on the notion of proper research conduct, also known as “good research practice”. The digitalization of research has introduced complex digital research outputs, such as software and data sets. Although first recommendations13 and policies14 exist, they are far from being widely adopted. It is still somewhat unclear how to translate good research practice into good research software practice, for example in terms of validity and reproducibility, but also pertaining to the responsible use of resources. The damage that failing to do so is causing both to the progress of the research community and to the credibility of academic research in society is becoming increasingly clear with the growth of the replication crisis - while the lack of universally agreed-upon and supported good research software practice is not the main reason for that crisis, it clearly is a contributing factor. Background While it is obvious that software qualifies as a potentially re-usable digital artifact, the additional benefit of not just reproducing a given scenario, but transferring software use to new problems, domains, and/or applications, justifies develop- ing research software with a long-term perspective as sustainable research software. Why sustainable research software in the first place? Page 5 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 • Lack of impact measures: It is unclear how to measure the impact of research software with respect to its quality, reusability, and benefit for the research community. This exceeds the implementation of research software citation (which is work in progress20,31,32,44), and pertains to sustainability and policy studies. Currently, creating and using sustainable research software is not sufficiently incentivized. To evaluate in which area this shortcoming should be addressed, we have identified the following challenges: • Lack of benefit for the individual: Currently, the primary motivation for sustainable research software is the common benefit, rather than the individual benefit. It is clearly beneficial for the research community as a whole to direct resources towards sustainable research software, as it enables better and more research by free- ing funds for domain research rather than (repetitive) software development. But the developers are often even at a disadvantage (e.g., they publish fewer papers5,6), which in turn prevents sustainable research software. • Infrastructure issues: Due to a lack of knowledge about how sustainability features impact the application of research software, there is not yet enough evidence for whether centralized or decentralized facilities should be favored to further research software sustainability45–47. Commonly, local infrastructure hinders cross-institutional collaboration, whereas cross-organizational infrastructures often suffer from lack of authentication and authorization implementations, or legal constraints. This in turn leads to a lack of infrastructure as a whole. • Lack of suitable incentive systems: Contributions to research that are not traditional text-based products (i.e., papers or monographs) are still not sufficiently rewarded, or not rewarded at all, due to the missing implementation of mandatory software citation20,24–32, among other reasons. Interestingly, one third of research software repositories have a lifespan (defined as the time from the first time any code was uploaded to the last contribution) of less than one day (median: 15 days11), indicating that many codes are only made available publicly for the publication in a journal (as increasingly encouraged or required by journals33 and associated with higher impact34) but are not maintained thereafter. • Legal issues: Many obstacles for research software per- tain to legal issues, such as applicable licensing and compatibility of licenses48, and decisions about license types. • Funding issues: Despite some individual initiatives49–52, funding for the creation, maintenance, and support of sustainable research software is still scarce. Why sustainable research software in the first place? During the next meeting with her PhD supervisor, Kim presents her collection of scripts. The supervisor encourages Kim to create a Boa library from them, as they will be very useful to other researchonomists. Thankfully, Kim’s project PI had applied for three RSE person months in their grant, so the project enlists an RSE from the central team. Over the next three months, Kim and the RSE work together to build the library, document it, test it, license it under the permissive Comanche license, update the TigLab repository to let others contribute, introduce automated builds for every code change via a continuous integration platform, and make the library citable. Finally, they release the first major version of the library, named hal9k and publish it through the university library’s software portal, where they get a DOI (Digital Object Identifier) for the version as well as a concept DOI for any future versions of the library. Working with the RSE, Kim has gained a good understanding of some methods in software engineering, and she’s thrilled because this also means she’ll be able to get a job with a local tech company once her fixed-term contract has run out. To make software-based research (and with that almost any research) reproducible, the used software must continue to exist. Furthermore, it must continue to be usable, understandable, and return consistent results (or potential changes to results and bug fixes must be clearly documented) in the evolving software and hardware environment. Moreover, the software should support reuse scenarios to avoid duplication of efforts and drain of resources. Therefore, if research software is publicly funded, it should be freely available under a FLOSS license. Kim passes her PhD - of which hal9k is an important part - with flying colors, and soon citations to her library start appearing in the researchonomic literature. To Kim’s surprise, she also reads a blog post about a citizen science maker project which has used hal9k to process researchonomic data measured in a neighborhood of her hometown. She is invited to give a talk at the local office of Siren, a global tech company, which look to adopt hal9k, and pay Kim a generous speaker honorarium. So generous in fact, that Kim can pay a student assistant for a full year from the money. Why sustainable research software in the first place? Addition- ally, existing models usually supply seed funding only, which disregards the support and maintenance steps in the software development lifecycle. Instead, a potential “market” is relied upon to support these, which may only develop long after the initial project has ended. With regard to the funding of infrastructure which underpins modern development approaches such as DevOps and continuous deployment, cloud infrastructure providers and their pricing models do not work well with current funding models, due to lack of knowledge of how to target them with traditional academic funding and budgeting, compliance issues, or rigid bureaucracy. • Lack of awareness: Research software sustainabil- ity and its importance is lacking visibility as well as acceptance35–38, and research software engineering in its implementation as sustainable software develop- ment and software maintenance is not sufficiently supported, both in Germany and beyond9,39,40. • Lack of expertise: Knowledge about how to create, maintain, and support sustainable research software is emerging41–43 but has not yet permeated related activities within organizations - specifically teaching, mentoring, and consultancy. This lack of expertise can also lead to divergence between software design and community uptake, e.g., if the software fails to meet the needs of the target group, or is insufficiently usable. RSEs combine sustainable software engineering expertise with experience in one or more research domains. • Slow adoption of research software engineering as a profession: Career options for research software work are not fully determined, although career paths are emerging in some regions. Initially, the RSE initiative in the UK has made progress in this area, and RSE groups have been installed in many institutions. In Germany, the US, and the Netherlands, this is still work in progress. It is also not yet determined how to match research software engineering roles in public institutions with industry roles53. • Heterogeneous research community: There are significant differences with respect to how software is developed, published, used, and valued in the different academic disciplines. Additionally, there is even hetero- geneity within a community in terms of application and approach. This also makes it hard to train researchers for sustainable software development, as beyond basic training in computational research such as provided by The Carpentries, advanced courses for research soft- ware engineering are not widely available (with the notable exception of the CodeRefinery project). Targeted curricula must be developed and updated regularly, and specialized instructors need to be trained. Stakeholder motivations for research software sustainability While a wide range of stakeholders share interest in sustain- able software, we argue that their individual motivation can differ quite significantly: Research funding organizations have inherent interest in – and directly benefit from – the existence of sustainable research software as it allows them to direct more resources towards actual research (rather than recreation of software) and increase return on investment. At the same time, funding organizations can create incentives for sustainable software by imposing policies that reflect the necessity of research software sustainability and creating respective funding opportunities. The general public benefits from research which supports the common good, in other terms: creates a better world, faster. Taxpayers have an interest in economical use of their tax money, to which duplicated or flawed efforts to create research software – in contrast to software reuse – is contrary. A subset of this group may be interested in sustainable, i.e., re-usable and understandable, software as part of citizen science. Domain researchers benefit from better software to do more, better, and faster research. Sustainable research software supports this through validated functionality (e.g., correct algorithms), the potential for reuse, and general availability. Sustainable software also potentially simplifies building upon previous research results by reusing the involved software to produce additional data or by extending the software’s function- ality. In light of recent updates to definitions of good research practice, sustainable research software also allows domain researchers to comply with guidelines and best practices. Addi- tionally, using software that is sustainable enough to establish itself as a standard tool in a field signifies inclusion in a research community. Less directly, researchers may benefit from the existence of sustainable standard tools as they yield stand- ard formats, which in themselves facilitate reuse of research data. Geopolitical units have a strategic interest to be independent of other geopolitical units to ensure that research can continue seamlessly regardless of geopolitical developments and ensuing embargoes on information flow. Reuse of sustainable software additionally frees up funding for uses other than software development. Well-established, sustainable software systems can also attract researchers and companies in the research and technology sector. Libraries (also registries, indices) benefit from sustainable research software, as it will undergo a formal publishing proc- ess and be properly described in its metadata. Libraries can extend their portfolio beyond text-based research objects and stake claims as organizations harnessing the digitalization of research. Stakeholder motivations for research software sustainability In turn, they help to increase visibility and discoverabil- ity for research software through their services and advance the competitiveness of their organization or geopolitical unit. In addition, libraries also use research software and would thus benefit directly from a more sustainable research software landscape. Last but not least, by using FLOSS research software, libraries could avoid expensive licenses and often insufficiently adapted commercial software. Research software engineers (RSEs) have an intrinsic inter- est in sustainable research software. They create better software for research, which enables more and better research. RSEs have an inherent interest in developing and working with high quality software, as part of professional ethics as well as good research practice. RSEs build their reputation on high quality software and software citation20,31, which will open up new career paths. Finally, for RSEs, creating sustainable research software is part of an attractive, intellectually challenging, and satisfying work environment. Infrastructure units, such as supercomputing facilities and university computing centers, benefit from sustainable software as it makes their daily work in terms of software installation and user support easier. Additionally, they can position them- selves at the forefront of research by bundling expertise on the creation and maintenance of sustainable research software and installing research software engineering teams. Research leaders as well as research performing organizations mainly focus on the economic aspects and management of research, i.e., available funds, people, and time employed to optimize research output. Both need to make sure that their employees continually improve their qualification and gener- ate impact to improve their standing in the various research communities and ensure continued funding. Overseeing and enabling the creation of sustainable research software advances their visibility in the field and makes their research endeavors both more future-proof and more easily traceable, reproduc- ible, and verifiable and thus more likely to attract additional resources (including human resources). Research performing organizations can additionally benefit from sustainable research software if it can be reused in other areas, creat- ing synergies between different research disciplines. These Industry benefits from sustainable research software, as the process of creating and maintaining research software produces a highly-skilled workforce. Depending on the employed licensing model, sustainable research software can also be adopted by industry partners to reduce cost in corporate research and development. Helping to sustain research software may also enable positive outreach for companies across industry and into society. F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Subsequently, we specify challenges towards satisfying the demands of the individual stakeholders. synergies typically free resources that can then be used in areas other than software development and maintenance. Finally, organizations can gain highly competitive positions in terms of funding and hiring opportunities, as well as a reputation for being on the cutting edge of research, through early adoption of research software engineering units, and the implementation of sustainable research software policy and practice. Why sustainable research software in the first place? • Heterogeneous research community: There are significant differences with respect to how software is developed, published, used, and valued in the different academic disciplines. Additionally, there is even hetero- geneity within a community in terms of application and approach. This also makes it hard to train researchers for sustainable software development, as beyond basic training in computational research such as provided by The Carpentries, advanced courses for research soft- ware engineering are not widely available (with the notable exception of the CodeRefinery project). Targeted curricula must be developed and updated regularly, and specialized instructors need to be trained. In summary, the necessary but resource-intensive practice of creating, maintaining, supporting, and funding sustainable research software is not yet sufficiently incentivized and enabled by research institutions and funding agencies, nor does it align well with the publish-or-perish culture that is still prominent in most fields. Therefore, it is necessary to comprehensively motivate sustainable research software practice. In the following, we identify stakeholders of research software54–56, and explicate their particular motivations for sustainable research software. Page 6 of 34 How to decide which software to sustain? Kim’s PI is happy because Kim writes a longer section on hal9k for the final project report and provides a software management plan alongside it, which ticks off a box in the template that the PI had previously worried about. The PI does not want to let Kim go and instead offers her to be co-PI on a follow-up project to test new methods on the data, and integrate them into hal9k as well. They are positive that such a project proposal has a good chance to be funded, as they can show impact of their first project via their university’s current research information system (CRIS) and through the number of citations of hal9k and the publications for which it was used. While they write the proposal, the faculty dean approaches the two to tell them that based on Kim’s work, they will now negotiate about two new RSEs for the central RSE team with the university’s provost for research and plan to consider candidates with a background in researchonomics. While an assessment based purely on quantitative metrics would allow for seemingly objective comparisons between pro- grams, the definition of valid and robust quantitative metrics that can be evaluated with reasonable effort is a major chal- lenge. On the other hand, a structured qualitative assessment with scores for groups of criteria can provide a middle ground. It is clear that both preparing an application for a review against these criteria from the applicant side as well as the evaluation by the reviewers requires significant effort. We believe that the added value significantly outweighs the invest- ment but appropriate resources need to be factored in. Sus- tainability of research software should be considered from the beginning for new projects. The criteria listed below, or a sub- set such as the “good enough” practices proposed by Wilson et al.43 and artifact review approaches58,59 are valuable throughout the development process (including early phases) for almost all types of research software applications. “Classical” research funding schemes should acknowledge the need to follow best practices during the development of new software and allow factoring in appropriate resources to design and implement for sustainability. In this section, we focus on the question which software to support in dedicated sustainability funding schemes. Stakeholder motivations for research software sustainability Independent (open source) developers can get involved in research software, even if they are not employed by a research institution. This can help them get in contact with other Page 7 of 34 Page 7 of 34 Page 7 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 developers in the field and may potentially lead to collaborations or job opportunities in research based on this extended experience. organize a fair and transparent review process. We believe that it is important that the review process is conducted by experts, or teams of experts, that have a strong background both on software engineering as well as on the domain-specific aspects, the latter because certain criteria often exist on a spectrum that is most likely shaped by the specific demands of the respective research community. organize a fair and transparent review process. We believe that it is important that the review process is conducted by experts, or teams of experts, that have a strong background both on software engineering as well as on the domain-specific aspects, the latter because certain criteria often exist on a spectrum that is most likely shaped by the specific demands of the respective research community. How to decide which software to sustain? For such sustained funding, only software in application class 2 or 3 as defined by Schlauch et al.60, i.e., with significant use beyond personal or institutional purposes, would likely be considered. Excellence as reflected in funded projects, publications, and software adoption, i.e., back- ing by a community, should be considered during selection. Nevertheless, we believe a good scheme should strike a balance between consolidating the field to few well-established software packages on one side and stimulating innovation and coopera- tion promoting diversity in terms of more than one monopolis- tic package on the other side. Last but not least, there is an inherent conflict between the long-term goals of sustainability funding software and the necessary reevaluation to monitor the state of the software over time. When they get the decision letter from the research funding organization, Kim and her co-PI are happy to learn that their new project has won the grant. The reviewers specifically point out the value of extending Kim’s Boa library to include the proposed new methods, as well as the significant reuse potential of hal9k for the researchonomic community as a direct effect of its well-engineered architecture and modularity. Additionally, they stress that it was really easy to evaluate the software due to the comprehensive test suite, documentation, and example data. In fact, during the first month of the new project, three other researchonomic research projects approach them to ask whether they can contribute to Kim’s library and offer to fund six months of RSE work for this. Kim uses this money to also parallelize hal9k together with the RSEs and works with her university’s computing center to offer it as a standard tool for researchonomic supercomputing. Requirements and challenges The sustained funding of all existing software efforts is not only impossible but would risk overly splintering the commu- nity and eventually become counterproductive to the efficiency of the research community. Therefore, it is important to agree on a list of transparent criteria that qualify a software prod- uct for sustained funding. We recognize that defining research software engineering criteria for software evaluation will also lead to activities aiming at optimizing scores to achieve these criteria. Hence, the criteria have to be designed such that all score-pushing effort truly advances the value of the software. Criteria that can be manipulated without effectively adding value, i.e., wasting resources, should be excluded. The list of criteria presented in this section could be the basis for a structured review process that facilitates an unbiased evalua- tion of software tools from various fields. Therefore, this list must be general enough to be applied to research software from various research disciplines while also respecting differences between fields (e.g. citation rates between humanities and life sciences). The challenge to do justice to a wide spectrum is e.g. reflected by suggesting criteria comprising different levels57. One of the major challenges in the endeavor to define a selec- tion scheme for sustainable funding of research software is to Selection criteria As mandatory criteria of software transparency and quality that have to be fulfilled, we consider (6) the public availability of the source code in both a code repository and an archive (for long term availability), developed using (7) version control with meaningful commit messages and linked to an issue tracker (ideally maintained, but at least mirrored on a public platform). (8) Documentation of the software needs to be publicly available comprising both user documentation (requirements, installation, getting started, user manual, release notes) and developer documentation (with a development guide and API documentation within the code, e.g. using Doxygen)67. (9) The license under which the soft- ware is distributed must be defined. Publicly funded software should be published under a Free/Libre Open Source Software (FLOSS) license by default, although exceptions to this might apply (e.g. excluding commercial use). (10) Dependencies on libraries and technologies must be defined. We acknowledge that some additional criteria have to be evaluated under consideration of the research domain. These comprise (11) the availability of examples (comprising input data and reference results), (12) mechanisms for extensibility (software modularity) as one aspect of software architecture68 and (13) interoperability (APIs / common and open data formats for input and output), (14) a test suite (including at least some of the following: unit tests, regression tests, integration tests, end-to-end tests, performance tests; ideally run in an auto- mated fashion in a continuous integration environment), (15) tagged releases (considering their frequency, and avail- ability for end users in terms of binary packages for major operating systems, or availability via package managers or containers), (16) no large-scale re-implementations for functionality for which good solutions already exist. Many of these aspects require appropriate infrastructure (see page 12). We drew inspiration from all these works and suggest a set of criteria on which to base reviews for sustainable fund- ing. This set comprises mandatory, hard criteria that we think have to be fulfilled across domains (highlighted in italics) and additional desirable, soft criteria that can be implemented to different degrees depending on the use case and domain- specific software development requirements. The soft criteria should be evaluated in a structured way by the reviewers with a specific response for each section rather than one running text. The fact that most of these criteria will be consid- ered in any software management plan (SMP) highlights its importance for sustainable research software. Usage and impact. Selection criteria Selection criteria Several evaluation schemes for research software have been proposed before and led to the formulation of first recommendations13,14. Gomez-Diaz & Recio suggested the CDUR scheme based on Citation, Dissemination (includ- ing aspects like license, web site, contact point), Use, and Research (output)61. Lamprecht et al. rephrased the FAIR data principles10 for research software12. Hasselbring et al. found that the adoption of FAIR principles is different between fields with an emphasis on reuse in computer science as opposed to a reproducibility focus in computational science11. Fehr et al. collected a set of best practices for the setup and publication of numerical experiments62. Jiménez et al. boiled it down to four Page 8 of 34 Page 8 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 best practices63: public source code, community registry, license, and governance. Hsu et al.64 proposed a framework of seven sus- tainability influences (outputs modified, code repository used, champion present, workforce stability, support from other organ- izations, collaboration/partnership, and integration with pol- icy). They found that the various outputs are widely accessible but not necessarily sustained or maintained. Projects with most sustainability influences often became institutionalized and met required needs of the community64. In the field of open source software, the CHAOSS (Community Health Ana- lytics Open Source Software) project has developed met- rics to evaluate sustainability. One objective of CHAOSS is to automatically generate project health reports based on software that evaluates the metrics, with most of the metrics already covered. The UK Software Sustainability Institute (SSI) suggested both a subjective tutorial-based and a more objective criteria-based software evaluation scheme65, the lat- ter being available as an online form. ROpenSci66 provides software reviews for R developers, which have been very successful in the community. The review criteria of the Jour- nal of Open Source Software (JOSS) focus on the aspects license, documentation, functionality, and tests. This list of essential items should be fulfilled by all research software that wants to beconsidered not only for publication but also for sustained funding. common software, would be a strong indicator of impact. (5) As community uptake and benefits are a central goal of sustained software funding, outreach and appropriate training material for new users of the software are essential. Software transparency and quality. Selection criteria Requirements qualifying software for sustained funding are (1) its use beyond a single research group, (2) the scientific relevance and validity of the software documented in at least one peer-reviewed scientific publication. Ideally a paper also describes the scope, performance, and design of the software. (3) The use of the software in pub- lications is a measure of impact but quantitative assessment brings about additional challenges27. Therefore, other, potentially domain-specific, impact measures, such as influence on pol- icy and practice as well as use in other software and products should be considered as well to evaluate relevance for academia and society. Considerable attendanceat training and networking events can be considered as a proof of use as well. (4) A market analysis needs to show that the software is important to a user base of relevant size and either unique or one of the main play- ers in a field with several existing solutions. Geographical or political aspects can be considered as well, e.g. to support the maintenance of a European solution. A convergence process of (parts of) a research community towards a specific software stack, i.e., documented transition of several research groups to a Maturity. The research software applying for sustained fund- ing must have already reached a certain level of maturity (typi- cally class 2 or 3 as defined by Schlauch et al.60). A mandatory requirement is (17) a comprehensive and up--to-date software management plan69. The software should (18) be maintainable with an appropriate amount of resources as detailed in a sustain- ability section of the software management plan. The software has (19) a well maintained website with a clearly defined point of contact and a communication channel to inform users about news regarding the software such as new releases. Besides an active user community, sustainable software requires (20) a group of developers (i.e., definitely more than 1 developer) doc- umented, e.g. by contributions to the code base or participation in documented, public discussions or issue tracking. Another criterion is (21) whether potential contributors are invited to par- ticipate in a clearly defined process (e.g., a CONTRIBUTING document). The group of developers should have defined a governance model for their project and easy ways for users to provide input regarding their needs. Requirements Possibly the most important demand is the need for an increase in recognition and awareness of research software as a first class citizen in research14,71,72. For sustainability of research soft- ware, long-term commitments of the respective software leads are crucial, but very few professional RSE profiles currently exist. In consequence, it is essential to create career paths for RSEs that are attractive and include permanency perspectives. While creating permanent positions in the German academic system below the faculty level is an actively discussed topic overall73, we specifically focus on the needs originating from the development and maintenance of research software here. As already mentioned, research software development not only requires domain expertise, but also software development education, skills, and competence. Currently, most of the domain researchers developing and maintaining domain-specific software technology have not received professional training on software development3,41. To enhance the productivity and sustainability of computer-based research, it is essential to integrate software development training into the education of domain researchers. Currently, a significant portion of the existing research software is developed by individuals or in small groups, primarily to serve their own requirements. This situation is unsatis- fying in terms of collaboration and inefficient in terms of several groups spending resources on generating similar or even the same functionality. To enable and promote syner- gies, it is important to allocate resources for research software development and to build communities, as described in 74. Recommendations Given the diversity in the software technology landscape, and the domain-specific software development cultures70, some of the above-mentioned criteria have to be evaluated against domain- specific requirements. Therefore, we highly recommend to base the selection process on a combination of (1) a software qual- ity-based review and (2) a domain-specific scientific review. In particular, the former should be ideally performed by a central institution (e.g. at funding bodies or other independent agen- cies such as a software sustainability institute). Only criteria for which improvement truly advances the value of the software should be considered in evaluation schemes, i.e. no criteria that can be gamed. After rejecting software not fulfilling the mandatory criteria in a first stage of the review process, the second stage of the selection process should be realized as a transparent procedure ideally allowing the reviewers to interact with the PIs of the software (e.g. remote meetings, forum-like discussions) and put the software quality and development efforts into the domain-specific context. The outcome of this sec- ond stage should be a structured review assessing each criterion explicitly and a rating for each of the dimensions Usage and impact, Software quality, and Maturity. For sustained software funding, it is important to audit the performance, relevance, impact, progress, and level of sustainability of funded software after reasonable time frames. Such a reevaluation should revisit the criteria under consideration of evolving software technology and scientific standards, without requiring a completely new proposal being submitted. We envision funding periods of 5 years to provide sufficient security for funded software projects, while allowing for adaptation of the portfolio of funded software to novel research directions and community needs. Failure to meet the reevaluation criteria should lead to the decision to phase-out sustainable funding. The phase-out process may come with a 1-year funding program based on a consolidation plan with clear goals regarding the archiving and preservation of the software, documentation, and all existing resources. Selection criteria Page 9 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Research relies on software and software relies on the people developing and maintaining it. Sustainable research requires sustainable software, and this in turn requires continuity for those who develop and maintain it. Recommendations F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 community. However, the current academic system in Germany does not provide a defined RSE role. Fixed-term positions are, at least currently within the German academic system, often effec- tively the end of a Research Software Engineer’s career path, sometimes even a dead end. The challenge here is the lack of available permanent positions within the non-professorial aca- demic faculty (“Mittelbau”) in Germany, compounded by a lack of access to these few permanent positions for RSEs. This in turn is due to the already mentioned lack of recognition for efforts concerning research software for faculty appointments within domain sciences. community. However, the current academic system in Germany does not provide a defined RSE role. Fixed-term positions are, at least currently within the German academic system, often effec- tively the end of a Research Software Engineer’s career path, sometimes even a dead end. The challenge here is the lack of available permanent positions within the non-professorial aca- demic faculty (“Mittelbau”) in Germany, compounded by a lack of access to these few permanent positions for RSEs. This in turn is due to the already mentioned lack of recognition for efforts concerning research software for faculty appointments within domain sciences. definition of research impact beyond traditional scientific publications to also include other impactful results. Not all researchers that think of themselves as RSEs pursue a fac- ulty position as their main career goal. However, permanent academic non-faculty positions are rare within the German academic system, also due to the lack of a defined RSE role. We recommend research institutions to leverage the benefit of dedi- cated RSEs by establishing attractive long-term career options in the academic environment. The long-term solution in order to gain sufficient software development skills should be education that is included early in the career path, ideally already at the Bachelor level. For the time being however, efforts involving workshops and seminars that provide easy access to hands-on training on software-related questions should be promoted and supported as much as possible. In order to develop sustainable software, researchers need to have the skills and expertise to build software that is easy to maintain and extend76. However, most of the researchers are self-taught developers3,41. How can research software be sustainably funded? As long as the necessary software skills within domain sciences are not yet wide-spread, building a network from those that have acquired relevant skills is difficult. Community efforts, that concentrate on questions regarding research software, can help to fill this gap. Examples of such efforts include the Software Carpentries, national and international RSE societies (e.g., within Germany deRSE e.V.). However, since research software is such an interdisciplinary topic, it is hard to get recognition and find funding within any specific discipline. As a result, existing communities often have to rely heavily on volunteers. This is challenging because despite benefits to domain science, volunteers hardly receive recognition for their work “back home”, i.e., within their domain, underlining again the importance of our first demand. Hal9k has grown into a widely used software in researchonomics, and Kim is proactively asked to apply for - and is subsequently awarded - a permanent RSE position at the institute for researchonomy at UofA, based on her work on the library. She works closely with the central RSE team, but mostly due to bureaucracy and the high demand for her library, Kim does not have enough time to maintain and further develop hal9k alone anymore. Together with the dean she develops a course for the researchonomics curriculum which teaches data processing with hal9k. As a lesson from her own career, she starts the course with sessions on the Hash shell, version control with Tig, Boa, and two whole sessions on basics of sustainable software development. This is very fruitful, and due to the implementation of a new research software funding scheme at UofA, Kim is able to hire one of the course students, who has shown great RSE skills, straight into a long-term position at her institute, where they focus on the maintenance and development of hal9k, work with the computing center to support hal9k-based supercomputing on a new, dedicated FGPA cluster, develop training materials for external users, and organize the yearly hal9k users and developers conference. Kim gets to travel the world to visit researchonomics groups who are using hal9k. F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Ideally, these skills have to be built into the domain science curricula, which could generally be done in two different ways (or a combination of them). One obvious solution attempt are additional courses that focus on these topics. The main challenge here is to decide which other topic(s) to possibly drop due to the limited volume of any given curriculum. A different approach is to incorporate software- related topics into existing domain science courses. While this would provide the benefit of show-casing the usage of specific software skills directly within the domain science, the challenge here is the amount of work necessary to change existing lecture material, let alone the need of the lecturers to acquire those skills themselves in the first place. It is important to provide an environment where communities can form and flourish by allocating resources for research software development and for building communities around it63,74,77. The identification with a community of like-minded people and personal action78 can lead to a permanent establishment of sustainable research software as a valuable research output. Thus, research institutions as well as funding agencies should not only be open-minded regarding existing volunteer organizations, but should actively promote the creation of such groups. How can research software be sustainably funded? Recommendations Increasing recognition and awareness is a challenge that calls for both immediate action and perseverance. Nevertheless, some measures will likely show positive effects comparatively soon. Similarly to plans for research data management, funding agencies should request that applicants include considerations about how software developed in a project can be sustained beyond the end of the funded project. A follow up on these plans during and after the project lifetime, i.e., a dedicated software management plan, is crucial. Challenges We are currently facing a lack of awareness for the importance of research software as discussed above. Moreover, there is little recognition for the efforts put into software development and maintenance. In consequence, software development in academic settings is mostly considered as a means to an end and sustain- ability is often not considered in project planning and grant proposals and contributes little to progressing research careers75. The main challenge here is the continued use of metrics that primarily leverage traditionally published articles and article citation numbers. Kim wants to broaden her research portfolio within researchonomics and applies for postdoctoral positions at other institutions. Her library hal9k is growing in popularity within researchonomics, and she wants to continue working on it. As her university has adopted an open science policy, hal9k is free software under a Free/Libre Open Source Software (FLOSS) license, and Kim is free to continue her work on the library even after moving away from UofA. Due to her involvement in the creation of hal9k as well as her previous success in attracting funding, Kim has the choice between multiple, attractive positions and decides to move to the researchonomics group at Eden University (EdU). She has already extended hal9k in multiple directions in the past and plans to continue this work at EdU. Her group leader at EdU would like to continue funding her but due to a law called the Fixed-term Research Contract Bill, EdU is not allowed to extend her contract, and neither third- party funding for her own position nor a permanent position are available. After having developed a now widely-used research tool, several publications in software and paper form, as well as having attracted funding, Kim finds herself looking for a job again. In academia, developers of research software are typically domain researchers, and in particular if new areas are explored, the software development process itself has research charac- ter. Obviously, developing research software requires not only domain knowledge but also software development skills, and the researchers leading the software development process are often domain experts with substantial software development experi- ence, making them extremely valuable members of the research Page 10 of 34 Page 10 of 34 Challenges Short-term engagement of (early career) researchers raises the question of how to maintain a constant level of expertise within a developer team and prevent knowledge drain concerning domain knowledge and software engineering skills. Conversely, the permanent engagement of qualified personnel requires to offer career perspectives, especially due to the fact that academia competes with industry for the same people. A challenge spe- cific to Germany is posed by the shortage of permanent positions and by the restrictions for temporary positions due to the German Wissenschaftszeitvertragsgesetz79. Computing centers and supercomputing facilities for research need to receive earmarked resources for the support of sustainable software development. This funding is necessary to provide continuous integration services, a hardware portfolio for development, testing and benchmarking software, as well as personnel for training domain researchers in software design and the proper usage of the services. The creation and maintenance of training materials for general research software engineering education and the software-specific documentation and tutorial creation needs to be reflected in funding opportunities. This can either happen by dedicating modules of research or software grants to providing support and the generation of training material, or by opening funding schemes focusing on interdisciplinary software devel- opment education. The latter may include research that looks at research software development as a process to analyze which measures, interactions, and team compositions make research software successful. Additionally, funding instruments fostering the formation of research software communities have to be established. Sustainable software development requires hardware tech- nology to develop, test, validate, and benchmark features in a continuous integration cycle. The challenge in this context is the persistent evolution of the hardware landscape. Hence, for creating an environment promoting sustainable software development, it is important to provide access to a wide hardware portfolio and to support a development cycle based on continuous integration. Expertise in sustainable research software development is a scarce resource, and training is heavily needed as one way of building up more expertise. However, while integrating interdisciplinary software engineering courses into the education curriculum can build up basic skills, some expertise is domain-specific and requires interinstitutional training activities. Furthermore, there exist no financial incentives for creating software-specific documentation and tutorials nor to provide other forms of support. Requirements Requirements Sustainable funding for research software boils down to funding the four main pillars enabling sustainable soft- ware development: (1) Personnel with expertise in research software development; (2) Infrastructure for developing, testing, validating, and benchmarking research software; (3) Training in software design and sustainable software development; and (4) Community management and events for creating synergies between research groups and software efforts. Another recommendation is aimed at decision makers concerning recruitment for academic positions: broaden the Page 11 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 to decide which software to sustain? may be entitled for sustained funding as long as they live up to the standards and remain a central component of the research landscape. Which infrastructure is needed to sustain research software? As the hal9k community grows, so does the need for infrastructure. Kim and her team collaborate with the National RSE Consortium to set up hal9k on the Consortium’s distributed TigHub instance and organize world-wide access to it via the NRSEC-AAI federation. Going forward, the Consortium’s Research Software Hub - a registry and Software Heritage Archive-based long-term repository for research software on a national level - ingests hal9k releases with complete metadata: citation information, the hal9k provenance graph and computational environment information, ORCID iDs, etc. and provides its own DOIs for versions under a concept (umbrella) DOI. The community reviews all code and documentation changes that are contributed to hal9k via the central TigHub instance. The Hub’s CI system Alfred builds, tests, and pushes new releases automatically to the registered supercomputing clusters. Community efforts become better and more streamlined by the day, as research software development training is now offered as part of most curricula, and skilled RSEs are now much easier to find and hire by research institutions. While the creation of research software communities is one major asset in sustaining research software technology, promot- ing this process requires the installation of new funding instru- ments. Traditionally, research grants are limited to rather short time frames and support personnel, material, hardware, and to a limited degree also travel and research visits. Creating a research software community however requires funding for community and training events as well as “virtual hardware” such as webspace, versioning systems, task-managing systems, and compute cycles. These demands can hardly be met without third-party funding45,80–82. Recommendation: creation of adequate funding schemes Funding is a crucial factor for sustaining research software. Currently available sources and instruments are not adequately shaped for the challenges and solutions outlined above. We recommend actions on the individual, organizational, and national level. Project management tools Research software is developed by individual researchers, in small teams within a single institution, or in larger teams distributed across multiple institutions. In particular if software development is distributed across institutions, there exists an urgent need for frameworks and tools enabling collaborative code development, software feature planning, and software management. As research software development typically includes bleeding-edge research and in some cases development that the researchers do not want to disclose for a certain time to preserve intellectual property, distributed research software development Research software is developed by individual researchers, in small teams within a single institution, or in larger teams distributed across multiple institutions. In particular if software development is distributed across institutions, there exists an urgent need for frameworks and tools enabling collaborative code development, software feature planning, and software management. As research software development typically includes bleeding-edge research and in some cases development that the researchers do not want to disclose for a certain time to preserve intellectual property, distributed research software development Existing project-focused funding instruments on the local, national, and international level need to be complemented with funding instruments specifically designed for research soft- ware development and sustained research software maintenance to make research software a first class citizen in the research landscape. For example, software projects enhancing research and fulfilling the sustainability criteria detailed in section How Page 12 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 also needs a global Authentication and Authorization Infrastruc- ture (AAI). We recommend the development and/or deployment of tools for distributed software development and software man- agement as central research infrastructure. An important aspect in this context is the cataloging of research software to reduce the duplication of development efforts. This can efficiently be realized by promoting the registration of all research software with a unique identifier and developing a tool that allows to explore the research software landscape. Research software contributors should have an ORCID iD to be uniquely iden- tifiable and referable. While some funding for such tools and software repositories is emerging (e.g. the bio.tools catalogue of bioinformatics tools funded as part of the European ELIXIR project83), a standardized extension of such efforts to the RSE community as a whole is necessary. However, as the experi- ences from ELIXIR demonstrate, this is a non-trivial effort that requires significant dedicated and long-term funding. Developer training, motivation, and knowledge exchange As elaborated, training in sustainable software development is key to achieve sustainability in research software. At the same time, it is not clear how such training should be facilitated and insti- tutionalized. Furthermore, for deriving software quality stand- ards, evaluating the quality of software, and providing a code review service, central resources are necessary that individuals and groups in the research software landscape can draw from. Especially in interdisciplinary environments, it would be helpful to have access to a meta software repository index, similar to what re3data86 does for research data repositories. We recommend the creation of such a meta index covering important (disciplinary) software indexes in order to ease discovery of relevant software locations. Evaluation of discovered software is an unsolved problem. Here, anonymous telemetry of usage may provide information for the selection of relevant software. Publishing software, their dependencies, and envi- ronment in containers may also ease evaluation and further reuse. These suggestions require significant investment in longterm infrastructure. When publishing research software it is recommended to make use of integration schemes like GitHub with Zenodo or local GitLab instances with publica- tion platforms. Such indices and publication outlets may benefit national federated research indexing & archiving systems, similar to the hierarchy of library catalogs87. We consider Software Carpentry and similar efforts like the creation of the Data Science Academy HIDA in the Helmholtz Association of German Research Centers helpful solutions to exchange and distribute knowledge. Local chapters of RSE groups and (inter-)national conferences will further foster networking and community building. We strongly recommend the creation of a national Software Sustainability Institute (involving funded positions to establish web platforms and training material) similar to the UK Software Sustainability Institute (SSI), which serves as a national contact for all aspects related to research software. The UK SSI also publishes best practice guidelines for research software engineering. Project management tools We consider GitLab or GitHub as collaborative working envi- ronments and repositories like Zenodo appropriate publica- tion platforms, because the latter mint DOIs, allow versioning and are publicly funded for long-term access. GitHub, Figshare, and Mendeley Data are examples of commercial enterprises with business cases in the background, which leverage research results. Besides the aforementioned metadata standards, it is advisable to document source code, e.g. using MarkDown (with Doxygen tooling). Metadata and citations play a role in beneficial tools like PIDgraph, DataCite.org, CrossRef, which utilize Persistent Identifiers (PIDs) like DOIs. Another solu- tion to discovery are (mostly) disciplinary software indices like swMATH or the Astronomy Source Code Library as well as language focused systems like CRAN for R. Most of them started as national endeavors and became platforms of global importance. For Germany, we assume that the Nationale Forschungsdateninfrastruktur (NFDI) will put effort into creat- ing or supporting discovery platforms at a central point that ease information retrieval. At the same time, all stakeholders should be aware of and counteract potential institutional “fear” of losing “their” data, software, and intellectual property. Developer training, motivation, and knowledge exchange Research software discovery and publication This means that in most cases of employed software developers and research staff, the institu- tion holds the rights of use for the software work. This is not automatically the case for students, freelancers, and individual external cooperation partners. Employment and service con- tracts with contributors could contain regulations regarding the transfer of rights of use. For researchers who conduct free research not subject to directives, in Germany the constitution guarantees freedom of research so that the rights of use for their work remains initially with the natural person. In addition to the rights of the people directly involved, other rights of third parties may also be relevant. Existing source code (e.g., other Free/Libre Open Source Software (FLOSS)), external libraries, and contributions from institutional cooperation partners are published and provided under certain licenses and their condi- tions must be observed (which, due to incompatibilities even among FLOSS licenses, may well mean that individually reusable pieces of software cannot be reused together or in a new con- text). The nature of research careers often brings additional complications to the chain of rights. It happens that research- ers take their software with them when they change institu- tions and develop it further during their career. Here, the former employer may be entitled to some rights of use. In third-party funded projects, in particular with industry but also with public funding, rules regarding rights of use are often defined. Last but not least, the software can also be affected by other (intellectual) property rights such as patents or trademarks. Software itself is usually not patentable but it may imple- ment a technical invention covered by patents. When using or distributing such software, an additional matching patent license may be necessary. Licenses exist (for example: GNU GPL v3) which automatically grant related patent licenses while using the software license. That should be considered when exploitation of the patent is planned. There are both local and global approaches to software con- servation. One solution to keep the software in an executable state by preserving its context and runtime environment is to use containers such as Docker. However, to archive the Docker containers, additional metadata should be added and stored with the software in an archive container format that allows exchange between repositories and exit strategies, such as the BagIt container format88. Research software discovery and publication Application or platform conservation is also achieved by conservational efforts where unmaintain- able (virtual) machines are sandboxed to keep the platform in a secure but running state. Other notable efforts in this direction include for example Singularity and Guix HPC. Another threat is losing project repositories on global platforms like Github or BitBucket. Here, global platforms like Software Heritage harvest those repositories and prevent loss by long-term archiving. Research software discovery and publication needed to reproduce results, they should also be archived with the software or the publication. Specialized and unique hard- ware like high performance computing resources can be part of the runtime environment, which may not be accessible in the future. To overcome this, an emulation of hardware may be a (challenging) solution. Emulation involves the encapsulation and distribution of the complete hardware and software stacks, including the operating system and driver interdependencies. This can result in intellectual property issues when offered as a service. Challenges and clarifications Clarification of rights. Software development is a creative activity. The main relevant law governing legal aspects is there- fore the copyright law. It regulates the rights and obligations of the parties involved. Chapter 8 of the German Act on Copyright and Related Rights (UrhG) contains specific provisions applicable to computer programs and is based on the EU computer programs directive. Copyright law protecting the creator of software in similar ways exist in nearly all legal systems. It is impor- tant for the identification of rights that software, in the sense of (German) law, includes not only the source code but also the design materials89. The challenge in the use, distribution, and commercialization of software is to determine the chain of rights and to identify all right holders. The owner of the copy- right is not necessarily the owner of the right of use. For Ger- many, the Copyright Act regulates the rights for employment relationships90. In such cases, the right of use is automatically transferred to the employer. This means that in most cases of employed software developers and research staff, the institu- tion holds the rights of use for the software work. This is not automatically the case for students, freelancers, and individual external cooperation partners. Employment and service con- tracts with contributors could contain regulations regarding the transfer of rights of use. For researchers who conduct free research not subject to directives, in Germany the constitution guarantees freedom of research so that the rights of use for their work remains initially with the natural person. In addition to the rights of the people directly involved, other rights of third parties may also be relevant. Research software discovery and publication Existing source code (e.g., other Free/Libre Open Source Software (FLOSS)), external libraries, and contributions from institutional cooperation partners are published and provided under certain licenses and their condi- tions must be observed (which, due to incompatibilities even among FLOSS licenses, may well mean that individually reusable pieces of software cannot be reused together or in a new con- text). The nature of research careers often brings additional complications to the chain of rights. It happens that research- ers take their software with them when they change institu- tions and develop it further during their career. Here, the former employer may be entitled to some rights of use. In third-party funded projects, in particular with industry but also with public funding, rules regarding rights of use are often defined. Last but not least, the software can also be affected by other (intellectual) property rights such as patents or trademarks. Software itself is usually not patentable but it may imple- ment a technical invention covered by patents. When using or distributing such software, an additional matching patent license may be necessary. Licenses exist (for example: GNU GPL v3) which automatically grant related patent licenses while using the software license. That should be considered when exploitation of the patent is planned. Liability Iss es of arrant and liabilit for fa lt soft are m st Challenges and clarifications Clarification of rights. Software development is a creative activity. The main relevant law governing legal aspects is there- fore the copyright law. It regulates the rights and obligations of the parties involved. Chapter 8 of the German Act on Copyright and Related Rights (UrhG) contains specific provisions applicable to computer programs and is based on the EU computer programs directive. Copyright law protecting the creator of software in similar ways exist in nearly all legal systems. It is impor- tant for the identification of rights that software, in the sense of (German) law, includes not only the source code but also the design materials89. The challenge in the use, distribution, and commercialization of software is to determine the chain of rights and to identify all right holders. The owner of the copy- right is not necessarily the owner of the right of use. For Ger- many, the Copyright Act regulates the rights for employment relationships90. In such cases, the right of use is automatically transferred to the employer. Research software discovery and publication Software preservation aims to extend the lifetime of software that is no longer actively maintained. There are different approaches, which vary in the effort required and the likelihood of success. Software archiving is one important aspect of software pres- ervation: the process of storing a copy of a software package so that it may be referred to in the future. The publication of a certain software version for reference in research articles requires simple ways to archive research software on a long-term basis. Furthermore, its integration with collaborative software development environments such as GitLab or GitHub and with publication repositories is needed to facilitate archiving of referenced software versions based on sustainable frameworks (e.g. Invenio for GitHub to Zenodo integration). Proper software publication and possibilities for the commu- nity to find existing software solutions for a given problem are a prerequisite to optimally exploit synergies and avoid redundant development. However, we observe that today, many funding proposals lack a thorough state-of-the-art report of software that could possibly be reused. This is most often caused by insufficient information retrieval strategies, lack of knowledge about relevant repositories, and an abundance of locations where software is collaboratively developed and stored84. Discovery requires publication in a globally accessible location with appropriate metadata, e.g. Citation File Format (CFF)85 and CodeMeta. Comprehensive metadata (e.g. contributors, contact, keywords, linked publications, etc.) and publishing platforms have to enable persistent citing, which in turn benefits research evaluation. Selection and curation of software (probably by a data/software librarian) for publication and discovery are certainly challenging. A challenge for software archiving is the need to (ideally) preserve the runtime environment and all dependencies of the software. This could improve reproducibility, especially when running the software in its original state. If research data are Page 13 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 needed to reproduce results, they should also be archived with the software or the publication. Specialized and unique hard- ware like high performance computing resources can be part of the runtime environment, which may not be accessible in the future. To overcome this, an emulation of hardware may be a (challenging) solution. Emulation involves the encapsulation and distribution of the complete hardware and software stacks, including the operating system and driver interdependencies. This can result in intellectual property issues when offered as a service. F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Ideas for solutions Ideas for solutions presented, and (if not implemented yet) develop the software policy of the research performing organization. As an example, with the help of on-boarding processes performed by the research software task force, RSEs should be able to keep the clearance of rights as simple as possible right from the start. This helps to avoid that - out of uncertainty and fear to make a legal mistake - some research groups end up not choosing any license at all, which may hinder reuse of the software. We suggest that the local task forces build a network with the other research performing organizations for exchange of ideas but also for generating a bottom-up strategy to organ- ize RSE standards for Germany and beyond and possibly be the origin of the aforementioned software sustainability institute. In order to meet the legal challenges mentioned, it is abso- lutely necessary for the software developer (team) to document the rights chain comprehensively during the software devel- opment (one possible solution is presented in the accompa- nying report91). Contributions of individual persons must be traceable and their (labor law) status must be named. At best, contracts with rules on the transfer of rights of use should be concluded before work begins. Declarations of assign- ment of rights can be made for existing works. License conditions for external contributions must be evaluated with regard to further rights of use and possible sub-licensing. Contracts and funding conditions must be conscientiously documented and analyzed with regard to rules on rights of use. In case that different parts of the software are based on different conditions and rights of third parties, individ- ual modules of the new software could be published under different licenses and merged accordingly. Conclusionsi We find that the research software ecosystem is notoriously lacking resources despite its strategic importance. If funding and support does not improve, the success story of science based on academic research software may be at stake. We recom- mend the installation of infrastructure that enables sustainable software development including platforms for collaboration, continuous integration, testing, discovery, and long-term preservation. We suggest the establishment of a nationwide institution similar to the Software Sustainability Institute (SSI) to provide project consulting and code review services as well as sustainable software development training. We think that sustainable software development should become an integral component of the universities’ teaching curriculum. We encourage the research funding bodies to reflect the licensing models for academic software development, and to decide whether the “public money, public code” paradigm justifies the requirement that all publicly funded software has to be publicly available under a Free/Libre Open Source Software (FLOSS) license. Ultimately, we strongly advise the implementation of funding schemes for sustainably supporting the development and maintenance of research software based on clear and transparent criteria, for creating incentives to produce high quality community software, and for enabling career paths as research software engineer (RSE). A national research software sustainability institute could be established. This institute supports local research software task forces and thereby respective researchers and research teams in the licensing of research software and related legal issues. For this purpose, a legal help desk will be set up, to which all members of their respective research performing organization can apply. Such a legal help desk should be seen as an infra- structural investment to avoid any uncertainty about re-use of existing research software and to support research-friendly licensing. If researchers want to publish the research software under a Free/Libre Open Source Software (FLOSS) license, the organization could bundle the necessary rights beforehand. This is particularly useful when teams of researchers, often international, write software. In addition, the sustainability institute may serve as a one-stop-shop for the licensing of research software. Glossary general public Lay people that do not necessarily have specific insight regarding a research domain. geopolitical units Governed public units, ranging from cities and councils, over federal states and countries, up to political unions such as the EU. In the context of this paper, the discussion usually focuses on the larger units (countries and political unions). Legal aspects More and more industrial partners enter the hal9k community, and they bring their lawyers. Together with UofA’s research software task force, the RSE team, the researchonomy institute, the corporate lawyers, and community representatives, Kim decides to create a foundation to govern hal9k and its environment: the Fullest Possible Use Foundation for Open Researchonomy, funded by the Ministry of Research and Education and a consortium of corporate partners. As a first step, they re-license hal9k under the OSI-approved MIT license. A common situation in research software creation is that the developer has no knowledge or awareness of legal aspects and therefore did not consider them early enough. As seen in Kim’s example, re-licensing later in the project can be not only legally, but also organizationally very tricky, in particular for projects which developed over many years and involved many contribu- tors from different organizations. Thus, we think the main legal demands for research software development are raising aware- ness and empowering all levels of responsible persons in academia (from researchers and RSEs over PIs to research performing organizations and research funding organizations) in legal aspects. This will hopefully lead to a general legal cer- tainty before, during, and after the research software develop- ment process and thus enable better options for collaborations between universities, non-commercial research institutions, and other national or international partners. Legal aspects always have to be considered regarding the relevant jurisdiction. Though similar issues arise in all jurisdictions, the follow- ing will focus on the European and specifically German legal framework. Liability. Issues of warranty and liability for faulty software must be taken into account. We consider the possibilities of contrac- tual limitation of liability in licenses. Full exclusions of liability are generally invalid in the German law. Limitations of liability usually depend on the form of distribution: The limitation options are larger if the rights of use are granted free of charge, e.g. provision “as is” as defined, e.g. in the BSD 3-clause license. Page 14 of 34 Recommendations We see it as an essential part of the sustainability of research to enable the free distribution of research software. There are a variety of open source software licensing models (ranging from permissive to copyleft; for further information, see tldrlegal, the ifrOSS Lizenz-Center, or Morin et al., 201248). The use of an FSF- or OSI-approved FLOSS license for example would enable a truly free model and also reduce legal issues. We recommend that research funding organiza- tions such as the DFG discuss if they expect publishing all funded software under these licenses, following the paradigm of “public money, public code”. If licenses such as Apache or MIT are applied, the research institutions may later still commercialize the software if appropriate. Such open source licensing is also beneficial for start-ups that intend to provide professional services for the software. Glossary domain researchers The people doing the research to advance knowledge in a field. References Research Software. arXiv: 1908.05986. 2019. Reference Source 12. Lamprecht AL, Garcia L, Kuzak M, et al.: Towards FAIR principles for research software. Data Sci. 2019; 3(1): 37–59. Publisher Full Text 13. Katerbow M, Feulner G: Recommendations on the development,use and provision of Research Software. 2018. Publisher Full Text 14. Scheliga K, Pampel H, Konrad U, et al.: Dealing with research software: Recommendations for best practices. Helmholtz Open Science Coordination Office. 2019. Publisher Full Text 15. Hatton L: The Chimera of Software Quality. Computer. 2007; 40(8): 104–103. Publisher Full Text 16. Chang G, Roth CB, Reyes CL, et al.: Retraction. Science. 2006; 314(5807): 1875–1875. PubMed Abstract \| Publisher Full Text 17. Matthews BW: Five retracted structure reports: inverted or incorrect? Protein Sci. 2007; 16(6): 1013–1016. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Kanewala U, Bieman JM: Techniques for testing scientific programs without an oracle. In: 2013 5th International Workshop on Software Engineering for Computational Science and Engineering (SE-CSE). 2013; 48–57. Reference Source 19. Vogel T, Druskat S, Scheidgen M, et al.: Challenges for Verifying and Validating Scientific Software in Computational Materials Science. In: International Workshop on Software Engineering for Science. 2019; 25–32. Publisher Full Text 20. Smith AM, Katz DS, Niemeyer KE, et al.: Software Citation Principles. PeerJ Comput Sci. 2016; 2: e86. Publisher Full Text 21. Merali Z: Computational science: ...Error. Nature. 2010; 467(7317): 775–777. PubMed Abstract \| Publisher Full Text 1. The Event Horizon Telescope Collaboration, Akiyama K, Alberdi A, et al.: First M87 Event Horizon Telescope Results. IV. Imaging the Central Supermassive Black Hole. Astrophys J. 2019; 875(1): L4. Publisher Full Text 2. Nowogrodzki A: How to support open-source software and stay sane. Nature. 2019; 571(7763): 133–134. PubMed Abstract \| Publisher Full Text 3. Philippe O, Hammitzsch M, Janosch S, et al.: softwaresaved/international- survey: Public release for 2018 results. 2019. Publisher Full Text 4. Hirsch JE: An index to quantify an individual’s scientific research output. Proc Natl Acad Sci U S A. 2005; 102(46): 16569–16572. PubMed Abstract \| Publisher Full Text \| Free Full Text 5. Bangerth W, Heister T: Quo Vadis, Scientific Software? SIAM News. 2014; 47(1): 8. Reference Source 6. Prins P, de Ligt J, Tarasov A, et al.: Toward effective software solutions for big biology. Nat Biotechnol. 2015; 33(7): 686–687. PubMed Abstract \| Publisher Full Text 7. Richardson C, Croucher M: Research Software Engineer: A New Career Track? 2018. 1. The Event Horizon Telescope Collaboration, Akiyama K, Alberdi A, et al.: First M87 Event Horizon Telescope Results. IV. Imaging the Central Supermassive Black Hole. Astrophys J. 2019; 875(1): L4. Publisher Full Text independent (open source) developers Project-external soft- ware developers who are not employed by the institution(s) carrying out the project. independent (open source) developers Project-external soft- ware developers who are not employed by the institution(s) carrying out the project. Also for legal aspects, we believe it is important that all (German) research performing organizations install a research software task force, especially in light of the new DFG Code of Conduct. Besides organization and bundling of techni- cal and infrastructural support for local RSEs and researchers (see previous sections), this group should organize a local legal help desk, organize educational offers e.g. for the legal topics industry Companies conducting research or profit from available academic research software which they can directly or indirectly apply to their field. Page 15 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 infrastructure units Computing centers of research bodies such as universities and other research centers, as well as high- performance computing facilities. Data availability Underlying data No underlying data are associated with this article. libraries (also registries, indices) Infrastructure units of research bodies such as universities, or independent organi- zations, which gather research outputs and their structured metadata, and provide indices, search, etc. Extended data Decision trees and documentation templates for the legal topics are available on Zenodo91: doi: 10.5281/zenodo.4327147 research funding organizations Public research funding bodies but potentially also companies, foundations, associations, etc. Author information research leaders Heads of research groups, such as professors and other people with staff responsibility. We are a group of software-providing researchers, RSEs, and infrastructural as well as legal supporters. Initially, a group of representatives of funded projects of funded projects of the first DFG sustainability call met during the first German RSE conference (deRSE19) in June 2019 in a grass-roots workshop on sustainable research software addressing the software-based research community. During this workshop, we realized that a lot of valuable experience and good ideas are present in the group, and we decided to start working on this paper together with other interested practition- ers. We followed the generous invitation of the DFG for the above-mentioned two-day meeting at the Robert Koch Institute in Berlin in November 2019 to sharpen the focus of this paper. research performing organizations Research groups, departments, faculties, research institutions (universities, national labs, cross-institutional research groups, etc.), umbrella organizations, such as Helmholtz-Gemeinschaft Deutscher Forschungszentren, Max-Planck-Gesellschaft zur Förderung der Wissenschaften, Leibniz-Gemeinschaft, etc. research software engineers (RSEs) People creating and maintaining research software; this group ranges from research-focused software developers, to software engineers with a focus on research; other definitions include other roles, such as research software managers. References Reference Source 8. Cohen J, Katz DS, Barker M, et al.: The Four Pillars of Research Software Engineering. IEEE Software. 2020; 38(1): 97–105. Publisher Full Text 9. Brett A, Croucher M, Haines R, et al.: Research Software Engineers: State of the Nation Report 2017. 2017. Publisher Full Text 10. Wilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3(1): 160018. PubMed Abstract \| Publisher Full Text \| Free Full Text 11. Hasselbring W, Carr L, Hettrick S, et al.: FAIR and Open Computer Science 12. Lamprecht AL, Garcia L, Kuzak M, et al.: Towards FAIR principles for research software. Data Sci. 2019; 3(1): 37–59. Publisher Full Text 2. Nowogrodzki A: How to support open-source software and stay sane. Nature. 2019; 571(7763): 133–134. 13. Katerbow M, Feulner G: Recommendations on the development,use and provision of Research Software. 2018. Publisher Full Text 3. Philippe O, Hammitzsch M, Janosch S, et al.: softwaresaved/international- survey: Public release for 2018 results. 2019. Publisher Full Text 14. Scheliga K, Pampel H, Konrad U, et al.: Dealing with research software: Recommendations for best practices. Helmholtz Open Science Coordination Office. 2019. Publisher Full Text 4. Hirsch JE: An index to quantify an individual’s scientific research output. Proc Natl Acad Sci U S A. 2005; 102(46): 16569–16572. PubMed Abstract \| Publisher Full Text \| Free Full Text 15. Hatton L: The Chimera of Software Quality. Computer. 2007; 40(8): 104–103. Publisher Full Text 5. Bangerth W, Heister T: Quo Vadis, Scientific Software? SIAM News. 2014; 47(1): 8. Reference Source 16. Chang G, Roth CB, Reyes CL, et al.: Retraction. Science. 2006; 314(5807): 1875–1875. PubMed Abstract \| Publisher Full Text 6. Prins P, de Ligt J, Tarasov A, et al.: Toward effective software solutions for big biology. Nat Biotechnol. 2015; 33(7): 686–687. PubMed Abstract \| Publisher Full Text 21. Merali Z: Computational science: ...Error. Nature. 2010; 467(7317): 775–777. PubMed Abstract \| Publisher Full Text 21. Merali Z: Computational science: ...Error. Nature. 2010; 467(7317): 775–777. PubMed Abstract \| Publisher Full Text Page 16 of 34 Page 16 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 22. Barnes N: Publish your computer code: it is good enough. Nature. 2010; 467(7317): 753. PubMed Abstract \| Publisher Full Text 46. References Loewe A, Seemann G, Wülfers EM, et al.: SuLMaSS - Sustainable Lifecycle Management for Scientific Software. E-Science-Tage 2019: Data to Knowledge. 2019. Publisher Full Text 23. Tse H: Computer code: more credit needed. Nature. 2010; 468(7320): 37. PubMed Abstract \| Publisher Full Text 24. Hafer L, Kirkpatrick AE: Assessing Open Source Software As a Scholarly Contribution. Commun ACM. 2009; 52(12): 126–129. Publisher Full Text 47. Druskat S, Krause T, Lüdeling A, et al.: Infrastrukturstrategien für nachhaltige Forschungssoftware in befristeten Projekten. deRSE19 - Conference for Research Software Engineers in Germany. Potsdam, Germany. 2019. 25. Howison J, Bullard J: Software in the Scientific Literature: Problems with Seeing, Finding, and Using Software Mentioned in the Biology Literature. J Assoc Inf Sci Technol. 2016; 67(9): 137–2155. Publisher Full Text Publisher Full Text 48. Morin A, Urban J, Sliz P: A Quick Guide to Software Licensing for the Scientist-Programmer. PLoS Comput Biol. 2012; 8(7): e1002598. PubMed Abstract \| Publisher Full Text \| Free Full Text 26. Li K, Yan E, Feng Y: How Is R Cited in Research Outputs? Structure, Impacts, and Citation Standard. J Informetr. 2017; 11(4): 989–1002. Publisher Full Text 49. Katz DS, Ramnath R: Looking at Software Sustainability and Productivity Challenges from NSF. 2015. arXiv: 1508.03348. Reference Source 27. Li K, Chen PY, Yan E: Challenges of measuring software impact through citations: An examination of the lme4 R package. J Informetr. 2019; 13(1): 449–461. f S 50. DFG: Nachhaltigkeit von Forschungssoftware. 2016. Reference Source Publisher Full Text 58. Hasselbring W, Carr L, Hettrick S, et al.: From FAIR research data toward FAIR and open research software. it - Information Technology. 2020; 62(1): 39–47. Publisher Full Text 35. Venters CC, Jay C, Lau LMS, et al.: Software Sustainability: The Modern Tower of Babel. �� In: Proceedings of the Third International Workshop on Requirements Engineering for Sustainable Systems Co-Located with 22nd International Conference on Requirements Engineering (RE 2014), Karlskrona, Sweden: CEUR-WS. 2014; 1216: 7–12. Reference Source 59. https://www.acm.org/publications/policies/artifact-review-badging. 60. Schlauch T, Meinel M, Haupt C: DLR Software Engineering Gui 60. Schlauch T, Meinel M, Haupt C: DLR Software Engineering Guidelines. Deutsches Zentrum für Luft- und Raumfahrt (DLR). 2018. Publisher Full Text 61. Gomez-Diaz T, Recio T: On the evaluation of research software: the CDUR procedure [version 2; peer review: 2 approved]. F1000Res. 2019; 8: 1353. PubMed Abstract \| Publisher Full Text \| Free Full Text 36. Goble C: Better Software, Better Research. IEEE Internet Comput. 2014; 18(5): 4–8. Publisher Full Text 62. Fehr J, Heiland J, Himpe C, et al.: Best practices for replicability, reproducibility and reusability of computer-based experiments exemplified by model reduction software. AIMS Mathematics. 2016; 1(3): 261–281. 37. Druskat S: A Proposal for the Measurement and Documentation of Research Software Sustainability in Interactive Metadata Repositories. �� In: Proceedings of the Fourth Workshop on Sustainable Software for Science: Practice and Experiences (WSSSPE4), Manchester, UK: CEUR-WS. 2016; 1686. Reference Source ll Text 68. Venters CC, Capilla R, Betz S, et al.: Software sustainability: Research and practice from a software architecture viewpoint. J Syst Software. 2018; 138: 174–188. Publisher Full Text 43. Wilson G, Bryan J, Cranston K, et al.: Good enough practices in scientific computing. PLoS Comput Biol. 2017; 13(6): e1005510. PubMed Abstract \| Publisher Full Text \| Free Full Text blisher Full Text 63. Jiménez RC, Kuzak M, Alhamdoosh M, et al.: Four simple recommendations to encourage best practices in research software [version 1; peer review: 3 approved]. F1000Res. 2017; 6: pii: ELIXIR-876. PubMed Abstract \| Publisher Full Text \| Free Full Text 38. Katz DS: Fundamentals of Software Sustainability. 2018. Reference Source 39. Akhmerov A, Cruz M, Drost N, et al.: Raising the Profile of Research Software: Recommendations for Funding Agencies and Research Institutions. NWO (The Netherlands Organisation for Scientific Research). 2019. Reference Source 64. Hsu L, Hutchison VB, Langseth ML: Measuring sustainability of seed-funded earth science informatics projects. PLoS One. 2019; 14(10): e0222807. PubMed Abstract \| Publisher Full Text \| Free Full Text 40. Casties R, Czmiel A, Damerow J, et al.: DH Research Software Engineers - For We Are Many. 2019. Reference Source 65. Jackson M, Crouch S, Baxter R: Software Evaluation Guide. 2019. Reference Source 66. rOpenSci; Anderson B, Chamberlain S, et al.: Software Peer Review, Why? What? In: rOpenSci Packages: Development, Maintenance, and Peer Review Zenodo. 2019. Publisher Full Text 41. Wilson G, Aruliah DA, Brown CT, et al.: Best practices for scientific computing. PLoS Biol. 2014; 12(1): e1001745. PubMed Abstract \| Publisher Full Text \| Free Full Text 45. Kuchinke W, Ohmann C, Stenzhorn H, et al.: Ensuring sustainability of software tools and services by cooperation with a research infrastructure. ource 57. Hong NC: Minimal information for reusable scientific software. In: 2nd Workshop on Sustainable Software for Science: Practice and Experiences (WSSSPE2). 2014. Publisher Full Text 34. Vandewalle P: Code Sharing Is Associated with Research Impact in Image Processing. Comput Sci Eng. 2012; 14(4): 42–47. Publisher Full Text ublisher Full Text 42. Stodden V, Miguez S: Best Practices for Computational Science: Software Infrastructure and Environments for Reproducible and Extensible Research. J Open Res Softw. 2014; 2(1): e21. Publisher Full Text 67. Lee BD: Ten simple rules for documenting scientific software. PLoS Comput Biol. 2018; 14(12): e1006561. PubMed Abstract \| Publisher Full Text \| Free Full Text erence Source 51. DFG: Qualitätssicherung von Forschungssoftware durch ihre nachhaltige Nutzbarmachung. 2019. Reference Source 28. Park H, Wolfram D: Research software citation in the Data Citation Index: Current practices and implications for research software sharing and reuse. J Informetr. 2019; 13(2): 574–582. Publisher Full Text 52. Chan Zuckerberg Initiative: Essential Open Source Software for Science. Reference Source 29. Pan X, Yan E, Cui M, et al.: How Important Is Software to Library and Information Science Research? A Content Analysis of Full-Text Publications. J Informetr. 2019; 13(1): 397–406. Publisher Full Text 53. Rodríguez-Sánchez F, Marwick B, Lazowska E, et al.: Academia’s failure to retain data scientists. Science. 2017; 355(6323): 357–358. PubMed Abstract \| Publisher Full Text 30. Doerr A, Rusk N, Vogt N, et al.: Giving Software Its Due. Nat Methods. 2019; 16(3): 207–207. PubMed Abstract \| Publisher Full Text 54. Katz DS, Druskat S, Haines R, et al.: The State of Sustainable Research Software: Learning from the Workshop on Sustainable Software for Science: Practice and Experiences (WSSSPE5.1). J Open Res Softw. 2019; 7(1): 11. Publisher Full Text 31. Druskat S: Software and Dependencies in Research Citation Graphs. Comput Sci Eng. 2020; 22(2): 8–21. Publisher Full Text \| Free Full Text 55. Druskat S, Katz DS: Mapping the Research Software Sustainability Space. In: 2018 IEEE 14th International Conference on E-Science (e-Science). 2018; 25–30. Publisher Full Text 32. Katz DS, Bouquin D, Hong NP, et al.: Software Citation Implementation Challenges. arXiv: 1905.08674. 2019. Reference Source 56. Ye Y, Boyce RD, Davis MK, et al.: Open Source Software Sustainability Models: Initial White Paper from the Informatics Technology for Cancer Research Sustainability and Industry Partnership Work Group. 2019. Reference Sourcei 33. Resnik DB, Morales M, Landrum R, et al.: Effect of impact factor and discipline on journal data sharing policies. Account Res. 2019; 26(3): 139–156. PubMed Abstract \| Publisher Full Text \| Free Full Text Publisher Full Text 69. SSI: Writing and using a software management plan. 2019. Reference Source 44. Li K, Lin X, Greenberg J: Software Citation, Reuse and Metadata Considerations: An Exploratory Study Examining LAMMPS. Proc Assoc Infor Sci Tech. 2016; 53(1): 1–10. Publisher Full Text 70. Johanson A, Hasselbring W: Software engineering for computational science: Past, present, future. Comput Sci Eng. 2018; 20(2): 90–109. Publisher Full Text 45. Kuchinke W, Ohmann C, Stenzhorn H, et al.: Ensuring sustainability of software tools and services by cooperation with a research infrastructure. 71��. Akhmerov A, Cruz M, Drost N, et al.: Making Research Software a First-Class Page 17 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 81. Aartsen W, Peeters P, Wagers S, et al.: Getting Digital Assets from Public- Private Partnership Research Projects through “The Valley of Death,” and Making Them Sustainable. Front Med (Lausanne). 2018; 5: 65. PubMed Abstract \| Publisher Full Text \| Free Full Text 82. Gabella C, Durinx C, Appel R: Funding knowledgebases: Towards a sustainable funding model for the UniProt use case [version 2; peer review: 3 approved]. F1000Res. 2018; 6: pii: ELIXIR-2051. PubMed Abstract \| Publisher Full Text \| Free Full Text 83. Ison J, Rapacki K, Ménager H, et al.: Tools and data services registry: a community effort to document bioinformatics resources. Nucleic Acids Res. 2016; 44(D1): D38–D47. PubMed Abstract \| Publisher Full Text \| Free Full Text 84. Struck A: Research Software Discovery: An Overview. In: 2018 IEEE 14th International Conference on e-Science IEEE. 2018. Publisher Full Text 85. Druskat S, Spaaks JH, Chue Hong N, et al.: Citation File Format (CFF) - Specifications. 2019. Publisher Full Text 86. re3data.org – Registry of Research Data Repositories. Publisher Full Text 87. Mönnich MW: KVK - a meta catalog of libraries. LIBER Quarterly. 2001; 11(2): 121–127. Publisher Full Text 88. Kunze J, Scancella J, Adams C, et al.: The bagIt file packaging format (v1. 0). RFC Editor. 2018; 8493. 89. Bundesministerium der Justiz und für Verbraucherschutz, § 69a subsection (1) UrhG. 2014. Reference Source 90. Bundesministerium der Justiz und für Verbraucherschutz, § 69b UrhG. 2014. Reference Source 91. Struck A, Loewe A, Achhammer E, et al.: A Guide for Publishing, Using, and Licensing Research Software in Germany. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4327147 81. Aartsen W, Peeters P, Wagers S, et al.: Getting Digital Assets from Public- Private Partnership Research Projects through “The Valley of Death,” and Making Them Sustainable. Publisher Full Text In: 2018 IEEE 14th International Conference on e-Science IEEE. 2018. Publisher Full Text 75. Science Guide: Room for everyone’s talent. 2019. Reference Source 85. Druskat S, Spaaks JH, Chue Hong N, et al.: Citation File Format (CFF) - Specifications. 2019. Publisher Full Text 76. Carver JC, Hong NPC, Thiruvathukal GK: Software engineering for science. CRC Press, 2016; 274. Reference Source 77. Iaffaldano G, Steinmacher I, Calefato F, et al.: Why do developers take breaks from contributing to OSS projects? A preliminary analysis. arXiv: 1903.09528. 2019. Reference Source 87. Mönnich MW: KVK - a meta catalog of libraries. LIBER Quarterly. 2001; 11(2): 121–127. Publisher Full Text 87. Mönnich MW: KVK - a meta catalog of libraries. LIBER Quarterly. 2001; 11(2): 121–127. Publisher Full Text F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Publisher Full Text Front Med (Lausanne). 2018; 5: 65. PubMed Abstract \| Publisher Full Text \| Free Full Text Citizen in Research. 2019. Publisher Full Text Citizen in Research. 2019. Publisher Full Text 72� �� . Hong NC: Making Software A First-Class Citizen. 2019. Publisher Full Text 73� ��ä�� . Vereinigung der Kanzlerinnen und Kanzler der Universitäten Deutschlands: Bayreuther Erklärung zu befristeten Beschäftigungsverhältnissen mit wissenschaftlichem und künstlerischem Personal in Universitäten. 2019. Reference Source 74��. Katz DS, McInnes LC, Bernholdt DE, et al.: Community Organizations: Changing the Culture in Which Research Software Is Developed and Sustained. Comput Sci Eng. 2019; 21(2): 8–24. Publisher Full Text 75. Science Guide: Room for everyone’s talent. 2019. Reference Source 76. Carver JC, Hong NPC, Thiruvathukal GK: Software engineering for science. CRC Press, 2016; 274. Reference Source 77. Iaffaldano G, Steinmacher I, Calefato F, et al.: Why do developers take breaks from contributing to OSS projects? A preliminary analysis. arXiv: 1903.09528. 2019. Reference Source 78. Allen A, Aragon C, Becker C, et al.: Engineering Academic Software (Dagstuhl Perspectives Workshop 16252). Dagstuhl Manifestos 2017; 6(1): 1–20. Reference Source 79. Bundesministerium der Justiz und für Verbraucherschutz: Gesetz über befristete Arbeitsverträge in der Wissenschaft. 2017. Reference Source 80. Chang V, Mills H, Newhouse S: From Open Source to long-term sustainability: Review of Business Models and Case studies. In: Proceedings of the UK e-Science All Hands Meeting 2007 University of Edinburgh/University of Glasgow (acting through the NeSC) 2007. Reference Source 72� �� . Hong NC: Making Software A First-Class Citizen. 2019. Publisher Full Text 82. Gabella C, Durinx C, Appel R: Funding knowledgebases: Towards a sustainable funding model for the UniProt use case [version 2; peer review: 3 approved]. F1000Res. 2018; 6: pii: ELIXIR-2051. PubMed Abstract \| Publisher Full Text \| Free Full Text 73� ��ä�� . Vereinigung der Kanzlerinnen und Kanzler der Universitäten Deutschlands: Bayreuther Erklärung zu befristeten Beschäftigungsverhältnissen mit wissenschaftlichem und künstlerischem Personal in Universitäten. 2019. Reference Source 83. Ison J, Rapacki K, Ménager H, et al.: Tools and data services registry: a community effort to document bioinformatics resources. Nucleic Acids Res. 2016; 44(D1): D38–D47. PubMed Abstract \| Publisher Full Text \| Free Full Text 74��. Katz DS, McInnes LC, Bernholdt DE, et al.: Community Organizations: Changing the Culture in Which Research Software Is Developed and Sustained. Comput Sci Eng. 2019; 21(2): 8–24. Publisher Full Text 84. Struck A: Research Software Discovery: An Overview. Version 2 © 2021 Hasselbring W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.5256/f1000research.25640.r62872 © 2020 Bast R. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Peer Review Current Peer Review Status: sher Full Text 78. Allen A, Aragon C, Becker C, et al.: Engineering Academic Software (Dagstuhl Perspectives Workshop 16252). Dagstuhl Manifestos 2017; 6(1): 1–20. Reference Source 88. Kunze J, Scancella J, Adams C, et al.: The bagIt file packaging format (v1. 0). RFC Editor. 2018; 8493. 89. Bundesministerium der Justiz und für Verbraucherschutz, § 69a subsection (1) UrhG. 2014. Reference Source 79. Bundesministerium der Justiz und für Verbraucherschutz: Gesetz über befristete Arbeitsverträge in der Wissenschaft. 2017. Reference Source 90. Bundesministerium der Justiz und für Verbraucherschutz, § 69b UrhG. 2014. Reference Source 80. Chang V, Mills H, Newhouse S: From Open Source to long-term sustainability: Review of Business Models and Case studies. In: Proceedings of the UK e-Science All Hands Meeting 2007 University of Edinburgh/University of Glasgow (acting through the NeSC) 2007. Reference Source 91. Struck A, Loewe A, Achhammer E, et al.: A Guide for Publishing, Using, and Licensing Research Software in Germany. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4327147 Page 18 of 34 Page 18 of 34 Willi Hasselbring Software Engineering Group, Kiel University, Kiel, Germany Thanks for delivering this revised version of your opinion article. I highly appreciate that you addressed all the concerns I had with the previous version, such that I can now fully approve your paper! Competing Interests: No competing interests were disclosed. Reviewer Expertise: Software Engineering I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Open Peer Review in Germany and beyond. in Germany and beyond. They examine the current state of research software sustainability and challenges in motivating sustainable research software development, selection criteria for funding, personnel, funding, infrastructure, and legal aspects, and offer recommendations for addressing these challenges. These sections are accompanied and with a story using a fictional character Kim which helps to relate these aspects to typical career stages of a research software engineer. The article is thoroughly researched, well-written, and offers an excellent overview of the challenges when building an environment for sustainable research software. Most of the discussed challenges and recommendations carry beyond Germany and are relevant and transferable to other countries. elow I give few (minor) suggestions for consideration when improving the manuscript Regarding the list of challenges under "Why sustainable research software in the first place?" (pages 5 and 6): (p g ) Infrastructure issues: One design choice that often limits the use or usability of local infrastructure resources is that they are often bound to institutional user accounts and thus limit collaboration possibilities with collaborators in other institutions and countries. On the other hand, pooling of infrastructure resources which could enable collaboration across organizations can be limited by lack of authentication and authorization infrastructure (AAI) or legal constraints. Later in the paper the authors indeed mention AAI (page 12) but this could already be pointed out and connected earlier. ○ Infrastructure issues: One design choice that often limits the use or usability of local infrastructure resources is that they are often bound to institutional user accounts and thus limit collaboration possibilities with collaborators in other institutions and countries. On the other hand, pooling of infrastructure resources which could enable collaboration across organizations can be limited by lack of authentication and authorization infrastructure (AAI) or legal constraints. Later in the paper the authors indeed mention AAI (page 12) but this could already be pointed out and connected earlier. ○ Legal issues: Not only licensing is an issue but legal constraints or uncertainty about legal boundaries and identity federation can also limit the deployment of infrastructure services. Often the deployment and operation of infrastructure services is given to technical teams who may lack the legal support or expertise to clarify legal and privacy terms for the storage of data and processing of data. Radovan Bast Radovan Bast Department of Information Technology, UiT The Arctic University of Norway, Tromsø, Norway In "An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action" the authors identify challenges for research software sustainability Department of Information Technology, UiT The Arctic University of Norway, Tromsø In "An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action" the authors identify challenges for research software sustainability Page 19 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 in Germany and beyond. Funding issues: The challenge is not only that funding is scarce but also that it does not align well with pricing models of cloud infrastructure providers. It can be easier for research groups to spend a larger chunk of the budget towards the end of a year for hardware compared to pay possibly relatively modest monthly fees for a cloud service, which however may not fit into the budget forms. These budget constraints may also limit the possibility of pooling resources and sharing them with other research groups. Software cloud infrastructure is often not considered at all in the proposal. There is also a resistance among some of my research colleagues to pay 20-50 USD/ month for an infrastructure service which is sometimes solved by reinventing the service locally "for free". Another mismatch between traditional funding models and support of software which "must continue to exist" to be sustainable (page 5), is the experience that it can take months or years until the software is picked up by other groups and contributions and questions start to roll in. But by that time the funding of the project stopped, the developer (team) may have already moved on to other positions and projects, and may not have the time to react and help, even though they still may have interest and the knowledge. Our traditional funding models consider the software to be "done" by the end of the project. Page 20 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Selection criteria for "How to decide which software to sustain?" (page 9): The authors mention "usage and impact", "software quality", as well as "maturity". But I would like to see also "openness and transparency" among these. The reason is that we can expect the research community to adapt to these or any metrics and we will over time observe what we measure. Any set of metrics could be criticized as to some extent being arbitrary but the advantage of including "openness and transparency" is that the community as whole would benefit from such a metric [Enrico Glerean, "Responsible conduct of research and questionable research practices", presentation, slide 471]. ○ Regarding "Who sustains research software?": The authors discuss the lack of recognition and awareness, as well as lack of career opportunities. References 1. Enrico Glerean: Responsible conduct of research and questionable research practices. 2018. Reference Source in Germany and beyond. It is also about respect and I was happy to see the sentence: "Not all researchers that think of themselves as RSEs pursue a faculty position as their main career goal." I have experienced that RSEs are sometimes regarded as those who somehow "failed" to obtain a faculty position whereas many RSEs have chosen this position over a faculty position because it was a better fit for their career goals. This misunderstanding can lead to a lack of respect towards this position and this career choice and can lead to excellent personnel leaving the academic environment towards commercial employment, possibly not primarily for financial reasons but sometimes to be more respected and recognized. ○ Archiving and software preservation (page 13: The authors mention Docker but also Singularity should be mentioned as a tool since it is getting traction in particular on many-user systems such as higher performance computing clusters. ○ Legal aspects (page 14): Re-licensing is mentioned in the story box and the text starts by pointing out that licensing is often not considered early enough in the project. Indeed re-licensing later in the project can be not only legally, but also organizationally very tricky, in particular for projects which developed over many years and involved many contributors in different organizations. This could be pointed out in the text as additional motivation to consider these very early in the project. ○ I very much like the recommendation of providing a legal help desk for research groups to avoid the problem that out of uncertainty and fear of making a legal mistake some research groups end up not choosing any license at all which may limit further reuse of the software. ○ The manuscript presents a decision tree for contributors (Figure 1) and also discusses contributor license agreements. It could be useful to point out that without clear policies or legal help desks, individuals or organizations may be hesitant to contribute to a project because they may not feel confident having enough knowledge or authority to sign such agreements and too many legal steps and question can also raise the barrier to contribute, in particular for smaller projects. Also here clear guidelines and a support desk can help removing these barriers. ○ References 1. Enrico Glerean: Responsible conduct of research and questionable research practices. 2018. I confirm that I have read this submission and believe that I have an appropria expertise to confirm that it is of an acceptable scientific standard. Author Response 17 Dec 2020 in Germany and beyond. Reference Source Page 21 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Is the topic of the opinion article discussed accurately in the context of the current literature? Yes Are all factual statements correct and adequately supported by citations? Yes Are arguments sufficiently supported by evidence from the published literature? Yes Are the conclusions drawn balanced and justified on the basis of the presented arguments? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: computational chemistry, research software engineering I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Axel Loewe, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany There is also a resistance among some of my research colleagues to pay 20-50 USD/ month for an infrastructure service which is sometimes solved by reinventing the service locally "for free". Another mismatch between traditional funding models and support of software which "must continue to exist" to be sustainable (page 5), is the experience that it can take months or years until the software is picked up by other groups and contributions and questions start to roll in. But by that time the funding of the project stopped, the developer (team) may have already moved on to other positions and projects, and may not have the time to react and help, even though they still may have interest and the knowledge. Our traditional funding models consider the software to be "done" by the end of the project. We thank the reviewer for these two comments. To address them, we have extended the “Funding issues” list item with a discussion of these issues. Funding issues: The challenge is not only that funding is scarce but also that it does not align well with pricing models of cloud infrastructure providers. It can be easier for research groups to spend a larger chunk of the budget towards the end of a year for hardware compared to pay possibly relatively modest monthly fees for a cloud service, which however may not fit into the budget forms. These budget constraints may also limit the possibility of pooling resources and sharing them with other research groups. Software cloud infrastructure is often not considered at all in the proposal. There is also a resistance among some of my research colleagues to pay 20-50 USD/ month for an infrastructure service which is sometimes solved by reinventing the service locally "for free". y g y Another mismatch between traditional funding models and support of software which "must continue to exist" to be sustainable (page 5), is the experience that it can take months or years until the software is picked up by other groups and contributions and questions start to roll in. But by that time the funding of the project stopped, the developer (team) may have already moved on to other positions and projects, and may not have the time to react and help, even though they still may have interest and the knowledge. election criteria for "How to decide which software to sustain?" (page 9): Selection criteria for "How to decide which software to sustain?" (page 9): The authors mention "usage and impact", "software quality", as well as "maturity". But I would like to see also "openness and transparency" among these. The reason is that we can expect the research community to adapt to these or any metrics and we will over time observe what we measure. Any set of metrics could be criticized as to some extent being arbitrary but the advantage of including "openness and transparency" is that the community as whole would benefit from such a metric [Enrico Glerean, "Responsible conduct of research and questionable research practices", presentation, slide 471]. We thank the reviewer for this suggestion and realized that indeed most aspects in this section (6- 10, 12, 13) are actually related to openness and transparency. Therefore, we changed the title of this section to “Software transparency and quality”. Axel Loewe, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany Axel Loewe, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany We thank you for the thorough review and constructive feedback regarding the manuscript. Below, we address the issues raised by you point-by-point. Our responses are set in italics. Regarding the list of challenges under "Why sustainable research software in the first place?" (pages 5 and 6): Regarding the list of challenges under "Why sustainable research software in the first place?" (pages 5 and 6): Infrastructure issues: One design choice that often limits the use or usability of local infrastructure resources is that they are often bound to institutional user accounts and thus limit collaboration possibilities with collaborators in other institutions and countries. On the other hand, pooling of infrastructure resources which could enable collaboration across organizations can be limited by lack of authentication and authorization infrastructure (AAI) or legal constraints. Later in the paper the authors indeed mention AAI (page 12) but this could already be pointed out and connected earlier. y p We thank the reviewer for their suggestion, and have included the mentioned issues in the respective list in the section “Why sustainable research software in the first place?”. Legal issues: Not only licensing is an issue but legal constraints or uncertainty about legal boundaries and identity federation can also limit the deployment of infrastructure services. Often the deployment and operation of infrastructure services is given to technical teams who may lack the legal support or expertise to clarify legal and privacy terms for the storage of data and processing of data. g p g We thank the reviewer for their suggestion, and have included the mentioned issues in the respective list in the section “Why sustainable research software in the first place?” Page 22 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Funding issues: The challenge is not only that funding is scarce but also that it does not align well with pricing models of cloud infrastructure providers. It can be easier for research groups to spend a larger chunk of the budget towards the end of a year for hardware compared to pay possibly relatively modest monthly fees for a cloud service, which however may not fit into the budget forms. These budget constraints may also limit the possibility of pooling resources and sharing them with other research groups. Software cloud infrastructure is often not considered at all in the proposal. Axel Loewe, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany Our traditional funding models consider the software to be "done" by the end of the project. We thank the reviewer for these two comments. To address them, we have extended the “Funding i ” li t it ith di i f th i getting traction in particular on many-user systems such as higher performance computing clusters. getting traction in particular on many-user systems such as higher performance computing clusters. We thank the reviewer for this suggestion and now also mention Singularity and GUIX. However, we are not aiming for an exhaustive list, as options change dynamically and might even be specific to certain research communities. Legal aspects (page 14): Re-licensing is mentioned in the story box and the text starts by pointing out that licensing is often not considered early enough in the project. Indeed re-licensing later in the project can be not only legally, but also organizationally very tricky, in particular for projects which developed over many years and involved many contributors in different organizations. This could be pointed out in the text as additional motivation to consider these very early in the project. We thank the reviewer to point this out and it also nicely fits into the message of increasing the awareness of legal aspects early on in the project. We have added the suggested sentence in the manuscript. I very much like the recommendation of providing a legal help desk for research groups to avoid the problem that out of uncertainty and fear of making a legal mistake some research groups end up not choosing any license at all which may limit further reuse of the software. Thank you for supporting this idea. We have further included your idea of avoiding any license out of a fear to make legal mistakes. The manuscript presents a decision tree for contributors (Figure 1) and also discusses contributor license agreements. It could be useful to point out that without clear policies or legal help desks, individuals or organizations may be hesitant to contribute to a project because they may not feel confident having enough knowledge or authority to sign such agreements and too many legal steps and question can also raise the barrier to contribute, in particular for smaller projects. Also here clear guidelines and a support desk can help removing these barriers. g We have decided to take the decision trees out of the manuscript to strengthen our point of publishing software under a FLOSS license. Instead, we published the decision trees together with documentation templates under a Creative Commons license via Zenodo: https://zenodo.org/record/4327148#.X9n6ui337OQ. In order to strengthen the point you addressed, we added some more details related to infrastructural investment. Competing Interests: No competing interests were disclosed. Reviewer Report 13 May 2020 https://doi.org/10.5256/f1000research.25640.r62873 Regarding "Who sustains research software?": g g The authors discuss the lack of recognition and awareness, as well as lack of career opportunities. It is also about respect and I was happy to see the sentence: "Not all researchers that think of themselves as RSEs pursue a faculty position as their main career goal." I have experienced that RSEs are sometimes regarded as those who somehow "failed" to obtain a faculty position whereas many RSEs have chosen this position over a faculty position because it was a better fit for their career goals. This misunderstanding can lead to a lack of respect towards this position and this career choice and can lead to excellent personnel leaving the academic environment towards commercial employment, possibly not primarily for financial reasons but sometimes to be more respected and recognized. We fully agree with the reviewer and thank them for the renewed confirmation that this is seen as problematic not only by the authors. Archiving and software preservation (page 13): Page 23 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Willi Hasselbring Software Engineering Group, Kiel University, Kiel, Germany The authors describe the state of the practice and current challenges for research software sustainability and suggest measures towards improvements that can solve these challenges. In particular, they propose to fund a German Software Sustainability Institute. The paper is the result of a community effort, with work undertaken during two workshops and subsequent collaborative work across the larger RSE community in Germany. The UK Software Sustainability Institute has already been established during a decade ( https://www.software.ac.uk/blog/2020-05-05-impact-institute-10-years). Thus, the idea of such an institute is not new, but it makes sense to take a specific look at the German situation. Besides universities, the German states (local and in particular federal) fund significant large-scale research associations (Helmholtz/DLR, Max-Planck, Leibniz). This is not the case for most other European states, at least not with a similar scale. Another specialty is the lack of long-term funding for research software engineers, as discussed by the authors. The paper is well-written and easy to read. I like the boxed story of Kim’s career path However, I’ve some suggestions for improving the paper: Concerning the statement “In order to support research, a sustainable software must be correct”, I suggest to include a short discussion of the test oracle problem for scientific software (see for instance https://doi.org/10.1109/SECSE.2013.66150991). ○ Concerning the discussion of “The list of criteria presented in this section could be the basis for a structured review process…” I suggest to include two additional initiatives for software review. The first is artifact evaluation in computer science conferences (the process is explained in https://doi.org/10.1515/itit-2019-00402). The second is the SPEC Research Group’s review process of tools for quantitative system evaluation and analysis (https://research.spec.org/tools/submission.html). ○ Concerning the discussion of “The list of criteria presented in this section could be the basis for a structured review process…” I suggest to include two additional initiatives for software review. The first is artifact evaluation in computer science conferences (the process is explained in https://doi.org/10.1515/itit-2019-00402). The second is the SPEC Research Group’s review process of tools for quantitative system evaluation and analysis (https://research.spec.org/tools/submission.html). https://doi.org/10.5256/f1000research.25640.r62873 © 2020 Hasselbring W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 24 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Willi Hasselbring ○ The authors write “We also argue that truly sustainable research software must ideally be published under a Free/Libre Open Source Software (FLOSS) license, and follow an open development model…” what I fully support (see for instance https://doi.org/10.1515/itit-2019-0040 2 The authors write We also argue that truly sustainable research software must ideally be published under a Free/Libre Open Source Software (FLOSS) license, and follow an open development model…” what I fully support (see for instance https://doi.org/10.1515/itit-2019-0040 2). However, later under the section heading “Legal aspects” this requirement is thwarted. I fully agree that legal aspects have to be considered, but the general bias of this section seems to be on commercial licensing of research software. For instance, the decision tree in Figure 1 starts with the question “Licensing planned?”. I assume that commercial licensing is meant, but this is not clear since the figures are not explained in the paper. Instead, the process should start with open sourcing the software. If licenses such as Apache or MIT are applied, the research institutions may later still commercialize the software if appropriate. Such open source licensing is also beneficial for start-ups, that intend to provide professional services for the software. My experience with technology transfer units of German universities and research institutes is that they do not understand the ideas of open source business models (see for instance https://doi.org/10.1109/MC.2019.28981633). Their focus is on patents and commercializing licenses, sometimes also on start-ups. Conversely, in the software industry, one major motivation for open sourcing software is on improving the quality of software. I cite from https://doi.org/10.1109/ICSAW.2017.114 : “the open-source approach has some psychological Page 25 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 effects: Developers show a tendency to apply higher quality standards if they know that the code will be publicly available.” For sustainability, quality is an important property of software. effects: Developers show a tendency to apply higher quality standards if they know that the code will be publicly available.” For sustainability, quality is an important property of software. The Figures 1-4 do more harm than good. They are daunting to researchers who intend to publish their code open source. These figures should be removed from the paper, they are useless without proper explanation. I suggest that the authors focus in the present paper on their main message (request for funding a German Software Sustainability Institute, which I fully support). Willi Hasselbring Figures 1-4 could be moved to a separate paper, enriched with proper explanation. References References 1. Kanewala U, Bieman JM: Techniques for testing scientific programs without an oracle. IEEE Xplore. 2013. Publisher Full Text 2. Hasselbring W, Carr L, Hettrick S, Packer H, et al.: From FAIR research data toward FAIR and open research software. it - Information Technology. 2020; 62 (1): 39-47 Publisher Full Text 3. Riehle D: The Innovations of Open Source. Computer. 2019; 52 (4): 59-63 Publisher Full Text 4. Hasselbring W, Steinacker G: Microservice Architectures for Scalability, Agility and Reliability in E-Commerce. IEEE. 2017. Publisher Full Text 5. Tools Submission Portal. SPEC Research Group. Reference Source References 1. Kanewala U, Bieman JM: Techniques for testing scientific programs without an oracle. IEEE Xplore. 2013. Publisher Full Text 2. Hasselbring W, Carr L, Hettrick S, Packer H, et al.: From FAIR research data toward FAIR and open research software. it - Information Technology. 2020; 62 (1): 39-47 Publisher Full Text 3. Riehle D: The Innovations of Open Source. Computer. 2019; 52 (4): 59-63 Publisher Full Text 4. Hasselbring W, Steinacker G: Microservice Architectures for Scalability, Agility and Reliability in E-Commerce. IEEE. 2017. Publisher Full Text 5. Tools Submission Portal. SPEC Research Group. Reference Source Are arguments sufficiently supported by evidence from the published literature? Partly Are the conclusions drawn balanced and justified on the basis of the presented arguments? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Software Engineering I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 17 Dec 2020 Axel Loewe, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany Thank you for the thorough review and constructive feedback regarding our manuscript. Below, Page 26 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 we address the issues raised by you point-by-point. Our responses are set in italics. Concerning the statement “In order to support research, a sustainable software must be correct”, I suggest to include a short discussion of the test oracle problem for scientific software (see for instance https://doi.org/10.1109/SECSE.2013.66150991). We thank the reviewer for this suggestion, and included a brief discussion of the test oracle problem as suggested, and additionally of further challenges to verification and validation, such as large configuration spaces and heterogeneous data (as discussed in e.g. https://doi.org/10.1109/SE4Science.2019.00010), and have suggested to implement the solutions mentioned in the literature. Concerning the discussion of “The list of criteria presented in this section could be the basis for a structured review process…” I suggest to include two additional initiatives for software review. The first is artifact evaluation in computer science conferences (the process is explained in https://doi.org/10.1515/itit-2019-00402). The second is the SPEC Research Group’s review process of tools for quantitative system evaluation and analysis ( h h l b i i h l problems and agree to your arguments. We like your suggested aspect of commercialization of FLOSS licensed software and included this aspect in the manuscript. problems and agree to your arguments. We like your suggested aspect of commercialization of FLOSS licensed software and included this aspect in the manuscript. The Figures 1-4 do more harm than good. They are daunting to researchers who intend to publish their code open source. These figures should be removed from the paper, they are useless without proper explanation. I suggest that the authors focus in the present paper on their main message (request for funding a German Software Sustainability Institute, which I fully support). Figures 1-4 could be moved to a separate paper, enriched with proper explanation. We thank the reviewer for this suggestion. Our initial thought was to place these decision trees in the supplemental material but did not realize that this is not the policy of f1000. The editorial team moved them into the main article. This is the reason why the decision trees appeared without additional information in the manuscript. We have now decided to take the Figures out and have published them together with documentation templates in a separate report via Zenodo unde a Creative Commons license, see , ttps://zenodo.org/record/4327148#.X9n6ui337OQ. This report is now cited in the F1000 anuscript. https://zenodo.org/record/4327148#.X9n6ui337OQ. This report is now cited in the F1000 manuscript. Competing Interests: No competing interests were disclosed. p p q y https://research.spec.org/tools/submission.html). p p g We thank the reviewer for this suggestion and included the artifact review approach in the introduction to the criteria section. The aspects of repeatability, reproducibility, and replicability are aimed more at the results of computational research rather than research software itself, we feel. Therefore, we didn’t include specific criteria in the list suggested to be used when evaluating research software for sustained funding. f f f g While the SPEC submission process is very clear, we could not find any concrete criteria applied during the review (except for requirements regarding the license). The authors write “We also argue that truly sustainable research software must ideally be published under a Free/Libre Open Source Software (FLOSS) license, and follow an open development model…” what I fully support (see for instance https://doi.org/10.1515/itit- 2019-00402). However, later under the section heading “Legal aspects” this requirement is thwarted. I fully agree that legal aspects have to be considered, but the general bias of this section seems to be on commercial licensing of research software. For instance, the decision tree in Figure 1 starts with the question “Licensing planned?”. I assume that commercial licensing is meant, but this is not clear since the figures are not explained in the paper. Instead, the process should start with open sourcing the software. If licenses such as Apache or MIT are applied, the research institutions may later still commercialize the software if appropriate. Such open source licensing is also beneficial for start-ups that intend to provide professional services for the software. My experience with technology transfer units of German universities and research institutes is that they do not understand the ideas of open source business models (see for instance https://doi.org/10.1109/MC.2019.28981633). Their focus is on patents and commercializing licenses, sometimes also on start-ups. Conversely, in the software industry, one major motivation for open sourcing software is on improving the quality of software. I cite from https://doi.org/10.1109/ICSAW.2017.114 : “the open-source approach has some psychological effects: Developers show a tendency to apply higher quality standards if they know that the code will be publicly available.” For sustainability, quality is an important property of software. p p q y https://research.spec.org/tools/submission.html). As further detailed below, we have moved the decision trees out of this manuscript as we see the As further detailed below, we have moved the decision trees out of this manuscript as we see the Page 27 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 problems and agree to your arguments. We like your suggested aspect of commercialization of FLOSS licensed software and included this aspect in the manuscript. think of them. Looking forward to your opinion and you feedback to my comments, if you like also gladly by telephone Best regards Dirk Feuchter Thank you very much for your comments regarding distribution issues, IP compliance and commercialization. We have generally subsumed these issues under the more general term “availability”. We believe that this implies that software is legally available only if it is licensed, either under an open source license or proprietarily. This impacts possible modes of distribution, but does not concern actual (commercial) distribution processes. As a community of Research Software Engineers, we strongly believe that publicly funded research software should be F(L)OSS-licensed per default, although we recognize that this may not always be possible (and hence have weakened “must” to “should” as you suggested in the respective paragraph under “Why sustainable research software in the first place”). We have further avoided any changes to the text that would weaken this point, as we see no central obstacles to making publicly funded research software open source in general. Contrarily, we do not accept that IP should override public interest (both intellectually and fiscally) here, some corner cases excluded. Concurrently, we purposefully do not focus on marketability and commercialization of research software. Instead, we see commercial opportunities, e.g., in the provision of services for a research software product, whereas the product itself should remain free and open source. p f f p Thank you, also, for notifying us of some errors in the text itself and the figures, which we are fixing in the next version. Below, we address some of your concrete suggestions in more detail: Abstract: I would like to suggest to extend „Research software must be sustainable in order to understand, replicate, reproduce, and…“ as follows „Research software must be sustainable in order to understand, replicate, reproduce, distribute and…“ We thank you for the comment. The development of research software does not focus on distribution of software results, which is more of a business aspect. The availability, regardless of the actual distribution, is noted in the next sentence of the abstract. Abstract: I would like to suggest to extend „Research software must be sustainable in order to understand, replicate, reproduce, and…“ as follows „Research software must be sustainable in order to understand, replicate, reproduce, distribute and…“ We thank you for the comment. The development of research software does not focus on distribution of software results, which is more of a business aspect. The availability, regardless of the actual distribution is noted in the next sentence of the abstract. Comments on this article Version 1 Author Response 17 Dec 2020 Author Response 17 Dec 2020 Axel Loewe, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany We thank you for your thorough review and constructive feedback regarding the manuscript. Below, we address the issues raised by you point-by-point. Our responses are set in italics. Dear Mr. Anzt, dear Mr. Loewe, dear Mr. Bach, dear Mr. Seemann, dear Elke, and dear Sven, as well as dear authors as yet unknown to me, I am working at KIT in the field of Innovation and Relations Management, especially Licensing of Intellectual Property Rights from KIT to Free Maket Economy. My particual focus is to Out-license Computerprograms to Third Parties and Industry. I am working at KIT in the field of Innovation and Relations Management, especially Licensing of Intellectual Property Rights from KIT to Free Maket Economy. My particual focus is to Out-license Computerprograms to Third Parties and Industry. Thank you for your great efforts with your extensive and intersting article including the nicely written and ever-recurring story of Kim. Thank you for your great efforts with your extensive and intersting article including the nicely written and ever-recurring story of Kim. Your FLOSS-based approach for sustainable software devlopment is holding immense savings potential. That’s great. Your FLOSS-based approach for sustainable software devlopment is holding immense savings potential. That’s great. Following are my comments to your article from the perspective of a TTO license manager, typically supporting RSEs in cases of proprietary licensing e.g. to spin-offs or industrial companies. Hence, most of my comments might go in a slightly different direction than the main focus of your article, but in my point of view these comments are complementary. Hence, I wonder what you Following are my comments to your article from the perspective of a TTO license manager, typically supporting RSEs in cases of proprietary licensing e.g. to spin-offs or industrial companies. Hence, most of my comments might go in a slightly different direction than the main focus of your article, but in my point of view these comments are complementary. Hence, I wonder what you Page 28 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 think of them. Looking forward to your opinion and you feedback to my comments, if you like also gladly by telephone Best regards Abstract: I would like to suggest to extend „In other words, software must be available, discoverable, usable, and adaptable to new needs, both now and in the future.“ as follows „In other words, software must be available, (IP-/FLOSS)compliant, discoverable, usable, and adaptable to new needs, both now and in the future.“ By „(IP-/FLOSS)compliant“ I mean in compliance with intellectual property of third-party suppliers, with the terms of free/libre open source licenses and with the aim to protect own intellectual property from unintended disclosure We thank you for the comment. As a community of RSEs we are aiming for FLOSS whenever feasible but Abstract: Page 29 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 nowledge that there are scenarios in which non-FLOSS licenses need to be considered. Suggestion of a new penultimate paragraph in subchapter „Stakeholder motivations for research software sustainability“ as follows: RSEs, Research leaders and research performing organisations are interested in software sustainability also in the sense that their (research)software is sustainable concerning legal compliance.This is an important issue distributing (research)software for both acaedmic puposes as well as commerical purposes. For the latter RSEs and their research leaders (typically contacting their TTO) are interested to marktet (parts of) their research software and/or additional connectable closed software (which they prevent from unintended disclosure) to a spin-off or an industrial company using a proprietary or a dual licensing model. For both, acaedmic and commerical purposes RSEs and their research leaders and their research performing organisation are interested that their (research) software plus any connectable closed software is compliant with intellectual property of third-party suppliers and compliant with the terms of free/libre open source licenses. An important stakeholder motivation is therefore „software sustainbility with the aim of clarification of all software rights ownerships“. We thank you for the suggestion While we aim for reusable software in terms of licenses we as a y g p We thank you for the suggestion. While we aim for reusable software in terms of licenses we as a community do not focus on commercial purposes. Please replace Subchapter heading„Challenges and clarifications Clarification of rights“by „Challenges and Clarification of rights“ We thank you for pointing this out and corrected the structure (section and subsection headings, respectively). The term "research institution" appears in the glossary as a duplicate. Therefore, "research institution" should be deleted in the brackets. Thank you, we have replaced it by “national labs”. as follows „In other words, software must be available, (IP-/FLOSS)compliant, discoverable, usable, and adaptable to new needs, both now and in the future.“ think of them. Looking forward to your opinion and you feedback to my comments, if you like also gladly by telephone Best regards Competing Interests: No competing interests were disclosed. Reader Comment 19 May 2020 Dirk Feuchter, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen and Karlsruhe, Germany Subject: Feedback to your article „An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action…“. Subject: Feedback to your article „An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action…“. Dear Mr. Anzt, dear Mr. Loewe, dear Mr. Bach, dear Mr. Seemann, dear Elke, and dear Sven, as well as dear authors as yet unknown to me, I am working at KIT in the field of Innovation and Relations Management, especially Licensing of Intellectual Property Rights from KIT to Free Maket Economy. My particual focus is to Out-license Computerprograms to Third Parties and Industry. Your FLOSS-based approach for sustainable software devlopment is holding immense savings Page 30 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 potential. That’s great. Following are my comments to your article from the perspective of a TTO license manager, typically supporting RSEs in cases of proprietary licensing e.g. to spin-offs or industrial companies. Hence, most of my comments might go in a slightly different direction than the main focus of your article, but in my point of view these comments are complementary. Hence, I wonder what you think of them. Looking forward to your opinion and you feedback to my comments, if you like also gladly by telephone Best regards Dirk Feuchter # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-01: Abstract: I would like to suggest to extend „Research software must be sustainable in order to understand, replicate, reproduce, and…“ as follows „Research software must be sustainable in order to understand, replicate, reproduce, distribute and…“ # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-02: Abstract: I would like to suggest to extend „In other words, software must be available, discoverable, usable, and adaptable to new needs, both now and in the future.“ # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-05:Why sustainable research software in the first place =>„Therefore, if research software is publicly funded, it should be freely available under a FLOSS license.„Therefore, if research software is publicly funded, it should be normally freely available under a FLOSS license. # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-06:Why sustainable research software in the first place =>I would like to suggest to extend„Legal issues: Many obstacles for research software pertain to legal issues, such as applicable licensing and compatibility of licenses45, and decisions about license types. as follows „Legal issues: Many obstacles for research software pertain to legal issues, such as IT law, copyright law, copyright notices and author attributions, applicable licensing and compatibility of licenses45, and decisions about license types. # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-07:Why sustainable research software in the first place =>„A subset of this group may be interested in …“.Which „group“ do you mean? => the „Taxpayers“? # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-08:Suggestion of a new penultimate paragraph in subchapter „Stakeholder motivations for research software sustainability“ as follows: RSEs, Research leaders and research performing organisations are interested in software sustainability also in the sense that their (research)software is sustainable concerning legal compliance.This is an important issue distributing (research)software for both acaedmic puposes as well as commerical purposes. For the latter RSEs and their research leaders (typically contacting their TTO) are interested to marktet (parts of) their research software and/or additional connectable closed software (which they prevent from unintended disclosure) to a spin-off or an industrial company using a proprietary or a dual licensing model. For both, acaedmic and commerical purposes RSEs and their research leaders and their research performing organisation are interested that their (research) software plus any connectable closed software is compliant with intellectual property of third-party suppliers and compliant with the terms of free/libre open source licenses. An important stakeholder motivation is therefore „software sustainbility with the aim of clarification of all software rights ownerships“. By „(IP-/FLOSS)compliant“ I mean in compliance with intellectual property of third-party suppliers, • with the terms of free/libre open source licenses and with the aim to protect own intellectual property from unintended disclosure • with the terms of free/libre open source licenses and with the aim to protect own intellectual property from unintended disclosure • # # # # # # # # # # # # # # # # # # # # # # #Comment-DF-03:Why sustainable research software in the first place =>I would like to suggest to extend„In order to support research, a sustainable software must be correct14, validatable, understandable, documented, publicly released,...“ as follows „In order to support research, a sustainable software must be correct14, (IP-/FLOSS) compliant* validatable, understandable, documented, publicly released,...“ *Concerning „(IP- /FLOSS)compliant“ please see upon # # # # # # # # # # # # # # # # # # # # # # # Page 31 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 Comment-DF-04:Why sustainable research software in the first place =>„We also argue that truly sustainable research software must ideallybe published under a Free/Libre Open Source Software (FLOSS) license, and…“„We also argue that truly sustainable research software should typically be published under a Free/Libre Open Source Software (FLOSS) license, and…“ • Comment-DF-04:Why sustainable research software in the first place =>„We also argue that truly sustainable research software must ideallybe published under a Free/Libre Open Source Software (FLOSS) license, and…“„We also argue that truly sustainable research software should typically be published under a Free/Libre Open Source Software (FLOSS) license, and…“ • # # # # # # # # # # # # # # # # # # # # # # #Comment-DF-10:In the heading such as „Why # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-12:In both Fig. 1 and „Legal aspects/Challenges and clarifications of rights“instead of „Subject to directives“ {weisungsgebunden dt.} from UrhG 69 bI propose to write „in execution of his duties {in Wahrnehmung seiner Aufgaben dt.}“ from UrhG 69b, in no way to constrain scientists, RSEs and Research leaders, but in order to free up the scope for decision-making and thus open up opportunities.[Regardless of that, I would translate the German "weisungsgebunden" from „UrhG 69 b“ with "bound by instructions"(short) or with "following the instructions given by his employer"(long)] # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-09:Abstract:I would like to suggest to extend„Failing to do so will threaten the quality and validity of research..“ as follows „Failing to do so will threaten the quality, Xi and validity of research..“ // X1= marketability or X2= distribution // As TTO-license-manager I personally would prefer X1 but X2 is fine as well Page 32 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 sustainable research software in the first place?“, one might prefix a chapter number, just as announced at the end of the introductory background: „This all leads to unmet requirements and unsolved challenges that we want to highlight in this paper by elaborating on (1)…(2)…(3)…(4)…(5)…(6)…“Hence, instead of„Why sustainable research software in the first place?“use„(1) Why sustainable research software in the first place?“and so on:(2) How to decide which software to sustain?(3) Who sustains research software?(4) How can research software be sustainably funded?(5) Which infrastructure is needed to sustain research software?(6) Legal aspects.That's a matter of taste, of course. sustainable research software in the first place?“, one might prefix a chapter number, just as announced at the end of the introductory background: „This all leads to unmet requirements and unsolved challenges that we want to highlight in this paper by elaborating on (1)…(2)…(3)…(4)…(5)…(6)…“Hence, instead of„Why sustainable research software in the first place?“use„(1) Why sustainable research software in the first place?“and so on:(2) How to decide which software to sustain?(3) Who sustains research software?(4) How can research software be sustainably funded?(5) Which infrastructure is needed to sustain research software?(6) Legal aspects.That's a matter of taste, of course. # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-11:Please replace Subchapter heading„Challenges and clarifications Clarification of rights“by „Challenges and Clarification of rights“ # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-13:In Subchapter „Conclusions“I would not write the following sentence, or at most I would write it differently.„We encourage the research funding bodies to reflect the licensing models for academic software development, and to decide whether the “public money, public code” paradigm justifies the requirement that all publicly funded software has to be publicly available under a Free/Libre Open Source Software (FLOSS) license.“ I can understand your point of view and this sentence. But isn't there a lack of perspective regarding those research leaders or RSE-teams, who are considering a foundation based on a proprietary license model regarding their software development or RSE teams whose software developments are taken over and marketed by industrial companies in or outside of Germany, without return to the research performing organizations and the corresponding RSE teams.Hence, an alternative formulation might be as follows: „We encourage the research funding bodies to reflect the licensing models for academic software development, and to suggest research performing organisations and their research leaders in the sense of sustainability to make their software typically publicly available under a FLOSS-license but also to take into consideration revenue-oriented approaches such as FLOSS business models or proprietrary licensing if applicable.“ • In my opinion, the final decision to license in and out as well as to transfer computer programs under FLOSS licenses should be the responsibility of the (authorised for this purpose by the Presidium/Board of Directors of the research performing organisation) Research Leaders, with whom the computerprogram developing RSEs should therefore consult. Furthermore, the RSEs and their research leaders ideally should have the opportunity at their research institution to Page 33 of 34 F1000Research 2021, 9:295 Last updated: 28 JAN 2021 contact (preferably at an early stage) science-supporting specialist departments such as Legal, TTO, research software local task forces or even a SSI-like nationwide institution and seek advice. # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-14:In Fig.3 it should read "Check (1)..." instead of "Check (2)..." in both the centre left and the top right. # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # Comment-DF-15: The term "research institution" appears in the glossary as a duplicate. Therefore, "research institution" should be deleted in the brackets. # # # # # # # # # # # # # # # # # # # # # # # Best regardsDirk Feuchter------------------------------------------------------------------Karlsruhe Institute of Technology (KIT)INNOVATION AND RELATIONS MANAGEMENT (IRM)Intellectual Property ManagementDr. Dirk Feuchter (Licenses)Hermann-von-Helmholtz-Platz 1D-76344 Eggenstein LeopoldshafenGermanyPhone: +49 721 608-2-3921E-Mail: dirk.feuchter at kit.eduWeb: https://www.irm.kit.edu/116_1500.php https://www.irm.kit.edu/english/91.php www.irm.kit.edu/ • Competing Interests: No competing interests were disclosed. Competing Interests: No competing interests were disclosed. 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https://openalex.org/W4220716003	https://advancesinsimulation.biomedcentral.com/track/pdf/10.1186/s41077-022-00204-5	English	null	A preliminary simulation-based qualitative study of healthcare students’ experiences of interprofessional primary care scenarios	Advances in simulation	2,022	cc-by	10,169	RESEARCH Open Access Lunde et al. Advances in Simulation (2022) 7:9 https://doi.org/10.1186/s41077-022-00204-5 Abstract Background: Introducing interprofessional education (IPE) in healthcare curricula can prepare students for health- care practices that have become increasingly complex. The use of simulation is promoted to support IPE. This study explores healthcare students’ experiences of participating in common, sub-acute patient scenarios that routinely occur in clinical practice in primary care. More specifically, it looks at how sub-acute patient scenarios from primary care can help develop interprofessional collaborative competence. Methods: Medical students (N = 10), master’s students in advanced geriatric nursing (N = 8) and bachelor’s students in nursing (N = 9) participated in the simulations. The students were in their last or second-to-last year of education. We conducted five semi-structured focus group interviews with the participants’ directly after the simulation training to elicit experiences related to the scenarios, the simulation and interprofessional collaboration. The transcripts were analysed using systematic text condensation. To supplement the focus group interviews, the students also completed the interprofessional collaborative competency attainment survey (ICCAS), which measures the students’ self-assessed interprofessional competence. Results: Three main themes emerged from the analysis of the focus group interviews: realism, uncertainty and reflection. The students emphasised the importance of authentic and recognisable scenarios. They said the vague and unspecific patient symptoms created uncertainty in the situation, making it difficult to understand the patient’s diagnosis. Despite that uncertainty, they described the experience as positive. Further, the students expressed that the simulation increased their confidence in interprofessional collaboration and prepared them for future work. The results from the ICCAS questionnaire showed that the students reported a subjective positive change in their inter- professional competence after participating in the scenarios. Conclusions: This study showed that simulation-based IPE with sub-acute primary care scenarios contributes to develop interprofessional collaborative competence in healthcare education. Sub-acute scenarios can supplement the more common approaches with acute care scenarios and aid in developing the collaborative competence required to work in healthcare teams. Keywords: Simulation, Interprofessional, Primary care, Healthcare students, Sub-acute scenarios, Focus group © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Interprofessional education (IPE) is a critical component in healthcare curricula and can help prepare students for healthcare practices that have become increasingly complex [1, 2]. However, there is no widespread educa- tional consensus on how to conduct IPE so that it better *Correspondence: lene.lunde@medisin.uio.no 1 Department of Nursing Science, Institute of Health and Society, Faculty of Medicine, University of Oslo, Oslo, Norway Full list of author information is available at the end of the article As a result, healthcare education needs to find approaches that expose students to interprofessional collaboration (IPC).h The use of simulation provides learning experiences where the students are placed in realistic and safe clini- cal situations [6]. A growing body of research promotes simulation as an educational strategy to support IPE in healthcare education [7–10]. Most simulation-based IPE experiences have focused on life-threatening, time- critical acute-care scenarios [11–14]. While it is impor- tant for healthcare students to learn and practice how to respond to severe, acute care scenarios, everyday clinical situations are rife with IPC. Shorter hospital stays and an increased emphasis on home care and ageing in place suggest that more patients with increasingly complex needs will require treatment in a primary care setting [15]. In contrast to most acute care algorithm-based sce- narios, sub-acute patient scenarios in primary care pro- vide the students with more time to solve a problem, but the actual clinical situation may be more complex. Intro- ducing simulation training of scenarios typical of primary care can therefore contribute to the students’ learning experiences of IPC. The simulations took place in a research laboratory at the University of Oslo. The simulation units were set up like rooms in nursing homes. The scenarios were cre- ated in collaboration with primary care health profes- sionals and comprised common medical conditions from primary care: an older patient convalescing at a nursing home following surgery for a hip fracture. The patient then developed symptoms of either a urinary tract infec- tion or pneumonia.h The students participated in both scenarios described in Additional file 2 during the simulation-based training. Two scenarios were conducted during each simulation- based training activity each preceded by a briefing and immediately followed by a debriefing [20]. The briefing provided an introduction to the simulation room, the available (technical) equipment and the patient simulator SimMan® by Laerdal Medical [21], as well as a reminder about confidentiality and an introduction to the sce- nario [22]. During the simulation, facilitators acted as the patient’s voice and answered the questions directed towards the patient. We instructed the students to act according to their distinct professional roles and future responsibilities. Each scenario lasted approximately 30 min. The debriefing took place directly after each sce- nario and lasted on average 25 min [23]. With this in mind, we developed simulation-based IPE with sub-acute patient scenarios that would commonly occur in clinical practice. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lunde et al. Advances in Simulation (2022) 7:9 Page 2 of 12 Page 2 of 12 Lunde et al. Advances in Simulation IPE simulation we developed is not yet implemented in our healthcare curricula. FG interviews were considered well suited to elicit experiences and views from the par- ticipants and encourage group dialogue after participat- ing in a joint experience such as simulation-based IPE [16]. A series of questions addressing experiences related to the scenarios, the simulation and IPC acted as a guide for the semi-structured interviews. The participants were encouraged to elaborate on topics they considered rel- evant and important (Additional file 1). In addition, to supplement the FG interviews, the students completed the Norwegian version of the interprofessional collabo- rative competency attainment survey (ICCAS). ICCAS captures the students’ self-assessment of their interpro- fessional competence and is validated across various set- tings and countries, including Norway [17–19].h prepares students to collaborate across healthcare disci- plines. Traditionally, healthcare students are educated in professional silos [3, 4]. As such, traditional teach- ing does not promote students’ interactions with other healthcare professions. It is a common assumption that students’ exposure to, and involvement in, teamwork occurs naturally in clinical practice and, consequently, prepares the students for working in interprofessional teams. However, there is no guarantee that without pur- poseful organisation, students will experience exemplary teamwork or even collaborate with other healthcare pro- fessionals or students during clinical practice [5]. Participants We recruited medical students, master’s students in advanced geriatric nursing and bachelor’s students in nursing through purposeful sampling. Educational lead- ers in universities in central Eastern Norway facilitated the recruitment. The inclusion criteria were healthcare students in the final semester of their last or second-to- last year of education because they had completed most of their clinical practice rotation and thus presumably would have skills competence sufficient to be capable of participating in IPC. Potential participants that met our The main aim of this article is to explore healthcare students’ experiences of participat- ing in the sub-acute patient scenarios. Specifically, we aimed to understand how the use of sub-acute patient scenarios from primary care could support the develop- ment of interprofessional collaborative competence. Strategies to enhance trustworthiness in the analysish Strategies to enhance trustworthiness in the analysis The authors are nurses (LL, AM), medical doctors (RBJ, EOR, AMB) and an educator (BM). Collectively, our experience combines primary care and medical educa- tions, as well as research, teaching, curriculum planning, workplace learning and simulation-based training. Our backgrounds might have influenced our preunderstand- ing of the simulation setting, the scenarios and the stu- dents’ experiences. However, having co-authors with different, yet complementary backgrounds might also help in ensuring the legitimacy of our interpretations [26]. By reporting the process of analysis and providing examples of codes, construction of condensates, syn- theses and themes in Table 2, we have brought a cer- tain transparency to the process. Through the research group’s collective reading and analysis, we have worked to enhance the trustworthiness of the results [26]. i The interviewers observed the simulations from behind a one-way mirror in the control room and did not inter- act with the students during the simulation. The FG interviews were audio-recorded and were exported to a secure data storage facility at the University of Oslo, then transcribed verbatim by LL. Research design and setting We conducted a qualitative study, using focus group (FG) interviews to capture experiences from students par- ticipating in IPE simulation sessions. This is part of an exploratory study exploring different aspects of simula- tion as a strategy for training healthcare students in IPC in future curricula development. We developed scenarios comprised of sub-acute situations from primary care. The Page 3 of 12 Lunde et al. Advances in Simulation (2022) 7:9 discussed, modified themes, reviewed abstractions and syntheses until reaching a consensus. inclusion criteria received information about the study from contact persons at the different universities. The lead author LL was also invited to several lectures to talk about the project to recruit participants. A total of 27 healthcare students agreed to participate, ranging from 21 to 49 years of age (mean 31), and 21 were female and six were male. All participants in the simulation train- ing took part in the FG interviews. Table 1 presents the details regarding the participants. We used the software NVivo12 to organise and struc- ture the data. As the analysis progressed, we organised the material into tables. Table 2 shows an example of the analysis. The ICCAS questionnaires were analysed using IBM SPSS Statistics Version 27. ICCAS comprised the interprofes- sional competency communication, collaboration, patient- and family-centred care, roles and responsibilities and conflict management. Since prior validation studies recom- mend analysing ICCAS at an overall level to address change in interprofessional competence [17, 18], we used paired t-test to determine the difference in perceived abilities in the mean overall pre- and post-score (range 1–5). We ana- lysed the differences in terms of Cohen d standardised effect size (“large” = values of ≥ 0.8, “moderate” = values between 0.79 and 0.50 and “small” values between 0.2 and 0.49) and 95% confidence limits [25]. To maintain the anonymity of the participants, gender and name were excluded from the transcripts, and abbre- viations were used, as can be seen in Table 1. The partici- pants were numbered in the order they appeared in the interviews (e.g. NS1). The FG interviews were numbered in the order they were conducted (e.g. FG1). Data analysis Th The transcripts from the FG interviews were analysed by systematic text condensation, in a four-step process [24]. First, we read the transcripts independently to get an overview and total impression and to identify prelimi- nary themes. Secondly, we collaboratively identified and sorted the meaning units into code groups. In the third step, we abstracted condensates from each code group. Finally, we created synthesised descriptions by recon- ceptualising the condensates and chose the quotes that would best represent the synthesised description (golden quotes). The initial steps were conducted by LL and AMB independently (step 1) and in collaboration (step 2). Then, LL drafted the first versions of condensates (step 3) and synthetisation (step 4) and translated the quotes into English. For each step of the analysis, the research group read the material independently, collaboratively Resultsh Three main themes emerged from the analysis of the FG interviews: realism, uncertainty and reflection. Within uncertainty, the sub-themes “unspecific situations”, “time to collaborate” and “room for communication” became apparent. In reflection, the sub-themes “opportunities not present in practice”, “developing confidence” and “better prepared for the future” emerged. Data collection d d We conducted the FG interviews in April 2019, just after the students had finished the simulations and com- pleted the ICCAS questionnaire, to avoid conflicts with study schedules. Each student was a member of one of 10 interprofessional teams during the simulations. Two teams participated in the simulation each day, and they joined the same FG, resulting in five FG interviews. The lead interviewers were members of the research group with experience in qualitative research and with doctoral degrees in nursing (AM) and medicine (AMB, EOR). Each FG interview lasted between 60 and 90 min and had five or six participants.h Unspecific situationh Although there was enough time to work on it, the clinical problem itself was less clear-cut. The students reported that they could not take any shortcuts because the symptoms were so vague. They had to discuss what they were unsure of and do a full clinical examination. The students expressed that in prior simulation experi- ences, they were usually provided with predefined ways of solving the problem, either through algorithms or checklists. In these scenarios, however, they experienced an ambiguous situation, where the right solution did not clearly stand out. They described it as they knew something was going on, but the unspecific clinical signs made the situation difficult to grasp and therefore difficult to analyse. AGN3 (FG2): If it is a cardiac arrest, pretty much everyone knows what to do, and you cooperate. But when it is so vague, you get a discussion of everyone’s knowledge, and it’s completely different. You get to use each other’s competence in a completely differ- ent way than if it was a very specific and dramatic situation. AGN3 (FG2): Very often it starts with the fact that you realise that there is something going on. Without having anything specific, everything is a bit vague. That’s what it’s often like. In contrast, the students found that a simulation solv- ing an acute care situation such as a cardiac arrest where they follow a predefined algorithm was more rehearsed and explicit as they would know what to do and how to react. Sub-acute scenarios provided the students with an opportunity to use each other’s competences in new ways. The vagueness, the students said, consequently led to another kind of insight of what the other students knew and how they could contribute, as they had to share their knowledge to expand on the problem. The simula- tion seemed to contribute to increased understanding of the competence the different educations provided, and how they could complement each other. Thus, when they combined their different perspectives, it helped reduce the uncertainty. This indicated that adding different The realisation that the patient’s situation was chang- ing encouraged the students to pay careful attention to the vague and undefinable signs that were found in the clinical examination. The students explained that espe- cially with elderly patients, the clinical signs might not be as apparent or lead to textbook solutions. Realism of the scenarioh The students recognised the scenarios as realistic, authentic and likely to be encountered in healthcare and, specifically, in primary care. MS9 (FG5): I especially think about the fact that it MS9 (FG5): I especially think about the fact that it Page 4 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Lunde et al. Advances in Simulation (2022) 7:9 was so relevant. The topics were important, and the situations ones that you would often experience. The students expressed that they were unaccustomed to these assessments, especially because there was no quick fix or easy solution. However, the students per- ceived this experience as positive because, in nursing homes, and primary care in general, they would often experience vague clinical situations. As such, it seemed that the scenarios were recognised as important learn- ing activities to prepare for real-life situations. The use of sub-acute scenarios shifted their focus to the inherently complex health services that are provided in primary care on a daily basis and appeared to renew the students’ understanding of the many different challenges that can occur. Furthermore, the students described the nursing home setting as recognisable and representative. They noted that to make the simulations authentic, you needed to have such surroundings. The students seemed to manage to conceptualise the scenario in a clearer way based on the information provided and the environmental set-up. AGN6 (FG4): And I also think that it was very good that we were told immediately that this is a nurs- ing home and this is the available equipment in the nursing home, and the doctor is present one day a week. This made it realistic. Time to collaborateh As such, the students emphasised that it was important to have authentic, recognisable scenarios and that having the setting and equipment described beforehand allowed them to better envision the scenario. Together, these statements illustrated that including information about setting, surroundings and available equipment as well as scenario description in the pre-briefing was important to prepare the students for the simulation. The students emphasised that having an adequate time to practice scenarios together in a calm setting offered the opportunity to ask additional questions, listen to one another and engage in group discussions to solve problems. MS4 (FG2): When you have so much time and it is quite calm, you have the opportunity to listen, and to ask, “What do you think? Is there anything we have not thought of?” Room for communication Based on their prior experience with simulation, the stu- dents said they expected an extraordinary situation even though they were prepared for sub-acute scenarios. The fact that the clinical condition in the scenario did not overwhelm them was highlighted as positive. Thus, it was possible to focus on the team’s interactions and commu- nication, which they deemed important. The students explained that it was not always possible to take an active part in collaboration in clinical prac- tice, and the possibility to interact with other students or healthcare personnel could be limited or non-existent. In clinical practice, they experienced that there was lit- tle time given over to reflect together with others. This appeared to illustrate that profession-specific learning goals in clinical practice are still the most common and that interprofessional activities where the students have time to reflect with others are scarce. MS5 (FG3): When it’s not medically precarious and acute, you get a little more time to actually commu- nicate. And that’s what’s most important. The students appreciated that the clinical condition did not decline rapidly, as it gave them more time to react and collaborate. They pointed out that in a medically complicated scenario, you could just as well end up with a situation where one team member dominates. NS9 (FG5): We know that, in practice, we can call the priest, social worker, or nutritionist and get them up there and then talk to them. But you may not know how you would collaborate with them in that meeting. You are doing that in here. What we do here is very important in shedding light on how we should collaborate. NS9 (FG5): We know that, in practice, we can call the priest, social worker, or nutritionist and get them up there and then talk to them. But you may not know how you would collaborate with them in that meeting. You are doing that in here. What we do here is very important in shedding light on how we should collaborate. NS10 (FG5): If it gets too complicated and there’s a dispute between the professions, and the one who speaks loudest overrides the rest of the group. Some just cave in and heed to the one who has the strong- est opinions. As the students pointed out, complicated cases could negatively affect the communication and collaboration. Unspecific situationh The presen- tation of vague and unspecific symptoms made the stu- dents think more broadly in their clinical assessments, as a symptom could be interpreted in several ways. Conse- quently, they were less certain of the patient’s diagnosis. MS6 (FG3): Because there were vague symptoms, you had to think a bit more broadly. You think there can always be something more to it. And, that this kind of assessment feels a little unfamiliar. Page 5 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Lunde et al. Advances in Simulation professional perspectives enhanced the joint discussion and thus increased their learning outcome. awareness and understanding of the situation together without merely pointing out what went wrong. MS10 (FG5): I absolutely believe that training in controlled settings where you get time to reflect after- wards has great value that is difficult to include in practice. Because in practice, you are dependent on a supervisor taking time to include reflection and a department with suitable conditions for reflection with others MS10 (FG5): I absolutely believe that training in controlled settings where you get time to reflect after- wards has great value that is difficult to include in practice. Because in practice, you are dependent on a supervisor taking time to include reflection and a department with suitable conditions for reflection with others Room for communication By sharing experiences and reflecting together, the stu- dents indicated that they got to know the competences of the other healthcare professions first hand. This was per- ceived as important for managing collaboration. The stu- dents described the simulation setting as a good way to become more aware of the roles and responsibility they would assume in their future work life. It also gave room to reflect on how to collaboratively solve problems, not just on the idea that collaboration was necessary. Expe- riencing the benefits of IPC may also lead to enhanced respect for each other’s profession. As such, the students voiced the importance of participating in training that enhances the quality of IPC. Opportunities not present in practice Several students talked about the simulation as being similar to practice and yet not so, especially regarding time to reflect during the simulation and in the debrief- ing. They highlighted that in these scenarios, they had time to talk through the clinical picture of the patient together and really listen to each other. In real-life prac- tice settings, they said it might be busy and chaotic, and opportunities for reflecting together and share pro- fession-specific knowledge about the patient were less available. AGN3 (FG2): I learned a lot from seeing what the others reflected on. Here you do the reflection together. You see what the different students see; there is not always room for that when you work. Developing confidence Th d id h The students said that participating in the simulation made them more aware of themselves for better or worse, in terms of how they behaved and dealt with situations. They described the experience as discovering themselves in a new way. Consequently, the experience appeared to develop their confidence to engage more actively in IPC. The students also emphasised that having the oppor- tunity to sit down together in the debriefing and reflect on what they did enhanced the learning outcome. In the debriefing, the students appreciated the possibility to talk about how they communicated and collaborated in the simulation sessions individually and as a team. They par- ticularly pointed out that they valued the focus on raising AGN4 (FG3): With simulation, I see that if I can talk to the medical student, then maybe I can talk to a real doctor. You see proof that it’s actually possible to AGN4 (FG3): With simulation, I see that if I can talk to the medical student, then maybe I can talk to a real doctor. You see proof that it’s actually possible to Page 6 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Discussioni In the findings reported here, the students emphasised the importance of authentic and recognisable scenarios. They described that the vague and unspecific symptoms in the scenarios created an uncertain situation where it was difficult to find a clear direction. The students repeatedly emphasised, however, that this experience was positive. They acknowledged, with some surprise, the complexity the sub-acute scenarios presented and the opportunity that arose for them to focus on collabora- tion and communication. Further, the students reported increased confidence and preparedness for future work. Our results from ICCAS also supported that partici- pating in the scenarios led to a positive change in self- assessed interprofessional competence. Furthermore, we discuss the potential for the sub-acute scenarios to pro- mote interprofessional collaborative learning opportuni- ties for healthcare students. Self‑reported interprofessional competence In addition to the material from the FG interviews, all 27 participants completed the ICCAS questionnaire. The results from the ICCAS questionnaire showed that after participating in the scenarios, the students reported a positive change in self-assessed interprofessional com- petence. There was a statistically significant increase in the mean sum score from pre-scores (mean = 3.64, SD = 0.65) to post-scores (mean = 4.4, SD = 0.3), t (26) = 6.67, p < .001 (two-tailed). The mean difference, 0.76, 95% CI [0.53, 0.99], represented a large effect of d = 1.29. NS5 (FG3): I learned today that I don’t have to be afraid. If I have some knowledge or something that I think of, with the patient in mind, I will just say it. NS5 (FG3): I learned today that I don’t have to be afraid. If I have some knowledge or something that I think of, with the patient in mind, I will just say it. The students explained that when they discussed together, they realised that they had an important role to play. Thus, the joint problem-solving activities the sce- narios provided seemed to increase their experience of themselves as important contributors to the interprofes- sional discussion. Consequently, the simulation experi- ence led to newfound confidence in the students’ abilities to participate and voice their opinions. This confidence appeared to reassure the students in their own role as healthcare professionals. When reassured in their own role, they managed to benefit from the others’ compe- tence and mutually create joint knowledge. talk to other professional groups. education. Consequently, this type of scenarios could prepare the students for future IPC. The students found that as the simulation progressed, they got more comfortable with expressing their opinion with the team, which made it possible to have a clinical conversation across professions. Solving the scenario, they explained, provided an opportunity for participating in discussions in a safe environment as equals. The crea- tion of a safe environment allowed the students to dare to present their perspectives and express their opinions. Better prepared for the futureh The students indicated that the experiences from the sce- narios would be long lasting because the simulation cre- ated practical memories they could recall later. MS5 (FG3): It’s the kind of experience that you can come back to and reflect on. You can call on it in dif- ferent settings and think, “Oh, yes, we did this that time.” Collaborative problem solving in a realistic setting A fi d f h d h Collaborative problem solving in a realistic setting An important finding from this study was the students’ positive response to the sub-acute scenarios, especially their seeing scenarios as authentic and realistic learn- ing situations. The recognisable scenarios, together with information about the setting and available equipment, were important factors in getting students to engage in the simulation. Considering the simulation activity as a social practice where learning is constructed in interac- tion between the participants, environment and equip- ment, it highlights the importance of pre-briefing to create a safe and recognisable environment for the stu- dents to interact in [22, 27, 28]. Thus, they seemed to manage to utilise the resources available in the room and frame the simulated situations into something manageable.hi The students said that taking part in the simulations would help them deal with similar situations in the future. Facing such issues in a safe environment dur- ing education gave the students a sense of assurance for future work. NS6 (FG4): If you could act through it in advance and be trained beforehand, you can handle it better later, in terms of how to talk to each other. Thus, the students reported that interprofessional col- laboration could become something familiar and man- ageable because of prior training. Participating in the scenarios seemed to provide the students with a clearer frame of reference for problem-solving in future situ- ations. Having useful experiences to refer could pro- vide security since they had faced such issues during This supports the findings showing that IPE has to be meaningful and relevant, with authentic activities, to be able to support interprofessional learning [4, 29]. Further, Page 7 of 12 Lunde et al. Advances in Simulation (2022) 7:9 for a learning experience to be of value and to prepare the students for future teamwork, structured opportunities for active engagement need to be made available [11, 30]. Thus, IPC experiences involving engagement and oppor- tunities to interact, rather than passive observation of teamwork, are found to have more impact on interpro- fessional learning and competence development [31–33].hi in the simulations. For students to be prepared for the expected collaboration, educators have to create spaces to train for IPC in healthcare education [15]. Learning opportunitiesh Through IPE-based simulation training of sub-acute situations, this study shows that the following learning potentials can be realised: establishing greater confidence in handling uncertain, sub-acute situations through IPC, understanding their own and others’ perspectives and competencies and strengthened confidence in their own IPC competencies and contributions for future work. These practice spaces for IPC emerge during the joint examination of the clinical situation and is strengthened through reflection.l l Reflecting on the simulation experience, especially the debriefing, is seen as a cornerstone in simulation-based training for students to reconstruct their experience into learning [27]. There are several ways of facilitating sce- nario debriefing [41, 42], making it important for educa- tors to make well-considered choice of debriefing strategy beforehand. In this study, the facilitators were instructed to follow the debriefing framework proposed by Rudolph et al. [23] where the focus is enhancing awareness and understanding of the situation. The framework highlights creating a safe learning environment where the students feel comfortable discussing successes and failures to understand and learn of their actions. The students in our study appreciated that the facilitators did not solely focus on what went wrong, but prompted questions, thoughts and opinions that engaged the students to contribute actively with their own reflections and perspectives on collaboration and communication. i When developing scenarios for simulation-based train- ing, careful consideration of the level of difficulty and complexity is necessary to optimise the learning oppor- tunities [27, 28, 36]. It is important to take into account that the students participating in the scenarios are there to train on competence they have not yet fully acquired [37]. Thus, a mismatch between the difficulty and com- plexity of the scenario and the students’ capacity to make sense of the scenario could compromise the learning opportunities. As the students explained, complicated cases can breed poor communication, as one team mem- ber may dominate. As such, scenarios where the patient’s condition is stable seem to provide students with more time and opportunity to emphasise team collaboration [38, 39].h In our study, the realistic but vague and unspecific signs and symptoms in the scenarios without a clear conclusion created uncertainty that challenged the stu- dents’ competence, their role understanding and task sharing. However, the uncertainty also mobilised their resources as they resolved the uncertainty by communi- cation and joint reflection in the simulation and during the debriefing. Collaborative problem solving in a realistic setting A fi d f h d h The founda- tion for fruitful learning spaces have to be laid in the pre- briefing to get the students to engage in the simulation and interact with the participants, scenario and environ- ment [22]. Without these spaces, it is difficult for health- care students to get to know one another and find ways of working together [40]. The practice space for IPC in the sub-acute scenarios seems to provide the opportunity for healthcare students to explore one another’s perspectives and use one another’s competencies interprofessionally. The unspecific symptoms presented in the scenarios created an uncertain situation for the students, where the patient’s problem or diagnosis was unclear. As such, the sub-acute scenarios exposed the students to the complexity often presented by this patient group, where accurate diagnosis can be difficult due to atypical symp- toms [34]. Since there was no detailed algorithm to fol- low, the outcome depended on the students’ capacity to discuss, identify signs and symptoms and use relevant knowledge to solve the patients’ main concerns. Students who actively share information, discuss and draw on one another’s resources and competencies seem to manage defining the patients’ concerns and prepare for future care in collaboration [35]. In our study, the students high- lighted that the relaxed pace of the scenarios, combined with a reasonable amount of time to complete them, made it possible to focus on the interactions and commu- nication within the team, to ask each other questions and discuss and reflect together without being overwhelmed. When students recognise the simulation-based activity as a safe environment, it can motivate them to perform at the edge of their expertise [22], which might enable them to expand on the learning activity and enhance their knowledge. In our scenarios, the students recognised the setting as a safe environment, which made them willing to ask questions, listen to reflections from others and contemplate on the best way forward together, although it might highlight skills deficiencies. Learning opportunitiesh As such, the development of IPC com- petence took place both during the scenario and in the debriefing. The quality of the debriefing seems as impor- tant for the development of IPC competence as the The students’ experiences of a collaborative learning potential in simulation seemed to come from the combi- nation of a realistic scenario and a practice space for IPC Lunde et al. Advances in Simulation (2022) 7:9 Page 8 of 12 Page 8 of 12 quality of the scenario since the debriefing is where the participants shift their perspective from the action to the reflection on actions and common experiences from the scenario [23]. This study suggests that the scenarios allowed for discussion and joint reflection and that the simulation training may lead to enhanced understand- ing of one another’s sense of competence and scope of practice. Most especially, the simulation provided an opportunity for equal discussions in a safe environment. This supports the findings suggesting that feeling safe in a learning situation fosters confidence in one’s role and willingness to participate in a team [8, 9, 12, 14]. Moreover, our results may indicate that the scenarios provided safe ways of developing interprofessional col- laborative competence where different perspectives are valued. Unequal power relations and hierarchical structures are seen as barriers for learning [43, 44]. We highlighted that everyone’s knowledge and perspective were necessary to solve the problem which seemed to promote a non-hierarchical learning environment and strengthen the students’ confidence in their interprofes- sional competence. which means that the necessary level of realism should be evaluated to create the required learning environ- ment [28]. In our study, we have shown that the stu- dents valued the authentic and realistic scenarios. Although the simulation was conducted in a simula- tion centre with a SimMan as the older nursing home patient, the student perceived the situation as realistic due to the authentic scenario description, convincing access to equipment, presentation of vague clinical signs and credible information provided in the medical record. This highlights that to create a realistic simu- lation experience—or the right amount of fidelity—it has to contain physical elements but also situations the students manage to make sense of and experience as relevant [28, 39]. Learning opportunitiesh In our study, albeit the fact that not everything was identical to practice, the abovemen- tioned factors contributed to create a context where the students experienced a sense of recognisability and, thus, engaged in the scenarios. Systematic IPE could be an advantage for future team- work, as the students explained that having experienced IPC, they felt prepared to contribute in future IPC situ- ations. The positive change in the students’ self-reported competence score supported that participating in the scenarios prepared the students for collaborative prac- tice. Other studies have also found that IPC training develops competence and enhances the ability to engage in future interprofessional teamwork in clinical practice [33]. Exposing healthcare students to IPE during educa- tion can result in more graduates with IPC competence, which in turn can promote a positive change towards fur- ther interprofessional collaborative healthcare practice. Thus, the IPC learning outcomes the students achieved in these scenarios could be transferable to other settings and situations. The students said that though similar to clinical practice, the simulated setting was also different. The joint discussions and reflections about the patients’ clinical picture they experienced during the simula- tion session were not usually encountered in clinical practice or work, neither was the structured debrief- ing. This might be seen as an educational paradox, in which students participate in IPE to prepare for future interprofessional practice that rarely takes place. Thus, it can be challenging to prepare students with IPC competencies if they do not find opportunities to practice in clinical work. Consequently, those students might not consider IPC as important in real-life clini- cal work [5]. At the same time, education institutions have a responsibility to include high-quality IPE, and thereby contribute to the quality of IPC in the future. Otherwise, newly graduated students risk entering their professions without the interprofessional col- laborative competence needed to work efficiently in future healthcare teams [2]. Strengths and limitations A strength of this study is that it expands simulation- based IPE as a strategy to prepare healthcare students for future IPC and shows the potential of adding sim- ulations of sub-acute primary care scenarios to IPE. We acknowledge that the participating students might be more positive about simulation and IPC than other students might. Reasons for non-participation were, however, mainly the lack of time and not getting time off from work or clinical practice. The simulations and FG interviews were conducted in 1 day to facili- tate participation and avoid study schedule conflicts. We do not know if the students would have shared the same viewpoints in the FG interview had they had time to process the experience over a longer period. How- ever, FG interviews provided us with detailed and rich descriptions of the students’ immediate experiences. The simulation-based experience offered a frame of reference for future problem solving. Thus, it seems that the realistic setting not only enhances learning, but also makes it more transferable for future situ- ations, confirming existing research which suggests that authentic, interactive and competence-building IPE experiences create lasting impressions [29, 30]. However, it is important to highlight that realism—or fidelity—does not mean that everything must be as found in practice, without exception. Simulation fidel- ity relates to the educational value of the simulation Page 9 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Lunde et al. Advances in Simulation Fig. 1 Simulation set-up and available equipment Fig. 1 Simulation set-up and available equipment Fig. 1 Simulation set-up and available equipment The students have not had the opportunity to comment on our interpretations, and we acknowledge that their interpretations or explanations of the transcripts may differ from ours. Although research promotes the use of simulation to support IPE, there are few studies with sub-acute scenarios from primary care. Thus, our study contributes to a new perspective on how to facilitate for IPE in healthcare education. These scenarios seem to be feasible for implementation in healthcare educa- tion. Adding observers with specific tasks related to observation of the simulation activity could be one way to scale up to accommodate real student numbers and consequently avoid inactive participants in the scenar- ios. Step 4 Step 4 Acknowledgements g We wish to thank all the medical students, master’s students in advanced geri- atric nursing and bachelor’s students in nursing who participated in this study. We also wish to thank Ragnhild Hellesø and Astri Letnes Janson for assisting in the focus group interviews. Abbreviations IPE: Interprofessional education; IPC: Interprofessional collaboration; MS: Medical student; AGN: Master’s students in advanced geriatric nursing; NS: Bachelor’s students in nursing; FG: Focus group. Conclusionsh The present study shows that simulation-based IPE with sub-acute primary care scenarios in healthcare educa- tion contributes to the development of the collaborative competence. The students valued the authentic scenarios and expressed that solving the scenarios increased their competence in IPC and prepared them for future work. The sub-acute scenarios, although complex in relation to the unspecific and vague symptoms, promoted collabo- rative learning opportunities for the students due to the authenticity and sufficient time to discuss and reflect. Introducing simulation-based IPE with a focus on pri- mary care scenarios can supplement more common acute care simulation approaches for developing the collabora- tive competence required to work in healthcare teams. Additional file 1. Additional file 2. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s41077-022-00204-5. Authors’ contributions LL, AMB, AM, EOR and RBJ contributed to the study conception, design, mate- rial preparation and data collection. All authors contributed to the analysis. LL wrote the first draft of the manuscript, and all authors contributed to the subsequent versions and the final manuscript. The authors read and approved the final manuscript. Step 3 Construction of artificial quotations (condensates) summarising several meaning units (first person) Identification and coding of mean- ing units (first person) Construction of artificial quotations (condensates) summarising several meaning units (first person) Syntheses of contents into main themes and sub-categories Choice of golden quotes (third person) Very realistic and similar to practice MS1 (FG1): “I am in nursing home practice now and had my first day yesterday. This could have been yes- terday! And it could be tomorrow.” NS2 (FG2): “I think they were really good cases. Because it’s the type of patient you would actually meet.” MS9 (FG5): “I especially think about the fact that it was so relevant. The topics were important, and the situ- ations ones that you would often experience.” MS1 (FG1): “I am in nursing home practice now and had my first day yesterday. This could have been yes- terday! And it could be tomorrow.” NS2 (FG2): “I think they were really good cases. Because it’s the type of patient you would actually meet.” MS9 (FG5): “I especially think about the fact that it was so relevant. The topics were important, and the situ- ations ones that you would often experience.” I am in practice at a nursing home and this could have been yesterday, or tomorrow. It felt very realistic and relevant for primary care. This is also the kind of patient you would typically meet in healthcare. We were told immediately that this is a nursing home and what equipment we had access to. Having been in a nursing home, the resources and their availability felt realistic. You need to have surroundings that feel realistic to make the simulation believable. Main theme: realism Sub category: recognition of realistic scenario and setting Strengths and limitations Advances in Simulation Table 2 Example of analysis Step 1 Step 2 Step General impression and identifica- tion of preliminary themes Identification and coding of mean- ing units (first person) Cons (con mea Table 2 Example of analysis Main theme: realism Sub category: recognition of realistic scenario and setting Main theme: realism Sub category: recognition of realistic scenario and setting The students recognised the scenarios as realistic, the situations as authentic situations and ones that they would likely encounter in healthcare, and specifically in primary care. The students also described the setting in a nursing home as recognisable and realistic. They high- lighted the necessity to have realistic surroundings that would make the simulation authentic. AGN6 (FG4): “And I also think that it was very good that we were told immediately that this is a nurs- ing home and this is the available equipment in the nursing home, and that the doctor is present one day a week. This made it realistic.” MS7 (FG4): “You need those sur- roundings to make it is as believable as possible.” Golden quotes: Golden quotes: MS9 (FG5): “I especially think about the fact that it was so relevant. The topics were important, and the situations ones that you would often experience.” AGN6 (FG4): “And I also think that it was very good that we were told immediately that this is a nursing home and this is the available equip- ment in the nursing home, and that the doctor is present one day a week. This made it realistic.” another scenario. The scenarios also have potential to be expanded to include other healthcare professions, which would have been an interesting opportunity for further study. Abbreviations Strengths and limitations Then, the students could take turn in taking part in a scenario and observing their peers taking part in The students have not had the opportunity to comment on our interpretations, and we acknowledge that their interpretations or explanations of the transcripts may differ from ours. Although research promotes the use of simulation to support IPE, there are few studies with sub-acute scenarios from primary care. Thus, our study contributes to a new perspective on how to facilitate for IPE in healthcare education. These scenarios seem to be feasible for implementation in healthcare educa- tion. Adding observers with specific tasks related to observation of the simulation activity could be one way to scale up to accommodate real student numbers and consequently avoid inactive participants in the scenar- ios. Then, the students could take turn in taking part in a scenario and observing their peers taking part in An interesting follow-up study could be to investigate how the students experienced the simulation after having entered healthcare as healthcare professionals. Although ICCAS added the students’ individual and anonymous assessment of their own competence, we have too small a sample size to evaluate the effect of the sub-acute scenarios. We performed the FG interviews with the whole interprofessional group and not divided by professions. This might have inhibited some partici- pants to speak freely, since they might be influenced by how they think they are expected to act in their future professional roles. However, since the students were in these groups for 1 day only, we consider it unlikely that this was a major problem. The analysis was based on the researchers’ interpretations of the transcripts. Table 1 Description of the participants a The AGN students have a minimum of 2 years of clinical experience as staff nurses before entering into the master’s programme N (%) Prior participation in simulation Prior participation in interprofessional simulation Yes, N (%) No, N (%) Yes, N (%) No, N (%) Total 27 (100) 22 (82) 5 (18) 7 (26) 20 (74) Medical students (MS) 10 (37) 8 (80) 2 (20) 2 (20) 8 (80) Master’s students in adv. geriatric nursing (AGN)a 8 (30) 6 (75) 2 (25) 2 (25) 6 (75) Bachelor’s students in nursing (NS) 9 (33) 8 (88.9) 1 (11.1) 3 (33.3) 6 (66.7) Page 10 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Lunde et al. Very realistic and similar to practice Funding Th F l g The Faculty of Medicine, University of Oslo, funded this study. Page 11 of 12 Lunde et al. Advances in Simulation (2022) 7:9 (2022) 7:9 Lunde et al. Advances in Simulation Consent for publication The students and facilitator in Fig. 1 have consented to the use of the picture in this article. y p 15. Thistlethwaite J. Interprofessional education: a review of context, learning and the research agenda. Med Educ. 2012;46(1):58–70. y 15. Thistlethwaite J. Interprofessional education: a review of c and the research agenda. Med Educ. 2012;46(1):58–70. 16. Malterud K. Fokusgrupper som forskningsmetode for medisin og helsefag [Focus groups as a research method for medicine and health sciences]. Oslo: Universitetsforlaget; 2012. 16. Malterud K. Fokusgrupper som forskningsmetode for medisin og helsefag [Focus groups as a research method for medicine and health sciences]. Oslo: Universitetsforlaget; 2012. Ethics approval and consent to participate 13. Akselbo I, Killingberg H, Aune I. Simulation as a pedagogical learning method for critical paediatric nursing in Bachelor of Nursing pro- grammes: a qualitative study. Adv Simul. 2020;5(1):24. 13. Akselbo I, Killingberg H, Aune I. Simulation as a pedagogical learning method for critical paediatric nursing in Bachelor of Nursing pro- grammes: a qualitative study. Adv Simul. 2020;5(1):24. The Norwegian Centre for Research Data approved the study (project number 60867). We obtained written, informed consent from the participants after providing oral and written information. 14. Costello M, Prelack K, Faller J, Huddleston J, Adly S, Doolin J. Student experiences of interprofessional simulation: findings from a qualitative study. J Interprof Care. 2018;32(1):95–7. Received: 17 May 2021 Accepted: 6 March 2022 Received: 17 May 2021 Accepted: 6 March 2022 Received: 17 May 2021 Accepted: 6 March 2022 20. INACSL Standards Committee, Watts PI, McDermott DS, Alinier G, Charnetski M, Ludlow J, et al. Healthcare Simulation Standards of Best Practice™: simulation design. Clin Simul Nurs. 2021;58:14–21. 21. Laerdal Medical. SimMan 3G Plus Stavanger: Norway; 2020. Avail- able from: https://www.laerdal.com/no/doc/86/SimMan. [cited 2020 November 5] Availability of data and materials respiratory therapy students: a mixed-method research study. Nurse Educ Today. 2021;99:104816. respiratory therapy students: a mixed-method research study. Nurse Educ Today. 2021;99:104816. The datasets generated and/or analysed during the current study are not publicly available due to the data corpus still being subject to analysis but are available from the corresponding author on reasonable request. Additional file 1: Interview guide; Additional file 2: Scenario description. y 11. Nichols A, Wiley S, Morrell BLM, Jochum JE, Moore ES, Carmack JN, et al. Interprofessional healthcare students’ perceptions of a simulation-based learning experience. J Allied Health. 2019;48(3):159–66. 11. Nichols A, Wiley S, Morrell BLM, Jochum JE, Moore ES, Carmack JN, et al. Interprofessional healthcare students’ perceptions of a simulation-based learning experience. J Allied Health. 2019;48(3):159–66. 12. Washington VL, Zakrajsek A, Myler L, Seurynck K, Holt S, Scazzero J. Blending interprofessional education and simulation learning: a mixed- methods study of an interprofessional learning experience with nursing and occupational therapy students. J Interprof Care. 2021:1–6. 12. Washington VL, Zakrajsek A, Myler L, Seurynck K, Holt S, Scazzero J. Blending interprofessional education and simulation learning: a mixed- methods study of an interprofessional learning experience with nursing and occupational therapy students. J Interprof Care. 2021:1–6. The authors declare that they have no competing interests. 17. Schmitz CC, Radosevich DM, Jardine P, Macdonald CJ, Trumpower D, Archibald D. The interprofessional collaborative competency attain- ment survey (ICCAS): a replication validation study. J Interprof Care. 2017;31(1):28–34. References 1. World Health Organization. Framework for action on interprofessional education & collaborative practice Geneva: WHO Press; 2010. Available from: http://apps.who.int/iris/bitstream/10665/70185/1/WHO_HRH_ HPN_10.3_eng.pdf?ua=1. [cited 2021 March 02] 1. World Health Organization. Framework for action on interprofessional education & collaborative practice Geneva: WHO Press; 2010. Available from: http://apps.who.int/iris/bitstream/10665/70185/1/WHO_HRH_ HPN_10.3_eng.pdf?ua=1. [cited 2021 March 02] 22. Rudolph JW, Raemer DB, Simon R. Establishing a safe container for learn- ing in simulation: the role of the presimulation briefing. Simul Healthc. 2014;9(6):339–49. 2. Frenk J, Chen LC-H, Bhutta ZA, Cohen J, Crisp N, Evans T, et al. Health professionals for a new century: transforming education to strengthen health systems in an interdependent world. Lancet. 2010;376(9756):1923–58. 23. Rudolph JW, Simon R, Dufresne RL, Raemer DB. Thereʼs no such thing as “nonjudgmental” debriefing: a theory and method for debriefing with good judgment. Simul Healthc. 2006;1(1):49–55. 24. Malterud K. Systematic text condensation: a strategy for qualitative analy- sis. Scand J Public Health. 2012;40(8):795–805. 3. World Health Organization. Transforming and scaling up health professionals’ education and training: World Health Organization guidelines 2013. Geneva: WHO Press; 2013. Available from: https:// www.who.int/hrh/resources/transf_scaling_hpet/en/. [cited 2021 March 10] 3. World Health Organization. Transforming and scaling up health professionals’ education and training: World Health Organization guidelines 2013. Geneva: WHO Press; 2013. Available from: https:// www.who.int/hrh/resources/transf_scaling_hpet/en/. [cited 2021 March 10] 25. Cohen J. Statistical power analysis for the behavioral sciences. 2nd ed. Hillsdale: Laurence Erlbaum; 1988. 26. Patton MQ. Qualitative research & evaluation methods: integrating theory and practice. 4th ed. Los Angeles: SAGE Publications; 2015. 4. Bell R, Fredland N. The use of theoretical frameworks guiding inter- professional simulation: an integrative review. Nurs Educ Perspect. 2020;41(3):141–5. 4. Bell R, Fredland N. The use of theoretical frameworks guiding inter- professional simulation: an integrative review. Nurs Educ Perspect. 2020;41(3):141–5. 27. Dieckmann P, Friis SM, Lippert A, Østergaard D. Goals, success factors, and barriers for simulation-based learning: a qualitative interview study in health care. Simul Gaming. 2012;43(5):627–47. 5. Thistlethwaite J, Forman RD, Matthews DL, Rogers DG, Steketee DC, Yass- ine DT. Competencies and frameworks in interprofessional education: a comparative analysis. Acad Med. 2014;89(6):869–75. 5. Thistlethwaite J, Forman RD, Matthews DL, Rogers DG, Steketee DC, Yass- ine DT. Competencies and frameworks in interprofessional education: a comparative analysis. Acad Med. 2014;89(6):869–75. 28. Chiniara G, Clark M, Jaffrelot M, Posner GD, Rivière É. Moving beyond fidelity. In: Chiniara G, editor. Clinical Simulation. 2nd ed: Academic Press; 2019. p. 539–54. 6. Gaba DM. Author details 1 Department of Nursing Science, Institute of Health and Society, Faculty of Medicine, University of Oslo, Oslo, Norway. 2 Department of Health Management and Health Economics, Institute of Health and Society, Faculty of Medicine, University of Oslo, Oslo, Norway. 3 Department of Communication and Psychology, Humanistic Faculty, Aalborg University, Aalborg, Denmark. 4 Department of General Practice, Institute of Health and Society, Faculty of Medicine, University of Oslo, Oslo, Norway. 18. Lunde L, Bærheim A, Johannessen A, Aase I, Almendingen K, Andersen IA, et al. Evidence of validity for the Norwegian version of the interprofes- sional collaborative competency attainment survey (ICCAS). J Interprof Care. 2020;35:1–8. 19. Archibald D, Trumpower D, Macdonald CJ. Validation of the interprofes- sional collaborative competency attainment survey (ICCAS). J Interprof Care. 2014;28(6):553–8. References The future vision of simulation in health care. Qual Saf Health Care. 2004;13:i2–10. 6. Gaba DM. The future vision of simulation in health care. Qual Saf Health Care. 2004;13:i2–10. 29. Naumann F, Mullins R, Cawte A, Beavis S, Musial J, Hannan-Jones M. Designing, implementing and sustaining IPE within an authentic clinical environment: the impact on student learning. J Interprof Care. 2020;35:1–7. 7. Bullard MJ, Fox SM, Wares CM, Heffner AC, Stephens C, Rossi L. Simulation-based interdisciplinary education improves intern attitudes and outlook toward colleagues in other disciplines. BMC Med Educ. 2019;19(1):276. 30. Gilligan C, Outram S, Levett-Jones T. Recommendations from recent graduates in medicine, nursing and pharmacy on improving interpro- fessional education in university programs: a qualitative study. BMC Med Educ. 2014;14(1):52. 8. Oxelmark L, Amorøe TN, Carlzon L, Rystedt H. Students’ understanding of teamwork and professional roles after interprofessional simulation: a qualitative analysis. Adv Simul. 2017;2:8. 31. O’Leary N, Salmon N, Clifford AM. ‘It benefits patient care’: the value of practice-based IPE in healthcare curriculums. BMC Med Educ. 2020;20(1):1–424. 9. Hamilton P, Coey-Niebel C, McCaig J, Zlotos L, Power A, Craig G, et al. Evaluation of inter-professional education (IPE) with medical, nursing and pharmacy students through a simulated IPL educational intervention. Int J Clin Pract. 2021;75(11):e14725. 32. van Lierop M, van Dongen J, Janssen M, Smeets H, van Bokhoven L, Moser A. Jointly discussing care plans for real-life patients: the poten- tial of a student-led interprofessional team meeting in undergraduate health professions education. Perspect Med Educ. 2019;8(6):372–7. 10. Kleib M, Jackman D, Duarte-Wisnesky U. Interprofessional simulation to promote teamwork and communication between nursing and 10. Kleib M, Jackman D, Duarte-Wisnesky U. Interprofessional simulation to promote teamwork and communication between nursing and Page 12 of 12 Lunde et al. Advances in Simulation (2022) 7:9 33. Brewer ML, Flavell HL. Teamwork, collaboration and networking: self- reported behavioural change following pre-licensure interprofessional clinical learning. J Interprof Care. 2020;34(2):184–92. 34. Karpa K, Graveno M, Brightbill M, Fox G, Kelly S, Lehman E, et al. Geriatric assessment in a primary care environment: a standardized patient case activity for interprofessional students. MedEdPORTAL. 2019;15(1):10844. 35. Lunde L, Moen A, Jakobsen RB, Rosvold EO, Brænd AM. Exploring health- care students’ interprofessional teamwork in primary care simulation scenarios: collaboration to create a shared treatment plan. BMC Med Educ. 2021;21(1):416. 36. Alinier G, Hssain I. Creating effective learning environments: the educa- tor’s perspective. In: Chiniara G, editor. Clinical Simulation. References 2nd ed: Academic Press; 2019. p. 217–27. 37. Husebø SE, Abrandt Dahlgren M, Edelbring S, Nordenström E, Nordahl Amorøe T, Rystedt H, et al. Reflecting on interprofessional simulation. In: Abrandt Dahlgren M, Rystedt H, Felländer-Tsai L, Nyström S, editors. Inter- professional simulation in health care. Professional and practice-based learning. Cham: Springer International Publishing; 2019. p. 139–71. 38. Abrandt Dahlgren M, Rystedt H, Felländer-Tsai L, Nyström S. Advancing simulation pedagogy and research. In: Abrandt Dahlgren M, Rystedt H, Felländer-Tsai L, Nyström S, editors. Interprofessional simulation in health care: materiality, embodiment, interaction. 26. Cham: Springer Interna- tional Publishing AG; 2019. p. 197–211. g p 39. Dieckmann P, Ringsted C. Pedagogy in simulation-based training in healthcare. In: Forrest K, McKimm J, Edgar S, editors. Essential simulation in clinical education. Oxford: Wiley; 2013. p. 43–58. 40. Schot E, Tummers L, Noordegraaf M. Working on working together. A systematic review on how healthcare professionals contribute to inter- professional collaboration. J Interprof Care. 2020;34(3):332–42. 41. Sawyer T, Eppich W, Brett-Fleegler M, Grant V, Cheng A. More than one way to debrief: a critical review of healthcare simulation debriefing meth- ods. Simul Healthc. 2016;11(3):209–17. 42. Levin H, Cheng A, Catena H, Chatfield J, Cripps A, Bissett W, et al. Debrief- ing frameworks and methods. In: Chiniara G, editor. Clinical Simulation. 2nd ed: Academic Press; 2019. p. 483–505. 43. Aase I, Aase K, Dieckmann P, Bjørshol CA, Hansen BS. Interprofessional communication in a simulation-based team training session in health- care: a student perspective. J Nurs Educ Pract. 2016;6(7):91. 44. Furr S, Lane SH, Martin D, Brackney DE. 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https://openalex.org/W4226232384	https://link.springer.com/content/pdf/10.1007/JHEP05(2022)138.pdf	English	null	A Green's basis for the bosonic SMEFT to dimension 8	null	2,021	cc-by	12,327	Published for SISSA by Springer Published for SISSA by Springer Received: January 12, 2022 Revised: April 6, 2022 Accepted: April 27, 2022 Published: May 20, 2022 Open Access, c⃝The Authors. Article funded by SCOAP3. Keywords: Eﬀective Field Theories, SMEFT, Speciﬁc BSM Phenomenology 4 Explicit form of the operators 4.1 Operators in the class Xφ2D4 4.1 Operators in the 4.1.1 X = B 4.1.2 X = W p 4.1.1 X = B 4.1.2 X = W 4.1.1 X = B 4.1.2 X = W 4.2 Operators in the class X2φ2D2 4.2 Operators in the class X2φ2D2 4.2.1 X2 = B2 4.2.2 X2 = W 2 4.2.3 X2 = WB 4.2.4 X2 = G2 4.3 Operators in the class X3D2 4.3 Operators in the class X3D2 4.3.1 X3 = W 2B 4.3.2 X3 = G2B 4.3.3 X3 = W 3 4.3.4 X3 = G3 4.4 Operators in the class X2D4 4.4 Operators in the class X2D4 4.4.1 X = B 4.4.2 X = W 4.4.3 X = G 5 On-shell relations A Green’s basis for the bosonic SMEFT to dimension 8 JHEP05(2022)138 Mikael Chala,a Álvaro Díaz-Carmonaa and Guilherme Guedesa,b aCAFPE and Departamento de Física Teórica y del Cosmos, Universidad de Granada, Campus de Fuentenueva, E-18071 Granada, Spain bLaboratório de Instrumentaçao e Física Experimental de Partículas, Departamento de Física da Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal E-mail: mikael.chala@ugr.es, aldiaz@ugr.es, gguedes@lip.pt E-mail: mikael.chala@ugr.es, aldiaz@ugr.es, gguedes@lip.pt Abstract: We present a basis of dimension-eight Green’s functions involving Standard Model (SM) bosonic ﬁelds, consisting of 86 new operators. Rather than using algebraic identities and integration by parts, we prove the independence of these interactions in momentum space, including discussion on evanescent bosonic operators. Our results pave the way for renormalising the SM eﬀective ﬁeld theory (SMEFT), as well as for performing matching of ultraviolet models onto the SMEFT, to higher order. To demonstrate the potential of our construction, we have implemented our basis in matchmakereft and used it to integrate out a heavy singlet scalar and a heavy quadruplet scalar up to one loop. We provide the corresponding dimension-eight Wilson coeﬃcients. Likewise, we show how our results can be easily used to simplify cumbersome redundant Lagrangians arising, for example, from integrating out heavy ﬁelds using the path-integral approach to matching. Keywords: Eﬀective Field Theories, SMEFT, Speciﬁc BSM Phenomenology ArXiv ePrint: 2112.12724 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP05(2022)138 1 Introduction 2 2 Theory and conventions 2 Theory and conventions 4 2 Theory and conventions 3 Oﬀ-shell independence in momentum space JHEP05(2022)138 4 Explicit form of the operators 1 Introduction Eﬀective ﬁeld theories (EFTs) are indispensable tools for tackling quantum ﬁeld theory (QFT) problems involving very diﬀerent mass scales, particularly when employing mass- independent renormalisation schemes such as MS. The Lagrangian of an EFT at a certain energy scale ˜µ is organised as a power expansion in operators of increasing energy dimension (and therefore decreasing relevance). If some observable at a much smaller energy ˜µ′ ≪˜µ is to be computed, the Lagrangian must be run down using the renormalisation group equations (RGEs). In doing so, one typically crosses energy thresholds corresponding to particles of mass M. The latter do not decouple in mass-independent schemes; decoupling must be instead enforced by integrating out M. In practice, this is done by requiring that the Lagrangians with and without the particles of mass M describe the same physics at ˜µ = M. This procedure is usually known as matching. JHEP05(2022)138 It is well known that EFT operators related by (non-singular) ﬁeld redeﬁnitions are physically equivalent [1, 2]. They give exactly the same S-matrix, which in turn requires all particles to be on the mass shell. However, when addressing the processes of running and matching, it is common practice to perform intermediate computations oﬀ-shell. In particular, for the matching this implies equating one-light-particle irreducible (oﬀ-shell) Green’s functions above and below ˜µ = M, rather than S-matrix elements. There are a variety of reasons for proceeding this way. 1. Within the traditional way of computing within QFT, namely à la Feynman, S-matrix elements are obtained from connected and amputated diagrams. The amount of these scales much faster than one-particle-irreducible (1PI) diagrams with the number of external legs, thus turning the calculations computationally very challenging. 2. In the path integral approach to matching [3–5], no oﬀ-shell Green’s functions are computed. However, the resulting EFT involves, in general, operators related not only by ﬁeld redeﬁnitions, but even by algebraic identities, integration by parts, etc. The easiest way to simplify this EFT is by matching it oﬀ-shell at tree level onto a set of independent Green’s functions whose reduction to a physical basis must be known in advance. 3. While helicity-amplitude methods have been proved powerful to address the com- putation of some anomalous dimensions strictly on-shell [6–15], to the best of our knowledge they are not yet mature enough to be used in a number of cases. 6 Some applications 6.1 Integrating out a scalar singlet to one loop 6.2 Integrating out a scalar quadruplet to one loop 6.3 Reduction of Lagrangian to a physical basis 6.1 Integrating out a scalar singlet to one loop 16 6.2 Integrating out a scalar quadruplet to one loop 17 6.3 Reduction of Lagrangian to a physical basis 18 7 Conclusions and future directions A Tables of operators 21 – 1 – 1 Introduction These include renormalisation triggered by operators with smaller number of legs than the resulting amplitude or mixing of amplitudes of diﬀerent mass dimensions. The price to pay for working oﬀ-shell, though, is that operators related by ﬁeld redeﬁ- nitions must be kept in the action. Still, those related by algebraic identities (including Fierz relations) or integration by parts must be considered equivalent. A complete set of independent operators (up to ﬁeld redeﬁnitions) is called a Green’s basis. – 2 – The Standard Model EFT (SMEFT) is arguably the most important EFT, and likely the most reliable extension of the SM.1 A Green’s basis for the SMEFT to dimension six was worked out in ref. [16]. On top of the 84 real terms appearing in the physical basis [17] (for one fermion family), it involves 8 bosonic and 73 fermionic new operators, thus a total of 81 redundant interactions. (Green’s bases are also known for the EFT of the SM with sterile neutrinos [18] to dimensions six, as well as for the EFT of the SM extended with an axion-like particle to dimension ﬁve [19].) However, there is by now convincing evidence that the dimension-six sector alone is not suﬃcient for making predictions within the SMEFT in a number of situations; see for exam- ple refs. [20–29] for details. Thus, in this article we elaborate a Green’s basis for the SMEFT to dimension eight. Given the large number of operators (1649 on top of the 993 physical ones of refs. [30] and [31]) and the technical diﬃculties, we restrict this work to bosonic interactions. We ﬁnd a total of 86 redundant operators that extend the 89 physical ones. JHEP05(2022)138 Let us notice that there are automatic tools to count independent oﬀ-shell operators, in particular Basisgen [32] and Sym2Int [33]. However, to the best of our knowledge, none can yet build the operators explicitly, leave apart reducing them to the physical basis. Even checking whether certain operators within a set of interactions can be related by integration by parts, Fierz and Bianchi identities, etc. can become extremely cumbersome when the operators involve several ﬁelds and derivatives. Thus, to simplify this task, we work in momentum space, where operator independence translates into the linear independence of 1PI tree-level amplitudes, conveniently written in terms of independent kinematic invariants. 1This claim is supported by the lack of evidence of new particles between the electroweak scale v ∼ 246 GeV and the TeV; as well as by the fact that the most tantalizing experimental anomalies, namely those in B decays, point to scales of several TeVs. 2 Theory and conventions Given that we focus only on the bosonic sector of the SMEFT, the relevant renormalisable SM Lagrangian reads simply: Given that we focus only on the bosonic sector of the SMEFT, the relevant renormalisable SM Lagrangian reads simply: LSM = −1 4GA µνGA µν −1 4W a µνW a µν −1 4BµνBµν (2.1) + (Dµφ)† (Dµφ) +µ2\|φ\|2 −λ\|φ\|4 . (2.1) We denote by W, B and G the electroweak gauge bosons and the gluon, respectively. We represent the Higgs doublet by φ = (ϕ+, ϕ0)T , and ˜φ = iσ2φ∗with σI (I = 1, 2, 3) being the Pauli matrices. Our convention for the covariant derivative is: We denote by W, B and G the electroweak gauge bosons and the gluon, respectively. We represent the Higgs doublet by φ = (ϕ+, ϕ0)T , and ˜φ = iσ2φ∗with σI (I = 1, 2, 3) being the Pauli matrices. Our convention for the covariant derivative is: JHEP05(2022)138 Dµ = ∂µ −ig1Y Bµ −ig2 σI 2 W I µ −ig3 λA 2 GA µ , (2.2) (2.2) where g1, g2 and g3 represent, respectively, the U(1)Y , SU(2)L and SU(3)c gauge couplings, Y stands for the hypercharge and λA are the Gell-Mann matrices. Also, as customary, we deﬁne dual tensors as eXµν = 1 2ϵµνρλXρλ, where our convention for the Levi-Civita symbol follows that of FeynRules and FormCalc [36], ϵ0123 = +1. The SMEFT extendes LSM with eﬀective operators of energy dimension d > 4, sup- pressed by powers of the cutoﬀΛ, above which the SMEFT is no longer a valid EFT. Ig- noring lepton number violation and operators of dimension higher than eight, the SMEFT Lagrangian reads: LSMEFT = LSM + X i c(6) i Λ2 O(6) i + X j c(8) j Λ4 O(8) j . (2.3) (2.3) Any set of physically independent dimension-six (bosonic) operators involve 15 terms. A widely-used such set is the Warsaw basis of ref. [17]. Likewise, the bosonic sector of the SMEFT to dimension eight comprises 89 independent physical couplings [30, 31]. In this work, though, we are interested in operators that are independent oﬀ-shell, namely up to ﬁeld redeﬁnitions. A basis of operators of this kind is known to dimension six; see ref. [16]. The remaining of the paper is devoted to discussing the corresponding dimension-eight set, the way we have obtained it and some applications. 2 Theory and conventions Hereafter, unless otherwise stated, independence of operators will implicitly mean oﬀ-shell independence. 1 Introduction One of the most appreciated implications of this approach is that integration by parts reduces simply to momentum conservation. Likewise, Bianchi (and other) identities manifest simply and automatically as relations between diﬀerent Feynman rules. This article is organised as follows. In section 2 we introduce the SMEFT Lagrangian, including notation and sign conventions. In section 3 we discuss the approach to establish- ing the oﬀ-shell independence of interactions in momentum space. We also make emphasis on subtleties arising in interactions involving pure four-dimensional objects such as the Levi-Civita symbol. In section 4 we provide, class by class, the operators comprising the bosonic Green’s basis. We also include the relevant information to cross-check their in- dependence in momentum space. In section 5 we provide the explicit reduction of the redundant operators to the physical basis. Finally, with the aim of demonstrating the potential of intertwining our results with automatic tools, in section 6 we integrate out a heavy scalar singlet as well as a heavy scalar quadruplet up to one loop to dimension eight using matchmakereft [34]. We also show how complicated Lagrangians arising from matching using functional methods can be trivially reduced to a physical basis using our Green’s set of operators together with standard tools such as FeynRules [35]. We conclude in section 7. Appendix A includes tables with all operators for an easier reading. As auxiliary material, we provide the operators in a FeynRules model. – 3 – 3 Oﬀ-shell independence in momentum space Let {Oi}i=1...N be N (potentially dependent) operators, and let A(a →b) be the 1PI amplitude for a given process a →b. The contribution of Oi to the latter can be written in terms of independent kinematics invariants {κα}α∈I where I is a collection of indices. Explicitly, at tree level: A(a →b) = ci X α∈I fi α(⃗g)κα , (3.1) (3.1) where f is simply a matrix that is function of ⃗g = (g1, g2, g3, λ), which collectively encodes the SM couplings. (No sum over i is implied.) where f is simply a matrix that is function of ⃗g = (g1, g2, g3, λ), which collectively encodes the SM couplings. (No sum over i is implied.) – 4 – – 4 – If two operators Oi and Oj, i ̸= j, describe diﬀerent oﬀ-shell physics, and precisely because the κ are independent, then there must exist at least one process for which the corresponding fi α = (fi α1, fi α2, . . .) and fj α = (fj α1, fj α2, . . .) are non-collinear vectors. In more generality, if there is one amplitude A such that the associate matrix M with elements (M)ij = fi j has rank N, then the operators {Oi}i=1...N are independent. (Clearly, the opposite is not true.) As a matter of example, let us consider the following dimension-eight six-Higgs oper- ators: O1 = (φ†φ)Dµ(φ†φ)Dµ(φ†φ) , (3.2) O2 = (φ†φ)2(D2φ†φ + φ†D2φ) , (3.3) O3 = (φ†φ)2Dµφ†Dµφ . (3.4) (3.2) JHEP05(2022)138 JHEP05(2022)138 (3.4) They are all hermitian. The 1PI amplitude for ϕ0(p1) →ϕ0(p2)ϕ+(p3)ϕ−(p4)ϕ+(p5)ϕ−(p6) reads: They are all hermitian. The 1PI amplitude for ϕ0(p1) →ϕ0(p2)ϕ+(p3)ϕ−(p4)ϕ+(p5)ϕ−(p6) reads: A = 2i c1(2κ13 + 2κ14 + 2κ15 + 2κ16 −2κ23 −2κ24 −2κ25 −2κ26 −κ34 −2κ35 −κ36 −κ45 −2κ46 −κ56) −4i c2(κ11 + κ22 + κ33 + κ44 + κ55 + κ66) + 2i c3(2κ12 −κ34 −κ36 −κ45 −κ56) , (3.5) (3.5) where κij = pi · pj. j j Clearly, the matrix M associated to this process has, in appearance, rank 3. Indeed, for example the sub-matrix ˆ M associated to the invariants κ11, κ12, κ13 looks like Clearly, the matrix M associated to this process has, in appearance, rank 3. 3 Oﬀ-shell independence in momentum space Indeed, for example the sub-matrix ˆ M associated to the invariants κ11, κ12, κ13 looks like ˆ M =   0 0 4i −4i 0 0 0 4i 0  =⇒3 ≥Rank(M) ≥Rank( ˆM) = 3 . (3.6) (3.6) However, the kinematic invariants that we have chosen are not independent, because by momentum conservation p1 = p2 + p3 + p4 + p5 + p6, and therefore κi1 can always be eliminated. Taking this into account, we get instead: A = 2i c1(2κ33 + 3κ43 + 2κ44 + 2κ53 + 3κ54 + 2κ55 + 3κ63 + 2κ64 + 3κ65 + 2κ66) −8i c2(κ22 + κ32 + κ33 + κ42 + κ43 + κ44 + κ52 + κ53 + κ54 + κ55 + κ62 + κ63 + κ64 + κ65 + κ66) + 2i c3(2κ22 + 2κ32 + 2κ42 −κ43 + 2κ52 −κ54 + 2κ62 −κ63 −κ65) . (3.7) (3.7) + 2i c3(2κ22 + 2κ32 + 2κ42 −κ43 + 2κ52 −κ54 + 2κ62 −κ63 −κ65) . (3.7) The corresponding matrix has only rank 2. This is clear from the fact that the ﬁrst and third lines in the equation above add to minus half the second one. Or in other words, O2 = −2(O1 +O3). At the level of the Lagrangian, this results from the fact that the three – 5 – operators are related by integration by parts up to a total derivative: operators are related by integration by parts up to a total derivative: Dµ (φ†φ)2Dµ(φ†φ) = 2(φ†φ)Dµ(φ†φ)Dµ(φ†φ) + (φ†φ)2(D2φ†φ + φ†D2φ) + 2(φ†φ)2Dµφ†Dµφ . (3.8) (3.8) Let us now study a slightly more elaborated example. Let us consider the following three operators: Let us now study a slightly more elaborated example. Let us consider the following three operators: O1 = Dµ(φ†φ)DνBµρBνρ , (3.9) O2 = (D2φ†φ + φ†D2φ)BνρBνρ , (3.10) O3 = Dµφ†DµφBνρBνρ . 3 Oﬀ-shell independence in momentum space (3.11) (3.9) JHEP05(2022)138 The amplitude for ϕ0(p1) →ϕ0(p2)B(p3)B(p4) takes the form: The amplitude for ϕ0(p1) →ϕ0(p2)B(p3)B(p4) takes the form: The amplitude for ϕ0(p1) →ϕ0(p2)B(p3)B(p4) takes the form: A = −ic1(κ3334 + 2κ3434 + κ3444 −κ′ 4333 −2κ′ 4334 −κ′ 4344) + 4ic2(2κ2234 + 2κ2334 + 2κ2434 + κ3334 + 2κ3434 + κ3444 −2κ′ 4322 −2κ′ 4323 −2κ′ 4324 −κ′ 4333 −2κ −2κ′ 4334 −κ4344) (3.12) −4ic3(κ2234 + κ2334 + κ2434 −κ′ 4322 −κ4323 −κ4324) ; (3.12) where we have removed p1 using momentum conservation, and the kinematic invariants are: κijkl = (ε3 · ε4)(pi · pj)(pk · pl) and κ′ ijkl = (ε3 · pi)(ε4 · pj)(pk · pl), with ε representing a polarization vector. The rank of the corresponding matrix is only 2, so one of the operators is a linear combination of the other two. In fact, from the expression above it is obvious to check that O1 = −1 4O2 −1 2O3. This result reﬂects the following Lagrangian relation: O1 = −Dµ(φ†φ)DµBρνBνρ −Dµ(φ†φ)DρBνµBνρ = −Dµ(φ†φ)DµBρνBνρ −Dµ(φ†φ)DνBµρBνρ = −Dµ(φ†φ)DµBρνBνρ −O1 ⇒O1 = −1 2Dµ(φ†φ)DµBρνBνρ = 1 2D2(φ†φ)BρνBνρ −1 2Dµ(φ†φ)BρνDµBνρ = 1 2D2(φ†φ)BρνBνρ −O1 ⇒O1 = 1 4D2(φ†φ)BρνBνρ = −1 4(D2φ†φ + φ†D2φ)BνρBνρ −1 4(2Dµφ†Dµφ)BνρBνρ = −1 4O2 −1 2O3 . (3.13) O1 = −Dµ(φ†φ)DµBρνBνρ −Dµ(φ†φ)DρBνµBνρ = −Dµ(φ†φ)DµBρνBνρ −Dµ(φ†φ)DνBµρBνρ = −Dµ(φ†φ)DµBρνBνρ −O1 (3.13) In the ﬁrst equality we have used the Bianchi identity DµBνρ + DνBρµ + DρBµν = 0; in the second we have renamed indices as ν ↔ρ in the last operator; in the ﬁfth equality we have integrated by parts the derivative acting on Bρν; while in the penultimate equality – 6 – we have simply expanded the derivative. In all steps, we have also taken into account that B is anti-symmetric: Bνρ = −Bρν. These purposely-easy-to-follow example calculations might look trivial to address also from the Lagrangian (position space) point of view. However, things get signiﬁcantly more complicated when not only a few but instead tens of operators are involved. More importantly, the examples above show how one can obtain the relations between depen- dent operators. However, demonstrating that several operators are independent, at the Lagrangian level, implies proving that there is no single combination of operations (inte- gration by parts, Bianchi, etc.) that can relate any of them. In momentum space, though, one only needs to check, still, the rank of the corresponding matrix. 2The identities above are not necessarily fulﬁlled in D > 4 space-time dimensions if the Levi-Civita symbol is replaced by a fully anti-symmetric rank-4 tensor. 3 Oﬀ-shell independence in momentum space Now, let us require that the three momenta (we have removed p1) and the two polariza- tion vectors can not be linearly independent in four space-time dimensions. In other words: (3.21) p4 = a1ε3 + a2ε4 + a3p2 + a4p3 (3.21) p4 = a1ε3 + a2ε4 + a3p2 + a4p3 r some real numbers ai, i = 1, . . . , 4. Upon using this constraint, the amplitude reads: A = (c1 + c2) a3κ342323 + a3(1 + a4)κ342323 + a4(1 + a4)κ342333 JHEP05(2022)138 + a1a3κ342332 + a1(1 + 2a4)κ342333 + a2a4κ342343 + a2 1κ342333 . (3.22) (3.22) sion, it is obvious that both operators are related (and identical). The n the two is actually an evanescent term which vanishes in D = 4 From this expression, it is obvious that both operators are related (and identical). The diﬀerence between the two is actually an evanescent term, which vanishes in D = 4. om this expression, it is obvious that both operators are related (and identical). The From this expression, it is obvious that both operators are related (and identical). Th diﬀerence between the two is actually an evanescent term which vanishes in D = 4 Equipped with this background, our strategy for ﬁnding minimal sets of Green’s op- erators in a given class (deﬁned by number of ﬁeld strength tensors, derivatives and Higgs ﬁelds) consists simply in building all possible operators in the class, computing their con- tribution to a single particular amplitude in which they are all independent,3 and ﬁnally eliminating operators whose removal does not decrease the rank of the system. From our point of view, automatising this procedure in momentum space is signiﬁcantly simpler than in position space. 4 Explicit form of the operators Following Basisgen and Sym2Int, there are 2 redundant operators in the class φ6D2, 10 in φ4D4, 1 in φ2D6, 4 in Xφ4D2, 44 in X2φ2D2, 6 in Xφ2D4, 16 in X3D2 and 3 in the class X2D4. Those in the ﬁrst four classes have been already presented in ref. [37]. Hence, we focus here on the remaining operators. For clarity, in the lists of interactions below, we include also the physical operators (in the classes in which they exist) as given in ref. [30], and with the same names. 3 Oﬀ-shell independence in momentum space JHEP05(2022)138 There is one last subtlety that one has to deal with in operators involving dual ﬁeld strength tensors. The fully anti-symmetric Levi-Civita symbol ϵµνρλ, which is a pure 4- dimensional object, fulﬁlls in particular the Schouten identity: (3.14) gµνϵαβγδ + gµαϵβγδν + gµβϵγδνα + gµγϵδναβ + gµδϵναβγ = 0 . (3.14) Related to this, the following relations involving an arbitrary rank-2 tensor T and two ﬁeld strength tensors X and F hold: T[µν]Xµ ρ eF νρ = T[µν] eXµ ρF νρ , (3.15) T{µν}Xµ ρ eF νρ = −T{µν} eXµ ρF νρ + 1 2T µ µ eXνρFνρ , (3.16) TµνXµρ eXν ρ = 1 4T µ µ Xνρ eXνρ , (3.17) (3.15) (3.17) where [µν] and {µν} denote, as usual, anti-symmetrisation and symmetrisation, respec- tively, with respect to the indices involved. Certainly, these relations are much less imme- diate to take care of at the level of amplitudes than, for example, momentum conservation. However, precisely because they are restrictions that hold only in D = 4 space-time dimen- sions,2 they can be automatically enforced upon requiring all Lorentz vectors (momenta and polarizations) involved in the amplitude to be four-vectors. In practice, we simply demand that ﬁve or more Lorentz vectors can not be linearly independent. Let us show how this works with a simple example. Let us consider the operators Let us consider the operators O1 = i(Dµφ†σIDνφ −Dνφ†σIDµφ)Bµ ρf W Iνρ , (3.18) O2 = i(Dµφ†σIDνφ −Dνφ†σIDµφ) eBµ ρW Iνρ . (3.19) (3.18) (3.19) (3.18) They are obviously related by the identity in eq. (3.15). They are obviously related by the identity in eq. (3.15). Now, let us compute the amplitude for ϕ0(p1) →ϕ0(p2)W 3(p3)B(p4). Already after imposing momentum conservation, we obtain: A = c1(−κ323443 −κ323444 + κ343424 + κ342334 + κ342344) + c2(−κ423433 −κ423434 −κ343423 + κ342433 + κ342434) ; (3.20) (3.20) 2The identities above are not necessarily fulﬁlled in D > 4 space-time dimensions if the Levi-Civita symbol is replaced by a fully anti-symmetric rank-4 tensor. – 7 – – 7 – where in this case, κijklmn = ϵ(εi, pj, pk, pl)(εm · pn) and κ′ ijklmn = ϵ(εi, εj, pk, pl)(pm · pn). Naively, one would conclude that the operators are independent, because the rank of the matrix of the system is obviously 2. 4.1 Operators in the class Xφ2D4 There are 3 real terms for X = B, and 3 more for X = W. In the ﬁrst case, it suﬃces to compute the amplitude for the process ϕ0(p1) →ϕ0(p2)B(p3), while in the second case only ϕ0(p1) →ϕ0(p2)W +(p2) is needed. 4.1.1 X = B 4.1.1 X = B O(1) Bφ2D4 = i(Dνφ†D2φ −D2φ†Dνφ)DµBµν , (4.1) O(2) Bφ2D4 = (Dνφ†D2φ + D2φ†Dνφ)DµBµν , (4.2) O(3) Bφ2D4 = i(DρDνφ†Dρφ −Dρφ†DρDνφ)DµBµν . (4.3) (4.1) (4.2) (4.3) 3For this we simply search for an amplitude whose associated rank equals the number of independent oﬀ-shell operators as provided by Sym2Int and Basisgen. – 8 – .2 X = W 4.1.2 X = W O(1) Wφ2D4 = i(Dνφ†σID2φ −D2φ†σIDνφ)DµW Iµν , (4.4) O(2) Wφ2D4 = (Dνφ†σID2φ + D2φ†σIDνφ)DµW Iµν , (4.5) O(3) Bφ2D4 = i(DρDνφ†σIDρφ −Dρφ†σIDρDνφ)DµW Iµν . (4.6) (4.6) 4.2 Operators in the class X2φ2D2 There are 12 independent operators for X2 = B2, 19 for X2 = W 2 and also 19 for X2 = WB. One can check the independence of the operators below by evaluating the amplitudes ϕ0(p1) →ϕ0(p2)B(p3)B(p4), ϕ0(p1) →ϕ0(p2)W +(p3)W −(p4) and ϕ0(p1) → ϕ+(p2)W +(p3)B(p4), respectively. JHEP05(2022)138 4.2.1 X2 = B2 4.2.1 X2 = B2 O(1) B2φ2D2 = (Dµφ†Dνφ)BµρBνρ , (4.7) O(2) B2φ2D2 = (Dµφ†Dµφ)BνρBνρ , (4.8) O(3) B2φ2D2 = (Dµφ†Dµφ)Bνρ eBνρ , (4.9) O(4) B2φ2D2 = (Dµφ†φ + φ†Dµφ)DνBµρBν ρ , (4.10) O(5) B2φ2D2 = i(φ†DµDνφ −DµDνφ†φ)BµρBν ρ , (4.11) O(6) B2φ2D2 = φ†φDµDνBµρBν ρ , (4.12) O(7) B2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµBµρBν ρ , (4.13) O(8) B2φ2D2 = (φ†Dνφ + Dνφ†φ)DµBµρBν ρ , (4.14) O(9) B2φ2D2 = (φ†D2φ + D2φ†φ)Bνρ eBνρ , (4.15) O(10) B2φ2D2 = i(φ†D2φ −D2φ†φ)Bνρ eBνρ (4.16) O(11) B2φ2D2 = (φ†Dνφ + Dνφ†φ)DµBµρ eBν ρ (4.17) O(12) B2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµBµρ eBν ρ . (4.18) 4.2.2 X2 = W 2 O(1) W 2φ2D2 = (Dµφ†Dνφ)W I µρW Iρ ν , (4.19) O(2) W 2φ2D2 = (Dµφ†Dµφ)W I νρW Iνρ , (4.20) O(3) W 2φ2D2 = (Dµφ†Dµφ)W I νρf W Iνρ , (4.21) O(4) W 2φ2D2 = iϵIJK(Dµφ†σIDνφ)W J µρW Kρ ν , (4.22) O(5) W 2φ2D2 = ϵIJK(Dµφ†σIDνφ)(W J µρf W Kρ ν −f W J µρW Kρ ν ) , (4.23) O(6) W 2φ2D2 = iϵIJK(Dµφ†σIDνφ)(W J µρf W Kρ ν + f W J µρW Kρ ν ) , (4.24) O(7) W 2φ2D2 = iϵIJK(φ†σIDνφ −Dνφ†σIφ)DµW Jµρf W K νρ , (4.25) O(1) B2φ2D2 = (Dµφ†Dνφ)BµρBνρ , (4.7) O(2) B2φ2D2 = (Dµφ†Dµφ)BνρBνρ , (4.8) O(3) B2φ2D2 = (Dµφ†Dµφ)Bνρ eBνρ , (4.9) O(4) B2φ2D2 = (Dµφ†φ + φ†Dµφ)DνBµρBν ρ , (4.10) O(5) B2φ2D2 = i(φ†DµDνφ −DµDνφ†φ)BµρBν ρ , (4.11) O(6) B2φ2D2 = φ†φDµDνBµρBν ρ , (4.12) O(7) B2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµBµρBν ρ , (4.13) O(8) B2φ2D2 = (φ†Dνφ + Dνφ†φ)DµBµρBν ρ , (4.14) O(9) B2φ2D2 = (φ†D2φ + D2φ†φ)Bνρ eBνρ , (4.15) O(10) B2φ2D2 = i(φ†D2φ −D2φ†φ)Bνρ eBνρ (4.16) O(11) B2φ2D2 = (φ†Dνφ + Dνφ†φ)DµBµρ eBν ρ (4.17) O(12) B2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµBµρ eBν ρ . 4.2 Operators in the class X2φ2D2 (4.18) 4.2.2 X2 = W 2 O(1) W 2φ2D2 = (Dµφ†Dνφ)W I µρW Iρ ν , (4.19) O(2) W 2φ2D2 = (Dµφ†Dµφ)W I νρW Iνρ , (4.20) O(3) W 2φ2D2 = (Dµφ†Dµφ)W I νρf W Iνρ , (4.21) O(4) W 2φ2D2 = iϵIJK(Dµφ†σIDνφ)W J µρW Kρ ν , (4.22) O(5) W 2φ2D2 = ϵIJK(Dµφ†σIDνφ)(W J µρf W Kρ ν −f W J µρW Kρ ν ) , (4.23) O(6) W 2φ2D2 = iϵIJK(Dµφ†σIDνφ)(W J µρf W Kρ ν + f W J µρW Kρ ν ) , (4.24) O(7) W 2φ2D2 = iϵIJK(φ†σIDνφ −Dνφ†σIφ)DµW Jµρf W K νρ , (4.25) O(1) B2φ2D2 = (Dµφ†Dνφ)BµρBνρ , (4.7) O(2) B2φ2D2 = (Dµφ†Dµφ)BνρBνρ , (4.8) O(3) B2φ2D2 = (Dµφ†Dµφ)Bνρ eBνρ , (4.9) O(4) B2φ2D2 = (Dµφ†φ + φ†Dµφ)DνBµρBν ρ , (4.10) O(5) B2φ2D2 = i(φ†DµDνφ −DµDνφ†φ)BµρBν ρ , (4.11) O(6) B2φ2D2 = φ†φDµDνBµρBν ρ , (4.12) O(7) B2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµBµρBν ρ , (4.13) O(8) B2φ2D2 = (φ†Dνφ + Dνφ†φ)DµBµρBν ρ , (4.14) O(9) B2φ2D2 = (φ†D2φ + D2φ†φ)Bνρ eBνρ , (4.15) O(10) B2φ2D2 = i(φ†D2φ −D2φ†φ)Bνρ eBνρ (4.16) O(11) B2φ2D2 = (φ†Dνφ + Dνφ†φ)DµBµρ eBν ρ (4.17) O(12) B2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµBµρ eBν ρ . 4.2 Operators in the class X2φ2D2 (4.18) X2 W 2 4.2.2 X2 = W 2 O(1) W 2φ2D2 = (Dµφ†Dνφ)W I µρW Iρ ν , (4.19) O(2) W 2φ2D2 = (Dµφ†Dµφ)W I νρW Iνρ , (4.20) O(3) W 2φ2D2 = (Dµφ†Dµφ)W I νρf W Iνρ , (4.21) O(4) W 2φ2D2 = iϵIJK(Dµφ†σIDνφ)W J µρW Kρ ν , (4.22) O(5) W 2φ2D2 = ϵIJK(Dµφ†σIDνφ)(W J µρf W Kρ ν −f W J µρW Kρ ν ) , (4.23) O(6) W 2φ2D2 = iϵIJK(Dµφ†σIDνφ)(W J µρf W Kρ ν + f W J µρW Kρ ν ) , (4.24) O(7) W 2φ2D2 = iϵIJK(φ†σIDνφ −Dνφ†σIφ)DµW Jµρf W K νρ , (4.25) – 9 – O(8) W 2φ2D2 = ϵIJKφ†σIφDνDµW Jµρf W Kν ρ , (4.26) O(9) W 2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµW Iµρf W Iν ρ , (4.27) O(10) W 2φ2D2 = (φ†Dνφ + Dνφ†φ)DµW Iµρf W Iν ρ , (4.28) O(11) W 2φ2D2 = (φ†Dνφ + Dνφ†φ)DµW IµρW Iν ρ , (4.29) O(12) W 2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµW IµρW Iν ρ , (4.30) O(13) W 2φ2D2 = φ†φDµW IµρDνW Iν ρ , (4.31) O(14) W 2φ2D2 = (Dµφ†φ + φ†Dµφ)W IνρDµW I νρ , (4.32) O(15) W 2φ2D2 = i(Dµφ†φ −φ†Dµφ)W IνρDµW I νρ , (4.33) O(16) W 2φ2D2 = (Dµφ†φ + φ†Dµφ)DµW Iνρf W I νρ , (4.34) O(17) W 2φ2D2 = i(Dµφ†φ −φ†Dµφ)DµW Iνρf W I νρ , (4.35) O(18) W 2φ2D2 = ϵIJK(φ†σIDνφ + Dνφ†σIφ)DµW JµρW K νρ , (4.36) O(19) W 2φ2D2 = iϵIJK(φ†σIDνφ −Dνφ†σIφ)DµW JµρW K νρ . (4.37) JHEP05(2022)138 4.2.3 X2 = W B 4.2.3 X2 = W B (4.55) (4 56) φ 4.2.4 X2 = G2 φ 4.2.4 X2 = G2 4.2.4 X2 = G2 O(1) G2φ2D2 = (Dµφ†Dνφ)GA µρGAνρ , (4.57) O(2) G2φ2D2 = (Dµφ†Dµφ)GA νρGAνρ , (4.58) O(3) G2φ2D2 = (Dµφ†Dµφ)GA νρ eGAνρ , (4.59) O(4) G2φ2D2 = (Dµφ†φ + φ†Dµφ)DνGAµρGAν ρ , (4.60) O(5) G2φ2D2 = i(φ†DµDνφ −DµDνφ†φ)GAµρGAν ρ , (4.61) O(6) G2φ2D2 = φ†φDµDνGAµρGAν ρ , (4.62) O(7) G2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµGAµρGAν ρ , (4.63) O(8) G2φ2D2 = (φ†Dνφ + Dνφ†φ)DµGAµρGAν ρ , (4.64) O(9) G2φ2D2 = (φ†D2φ + D2φ†φ)GAνρ eGAν ρ , (4.65) O(10) G2φ2D2 = i(φ†D2φ −D2φ†φ)GAνρ eGA νρ (4.66) O(11) G2φ2D2 = (φ†Dνφ + Dνφ†φ)DµGAµρ eGAν ρ (4.67) O(12) G2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµGAµρ eGAν ρ . (4.68) O(1) G2φ2D2 = (Dµφ†Dνφ)GA µρGAνρ , (4.57) O(2) G2φ2D2 = (Dµφ†Dµφ)GA νρGAνρ , (4.58) O(3) G2φ2D2 = (Dµφ†Dµφ)GA νρ eGAνρ , (4.59) O(4) G2φ2D2 = (Dµφ†φ + φ†Dµφ)DνGAµρGAν ρ , (4.60) O(5) G2φ2D2 = i(φ†DµDνφ −DµDνφ†φ)GAµρGAν ρ , (4.61) O(6) G2φ2D2 = φ†φDµDνGAµρGAν ρ , (4.62) O(7) G2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµGAµρGAν ρ , (4.63) O(8) G2φ2D2 = (φ†Dνφ + Dνφ†φ)DµGAµρGAν ρ , (4.64) O(9) G2φ2D2 = (φ†D2φ + D2φ†φ)GAνρ eGAν ρ , (4.65) O(10) G2φ2D2 = i(φ†D2φ −D2φ†φ)GAνρ eGA νρ (4.66) O(11) G2φ2D2 = (φ†Dνφ + Dνφ†φ)DµGAµρ eGAν ρ (4.67) O(12) G2φ2D2 = i(φ†Dνφ −Dνφ†φ)DµGAµρ eGAν ρ . (4.68) JHEP05(2022)138 4.2.3 X2 = W B 4.2.3 X2 = W B O(1) WBφ2D2 = (Dµφ†σIDµφ)BνρW Iνρ , (4.38) O(2) WBφ2D2 = (Dµφ†σIDµφ)Bνρf W Iνρ , (4.39) O(3) WBφ2D2 = i(Dµφ†σIDνφ)(BµρW I ρ ν −BνρW I ρ µ ) , (4.40) O(4) WBφ2D2 = (Dµφ†σIDνφ)(BµρW I ρ ν + BνρW I ρ µ ) , (4.41) O(5) WBφ2D2 = i(Dµφ†σIDνφ)(Bµρf W I ρ ν −Bνρf W I ρ µ ) , (4.42) O(6) WBφ2D2 = (Dµφ†σIDνφ)(Bµρf W I ρ ν + Bνρf W I ρ µ ) , (4.43) O(7) WBφ2D2 = i(φ†σIDµφ −Dµφ†σIφ)DµBνρW I νρ , (4.44) O(8) WBφ2D2 = (φ†σIDνφ + Dνφ†σIφ)DµBµρW I νρ , (4.45) O(9) WBφ2D2 = i(φ†σIDνφ −Dνφ†σIφ)DµBµρW I νρ , (4.46) O(10) WBφ2D2 = (φ†σIφ)DµBµρDνW Iνρ , (4.47) O(11) WBφ2D2 = (Dνφ†σIφ + φ†σIDνφ)BµρDµW Iνρ , (4.48) O(12) WBφ2D2 = i(Dνφ†σIφ −φ†σIDνφ)BµρDµW Iνρ , (4.49) O(13) WBφ2D2 = (φ†σIφ)BµρDνDµW Iνρ , (4.50) O(14) WBφ2D2 = i(Dνφ†σIφ −φ†σIDνφ)DµBµρf W Iνρ , (4.51) O(15) WBφ2D2 = i(φ†σIDµφ −Dµφ†σIφ)DµBνρf W Iνρ , (4.52) O(16) WBφ2D2 = (φ†σIφ)(D2Bνρ)f W I νρ , (4.53) O(17) WBφ2D2 = (φ†σIφ)(DρDµW Iµν) eBνρ , (4.54) O(1) WBφ2D2 = (Dµφ†σIDµφ)BνρW Iνρ , (4.38) O(2) WBφ2D2 = (Dµφ†σIDµφ)Bνρf W Iνρ , (4.39) O(3) WBφ2D2 = i(Dµφ†σIDνφ)(BµρW I ρ ν −BνρW I ρ µ ) , (4.40) O(4) WBφ2D2 = (Dµφ†σIDνφ)(BµρW I ρ ν + BνρW I ρ µ ) , (4.41) O(5) WBφ2D2 = i(Dµφ†σIDνφ)(Bµρf W I ρ ν −Bνρf W I ρ µ ) , (4.42) O(6) WBφ2D2 = (Dµφ†σIDνφ)(Bµρf W I ρ ν + Bνρf W I ρ µ ) , (4.43) O(7) WBφ2D2 = i(φ†σIDµφ −Dµφ†σIφ)DµBνρW I νρ , (4.44) O(8) WBφ2D2 = (φ†σIDνφ + Dνφ†σIφ)DµBµρW I νρ , (4.45) O(9) WBφ2D2 = i(φ†σIDνφ −Dνφ†σIφ)DµBµρW I νρ , (4.46) O(10) WBφ2D2 = (φ†σIφ)DµBµρDνW Iνρ , (4.47) O(11) WBφ2D2 = (Dνφ†σIφ + φ†σIDνφ)BµρDµW Iνρ , (4.48) O(12) WBφ2D2 = i(Dνφ†σIφ −φ†σIDνφ)BµρDµW Iνρ , (4.49) O(13) WBφ2D2 = (φ†σIφ)BµρDνDµW Iνρ , (4.50) O(14) WBφ2D2 = i(Dνφ†σIφ −φ†σIDνφ)DµBµρf W Iνρ , (4.51) O(15) WBφ2D2 = i(φ†σIDµφ −Dµφ†σIφ)DµBνρf W Iνρ , (4.52) O(16) WBφ2D2 = (φ†σIφ)(D2Bνρ)f W I νρ , (4.53) O(17) WBφ2D2 = (φ†σIφ)(DρDµW Iµν) eBνρ , (4.54) – 10 – O(18) WBφ2D2 = i(Dνφ†σIφ −φ†σIDνφ) eBµρDµW I νρ , (4.55) O(19) WBφ2D2 = (Dνφ†σIφ + φ†σIDνφ) eBµρDµW I νρ . (4.56) O(18) WBφ2D2 = i(Dνφ†σIφ −φ†σIDνφ) eBµρDµW I νρ , O(19) WBφ2D2 = (Dνφ†σIφ + φ†σIDνφ) eBµρDµW I νρ . 4.4 Operators in the class X2D4 4.4 Operators in the class X2D4 In this class, there is only 1 operator per category, X = B, W, G. So the independence of operators is obvious. 4.4.1 X = B OB2D4 = (DσDµBµν)(DσDρBρν) . (4.85) 4.4.1 X = B OB2D4 = (DσDµBµν)(DσDρBρν) . (4.85) (4.85) 4.4.2 X = W OW 2D4 = (DσDµW Iµν)(DσDρW I ρν) . (4.86) (4.86) 4.4.3 X = G OG2D4 = (DσDµGAµν)(DσDρGA ρν) . (4.87) 4.3 Operators in the class X3D2 (4.80) 4.3.4 X3 = G3 JHEP05(2022)138 O(1) G3D2 = fABCGA µνDρGBµνDσGCρσ , (4.81) O(2) G3D2 = fABCGA µνDρGBρµDσGCσν , (4.82) O(3) G3D2 = fABC eGA µνDρGBµνDσGCρσ , (4.83) O(4) G3D2 = fABC eGA µνDρGBρµDσGCσν , (4.84) O(1) G3D2 = fABCGA µνDρGBµνDσGCρσ , (4.81) O(2) G3D2 = fABCGA µνDρGBρµDσGCσν , (4.82) O(3) G3D2 = fABC eGA µνDρGBµνDσGCρσ , (4.83) O(4) G3D2 = fABC eGA µνDρGBρµDσGCσν , (4.84) h l X2D4 4.3 Operators in the class X3D2 In this case, there are 4 operators for each of the combinations X3 = W 2B, X3 = G2B, X3 = W 3 and X3 = G3. The (CP-conserving) W 3 and G3 operators were previously presented in ref. [38]. For the test, again, only one amplitude is needed for each combination to manifest their independence. For example: B(p1) →W +(p2)W −(p3) and B(p1) → G(p2)G(p3). 4.3.1 X3 = W 2B 4.3.1 X3 = W 2B O(1) W 2BD2 = BµνDρW IµνDσW Iρσ , (4.69) O(2) W 2BD2 = Bµν(D2W Iµρ)W Iν ρ , (4.70) O(3) W 2BD2 = eBµνDρW IµνDσW Iρσ , (4.71) O(4) W 2BD2 = eBµν(D2W Iµρ)W Iν ρ . (4.72) 4.3.2 X3 = G2B O(1) G2BD2 = BµνDρGAµνDσGAρσ , (4.73) O(2) G2BD2 = Bµν(D2GAµρ)GAν ρ , (4.74) O(3) G2BD2 = eBµνDρGAµνDσGAρσ , (4.75) O(4) G2BD2 = eBµν(D2GAµρ)GAν ρ . (4.76) 4.3.1 X = W B O(1) W 2BD2 = BµνDρW IµνDσW Iρσ , (4.69) O(2) W 2BD2 = Bµν(D2W Iµρ)W Iν ρ , (4.70) O(3) W 2BD2 = eBµνDρW IµνDσW Iρσ , (4.71) O(4) W 2BD2 = eBµν(D2W Iµρ)W Iν ρ . (4.72) 4.3.2 X3 = G2B O(1) G2BD2 = BµνDρGAµνDσGAρσ , (4.73) O(2) G2BD2 = Bµν(D2GAµρ)GAν ρ , (4.74) O(3) G2BD2 = eBµνDρGAµνDσGAρσ , (4.75) O(4) G2BD2 = eBµν(D2GAµρ)GAν ρ . (4.76) – 11 – 4.3.3 X3 = W 3 4.3.3 X3 = W 3 O(1) W 3D2 = ϵIJKW I µνDρW JµνDσW Kρσ , (4.77) O(2) W 3D2 = ϵIJKW I µνDρW JρµDσW Kσν , (4.78) O(3) W 3D2 = ϵIJK f W I µνDρW JµνDσW Kρσ , (4.79) O(4) W 3D2 = ϵIJK f W I µνDρW JρµDσW Kσν . (4.80) 4.3.4 X3 = G3 O(1) W 3D2 = ϵIJKW I µνDρW JµνDσW Kρσ , (4.77) O(2) W 3D2 = ϵIJKW I µνDρW JρµDσW Kσν , (4.78) O(3) W 3D2 = ϵIJK f W I µνDρW JµνDσW Kρσ , (4.79) O(4) W 3D2 = ϵIJK f W I µνDρW JρµDσW Kσν . (4.80) 4.3.4 X3 = G3 O(1) W 3D2 = ϵIJKW I µνDρW JµνDσW Kρσ , (4.77) O(2) W 3D2 = ϵIJKW I µνDρW JρµDσW Kσν , (4.78) O(3) W 3D2 = ϵIJK f W I µνDρW JµνDσW Kρσ , (4.79) O(4) W 3D2 = ϵIJK f W I µνDρW JρµDσW Kσν . 5 On-shell relations Tables 1, 2, 3 and 4 show all bosonic operators comprising the Green’s basis for the dimension-eight SMEFT. Those in gray are redundant on-shell. They can be reduced to the physical basis of ref. [30] by using the SM equations of motion (EOM). Ignoring fermions, the latter read simply: D2φi = µ2φi −2λ(φ†φ)φi , (5.1) ∂νBµν = g1 2 (φ†iDµφ −Dµφ†iφ) , (5.2) DνW I µν = g2 2 (φ†iDµσIφ −φ†iσIDµφ) . (5.3) (5.1) (5.2) (5.3) – 12 – The following relations are also useful: The following relations are also useful: The following relations are also useful: [Dµ, Dν]φ = −ig1 2 Bµνφ −ig2 2 σIW I µνφ , (5.4) [Dµ, Dν]W Iρλ = g2ϵIJKW J µνW Kρλ , (5.5) [Dµ, Dν]GAρλ = g3fABCGB µνGCρλ . (5.6) (5.4) (5.5) (5.6) (5.6) The operators O(5) φ4 ,O(7) φ4 ,O(9) φ4 ,O(13) φ4 , O(4) φ6 , O(2) Bφ2D4, O(2) Wφ2D4, O(5) Wφ2D4, O(5) Wφ4D2, O(5) B2φ2D2, O(7) B2φ2D2, O(10) B2φ2D2, O(12) B2φ2D2, O(15) W 2φ2D2, O(17) W 2φ2D2, O(5) G2φ2D2, O(7) G2φ2D2, O(8) G2φ2D2, O(10) G2φ2D2, O(11) G2φ2D2, O(12) G2φ2D2, O(1) G2BD2, O(2) G2BD2, O(3) G2BD2, O(4) G2BD2, O(1) G3D2, O(2) G3D2, O(3) G3D2, O(4) G3D2 and OG2D4 vanish on-shell (up to fermionic interactions). The rest shift the Wilson coeﬃ- cients of physical operators, which ﬁnally read as follows: The operators O(5) φ4 ,O(7) φ4 ,O(9) φ4 ,O(13) φ4 , O(4) φ6 , O(2) Bφ2D4, O(2) Wφ2D4, O(5) Wφ2D4, O(5) Wφ4D2, O(5) B2φ2D2, O(7) B2φ2D2, O(10) B2φ2D2, O(12) B2φ2D2, O(15) W 2φ2D2, O(17) W 2φ2D2, O(5) G2φ2D2, O(7) G2φ2D2, O(8) G2φ2D2, O(10) G2φ2D2, O(11) G2φ2D2, O(12) G2φ2D2, O(1) G2BD2, O(2) G2BD2, O(3) G2BD2, O(4) G2BD2, O(1) G3D2, O(2) G3D2, O(3) G3D2, O(4) G3D2 and OG2D4 vanish on-shell (up to fermionic interactions). 5 On-shell relations (5.43) c(4) Wφ4D2 →c(4) Wφ4D2 + c(3) W 2BD2g1g2 2 + c(4) W 2BD2g1g2 4 −c(8) W 2φ2D2g2 +c(9) W 2φ2D2g2 +c(14) WBφ2D2g1 −c(15) WBφ2D2g1 − c(18) WBφ2D2g1 2 , (5.41) (5.41) 2 c(1) Bφ4D2 →c(1) Bφ4D2 +cB2D4g3 1 −c(6) B2φ2D2g1 +c(8) B2φ2D2g1 − c(3) Bφ2D4g2 1 2 −3cW 2D4g1g2 2 + 3c(11) WBφ2D2g2 2 +3c(13) WBφ2D2g2 + 3c(3) Wφ2D4g1g2 2 , (5. c(1) Bφ4D2 →c(1) Bφ4D2 +cB2D4g3 1 −c(6) B2φ2D2g1 +c(8) B2φ2D2g1 − c(3) Bφ2D4g2 1 2 −3cW 2D4g1g2 2 (11) (3) c(1) Bφ4D2 →c(1) Bφ4D2 +cB2D4g3 1 −c(6) B2φ2D2g1 +c(8) B2φ2D2g1 − c(3) Bφ2D4g2 1 2 −3cW 2D4g1g2 2 + 3c(11) WBφ2D2g2 2 +3c(13) WBφ2D2g2 + 3c(3) Wφ2D4g1g2 2 , (5.42) c(2) Bφ4D2 →c(2) Bφ4D2 +c(11) B2φ2D2g1 −3c(19) WBφ2D2g2 −3c(17) WBφ2D2g2 . (5.43) (5.42) (5.43) JHEP05(2022)138 Wilson coeﬃcients absent in the equations above are not modiﬁed by redundant interac- tions. This concludes the reduction of the redundant operators to the physical basis. One more important remark is in order, though. The physical operators also get corrections from the removal of redundant dimension-six interactions that, due to our focus on the dimension-eight sector, we do not specify. Eliminating these operators by applying the dimension-six SMEFT EOM is only valid up to linear order in the redundant Wilson co- eﬃcients [39]. For calculating the RGEs at one-loop accuracy, one can therefore apply directly the EOMs as the neglected quadratic eﬀects are formally two-loop corrections [37]; however, if the redudant contributions arise at tree-level — which can happen in the cal- culation of the matching conditions of a UV theory onto the SMEFT — the dimension-six redundant operators must be removed by proper ﬁeld redeﬁnitions. This exercise will be addressed elsewhere. Furthermore, let us note that due to the µ proportional term in the Higgs EOM, eq. (5.1), the removal of the redundant dimension-eight operators also results in a shift of the dimension-six physical Wilson coeﬃcients. While not written in the manuscript, we also provide these contributions (in addition to the ones presented in the paper) in the Warsaw basis [17] in an auxiliary notebook in the supplementary material. 5 On-shell relations The rest shift the Wilson coeﬃ- cients of physical operators, which ﬁnally read as follows: JHEP05(2022)138 cφ8 →cφ8 −1 2cB2D4g2 1g2 2λ+ 1 2c(8) B2φ2D2g2 1λ+2c(1) Bφ2D4g1λ2 + 1 4c(3) Bφ2D4g1g2 2λ −2c(3) Bφ2D4g1λ2 +c(3) Bφ4D2g1λ+4c(10) φ4 λ2 +4c(11) φ4 λ2 −4c(12) φ4 λ2 +8c(8) φ4 λ2 −cW 2D4g2 1g2 2λ−1 2cW 2D4g4 2λ+ 1 2c(11) W 2φ2D2g2 2λ−1 2c(13) W 2φ2D2g2 2λ−c(19) W 2φ2D2g2 2λ −c(1) W 3D2g3 2λ+ 1 2c(2) W 3D2g3 2λ−1 2c(10) WBφ2D2g1g2λ+ c(11) WBφ2D2g1g2λ 4 +c(13) WBφ2D2g1g2λ +2c(1) Wφ2D4g2λ2 + 1 2c(3) Wφ2D4g2 1g2λ−2c(3) Wφ2D4g2λ2 +c(6) Wφ4D2g2λ+ c(7) Wφ4D2g2λ 2 −4c(3) φ6 λ−cφ2 g2 1λ2 +g2 2λ2 +32λ3 , (5.7) (5.7) c(1) φ6 →c(1) φ6 +cB2D4g2 1g2 2 − 3c(8) B2φ2D2g2 1 4 −3c(1) Bφ2D4g1λ−1 2c(3) Bφ2D4g1g2 2 +3c(3) Bφ2D4g1λ − 3c(3) Bφ4D2g1 2 + 3 2cφ2g2 1λ+ 5 2cφ2g2 2λ+8cφ2λ2 +4c(12) φ4 λ−4c(4) φ4 λ−2c(6) φ4 λ + 3 2cW 2D4g2 1g2 2 + 5cW 2D4g4 2 4 − 5c(11) W 2φ2D2g2 2 4 + 5c(13) W 2φ2D2g2 2 4 + 5c(19) W 2φ2D2g2 2 2 + 5c(1) W 3D2g3 2 2 −5c(2) W 3D2g3 2 4 + 3c(10) WBφ2D2g1g2 4 − c(11) WBφ2D2g1g2 4 − 3c(13) WBφ2D2g1g2 2 + c(8) WBφ2D2g1g2 4 −5c(1) Wφ2D4g2λ−3 4c(3) Wφ2D4g2 1g2 +5c(3) Wφ2D4g2λ− 5c(6) Wφ4D2g2 2 −c(7) Wφ4D2g2 , (5.8) (5.8) c(2) φ6 →c(2) φ6 + 1 4cB2D4g2 1g2 2 − c(8) B2φ2D2g2 1 2 −2c(1) Bφ2D4g1λ−1 8c(3) Bφ2D4g1g2 2 +2c(3) Bφ2D4g1λ −c(3) Bφ4D2g1 +cφ2g2 1λ+2c(12) φ4 λ−2c(6) φ4 λ+cW 2D4g2 1g2 2 + c(10) WBφ2D2g1g2 2 − c(8) WBφ2D2g1g2 4 − 3c(11) WBφ2D2g1g2 8 −c(13) WBφ2D2g1g2 −1 2c(3) Wφ2D4g2 1g2 c(7) Wφ4D2g2 (5 9) (5.9) – 13 – c(1) φ4 →c(1) φ4 +cB2D4g2 1 −c(3) Bφ2D4g1 −cW 2D4g2 2 +c(3) Wφ2D4g2 , (5.10) c(2) φ4 →c(2) φ4 −cB2D4g2 1 +c(3) Bφ2D4g1 −cW 2D4g2 2 +c(3) Wφ2D4g2 , (5.11) c(3) φ4 →c(3) φ4 +2cW 2D4g2 2 −2c(3) Wφ2D4g2 , (5.12) c(1) G3φ2 →c(1) G3φ2 +g3c(6) G2φ2D2 , (5.13) c(1) W 3φ2 →c(1) W 3φ2 −c(1) W 3D2g2 2 2 , (5.14) c(2) W 3φ2 →c(2) W 3φ2 −c(3) W 3D2g2 2 2 , (5.15) c(1) W 2Bφ2 →c(1) W 2Bφ2 −c(1) W 3D2g1g2 2 + c(11) WBφ2D2g2 2 +c(13) WBφ2D2g2 , (5.16) c(2) W 2Bφ2 →c(2) W 2Bφ2 + c(3) W 3D2g1g2 4 + c(19) WBφ2D2g2 2 , (5.17) c(1) G2φ4 →c(1) G2φ4 +λc(4) G2φ2D2 , (5.18) c(2) G2φ4 →c(2) G2φ4 −4λc(9) G2φ2D2 , (5.19) c(1) W 2φ4 →c(1) W 2φ4 −1 8cB2D4g2 1g2 2 + 1 16c(3) Bφ2D4g1g2 2 −cW 2D4g4 2 8 + c(11) W 2φ2D2g2 2 4 +2c(14) W 2φ2D2λ− c(19) W 2φ2D2g2 2 2 −c(1) W 3D2g3 2 2 + c(2) W 3D2g3 2 4 − c(11) WBφ2D2g1g2 16 − c(8) WBφ2D2g1g2 8 + c(7) Wφ4D2g2 8 −1 2cφ2g2 2λ, (5.20) c(2) W 2φ4 →c(2) W 2φ4 + c(10) W 2φ2D2g2 2 4 +2c(16) W 2φ2D2λ− c(7) W 2φ2D2g2 2 2 + c(3) W 3D2g3 2 2 + c(4) W 3D2g3 2 4 + c(16) WBφ2D2g1g2 4 + c(19) WBφ2D2g1g2 16 , (5.21) c(3) W 2φ4 →c(3) W 2φ4 + 1 16c(3) Bφ2D4g1g2 2 + c(7) Wφ4D2g2 8 + c(8) WBφ2D2g1g2 8 + c(11) WBφ2D2g1g2 16 , (5.22) c(4) W 2φ4 →c(4) W 2φ4 − c(16) WBφ2D2g1g2 4 − c(19) WBφ2D2g1g2 16 , (5.23) c(1) WBφ4 →c(1) WBφ4 − c(6) B2φ2D2g1g2 4 + c(8) B2φ2D2g1g2 4 + 1 8c(3) Bφ2D4g2 1g2 −1 2cW 2D4g1g3 2 + c(11) W 2φ2D2g1g2 4 − c(19) W 2φ2D2g1g2 2 −1 2c(1) W 3D2g1g2 2 + 1 4c(2) W 3D2g1g2 2 + c(11) WBφ2D2g2 2 8 +c(11) WBφ2D2λ+ c(13) WBφ2D2g2 2 4 + 1 4c(3) Wφ2D4g1g2 2 + c(7) Wφ4D2g1 4 −cφ2g1g2λ, (5.24) c(1) φ4 →c(1) φ4 +cB2D4g2 1 −c(3) Bφ2D4g1 −cW 2D4g2 2 +c(3) Wφ2D4g2 , (5.10) c(2) φ4 →c(2) φ4 −cB2D4g2 1 +c(3) Bφ2D4g1 −cW 2D4g2 2 +c(3) Wφ2D4g2 , (5.11) c(3) φ4 →c(3) φ4 +2cW 2D4g2 2 −2c(3) Wφ2D4g2 , (5.12) c(1) G3φ2 →c(1) G3φ2 +g3c(6) G2φ2D2 , (5.13) c(1) W 3φ2 →c(1) W 3φ2 −c(1) W 3D2g2 2 2 , (5.14) c(2) W 3φ2 →c(2) W 3φ2 −c(3) W 3D2g2 2 , (5.15) φ φ W D g2 Wφ D g ( ) c(1) G3φ2 →c(1) G3φ2 +g3c(6) G2φ2D2 , (5.13) c(1) W 3φ2 →c(1) W 3φ2 −c(1) W 3D2g2 2 2 , (5.14) c(2) W 3φ2 →c(2) W 3φ2 −c(3) W 3D2g2 2 2 , (5.15) c(1) W 2Bφ2 →c(1) W 2Bφ2 −c(1) W 3D2g1g2 2 + c(11) WBφ2D2g2 2 +c(13) WBφ2D2g2 , (5.16) c(2) W 2Bφ2 →c(2) W 2Bφ2 + c(3) W 3D2g1g2 4 + c(19) WBφ2D2g2 2 , (5.17) c(1) G2φ4 →c(1) G2φ4 +λc(4) G2φ2D2 , (5.18) c(2) G2φ4 →c(2) G2φ4 −4λc(9) G2φ2D2 , (5.19) c(1) W 2φ4 →c(1) W 2φ4 −1 8cB2D4g2 1g2 2 + 1 16c(3) Bφ2D4g1g2 2 −cW 2D4g4 2 8 + c(11) W 2φ2D2g2 2 4 +2c(14) W 2φ2D2λ− c(19) W 2φ2D2g2 2 2 −c(1) W 3D2g3 2 2 + c(2) W 3D2g3 2 4 − c(11) WBφ2D2g1g2 16 c(8) g1g2 c(7) g2 1 JHEP05(2022)138 (5.20) (5.21) (5.24) – 14 – c(2) WBφ4 →c(2) WBφ4 + c(11) B2φ2D2g1g2 4 + c(10) W 2φ2D2g1g2 4 − c(7) W 2φ2D2g1g2 2 + 1 2c(3) W 3D2g1g2 2 + 1 4c(4) W 3D2g1g2 2 − c(19) WBφ2D2g2 2 4 +c(19) WBφ2D2λ− c(17) WBφ2D2g2 2 4 , (5.25) c(1) B2φ4 →c(1) B2φ4 + cB2D4g4 1 8 +c(4) B2φ2D2λ− c(6) B2φ2D2g2 1 4 + c(8) B2φ2D2g2 1 4 −3 8cW 2D4g2 1g2 2 + c(11) WBφ2D2g1g2 8 + c(13) WBφ2D2g1g2 4 + 1 4c(3) Wφ2D4g2 1g2 −1 2cφ2g2 1λ, (5.26) c(2) B2φ4 →c(2) B2φ4 + c(11) B2φ2D2g2 1 4 −4c(9) B2φ2D2λ− c(19) WBφ2D2g1g2 4 − c(17) WBφ2D2g1g2 4 , (5.27) c(2) G2φ2D2 →c(2) G2φ2D2 −1 2c(4) G2φ2D2 , (5.28) c(2) W 2φ2D2 →c(2) W 2φ2D2 −c(14) W 2φ2D2 , (5.29) c(3) W 2φ2D2 →c(3) W 2φ2D2 −c(16) W 2φ2D2 , (5.30) c(4) W 2φ2D2 →c(4) W 2φ2D2 −2c(1) W 3D2g2 , (5.31) c(6) W 2φ2D2 →c(6) W 2φ2D2 −c(3) W 3D2g2 , (5.32) c(1) WBφ2D2 →c(1) WBφ2D2 − c(11) WBφ2D2 2 , (5.33) c(2) WBφ2D2 →c(2) WBφ2D2 − c(19) WBφ2D2 2 , (5.34) c(3) WBφ2D2 →c(3) WBφ2D2 −c(1) W 2BD2g2 +c(2) W 2BD2g2 +c(12) WBφ2D2 +2c(7) WBφ2D2 , (5.35) c(5) WBφ2D2 →c(5) WBφ2D2 −c(3) W 2BD2g2 + 3c(4) W 2BD2g2 2 +2c(15) WBφ2D2 +c(18) WBφ2D2 , (5.36) c(2) B2φ2D2 →c(2) B2φ2D2 − c(4) B2φ2D2 2 , (5.37) c(1) Wφ4D2 →c(1) Wφ4D2 −cB2D4g2 1g2 + c(3) Bφ2D4g1g2 2 −cW 2D4g3 2 +c(11) W 2φ2D2g2 −4c(19) W 2φ2D2g2 −4c(1) W 3D2g2 2 +2c(2) W 3D2g2 2 − c(11) WBφ2D2g1 2 −c(8) WBφ2D2g1 − c(3) Wφ2D4g2 2 2 +2c(7) Wφ4D2 , (5.38) c(2) Wφ4D2 →c(2) Wφ4D2 +c(10) W 2φ2D2g2 −4c(7) W 2φ2D2g2 +4c(3) W 3D2g2 2 +2c(4) W 3D2g2 2 +2c(16) WBφ2D2g1 + c(19) WBφ2D2g1 2 , (5.39) c(3) Wφ4D2 →c(3) Wφ4D2 + c(1) W 2BD2g1g2 2 +c(12) W 2φ2D2g2 +c(18) W 2φ2D2g2 − c(12) WBφ2D2g1 2 −c(7) WBφ2D2g1 −c(9) WBφ2D2g1 , (5.40) (5.25) JHEP05(2022)138 c(1) Wφ4D2 →c(1) Wφ4D2 −cB2D4g2 1g2 + c(3) Bφ2D4g1g2 2 −cW 2D4g3 2 +c(11) W 2φ2D2g2 −4c(19) W 2φ2D2g2 −4c(1) W 3D2g2 2 +2c(2) W 3D2g2 2 − c(11) WBφ2D2g1 2 −c(8) WBφ2D2g1 − c(3) Wφ2D4g2 2 2 +2c(7) Wφ4D2 , (5.38) c(2) Wφ4D2 →c(2) Wφ4D2 +c(10) W 2φ2D2g2 −4c(7) W 2φ2D2g2 +4c(3) W 3D2g2 2 +2c(4) W 3D2g2 2 +2c(16) WBφ2D2g1 + c(19) WBφ2D2g1 2 , (5.39) c(3) Wφ4D2 →c(3) Wφ4D2 + c(1) W 2BD2g1g2 2 +c(12) W 2φ2D2g2 +c(18) W 2φ2D2g2 − c(12) WBφ2D2g1 2 −c(7) WBφ2D2g1 −c(9) WBφ2D2g1 , (5.40) (5.38) (5.38) + Wφ4D2 , (5 38) c(2) Wφ4D2 →c(2) Wφ4D2 +c(10) W 2φ2D2g2 −4c(7) W 2φ2D2g2 +4c(3) W 3D2g2 2 +2c(4) W 3D2g2 2 +2c(16) WBφ2D2g1 + c(19) WBφ2D2g1 2 , (5.39) c(3) Wφ4D2 →c(3) Wφ4D2 + c(1) W 2BD2g1g2 2 +c(12) W 2φ2D2g2 +c(18) W 2φ2D2g2 − c(12) WBφ2D2g1 2 −c(7) WBφ2D2g1 −c(9) WBφ2D2g1 , (5.40) (5.39) (5.40) (5.40) −c(9) WBφ2D2g1 , (5.40) – 15 – c(4) Wφ4D2 →c(4) Wφ4D2 + c(3) W 2BD2g1g2 2 + c(4) W 2BD2g1g2 4 −c(8) W 2φ2D2g2 +c(9) W 2φ2D2g2 +c(14) WBφ2D2g1 −c(15) WBφ2D2g1 − c(18) WBφ2D2g1 2 , (5.41) c(1) Bφ4D2 →c(1) Bφ4D2 +cB2D4g3 1 −c(6) B2φ2D2g1 +c(8) B2φ2D2g1 − c(3) Bφ2D4g2 1 2 −3cW 2D4g1g2 2 + 3c(11) WBφ2D2g2 2 +3c(13) WBφ2D2g2 + 3c(3) Wφ2D4g1g2 2 , (5.42) c(2) Bφ4D2 →c(2) Bφ4D2 +c(11) B2φ2D2g1 −3c(19) WBφ2D2g2 −3c(17) WBφ2D2g2 . 6.1 Integrating out a scalar singlet to one loop Let us extend the SM with a heavy singlet scalar S ∼(1, 1)0. The numbers within paren- theses and the sub-index indicate the SU(3)c and SU(2)L quantum numbers and the hy- percharge, respectively. We assume a Z2 symmetry S →−S, so that the new physics Lagrangian reads: LNP = 1 2(DµS)(DµS) −1 2m2 SS2 −λSφS2φ†φ −λSS4 . (6.1) (6.1) Because of the Z2 symmetry, all eﬀective operators arise ﬁrst at one loop. Hence, the contribution from redundant dimension-six interactions to the dimension-eight terms upon ﬁeld redeﬁnitions are formally two-loop corrections and therefore negligible within our order of calculation. Consequently, eqs. (5.7)–(5.43) are valid without any further corrections. Because of the Z2 symmetry, all eﬀective operators arise ﬁrst at one loop. Hence, the contribution from redundant dimension-six interactions to the dimension-eight terms upon ﬁeld redeﬁnitions are formally two-loop corrections and therefore negligible within our order of calculation. Consequently, eqs. (5.7)–(5.43) are valid without any further corrections. – 16 – Thus, we implement this model, together with our Green’s basis of operators (including the on-shell relations in eqs. (5.7)–(5.43)) in matchmakereft [34]. Automatically, we get the following dimension-eight Wilson coeﬃcients: c(1) φ6 Λ4 = 1 1920 m4 S π2 λ2 Sφ (5λSφ −8λ) , (6.2) c(3) φ4 Λ4 = 1 960 m4 S π2 λ2 Sφ . (6.3) (6.2) (6.3) For simplicity, we have taken the limit g2 →0. (Also, we have not computed the Wilson coeﬃcient of Oφ8.) To the best of our knowledge, this is the ﬁrst one-loop computation of the matching of a scalar singlet onto the bosonic SMEFT to dimension-eight. JHEP05(2022)138 For simplicity, we have taken the limit g2 →0. (Also, we have not computed the Wilson coeﬃcient of Oφ8.) To the best of our knowledge, this is the ﬁrst one-loop computation of the matching of a scalar singlet onto the bosonic SMEFT to dimension-eight. 6.2 Integrating out a scalar quadruplet to one loop We are also in agreement with the (loop produced) c(1) B2 and c(2) B2 previously reported in refs. [38, 40]. 6.2 Integrating out a scalar quadruplet to one loop In this case, we consider the SM extended with a scalar SU(2)L quadruplet with Y = 1/2 and mass mΘ. We name it as Θ. The relevant new physics Lagrangian is: LNP = DµΘ†DµΘ −m2 ΘΘ†Θ −λΘ(φ†σIφ)Cα Iβ ˜φβϵαγΘγ + h.c. . (6.4) (6.4) (For simplicity, we are ignoring other quartic terms.) The Cα Iβ symbol represents the Clebsh-Gordan needed to single out the SU(2)L singlet from the contraction of a quadru- plet, a doublet and a triplet. At tree level and dimension six, only the operator (φ†φ)3 is generated. (Therefore, once more, indirect contributions to the dimension-eight Wilson coeﬃcients can be obtained simply from eqs. (5.7)–(5.43).) At dimension eight, and again in the limit g2 →0, we obtain: c(1) B4 Λ4 = 7g4 1 92160 m4 Θ π2 , (6.5) c(2) B4 Λ4 = g4 1 92160 m4 Θ π2 , (6.6) c(1) φ6 Λ4 = \|λΘ\|2 3 m2 Θ + −6440 g2 1 \|λΘ\|2 + 103040 \|λΘ\|2λ 80640 m4 Θ π2 , (6.7) c(2) φ6 \|λΘ\|2 + +3640 g2 1\|λΘ\|2 −655200 \|λΘ\|2 λ (6 8) c(1) B4 Λ4 = 7g4 1 92160 m4 Θ π2 , (6.5) c(2) B4 Λ4 = g4 1 92160 m4 Θ π2 , (6.6) c(1) φ6 Λ4 = \|λΘ\|2 3 m2 Θ + −6440 g2 1 \|λΘ\|2 + 103040 \|λΘ\|2λ 80640 m4 Θ π2 , (6.7) c(2) φ6 Λ4 = −\|λΘ\|2 2 m2 Θ + +3640 g2 1\|λΘ\|2 −655200 \|λΘ\|2 λ 483840 m4 Θ π2 , (6.8) c(1) φ4 Λ4 = 4480 \|λΘ\|2 −3g4 1 40320 m4 Θ π2 , (6.9) c(2) φ4 Λ4 = 3g4 1 + 1120 \|λΘ\|2 40320 m4 Θ π2 , (6.10) c(3) φ4 Λ4 = − \|λΘ\|2 18 m4 Θ π2 , (6.11) (6.5) (6.6) – 17 – c(1) B2φ4 Λ4 = 1960 g2 1\|λΘ\|2 −3g6 1 322560 m4 Θ π2 , (6.12) c(1) Bφ4D2 Λ4 = − g5 1 13440 m4 Θ π2 . (6.13) (6.12) (6.13) The tree-level contribution to c(1) φ6 and c(2) φ6 had been previously computed in ref. [30], and we agree with the result therein. We are also in agreement with the (loop produced) c(1) B2 and c(2) B2 previously reported in refs. [38, 40]. The tree-level contribution to c(1) φ6 and c(2) φ6 had been previously computed in ref. [30], and we agree with the result therein. 6.3 Reduction of Lagrangian to a physical basis JHEP05(2022)138 Functional methods comprise a very powerful tool to match UV models onto EFTs, by literally integrating over the heavy dynamical ﬁelds in the path integral [3–5]. The advan- tage of this approach with respect to matching 1PI amplitudes in Feynman diagrams is that no basis of EFT operators (neither Green’s nor physical) must be known in advance to complete the calculation. The drawback, though, is that the resulting EFT Lagrangian is highly redundant, in- volving operators related by EOM, integration by parts and algebraic identities. Operators can be even connected by a re-labeling of dummy indices. (However simple this might look, they can be hard to diﬀerentiate in a more or less automatic fashion.) With the help of SuperTracer [41], we have checked the monstruosity of the EFT Lagrangian resulting from integrating out heavy ﬁelds to one loop in elaborated models. However, the cumbersone mixture of EFT interactions can be already appreciated in simple models and even at tree level. Let us consider, for example, an extension of the SM with a heavy real vector triplet W ∼(1, 3)0, with Lagrangian: LNP = 1 2 DµW† νDνWµ −DµW† νDµWν +m2 WW† µWµ +(gφ WWµφI†σIiDµφ+h.c.) . (6.1 At tree level, the eﬀective action is given by the UV action evaluated on W = Wc, namely the classical conﬁguration that solves the W EOM. We compute this to order 1/m4 W using MatchingTools [42], obtaining: L(8) EFT= (gφ W)2 m4 W 2(Dµφ†Dνφ)(Dµφ†Dνφ)+4(Dνφ†DνDµφ)(Dµφ†φ)−2(Dµφ†Dνφ)(φ†DµDνφ) −4(Dµφ†φ)(DµDνφ†Dνφ)+2(Dµφ†Dνφ)(DµDνφ†φ)−4(Dµφ†Dµφ)(Dνφ†Dνφ) +2(Dµφ†Dνφ)(Dνφ†Dµφ)+1 2(φ†DµDνφ)(φ†DµDνφ)−2(DνDρφ†DνDρφ)(φ†φ) +(DµDνφ†φ)(φ†DµDνφ)−4(φ†Dρφ)(Dνφ†DρDνφ)+2(φ†DνDµφ)(Dµφ†Dνφ) +1 2(DµDνφ†φ)(DµDνφ†φ)+4(DρDνφ†Dρφ)(Dνφ†φ)−2(DνDµφ†φ)(Dµφ†Dνφ) −1 2(φ†DνDµφ)(φ†DµDνφ)+2(DρDνφ†DνDρφ)(φ†φ)−(DνDµφ†φ)(φ†DµDνφ) −1 2(DνDµφ†φ)(DµDνφ†φ) . L(8) EFT= (gφ W)2 m4 W 2(Dµφ†Dνφ)(Dµφ†Dνφ)+4(Dνφ†DνDµφ)(Dµφ†φ)−2(Dµφ†Dνφ)(φ†DµDνφ) −4(Dµφ†φ)(DµDνφ†Dνφ)+2(Dµφ†Dνφ)(DµDνφ†φ)−4(Dµφ†Dµφ)(Dνφ†Dνφ) +2(Dµφ†Dνφ)(Dνφ†Dµφ)+1 2(φ†DµDνφ)(φ†DµDνφ)−2(DνDρφ†DνDρφ)(φ†φ) +(DµDνφ†φ)(φ†DµDνφ)−4(φ†Dρφ)(Dνφ†DρDνφ)+2(φ†DνDµφ)(Dµφ†Dνφ) +1 2(DµDνφ†φ)(DµDνφ†φ)+4(DρDνφ†Dρφ)(Dνφ†φ)−2(DνDµφ†φ)(Dµφ†Dνφ) −1 2(φ†DνDµφ)(φ†DµDνφ)+2(DρDνφ†DνDρφ)(φ†φ)−(DνDµφ†φ)(φ†DµDνφ) −1 2(DνDµφ†φ)(DµDνφ†φ) . – 18 – (The dimension-six piece can be checked in ref. [39, 43], from where we borrow notation.) We reproduce precisely the diﬀerent dummy indices resulting from the automatic calcu- lation. It is apparent that this Lagrangian can not easily (at least not immediately) be reduced to a physical basis by hand. 6.3 Reduction of Lagrangian to a physical basis ( ) However, one could simply (and automatically) export L(8) EFT to FeynArts with the help of FeynRules, compute the relevant 1PI tree-level oﬀ-shell amplitudes and project the results onto our basis.4 Proceeding this way, we obtain: L(8) EFT = (gφ W)2 m4 W 2O(1) φ4 + 2O(2) φ4 −4O(3) φ4 −1 4g2 2O(1) W 2φ4 + 1 2g1g2O(1) WBφ4 + 3 4g2 1O(1) B2φ4 −2g2O(1) Wφ4D2 + 6g1O(1) Bφ4D2 + 2g1O(3) Bφ4D2 . (6.15) JHEP05(2022)138 (6.15) Thus, even matching computations performed using functional methods can beneﬁt from knowing a basis of independent Green’s functions. Thus, even matching computations performed using functional methods can beneﬁt from knowing a basis of independent Green’s functions. 4Matchmakereft includes also a single instruction to perform this action automatically. 7 Conclusions and future directions (A more mundane albeit technically important property of our basis is that all the eﬀective operators in there can be exported to FeynArts using FeynRules, which is not always the case.) This simpliﬁes notably the calculation, because these operators generate amplitudes with three polarization vectors and ﬁve diﬀerent momenta, from which three vectors must be therefore projected onto the remaining four independent ones. (A more mundane albeit technically important property of our basis is that all the eﬀective operators in there can be exported to FeynArts using FeynRules, which is not always the case.) Renormalising the bosonic sector of the SMEFT to dimension eight (thus concluding the eﬀort initiated in ref. [37]) is in fact an avenue we have already started to explore on the basis of this work. Another future direction of our current work includes classifying all independent bosonic evanescent operators (and projecting them onto the physical basis in four dimensions). This will simplify the matching of UV models onto the SMEFT using tools based on diagrammatic calculations, since the Levi-Civita symbol can be assumed to be simply a totally anti-symmetric tensor without further structure inherited from the four-dimensional space-time. (In fact, for performing the matching in section 6.2, we augmented the basis with the operator O = BµνBνρBρσBσµ, which in D = 4 fullﬁlls O = 1 2O(1) B4 + 1 4O(2) B4.) JHEP05(2022)138 Connected to this, one more avenue that we aim to pursue in the near future is using matchmakereft to analyse positivity bounds on SMEFT X2φ2D2 operators (ﬁrst presented in ref. [46]) in models in which low-momentum 2 →2 amplitudes are not necessarily well approximated by the EFT at tree level. Such models involve in general heavy ﬁelds with linear interactions, which can be integrated out only if loops involving heavy-light particles are calculated. To the best of our knowledge, other tools for matching, such as Codex [47], do not include yet these loops, particularly for dimension-eight computations. The computation of the ﬁeld redeﬁnitions needed to remove the redundant operators from a dimension-six Green’s basis and its impact on the physical Wilson coeﬃcients at dimension-eight is also a future path to follow. 7 Conclusions and future directions Oﬀ-shell calculations are common practice within eﬀective quantum ﬁeld theories. They have the advantage that they involve a substantially smaller amount of Feynman diagrams than calculations of physical (on-shell) S-matrix elements. In contrast, they require intro- ducing redundant interactions in the Lagrangian, including nonphysical terms that vanish under ﬁeld redeﬁnitions. Concentrating on the SMEFT, it would then be desirable to have a complete set of operators independent oﬀshell, so neither related by algebraic identities nor by integration by parts. Any such set of interactions is called a Green’s basis [44]; a particular realisation to dimension six was built in ref. [16]. In this paper, we have constructed the bosonic dimension-eight counterpart, which consists of 86 new interactions. One important aspect of our approach has been working in momentum space to estab- lish the oﬀ-shell independence of operators, thus avoiding the otherwise cumbersome oper- ations needed at the level of the Lagrangian when the interactions involve many ﬁelds and derivatives. In particular, integration by parts amounts simply to removing one momentum in 1PI amplitudes. Other relations, such as four-dimensional constraints resulting from con- tractions of the Levi-Civita symbol, are harder to enforce systematically at the level of am- plitudes, but we have shown that they can be accounted for by requiring that at most four Lorentz vectors (momenta or polarisations) are linearly independent in four dimensions. Our Green’s basis is obviously not unique. Inﬁnitely many other combinations of operators could be considered, in particular bases in which the redundant interactions are related to physical ones by EOM through simpler relations than those in eqs. (5.7)– (5.43). One advantage of the one presented here, though, is that the renormalisation of the X3φ2 operators (which will be presented elsewhere [45]) can be carried out without necessarily projecting the contractions of the Levi-Civita symbol onto four dimensions. – 19 – This simpliﬁes notably the calculation, because these operators generate amplitudes with three polarization vectors and ﬁve diﬀerent momenta, from which three vectors must be therefore projected onto the remaining four independent ones. Acknowledgments We are grateful to Jose Santiago for sharing (and for instructing us on) matchmakereft, as well as for discussions and for comments on the manuscript. We are also enormously thank- ful to Renato Fonseca for the many enlightening discussions and for help with Sym2Int. MC would also like to thank Christopher Murphy for clarifying some calculations in ref. [30]. MC and ADC are supported by SRA under grant PID2019-106087GB-C21/C22 (10.13039/501100011033). MC is also supported by the Junta de Andalucía grants FQM 101, A-FQM-211-UGR18 and P18-FR-4314 (FEDER), as well as by the Spanish MINECO under the Ramón y Cajal programme. ADC is also supported by the Spanish MINECO under the FPI programme. GG acknowledges support by LIP (FCT, COMPETE2020- Portugal2020, FEDER, POCI-01-0145-FEDER-007334) as well as by FCT under project CERN/FIS-PAR/0024/2019 and under the grant SFRH/BD/144244/2019. – 20 – A Tables of operators A Tables of operators A Tables of operators A Operator Notation Operator Notation φ8 (φ†φ)4 Oφ8 φ6D2 (φ†φ)2(Dµφ†Dµφ) O(1) φ6 (φ†φ)(φ†σIφ)(Dµφ†σIDµφ) O(2) φ6 (φ†φ)2(φ†D2φ + h.c.) O(3) φ6 (φ†φ)2Dµ(φ†i←→ D µφ) O(4) φ6 φ4D4 (Dµφ†Dνφ)(Dνφ†Dµφ) O(1) φ4 (Dµφ†Dνφ)(Dµφ†Dνφ) O(2) φ4 (Dµφ†Dµφ)(Dνφ†Dνφ) O(3) φ4 Dµφ†Dµφ(φ†D2φ + h.c.) O(4) φ4 Dµφ†Dµφ(φ†iD2φ + h.c.) O(5) φ4 (Dµφ†φ)(D2φ†Dµφ) + h.c. O(6) φ4 (Dµφ†φ)(D2φ†iDµφ) + h.c. O(7) φ4 (D2φ†φ)(D2φ†φ) + h.c. O(8) φ4 (D2φ†φ)(iD2φ†φ) + h.c. O(9) φ4 (D2φ†D2φ)(φ†φ) O(10) φ4 (φ†D2φ)(D2φ†φ) O(11) φ4 (Dµφ†φ)(Dµφ†D2φ) + h.c. O(12) φ4 (Dµφ†φ)(Dµφ†iD2φ) + h.c. O(13) φ4 X3φ2 fABC(φ†φ)GA,ν µ GB,ρ ν GC,µ ρ O(1) G3φ2 fABC(φ†φ)GA,ν µ GB,ρ ν ˜GC,µ ρ O(2) G3φ2 ϵIJK(φ†φ)W Iν µ W Jρ ν W Kµ ρ O(1) W 3φ2 ϵIJK(φ†φ)W Iν µ W Jρ ν f W Kµ ρ O(2) W 3φ2 ϵIJK(φ†σIφ)B ν µ W Jρ ν W Kµ ρ O(1) W 2Bφ2 ϵIJK(φ†σIφ)( eBµνW J νρW Kρ µ + BµνW J νρf W Kρ µ ) O(2) W 2Bφ2 X2φ4 (φ†φ)2GA µνGAµν O(1) G2φ4 (φ†φ)2 eGA µνGAµν O(2) G2φ4 (φ†φ)2W I µνW Iµν O(1) W 2φ4 (φ†φ)2f W I µνW Iµν O(2) W 2φ4 (φ†σIφ)(φ†σJφ)W I µνW Jµν O(3) W 2φ4 (φ†σIφ)(φ†σJφ)f W I µνW Jµν O(4) W 2φ4 (φ†φ)(φ†σIφ)W I µνBµν O(1) WBφ4 (φ†φ)(φ†σIφ)f W I µνBµν O(2) WBφ4 (φ†φ)2BµνBµν O(1) B2φ4 (φ†φ)2 eBµνBµν O(2) B2φ4 Xφ2D4 i(Dνφ†σID2φ −D2φ†σIDνφ)DµW Iµν O(1) Wφ2D4 (Dνφ†σID2φ + D2φ†σIDνφ)DµW Iµν O(2) Wφ2D4 i(DρDνφ†σIDρφ −Dρφ†σIDρDνφ)DµW Iµν O(3) Wφ2D4 i(Dνφ†D2φ −D2φ†Dνφ)DµBµν O(1) Bφ2D4 (Dνφ†D2φ + D2φ†Dνφ)DµBµν O(2) Bφ2D4 i(DρDνφ†Dρφ −Dρφ†DρDνφ)DµBµν O(3) Bφ2D4 Xφ4D2 i(φ†φ)(Dµφ†σIDνφ)W I µν O(1) Wφ4D2 i(φ†φ)(Dµφ†σIDνφ)f W I µν O(2) Wφ4D2 iϵIJK(φ†σIφ)(Dµφ†σJDνφ)W K µν O(3) Wφ4D2 iϵIJK(φ†σIφ)(Dµφ†σJDνφ)f W K µν O(4) Wφ4D2 (φ†φ)DνW Iµν(Dµφ†σIφ + h.c.) O(5) Wφ4D2 (φ†φ)DνW Iµν(Dµφ†iσIφ + h.c.) O(6) Wφ4D2 ϵIJK(Dµφ†σIφ)(φ†σJDνφ)W Kµν O(7) Wφ4D2 i(φ†φ)(Dµφ†Dνφ)Bµν O(1) Bφ4D2 i(φ†φ)(Dµφ†Dνφ) eBµν O(2) Bφ4D2 (φ†φ)DνBµν(Dµφ†iφ + h.c.) O(3) Bφ4D2 φ2D6 D2φ†DµDνDµDνφ+h.c. Oφ2 Table 1. Green’s basis of operators, part I. Operators in gray are redundant. JHEP05(2022)138 Table 1. Green’s basis of operators, part I. Operators in gray are redundant. – 21 – JHEP05(2022)138 Table 2. Green’s basis of operators, part II. Table 3. Green’s basis of operators, part III. A Tables of operators – 22 – Operator Notation Operator Notation X4, X3X′ (GA µνGAµν)(GB ρσGBρσ) Q(1) G4 (GA µν eGAµν)(GB ρσ eGBρσ) Q(2) G4 (GA µνGBµν)(GA ρσGBρσ) Q(3) G4 (GA µν eGBµν)(GA ρσ eGBρσ) Q(4) G4 (GA µνGAµν)(GB ρσ eGBρσ) Q(5) G4 (GA µνGBµν)(GA ρσ eGBρσ) Q(6) G4 dABEdCDE(GA µνGBµν)(GC ρσGDρσ) Q(7) G4 dABEdCDE(GA µν eGBµν)(GC ρσ eGDρσ) Q(8) G4 dABEdCDE(GA µνGBµν)(GC ρσ eGDρσ) Q(9) G4 (W I µνW Iµν)(W J ρσW Jρσ) Q(1) W 4 (W I µν f W Iµν)(W J ρσ f W Jρσ) Q(2) W 4 (W I µνW Jµν)(W I ρσW Jρσ) Q(3) W 4 (W I µν f W Jµν)(W I ρσ f W Jρσ) Q(4) W 4 (W I µνW Iµν)(W J ρσ f W Jρσ) Q(5) W 4 (W I µνW Jµν)(W I ρσ f W Jρσ) Q(6) W 4 (BµνBµν)(BρσBρσ) Q(1) B4 (Bµν eBµν)(Bρσ eBρσ) Q(2) B4 (BµνBµν)(Bρσ eBρσ) Q(3) B4 dABC(BµνGAµν)(GB ρσGCρσ) Q(1) G3B dABC(Bµν eGAµν)(GB ρσ eGCρσ) Q(2) G3B dABC(Bµν eGAµν)(GB ρσGCρσ) Q(3) G3B dABC(BµνGAµν)(GB ρσ eGCρσ) Q(4) G3B X2X′2 (W I µνW Iµν)(GA ρσGAρσ) Q(1) G2W 2 (W I µν f W Iµν)(GA ρσ eGAρσ) Q(2) G2W 2 (W I µνGAµν)(W I ρσGAρσ) Q(3) G2W 2 (W I µν eGAµν)(W I ρσ eGAρσ) Q(4) G2W 2 (W I µν f W Iµν)(GA ρσGAρσ) Q(5) G2W 2 (W I µνW Iµν)(GA ρσ eGAρσ) Q(6) G2W 2 (W I µνGAµν)(W I ρσ eGAρσ) Q(7) G2W 2 (BµνBµν)(GA ρσGAρσ) Q(1) G2B2 (Bµν eBµν)(GA ρσ eGAρσ) Q(2) G2B2 (BµνGAµν)(BρσGAρσ) Q(3) G2B2 (Bµν eGAµν)(Bρσ eGAρσ) Q(4) G2B2 (Bµν eBµν)(GA ρσGAρσ) Q(5) G2B2 (BµνBµν)(GA ρσ eGAρσ) Q(6) G2B2 (BµνGAµν)(Bρσ eGAρσ) Q(7) G2B2 (BµνBµν)(W I ρσW Iρσ) Q(1) W 2B2 (Bµν eBµν)(W I ρσ f W Iρσ) Q(2) W 2B2 (BµνW Iµν)(BρσW Iρσ) Q(3) W 2B2 (Bµν f W Iµν)(Bρσ f W Iρσ) Q(4) W 2B2 (Bµν eBµν)(W I ρσW Iρσ) Q(5) W 2B2 (BµνBµν)(W I ρσ f W Iρσ) Q(6) W 2B2 (BµνW Iµν)(Bρσ f W Iρσ) Q(7) W 2B2 Table 3. Green’s basis of operators, part III. JHEP05(2022)138 Table 3. Green’s basis of operators, part III. A Tables of operators – 23 – Operator Notation Operator Notation X3D2 BµνDρW IµνDσW Iρσ O(1) W 2BD2 Bµν(D2W Iµρ)W Iν ρ O(2) W 2BD2 eBµνDρW IµνDσW Iρσ O(3) W 2BD2 eBµν(D2W Iµρ)W Iν ρ O(4) W 2BD2 BµνDρGAµνDσGAρσ O(1) G2BD2 Bµν(D2GAµρ)GAν ρ O(2) G2BD2 eBµνDρGAµνDσGAρσ O(3) G2BD2 eBµν(D2GAµρ)GAν ρ O(4) G2BD2 ϵIJKW I µνDρW JµνDσW Kρσ O(1) W 3D2 ϵIJKW I µνDρW JρµDσW Kσν O(2) W 3D2 ϵIJK f W I µνDρW JµνDσW Kρσ O(3) W 3D2 ϵIJK f W I µνDρW JρµDσW Kσν O(4) W 3D2 fABCGA µνDρGBµνDσGCρσ O(1) G3D2 fABCGA µνDρGBρµDσGCσν O(2) G3D2 fABC eGA µνDρGBµνDσGCρσ O(3) G3D2 fABC eGA µνDρGBρµDσGCσν O(4) G3D2 X2D4 (DσDµBµν)(DσDρBρν) OB2D4 (DσDµW Iµν)(DσDρW I ρν) OW 2D4 (DσDµGAµν)(DσDρGA ρν) OG2D4 Table 4. Green’s basis of operators, part IV. JHEP05(2022)138 Table 4. Green’s basis of operators, part IV. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References Baratella, C. Fernandez, B. von Harling and A. Pomarol, Anomalous dimensions of eﬀective theories from partial waves, JHEP 03 (2021) 287 [arXiv:2010.13809] [INSPIRE]. [13] M. Jiang, T. Ma and J. Shu, Renormalization group evolution from on-shell SMEFT, JHEP 01 (2021) 101 [arXiv:2005.10261] [INSPIRE]. JHEP05(2022)138 [14] M. Accettulli Huber and S. De Angelis, Standard model EFTs via on-shell methods, JHEP 11 (2021) 221 [arXiv:2108.03669] [INSPIRE]. [15] P. Baratella, D. Haslehner, M. Ruhdorfer, J. Serra and A. Weiler, RG of GR from on-shell amplitudes, JHEP 03 (2022) 156 [arXiv:2109.06191] [INSPIRE]. [16] V. Gherardi, D. Marzocca and E. Venturini, Matching scalar leptoquarks to the SMEFT at one loop, JHEP 07 (2020) 225 [Erratum ibid. 01 (2021) 006] [arXiv:2003.12525] [INSPIRE]. [17] B. Grzadkowski, M. Iskrzynski, M. Misiak and J. Rosiek, Dimension-six terms in the standard model lagrangian, JHEP 10 (2010) 085 [arXiv:1008.4884] [INSPIRE]. [18] M. Chala and A. Titov, One-loop matching in the SMEFT extended with a sterile neutrino, JHEP 05 (2020) 139 [arXiv:2001.07732] [INSPIRE]. [19] M. Chala, G. Guedes, M. Ramos and J. Santiago, Running in the ALPs, Eur. Phys. J. C 81 (2021) 181 [arXiv:2012.09017] [INSPIRE]. [20] C. Hays, A. Martin, V. Sanz and J. Setford, On the impact of dimension-eight SMEFT operators on Higgs measurements, JHEP 02 (2019) 123 [arXiv:1808.00442] [INSPIRE]. [21] M. Chala, C. Krause and G. Nardini, Signals of the electroweak phase transition at colliders and gravitational wave observatories, JHEP 07 (2018) 062 [arXiv:1802.02168] [INSPIRE]. [22] G. Panico, A. Pomarol and M. Riembau, EFT approach to the electron electric dipole moment at the two-loop level, JHEP 04 (2019) 090 [arXiv:1810.09413] [INSPIRE]. [23] J. Ellis, S.-F. Ge, H.-J. He and R.-Q. Xiao, Probing the scale of new physics in the ZZγ coupling at e+e−colliders, Chin. Phys. C 44 (2020) 063106 [arXiv:1902.06631] [INSPIRE]. [24] S. Alioli, R. Boughezal, E. Mereghetti and F. Petriello, Novel angular dependence in Drell-Yan lepton production via dimension-8 operators, Phys. Lett. B 809 (2020) 135703 [arXiv:2003.11615] [INSPIRE]. [25] J. Gu, L.-T. Wang and C. Zhang, An unambiguous test of positivity at lepton colliders, arXiv:2011.03055 [INSPIRE]. [26] J. Ellis, H.-J. He and R.-Q. Xiao, Probing new physics in dimension-8 neutral gauge couplings at e+e−colliders, Sci. China Phys. Mech. Astron. 64 (2021) 221062 [arXiv:2008.04298] [INSPIRE]. [27] C. Hays, A. Helset, A. Martin and M. Trott, Exact SMEFT formulation and expansion to O(v4/Λ4), JHEP 11 (2020) 087 [arXiv:2007.00565] [INSPIRE]. [28] T. Corbett, A. Helset, A. Martin and M. References [1] J.S.R. Chisholm, Change of variables in quantum ﬁeld theories, Nucl. Phys. 26 (1961) 469 [INSPIRE]. [1] J.S.R. Chisholm, Change of variables in quantum ﬁeld theories, Nucl. Phys. 26 (1961) 469 [INSPIRE]. [1] J.S.R. Chisholm, Change of variables in quantum ﬁeld theories, Nucl. Phys. 26 (1961) 469 [INSPIRE]. [2] S. Kamefuchi, L. O’Raifeartaigh and A. Salam, Change of variables and equivalence theorems in quantum ﬁeld theories, Nucl. Phys. 28 (1961) 529 [INSPIRE]. [3] M.K. Gaillard, The eﬀective one loop Lagrangian with derivative couplings, Nucl. Phys. B 268 (1986) 669 [INSPIRE]. [3] M.K. Gaillard, The eﬀective one loop Lagrangian with derivative couplings, Nucl. Phys. B 268 (1986) 669 [INSPIRE]. [4] O. Cheyette, Eﬀective action for the standard model with large Higgs mass, Nucl. Phys. B 297 (1988) 183 [INSPIRE]. [4] O. Cheyette, Eﬀective action for the standard model with large Higgs mass, Nucl. Phys. B 297 (1988) 183 [INSPIRE]. [5] B. Henning, X. Lu and H. Murayama, How to use the standard model eﬀective ﬁeld theory, JHEP 01 (2016) 023 [arXiv:1412.1837] [INSPIRE]. [5] B. Henning, X. Lu and H. Murayama, How to use the standard model eﬀective ﬁeld theory, JHEP 01 (2016) 023 [arXiv:1412.1837] [INSPIRE]. [6] S. Caron-Huot and M. Wilhelm, Renormalization group coeﬃcients and the S-matrix, JHEP 12 (2016) 010 [arXiv:1607.06448] [INSPIRE]. [6] S. Caron-Huot and M. Wilhelm, Renormalization group coeﬃcients and the S-matrix, JHEP 12 (2016) 010 [arXiv:1607.06448] [INSPIRE]. [7] Z. Bern, J. Parra-Martinez and E. Sawyer, Nonrenormalization and operator mixing via on-shell methods, Phys. Rev. Lett. 124 (2020) 051601 [arXiv:1910.05831] [INSPIRE]. [7] Z. Bern, J. Parra-Martinez and E. Sawyer, Nonrenormalization and operator mixing via on-shell methods, Phys. Rev. Lett. 124 (2020) 051601 [arXiv:1910.05831] [INSPIRE]. [8] G. Yang, On-shell methods for form factors in N = 4 SYM and their applications, Sci. China Phys. Mech. Astron. 63 (2020) 270001 [arXiv:1912.11454] [INSPIRE]. [8] G. Yang, On-shell methods for form factors in N = 4 SYM and their applications, Sci. China Phys. Mech. Astron. 63 (2020) 270001 [arXiv:1912.11454] [INSPIRE]. – 24 – [9] P. Baratella, C. Fernandez and A. Pomarol, Renormalization of higher-dimensional operators from on-shell amplitudes, Nucl. Phys. B 959 (2020) 115155 [arXiv:2005.07129] [INSPIRE]. [10] Z. Bern, J. Parra-Martinez and E. Sawyer, Structure of two-loop SMEFT anomalous dimensions via on-shell methods, JHEP 10 (2020) 211 [arXiv:2005.12917] [INSPIRE]. [11] J. Elias Miró, J. Ingoldby and M. Riembau, EFT anomalous dimensions from the S-matrix, JHEP 09 (2020) 163 [arXiv:2005.06983] [INSPIRE]. [12] P. References Trott, EWPD in the SMEFT to dimension eight, JHEP 06 (2021) 076 [arXiv:2102.02819] [INSPIRE]. [29] M. Ardu and S. Davidson, What is leading order for LFV in SMEFT?, JHEP 08 (2021) 002 [arXiv:2103.07212] [INSPIRE]. – 25 – [30] C.W. Murphy, Dimension-8 operators in the standard model eﬀective ﬁeld theory, JHEP 10 (2020) 174 [arXiv:2005.00059] [INSPIRE]. [31] H.-L. Li, Z. Ren, J. Shu, M.-L. Xiao, J.-H. Yu and Y.-H. Zheng, Complete set of dimension-eight operators in the standard model eﬀective ﬁeld theory, Phys. Rev. D 104 (2021) 015026 [arXiv:2005.00008] [INSPIRE]. [32] J.C. Criado, BasisGen: automatic generation of operator bases, Eur. Phys. J. C 79 (2019) 256 [arXiv:1901.03501] [INSPIRE]. [33] R.M. Fonseca, Enumerating the operators of an eﬀective ﬁeld theory, Phys. Rev. D 101 (2020) 035040 [arXiv:1907.12584] [INSPIRE]. [34] A. Carmona, A. Lazopoulos, P. Olgoso and J. Santiago, Matchmakereft: automated tree-level and one-loop matching, arXiv:2112.10787 [INSPIRE]. JHEP05(2022)138 [35] A. Alloul, N.D. Christensen, C. Degrande, C. Duhr and B. Fuks, FeynRules 2.0 — a complete toolbox for tree-level phenomenology, Comput. Phys. Commun. 185 (2014) 2250 [arXiv:1310.1921] [INSPIRE]. [36] T. Hahn and M. Pérez-Victoria, Automatized one loop calculations in four-dimensions and D-dimensions, Comput. Phys. Commun. 118 (1999) 153 [hep-ph/9807565] [INSPIRE]. [37] M. Chala, G. Guedes, M. Ramos and J. Santiago, Towards the renormalisation of the standard model eﬀective ﬁeld theory to dimension eight: bosonic interactions I, SciPost Phys. 11 (2021) 065 [arXiv:2106.05291] [INSPIRE]. [37] M. Chala, G. Guedes, M. Ramos and J. Santiago, Towards the renormalisation of the standard model eﬀective ﬁeld theory to dimension eight: bosonic interactions I, SciPost Phys. 11 (2021) 065 [arXiv:2106.05291] [INSPIRE]. [38] J. Quevillon, C. Smith and S. Touati, Eﬀective action for gauge bosons, Phys. Rev. D 99 (2019) 013003 [arXiv:1810.06994] [INSPIRE]. [38] J. Quevillon, C. Smith and S. Touati, Eﬀective action for gauge bosons, Phys. Rev. D 99 (2019) 013003 [arXiv:1810.06994] [INSPIRE]. [39] J.C. Criado and M. Pérez-Victoria, Field redeﬁnitions in eﬀective theories at higher orders, JHEP 03 (2019) 038 [arXiv:1811.09413] [INSPIRE]. [39] J.C. Criado and M. Pérez-Victoria, Field redeﬁnitions in eﬀective theories at higher orders, JHEP 03 (2019) 038 [arXiv:1811.09413] [INSPIRE]. [40] G.N. Remmen and N.L. Rodd, Consistency of the standard model eﬀective ﬁeld theory, JHEP 12 (2019) 032 [arXiv:1908.09845] [INSPIRE]. [40] G.N. Remmen and N.L. Rodd, Consistency of the standard model eﬀective ﬁeld theory, JHEP 12 (2019) 032 [arXiv:1908.09845] [INSPIRE]. [41] J. Fuentes-Martin, M. König, J. Pagès, A.E. Thomsen and F. References Wilsch, SuperTracer: a calculator of functional supertraces for one-loop EFT matching, JHEP 04 (2021) 281 [arXiv:2012.08506] [INSPIRE]. [42] J.C. Criado, MatchingTools: a python library for symbolic eﬀective ﬁeld theory calculations, Comput. Phys. Commun. 227 (2018) 42 [arXiv:1710.06445] [INSPIRE]. [43] J. de Blas, J.C. Criado, M. Pérez-Victoria and J. Santiago, Eﬀective description of general extensions of the standard model: the complete tree-level dictionary, JHEP 03 (2018) 109 [arXiv:1711.10391] [INSPIRE]. [44] M. Jiang, N. Craig, Y.-Y. Li and D. Sutherland, Complete one-loop matching for a singlet scalar in the standard model EFT, JHEP 02 (2019) 031 [Erratum ibid. 01 (2021) 135] [arXiv:1811.08878] [INSPIRE]. [45] S. Bakshi Das, M. Chala, A. Díaz-Carmona and G. Guedes, Towards the renormalisation of the standard model eﬀective ﬁeld theory to dimension eight: bosonic interactions II, arXiv:2205.03301 [INSPIRE]. [46] Q. Bi, C. Zhang and S.-Y. Zhou, Positivity constraints on aQGC: carving out the physical parameter space, JHEP 06 (2019) 137 [arXiv:1902.08977] [INSPIRE]. [47] S. Das Bakshi, J. Chakrabortty and S.K. Patra, CoDEx: Wilson coeﬃcient calculator connecting SMEFT to UV theory, Eur. Phys. J. C 79 (2019) 21 [arXiv:1808.04403] [INSPIRE]. – 26 –
https://openalex.org/W4381433996	https://amu.hal.science/hal-04171966/document	English	null	Viticulture extension in response to global climate change drivers – lessons from the past and future projections	Climate of the past	2,023	cc-by	20,621	To cite this version: Joel Guiot, Nicolas Bernigaud, Alberte Bondeau, Laurent Bouby, Wolfgang Cramer. Viticulture extension in response to global climate change drivers – lessons from the past and future projections. Climate of the Past, 2023, 19 (6), pp.1219-1244. 10.5194/cp-19-1219-2023. hal-04171966 Distributed under a Creative Commons Attribution 4.0 International License Viticulture extension in response to global climate change drivers – lessons from the past and future projections Joel Guiot1, Nicolas Bernigaud1, Alberte Bondeau2, Laurent Bouby3, and Wolfgang Cramer2 1Aix-Marseille Université, CNRS, IRD, INRAE, CEREGE, Aix-en-Provence, France 2Institut Méditerranéen de Biodiversité et d’Écologie marine et continentale (IMBE), Aix Marseille Université, CNRS, IRD, Avignon Université, Aix-en-Provence, France 3ISEM, Université Montpellier, CNRS, IRD, EPHE, Montpellier, France Correspondence: Joel Guiot (guiot@cerege.fr) Correspondence: Joel Guiot (guiot@cerege.fr) Received: 15 December 2022 – Discussion started: 3 January 2023 Revised: 11 May 2023 – Accepted: 12 May 2023 – Published: 20 June 2023 Abstract. The potential areal extent of agricultural crops is sensitive to climate change and its underlying drivers. To dis- tinguish between the drivers of past variations in the Mediter- ranean viticulture extension since Early Antiquity and im- prove projections for the future, we propose an original at- tribution method based on an emulation of ofﬂine coupled climate and ecosystem models. The emulator connects the potential productivity of grapevines to global direct and in- direct climate drivers, notably orbital parameters, solar and volcanic activities, demography, and greenhouse gas con- centrations. This approach is particularly useful to place the evolution of future agrosystems in the context of their past variations. We found that variations in potential area for viticulture during the last 3 millennia in the Mediterranean Basin were mainly due to volcanic activity, while the ef- fects of solar activity and orbital changes were negligible. In the future, as expected, the dominating factor is the in- crease in greenhouse gases, causing signiﬁcantly drier con- ditions and thus major difﬁculties for viticulture in Spain and North Africa. These constraints will concern signiﬁcant areas of the southern Mediterranean Basin when global warming exceeds + 2 ◦C above preindustrial conditions. Our experi- ments showed that even intense volcanic activity compara- ble to that of the Samalas – sometimes considered to be the starting point of the Little Ice Age in the mid-13th century – would not decrease aridity and so not slow down this de- cline in viticulture extension in the southern margin of the Mediterranean area. This result does not conﬁrm the idea of geoengineering that solar radiation modiﬁcation (SRM) is an efﬁcient option to limit future global warming. HAL Id: hal-04171966 https://amu.hal.science/hal-04171966v1 Submitted on 27 Jul 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. 2 Material and methods As ESMs are an imperfect representation of reality, our ap- proach has the same limitations even if the model param- eters are carefully chosen (Cruciﬁx, 2012). Cruciﬁx (2012) developed the idea that a Bayesian framework is adequate to stimulate the modeling of the distance between the ESM and reality. So ESMs produce a certain vision of the world that needs to converge to the reality represented by the paleodata. This procedure is called data assimilation, which provides a way to reduce the uncertainties. The global drivers of the studied changes are anthro- pogenic, including greenhouse gas (GHG) emissions, land use and cover changes, population density, and economic production, and natural, including the Earth’s orbit as well as volcanic and solar activity. They are the boundary condi- tions of Earth system models (ESM) (Kay et al., 2014). The ecosystem processes are assessed using impact models (IMs) coupled to these ESMs (Franklin et al., 2016; Warszawski et al., 2014; Frieler et al., 2017). In most cases, coupling is of- ﬂine because (i) the spatial scales of ecosystem processes are much ﬁner than are those of ESMs, thus making a scale trans- fer necessary (Su et al., 2016); (ii) a climate simulation of a given ESM is interpreted as one realization out of a set of possibilities determined by the boundary conditions and the characteristics of the ESM, thus making it necessary to work on ensembles of models to be representative of the climate system (Kay et al., 2014); and (iii) each ESM has intrinsic biases that must be corrected before it can be used to drive the ecosystem model (Williamson et al., 2015). The concept of our method and the data used are presented in Fig. 1. Global forcings are the inputs of low-resolution ESMs. Step 1 involves adapting output ﬁelds to a common high-resolution grid using statistical downscaling. This step is also useful to correct the systematic biases of the ESM simulations. Step 2 involves applying the vegetation model BIOME4 to the high-resolution ﬁelds to calculate ecosystem variables. Steps 1 and 2 are repeated for each ESM simula- tion available. These simulations based on different forcings and different models provide a large and diversiﬁed calibra- tion dataset. They are a guarantee that the emulator is in some way more robust than the ESM simulations taken separately. 1 Introduction Our scientiﬁc question is related to the attribution of viticul- ture, which has had an economic role in the Mediterranean Basin since antiquity, extension changes to any natural or an- thropogenic drivers. The cultivation and domestication of the grapevine began between the 7th and 4th millennia before the common era (BCE) between the eastern Mediterranean and Caspian areas and spread to Egypt, the Middle East, and the entire Mediterranean (Terral et al., 2010; Bouby et al., 2021). Introduced in the Gaul region (i.e., roughly France and sur- rounding regions) by Greek colonists ca. 600 BCE, around the time they settled Marseilles, viticulture was initially lim- ited to Mediterranean Gaul (Bouby et al., 2014). Vineyards expanded into the northern part of Gaul in the 1st century CE, where wine production developed quite considerably in the following centuries up to the Paris region, the Rhine and Moselle valleys (Brun, 2010), and even in southern England (Brown et al., 2001). One hypothesis behind this expansion is the climate warming during the Roman Climatic Optimum (RCO) (Mccormick et al., 2012). These climate variations are driven by global forcing variables such as solar or vol- canic activity (Wanner et al., 2008; Brayshaw et al., 2010; Fuks et al., 2017). After the RCO, the temperature decreased signiﬁcantly and Gaul entered the so-called Late Antique Lit- tle Ice Age, or LALIA (536–660 CE) (Büntgen et al., 2016). This change may have been triggered by several large vol- canic eruptions at 536, 540, and 547 CE (Sigl et al., 2015). This assumption remains difﬁcult to prove because of the limited historical and archeological sources. In any case, the Published by Copernicus Publications on behalf of the European Geosciences Union. J. Guiot et al.: Viticulture extension in response to global climate change drivers 1220 500 to 900 CE period remained relatively cold with oscillat- ing precipitation changes in the region (Reale and Dirmeyer, 2000). In Europe, the following Medieval Climate Anomaly (MCA, approx. 900–1200 CE) (Luterbacher et al., 2016) was likely of intensity comparable to the RCO. The Little Ice Age (LIA, approx. 1250–1850 CE) was a period of alpine glacier advance (Holzhauser et al., 2005), again marked by several large volcanic eruptions, in particular that of the Samalas (In- donesia) in 1257 (Lavigne et al., 2013). and Medieval periods with a regression of the grape cultiva- tion. 2 Material and methods Calibration of the emulator (step 3) is done by spatial re- gression of the ecosystem variables on the forcing variables. Step 4 involves the application of the emulator to the forcings of new time slices or future scenarios. During this step, the annual temperature and precipitation results for the past time slice were compared to paleodata to identify the amplitude of the volcanic and solar activity effects (data assimilation). Step 5 is the independent validation of the emulator using tree-ring data. Even if human innovation and colonization were also re- sponsible for the expansion of viticulture, the climate must be suitable for grape cultivation and thus remains a control vari- able. As soon as the climate changes to worse conditions for wine growth, it becomes a driver of a decline in viticulture. Such ﬂuctuations are particularly noticeable near the north- ern range limit of wine growth during the end of the Roman 1 Introduction Although we focus our viticultural analysis on the Gaul region (France and surrounding countries), we need to en- large the area to the entire Mediterranean and European sur- rounding region to robustly capture the relationships between global drivers and viticulture extension. For the same reason, we use a large diversity of time slices of the past (Last Glacial Maximum, mid-Holocene, last millennium) and of the future up to 2100 according to several scenarios. The widely diverse situations used for calibration made it possible to produce a robust emulator that was effective for extrapolating a wide range of past and future climate states. This method is appro- priate to establish a bridge between past variations of a given agrosystem and its future evolution. Once calibrated, the em- ulator is of great help to efﬁciently support decisions in the domain of impacts of climatic change. On timescales of centuries and millennia, quality and yield of agricultural crops are strongly affected by climate ﬂuctu- ations. The nature of the change depends on external forc- ings and internal feedbacks of the climate system, which pro- duce different spatial and seasonal patterns of the main vari- ables in the atmospheric environment. The main objective of this paper is to develop an innovative solution to statisti- cally model the impact of changing climate forcing on veg- etation over several millennia using the grapevine (a major crop of the Mediterranean and European region) as an ex- ample. We mimic this impact model on the basis of a large ensemble of existing model simulations using statistical re- lationships much faster to compute (Kennedy and O’Hagan, 2000). Based on a large range of climate states from high- resolution simulations with coupled Earth system models for the last glacial period to future global warming scenarios, this approach, called a “emulator”, provides robust results and can be applied to a large range of ecosystem processes under different conditions. J. Guiot et al.: Viticulture extension in response to global climate change drivers Low-density hashes in the trapezes indicate low resolution of the corresponding data, and high-density hashes correspond to higher resolution. i. Orbital parameters: eccentricity (ecc) and obliquity of the ecliptic (obl) as well as solar longitude at the per- ihelion (ω), or more precisely the climate precession pr = ecc × sin(ω). Nevertheless, to avoid redundancy, we use as forcing variables the three basic parame- ters (ecc, obl, ω). They drive the orbit of the Earth and inﬂuence its climate by modulating the solar en- ergy intercepted by the planet and its seasonal distribu- tion at timescales above thousands of years (Berger and Loutre, 1991). They had a major impact on the Earth’s climate from the last glacial period to the Holocene. At the century timescale, the orbital parameters do not operate signiﬁcantly, so the values of the last millen- nium can be linearly interpolated between the values at 1000 yr BP and the present values, and the values of the 21st century can be set to the present values. i. Orbital parameters: eccentricity (ecc) and obliquity of the ecliptic (obl) as well as solar longitude at the per- ihelion (ω), or more precisely the climate precession pr = ecc × sin(ω). Nevertheless, to avoid redundancy, we use as forcing variables the three basic parame- ters (ecc, obl, ω). They drive the orbit of the Earth and inﬂuence its climate by modulating the solar en- ergy intercepted by the planet and its seasonal distribu- tion at timescales above thousands of years (Berger and Loutre, 1991). They had a major impact on the Earth’s climate from the last glacial period to the Holocene. At the century timescale, the orbital parameters do not operate signiﬁcantly, so the values of the last millen- nium can be linearly interpolated between the values at 1000 yr BP and the present values, and the values of the 21st century can be set to the present values. Figure 1. Diagram of the different steps in the emulator approach; the ecosystem variables, related to bioclimate and net primary pro- ductivity, are deﬁned in Table 1. Low-density hashes in the trapezes indicate low resolution of the corresponding data, and high-density hashes correspond to higher resolution. ii. Greenhouse gas (GHG) concentration: carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O). J. Guiot et al.: Viticulture extension in response to global climate change drivers 1221 . The biome types simulated by BIOME4 and output variables used for the paper; note that there is no correspondence between the umns. No. Biome names Output variables 1 Tropical evergreen forest NPPtot: total net primary production 2 Tropical semi-deciduous forest Dominant PFT (plant function type) 3 Tropical deciduous forest/woodland AET actual evapotranspiration (mm) 4 Temperate deciduous forest MTCO mean temperature of the coldest month (◦C) 5 Temperate conifer forest MTWA mean temperature of the warmest month (◦C) 6 Warm mixed forest E/PE actual over potential evapotranspiration 7 Cool mixed forest GDD5 growing degree days above 5 ◦C (◦d−1) 8 Cool conifer forest TANN annual mean temperature (◦C) 9 Cold mixed forest PANN annual sum of precipitation (mm) 10 Evergreen taiga/montane forest 11 Deciduous taiga/montane forest 12 Tropical savanna NPP net primary production of following PFTs 13 Tropical xerophytic shrubland Tet: tropical evergreen trees 14 Temperate xerophytic shrubland Trt: tropical drought–deciduous trees (raingreen) 15 Temperate sclerophyll woodland Tbe: temperate broadleaved evergreen trees 16 Temperate broadleaved savanna Tst: temperate deciduous trees 17 Open conifer woodland Ctc: cool conifer trees 18 Cool desert Bec: boreal evergreen trees 19 Tropical grassland Bst: boreal deciduous trees 20 Temperate grassland tg: C3/C4 temperate grass plant type 21 Hot desert Trg4: C4 tropical grass plant type 22 Steppe tundra Wds: C3/C4 woody desert plant type 23 Shrub tundra Tsg: tundra shrub type 24 Dwarf shrub tundra Ch: cold herbaceous type 25 Prostrate shrub tundra Lf: lichen/forb type 26 Cushion forb lichen moss tundra 27 Barren 28 Land ice Table 1. The biome types simulated by BIOME4 and output variables used for the paper; note that there is no correspondence between the two columns. Table 1. The biome types simulated by BIOME4 and output variables used for the paper; note that there is no correspondence between the two columns. NPP net primary production of following PFTs Tet: tropical evergreen trees Trt: tropical drought–deciduous trees (raingreen) Tbe: temperate broadleaved evergreen trees Tst: temperate deciduous trees Ctc: cool conifer trees Bec: boreal evergreen trees Bst: boreal deciduous trees tg: C3/C4 temperate grass plant type Trg4: C4 tropical grass plant type Wds: C3/C4 woody desert plant type Tsg: tundra shrub type Ch: cold herbaceous type Lf: lichen/forb type Figure 1. Diagram of the different steps in the emulator approach; the ecosystem variables, related to bioclimate and net primary pro- ductivity, are deﬁned in Table 1. 2.1 The forcing variables Climate forcings or drivers are perturbations imposed on the Earth’s energy balance. They are mainly the following. https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 2.3 The future climate data (used for calibration) The future periods are determined by increasingly high GHG concentrations (depending on the scenario, see below), and the other forcing variables were set to the PI value. The sim- ulations were archived by the Coupled Model Intercompari- son Project (CMIP). We used simulations of CMIP5 (Taylor J. Guiot et al.: Viticulture extension in response to global climate change drivers Volcano activity (V ) represented by the effective aerosol radius deduced from the aerosol optical depth from ice core sulfate records from both polar regions for the last millennium (Crowley and Unterman, 2013) (Fig. 2). Its value is 0.2 when there is no eruption. The maxi- mum value (0.8) was found in 1258, the year after the Samalas eruption (Lavigne et al., 2013). The 21 ka, 6 ka, and future values were set to the preindustrial values. The simulations were archived by the Paleoclimate Mod- elling Intercomparison Project (PMIP), which produced snapshot simulations of the mid-Holocene (6 ka BP) and Last Glacial Maximum (21 ka BP) time slices to study the ef- fects of insolation, greenhouse gases, and massive ice sheets on the climate (https://pmip3.lsce.ipsl.fr/overview/, last ac- cess: 15 June 2023) (Braconnot et al., 2012). The models used for intercomparisons were coupled atmosphere–ocean– vegetation models with various levels of complexity and res- olution (Table 2). v. Solar activity (S) is inferred from 14C and 10Be records, the proxy for the total solar irradiance (TSI) (Muscheler et al., 2007) for the last millennium, and varies from 0 to 1200 MeV (Fig. 2). The 21 ka, 6 ka, and future values were set to the preindustrial values. J. Guiot et al.: Viticulture extension in response to global climate change drivers The effect of these GHGs has been signiﬁcant from the glacial to interglacial periods and has a major effect for the current century (see, for example, Fig. 2 for CO2). Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers 1222 Figure 2. Natural (volcanic activity and solar activity) and anthropogenic forcing (CO2) for the last millennium: (a) the effective aerosol radius, which is directly related to the aerosol optical depth, is calculated by Crowley and Unterman (2013); (b) the proxy indicating the total solar irradiance (TSI), i.e., the solar modulation function in MeV based on 14C (Muscheler et al., 2007); and the (c) atmospheric CO2 concentration (in ppm). The red horizontal lines represent the mean values of the ﬁve selected periods. Figure 2. Natural (volcanic activity and solar activity) and anthropogenic forcing (CO2) for the last millennium: (a) the effective aerosol radius, which is directly related to the aerosol optical depth, is calculated by Crowley and Unterman (2013); (b) the proxy indicating the total solar irradiance (TSI), i.e., the solar modulation function in MeV based on 14C (Muscheler et al., 2007); and the (c) atmospheric CO2 concentration (in ppm). The red horizontal lines represent the mean values of the ﬁve selected periods. iii. The world population values taken from the Hyde 3.1 database for the past periods from Klein Goldewijk et al. (2011) and for the future periods from van Vuuren et al. (2011). The population varied from less than 106 at the Last Glacial Maximum (LGM) to 7 × 109 in 2010 and is projected to be between 9 × 109 and 12.3 × 109 in 2100 according to the scenario. determined by a low ecc; (2) the mid-Holocene (6 ka) charac- terized by GHG close to that of the preindustrial period, low ecc and pr, and a large obl increasing the seasonality of the climate; (3) ﬁve periods of the last millennium (Fig. 2) repre- senting various combinations of S and V forcings, including V −S+ (1200–1220), V + −S (1255–1320), V −S+ (1380– 1420), V −S + + (1720–1780), and V + S+ (1810–1840); and (4) the preindustrial (PI) reference period with interme- diate GHG concentrations, low ecc, medium obl, medium pr, and large V and S. iv. 2.2 The past time climate data (used for calibration) The past time periods retained for calibration are (1) the Last Glacial Maximum (21 ka) characterized by very low green- house gases (GHGs), low ecc, and obl and a pr close to zero Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers The +3 ◦C global warming is reached with a CO2 con- centration of 600 ppm and is reached under scenario RCP8.5 in approximately 2060 (Fig. 3). iii. The +3 ◦C global warming is reached with a CO2 con- centration of 600 ppm and is reached under scenario RCP8.5 in approximately 2060 (Fig. 3). iv. The +5 ◦C global warming is reached with a CO2 con- centration of 900 ppm and is reached under scenario RCP8.5 in about 2090 (Fig. 3). iv. The +5 ◦C global warming is reached with a CO2 con- centration of 900 ppm and is reached under scenario RCP8.5 in about 2090 (Fig. 3). We obtained the corresponding CH4 and N2O concentrations and population sizes from the boundary condition database of CMIP5. The other future forcings are set to the present values. Following Giorgi and Lionello (2008), the study area was divided into nine grid boxes (Fig. 4). We added a 10th box corresponding to the Gallia Narbonensis Roman province (south of France), the key area for the introduction of viti- culture in Gaul. J. Guiot et al.: Viticulture extension in response to global climate change drivers 1223 Table 2. CMIP5 and PMIP3 simulations used in the paper. The ﬁrst column is the code of model, and the second indicates the institute which built the model; the other columns provide the availability of the scenarios (RCP) or time slices. Hist.: historical period (last millennium), MidHol: mid-Holocene (6 kyr BP), LGM: Last Glacial Maximum (21 kyr BP). Table 2. CMIP5 and PMIP3 simulations used in the paper. The ﬁrst column is the code of model, and the second indicates the institute which built the model; the other columns provide the availability of the scenarios (RCP) or time slices. Hist.: historical period (last millennium), MidHol: mid-Holocene (6 kyr BP), LGM: Last Glacial Maximum (21 kyr BP). MidHol: mid-Holocene (6 kyr BP), LGM: Last Glacial Maximum (21 kyr BP). Model Institute RCP2.6 RCP4.5 RCP8.5 Hist. MidHol LGM bcc-csm1-1 Beijing Climate Center, China Meteorological X X X X Administration bcc-csm1-1-m Beijing Climate Center, China Meteorological X X X Administration CCSM4 Community Climate System Model Contributors, X X UCAR-NSF-DoE-NASA, USA CCSM4b Community Climate System Model Contributors, X X UCAR-NSF-DoE-NASA, USA CanESM2 Canadian Centre for Climate Modelling, Canada X X X CESM1-BGC Community Earth System Model Contributors, X NSF-DoE-NCAR, USA CSIRO-Mk3 CSIRO Marine and Atmospheric Research, X Victoria, Australia CSIRO-Mk3L CSIRO Marine and Atmospheric Research, X X Victoria, Australia CMCC-CM Centro Euro-Med per Cambiamenti Climatici, X X Italy CNRM-CM5 Centre National de Recherches Météorologiques/ X X X X X Centre Européen de Recherche et Formation Avancée en Calcul Scientiﬁque, France COSMOS-ASO X FGOALS-g2 LASG, Institute of Atmospheric Physics, Chinese X Academy of Sciences and CESS, Tsinghua University, China FGOALS-s2 LASG, Institute of Atmospheric Physics, Chinese X X Academy of Sciences and CESS, Tsinghua University, China GFDL-CM3 NOAA Geophysical Fluid Dynamics Laboratory, X X USA GISS-E2-H NASA Goddard Institute for Space Studies, USA X X X GISS-E2-H-CC NASA Goddard Institute for Space Studies, USA GISS-E2-R NASA Goddard Institute for Space Studies, USA X X X X X GISS-E2-Rb NASA Goddard Institute for Space Studies, USA X HadCM3 Met-Ofﬁce – Hadley Center, UK X HadGEM2-AO Met-Ofﬁce – Hadley Center, UK X X X HadGEM2-ES Met-Ofﬁce – Hadley Center, contributed by X Instituto Nacional de Pesquisas Espaciais, Spain inmcm4 Inst. For Numerical Mathematics, Russia X X IPSL-CM5A-LR Institut Pierre-Simon Laplace, France X X X X X IPSL-CM5A-MR Institut Pierre-Simon Laplace, France X X X https://doi.org/10.5194/cp-19-1219-2023 J. J. Guiot et al.: Viticulture extension in response to global climate change drivers Guiot et al.: Viticulture extension in response to global climate change drivers J. Guiot et al.: Viticulture extension in response to global climate change drivers 1224 Table 2. Continued. Table 2. Continued. able 2. Continued. Model Institute RCP2.6 RCP4.5 RCP8.5 Hist. MidHol LGM IPSL-CM5B-LR Institut Pierre-Simon Laplace, France MIROC-ESM University of Tokyo, National Institute for X X Environmental Studies, and Japan Agency for Marine-Earth Science and Technology MPI-ESM-LR Max-Planck Inst. für Meteorologie, Germany X X X MPI-ESM-P Max-Planck Inst. für Meteorologie, Germany X X MPI-ESM-MR Max-Planck Inst. für Meteorologie, Germany X X X MRI-CGCM3 Meteorological Research Institute, Japan X X X X X NorESM1-M Norwegian Climate Centre X X X NorESM1-ME Norwegian Climate Centre X X X Nb simulations 16 18 16 2 13 11 440 ppm; Fig. 3 shows that the average model simula- tion reaches this value under scenario RCP2.6 in ap- proximately 2040. 440 ppm; Fig. 3 shows that the average model simula- tion reaches this value under scenario RCP2.6 in ap- proximately 2040. et al., 2012). These include both (i) century-scale integra- tions, which usually start from a preindustrial control, and climate predictions until the end of the 21st century, as well as (ii) near-term integrations for the next 10 to 30 years, which are initialized using observed ocean and sea-ice con- ditions. The CMIP5 climate change projections are driven by emission scenarios divided into four classes referred to as Representative Concentration Pathways (RCPs) (Moss et al., 2010). The high-emissions scenario, named RCP8.5 or “busi- ness as usual”, refers to a high radiative forcing of emissions at the end of the 21st century (8.5 W m−2). The low-emission scenario, which roughly represents that of the Paris Agree- ment, reaches its maximum value near the middle of the cen- tury before decreasing to a level of 2.6 W m−2 at the end of the century. We only used the intermediate scenario RCP4.5, which refers to a radiative forcing maintained at values of 4.5 W m−2 at the end of the 21st century. We used 10 time slices interpolated at a 10-year steps (2010, 2020, .. . , 2100). The forcing variables for the future scenarios were obtained from the website http://tntcat.iiasa.ac.at/RcpDb/ (last access: 15 June 2023). ii. The +2 ◦C global warming is reached with a CO2 con- centration of 480 ppm and is reached under scenario RCP4.5 in approximately 2050 (Fig. 3). iii. J. Guiot et al.: Viticulture extension in response to global climate change drivers 1225 Table 3. Typical past periods used to calibrate our emulator. The paleoclimatic and societal information is found in the literature given in the references from the late Holocene to the present. Table 3. Typical past periods used to calibrate our emulator. The paleoclimatic and societal information is found in the literature given in the references from the late Holocene to the present. yp p p p g erences from the late Holocene to the present. Time slices and labels Location Climate Societal events References 4200 (4200–3900 yr BP) Levant, Mesopotamia, Drought Collapse of Akkadian Kaniewski et al. (2018), Sicily empire in Mesopotamia Weiss and Bradley (2001) Magny et al. (2013) 3200 (3300–2900 yr BP) The Levant, Anatolia, Drought Collapse/decline of Roberts et al. (2011), Aegean, Egypt Aegean, Hittite, Kaniewski et al. (2015), Palestinian, Egyptian, Neumann and Parpola Mesopotamia Babylonian civilizations (1987) 2500 (2600–2400 yr BP) West Med., France Cold Early Iron Age Finné et al. (2011), Magny et al. (2013), Maghreb Dry Leveau (2009) 2000 (2100–1800 yr BP) West Med., France, Warm, wet Roman Climate Optimum Mccormick et al. (2012) Maghreb (RCO), expansion of the Büntgen et al. (2016) empire 1300 (1410–1290 yr BP West Med., Alps Cold Late Antique Little Ice Büntgen et al. (2016), or AD 536–660) Age (LALIA) (migrations, pandemics, social turmoil) Mesopotamia, Iran Dry Demise of Sasanians Shariﬁet al. (2015) 1000 (1150–650 yr BP Europe, Alps, Warm Medieval Climate Anomaly Telelis (2008), Büntgen et or AD 800–1300) (MCA) al. (2011), Izdebski et al. East dry (2016), Finné et al. (2011) 700 (700–600 yr BP Europe, Alps Cold Beginning of Little Ice Büntgen et al. (2011), or AD 1250–1350) Age (LIA); famine, black Luterbacher et al. (2016) death 200 (300–230 yr BP Europe, Alps Cold wet Max of Little Ice Age Magny et al. (2013), or 1650–1720) (LIA); famines Büntgen et al. (2011), Spain Dry Magny et al. (2013) Present (1961–1990) All areas Warm and CRU data (Jones et al., dry 2012) resolution dataset of surface climate over global land areas (New et al., 2002) (which has a 10 min resolution and was aggregated at a 30 min resolution). This climatology is based on the 1961–1990 period. Each low-resolution (LR) anomaly ﬁeld provided by the ESM is downscaled to the HR grid by the bilinear interpolator of platform R (function interp. sur- face of package ﬁelds). J. Guiot et al.: Viticulture extension in response to global climate change drivers This HR anomaly ﬁeld is added to the 1961–1990 climatological HR ﬁeld of New et al. (2002). As the simulations are corrected by the differences between con- trol simulations and observations, the main systematic biases of the model are removed. For the PMIP3 simulations (LGM, MidHol, and historical), the control is the preindustrial simu- lation (PI), so we use the 1900–1931 climatology very close to the PI (1861–1890) used by the recent IPCC reports (Allen et al., 2018). Figure 3 presents the global mean temperature for each period and scenario considered. 2.4 The application data We also considered two other scenarios based on bound- ary conditions of +5 ◦C except for volcanic and solar activ- ity. The idea is to assess whether volcanic and solar condi- tions typical of the Little Ice Age can moderate the effect of the strong increase in GHG concentrations. The ﬁrst ad- ditional scenario (labeled +5 CV+) was assigned very sub- stantial volcanic activity (0.8), the highest value in the last millennium, and low solar activity (i.e., 100). In contrast, the second additional scenario (labeled +5 CV−) was assigned low volcanic activity (0.1) and high solar activity (700), cor- responding to those of the MCA. The forcing values are listed in Table 2. We used our emulator to analyze the response of key ecosys- tem variables to global forcing. We focused on past periods that were marked by important climate and societal changes (Table 3). The present time slice was deﬁned by the mean values for the 1961–1990 period. For the future, instead of using time slices, we deﬁned the scenarios according to the global temperature signal simulated in the different models using the relationship between global warming and CO2 con- centration (Guiot and Cramer, 2016). i. The +1.5 ◦C global warming recommended by the Paris Agreement is reached with a CO2 concentration of https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers J. Guiot et al.: Viticulture extension in response to global climate change drivers 1226 J. Guiot et al.: Viticulture extension in response to global climate change drivers Figure 3. Global annual temperature anomalies (T ) simulated by the various models of PMIP3 and CMIP5, according to the reference period average (1961–1990). The left graphic represents the past simulations, and the right graphic represents the present and future projections for three scenarios (RCP2.6 in green, RCP4.5 in orange, RCP8.5 in red). Each dot represents one model simulation. The vertical scales are shifted for better readability. The reference is given by the New et al. (2002) dataset calculated on the 1961–1990 period. The anomalies versus the preindustrial period are obtained by adding approximately 0.5 ◦C to the 1961–1990 anomalies. Figure 3. Global annual temperature anomalies (T ) simulated by the various models of PMIP3 and CMIP5, according to the reference period average (1961–1990). The left graphic represents the past simulations, and the right graphic represents the present and future projections for three scenarios (RCP2.6 in green, RCP4.5 in orange, RCP8.5 in red). Each dot represents one model simulation. The vertical scales are shifted for better readability. The reference is given by the New et al. (2002) dataset calculated on the 1961–1990 period. The anomalies versus the preindustrial period are obtained by adding approximately 0.5 ◦C to the 1961–1990 anomalies. Figure 4. Map of the 10 boxes deﬁned in the Mediterranean region. Figure 4. Map of the 10 boxes deﬁned in the Mediterranean region. 2.5 Downscaling (step 1) ESM resolution is too coarse (> 100 km depending on the model) to assess the impact and risk of climate changes on ecosystems or human systems. Different approaches can be used to derive higher-resolution information. We use a sta- tistical downscaling (SD) based on statistical relationships that link large-scale atmospheric variables with local and regional climate variables and that are applied to coarser- resolution model simulations (Su et al., 2016; Grouillet et al., 2016; Levavasseur et al., 2011), thereby providing higher- resolution estimates of climate variables. SD methods are among the less computationally expensive downscaling tech- niques. The SD that we use assumes the hypothesis that the ﬁelds of climate anomalies do not depend on the grid resolution. So, they can be interpolated at a ﬁner resolution than the ini- tial resolution. We use a common 0.5◦high-resolution (HR) grid for each climate simulation. It is provided by the high- Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 1226 2.7 The calibration of the emulator (step 3) The numerous model outputs available in the CMIP and PMIP databases (Table 2) are used to calibrate robust statis- tical approximations of coupled ESMs and BIOME4, called the emulator (Kennedy and O’Hagan, 2000). Various emula- tors are used in climate science (Tran et al., 2016; Zhu et al., 2015; Rougier and Goldstein, 2014; Castruccio et al., 2014), including in paleoclimatology (Strassmann and Joos, 2018; Joos et al., 1996; Bounceur et al., 2015). Our approach is the ﬁrst ESM-independent emulator because it is calibrated us- ing a large set of model simulations under very different sce- narios. The calibration is based on a geographically weighted regression (Brunsdon et al., 1998), whereby the ecosystem variables are expressed as functions of the global forcing variables. The emulator is trained on an ensemble of simulations performed on data sufﬁciently browsing the plausible input space. The more the input space is ﬁlled, the more robust the emulator will be. The fact that we have extended the range of forcing parameters to past, present, and future scenarios is an excellent way to ﬁll this input space. Even if they are not a fully exhaustive sampling, it is reasonable to assume that they represent a model population (Chandler, 2013). The emulator is used to estimate the conditional probability distribution of the ecosystem variables (Table 1) in each location or biome based on the forcing variables (Table 4). The total number of simulations available for 18 time slices and three scenarios (for the 2020 to 2100 time slices) from 2 to 18 models is 582. As each one simulates climate for 65 559 grid points of the globe (after downscaling), the total number of available observations is ∼4×107. Because of their strong spatial correlation, it is not necessary to use all the grid points. To have a balance between the differ- ent biomes represented on the Earth, for each time slice and each model, we randomly draw a subset of grid points in each biome proportional to its representativity (the proportion of For any choice of input q vector x (forcings), the climate simulator is y(xs) = (y1(x,s), ..., ym(x,s)), where m is the number of (bio)climatic variables whose response is to be analyzed and s is the location where the climate variables must be estimated. There are deterministic and possibly non- linear functions. 2.6 Vegetation modeling (BIOME4, step 2) bration, the use of a single ecosystem model is required to ensure that the same ecosystem variables are calculated for all simulations. The ecosystem model is coupled ofﬂine to downscaled climate outputs and provides indications of land vegeta- tion structure and productivity. Here, we used a process- based equilibrium vegetation model, BIOME4 (Kaplan et al., 2002), which has been successfully applied to similar ques- tions before (Guiot and Cramer, 2016). While a wide range of climate simulations are necessary to ensure robust cali- BIOME4 simulates some of the most important processes related to vegetation at the scale of the ecosystem, specif- ically photosynthesis, respiration, and evapotranspiration. It predicts structure and productivity of broad-scale land ecosystems from monthly temperature and rainfall values as https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 1227 J. Guiot et al.: Viticulture extension in response to global climate change drivers erties to run very fast and provide an uncertainty description for the whole plausible input space (i.e., conform to physics, internally consistent and reasonable; Amara, 1991): well as annual CO2 concentration. It uses a photosynthesis scheme that simulates acclimation of plants to changed atmo- spheric CO2 by optimization of nitrogen allocation to foliage and by accounting for the effects of CO2 on net assimilation, stomatal conductance, leaf area index (LAI), and ecosystem water balance. BIOME4 is based on a sufﬁciently simple de- scription of ecophysiological processes to allow broad-scale application. It represents substantial advantages over niche models because it has not been tuned to reproduce present- day potential vegetation, but rather to correctly simulate the main processes underlying the potential vegetation distribu- tion, which are assumed to have been similar throughout the Holocene. BIOME4 does not account for human land use. yj(x,s) = Xβj(s) + ej(s) j = 1, ···, m;s = 1, ..., n, (1) (1) where s indicates the grid point out of a total of n points, yj(x,s) is the anomaly of the climatic variable j (out of m) at location s, i.e., the climate variable at time t minus the cli- mate variable at time 0 (present), X is a matrix with v speci- ﬁed columns of the input global forcings (they do not depend on the location s), and e(s) is a stationary Gaussian vari- able N(0,σ 2 j ). This can be considered a least-square problem wherein the coefﬁcients βj(s) are estimated to minimize the sum of the squared errors. 2.6 Vegetation modeling (BIOME4, step 2) These coefﬁcients can be written as The inputs of the model are monthly average values of temperature, precipitation and sunshine percentage, atmo- spheric CO2 concentration, and soil texture. The tempera- ture and precipitation variables are directly obtained from the simulations downscaled at the 0.5◦grid. The sunshine percentages are obtained by linear regression on temperature and precipitation (Guiot et al., 2000). The absolute minimum temperature is derived from the mean temperature of the coldest month according to the quadratic equation of Prentice et al. (1992). The soil data are provided by the FAO (Zobler, 1986). The CO2 atmospheric concentration values are those used by CMIP5 and PMIP3 as boundary conditions for their simulations. as ˆβj(s) = XT W(s)X −1 XT W(s)yj, (2) (2) (2) with W(s) as a matrix of weights speciﬁc to location s such that observations nearer to s are given greater weight than ob- servations further away, and T is the transpose symbol. The estimation of βj(s) in location s is provided by weighting all the observations according to their distance from s, dis. The method is called geographically weighted regression (GWR) (Brunsdon et al., 1998). We use a bi-square weighting func- tion so that W(s) is the diagonal matrix with elements with W(s) as a matrix of weights speciﬁc to location s such that observations nearer to s are given greater weight than ob- servations further away, and T is the transpose symbol. The estimation of βj(s) in location s is provided by weighting all the observations according to their distance from s, dis. The method is called geographically weighted regression (GWR) (Brunsdon et al., 1998). We use a bi-square weighting func- tion so that W(s) is the diagonal matrix with elements The outputs include net primary production (NPP) of each of the potentially occurring 13 plant functional types (PFTs), the total NPP, and the corresponding “biome type” from a set of 28 broad categories (Table 1). The model also provides several bioclimatic variables (Table 1). wis = 1 − dis h 2!2 if dis < h and 0 if dis > h. (3) (3) Here, h is called the bandwidth. We use the function gwr.basic of the package gw.model on R (Lu et al., 2014). We have chosen h = 8 (in longitude and latitude degree units). J. Guiot et al.: Viticulture extension in response to global climate change drivers 1228 Table 4. The nine global forcing variables and their values for the different periods considered. The global forcing variables are the predictors of the emulator; we give their values for the time slices used in the calibration: GHG atmospheric concentration (carbon dioxide, methane, nitrogen protoxide) in parts per million (ppm), Earth orbit parameters (eccentricity Ecc, obliquity Obl, omega ω), population in millions of people (M), volcanic activity (Volc), and solar activity (Sol, in MeV). Every column is one out of the v forcings of Eq. (1) (columns of X). For columns CO2, CH4, N2O, and population, we use observations until 2010 (see Sect. 2.1) and the values used by the three scenarios (RCP2.6, RCP4.5, RCP8.5) for projections (2020 to 2100). Table 4. The nine global forcing variables and their values for the different periods considered. The global forcing variables are the predictors of the emulator; we give their values for the time slices used in the calibration: GHG atmospheric concentration (carbon dioxide, methane, nitrogen protoxide) in parts per million (ppm), Earth orbit parameters (eccentricity Ecc, obliquity Obl, omega ω), population in millions of people (M), volcanic activity (Volc), and solar activity (Sol, in MeV). Every column is one out of the v forcings of Eq. (1) (columns of X). For columns CO2, CH4, N2O, and population, we use observations until 2010 (see Sect. 2.1) and the values used by the three scenarios (RCP2.6, RCP4.5, RCP8.5) for projections (2020 to 2100). Table 4. The nine global forcing variables and their values for the different periods considered. The global forcing variables are the predictors of the emulator; we give their values for the time slices used in the calibration: GHG atmospheric concentration (carbon dioxide, methane, nitrogen protoxide) in parts per million (ppm), Earth orbit parameters (eccentricity Ecc, obliquity Obl, omega ω), population in millions of people (M), volcanic activity (Volc), and solar activity (Sol, in MeV). Every column is one out of the v forcings of Eq. (1) (columns of X). For columns CO2, CH4, N2O, and population, we use observations until 2010 (see Sect. 2.1) and the values used by the three scenarios (RCP2.6, RCP4.5, RCP8.5) for projections (2020 to 2100). J. Guiot et al.: Viticulture extension in response to global climate change drivers Time CO2 CH4 N2O Ecc Obl Omega Population Volc Sol period ω 21k 185 350 200 0.0196 23.4 −213 2M 0.3 270 6k 280 650 270 0.0190 24.2 −27 28M 0.3 270 e 1200 280 650 270 0.0170 23.6 85 394M 0.2 400 1255 280 650 270 0.0170 23.6 85 396M 0.4 200 1380 280 650 270 0.0170 23.6 85 390M 0.2 200 1720 280 650 270 0.0170 23.6 85 768M 0.2 600 1810 280 650 270 0.0167 23.4 102 1082M 0.5 400 2006 379 1754 319 0.0167 23.4 102 6542M 0.3 270 2010 389 1773 323 0.0167 23.4 102 6958M 0.3 270 2020 412, 412, 416 1731, 1801, 1824 329, 330, 332 0.0167 23.4 102 7510M, 7505M, 7530M 0.3 270 2030 431, 435, 449 1600, 1830, 2132 334, 337, 342 0.0167 23.4 102 8200M, 8180M, 8800M 0.3 270 2040 440, 461, 489 1527, 1842, 2399 339, 344, 354 0.0167 23.4 102 8800M, 8500M, 9400M 0.3 270 2050 443, 487, 541 1452, 1833, 2740 342, 351, 367 0.0167 23.4 102 9000M, 8900M, 10400M 0.3 270 2060 442, 509, 604 1365, 1801, 3076 343, 356, 381 0.0167 23.4 102 9100M, 9000M,10700M 0.3 270 2070 437, 524, 677 1311, 1745, 3322 344, 361, 394 0.0167 23.4 102 9150M, 9050M, 11500M 0.3 270 2080 432, 531, 758 1285, 1672, 3490 344,365,408 0.0167 23.4 102 9150M, 9050M, 11900M 0.3 270 2090 426, 534, 845 1268, 1614, 3639 344, 369, 421 0.0167 23.4 102 9150M, 8950M, 12050M 0.3 270 2100 421, 538, 936 1254, 1576, 3751 344, 372, 435 0.0167 23.4 102 9050M, 8800M, 12300M 0.3 270 Table 5. Aggregation of the plant functional types (PFTs). The 13 original PFTs are deﬁned in Table 1. grid points with that biome). So, the calibration is done on about 3×105 observations (the number is slightly lower than expected because some biomes are absent in some simula- tions). J. Guiot et al.: Viticulture extension in response to global climate change drivers g Aggregated PFT (aPFT) original PFT (PFT) Tropical trees (trt) tet, trt Temperate broadleaved evergreen trees (tbe) tbe Temperate trees (tet) tst, ctc Boreal trees (bot) bec, bst C3/C4 temperate grass plant type (teg) tg C4 tropical grass plant type (trg4) trg4 C3/C4 woody desert plant type (wds) wds Tundra grass/shrub (tug) tsg, ch, lf As the global forcing variables have the same value for all the grid points for a given time slice and scenario, this may produce problems for the computation of the emulator; we have then added to them a small noise value (a Gaussian value with mean 0 and standard deviation equal to the initial standard deviation divided by 100). Because of collinearity between the predictors (the global forcing variables), their dimensionality is reduced using prin- cipal components (PCs) of the normalized variables. The nine variables are reduced into ﬁve PCs together explaining 89 % of their total variance. The ﬁrst PC is mainly related to the greenhouse gases and the global population, which is strongly correlated with them. PC3 shows the opposition be- tween solar and volcanic activity. The other ones are difﬁcult to interpret. reconstructed variance higher than 10 % (column R2 (lm)), which clearly shows that a single linear model is not adapted to our objective. The GWR model is much better as half of the bioclimatic variables have R2 higher than 0.50, but at the cost of a much larger effective number of parameters (ENPs) (ENP ∼1311 vs. 6 for the global linear model). As we have a very high number of observations (∼3×105), the GWRs re- main very signiﬁcant. This is illustrated in Fig. 5 by the maps of the spatial distribution of the regression coefﬁcients (they are standardized by their standard error, which comes to the t value). The degree of smoothing is deﬁned by the bandwidth h (here set to 8). With a lower h, the patterns should be much The 13 PFTs are aggregated into eight PFTs, represent- ing the main types of vegetation across the world (Table 5). This enables reducing the dimension of the ecosystem vari- able vector. The number of variables to be estimated by the emulator is ﬁnally reduced to 17 (predictands in Table 6). For comparison, we ﬁrst apply a simple linear model to the data. Table 6 shows that most of the bioclimatic variables are not well-ﬁtted. 2.7 The calibration of the emulator (step 3) This model is a statistical representation of the ESM + BIOME4 model, with the very interesting prop- https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers 1229 Table 6. The t values of the linear regression between the PFT and bioclimatic variables as well as the ﬁve PCs of the forcing variables with the R2 (in %) of the linear model and the GWR model. For the latter, the coefﬁcients cannot be displayed in a table and are represented as maps in Fig. 5. Table 6. The t values of the linear regression between the PFT and bioclimatic variables as well as the ﬁve PCs of the forcing variables with the R2 (in %) of the linear model and the GWR model. For the latter, the coefﬁcients cannot be displayed in a table and are represented as maps in Fig. 5. Predictands Intercept PC1 PC2 PC3 PC4 PC5 R2 (lm) R2 (GWR) NPPtot 132 194 −53 45 −20 −7 36 59 trt 61 98 −25 24 −11 −13 13 78 tbe 37 42 −15 14 −7 −5 03 25 tet 106 145 −44 39 −21 −15 25 61 bot −27 −3 18 −14 8 0 01 29 teg 110 154 −36 32 −17 −16 26 74 trg4 52 77 −22 23 −15 −25 09 52 wds 290 380 −146 120 −61 2 71 88 tug −19 −31 5 −9 7 19 02 19 AET 23 48 9 5 −5 −55 07 28 MTCO 121 310 −28 58 −43 −99 59 76 MTWA 299 387 −100 104 −65 −107 71 77 E/PE 20 4 −4 11 −11 −22 01 16 P −E 28 −4 −19 13 −8 8 01 17 GDD5 309 488 −88 109 −71 −110 78 87 TANN 279 486 −66 97 −67 −138 78 88 PANN 36 39 −5 14 −10 −42 05 28 patchier and the ENPs should become much larger, thereby diminishing the prediction capability of the model. large diversity of model simulations, in contrast to a single ESM. The third source of errors is the variance of the error terms ej(s) of Eq. (1): the statistical model does not perfectly ﬁt the data. This prompted us to converge the emulator esti- mates to the real world represented by the paleoclimate data. This is known as paleodata assimilation (Goosse et al., 2012). This led to adjusting a few parameters known to have a large uncertainty to optimize temperature and precipitation using information given by proxies (Table 7). J. Guiot et al.: Viticulture extension in response to global climate change drivers The interpretation is not straightforward because the pre- dictors are not the forcing parameters but their PCs. Never- theless, a partial interpretation is possible by using the corre- lation of the PCs with the forcings. For example, PC1 is cor- related with the greenhouse gases (GHGs) and the population size, and Fig. 5 (Tann/PC1) shows that the maximum impact of the GHGs on temperature is in Russia. Figure 5 (Pan- n/PC1) shows that the GHGs have a negative effect on the precipitation around the Mediterranean and northern Africa and a positive effect on Russia. The decrease in precipitation according to the increase in GHGs is a well-known feature in the Mediterranean (Giorgi and Lionello, 2008). Figure 5 also shows that the annual temperature (Tann/Pred vs. Obs) is well-emulated with a strong correlation with CO2. It is also true, but to a lesser extent, for NPPtot. The annual precipita- tion estimates have a much lower variance than the observa- tions. These uncertain parameters are (i) local impact of volcanic activity, (ii) local impact of solar activity, (iii) a possible global bias for the temperature simulation (δT ) and (iv) for the precipitation simulation (δP), and (v) the standard error of temperature (σT ) and (vi) of precipitation (σP ). Param- eters (iii) and (iv) are related to a possible global ESM bias and are then independent of the geographical position. Pa- rameters (v) and (vi) express the mean quadratic difference between observations and simulations and then contain the ESM mean error and the emulator mean error (after remov- ing the effects of biases δT or δP ). The reconstructed annual temperature and precipitation values are expressed as anomalies relative to the present day for each time slice and each box given in Table 7. It shows that the ﬁrst two time slices (4200 and 3200 yr BP) were characterized by dry conditions in the eastern Mediterranean Basin (boxes Lev, Ana). All the other time slices are charac- terized by temperature changes. J. Guiot et al.: Viticulture extension in response to global climate change drivers Indeed, only 9 of 17 have a proportion of Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers 1230 Figure 5. Table 7. Proxy-based reconstruction of annual temperature (TANN, ◦C) and annual precipitation (PANN, mm). The values are expressed as anomalies from the preindustrial period for the 10 spatial boxes and the nine time slices, obtained from pollen (Guiot and Kaniewski, 2015) and corrected or made precise as indicated in Table 3. 2.8 Paleodata assimilation (step 4) The climatic data estimated by the emulator have biases and uncertainties likely larger than those of the ESMs which have generated the calibration data. The ﬁrst source of errors of the emulator is the same as the source of errors of the ESMs, which are imperfect representations of the real world. The second source of errors comes from the oversimpliﬁcation, which consists of replacing a complex model by a statisti- cal model, in considering that a few global forcings are able to generate the complex observed climate ﬁeld. It is partly compensated for by the fact that the emulator is based on a The statistical simpliﬁcation of the emulator makes it pos- sible to run it thousands of times in a relatively short compu- tational time as required by the assimilation methods (Wid- mann et al., 2010). We use a Bayesian approach called the https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers https://doi.org/10.5194/cp-19-1219-2023 2.9 The viticulture index (VI) 2.9 The viticulture index (VI) Markov chain Monte Carlo (MCMC) method, which makes it possible to converge towards the best parameters in the sense of probability distribution (Hargreaves and Annan, 2002). We subsequently applied our emulator to the question of how viticulture has evolved in the Mediterranean region and in response to which climatic stimuli and global forc- ing. Numerous bioclimate indices have been published to delimit viticultural zones in the world (Tonietto and Car- bonneau, 2004; Santos et al., 2012; Howell, 2001). Among them, the following are cited: (1) the sum of degree days above 10 ◦C during the growing season or the heliothermal index of Huglin (HI), (2) the number of days with a min- imum temperature below −17 ◦C, which is very important for the grapes growing in continental climates, (3) the min- imum temperature of September (cool night index) impor- tant for the ripening, (4) the sum of the growing season of the monthly temperature multiplied by monthly precipitation (Hyl), and (5) the drought stress index (DI) related to the po- tential water balance of the soil during the growing season. Malheiro et al. (2010) proposed a composite index (CompI) calculated based on the ratio of years simultaneously satis- fying four criteria (HI > 1400 degree days), DI > −100 mm, Hyl < 5100 ◦C mm−1, and Tmin > −17 ◦C). Starting from the prior probability distributions of the six parameters, available climate reconstructions and model out- puts provide estimates of their posterior probability distribu- tions. For each time period, these six parameters were opti- mized to provide the best posterior probability distribution of mean temperature and precipitation at the centers of the 10 Mediterranean boxes. The prior probability distributions are given by uniform distributions delimited by the ±d (Table 8) for the volcanic and solar activity. The prior distribution of the other parameters was assumed to be uniform within wide ranges so that they were considered noninformative for the purpose of this study. From Tables 3 and 7, we consider the 4200 and 3200 yr BP time slices to provide robust paleoclimatic information for precipitation and the other periods to provide robust informa- tion for temperature. Both the precipitation and temperature signals were robust for the present time slice. Even if we ulti- mately calculate both temperature and precipitation, only the robust variable(s) is (are) used to constrain the emulator. J. Guiot et al.: Viticulture extension in response to global climate change drivers Guiot et al.: Viticulture extension in response to global climate change drivers 1 Figure 5. Markov chain Monte Carlo (MCMC) method, which makes it possible to converge towards the best parameters in the sense of probability distribution (Hargreaves and Annan, 2002). 2.9 The viticulture index (VI) We subsequently applied our emulator to the questio ho itic lt re has e ol ed in the Mediterranean re Figure 5. Figure 5. Table 7. Proxy-based reconstruction of annual temperature (TANN, ◦C) and annual precipitation (PANN, mm). The values are expressed as anomalies from the preindustrial period for the 10 spatial boxes and the nine time slices, obtained from pollen (Guiot and Kaniewski, 2015) and corrected or made precise as indicated in Table 3. TANN WMa EMa Lev Ana CMed Ibe Fra Alp Balk Narb 4200 BP 0.14 0.14 0.02 −0.48 −0.26 0.26 0.62 0.26 -0.6 0.62 3200 BP −0.08 −0.08 −0.15 −0.04 −0.08 −0.02 0.16 0.05 −0.2 0.16 2500 BP 0.24 0.24 0.13 −0.12 −0.2 −0.13 −1.22 −1 −1 −1.22 2000 BP 0.04 0.04 0.18 0.18 0.5 0.5 0.8 0.5 −0.02 0.5 1300 BP 0.03 0.03 −0.14 0.09 −0.25 −0.2 −0.43 −0.43 −0.68 −0.43 1000 BP −0.07 −0.07 −0.17 −0.07 0.07 0.09 0.54 0.41 −0.25 0.54 700 BP −0.07 −0.07 −0.25 0.27 0.09 −0.5 −0.8 −0.8 0.25 −0.8 200 BP −0.3 −0.3 −0.33 −0.15 −0.3 −0.3 −0.5 −0.5 −0.5 −0.5 Present 1.4 1.3 1.2 1.4 1.4 1 1.2 1.5 1.5 1.2 PANN WMa EMa Lev Ana CMed Ibe Fra Alp Balk Narb 4200 BP 48.4 48.4 −80 −88.7 29.94 40.83 18.11 51.92 42.84 18.11 3200 BP −6.84 −6.84 −33 −50.04 4.11 5.13 41.03 34.74 2.82 41.03 2500 BP 61.33 61.33 −34.03 −14.8 25.58 43.78 −8.9 35.35 3.02 −8.9 2000 BP −11.18 −11.18 4.23 10.3 34.61 −2.78 22.4 47.35 10.2 22.4 1300 BP 20.41 20.41 −11.98 −5.78 67.64 27.34 48.13 94.55 36.31 48.13 1000 BP −8.72 −8.72 −24.36 −26.77 40.28 −0.45 24.36 51 39.21 24.36 700 BP 37.51 37.51 −37.45 −7.43 1.94 25.73 −9.59 16.18 −29.73 −9.59 200 BP 68.77 68.77 −37.65 −3.47 −2.94 63.72 48.56 21.99 −12.64 48.56 Present −40 −20 −20 0 −40 −40 −50 0 0 −20 https://doi.org/10.5194/cp-19-1219-2023 https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers 1231 J. Guiot et al.: Viticulture extension in response to global climate change drivers Figure 5. Markov chain Monte Carlo (MCMC) method, which makes it possible to converge towards the best parameters in the sense of probability distribution (Hargreaves and Annan, 2002). Starting from the prior probability distributions of the six parameters, available climate reconstructions and model out- puts provide estimates of their posterior probability distribu- tions. For each time period, these six parameters were opti- mized to provide the best posterior probability distribution of mean temperature and precipitation at the centers of the 10 Mediterranean boxes. The prior probability distributions are given by uniform distributions delimited by the ±d (Table 8) for the volcanic and solar activity. The prior distribution of the other parameters was assumed to be uniform within wide ranges so that they were considered noninformative for the purpose of this study. From Tables 3 and 7, we consider the 4200 and 3200 yr BP time slices to provide robust paleoclimatic information for precipitation and the other periods to provide robust informa- tion for temperature. Both the precipitation and temperature signals were robust for the present time slice. Even if we ulti- mately calculate both temperature and precipitation, only the robust variable(s) is (are) used to constrain the emulator. 2.9 The viticulture index (VI) We subsequently applied our emulator to the how viticulture has evolved in the Mediterran and in response to which climatic stimuli and ing. Numerous bioclimate indices have been p delimit viticultural zones in the world (Toniet bonneau, 2004; Santos et al., 2012; Howell, 200 them, the following are cited: (1) the sum of above 10 ◦C during the growing season or the h index of Huglin (HI), (2) the number of days imum temperature below −17 ◦C, which is ver for the grapes growing in continental climates, imum temperature of September (cool night in tant for the ripening, (4) the sum of the growin the monthly temperature multiplied by monthly p (Hyl), and (5) the drought stress index (DI) relate tential water balance of the soil during the grow Malheiro et al. (2010) proposed a composite ind calculated based on the ratio of years simultane fying four criteria (HI > 1400 degree days), DI > Hyl < 5100 ◦C mm−1, and Tmin > −17 ◦C). Some of the climate variables needed for th were not available from the BIOME4 outputs J. https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers 1232 Figure 5. Local (longitude × latitude) response of a few (bio)climatic variables to the PCs of forcing variables. The colors represent the t values (t-val) of the response (regression coefﬁcient / standard error). Maps 1 to 5 represent the ﬁrst ﬁve PCs. Map 6 represents the R2 of the GWRs. Maps 7 and 8 represent the estimates for low-CO2 time slices (< 290 ppm, i.e., LGM and mid-Holocene) and high-CO2 time slices (> 600 ppm, second half of 21st century with scenario RCP8.5), respectively. The last graphic represents the estimates as a function of the observations: blue dots are for observations with CO2 < 250 ppm, green dots for CO2 belonging to [250, 400] ppm, orange dots for [400, 600] ppm, and red dots for CO2 > 600 ppm. The ﬁrst series of maps is for the net primary production of the ecosystem (NPPtot); the second is for annual temperature (Tann), and the last one is for annual precipitation (Pann). Figure 5. Local (longitude × latitude) response of a few (bio)climatic variables to the PCs of forcing variables. The colors represent the t values (t-val) of the response (regression coefﬁcient / standard error). Maps 1 to 5 represent the ﬁrst ﬁve PCs. Map 6 represents the R2 of the GWRs. Maps 7 and 8 represent the estimates for low-CO2 time slices (< 290 ppm, i.e., LGM and mid-Holocene) and high-CO2 time slices (> 600 ppm, second half of 21st century with scenario RCP8.5), respectively. The last graphic represents the estimates as a function of the observations: blue dots are for observations with CO2 < 250 ppm, green dots for CO2 belonging to [250, 400] ppm, orange dots for [400, 600] ppm, and red dots for CO2 > 600 ppm. The ﬁrst series of maps is for the net primary production of the ecosystem (NPPtot); the second is for annual temperature (Tann), and the last one is for annual precipitation (Pann). Table 8. Forcing variables used for the 15 time slices and scenarios. The forcing greenhouse gas atmospheric concentrations (CO2, CH4, N2O), orbital parameters (ecc: eccentricity, Obl: obliquity, ω), population size (POP) in number of people, volcanic activity with uncer- tainty (d), and solar activity with uncertainty (d). 2.9 The viticulture index (VI) Some of the climate variables needed for these indices were not available from the BIOME4 outputs. However, other variables, such as those associated with the net primary https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers The blue line is the perfect estimate line (R = 1), and the black line is the regression line between the simu- lations and proxy reconstructions. For x = NPPtrop, the net primary production of tropical plants, the interval [xmin, xmax] is [0, 10 kg C m−2]. For x = NPP, the total ecosystem net primary production, it is [500, 1000 kg C m−2]. For x = Pann, the total annual pre- cipitation, it is [400, 800 mm]. For x = MTWA, the mean temperature of the warmest month, it is [18, 23 ◦C]. For x = MTCO, the mean temperature of the coldest month, it is [3, 12 ◦C]. The lower limit of 3 ◦C corresponds to a mini- mum mean monthly temperature with no chance of having an absolute daily minimum below −17 ◦C. For x = α, the actual to potential evapotranspiration ratio, it is [30 %, 60 %]. and solar activities have no signiﬁcant impact, which is eas- ily explained by the dominant GHG effect (not used for con- straining the assimilation). The impact of solar activity is not clear, as there is no signiﬁcant difference between cold and warm periods or between the dry and wet periods. The temperature bias (δT independent of the spatial variability) is estimated to be between 0.6 and 1.6 ◦C for the periods between 2500 and 200 yr BP and is not signiﬁcantly differ- ent from zero for the dry periods and for the present. The δP estimates were not signiﬁcant for any time period. Fig- ure 6 presents the overall correlations between the emulator outputs and the proxy-based reconstructions. A good emula- tion should be indicated by good coherency of both blue and black lines and a low variance of the dots around the black line. So, the temperature was particularly well-simulated by the emulator with a squared correlation (R2) of 0.76, but with an overestimation of the low temperatures. As expected, pre- cipitation is less well-simulated with a signiﬁcant R2 of 0.22 with an underestimation of the large (negative or positive) anomalies. All factors except those related to NPPtrop assume that vine growth is limited only by the lower values and not by the higher values. Because there is no possible viticulture in the tropics (where temperature is not cold enough for an appro- priate dormancy), 1 −INPPtrop is limited by its upper value. J. Guiot et al.: Viticulture extension in response to global climate change drivers 1233 Figure 6. Temperature and precipitation anomalies for the 10 Mediterranean boxes estimated by data assimilation versus data reconstructed from proxies. Temperature dots represent the mean annual temperature in the 10 boxes for the 11 periods from 2500 yr BP until the present, and precipitation dots represent the total annual precipitation in the two oldest periods (4200 and 3200 yr BP) and in the present. The vertical bars are the 90 % con- ﬁdence intervals (CIs). The blue line is the perfect estimate line (R = 1), and the black line is the regression line between the simu- lations and proxy reconstructions. production of plant types, are very interesting because they include the CO2 effect on photosynthesis and water use efﬁ- ciency. Considering only rain-fed viticulture, we propose the following index, VI: production of plant types, are very interesting because they include the CO2 effect on photosynthesis and water use efﬁ- ciency. Considering only rain-fed viticulture, we propose the following index, VI: VI = 1 −INPPtrop · INPP · IPann · IMTWA · IMTCO · Iα, (4) where each of these factors denoted Ix follows the function VI = 1 −INPPtrop · INPP · IPann · IMTWA · IMTCO · Iα, (4) where each of these factors denoted Ix follows the function VI = 1 −INPPtrop · INPP · IPann · IMTWA · IMTCO · Iα, (4) (4) where each of these factors denoted Ix follows the function Ix = 0 for x < xmin Ix = 1 for x > xmax Ix = x −xmin (xmax −xmin) for xmin ≤x ≤xmax. Ix = 0 for x < xmin Ix = 0 for x < xmin Ix = 1 for x > xmax Ix = 1 for x > xmax Ix = x −xmin (xmax −xmin) for xmin ≤x ≤xmax. Figure 6. Temperature and precipitation anomalies for the 10 Mediterranean boxes estimated by data assimilation versus data reconstructed from proxies. Temperature dots represent the mean annual temperature in the 10 boxes for the 11 periods from 2500 yr BP until the present, and precipitation dots represent the total annual precipitation in the two oldest periods (4200 and 3200 yr BP) and in the present. The vertical bars are the 90 % con- ﬁdence intervals (CIs). J. Guiot et al.: Viticulture extension in response to global climate change drivers p This approach is a static approach as it is based on an equilibrium vegetation model only based on the mean state of the climate. The variability of the climate and therefore the extreme values of some variables are not considered. We are aware of these limitations, and it is the reason why our interpretations will only concern the potential extension of viticulture. Other weaknesses of the approach are the im- possibility to consider the diversity of cultivars, the adapta- tion of grapes to frost, and the risk of biotic damage to the grapevines. This is a niche model approach of the same na- ture as those used in the literature cited at the beginning of this section. This oversimpliﬁed approach nevertheless has the advantage of providing a framework for the past and future trends and of being easily applicable to the past for which the data are rather limited. 3 Results This assimilation process was applied to simulate the an- nual temperature and precipitation for the 10 boxes and the 13 time slices and scenarios. The spatial patterns of the re- constructions and simulations are consistent, except for the temperatures at 4200 and 3200 yr BP. (Fig. 7). Therefore, global forcing variables can drive not only the mean cli- mate evolution but also the spatial patterns reconstructed by the proxy data in the Mediterranean. Volcanic activ- J. Guiot et al.: Viticulture extension in response to global climate change drivers Period CO2 CH4 N2O Ecc Obl Omega POP Volc [δ] Sol [δ] 4200 280 650 270 0.0181 23.9 34 64 138 0.2 [0.2] 200 [200] 3200 280 650 270 0.0178 23.8 45 101 664 0.2 [0.2] 200 [200] 2500 280 650 270 0.0175 23.75 65 146 283 0.6 [0.2] 270 [200] 2000 280 650 270 0.0175 23.7 68 188 239 0.2 [0.2] 400 [300] 1300 280 650 270 0.0174 23.65 75 218 535 0.6 [0.2] 270 [200] 1000 280 650 270 0.0173 23.65 75 295 040 0.2 [0.2] 400 [300] 700 280 650 270 0.0175 23.5 90 396 811 0.6 [0.2] 270 [200] 200 280 650 270 0.0169 23.4 95 813 664 0.6 [0.2] 270 [200] Present 400 1730 330 0.0167 23.45 102 7 500 000 0.2 [0.2] 600 [300] +1.5C 440 1527 339 0.0167 23.45 102 8 200 000 0.2 [0.2] 270 [200] +2C 487 1833 350 0.0167 23.45 102 8 200 000 0.2 [0.2] 270 [200] +3C 603 3076 381 0.0167 23.45 102 8 200 000 0.2 [0.2] 270 [200] +5C 900 3700 430 0.0167 23.45 102 12 300 000 0.2 [0.2] 270 [200] 5 CV+ 900 3700 430 0.0167 23.45 102 12 300 000 0.8 [0.2] 100 [200] 5 CV− 900 3700 430 0.0167 23.45 102 12 300 000 0.1 [0.2] 700 [200] https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 3.1 Paleodata assimilation From the posterior distributions (Table 9), it appears that the simulated volcanic activity impacts were the strongest during the cold periods (2500, 1300, 700, and 200 yr BP) and rather weak during the dry periods (4200 and 3200 yr BP) and warm periods (2000 and 1000 yr BP). The conﬁdence intervals are signiﬁcant as they do not overlap. For the present, volcanic https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers Volc Volc.CI Solar Solar.CI δT δT · CI σT σT · CI δP δP · CI σP σP · CI 4200 0.1 [0.1, 0.27] 270 [153, 387] 0.3 [−1.7, 1.7] 0.8 [0.4, 4.7] −15 [−19, 3] 81 [70, 81] 3200 0.15 [0.1, 0.33] 267 [190, 387] −1 [−1.7, 1.7] 2.6 [0.4, 4.7] −20 [−20, −7] 81 [66, 81] 2500 0.4 [0.4, 0.75] 304 [103, 444] 0.6 [0.2, 0.9] 2,1 [1.8, 3.5] 16 [−17, 18] 25 [5, 77] 2000 0.17 [0.1, 0.38] 106 [153, 642] 1.6 [1.1, 1.9] 0.6 [0.9, 2.8] −15 [−18, 18] 53 [5, 75] 1300 0.4 [0.4, 0.76] 324 [121, 452] 1 [0.5, 1.3] 1 [1.1, 2.9] −9 [−18, 18] 45 [6, 76] 1000 0.22 [0.1, 0.39] 141 [150, 660] 1.3 [0.9, 1.7] 0.9 [0.9, 2.7] −3 [−18, 17] 5 [6, 76] 700 0.62 [0.4, 0.78] 468 [109, 453] 1.2 [0.8, 1.6] 0,8 [0.9, 2.7] −5 [−18, 17] 25 [5, 76] 200 0.41 [0.4, 0.77] 450 [97, 458] 1,2 [0.7, 1.5] 0.3 [0.3, 2.6] 4 [−18, 17] 49 [5, 76] Present 0.36 [0.15, 0.4] 345 [307, 548] 0.7 [−0.3, 1.6] 0.3 [0.2, 4.6] −14 [−18.7, 10] 57 [36, 79] 700 0.62 [0.4, 0.78] 468 [109, 453] 1.2 [0.8, 1.6] 0,8 [0.9, 2.7] −5 [−18, 17] 25 [5, 76] 200 0.41 [0.4, 0.77] 450 [97, 458] 1,2 [0.7, 1.5] 0.3 [0.3, 2.6] 4 [−18, 17] 49 [5, 76] Present 0.36 [0.15, 0.4] 345 [307, 548] 0.7 [−0.3, 1.6] 0.3 [0.2, 4.6] −14 [−18.7, 10] 57 [36, 79] Figure 7. Spatial distribution of the temperature and precipitation anomalies for the past and present time slices. The pairs of symbols are simulations (left symbols) and observations or pollen reconstructions (right symbols). The colors are used for the temperature anomalies. Triangles are used for negative precipitation anomalies and circles for the positive precipitation anomalies. The size of the triangles and circles is proportional to the absolute value of the precipitation anomalies. Figure 7. Spatial distribution of the temperature and precipitation anomalies for the past and present time slices. The pairs of symbols are simulations (left symbols) and observations or pollen reconstructions (right symbols). The colors are used for the temperature anomalies. Triangles are used for negative precipitation anomalies and circles for the positive precipitation anomalies. The size of the triangles and circles is proportional to the absolute value of the precipitation anomalies. Figure 7. J. Guiot et al.: Viticulture extension in response to global climate change drivers 1234 Table 9. Posterior distribution of the parameters. The estimates are given by the best ﬁt, and the CI is the 90 % conﬁdence interval calculated on the 10 % best ﬁts. Volc index has no units; the solar index is in megaelectron volts (MeV), T is in degrees Celsius (◦C), and P is in millimeters per year (mm yr−1). Table 9. Posterior distribution of the parameters. The estimates are given by the best ﬁt, and the CI is the 90 % conﬁdence interval calculated on the 10 % best ﬁts. Volc index has no units; the solar index is in megaelectron volts (MeV), T is in degrees Celsius (◦C), and P is in millimeters per year (mm yr−1). Volc Volc.CI Solar Solar.CI δT δT · CI σT σT · CI δP δP · CI σP σP · CI 4200 0.1 [0.1, 0.27] 270 [153, 387] 0.3 [−1.7, 1.7] 0.8 [0.4, 4.7] −15 [−19, 3] 81 [70, 81] 3200 0.15 [0.1, 0.33] 267 [190, 387] −1 [−1.7, 1.7] 2.6 [0.4, 4.7] −20 [−20, −7] 81 [66, 81] 2500 0.4 [0.4, 0.75] 304 [103, 444] 0.6 [0.2, 0.9] 2,1 [1.8, 3.5] 16 [−17, 18] 25 [5, 77] 2000 0.17 [0.1, 0.38] 106 [153, 642] 1.6 [1.1, 1.9] 0.6 [0.9, 2.8] −15 [−18, 18] 53 [5, 75] 1300 0.4 [0.4, 0.76] 324 [121, 452] 1 [0.5, 1.3] 1 [1.1, 2.9] −9 [−18, 18] 45 [6, 76] 1000 0.22 [0.1, 0.39] 141 [150, 660] 1.3 [0.9, 1.7] 0.9 [0.9, 2.7] −3 [−18, 17] 5 [6, 76] 700 0.62 [0.4, 0.78] 468 [109, 453] 1.2 [0.8, 1.6] 0,8 [0.9, 2.7] −5 [−18, 17] 25 [5, 76] 200 0.41 [0.4, 0.77] 450 [97, 458] 1,2 [0.7, 1.5] 0.3 [0.3, 2.6] 4 [−18, 17] 49 [5, 76] Present 0.36 [0.15, 0.4] 345 [307, 548] 0.7 [−0.3, 1.6] 0.3 [0.2, 4.6] −14 [−18.7, 10] 57 [36, 79] Figure 7. Spatial distribution of the temperature and precipitation anomalies for the past and present time slices. The pairs of symbols are simulations (left symbols) and observations or pollen reconstructions (right symbols). The colors are used for the temperature anomalies. Triangles are used for negative precipitation anomalies and circles for the positive precipitation anomalies. The size of the triangles and circles is proportional to the absolute value of the precipitation anomalies. J. Guiot et al.: Viticulture extension in response to global climate change drivers 1235 Figure 8. Spatial distribution of the temperature and precipitation anomalies simulated for future time slices (scenarios). The colors are used for the temperature anomalies. Triangles are used for negative precipitation anomalies and circles for positive precipitation anomalies. The size of the triangles and circles is proportional to the absolute value of the precipitation anomalies. Figure 8. Spatial distribution of the temperature and precipitation anomalies simulated for future time slices (scenarios). The colors are used for the temperature anomalies. Triangles are used for negative precipitation anomalies and circles for positive precipitation anomalies. The size of the triangles and circles is proportional to the absolute value of the precipitation anomalies. To answer the question of whether volcanic activity can mitigate the impact of high GHG emissions, Fig. 8 shows that the temperature distributions of +5 CV+ and 5 CV−are quite similar, meaning that the cooling effect of volcanoes is relatively low in a large GHG emission scenario. The pre- cipitation distributions of +5 CV+ and 5 CV−are similar in the western Mediterranean, and +5 CV+ produced slightly higher precipitation anomalies than +5 CV−in the eastern Mediterranean. are not fully similar, a visual comparison shows pretty good agreement (Fig. 9). So for 2000 BP, Iberia and central Europe were wet in both maps, and no PDSI data were available for the southern and eastern Mediterranean. For 1300 BP, all of Europe was dry in both maps except eastern Spain, which was as wet as at 2000 BP. For 1000 BP, the area surrounding the sea was wet except Greece in both maps, and for 700 BP all areas were wetter than 1000 BP in both maps. J. Guiot et al.: Viticulture extension in response to global climate change drivers Spatial distribution of the temperature and precipitation anomalies for the past and present time slices. The pairs of symbols are simulations (left symbols) and observations or pollen reconstructions (right symbols). The colors are used for the temperature anomalies. Triangles are used for negative precipitation anomalies and circles for the positive precipitation anomalies. The size of the triangles and circles is proportional to the absolute value of the precipitation anomalies. Future projections (Fig. 8) show general warming for the four scenarios (+1.5C, +2C, +3C, +5C) that is particularly strong for +3C and +5C scenarios. The local warming is less strong in the areas inﬂuenced by the Atlantic Ocean (France and the Iberian Peninsula). For precipitation, the sig- nal is weak for +1.5C and +2C with drier and wetter zones and clearer for +3C and +5C (all the symbols are triangles, i.e., negative). Combined with the warming, it is undeniable that water stress will increase considerably with +3C and +5C scenarios. ity seems to be the main driver for the cold periods of the Iron Age (2500 yr BP), the LALIA (1300 yr BP), the LIA (700, 200 yr BP), and the warm periods of the RCO (2000 yr BP) and MCA (1000 yr BP). The main driver of the present warming and drying (present time in Fig. 7) is GHG forcing. For the 4200 and 3200 yr BP periods, the volcanic and solar activity drivers do not seem to be plausible expla- nations for the droughts, at least according to our emulator, and likely the ESMs; this also explains the absence of agree- ment. Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers 1236 Figure 9. Comparison of E/PE reconstructed in this paper with in- dependently reconstructed Palmer Drought Severity Index (PDSI). The PDSI anomalies are based on the 1928–1978 reference period and are reconstructed from tree rings (Cook et al., 2015). E/PE is the ratio of actual to potential evapotranspiration obtained from the emulator (in %). Figure 10. Distribution of the wine sites: (a) European wine re- gions (circles) with the corresponding CompI value for the period of 1980–2009: from Fig. 1e of Fraga et al. (2013). (b) Simulation using the viticulture index calculated using 1961–1990 climate data. Figure 9. Comparison of E/PE reconstructed in this paper with in- dependently reconstructed Palmer Drought Severity Index (PDSI). The PDSI anomalies are based on the 1928–1978 reference period and are reconstructed from tree rings (Cook et al., 2015). E/PE is the ratio of actual to potential evapotranspiration obtained from the emulator (in %). Figure 10. Distribution of the wine sites: (a) European wine re- gions (circles) with the corresponding CompI value for the period of 1980–2009: from Fig. 1e of Fraga et al. (2013). (b) Simulation using the viticulture index calculated using 1961–1990 climate data. Figure 11. Diagram of the application of the emulator to recon- struct and predict the viticulture extension. threshold of 3 ◦C is likely overestimated by about 5 ◦C. So, we consider this index to adequately represent the macrocli- mate of potential vine distribution in western Europe and the Mediterranean region. The variables needed for viticulture in Eq. (4) are provided by the emulator for all time slices studied in the past time slices and for future scenarios. The procedure is sketched in Fig. 11. We modiﬁed the global forcings according to the values of Table 3 for the past and Table 2 for the future, and we thereby simulated, by applying the emulator, viticul- ture extension maps (Fig. 12). Figure 12 shows that during the dry time slices of 4200 and 3200 yr BP, the suitable ar- eas were located between 34 and 46◦N latitudes. During the cold periods (2500, 1300, 700, and 200 yr BP), they occupied approximately the same area between 34 and 47◦N. During the warmer periods (2000 and 1000 yr BP), the southern limit did not change much, but the northern limit reached 49◦N, implying that most of Gaul was suitable for viticulture, as al- ready shown by Bernigaud et al. (2021). 3.3 Independent validation Additional independent validation was completed through comparisons with a tree-ring-based reconstruction of the Palmer Drought Severity Index (PDSI) (Cook et al., 2015) for the last 2 millennia, for which tree-ring data are avail- able. The PDSI reﬂects the spring–summer soil moisture conditions. We compared this variable with the reconstruc- tion of the E/PE (ratio of actual to potential evapotranspira- tion in %) variable provided by BIOME4. E/PE is a mois- ture index which is equal to zero when the soil is fully dry and 100 when it is fully wet. The range of the PDSI is usu- ally between −6 and 6 units. Negative values correspond to conditions drier than normal. Considering that both indices Applied to the mean climate of 1980–2009, the viticulture in- dex (VI) approximately reproduces the area in Europe where viticulture is present (Fraga et al., 2013) (Fig.10). As shown in Fig. 10a, the simulations give approximately the same lim- its, with some noticeable exceptions in Germany as well as the Alpine and Balkan regions, which are likely too continen- tal to pass the MTCO criterion. This is the main limitation of our analysis. The presence of viticulture greatly depends on local factors such as valleys, slopes, and soil, which are not accounted for in our analysis. More generally, these dis- crepancies show that our index is not calibrated for continen- tal regions with winters colder than 3 ◦C (on average). This https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers J. Guiot et al.: Viticulture extension in response to global climate change drivers The present map is warmer than the preindustrial period (200 yr BP slice), which suggests that viticulture is now at its maximum potential ex- tension in 4000 years, up to 51◦N. Because of the drier con- Figure 11. Diagram of the application of the emulator to recon- struct and predict the viticulture extension. ditions, the southern limit has shifted from 34 to 35◦N. Note that these variations only depend on climate changes, with- out any consideration of other factors such as types of soils, which are very important for wine quality. In the future projections, the northern extension of poten- tial viticulture should shift from 51◦N (+1.5C scenario) to 53◦N (+3C scenario) and even above 55◦N (for the +5C Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers The curves represent three bands of latitude: the south- ern band with latitude < 37◦N in red, the center band with lati- tudes between 37 and 44◦N in green, and the northern band with latitudes between 44 and 48◦N in blue). The thin lines show the results for the boxes corresponding to the Iberian Peninsula, Anato- lia, and France (deﬁnition in Fig. 4). The scenarios and time slices are deﬁned in Table 8. The x axis gives the center of the time slices considered or the future scenario. 3. The effect of this volcanic forcing has a clear spatial pat- tern across the Mediterranean Basin. From 2500 yr BP to the present, temperature variations were more signif- icant in the north and in the west than in the southeast (Fig. 7). Fischer et al. (2007) found that northern and western Europe were the coldest and driest areas af- ter an eruption. They also found that southern Europe, North Africa, and the Levant have experienced milder and wetter weather than at present. Part of this pattern might be due to regional feedbacks of vegetation on the climate. Some climate model simulations have shown (Reale and Dirmeyer, 2000) that wetter vegetation dur- ing the RCO may have induced a northward shift of the Intertropical Convergence Zone during the summer over the African continent with an increase in mois- ture in North Africa and Iberia, as well as a decrease in the central Mediterranean (Reale and Shukla, 2000). In the future, the southeast should be relatively less dry and warmer than the northwest, especially in summer, which is consistent with our results (Fig. 8) (Giorgi and Lionello, 2008). Iberian Peninsula. In contrast, potential productive areas will likely expand at latitudes above 50◦N, in the Balkans, and in the Alps, which is a signiﬁcant change considering that our approach underestimates the grapevine potential of continen- tal climates. For the two additional scenarios of high emissions com- bined with extreme volcanic and solar activities, the ques- tion is whether an entirely hypothetical set of strong volcanic events could slow down the decline in viticulture in the south. The answer is slightly positive for Spain, Italy, Greece, and Turkey (green curve increases for +5 CV+ and decreases for +5 CV−), but it is negative in North Africa and the Levant (red curve) because the water stress should remain too sub- stantial. J. Guiot et al.: Viticulture extension in response to global climate change drivers Figure 13. Synthesis of the evolution of viticulture in the Mediter- ranean area. The curves represent three bands of latitude: the south- ern band with latitude < 37◦N in red, the center band with lati- tudes between 37 and 44◦N in green, and the northern band with latitudes between 44 and 48◦N in blue). The thin lines show the results for the boxes corresponding to the Iberian Peninsula, Anato- lia, and France (deﬁnition in Fig. 4). The scenarios and time slices are deﬁned in Table 8. The x axis gives the center of the time slices considered or the future scenario. 2. We showed that the major climatic variations of the last millennia in the Mediterranean Basin can be attributed to volcanic activity, whereas the effects of solar activ- ity were negligible. The effects of volcanic and solar activities have been largely debated, as the reconstruc- tion of temperatures in the last millennium from tree rings has often shown less signiﬁcant cooling than the model simulations (Mann et al., 2012). A more recent tree-based reconstruction (Stoffel et al., 2015) showed substantial summer cooling after the Samalas eruption in 1257 and that of Tambora in 1816, but less intense than simulated. Luterbacher et al. (2016) showed that solar activity had a relatively weak inﬂuence on the Eu- ropean summer temperatures. Thus, our results are in line with the state of the art in the scientiﬁc literature. 2. We showed that the major climatic variations of the last millennia in the Mediterranean Basin can be attributed to volcanic activity, whereas the effects of solar activ- ity were negligible. The effects of volcanic and solar activities have been largely debated, as the reconstruc- tion of temperatures in the last millennium from tree rings has often shown less signiﬁcant cooling than the model simulations (Mann et al., 2012). A more recent tree-based reconstruction (Stoffel et al., 2015) showed substantial summer cooling after the Samalas eruption in 1257 and that of Tambora in 1816, but less intense than simulated. Luterbacher et al. (2016) showed that solar activity had a relatively weak inﬂuence on the Eu- ropean summer temperatures. Thus, our results are in line with the state of the art in the scientiﬁc literature. Figure 13. Synthesis of the evolution of viticulture in the Mediter- ranean area. J. Guiot et al.: Viticulture extension in response to global climate change drivers The effect is also positive in the northern band and Turkey (the blue and cyan curves increase for +5 CV+ and decrease for +5 CV−). In all the cases, the effect was clearly too small to compensate for the GHG effect. 4. Our simulations of climate change effect on viticulture are mostly concerned with larger-scale regional produc- tion systems since they would require nearly full-time engagement of wine growers with high certainty of pro- duction every year, sufﬁcient for speculative trade. For smaller domains and local consumption, it may have also been possible to produce wine under degraded or unstable weather conditions. For example, in England, viticulture continued to be practiced on land owned by the church, even as risks increased due to a wetter cli- mate, with cooler summers and milder winters (Clout, 2013). In most wine regions in western Europe, par- ticularly in France, the grape harvest dates were ad- vanced after the LIA and particularly after the 1940s (Le Roy Ladurie et al., 2006). For example, from 1945 to the beginning of the 21st century, in Chateauneuf du Pape (southern Rhone Valley, France) the harvest date 4. Our simulations of climate change effect on viticulture are mostly concerned with larger-scale regional produc- tion systems since they would require nearly full-time engagement of wine growers with high certainty of pro- duction every year, sufﬁcient for speculative trade. For smaller domains and local consumption, it may have also been possible to produce wine under degraded or unstable weather conditions. For example, in England, viticulture continued to be practiced on land owned by the church, even as risks increased due to a wetter cli- mate, with cooler summers and milder winters (Clout, 2013). In most wine regions in western Europe, par- ticularly in France, the grape harvest dates were ad- vanced after the LIA and particularly after the 1940s (Le Roy Ladurie et al., 2006). For example, from 1945 to the beginning of the 21st century, in Chateauneuf du Pape (southern Rhone Valley, France) the harvest date J. Guiot et al.: Viticulture extension in response to global climate change drivers 1237 p g g Figure 12. Distribution of the viticulture index (Vit. Index) for several time slices of the past (in yr BP) and future scenarios (expresse anomalies of global temperature vs. preindustrial reference). The index is labeled VI and is explained in Eq. (4). White areas indicate w it is impossible to cultivate vine. eral time slices of the past (in yr BP) and future scenarios (expressed as dex is labeled VI and is explained in Eq. (4). White areas indicate where Figure 12. Distribution of the viticulture index (Vit. Index) for several time slices of the past (in yr BP) and future scenarios (expressed as anomalies of global temperature vs. preindustrial reference). The index is labeled VI and is explained in Eq. (4). White areas indicate where it is impossible to cultivate vine. The results are summarized in Fig. 13. In the past, only the warm regions of the southern band (29–37◦N) had a suit- able wine-growing area equivalent to the present. The cen- tral geographical band (37–44◦N) and the northern one (44– 48◦N) underwent sudden changes in the viticulture area, ﬁrst from the cool Iron Age (2500 yr BP) to the RCO (2000 yr BP) and later from the end of the LIA (200 yr BP) to the present. The cold periods are all characterized by a decline in viti- culture at latitudes above 37◦N. For the future projections, northward displacements are likely to be drastic from +3 ◦C global warming. Viticulture potential will likely disappear from North Africa and is set to decrease drastically in the scenario). This would allow viticulture to be possible up to central England, but it would regress in the south due to stronger water stress. In the Iberian Peninsula, only the At- lantic coast should be suitable for cultivating wine grapes un- less there is signiﬁcant irrigation. These projected unfavor- able conditions were in agreement with Fraga et al. (2013) based on other viticulture indices. The 5 CV+ and 5 CV− scenarios appear quite like the 5C scenario, indicating that the effect of high or low volcanic activities should have a weak effect on the potential distribution of viticulture in com- parison to a strong GHG forcing. https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 1238 5 Conclusion We demonstrated the efﬁciency of a statistical emulator based on multiple ESMs and calibrated over a large range of forcing and climate states to link the production of a re- gionally important crop (grapes) to global climate forcing variables. There are two main methodological innovations: (i) past climate simulations are used together with future ones to calibrate a robust emulator, and (ii) this emulator is in- dependent of any single model because it captures what is common among all the available model simulations. But it remains an emulator which captures no more than the infor- mation contained in the ESMs. . Major difﬁculties are forecast for 21st century viticul- ture in Spain and North Africa. These are particularly important for global warming levels of +3 ◦C and more. Caffarra and Eccel (2011) showed that the projected warming should make some mountain sites at approx- imately 1000 m climatically suitable for viticulture be- fore the end of this century. The MCA limit will cer- tainly be passed. However, other factors could become limiting, such as excessively mild winters that enable pest attacks and infections, lack of expertise in vine growing and wine making, and products that are more expensive than the current Mediterranean wines (Clout, 2013). Other limitations are extreme events (late frost, ﬂooding). More frequent and more intense heatwaves will no longer be favorable to viticulture at the present southern Mediterranean limit of its niche. These factors are not considered in our approach. A recent study con- sidering grape varieties showed (Sgubin et al., 2022) that, for global temperature anomalies below 2 ◦C, the mean relative area loss was estimated to be 3.9 % ◦C−1, while for higher values of global warming this loss trend is estimated to be a much larger rate of 17.1 % ◦C−1. This conﬁrms the existence of a safe limit below 2 ◦C of global warming for the European wine-making sector, above which adaptation might become far more chal- lenging. Using it on past time slices, we showed that volcanic ac- tivity is a good predictor of the past temperature variations in the Mediterranean Basin and consequently of the viti- culture productivity. During the warm phases of historical times (the Roman Climate Optimum and the Medieval Cli- mate Anomaly) characterized by low volcanic activity, the viticulture area has shifted northward from 47 to 49◦N. 4 Discussion The discussion considers six points, which concern method- ological innovations as well as contributions to climate sci- ence and viticulture science. 1. We used an emulator approach which recently became popular in the ESM community. In some ways, it has also been used with paleoclimatic data (Cruciﬁx, 2012; Joos et al., 1996),= and to emulate a climate–vegetation system (Foley et al., 2016). But this study is the ﬁrst to use an ensemble of climate models, along with several forcing variables provided by past, present, and future states, to project vegetation conditions from global cli- mate drivers. This has involved solving several technical issues. Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers 1239 global warming (Turkey, northern Europe, the Alps, and the Balkans) and negligible in North Africa. IPCC has assessed the literature concerning the question of whether volcanic eruptions could be analogous for geo- engineering proposals for climate mitigation (Myhre et al., 2013). Independent of their possible negative side effects, solar radiation modiﬁcation (SRM), consisting of injecting sulfur aerosols in the stratosphere like a vol- cano does, is likely not an efﬁcient technique to cool the atmosphere. Indeed, our results show that very strong volcanic activity, even accompanied by low solar activ- ity, should not have any signiﬁcant effects on viticulture extension in comparison to the radiative forcing from anthropogenic GHG emissions. advanced on average from 1 October to 11 Septem- ber (Ganichot, 2002). This change is related to sum- mer warming, but factors related to wine quality, agri- cultural practices, and alcohol content regulation may induce a bias in the interpretation (de Cortázar-Atauri et al., 2010). In the Languedoc region, the potential al- cohol content increased by two degrees from 1984 to 2013 (van Leeuwen and Darriet, 2016). Even if the al- cohol content does not exactly reﬂect the grape yields, earlier harvest dates with a higher sugar content have clearly been related to improved conditions of grape cultivation since the LIA, which is also related to in- creased productivity. Another climate-sensitive symbol of Mediterranean agriculture is olive trees. Moriondo et al. (2013) found three characteristic periods in olive growing, namely the RCO (300 BCE to 400 CE) and the MCA (900 CE to 1200 CE), during which olive-growing areas expanded northward, and the LIA (1400 CE to 1900 CE) during which a contraction was reported. 4 Discussion This is consistent with the variations in viticulture, at least in western Europe and the Mediterranean area as our VI is not properly calibrated for strongly continental cli- mates. References Allen, M. R., Dube, O. P., Solecki, W., Aragón-Durand, F., Cramer, W., Humphreys, S., Kainuma, M., Kala, J., Mahowald, N., Mu- lugetta, Y., Perez, R., Wairiu, M., and Zickfeld, K.: Framing and Context, in: Global Warming of 1.5 ◦C, in: An IPCC Spe- cial Report on the impacts of global warming of 1.5 ◦C above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, edited by: Masson-Delmotte, V., Zhai, P., Pörtner, H.-O., Roberts, D., Skea, J., Shukla, P. R., Pi- rani, A., Moufouma-Okia, W., Péan, C., Pidcock, R., Connors, S., Matthews, J. B. R., Chen, Y., Zhou, X., Gomis, M. I., Lon- noy, E., Maycock, T., Tignor, M., and Water, T., Cambridge Uni- versity Press, Cambridge, UK and New York, NY, USA, 49–92, https://doi.org/10.1017/9781009157940.003, 2018. Büntgen, U., Tegel, W., Nicolussi, K., McCormick, M., Frank, D., Trouet, V., Kaplan, J. O., Herzig, F., Heussner, K.-U., Wanner, H., Luterbacher, J., and Esper, J.: 2500 years of European cli- mate variability and human susceptibility, Science, 331, 578– 582, https://doi.org/10.1126/science.1197175, 2011. Büntgen, U., Myglan, V. S., Ljungqvist, F. C., Mccormick, M., Di Cosmo, N., Sigl, M., Jungclaus, J., Wagner, S., Krusic, P. J., Esper, J., Kaplan, J. O., De Vaan, M. A. C., Luterbacher, J., Wacker, L., and Tegel, W.: Cooling and societal change during the Late Antique Little Ice Age from 536 to around 660 AD, Nat. Geosci., 9, 231–236, https://doi.org/10.1038/NGEO2652, 2016. Caffarra, A. and Eccel, E.: Projecting the impacts of climate change on the phenology of grapevine in a mountain area, Aust. J. Grape Wine Res., 17, 52–61, https://doi.org/10.1111/j.1755- 0238.2010.00118.x, 2011. Amara, R.: Views on futures research methodology, Futures, 23, 645–649, https://doi.org/10.1016/0016-3287(91)90085-G, 1991. Castruccio, S., McInerney, D. J., Stein, M. L., Crouch, F. L., Jacob, R. L., and Moyer, E. J.: Statistical emulation of climate model projections based on precomputed GCM runs, J. Climate, 27, 1829–1844, https://doi.org/10.1175/JCLI-D-13-00099.1, 2014. Berger, A. and Loutre, M. F.: Insolation values for the climate of the last 10,000,000 years, Quaternary Sci. Rev., 10, 297–317, 1991. Bernigaud, N., Bondeau, A., and Guiot, J.: Understanding the de- velopment of viticulture in Roman Gaul during and after the Roman climate optimum: The contribution of spatial analysis and agro-ecosystem modeling, J. Archaeol. Sci. Rep., 38, 1–9, https://doi.org/10.1016/j.jasrep.2021.103099, 2021. Chandler, R. E.: Exploiting strength, discounting weak- ness: Combining information from multiple climate simulators, Philos. T. Roy. Soc. J. Guiot et al.: Viticulture extension in response to global climate change drivers 1240 study and reviewed and edited the paper. WC reviewed and edited the paper. Bouby, L., Wales, N., Jalabadze, M., Rusishvili, N., Bonhomme, V., Ramos-Madrigal, J., Evin, A., Ivorra, S., Lacombe, T., Pag- noux, C., Boaretto, E., Gilbert, M. T. P., Bacilieri, R., Lord- kipanidze, D., and Maghradze, D.: Tracking the history of grape cultivation in Georgia combining geometric morphomet- rics and ancient DNA, Veg. Hist. Archaeobot., 30, 63–76, https://doi.org/10.1007/s00334-020-00803-0, 2021. Competing interests. The contact author has declared that none of the authors has any competing interests. Bounceur, N., Cruciﬁx, M., and Wilkinson, R. D.: Global sensitivity analysis of the climate-vegetation system to astronomical forc- ing: An emulator-based approach, Earth Syst. Dynam., 6, 205– 224, https://doi.org/10.5194/esd-6-205-2015, 2015. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Braconnot, P., Harrison, S. P., Kageyama, M., Bartlein, P. J., Masson-Delmotte, V., Abe-Ouchi, A., Otto-Bliesner, B., and Zhao, Y.: Evaluation of climate models us- ing palaeoclimatic data, Nat. Clim. Change, 2, 417–424, https://doi.org/10.1038/nclimate1456, 2012. Acknowledgements. Charles La Via edited the English in an ear- lier version of the paper. Brayshaw, D. J., Hoskins, B., and Black, E.: Some physical drivers of changes in the winter storm tracks over the North Atlantic and Mediterranean during the Holocene, Philos. T. Roy. Soc. A, 368, 5185–5223, https://doi.org/10.1098/rsta.2010.0180, 2010. Financial support. This work has been funded by the Excellence Initiative of Aix-Marseille University A∗MIDEX, a French “In- vestissements d’Avenir” program (project RDMED), and the Labex OT-Med project (project ANR-11-LABEX-0061). Brown, A. G., Meadows, I., Turner, S. D., and Mattingley, D.: Ro- man vineyards in Britain: stratigraphic and palynological data from Wollaston in the Nene Valley, England, Antiquity, 75, 745– 757, 2001. Review statement. This paper was edited by Martin Claussen and reviewed by two anonymous referees. Brun, J.-P.: 15. Viticulture et oléiculture en Gaule, in: Comment les Gaules devinrent romaines, edited by: Ouzoulias, P., La Décou- verte, Paris, 231–253, 2010. Brunsdon, C., Fotheringham, S., and Charlton, M.: Geographically weighted regression – Modelling spatial non-stationarity, J. Roy. Stat. Soc. Ser. D, 47, 431–443, https://doi.org/10.1111/1467- 9884.00145, 1998. 5 Conclusion This historical limit has already been passed at the present time as it has already shifted to 51◦N, and with global warming of +3 ◦C, it should pass the 53◦N limit. Even if, in the warm periods of the past, North African and Iberian farmers never had real difﬁculties in cultivating grapes, they will meet large difﬁculties with global warming of +3 ◦C or more, except on the Atlantic margin. Moreover, our sensitivity experiments show that even intense volcanic activity is not sufﬁcient to stop this decline. Code and data availability. All data, codes, and materials used in the analyses are available at https://github.com/douane7/C5P3_ B4_emul.git (douane7, 2023). 6. It is not very likely that intense volcanic activity could slow down this decline because (despite the many other negative consequences of such events) the beneﬁcial climatic effects of this activity would be highest in regions with increased wine-growing potential under 6. It is not very likely that intense volcanic activity could slow down this decline because (despite the many other negative consequences of such events) the beneﬁcial climatic effects of this activity would be highest in regions with increased wine-growing potential under Author contributions. JG designed the study, did the formal analyses, performed the visualisations, and wrote the draft of the paper. AB, NB, and LB participated in the conceptualisation of the https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers Earth Sci., 3, 28, https://doi.org/10.3389/feart.2015.00028, 2015. douane7: C5P3_B4_emul, GitHub [code and data set], https:// github.com/douane7/C5P3_B4_emul (last access: 16 June 2023), 2023. Guiot, J., Torre, F., Jolly, D., Peyron, O., Boreux, J. J. J., Cheddadi, R., Borreux, J. J., and Cheddadi, R.: Inverse vegetation model- ing by Monte Carlo sampling to reconstruct paleoclimate under changed precipitation seasonality and CO2 conditions: applica- tion to glacial climate in Mediterranean region, Ecol. Model., 127, 119–140, https://doi.org/10.1016/S0304-3800(99)00219-7, 2000. Finné, M., Holmgren, K., Sundqvist, H. S., Weiberg, E., and Lind- blom, M.: Climate in the eastern Mediterranean, and adjacent regions, during the past 6000 years – A review, J. Archaeol. Sci., 38, 3153–3173, https://doi.org/10.1016/j.jas.2011.05.007, 2011. Fischer, E. a., Luterbacher, J., Zorita, E., Tett, S. F. B., Casty, C., and Wanner, H.: European climate response to tropical volcanic eruptions over the last half millennium, Geophys. Res. Lett., 34, 1–6, https://doi.org/10.1029/2006GL027992, 2007. Hargreaves, J. and Annan, J.: Assimilation of paleo-data in simple Earth system model, Clim. Dynam., 19, 371–381, https://doi.org/10.1007/s00382-002-0241-0, 2002. Foley, A. M., Holden, P. B., Edwards, N. R., Mercure, J.-F., Salas, P., Pollitt, H., and Chewpreecha, U.: Climate model emulation in an integrated assessment framework: a case study for mitigation policies in the electricity sector, Earth Syst. Dynam., 7, 119–132, https://doi.org/10.5194/esd-7-119-2016, 2016. Holzhauser, H., Magny, M., and Zumbuhl, H. J.: Glacier and lake-level variations in west-central Eu- rope over the last 3500 years, Holocene, 15, 789–801, https://doi.org/10.1191/0959683605hl853ra, 2005. Howell, S. G.: Sustainable Grape Productivity and the Growth- Yield Relationship, Am. J. Enol. Vitic., 52, 165–174, 2001. Fraga, H., Malheiro, A. C., Moutinho-Pereira, J., and Santos, J. A.: Future scenarios for viticultural zoning in Europe: Ensemble projections and uncertainties, Int. J. Biometeorol., 57, 909–925, https://doi.org/10.1007/s00484-012-0617-8, 2013. Izdebski, A., Holmgren, K., Weiberg, E., Stocker, S. R., Bunt- gen, U., Florenzano, A., Gogou, A., Leroy, S. A. G., Luter- bacher, J., Martrat, B., Masi, A., Mercuri, A. M., Mon- tagna, P., Sadori, L., Schneider, A., Sicre, M. A., Trianta- phyllou, M., and Xoplaki, E.: Realising consilience: How better communication between archaeologists, historians and natural scientists can transform the study of past climate change in the Mediterranean, Quaternary Sci. Rev., 136, 5–22, https://doi.org/10.1016/j.quascirev.2015.10.038, 2016. Franklin, J., Serra-Diaz, J. M., Syphard, A. D., and Re- gan, H. M.: Global change and terrestrial plant commu- nity dynamics, P. Natl. Acad. Sci. USA, 113, 3725–3734, https://doi.org/10.1073/pnas.1519911113, 2016. Frieler, K., Lange, S., Piontek, F., Reyer, C. P. J. Guiot et al.: Viticulture extension in response to global climate change drivers J. Guiot et al.: Viticulture extension in response to global climate change drivers 1241 and coins: consiliences of palaeoclimate and economy in the Late Antique southern Levant, Levant, 49, 205–223, https://doi.org/10.1080/00758914.2017.1379181, 2017. Hayles, L., Baillie, M., Baittinger, C., Bleicher, N., Bonde, N., Brown, D., Carrer, M., Cooper, R., Eùfar, K., DIttmar, C., Esper, J., Griggs, C., Gunnarson, B., Günther, B., Gutierrez, E., Haneca, K., Helama, S., Herzig, F., Heussner, K. U., Hof- mann, J., Janda, P., Kontic, R., Köse, N., Kyncl, T., Levaniè, T., Linderholm, H., Manning, S., Melvin, T. M., Miles, D., Neuwirth, B., Nicolussi, K., Nola, P., Panayotov, M., Popa, I., Rothe, A., Seftigen, K., Seim, A., Svarva, H., Svoboda, M., Thun, T., Timonen, M., Touchan, R., Trotsiuk, V., Trouet, V., Walder, F., Wany, T., Wilson, R., and Zang, C.: Old World megadroughts and pluvials during the Common Era, Sci. Adv., 1, 37, https://doi.org/10.1126/sciadv.1500561, 2015. Ganichot, B.: Évolution de la date des vendanges dans les Côtes du Rhône méridionales, in: Actes des 6e Rencontres rhodaniennes, Institut Rhodanien, Orange, France, 38–41, 2002. Giorgi, F. and Lionello, P.: Climate change projections for the Mediterranean region, Global Planet. Change, 63, 90–104, https://doi.org/10.1016/j.gloplacha.2007.09.005, 2008. Goosse, H., Guiot, J., Mann, M. E. M. E., Dubinkina, S., and Sallaz-Damaz, Y.: The medieval climate anomaly in Europe: Comparison of the summer and annual mean signals in two reconstructions and in simulations with data assimilation, Global Planet. Change, 84–85, 35–47, https://doi.org/10.1016/j.gloplacha.2011.07.002, 2012. Crowley, T. J. and Unterman, M. B.: Technical details concern- ing development of a 1200 yr proxy index for global volcanism, Earth Syst. Sci. Data, 5, 187–197, https://doi.org/10.5194/essd- 5-187-2013, 2013. Grouillet, B., Ruelland, D., Ayar, P. V., and Vrac, M.: Sensitivity analysis of runoff modeling to statistical downscaling models in the western Mediterranean, Hydrol. Earth Syst. Sci., 20, 1031– 1047, https://doi.org/10.5194/hess-20-1031-2016, 2016. Cruciﬁx, M.: Traditional and novel approaches to palaeo- climate modelling, Quaternary Sci. Rev., 57, 1–16, https://doi.org/10.1016/j.quascirev.2012.09.010, 2012. Guiot, J. and Cramer, W.: Climate change: The 2015 Paris Agree- ment thresholds and Mediterranean basin ecosystems, Science, 354, 4528–4532, https://doi.org/10.1126/science.aah5015, 2016. de Cortázar-Atauri, I. G., Daux, V., Garnier, E., Yiou, P., Viovy, N., Seguin, B., Boursiquot, J. M., Parker, A. K., va Leeuwen, C., and Chuine, I.: Climate reconstructions from grape harvest dates: Methodology and uncertainties, Holocene, 20, 599–608, https://doi.org/10.1177/0959683609356585, 2010. Guiot, J. and Kaniewski, D.: The Mediterranean Basin and Southern Europe in a warmer world: What can we learn from the past?, Front. References A, 371, 20120388, https://doi.org/10.1098/rsta.2012.0388, 2013. Bouby, L., Marinval, P., and Terral, J.-F.: From secondary to speculative ProductIon? the Protohistory history of viticulture in Southern France, in: Plants and People: choices and diver- sity through time, edited by: Chevalier, A., Marinova, E., and Chocarro, L. P., Oxbow Book, London, Philadelphia, 175–181, https://doi.org/10.2307/1309151, 2014. Clout, H.: An Overview of the Fluctuating Fortunes of Viticulture in England and Wales, EchoGeo, 23, https://doi.org/10.4000/echogeo.13333, 2013. Cook, E. R., Seager, R., Kushnir, Y., Briffa, K. R., Büntgen, U., Frank, D., Krusic, P. J., Tegel, W., Schrier, G. Vander, Andreu- https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers J. Guiot et al.: Viticulture extension in response to global climate change drivers 1242 Ljungqvist, F. C. C., Büntgen, U., Zorita, E., Wagner, S., Es- per, J., McCarroll, D., Toreti, A., Frank, D., Jungclaus, J. H. H., Barriendos, M., Bertolin, C., Bothe, O., Brázdil, R., Camuffo, D., Dobrovolný, P., Gagen, M., García-Bustamante, E., Ge, Q., Gómez-Navarro, J. J. J., Guiot, J., Hao, Z., Hegerl, G. C. C., Holmgren, K., Klimenko, V. V. V, Martín-Chivelet, J., Pﬁster, C., Roberts, N., Schindler, A., Schurer, A., Solomina, O., Von Gunten, L., Wahl, E., Wanner, H., Wetter, O., Xoplaki, E., Yuan, N., Zanchettin, D., Zhang, H., and Zerefos, C.: European sum- mer temperatures since Roman times, Environ. Res. Lett., 11, 024001, https://doi.org/10.1088/1748-9326/11/2/024001, 2016. of complex oceanic and biospheric models of anthropogenic car- bon uptake, Tellus B, 48, 397–417, 1996. Kaniewski, D., Guiot, J., and Van Campo, E.: Drought and soci- etal collapse 3200 years ago in the Eastern Mediterranean: A review, Wiley Interdisciplin. Rev. Clim. Change, 6, 369–382, https://doi.org/10.1002/wcc.345, 2015. Kaniewski, D., Marriner, N., Cheddadi, R., Guiot, J., and Van Campo, E.: The 4.2 ka BP event in the Levant, Clim. Past, 14, 1529–1542, https://doi.org/10.5194/cp-14-1529-2018, 2018. Kaplan, J. O., Prentice, I. C., and Buchmann, N.: The stable car- bon isotope composition of the terrestrial biosphere: Modeling at scales from the leaf to the globe, Global Biogeochem. Cy., 16, 8-1–8-11, https://doi.org/10.1029/2001gb001403, 2002. Magny, M., Combourieu-Nebout, N., De Beaulieu, J. L., Bout- Roumazeilles, V., Colombaroli, D., Desprat, S., Francke, A., Joannin, S., Ortu, E., Peyron, O., Revel, M., Sadori, L., Siani, G., Sicre, M. A., Samartin, S., Simonneau, A., Tinner, W., Vanniere, B., Wagner, B., Zanchetta, G., Anselmetti, F., Brugiapaglia, E., Chapron, E., Debret, M., Desmet, M., Didier, J., Essallami, L., Galop, D., Gilli, A., Haas, J. N., Kallel, N., Millet, L., Stock, A., Turon, J. L., and Wirth, S.: North-south palaeohydrological contrasts in the central mediterranean during the holocene: Ten- tative synthesis and working hypotheses, Clim. Past, 9, 2043– 2071, https://doi.org/10.5194/cp-9-2043-2013, 2013. Kay, J. E., Deser, C., Phillips, A., Mai, A., Hannay, C., Strand, G., Arblaster, J. M., Bates, S. C., Danabasoglu, G., Edwards, J., Holland, M., Kushner, P., Lamarque, J. J. Guiot et al.: Viticulture extension in response to global climate change drivers F., Lawrence, D., Lindsay, K., Middleton, A., Munoz, E., Neale, R., Oleson, K., Polvani, L., and Vertenstein, M.: The Community Earth Sys- tem Model (CESM) Large Ensemble Project: A Community Re- source for Studying Climate Change in the Presence of Inter- nal Climate Variability, B. Am. Meteorol. Soc., 96, 1333–1349, https://doi.org/10.1175/BAMS-D-13-00255.1, 2014. Kennedy, M. C. and O’Hagan, A.: Predicting the output from a complex computer code when fast approximations are available, Biometrika, 87, 1–13, https://doi.org/10.1093/biomet/87.1.1, 2000. Malheiro, A. C., Santos, J. A., Fraga, H., and Pinto, J. G.: Climate change scenarios applied to viticultural zoning in Europe, Clim. Res., 43, 163–177, https://doi.org/10.3354/cr00918, 2010. Mann, M. E., Fuentes, J. D., and Rutherford, S.: Underes- timation of volcanic cooling in tree-ring-based reconstruc- tions of hemispheric temperatures, Nat. Geosci., 5, 202–205, https://doi.org/10.1038/ngeo1394, 2012. Klein Goldewijk, K., Beusen, A., Van Drecht, G., and De Vos, M.: The HYDE 3.1 spatially explicit database of human-induced global land-use change over the past 12,000 years, Global Ecol. Biogeogr., 20, 73–86, https://doi.org/10.1111/j.1466- 8238.2010.00587.x, 2011. Mccormick, M., Büntgen, U., Cane, M. A., Cook, E. R., Harper, K., Huybers, P., Litt, T., Manning, S. W., Mayewski, P. A., More, A. F. M., Nicolussi, K., and Tegel, W.: Climate Change During & After the Roman Empire, J. Interdiscip. Hist., xliii, 169–220, https://doi.org/10.1162/JINH_a_00379, 2012. Lavigne, F., Degeai, J.-P., Komorowski, J.-C., Guillet, S., Robert, V., Lahitte, P., Oppenheimer, C., Stoffel, M., Vidal, C. M., Surono, Pratomo, I., Wassmer, P., Hajdas, I., Hadmoko, D. S., and de Belizal, E.: Source of the great A.D. 1257 mys- tery eruption unveiled, Samalas volcano, Rinjani Volcanic Com- plex, Indonesia, P. Natl. Acad. Sci. USA, 110, 16742–16747, https://doi.org/10.1073/pnas.1307520110, 2013. Moriondo, M., Trombi, G., Ferrise, R., Brandani, G., Dibari, C., Ammann, C. M., Lippi, M. M., and Bindi, M.: Olive trees as bio-indicators of climate evolution in the Mediterranean Basin, Global Ecol. Biogeogr., 22, 818–833, https://doi.org/10.1111/geb.12061, 2013. Le Roy Ladurie, E., Daux, V., and Luterbacher, J.: Vignes et ven- danges des XIV–XXIe siècles, 25e année, Hist. économie so- ciété, 421–436, https://doi.org/10.3917/hes.063.0421, 2006. Moss, R. H., Edmonds, J. a, Hibbard, K. a, Manning, M. R., Rose, S. K., van Vuuren, D. P., Carter, T. R., Emori, S., Kainuma, M., Kram, T., Meehl, G. a, Mitchell, J. F. B., Nakicenovic, N., Ri- ahi, K., Smith, S. J., Stouffer, R. J., Thomson, A. M., Weyant, J. P., and Wilbanks, T. J. Guiot et al.: Viticulture extension in response to global climate change drivers O., Schewe, J., Warszawski, L., Zhao, F., Chini, L., Denvil, S., Emanuel, K., Geiger, T., Halladay, K., Hurtt, G., Mengel, M., Murakami, D., Ostberg, S., Popp, A., and Riva, R.: Assessing the impacts of 1.5 ◦C global warming – simulation protocol of the Inter-Sectoral Impact Model Intercomparison Project (ISIMIP2b), Geosci. Model Dev., 10, 4321–4345, https://doi.org/10.5194/gmd-10- 4321-2017, 2017. Jones, P. D., Lister, D. H., Osborn, T. J., Harpham, C., Salmon, M., and Morice, C. P.: Hemispheric and large-scale land- surface air temperature variations: An extensive revision and an update to 2010, J. Geophys. Res.-Atmos., 117, D05127, https://doi.org/10.1029/2011JD017139, 2012. Joos, F., Bruno, M., Fink, R., Siegenthaler, U., Stocker, T., LeQuere, C., and Sarmiento, J. L.: An efﬁcient and accurate representation Fuks, D., Ackermann, O., Ayalon, A., Bar-Matthews, M., Bar-Oz, G., Levi, Y., Maeir, A. M., Weiss, E., Zilber- man, T., and Safrai, Z.: Dust clouds, climate change Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers and Goldstein, M.: Climate Simulators and Cli- mate Projections, Annu. Rev. Stat. Appl., 1, 103–123, https://doi.org/10.1146/annurev-statistics-022513-115652, 2014. Tonietto, J. and Carbonneau, A.: A multicriteria cli- matic classiﬁcation system for grape-growing re- gions worldwide, Agr. Forest Meteorol., 124, 81–97, https://doi.org/10.1016/j.agrformet.2003.06.001, 2004. Santos, J. A., Malheiro, A. C., Pinto, J. G., and Jones, G. V.: Macroclimate and viticultural zoning in Europe: Ob- served trends and atmospheric forcing, Clim. Res., 51, 89–103, https://doi.org/10.3354/cr01056, 2012. Tran, G. T., Oliver, K. I. C., Sóbester, A., Toal, D. J. J., Holden, P. B., Marsh, R., Challenor, P., and Edwards, N. R.: Build- ing a traceable climate model hierarchy with multi-level em- ulators, Adv. Stat. Climatol. Meteorol. Oceanogr., 2, 17–37, https://doi.org/10.5194/ascmo-2-17-2016, 2016. Sgubin, G., Swingedouw, D., Mignot, J., Alan, G., Benjamin, G., Harilaos, B., Thomas, L., Pieri, P., García, I., Ollat, A. N., and Van Leeuwen, C.: Linear loss of suitable wine regions over Eu- rope in response to increasing global warming, Global Change Biol., 29, 808–826, https://doi.org/10.1111/gcb.16493, 2022. van Leeuwen, C. and Darriet, P.: The Impact of Climate Change on Viticulture and Wine Quality, J. Wine Econ., 11, 150–167, https://doi.org/10.1017/jwe.2015.21, 2016. Shariﬁ, A., Pourmand, A., Canuel, E. A., Ferer-Tyler, E., Peter- son, L. C., Aichner, B., Feakins, S. J., Daryaee, T., Djamali, M., Beni, A. N., Lahijani, H. A. K., and Swart, P. K.: Abrupt climate variability since the last deglaciation based on a high-resolution, multi-proxy peat record from NW Iran: The hand that rocked the Cradle of Civilization?, Quaternary Sci. Rev., 123, 215–230, https://doi.org/10.1016/j.quascirev.2015.07.006, 2015. van Vuuren, D. P., Isaac, M., Kundzewicz, Z. W., Arnell, N., Barker, T., Criqui, P., Berkhout, F., Hilderink, H., Hinkel, J., Hof, A., Kitous, A., Kram, T., Mechler, R., and Scrieciu, S.: The use of scenarios as the basis for combined assessment of climate change mitigation and adaptation, Global Environ. Change, 21, 575–591, https://doi.org/10.1016/j.gloenvcha.2010.11.003, 2011. Sigl, M., Winstrup, M., McConnell, J. R., Welten, K. C., Plun- kett, G., Ludlow, F., Büntgen, U., Caffee, M., Chellman, N., Dahl-Jensen, D., Fischer, H., Kipfstuhl, S., Kostick, C., Maselli, O. J., Mekhaldi, F., Mulvaney, R., Muscheler, R., Pasteris, D. R., Pilcher, J. R., Salzer, M., Schüpbach, S., Steffensen, J. P., Vinther, B. M., and Woodruff, T. E.: Timing and climate forc- ing of volcanic eruptions for the past 2,500 years, Nature, 523, 543–549, https://doi.org/10.1038/nature14565, 2015. Wanner, H., Beer, J., Bütikofer, J., Crowley, T. J. Guiot et al.: Viticulture extension in response to global climate change drivers 1243 University Press, Cambridge, UK and New York, NY, USA, 659– 740, https://doi.org/10.1017/CBO9781107415324.018, 2013. University Press, Cambridge, UK and New York, NY, USA, 659– 740, https://doi.org/10.1017/CBO9781107415324.018, 2013. Strassmann, K. M. and Joos, F.: The Bern Simple Climate Model (BernSCM) v1.0: An extensible and fully documented open- source re-implementation of the Bern reduced-form model for global carbon cycle-climate simulations, Geosci. Model Dev., 11, 1887–1908, https://doi.org/10.5194/gmd-11-1887-2018, 2018. Neumann, J. and Parpola, S.: Climatic Change and the Eleventh- Tenth-Century Eclipse of Assyria and Babylonia, J. Near East. Stud., 46, 161–182, 1987. New, M., Lister, D., Hulme, M., and Makin, I.: A high-resolution data set of surface climate over global land areas, Clim. Res., 21, 1–25, https://doi.org/10.3354/cr021001, 2002. Su, B., Huang, J., Gemmer, M., Jian, D., Tao, H., Jiang, T., and Zhao, C.: Statistical downscaling of CMIP5 multi-model ensemble for projected changes of climate in the Indus River Basin, Atmos. Res., 178, 138–149, https://doi.org/10.1016/j.atmosres.2016.03.023, 2016. Prentice, I. C., Cramer, W., Harrison, S. P., Leemans, R., Monserud, R. A., and Solomon, A. M.: A global biome model based on plant physiology and dominance, soil properties and climate, J. Bio- geogr., 19, 117–134, 1992. Taylor, K. E., Stouffer, R. J., and Meehl, G. A.: An overview of CMIP5 and the experiment design, B. Am. Meteorol. Soc., 93, 485–498, https://doi.org/10.1175/BAMS-D-11-00094.1, 2012. Reale, O. and Dirmeyer, P.: Modeling the effects of vegetation on Mediterranean climate during the Roman Classical Period. Part I: Climate history and model sensitivity, Global Planet. Change, 25, 163–184, https://doi.org/10.1016/S0921-8181(00)00002-3, 2000. Telelis, I.: Climatic Fluactions in the Eastern Mediterranean and the Middle East AD 300–1500 from Byzantine Doc- umentary and Proxy Physical Paleoclimatic Evidence – A Comparison, Jahrb. Österreich. Byzantin., 58, 167–208, https://doi.org/10.1553/joeb58s167, 2008. Reale, O. and Shukla, J.: Modeling the effects of vegetation on Mediterranean climate during the Roman Classical Period: Part II. Model simulation, Global Planet. Change, 25, 185–214, https://doi.org/10.1016/S0921-8181(00)00003-5, 2000. Terral, J.-F., Tabard, E., Bouby, L., Ivorra, S., Pastor, T., Figueiral, I., Picq, S., Chevance, J.-B., Jung, C., Fabre, L., Tardy, C., Com- pan, M., Bacilieri, R., Lacombe, T., and This, P.: Evolution and history of grapevine (Vitis vinifera) under domestication: new morphometric perspectives to understand seed domestica- tion syndrome and reveal origins of ancient European cultivars, Ann. Bot., 105, 443–455, https://doi.org/10.1093/aob/mcp298, 2010. Roberts, N., Brayshaw, D., Kuzucuo˘glu, C., Perez, R., and Sadori, L.: The mid-Holocene climatic transition in the Mediterranean: causes and consequences, Holocene, 21, 3–13, https://doi.org/10.1177/0959683610388058, 2011. Rougier, J. J. Guiot et al.: Viticulture extension in response to global climate change drivers J.: The next generation of scenarios for climate change research and assessment, Nature, 463, 747–756, https://doi.org/10.1038/nature08823, 2010. Levavasseur, G., Vrac, M., Roche, D. M., Paillard, D., Martin, A., and Vandenberghe, J.: Present and LGM permafrost from climate simulations: Contribution of statistical downscaling, Clim. Past, 7, 1225–1246, https://doi.org/10.5194/cp-7-1225-2011, 2011. Leveau, P.: Les conditions environnementales dans le nord de l’Afrique à l’époque romaine. Contribution historiographique à l’histoire du climat et des relations homme/milieu, in: Sociétés et climats dans l’Empire romain. Pour une perspective historique et systémique de la gestion des ressources en eau dans l’Empire ro- main, edited by: Hermon, E., Editoriale Scientiﬁca, Naples, 309– 348, 2009. Muscheler, R., Joos, F., Beer, J., Müller, S. A., Vonmoos, M., and Snowball, I.: Solar activity during the last 1000 yr inferred from radionuclide records, Quaternary Sci. Rev., 26, 82–97, https://doi.org/10.1016/j.quascirev.2006.07.012, 2007. Myhre, G., Shindell, D., Bréon, F.-M., Collins, W., Fuglestvedt, J., Huang, J., Koch, D., Lamarque, J.-F., Lee, D., Mendoza, B., Nakajima, T., Robock, A., Stephens, G., Takemura, T., and Zhang, H.: Anthropogenic and Natural Radiative Forcing, in: Climate Change 2013: The Physical Science Basis, Contribution of Working Group I to the Fifth Assessment Report of the Inter- governmental Panel on Climate Change, edited by: Stocker, T. F., Qin, D., Plattner, G.-K., Tignor, M., Allen, S. K., Boschung, J., Nauels, A., Xia, Y., Bex, V., and Midgley, P. M., Cambridge Lu, B., Charlton, M., Harris, P., and Fotheringham, A. S.: Geographically weighted regression with a non- Euclidean distance metric: A case study using hedonic house price data, Int. J. Geogr. Inf. Sci., 28, 660–681, https://doi.org/10.1080/13658816.2013.865739, 2014. Luterbacher, J., Werner, J. P. P., Smerdon, J. E. E., Fernández- Donado, L., González-Rouco, F. J. J., Barriopedro, D., Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 g g p g g Zobler, L.: A world soil ﬁle grobal climate modeling, NASA Tech. Memo 32, NASA, 1986. J. Guiot et al.: Viticulture extension in response to global climate change drivers J., Cubasch, U., Flückiger, J., Goosse, H., Grosjean, M., Joos, F., Kaplan, J. O., Küttel, M., Müller, S. A., Prentice, I. C., Solomina, O., Stocker, T. F., Tarasov, P., Wagner, M., and Widmann, M.: Mid- to Late Holocene climate change: an overview, Quaternary Sci. Rev., 27, 1791–1828, https://doi.org/10.1016/j.quascirev.2008.06.013, 2008. Warszawski, L., Frieler, K., Huber, V., Piontek, F., Serdeczny, O., and Schewe, J.: The Inter-Sectoral Im- pact Model Intercomparison Project (ISI-MIP): project framework, P. Natl. Acad. Sci. USA, 111, 3228–3232, https://doi.org/10.1073/pnas.1312330110, 2014. Stoffel, M., Khodri, M., Corona, C., Guillet, S., Poulain, V., Bekki, S., Guiot, J., Luckman, B. H., Oppenheimer, C., Lebas, N., Beniston, M., and Masson-Delmotte, V.: Esti- mates of volcanic-induced cooling in the Northern Hemi- sphere over the past 1,500 years, Nat. Geosci., 8, 784–788, https://doi.org/10.1038/ngeo2526, 2015. Weiss, H. and Bradley, R. S.: What Drives Societal Collapse?, Sci- ence, 291, 609–611, 2001. https://doi.org/10.5194/cp-19-1219-2023 Clim. Past, 19, 1219–1244, 2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023 J. Guiot et al.: Viticulture extension in response to global climate change drivers 1244 Widmann, M., Goosse, H., van der Schrier, G., Schnur, R., and Barkmeijer, J.: Using data assimilation to study extratropical Northern Hemisphere climate over the last millennium, Clim. Past, 6, 627–644, https://doi.org/10.5194/cp-6-627-2010, 2010. Zhu, Q., Peng, C., Chen, H., Fang, X., Liu, J., Jiang, H., Yang, Y., and Yang, G.: Estimating global natural wetland methane emis- sions using process modelling: Spatio-temporal patterns and con- tributions to atmospheric methane ﬂuctuations, Global Ecol. Bio- geogr., 24, 959–972, https://doi.org/10.1111/geb.12307, 2015. p g p Williamson, D., Blaker, A. T., Hampton, C., and Salter, J.: Identifying and removing structural biases in climate mod- els with history matching, Clim. Dynam., 45, 1299–1324, https://doi.org/10.1007/s00382-014-2378-z, 2015. g g p g g Zobler, L.: A world soil ﬁle grobal climate modeling, NASA Tech. Memo 32, NASA, 1986. Clim. Past, 19, 1219–1244, 2023 https://doi.org/10.5194/cp-19-1219-2023
https://openalex.org/W3154462684	https://www.journalrmc.com/index.php/JRMC/article/download/1612/740	English	null	Do Pruritus and Urticaria Predict For Response to Antihistamine Therapy in COVID-19 Patients with Pulmonary Symptoms?	Journal of Rawalpindi Medical College/Journal Rawalpindi Medical College	2,021	cc-by-sa	1,481	Access Online: g Source: Nil she was started on hydroxyzine 25mg q.d. and topical triamcinolone acetonide 0.1%. A first-generation antihistamine was selected to address both pruritus and associated insomnia. Additionally, prednisone was discontinued as there was no response to the medication. The patient experienced relief of urticaria within 4 hours after starting hydroxyzine and reported improvement of her dyspnea by the following morning. A SARS-CoV-2 test performed at the urgent care clinic was positive for the detection of viral antigen. The patient’s symptoms completely resolved within 5 days after initiating hydroxyzine. The signs and symptoms of mast cell activation including urticaria and pruritus and response to antihistamines suggest that mast cells and their chemical mediators play a significant role in the pathophysiology of some COVID-19 patients. While case reports have described improvement of urticaria and other mast cell-mediated symptoms in COVID-19 patients following the administration of antihistamines, these cases were co- founded by the concomitant addition of new medications; this issue is less relevant in our report.1 3 3 3 Journal of Rawalpindi Medical College (JRMC); 2020; 25(1): 3-5 Letter to the Editor Letter to the Editor Do Pruritus and Urticaria Predict For Response to Antihistamine Therapy in COVID-19 Patients with Pulmonary Symptoms? Alexandra Rubin1, Mahin Alamgir2, Babar K Rao3 1,2,3 Center for Dermatology, Rutgers Robert Wood Johnson Medical School, Somerset, NJ 08901. Author’s Contribution 1,3 Conception of study 1,2 Analysis/Interpretation/Discussion 1,2,3 Manuscript Writing 3 Critical Review 1,2 Facilitation and Material analysis Corresponding Author Dr. Alexandra Rubin, Center for Dermatology, Rutgers Robert Wood Johnson Medical School, Somerset, NJ 08901 Email: alirubin@rwjms.rutgers.edu Article Processing Received: 14/01/2021 Accepted: 18/03/2021 Do Pruritus and Urticaria Predict For Response to Antihistamine Therapy in COVID-19 Patients with Pulmonary Symptoms? Alexandra Rubin1, Mahin Alamgir2, Babar K Rao3 1,2,3 Center for Dermatology, Rutgers Robert Wood Johnson Medical School, Somerset, NJ 08901. Author’s Contribution 1,3 Conception of study 1,2 Analysis/Interpretation/Discussion 1,2,3 Manuscript Writing 3 Critical Review 1,2 Facilitation and Material analysis Corresponding Author Dr. Alexandra Rubin, Center for Dermatology, Rutgers Robert Wood Johnson Medical School, Somerset, NJ 08901 Email: alirubin@rwjms.rutgers.edu Article Processing Received: 14/01/2021 Accepted: 18/03/2021 Do Pruritus and Urticaria Predict For Response to Antihistamine Therapy in COVID-19 Patients with Pulmonary Symptoms? Alexandra Rubin1, Mahin Alamgir2, Babar K Rao3 3 Center for Dermatology, Rutgers Robert Wood Johnson Medical School, Somerset, NJ 08901. Author’s Contribution 3 Conception of study 2 Analysis/Interpretation/Discussion 2,3 Manuscript Writing Critical Review 2 Facilitation and Material analysis Corresponding Author Dr. Alexandra Rubin, Center for Dermatology, Rutgers Robert Wood Johnson Medical School, Somerset, NJ 08901 Email: alirubin@rwjms.rutgers.edu Article Processing Received: 14/01/2021 Accepted: 18/03/2021 ite this Article: Rubin A Alamgir M Rao B K Conflict of Interest: Nil Access Online: 1,2,3 Center for Dermatology, Rutgers Robert Wood Joh Author’s Contribution 1,3 Conception of study 1,2 Analysis/Interpretation/Discussion 1,2,3 Manuscript Writing 3 Critical Review 1,2 Facilitation and Material analysis Corresp Dr. Alexa Center for Rutgers R Somerset, Email: alir Cite this Article: Rubin, A, Alamgir, M., Rao, B.K. Do Pruritus and Urticaria Predict For Response to Antihistamine Therapy in COVID-19 Patients with Pulmonary Symptoms? Journal of Rawalpindi Medical College. 30 Dec. 2020; 24(4): 3-5. DOI: https://doi.org/ 10.37939/jrmc.v25i1.1612 Article Processing Received: 14/01/2021 Accepted: 18/03/2021 Author’s Contribution 1,3 Conception of study 1,2 Analysis/Interpretation/Discussion 1,2,3 Manuscript Writing 3 Critical Review 1,2 Facilitation and Material analysis Conflict of Interest: Nil Funding Source: Nil Access Online: Dear Editor, Skin manifestations and respiratory symptoms are commonly seen in patients with COVID-19. While infection by SARS-CoV-2 and an accompanying cytokine storm are proposed to account for respiratory symptoms, additional inflammatory mediators including histamine may also contribute. In this regard, we report the resolution of dyspnea associated with urticaria in a 61-year-old female patient acutely infected with SARS-CoV-2. The patient’s medical history is significant for idiopathic thrombocytopenic purpura, hypertension, and gastroesophageal reflux disease, and her long-standing medications consisted of eltrombopag, hydrochlorothiazide, lisinopril, and pantoprazole. The patient had no prior history of urticaria, angioedema, or respiratory illness. Due to the severity of her symptoms which developed over the course of a few days, the patient was referred to an urgent care clinic where intravenous methylprednisolone was administered, and tapering doses of prednisone were prescribed starting at 50mg. Nevertheless, all of her symptoms, particularly the urticaria (Figure 1 a,b), continued to worsen, prompting her to visit our dermatology clinic where 4 4 Journal of Rawalpindi Medical College (JRMC); 2020; 25(1): 3-5 4 Journal of Rawalpindi Medical College (JRMC); 2020; 25(1): 3-5 Figure 1: Wide-spread urticarial rash involving the chest (a) and arms (b) Figure 1: Wide-spread urticarial rash involving the chest (a) and arms (b) Figure 1: Wide-spread urticarial rash involving the chest (a) and arms (b) Figure 1: Wide-spread urticarial rash involving the chest (a) and arms (b) in rates of intubation, mortality, and length of hospital stay.8 Additionally, a retrospective study of 1,620 COVID-19 patients who received the H2 receptor antagonist famotidine, showed a statistically significant reduction in the rates of intubation and death.9 in rates of intubation, mortality, and length of hospital stay.8 Additionally, a retrospective study of 1,620 COVID-19 patients who received the H2 receptor antagonist famotidine, showed a statistically significant reduction in the rates of intubation and death.9 Urticaria is a common dermatologic manifestation of COVID-19 patients, with an incidence of 19% in a prospective study of 375 patients.2 No correlation between urticaria and disease severity, or timing of onset has been described.3 Possible etiologies of urticaria in COVID-19 patients include a reaction to viral infection, medications, and stress. Dear Editor, The SARS- CoV-2 virus itself may directly activate mast cells via toll-like receptors or signaling molecules such as interferon type 1, TNF-α, and/or chemokines.4 It is thought that mast cell degranulation may either be caused directly by the virus or indirectly through viral stimulation of complement fragments C3a and C5a activating G-protein-coupled receptors.4 Based on the observations that urticaria and pruritus may accompany pulmonary symptoms, we postulate these patients may better respond to antihistamine therapy. Therefore, we suggest these dermatologic findings are incorporated into the analysis plans of clinical studies assessing antihistamines in COVID-19 patients with pulmonary symptoms. Further, we suggest considering antihistamines as a component of the first-line treatment for COVID-19 patients with urticaria. While cytokine storms, and more recently, bradykinin storms, are proposed as mediators in the pathophysiology of COVID-19, mast cells and histamine may also contribute to the development of pulmonary manifestations of COVID-19 patients.5 The clinical course of our patient indicates her respiratory symptoms appeared solely related to SARS-CoV-2 induced urticaria. Release of histamine, prostaglandin-D2, and leukotriene-C4 by mast cells cause bronchoconstriction.6 Further, the release of mast cell cytokines such as IL-1 and IL-6 contribute to the development of pulmonary and systemic inflammation, as well as urticaria in COVID-19 patients.3,4,6,7 Preliminary clinical trial reports suggest antihistamine treatment mitigates pulmonary symptoms in COVID-19 patients. Trials of dual- histamine receptor blockade with cetirizine-famotidine in hospitalized COVID-19 patients with severe pulmonary symptoms resulted in marked reductions g py J 4. Criado PR, Pagliari C, Criado RF, Marques GF, Belda Jr W. What the physicians should know about mast cells, dendritic cells, urticaria, and omalizumab during COVID19 or asymptomatic infections due to SARSCoV2?. Dermatologic Therapy. 2020 Jan 1:e14068. p g j 3. Kaushik A, Parsad D, Kumaran MS. Urticaria in the times of COVID19. Dermatologic Therapy. 2020 Jun 12. References 1. Henry D, Ackerman M, Sancelme E, Finon A, Esteve E. Urticarial eruption in COVID19 infection. Journal of the European Academy of Dermatology and Venereology. 2020 Jun; 34(6):e244-5 https://doi.org/10.1111/jdv.16472. p g j 2. Galván Casas C, Catala AC, Carretero Hernández G, RodríguezJiménez P, FernándezNieto D, RodríguezVilla Lario A, et al. Classification of the cutaneous manifestations of COVID19: a rapid prospective nationwide consensus study in Spain with 375 cases. British Journal of Dermatology. 2020 Jul; 183(1):71-7. https://doi.org/10.1111/bjd.19163 p g j 3. Kaushik A, Parsad D, Kumaran MS. Urticaria in the times of COVID19. Dermatologic Therapy. 2020 Jun 12. 5 Journal of Rawalpindi Medical College (JRMC); 2020; 25(1): 3-5 5. Garvin MR, Alvarez C, Miller JI, Prates ET, Walker AM, Amos BK, et al. A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm. Elife. 2020 Jul 7; 9:e59177. 6. Kempuraj D, Selvakumar GP, Ahmed ME, Raikwar SP, Thangavel R, Khan A, et al. COVID-19, mast cells, cytokine storm, psychological stress, and neuroinflammation. The Neuroscientist. 2020 Oct;26(5-6):402-14. 7. de Medeiros VLS, Silva LFT. Follow-up of skin lesions during the evolution of COVID-19: a case report [published online ahead of print, 2020 May 14]. Arch Dermatol Res. 2020;1-4. y 8. Hogan Ii RB, Hogan Iii RB, Cannon T, Rappai M, Studdard J, Paul D, et al. Dual-histamine receptor blockade with cetirizine- famotidine reduces pulmonary symptoms in COVID-19 patients. Pulmonary Pharmacology & Therapeutics. 2020 Aug 1; 63:101942. 9. Freedberg DE, Conigliaro J, Wang TC, Tracey KJ, Callahan MV, Abrams JA, et al. Famotidine use is associated with improved clinical outcomes in hospitalized COVID-19 patients: a propensity score matched retrospective cohort study. Gastroenterology. 2020 Sep 1;159(3):1129- 31.doi:10.1053/j.gastro.2020.05.053
https://openalex.org/W1980948587	https://europepmc.org/articles/pmc3901444?pdf=render	English	null	Internet-delivered eye movement desensitization and reprocessing (iEMDR): an open trial	F1000Research	2,013	cc-by	8,529	RESEARCH ARTICLE Internet-delivered eye movement desensitization and reprocessing (iEMDR): an open trial [v2; ref status: indexed, http://f1000r.es/zr] Open Peer Review , Veterans Affairs Medical Alyssa Boasso Center Boston USA , Swinburne University of Jo Abbott Technology Australia Discuss this article (0) Comments 2 1 Abstract Recent research indicates internet-delivered cognitive behavioural therapy (iCBT) can reduce symptoms of post traumatic stress disorder (PTSD). This study examined the efficacy of an internet-delivered treatment protocol that combined iCBT and internet-delivered eye movement desensitization and reprocessing (iEMDR), in an uncontrolled trial. Eleven of the 15 participants completed post-treatment questionnaires. Large effect sizes were found from pre-treatment to 3-month follow-up ( = 1.03 – 1.61) on clinician-assessed and d self-reported measures of PTSD, anxiety and distress, with moderate effect sizes ( = 0.59 – 0.70) found on measures of depression and disability. At d post-treatment, 55% of the participants no longer met criteria for PTSD and this was sustained at follow-up. Symptom worsening occurred in 3 of 15 (20%) of the sample from pre- to post-treatment; however, these participants reported overall symptom improvement by follow-up. Future research directions for iEMDR are discussed. Referee Status: Invited Referees version 2 published 07 May 2013 version 1 published 06 Mar 2013 1 2 report report report 06 Mar 2013, :79 (doi: ) First published: 2 10.12688/f1000research.2-79.v1 07 May 2013, :79 (doi: ) Latest published: 2 10.12688/f1000research.2-79.v2 v2 06 Mar 2013, :79 (doi: ) First published: 2 10.12688/f1000research.2-79.v1 07 May 2013, :79 (doi: ) Latest published: 2 10.12688/f1000research.2-79.v2 v2 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 Jay Spence ( ) Corresponding author: jay@jayspence.com.au Spence J, Titov N, Johnston L How to cite this article: et al. Internet-delivered eye movement desensitization and reprocessing (iEMDR): 2013, :79 (doi: ) an open trial [v2; ref status: indexed, ] http://f1000r.es/zr F1000Research 2 10.12688/f1000research.2-79.v2 © 2013 Spence J . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver The author(s) declared that no grants were involved in supporting this work. Grant information: Competing interests: There are no competing interests for any author. 06 Mar 2013, :79 (doi: ) First published: 2 10.12688/f1000research.2-79.v1 21 Mar 2013, :79 (doi: ) First indexed: 2 10.12688/f1000research.2-79.v1 Abstract Recent research indicates internet-delivered cognitive behavioural therapy (iCBT) can reduce symptoms of post traumatic stress disorder (PTSD). This study examined the efficacy of an internet-delivered treatment protocol that combined iCBT and internet-delivered eye movement desensitization and reprocessing (iEMDR), in an uncontrolled trial. Eleven of the 15 participants completed post-treatment questionnaires. Large effect sizes were found from pre-treatment to 3-month follow-up ( = 1.03 – 1.61) on clinician-assessed and d self-reported measures of PTSD, anxiety and distress, with moderate effect sizes ( = 0.59 – 0.70) found on measures of depression and disability. At d post-treatment, 55% of the participants no longer met criteria for PTSD and this was sustained at follow-up. Symptom worsening occurred in 3 of 15 (20%) of the sample from pre- to post-treatment; however, these participants reported overall symptom improvement by follow-up. Future research directions for iEMDR are discussed. version 2 published 07 May 2013 version 1 published 06 Mar 2013 report report report Jay Spence ( ) Corresponding author: jay@jayspence.com.au Spence J, Titov N, Johnston L How to cite this article: et al. Internet-delivered eye movement desensitization and reprocessing (iEMDR): 2013, :79 (doi: ) an open trial [v2; ref status: indexed, ] http://f1000r.es/zr F1000Research 2 10.12688/f1000research.2-79.v2 © 2013 Spence J . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver The author(s) declared that no grants were involved in supporting this work. Grant information: Competing interests: There are no competing interests for any author. 06 Mar 2013, :79 (doi: ) First published: 2 10.12688/f1000research.2-79.v1 21 Mar 2013, :79 (doi: ) First indexed: 2 10.12688/f1000research.2-79.v1 Jay Spence ( ) Corresponding author: jay@jayspence.com.au Spence J, Titov N, Johnston L How to cite this article: et al. Internet-delivered eye movement desensitization and reprocessing (iEMDR): 2013, :79 (doi: ) an open trial [v2; ref status: indexed, ] http://f1000r.es/zr F1000Research 2 10.12688/f1000research.2-79.v2 © 2013 Spence J . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The study was approved by the Macquarie University Human Research Ethics Committee (HREC#: 5201100382). Participants provided informed consent. The trial is registered with the Australian and New Zealand Clinical Trials registry as ACTRN12611000151932. The study was approved by the Macquarie University Human Research Ethics Committee (HREC#: 5201100382). Participants provided informed consent. The trial is registered with the Australian and New Zealand Clinical Trials registry as ACTRN12611000151932. We would like to thank the reviewers for their comments on this manuscript, which has been edited to address their feedback concerning following points: (i) the rationale for the development of iEMDR has been clarified; (ii) the participant flow process has been clarified; (iii) the process of anchoring the positive belief followed a standardized procedure, which has now been referenced more clearly; (iv) the section comparing findings to the face-to-face literature has been edited to highlight the differences between mixed trauma and single incident trauma samples; (v) symptom worsening has been clarified; (vi) references have been updated; (vii) telephone support with a specialist PTSD therapist has been emphasized to highlight that this was not a purely internet-based treatment; (viii) the basis of the findings (completer analyses) has been clarified; and, (ix) significance indicators have been added to the tables. Participants and recruitmentl p Participant flow is shown in Figure 1. Participants were recruited from visitors to a research website that evaluates internet-elivered treatments (www.ecentreclinic.org). During the recruitment period, which ran over 2 weeks during June 2011, 32 individuals applied and 15 met the following inclusion criteria: (i) self-identified as having a principal complaint of PTSD as indicated by total scores above a clinical cut-off recommended to indicate probable diagno- sis of PTSD15 (defined as > 44 on the PTSD Checklist (PCL-C)16) as well as a confirmed primary diagnosis of PTSD determined by clinician-administered interview using the PTSD Symptom Scale- Interview (PSS–I)17; (ii) at least one month had elapsed since the primary trauma; (iii) no psychotherapy for PTSD during the treat- ment period (however, supportive group and individual counselling that did not specifically target PTSD symptoms was permitted); (iv) if using psychotropic medication, no change in dosage or type of medication 1 month prior to or during treatment; (v) a resident of Australia, (vi) at least 18 years of age, (vii) had computer and internet access, (viii) not currently experiencing a psychotic mental illness, extreme current symptoms of depression (defined as a total score > 22 or responding > 2 to Question 9 (suicidal ideation) on the Patient Health Questionnaire - 9 Item (PHQ-9)18, current suicidal intent and plan, or highly dissociative (defined as a total score above 22) on the Dissociative Experiences Scale – Brief Version (DES-B))19. See referee reports See referee reports Abstract Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver The author(s) declared that no grants were involved in supporting this work. Grant information: Competing interests: There are no competing interests for any author. 06 Mar 2013, :79 (doi: ) First published: 2 10.12688/f1000research.2-79.v1 21 Mar 2013, :79 (doi: ) First indexed: 2 10.12688/f1000research.2-79.v1 pence J, Titov N, Johnston L et al. Internet-delivered eye movement desensitization and reprocessing (iEMDR): 2013, :79 (doi: ) us: indexed, ] http://f1000r.es/zr F1000Research 2 10.12688/f1000research.2-79.v2 Page 1 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 Introduction Results of meta-analyses indicate that both trauma-focused cognitive behavioural therapy (TF-CBT) and eye movement desensitization and reprocessing (EMDR)1 are effective in reducing PTSD symp- toms. However, barriers to accessing these treatments include stigma, cost, distance, low mental health literacy, and long waiting lists2,3. Internet-delivered psychological treatments may increase access to psychological therapy4. TF-CBT has been delivered via the internet and has shown promise in significantly reducing PTSD symptoms in military personnel5,6, university students7,8, and community sam- ples in the U.S.7, Holland9, Iraq10, Australia11,12 and German-speak- ers in Europe13. For example, in a previous study12 we evaluated an internet-delivered TF-CBT (iCBT) protocol with Australian adults with a primary diagnosis of PTSD. We found large within-group effect sizes (ESs) and small-to-moderate between-group ESs on measures of PTSD symptoms, depression, anxiety and disability, in a treatment group relative to a control condition. Changes from Version 1 The study was approved by the Macquarie University Human Research Ethics Committee (HREC#: 5201100382). Participants provided informed consent. The trial is registered with the Australian and New Zealand Clinical Trials registry as ACTRN12611000151932. Measures The primary outcome measures were severity of symptoms of PTSD, measured by the PSS-I and the PCL-C. The PSS-I17 is a 17-item semi-structured clinician-administered interview based on the DSM-IV criteria for PTSD. The PCL-C16 is also a 17-item, self- report scale of PTSD symptoms based on the DSM-IV criteria for PTSD. Time burdens on participants could hinder effective dissemination of internet treatments for PTSD. This study investigated the effi- cacy of internet-delivered EMDR (iEMDR) on the basis of meta- analytic findings showing that outcomes from face-to-face EMDR are equivalent to TF-CBT with the important distinction that TF- CBT required approximately 23 hours (SD = 11) of homework while EMDR required only 3 hours (SD = 4)14. iEMDR may offer an alternative model of remote treatment for PTSD to iCBT. The present study aimed to explore the acceptability and efficacy of iEMDR when used in conjunction with an iCBT protocol (iCBT/ iEMDR course), and evaluated using an open trial design. To increase generalizability of results, inclusion criteria were consistent with those of outpatient services. The primary hypothesis was that the iCBT/iEMDR course would be associated with significant improvements in PTSD symptoms, depression, anxiety, distress, and disability. Secondary outcomes measures included the Generalized Anxiety Dis- order 7-Item Scale (GAD-7, which measures anxiety)20, the PHQ-9 (which measures depression)18, the Mini International Neuropsy- chiatric Interview (MINI; which was used to determine the presence of a major depressive episode, panic, agoraphobia, social phobia, obsessive compulsive disorder, and generalized anxiety disorder)21, the Kessler 10 Item22 (K10; which measures general distress), and the Sheehan Disability Scale23 (SDS, which measures impairment in psychosocial functioning). Traumatic experiences were assessed using the Life Events Checklist (LEC)24, which provides a list of traumatic events and assesses the occurrence rates of common Criterion A1 (life-threatening) traumas according to the DSM-IV. Additional outcomes included completion rates (percentage of participants who read the six online lessons of the iCBT/iEMDR course within the six weeks of the course), and treatment satisfac- tion (percentage who reported feeling satisfied with the program or who would recommend it to a friend). Secondary hypotheses were that the treatment would be rated as accept- able to participants and would not be associated with adverse events. Page 2 of 11 Page 2 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 Figure 1. Participant flow chart. iEMDR: Internet-delivered eye movement desensitization and reprocessing. PHQ-9: Patient Health Questionnaire – 9 Item. Measures MINI: MINI International Neuropsychiatric Interview. DES-B: Dissociative Experiences Scale – Brief Version. 32 individuals applied for the iEMDR Course (between 14/06/2011 – 30/06/2011) Unsuccessful Application (n=9) Met inclusion criteria (n=23) Could not contact (n=1) Unsuccessful Diagnostic Interview (n=7) Treatment Group (n=15) Eligible for Post-Treatment ITT Analysis (n=15) Eligible for Follow up ITT Analysis (n=15) Didn’t complete all lessons (n=4) Eligible for Post-Treatment Completer Analysis Eligible for Follow up Completer Analysis Completed Clinician-assessed measures (n=12): Completed Clinician-assessed measures (n=9): Completed all lessons: n = 10 Completed all lessons: n = 8 Completed all lessons: n = 10 Completed all lessons: n = 8 Completed 2 lessons: n = 2 Completed 2 lessons: n = 1 Completed 2 lessons: n = 1 Completed 1 lesson: n = 1 Completed Self-report measures (n=11): Completed Self-report measures (n=9): Completed lesson 1 only (n=1) Completed lessons 1 - 2 (n=3) Completed Pre-Treatment Questionnaires and Started Lesson 1 (n=15) Did not meet diagnostic criteria (n=5) Scored over cut-off point on DES-B (n=1) Withdrew application (n=1) Completed telephone interview with MINI 5.0 (n=22) Severe depressive symptoms on PHQ-9 (n=3) Did not complete the application (n=3) Currently receiving CBT (n=1) Under 18 (n=1) Not Australian resident (n=1) 32 individuals applied for the iEMDR Course (between 14/06/2011 – 30/06/2011) Met inclusion criteria (n=23) Completed telephone interview with MINI 5.0 (n=22) Unsuccessful Diagnostic Interview (n=7) Did not meet diagnostic criteria (n=5) Scored over cut-off point on DES-B (n=1) Withdrew application (n=1) Didn’t complete all lessons (n=4) Completed lesson 1 only (n=1) Completed lessons 1 - 2 (n=3) Eligible for Post-Treatment ITT Analysis (n=15) Eligible for Post-Treatment Completer Analysis Completed Clinician-assessed measures (n=12): Completed all lessons: n = 10 Completed all lessons: n = 10 Completed 2 lessons: n = 2 Completed 2 lessons: n = 1 Completed Self-report measures (n=11): Eligible for Post-Treatment Completer Analysis Eligible for Follow up ITT Analysis (n=15) Eligible for Follow up Completer Analysis Completed Clinician-assessed measures (n=9): Completed all lessons: n = 8 Completed all lessons: n = 8 Completed 2 lessons: n = 1 Completed 1 lesson: n = 1 Completed Self-report measures (n=9): Figure 1. Participant flow chart. iEMDR: Internet-delivered eye movement desensitization and reprocessing. PHQ-9: Patient Health Questionnaire – 9 Item. MINI: MINI International Neuropsychiatric Interview. DES-B: Dissociative Experiences Scale – Brief Version. Figure 1. Participant flow chart. iEMDR: Internet-delivered eye movement desensitization and reprocessing. PHQ-9: Patient Health Questionnaire – 9 Item. Therapist One Clinical Psychologist (JS) provided all clinical contact with participants, which occurred via weekly telephone calls or secure email. The clinician had received Level I and II training in EMDR by a certified EMDR instructor, and had two years experience in administering iCBT and in facilitating EMDR in face-to-face treatment. The clinician was supervised by NT. Baseline data The mean age of participants was 47 years (SD = 10.4), and 10/15 (66%) were women. Ten of 15 participants (67%) reported being either married or in a de facto relationship, 4/15 (27%) reported being separated or widowed and 1/15 (7%) reported being single or never married. Four of fifteen (27%) had a tertiary education, 9/15 (60%) reported having a post-high school certificate and 2/15 (13%) reported as having year 10 high school level education. One participant (7%) was in full-time employment, eight (53%) were employed part-time or studying and six (40%) reported being unemployed, retired, or disabled. Fourteen of fifteen participants (93%) reported having had previous mental health treatment and 10/15 (67%) reported taking medication related to their symptoms of anxiety or depression. One half (5/10) of the participants who completed post-treatment questionnaires reported that they were receiving individual or group supportive counselling during the treatment period that was not specifically directed at the treat- ment of PTSD symptoms (mean sessions = 3; SD = 2.1). Between post-treatment and follow-up, 25% (2/8) of respondents reported receiving ongoing supportive therapy (not specifically for PTSD) and 13% (1/8) commenced treatment with a psychologist spe- cifically for PTSD (mean sessions = 4; SD = 3.5). There were no reported medication changes during the course. One quarter (2/8) of respondents reported changing their medication post- treatment. Five participants (33%) who reported not having used self-guided EMDR by mid-treatment were contacted and offered a second EMDR session guided by the therapist via telephone. None elected to participate in further EMDR, citing that EMDR had led to an increase in re-experiencing symptoms. iEMDR was conducted using a web-based EMDR tool (http:// www.rapidtables.com/tool/EMDR.htm). The initial session of EMDR was conducted with the support of the therapist (JS) who guided participants by telephone through the procedure while they accessed the web-based EMDR tool. Further therapist-guided EMDR was provided as requested. Participants who reported not having used self-guided EMDR by mid-treatment were contacted and offered a second guided EMDR session. Instructions for work- ing with blockages to processing were provided in an additional resource one week after giving the iEMDR instructions. Results iEMDR intervention: The EMDR intervention follows the standard EMDR treatment protocol by Shapiro26 with the following adaptations for self-directed use via the internet: the protocol was divided into a desensitisation phase (weeks 2–4) followed by a phase aimed at anchoring the positive belief (weeks 5–6). The desensitiza- tion phase followed Shapiro’s protocol for reducing the Subjective Units of Distress (SUDS) rating to less than 2. Anchoring the positive belief also followed Shapiro’s protocol26 until the Validity of Cogni- tion (VoC) rating was above 5. Participants were instructed to anchor the positive belief in week 5 of the course only for trauma memories that were no longer distressing (SUDS < 2). Intervention The iCBT/iEMDR course is a six lesson online intervention utilising evidence-based principles of TF-CBT25 and EMDR26. The TF-CBT components were similar to those used in a previous internet-based CBT program for PTSD12. The course comprises text-based infor- mation and instructions and educational case stories. Pre- to post-treatment and pre-treatment to follow-up changes in questionnaire scores were analysed using paired-sample t-tests. Effect sizes (Cohen’s d)27 were calculated based on the pooled standard deviation. All analyses were performed in PASW version 18.0 (SPSS, Inc., Chicago, IL). Lesson 1 of the iCBT/iEMDR course includes information about the causes, symptomatology and neurobiology of PTSD, how cognitive, behavioural, and physical symptoms maintain PTSD, and provides instructions for physiological de-arousal strategies. Lesson 2 provides the rationale for using EMDR and detailed instructions about a self-guided iEMDR process. Lesson 3 describes cognitive restructuring strategies. Lesson 4 provides more detail on how to use cognitive restructuring for common trauma-related cog- nitions. Lesson 5 describes avoidance and safety behaviours and the principles of graded exposure. Lesson 6 describes the principles of relapse prevention. Changes in prevalence of PTSD and comorbid disorders were calculated based on the results of telephone administered diagnos- tic interviews administered at pre-treatment, post-treatment and follow-up. To measure adverse events we used Tarrier’s28 definitions of treat- ment worsening, defined as any increase in symptom scores greater than zero from pre- to post-treatment or follow up, and defined serious adverse events as self-reported hospitalizations, suicide attempts, or onset of substance abuse due to treatment. Measures MINI: MINI International Neuropsychiatric Interview. DES-B: Dissociative Experiences Scale – Brief Version. Page 3 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 data (i.e. baseline-observation-carried-forward model; BOCF). data (i.e. baseline-observation-carried-forward model; BOCF). Acceptability Primary outcome measures. A paired-sample t-test comparing pre- and post-treatment scores for the ITT sample revealed signifi- cant reductions on the PSS-I (t14 = 3.50, p = 0.004), and this was maintained at follow up (t14 = 4.59, p < 0.0001). Scores on the PCL-C did not significantly improve from pre- to post-treatment (t14 = 2.12, p = 0.053). However, at follow-up, scores on the PCL-C had signifi- cantly improved from pre-treatment (t14 = 17.76, p < 0.0001). At post-treatment, 6/11 (55%) reported that they were very satisfied with the course, one participant (9%) was mostly satisfied, and 4/11 were neutral or somewhat satisfied. None of the participants reported being dissatisfied with the course. Nine of 11 (82%) reported they would recommend this course to a friend with PTSD. EMDR open trial data set, Spence et al. 2013 2 Data Files http://dx.doi.org/10.6084/m9.figshare.639181 Discussion This study explored the feasibility of a combined iCBT/iEMDR course for treating PTSD in adults using an open-trial design. The EMDR open trial data set, Spence et al. 2013 2 Data Files http://dx.doi.org/10.6084/m9.figshare.639181 Secondary outcome measures. Paired sample t-tests for the ITT sample revealed significant reductions between pre- and post-treatment on the K10 (t14 = 2.20, p = 0.046) but not on the PHQ-9 (t14 = 2.12, p = 0.053), GAD-7 (t14 = 2.02, p = 0.063), or SDS (t14 = 1.22, p = 0.281). There was a significant differ- ence between pre-treatment and follow-up scores on the PHQ-9 (t14 = 2.46, p = 0.027), GAD-7 (t14 = 2.90, p = 0.012), K10 (t14 = 3.10, p = 0.008), but not on the SDS (t14 = 2.08, p = 0.056). Intention-to-treat (ITT) analysis Intention-to-treat (ITT) analysis Effect sizes had occurred 32.8 years prior (SD = 12.5). The average age at which the primary trauma occurred was 13.3 years (SD = 12.9). According to the LEC, participants reported having experienced an average of 9.2 types of trauma during their lifetime. The most common was physical assault (13/15; 87%), followed by assault with a weapon (12/15; 80%), and other unwanted or uncomfortable sexual experience (12/15; 80%). had occurred 32.8 years prior (SD = 12.5). The average age at which the primary trauma occurred was 13.3 years (SD = 12.9). According to the LEC, participants reported having experienced an average of 9.2 types of trauma during their lifetime. The most common was physical assault (13/15; 87%), followed by assault with a weapon (12/15; 80%), and other unwanted or uncomfortable sexual experience (12/15; 80%). Using the ITT analysis, from pre-treatment to post-treatment a large within-group effect size was found on the PSS-I. Moderate within- group effects were found on the GAD-7, PHQ-9, and K10. A small effect size was reported on the SDS. From pre-treatment to follow- up, large effect sizes were found on the PSS-I, PCL-C, and GAD-7, and moderate effect sizes for the PHQ-9, and SDS. Attrition l The flow is shown in Figure 1. Eleven participants (73%) completed all six lessons. One participant completed a single lesson and did not complete further assessments. Three participants completed two les- sons, one of whom completed post-treatment and follow up assess- ments and one who completed post-treatment assessments only. One participant completed six lessons, but not the post-treatment or follow-up assessments. Twelve participants completed clinician- assessed post-treatment interviews although one of these partici- pants did not complete the self-report questionnaires. Nine participants completed follow-up questionnaires, including the abovementioned participant who had only completed two lessons. There were no pre- treatment differences between completers and non-completers on the PSS-I, PCL-C or the GAD-7 at pre-treatment. Clinical significance Based on the results of the clinician and telephone-administered PSS-I, 6/11 (55%) participants no longer met criteria for PTSD at post-treatment and 5/9 (56%) no longer had PTSD at follow-up. Based on an ITT approach with the BOCF, 5/15 (33%) no longer met criteria for PTSD at post-treatment and follow-up. With regard to co-morbid diagnoses for completers as measured by clinician-administered MINI, the average number of co-morbid diagnoses reduced from 2.5 (SD = 2.0) at intake to 1.2 (SD = 1.0) at post-treatment, and further reduced to 0.6 (SD = 1.6) at follow- up. According to an ITT analysis the average number of co-morbid diagnoses reduced from 2.5 (SD = 1.7) at intake to 1.4 (SD = 0.9) at post-treatment, and 1.1 (SD = 1.1) at follow-up. Completer analysis i Primary outcome measures. Primary outcome scores for com- pleters improved from pre- to post-treatment as shown in Table 1. Paired-sample t-tests revealed significant reductions on the PSS-I (t10 = 3.66, p = 0.004) and PCL-C (t10 = 2.73, p = 0.021) between pre- and post-treatment, and between pre-treatment and follow-up (PSS-I: t10 = 4.90, p = 0.001; PCL-C: t10 = 4.26, p = 0.002). Adverse events Three participants reported symptom worsening as defined by Tarrier28 and no participants reported serious adverse events. Of the participants who completed post-treatment questionnaires, three participants showed symptom worsening between pre- and post-treatment on the PCL-C, and one of these had dropped out of treatment after the third lesson. All three improved between post-treatment and follow-up such that no participants worsened between pre-treatment and follow-up. No participants worsened on the PSS-I between any time points. Secondary outcome measures. Paired sample t-tests between pre- and post-treatment indicated significant reductions for completers on the PHQ-9 (t9 = 2.66, p = 0.026), GAD-7 (t9 = 2.31, p = 0.047), K10 (t9 = 2.49, p = 0.034), but not on the SDS (t9 = 1.66, p = 0.131). Signifi- cant reductions were reported between pre-treatment and follow-up on the PHQ-9 (t7 = 3.13, p = 0.017), GAD-7 (t7 = 4.16, p = 0.004), K10 (t7 = 3.95, p = 0.006), and SDS (t7 = 4.15, p = 0.004). Trauma history Th Primary analyses were conducted using data only from question- naire completers, defined as those who completed treatment, post- treatment or follow-up questionnaires. A secondary set of analyses was performed using an intention-to-treat (ITT) model where missing data were addressed by carrying forward the first available The most common reported primary trauma was childhood sexual abuse (9/15; 60%), followed by childhood physical abuse (2/15; 13%), domestic violence as an adult (2/15; 13%), witnessing domestic violence as a child (1/15; 7%), captivity (1/15; 7%) and life threatening illness (1/15; 7%). On average, the primary trauma Page 4 of 11 Page 4 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 Effect sizes Using the completer analysis, large effect sizes were reported on the PSS-I, PCL-C, GAD-7, and K10 at post-treatment and a moderate effect size was reported on the PHQ-9 and SDS (Table 1). Large effect sizes were reported on all measures between pre-treatment and follow-up. Discussion This study explored the feasibility of a combined iCBT/iEMDR course for treating PTSD in adults using an open-trial design. The Page 5 of 11 Page 5 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 Table 1. Descriptive statistics and within-group effects on symptom measures at each assessment. Measure Pre-treatment Mean (SD) Post-treatment Mean (SD) Follow-up Mean (SD) Within-group effect size Pre- to post-treatment (95% CI) Pre-treatment to follow-up (95% CI) PSS-I Completers 31.6 (4.7) 19.2 (9.9) 17.1 (8.5) 1.61 (0.65–2.47)* 2.32 (1.16–3.31)* ITT 31.6 (4.7) 22.0 (9.8) 21.5 (8.6) 1.25 (0.44–2.00)* 1.45 (0.61–2.21)* PCL-C Completers 59.0 (11.2) 46.9 (14.9) 43.1 (13.3) 0.95 (0.08–1.76)* 1.33 (0.35–2.22)* ITT 59.0 (11.2) 50.1 (13.3) 48.1 (11.5) 0.73 (-0.03–1.44)† 0.96 (0.18–1.69)* GAD-7 Completers 14.1 (4.4) 9.3 (4.7) 8.0 (4.0) 1.06 (0.18–1.87)* 1.42 (0.42–2.31)* ITT 14.1 (4.4) 11.1 (5.2) 9.9 (3.8) 0.62 (-0.13–1.34) 1.00 (0.22–1.73)* PHQ-9 Completers 15.3 (4.2) 11.7 (6.2) 11.3 (5.5) 0.70 (-0.14–1.50)* 0.86 (-0.06–1.72)* ITT 15.3 (4.2) 12.1 (5.3) 11.8 (4.7) 0.66 (-0.10–1.37) 0.78 (0.02–1.50)* SDS Completers 21.3 (5.5) 16.6 (10.8) 12.8 (8.9) 0.59 (-0.24–1.39) 1.26 (0.29–2.1)* ITT 21.3 (5.5) 18.3 (9.9) 16.6 (8.8) 0.37 (-0.36–1.09) 0.65 (-0.1–1.36)† K-10 Completers 32.2 (5.5) 25.8 (7.3) 24.0 (8.0) 1.03 (0.5–1.84)* 1.28 (0.30–2.16)* ITT 32.2 (5.5) 27.7 (6.7) 26.9 (6.7) 0.73 (-0.02–1.45)* 0.85 (0.08–1.57)* Note: Intention-to-treat (ITT) model (n=15) was employed with pre-treatment scores carried forward if post-treatment or follow-up data were not available. Completer data were available for 10 participants at post-treatment and 8 at follow-up. Abbreviations: PSS-I: PTSD Symptom Scale – Interview Version; PCL-C: PTSD Checklist – Civilian Version; GAD-7: Generalised Anxiety Disorder 7-Item; PHQ-9: Patient Health Questionnaire – 9 Item; K10: Kessler 10 Item; SDS: Sheehan Disability Scale. *p < 0.05; †p < 0.06. Table 1. Descriptive statistics and within-group effects on symptom measures at each assessment. results indicated significantly reduced symptoms of PTSD, depres- sion, anxiety, distress, and disability between pre-treatment and three-month follow-up according to an analysis of completers. By post-treatment, 55% of the participants no longer met criteria for PTSD, and the number of comorbid diagnoses had halved. These reductions indicate that PTSD can be treated via the internet using a combination of CBT and EMDR techniques when telephone support from a specialist therapist is also included. With respect to acceptability, this protocol was moderately tolerated, indicating that improvements would be required for further use of this intervention. Author contributions The absence of a waitlist control condition means that the improve- ments could have been the results of time, repeated measurement or other non-specific effects. The design did not allow determination of whether the effects were due to the iCBT or iEMDR compo- nents. The small sample size composed of a high number of multi- ply traumatized, childhood sexual abuse survivors may not apply to other PTSD populations. JS and NT Conceived the study and design, analysed and inter- preted the data, and drafted the article. LJ, BFD, BW, MT and JZ Contributed to the design of the study and the revision of the manuscript. Competing interests There are no competing interests for any author. Discussion motor vehicle accident survivors32 indicating that outcomes could potentially be improved if future iCBT treatments are tailored and delivered to a specific trauma population. In our trial, 3/15 (20%) reported symptom worsening according to the PCL-C score between pre- and post-treatment, although all three reported treatment gains by follow-up. Although no serious adverse events (e.g., hospitalizations, suicide attempts, relapse to substance use) occurred during the program, an increase in re-experiencing symptoms (such as intrusive thoughts and increased emotional/ physiological reactivity when reminded about the event) following iEDMR lead three participants (20%) to discontinue and five (33%) to cease using iEMDR. This potentially contributed to the higher attrition, moderate acceptability, and limited course and question- naire completion rates, relative to our earlier study. Although this may have been due to exposure-based components such as iEMDR and in vivo exposure, it is important to note that investigations of symptom deterioration and adverse events from the face-to-face literature have failed to indicate differences between exposure- and nonexposure- based treatments33. Furthermore, symptom exacerbation is no higher than reported in waiting lists nor is it greater than the error rates of the instruments used to detect adverse events34,35. Compared with ITT data from our previous trial12, the within-group effect size (ES) on the PCL-C was lower at post-treatment and follow-up. These differences may be due to changes to the proto- col, the use of a patient sample composed primarily of childhood sexual abuse survivors, or due to the influence of attrition on the ITT analysis as a result of using a small sample. According to out- comes from the PCL-C, these results compare favourably to other studies that used the same measure with mixed trauma samples in both face to face29,30 and online interventions11,31. However, results compared less favourably with face to face TF-CBT treatment of Page 6 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 ( ) PubMed Abstract \| Publisher Full Text 5. Litz BT, Bryant R, Williams L, et al.: A therapist-assisted internet self-help program for traumatic stress. Professional Psychology: Research and Practice. 2004; 35(6): 628–34. P bli h F ll T Grant information The authors would like to thank the New South Wales Institute for Psychiatry (NSWIOP) for funding this research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. y y ; ( ) PubMed Abstract \| Publisher Full Text 16. Weathers F, Litz BT, Herman D, et al.: The PTSD checklist (PCL): Reliability, validity, and diagnostic utility. Annual Conference of the International Society for Traumatic Stress Studies; San Antonio, TX 1993. 4. Ruzek JI, Rosen RC: Disseminating evidence-based treatments for PTSD in organizational settings: A high priority focus area. Behav Res Ther. 2009; 47(11): 980–9. 17. Foa EB, Riggs DS, Dancu CV, et al.: Reliability and validity of a brief instrument for assessing post-traumatic stress disorder. J Trauma Stress. 1993; 6(4): 459–73. 1. Bisson J, Andrew M: Psychological treatment of post-traumatic stress disorder (PTSD). Cochrane Database Syst Rev. 2007; (3): CD003388. PubMed Abstract \| Publisher Full Text Conclusions The results of this small feasibility study indicate that the combined iCBT/iEMDR protocol is potentially efficacious. The magnitude of gains did not appear to be as large as our previous study, although these may have been attenuated by differences in the sample and iCBT protocol. These results indicate that future research of the relative benefits of iCBT/iEMDR is warranted. PubMed Abstract \| Publisher Full Text 10. Wagner B, Schulz W, Knaevelsrud C: Efficacy of an Internet-based intervention for posttraumatic stress disorder in Iraq: a pilot study. Psychiatry Res. 2012; 195(1–2): 85–8. PubMed Abstract \| Publisher Full Text 23. Sheehan DV: The Anxiety Disease. New York, NY: Scribner; 1983. Reference Source Publisher Full Text 18. Kroenke K, Spitzer RL, Williams JB: The PHQ-9: validity of a brief depression severity measure. J Gen Intern Med. 2001; 16(9): 606–13. PubMed Abstract \| Publisher Full Text \| Free Full Text ; ( ) PubMed Abstract \| Publisher Full Text 7. Hirai M, Clum GA: An internet-based self-change program for traumatic event related fear, distress, and maladaptive coping. J Trauma Stress. 2005; 18(6): 631–6. PubMed Abstract \| Publisher Full Text 20. Spitzer RL, Kroenke K, Williams JBW, et al.: A brief measure for assessing generalized anxiety disorder: the GAD-7. Arch Intern Med. 2006; 166(10): 1092–7. ( ) PubMed Abstract \| Publisher Full Text 11. Klein B, Mitchell J, Gilson K, et al.: A therapist-assisted Internet-based CBT intervention for posttraumatic stress disorder: preliminary results. Cogn Behav Ther. 2009; 38(2): 121–31. PubMed Abstract \| Publisher Full Text 24. Gray MJ, Litz BT, Hsu JL, et al.: Psychometric properties of the life events checklist. Assessment. 2004; 11(4): 330–41. PubMed Abstract \| Publisher Full Text 25. Foa EB, Hembree EA, Rothbaum BO: Prolonged exposure therapy for PTSD: Emotional processing of traumatic experiences: Therapist guide. Oxford University Press, USA; 2007. Reference Source 12. Spence J, Titov N, Dear BF, et al.: Randomized controlled trial of Internet- delivered cognitive behavioral therapy for posttraumatic stress disorder. Depress Anxiety. 2011; 28(7): 541–50. PubMed Abstract \| Publisher Full Text r Full Text 6. Possemato K, Ouimette P, Knowlton P: A brief self-guided telehealth intervention for post-traumatic stress disorder in combat veterans: a pilot study. J Telemed Telecare. 2011; 17(5): 245–50. PubMed Abstract \| Publisher Full Text 19. Dalenberg C, Carlson E: editors. New versions of the Dissociative Experiences Scale: The DES-R (revised) and the DES-B (brief). Annual meeting of the International Society for Traumatic Stress Studies, November, Montreal, Quebec; 2010. her Full Text 3. Trusz SG, Wagner AW, Russo J, et al.: Assessing barriers to care and readiness for cognitive behavioral therapy in early acute care PTSD interventions. Psychiatry. 2011; 74(3): 207–23. 15. Blanchard EB, Jones-Alexander J, Buckley TC, et al.: Psychometric properties of the PTSD checklist (PCL). Behav Res Ther. 1996; 34(8): 669–73. PubMed Abstract \| Publisher Full Text PubMed Abstract \| Publisher Full Text 8. Littleton H, Buck K, Rosman L, et al.: From Survivor to Thriver: A Pilot Study of an Online Program for Rape Victims. Cogn Behav Pract. 2012; 19(2): 315–27. PubMed Abstract \| Publisher Full Text \| Free Full Text 21. Sheehan DV, Lecrubier Y, Sheehan KH, et al.: The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry. 1998; 59(Suppl 20): 22–33. PubMed Abstract References 1. Bisson J, Andrew M: Psychological treatment of post-traumatic stress disorder (PTSD). Cochrane Database Syst Rev. 2007; (3): CD003388. PubMed Abstract \| Publisher Full Text 2. Spence J, Titov N, Solley K, et al.: Characteristics and treatment preferences of people with symptoms of posttraumatic stress disorder: an internet survey. PLoS One. 2011; 6(7): e21864. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Trusz SG, Wagner AW, Russo J, et al.: Assessing barriers to care and readiness for cognitive behavioral therapy in early acute care PTSD interventions. Psychiatry. 2011; 74(3): 207–23. PubMed Abstract \| Publisher Full Text 4. Ruzek JI, Rosen RC: Disseminating evidence-based treatments for PTSD in organizational settings: A high priority focus area. Behav Res Ther. 2009; 47(11): 980–9. PubMed Abstract \| Publisher Full Text 5. Litz BT, Bryant R, Williams L, et al.: A therapist-assisted internet self-help program for traumatic stress. Professional Psychology: Research and Practice. 2004; 35(6): 628–34. Publisher Full Text 6. Possemato K, Ouimette P, Knowlton P: A brief self-guided telehealth intervention for post-traumatic stress disorder in combat veterans: a pilot study. J Telemed Telecare. 2011; 17(5): 245–50. PubMed Abstract \| Publisher Full Text 7. Hirai M, Clum GA: An internet-based self-change program for traumatic event related fear, distress, and maladaptive coping. J Trauma Stress. 2005; 18(6): 631–6. PubMed Abstract \| Publisher Full Text 8. Littleton H, Buck K, Rosman L, et al.: From Survivor to Thriver: A Pilot Study of an Online Program for Rape Victims. Cogn Behav Pract. 2012; 19(2): 315–27. PubMed Abstract \| Publisher Full Text \| Free Full Text 9. Lange A, Rietdijk D, Hudcovicova M, et al.: Interapy: a controlled randomized trial of the standardized treatment of posttraumatic stress through the internet. J Consult Clin Psychol. 2003; 71(5): 901–9. PubMed Abstract \| Publisher Full Text 10. Wagner B, Schulz W, Knaevelsrud C: Efficacy of an Internet-based intervention for posttraumatic stress disorder in Iraq: a pilot study. Psychiatry Res. 2012; 195(1–2): 85–8. PubMed Abstract \| Publisher Full Text 11. Klein B, Mitchell J, Gilson K, et al.: A therapist-assisted Internet-based CBT intervention for posttraumatic stress disorder: preliminary results. Cogn Behav Ther. 2009; 38(2): 121–31. PubMed Abstract \| Publisher Full Text 12. Spence J, Titov N, Dear BF, et al.: Randomized controlled trial of Internet- delivered cognitive behavioral therapy for posttraumatic stress disorder. Depress Anxiety. 2011; 28(7): 541–50. PubMed Abstract \| Publisher Full Text 13. ( ) PubMed Abstract \| Publisher Full Text \| Free Full Text 9. Lange A, Rietdijk D, Hudcovicova M, et al.: Interapy: a controlled randomized trial of the standardized treatment of posttraumatic stress through the internet. J Consult Clin Psychol. 2003; 71(5): 901–9. PubMed Abstract \| Publisher Full Text 22. Kessler RC, Andrews G, Colpe LJ, et al.: Short screening scales to monitor population prevalences and trends in non-specific psychological distress. Psychol Med. 2002; 32(6): 959–76. PubMed Abstract \| Publisher Full Text References Knaevelsrud C, Maercker A: Internet-based treatment for PTSD reduces distress and facilitates the development of a strong therapeutic alliance: a randomized controlled clinical trial. BMC Psychiatry. 2007; 7: 13. PubMed Abstract \| Publisher Full Text \| Free Full Text 14. 14. Ho MSK, Lee CW: Cognitive behaviour therapy versus eye movement desensitization and reprocessing for post-traumatic disorder – is it all in the homework then? Revue Européenne de Psychologie Appliquée/European Review of Applied Psychology. 2012; 62(4): 253–260. Publisher Full Text 2. Spence J, Titov N, Solley K, et al.: Characteristics and treatment preferences of people with symptoms of posttraumatic stress disorder: an internet survey. PLoS One. 2011; 6(7): e21864. PubMed Abstract \| Publisher Full Text \| Free Full Text PubMed Abstract \| Publisher Full Text 26. Shapiro F: Eye movement desensitization and reprocessing: Basic principles, protocols, and procedures. The Guilford Press; 2001. Reference Source 13. Knaevelsrud C, Maercker A: Internet-based treatment for PTSD reduces distress and facilitates the development of a strong therapeutic alliance: a randomized Page 7 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 27. Cohen J: A power primer. Psychol Bull. 1992; 112(1): 155–9. PubMed Abstract \| Publisher Full Text 28. Tarrier N, Pilgrim H, Sommerfield C, et al.: A randomized trial of cognitive therapy and imaginal exposure in the treatment of chronic posttraumatic stress disorder. J Consult Clin Psychol. 1999; 67(1): 13–8. PubMed Abstract \| Publisher Full Text 29. Monson CM, Schnurr PP, Resick PA, et al.: Cognitive processing therapy for veterans with military-related posttraumatic stress disorder. J Consult Clin Psychol. 2006; 74(5): 898–907. PubMed Abstract \| Publisher Full Text 30. Schnurr PP, Friedman MJ, Engel CC, et al.: Cognitive behavioral therapy for posttraumatic stress disorder in women: A randomized controlled trial. JAMA. 2007; 297(8): 820–30. PubMed Abstract \| Publisher Full Text 31. Klein B, Mitchell J, Abbott J, et al.: A therapist-assisted cognitive behavior therapy internet intervention for posttraumatic stress disorder: Pre-, post- and 3-month follow-up results from an open trial. J Anxiety Disord. 2010; 24(6): 635–44. PubMed Abstract \| Publisher Full Text 32. Blanchard EB, Hickling EJ, Devineni T, et al.: A controlled evaluation of cogn behaviorial therapy for posttraumatic stress in motor vehicle accident survivors. Behav Res Ther. 2003; 41(1): 79–96. PubMed Abstract \| Publisher Full Text 33. Taylor S, Thordarson DS, Fedoroff IC, et al.: Comparative efficacy, speed, an adverse effects of three PTSD treatments: Exposure therapy, EMDR, and relaxation training. J Consult Clin Psychol. 2003; 71(2): 330–8. PubMed Abstract \| Publisher Full Text 34. Hembree EA, Foa EB, Dorfan NM, et al.: Do patients drop out prematurely fr exposure therapy for PTSD? J Trauma Stress. 2003; 16(6): 555–62. PubMed Abstract \| Publisher Full Text 35. Neuner F: Safety First? Trauma Exposure in PTSD. Exposure Therapy. 2012; 299–312. Publisher Full Text 27. Cohen J: A power primer. Psychol Bull. 1992; 112(1): 155–9. PubMed Abstract \| Publisher Full Text 28. Tarrier N, Pilgrim H, Sommerfield C, et al.: A randomized trial of cognitive therapy and imaginal exposure in the treatment of chronic posttraumatic stress disorder. J Consult Clin Psychol. 1999; 67(1): 13–8. PubMed Abstract \| Publisher Full Text 29. 31. Klein B, Mitchell J, Abbott J, et al.: A therapist-assisted cognitive behavior therapy internet intervention for posttraumatic stress disorder: Pre-, post- and 3-month follow-up results from an open trial. J Anxiety Disord. 2010; 24(6): I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. No competing interests were disclosed. Competing Interests: Version 2 04 March 2014 Referee Report PubMed Abstract \| Publisher Full Text Monson CM, Schnurr PP, Resick PA, et al.: Cognitive processing therapy for veterans with military-related posttraumatic stress disorder. J Consult Clin Psychol. 2006; 74(5): 898–907. PubMed Abstract \| Publisher Full Text 30. Schnurr PP, Friedman MJ, Engel CC, et al.: Cognitive behavioral therapy for posttraumatic stress disorder in women: A randomized controlled trial. JAMA. 2007; 297(8): 820–30. PubMed Abstract \| Publisher Full Text 31. Klein B, Mitchell J, Abbott J, et al.: A therapist-assisted cognitive behavior therapy internet intervention for posttraumatic stress disorder: Pre-, post- and 3-month follow-up results from an open trial. J Anxiety Disord. 2010; 24(6): 635–44. PubMed Abstract \| Publisher Full Text 32. Blanchard EB, Hickling EJ, Devineni T, et al.: A controlled evaluation of cognitive behaviorial therapy for posttraumatic stress in motor vehicle accident survivors. Behav Res Ther. 2003; 41(1): 79–96. PubMed Abstract \| Publisher Full Text 33. Taylor S, Thordarson DS, Fedoroff IC, et al.: Comparative efficacy, speed, and adverse effects of three PTSD treatments: Exposure therapy, EMDR, and relaxation training. J Consult Clin Psychol. 2003; 71(2): 330–8. PubMed Abstract \| Publisher Full Text 34. Hembree EA, Foa EB, Dorfan NM, et al.: Do patients drop out prematurely from exposure therapy for PTSD? J Trauma Stress. 2003; 16(6): 555–62. PubMed Abstract \| Publisher Full Text 35. Neuner F: Safety First? Trauma Exposure in PTSD. Exposure Therapy. 2012; 299–312. Publisher Full Text 27. Cohen J: A power primer. Psychol Bull. 1992; 112(1): 155–9. PubMed Abstract \| Publisher Full Text 28. Tarrier N, Pilgrim H, Sommerfield C, et al.: A randomized trial of cognitive therapy and imaginal exposure in the treatment of chronic posttraumatic stress disorder. J Consult Clin Psychol. 1999; 67(1): 13–8. PubMed Abstract \| Publisher Full Text 29. Monson CM, Schnurr PP, Resick PA, et al.: Cognitive processing therapy for veterans with military-related posttraumatic stress disorder. J Consult Clin Psychol. 2006; 74(5): 898–907. PubMed Abstract \| Publisher Full Text 30. Schnurr PP, Friedman MJ, Engel CC, et al.: Cognitive behavioral therapy for posttraumatic stress disorder in women: A randomized controlled trial. JAMA. 2007; 297(8): 820–30. PubMed Abstract \| Publisher Full Text 31. Klein B, Mitchell J, Abbott J, et al.: A therapist-assisted cognitive behavior therapy internet intervention for posttraumatic stress disorder: Pre-, post- and 3-month follow-up results from an open trial. J Anxiety Disord. 2010; 24(6): Page 8 of 11 Page 8 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 Jo Abbott S i b U Jo Abbott Swinburne University of Technology, Hawthorn, Victoria, Australia Jo Abbott Swinburne University of Technology, Hawthorn, Victoria, Australia Swinburne University of Technology, Hawthorn, Victoria, Australia This is an important study in that in that it is exploring the feasibility of an online intervention for PTSD that includes eye movement desensitization as well as CBT. These early stage research studies evaluating novel interventions are important prior to controlled trials and it will be interesting to see how future research can identify the benefits of each component of the intervention. There were a couple of parts of the flow chart that could be a bit clearer – I too didn't follow where the 16 participants meeting all criteria came from. I also couldn't follow in the flow chart where the following information in the Attrition section fitted: “…one participant completed six lessons, but not the post-treatment assessments”, “Ten participants completed post-treatment questionnaires while eight completed follow-up questionnaires.” Regarding reference 11 (Klein et al., 2009) – there is a more recent paper about this same trial that includes follow-up data ( ) – of relevance given the comparison made to this trial. Klein et al., 2010 I think it is relevant to mention in the discussion that this was a telephone-assisted intervention (not entirely internet-delivered). I thought that the interpretation of the results would be facilitated if the authors made comment on which of the analyses (ITT or completer) they were placing weight on in drawing their conclusion and why. For example, in the discussion they state that “The results indicated significantly reduced symptoms of PTSD, depression, anxiety, distress, and disability between pre-treatment and three-month follow-up.”, but the disability measure was only significantly different between the groups when using completer analyses. It would also be useful if Table 1 also noted which of the mean differences were significant (rather than the reader needing to refer back to the text). I was also interested to know more about the worsening symptoms that some participants reported. Also, the Discussion states that 3/15 participants reported worsening symptoms but in the Results it reads as though five participants stopped using EMDR because they said it “led to an increase in re-experiencing symptoms”. doi:10.5256/f1000research.309.r857 doi:10.5256/f1000research.309.r857 Version 1 21 March 2013 Referee Report 21 March 2013 Referee Report I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. No competing interests were disclosed. Competing Interests: 01 July 2013 Referee Report doi:10.5256/f1000research.1287.r1032 Jo Abbott Swinburne University of Technology, Hawthorn, Victoria, Australia Swinburne University of Technology, Hawthorn, Victoria, Australia The authors’ changes have made have made the paper stronger. I have provided a few minor points of feedback but think the paper acceptable as it is overall, a good feasible evaluation of an innovative online intervention. The rationale for testing iEMDR is very clear but the rationale for combining iEMDR and i-TF-CBT could be stronger. In the 3 paragraph of the introduction the authors cite evidence that iEMDR is as effective as TF-CBT and can be delivered in a shorter period of time. So this provides a rationale for testing iEMDR but the addition of CBT components does not clearly follow as a rationale. rd As I suggested previously the paper that includes follow-up assessment data is more Klein . 2010 et al relevant to cite than the (reference number 11) paper that reports only pre- and Klein 2009 et al. post-assessment data for the same trial. There is one sentence under “Adverse events” in the “Results”section that doesn’t seem to clearly follow from the previous sentence: “All three improved between post-treatment and follow-up such that no participants worsened between pre-treatment and follow-up”. However, the changes made to the “Discussion” section about symptoms worsening has made the findings on worsening symptoms much clearer. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Page 9 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 No competing interests were disclosed. Competing Interests: No competing interests were disclosed. Competing Interests: No competing interests were disclosed. Competing Interests: 11 March 2013 Referee Report No competing interests were disclosed. Competing Interests: No competing interests were disclosed. Competing Interests: Author Response 24 Mar 2013 , Macquarie University, Australia Jay Spence , Macquarie University, Australia Jay Spence , Macquarie University, Australia Jay Spence Dear Dr Abbott, Thank you for your feedback. We have addressed your suggestions in an updated manuscript that has recently been uploaded. We would welcome any other edits that you feel are Page 10 of 11 F1000Research 2013, 2:79 Last updated: 05 MAR 2015 required if they are not adequately addressed in the revised publication. Thank you again for your time in improving this paper. Kind regards, Jay Spence doi:10.5256/f1000research.309.r825 doi:10.5256/f1000research.309.r825 Alyssa Boasso Veterans Affairs Medical Center Boston, Boston, MA, USA Overall, the writing is succinct and clear. The analyses are appropriate given the study design and the conclusions are justified. There are a few minor areas where further explanation or clarification is needed, these are detailed below. What is the rationale for combining the two therapies? How is iEMDR expected to add to the effectiveness of pre-existing iCBT protocols? What is the rationale for combining the two therapies? How is iEMDR expected to add to the effectiveness of pre-existing iCBT protocols? The section on recruitment does not explain how 23 people qualified, but only 16 were enrolled. In the iEMDR Intervention subsection, the authors state, “the positive belief” but do not previously define it. Also, how many people were provided special therapist-guided EMDR? Is there conjecture about how additional sessions might have affected outcomes? In the discussion section, the authors state “these results compare favourably to a similar study...” How is the study similar? Clarifying this may help contrast your findings with the following study which used motor vehicle accident survivors. In the discussion section, the authors state “these results compare favourably to a similar study...” How is the study similar? Clarifying this may help contrast your findings with the following study which used motor vehicle accident survivors. The section on worsening symptoms should address whether other similar studies have encountered the same problem? Also, is there data from the experiment that suggests which aspect of the therapy may be contributing to this issue? The section on worsening symptoms should address whether other similar studies have encountered the same problem? Also, is there data from the experiment that suggests which aspect of the therapy may be contributing to this issue? I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. No competing interests were disclosed. Competing Interests: Page 11 of 11
https://openalex.org/W2979819392	https://kau.diva-portal.org/smash/get/diva2:1360355/FULLTEXT02	English	null	A lattice model for active-passive pedestrian dynamics: a quest for drafting effects	Mathematical biosciences and engineering	2,020	cc-by	10,060	* Correspondence: Email: thikimthoa.thieu@gssi.it. Abstract: We study the pedestrian escape from an obscure room using a lattice gas model with two species of particles. One species, called passive, performs a symmetric random walk on the lattice, whereas the second species, called active, is subject to a drift guiding the particles towards the exit. The drift mimics the awareness of some pedestrians of the geometry of the room and of the location of the exit. We provide numerical evidence that, in spite of the hard core interaction between particles – namely, there can be at most one particle of any species per site – adding a fraction of active particles in the system enhances the evacuation rate of all particles from the room. A similar eﬀect is also observed when looking at the outgoing particle ﬂux, when the system is in contact with an external particle reservoir that induces the onset of a steady state. We interpret this phenomenon as a discrete space counterpart of the drafting eﬀect typically observed in a continuum set–up as the aerodynamic drag experienced by pelotons of competing cyclists. Keywords: pedestrian dynamics; evacuation; obscure room; simple exclusion dynamics; particle currents; drafting Emilio N. M. Cirillo1, Matteo Colangeli2, Adrian Muntean3 and T. K. Thoa Thieu4, p pp g g p y 2 Department of Information Engineering, Computer Science and Mathematics, University of L’Aquila, Italy 3 Department of Mathematics and Computer Science, Karlstad University, Sweden 4 Department of Mathematics, Gran Sasso Science Institute, L’Aquila, Italy 3 Department of Mathematics and Computer Science, Karlstad University, Sweden 4 D t t f M th ti G S S i I tit t L’A il It l 3 Department of Mathematics and Computer Science, Karlstad University, Sweden Department of Mathematics and Computer Science, Karlstad University, Sweden 4 Department of Mathematics, Gran Sasso Science Institute, L’Aquila, Italy 4 Department of Mathematics, Gran Sasso Science Institute, L’Aquila, Italy http://www.aimspress.com/journal/MBE http://www.aimspress.com/journal/MBE http://www.diva-portal.org http://www.diva-portal.org This is the published version of a paper published in Mathematical Biosciences and Engineering. Citation for the original published paper (version of record): Cirillo, E., Colangeli, M., Muntean, A., Thieu, T K. (2020) A lattice model for active–passive pedestrian dynamics: a quest for drafting effects Mathematical Biosciences and Engineering, 17(1): 460-477 https://doi.org/10.3934/mbe.2020025 c 2020 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0) http://urn.kb.se/resolve?urn=urn:nbn:se:kau:diva-75257 MBE, 17(1): 460–477. DOI: 10.3934/mbe.2020025 Received: 19 July 2019 Accepted: 10 October 2019 Published: 14 October 2019 1. Introduction This work is part of a larger recent research initiative oriented towards investigating the evacuation behavior of crowds of pedestrians where the geometry in which the dynamics takes place is partly unknown and possibly also with limited visibility. Such scenarios are encountered for instance when catastrophic situations occur in urban environments (e.g., in large oﬃce spaces), in tunnels, within underground spaces and/or in forests in ﬁre. We refer the reader, for instance, to [1–5] and references 461 cited therein, as well as to our previous results; see e.g. [6]. Experimental information in this context is provided, for instance, in [7] and [8] (also in connection with what is usually referred to as ”faster- is-slower” eﬀect). In the current framework, our hypothesis is that the crowd under consideration is always heterogeneous in the sense that some part of the population is well informed about the details of the geometry of the location and corresponding exits as well as of the optimal escape routes and consequently adapts its motion strategy, while the rest of the population has a passive attitude and move without following a precise strategy. This is exactly the standing assumption we have investigated in [9,10] for a dynamics in smoke scenario. It turns out that in situations where the information can only diﬃcultly be transmitted from pedes- trian to pedestrian (like when large crowds are present and/or if the geometry of the evacuation is largely unknown or invisible and/or groups are not able to act rationally), the use of leaders to guide crowds towards the exists might not always be possible, or it works ineﬃciently. In such cases, leader- ship is not essential to speed up evacuation∗. So, what can then be still done to improve evacuations for such unfavorable conditions, i.e. to decrease the evacuation time of the overall crowd? One of the main points we want to make here is the following: Even if information cannot be transmitted within the crowd, simply having a suitable fraction of informed pedestrians speeds up the overall evacuation time. We study the typical time needed by a heterogeneous crowd of pedestrians to escape a dark room. The heterogeneity of the crowd is incorporated in the fact that we consider two pedestrian species, the active and the passive one, i.e., those who know the location of the exit and those who do not. ∗This is contrary to situations like those described on [17], where leadership is an eﬃcient crowd management mecha- nism. Yet, often in pedestrian crowds there are no obvious leaders, such as those present, for instance, in political demon- strations. There is contrary evidence concerning the eﬃciency of the leadership in managing crowds. For instance, some- times artists (who could be seen as leaders of crowds) have wrongly been accused of working against safety personnel or po- lice. A famous example is that of the band Pearl Jam at the Roskilde Festival in 2001. A number of people died and re- sponsibility was placed on the band and their actions. The inquiry that came later then altered this view. See some informa- tion on this matter can be found via https://uproxx.com/music/pearl-jam-roskilde/. Another example dates back to the 1989 Hillsborough Disaster. Using Hillsborough literature, police accused the crowds in the wake of the tragic event but, al- most 30 years later, all accusations were dropped and blame was heavily placed on police and organisation. This article seems to cover it: https://www.tandfonline.com/doi/full/10.1080/1466097042000235209?casa_token=O7xM8m5HQScAAAAA: AU8DNl8LPnuSv5a01w0iw3WXEXA2AHDE95M7szy6WAkzoql4v0c2Niv44wQK5WC1eDg_qYbNFJPLLA. Mathematical Biosciences and Engineering 2. The model The room is the square lattice Λ = {1, . . . , L} × {1, . . . , L} ⊂Z2 of side length L, with L an odd positive integer number, see Figure 1. An element x = (x1, x2) of the room Λ is called site or cell. Two sites x, y ∈Λ are said nearest neighbor if and only if \|x −y\| = 1. We conventionally call horizontal the ﬁrst coordinate axis and vertical the second one. The words left, right, up, down, top, bottom, above, below, row, and column will be used accordingly. We call exit a set of wex pairwise adjacent sites, with wex an odd positive integer smaller than L, of the top row of the room Λ symmetric with respect to its median column. In other words, the exit is a centered slice of the top row of the room mimicking the presence of an exit door. The number wex will be called width of the exit. Inside the top part of the room we deﬁne a rectangular interaction zone, namely, the visibility region V, made of the ﬁrst Lv top rows of Λ, with the positive integer Lv ≤L called depth of the visibility region. By writing Lv = 0, we refer to the case in which no visibility region is considered. ✛ L ✲ ❄ L ✻ ❄ Lv ✻ ✛ wex ✲ ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t ✛✲ ❄ ✻ t Figure 1. Schematic representation of our lattice model. Blue and red disks denote passive and active particles, respectively. The rectangle of sites delimited by the red contour denotes the exit. Black and red arrows (color online) denote transitions performed with rates 1 and 1 + ε, respectively. Figure 1. Schematic representation of our lattice model. Blue and red disks denote passive and active particles, respectively. The rectangle of sites delimited by the red contour denotes the exit. Black and red arrows (color online) denote transitions performed with rates 1 and 1 + ε, respectively. 1. Introduction Using a lattice–type model, we show that the presence of pedestrians aware of the location of the exit helps the unaware companions to ﬁnd the exit of the room even in absence of any information exchange among them. This eﬀect will be called drafting and it has a twofold interpretation: i) active particles, quickly moving towards the exit, will leave a wake of empty sites in which the unaware particles can jump in, so that they are guided to the exit; ii) active pedestrians, in their rational motion towards the exit, push their passive companions to the exit as well. People belonging to the same group do not necessarily have to move together or coherently, for they only share the same dynamical rules. In other words, the motion of one pedestrian is not directly aﬀected by the motion of the pedestrians belonging to the same group. The sole interaction we consider here is the hard–core repulsion between any pair of pedestrians, regardless of their group. In this sense the situation we have in mind is diﬀerent from the one considered in some experiments, where people in the same social group move coherently and not independently [7]. We refer the reader to for instance to [11–13] for some level of details on crowd dynamics modelling and to [14–16] for relevant works regarding the handling of the presence of the obstacles and the way they aﬀect the pedestrian motion. Mind also that similar situations appear frequently also in soft matter physics cf. e.g. [18]. Volume 17, Issue 1, 460–477. 462 The reminder of the paper is organized as follows. In Section 2 we deﬁne the model. Section 3 is devoted to the study of the evacuation time. In Section 4 a slightly modiﬁed version of the model is considered, so that a not trivial stationary state is reached. For such a model the stationary exit ﬂux is thus studied. In Section 5, we summarize our conclusions and give a glimpse of possible further research. 2. The model What concerns this precise setup, the dynamics is incorporated in the continuous time Markov chain η(t) on Ωwith rates c(η, η′) deﬁned as follows: Let ε ≥0 be the drift; for any pair x = (x1, x2), y = (y1, y2) of nearest neighbor sites in Λ we set ϵ(x, y) = 0, excepting the following cases in which we set ϵ(x, y) = ε: ∈V and y2 = x2 + 1, namely, x and y belong to the visibility region and x is below y; – x, y ∈V and y2 = x2 + 1, namely, x and y belong to the visibility region and x is below – x, y ∈V and y1 = x1 +1 < (L +1)/2, namely, x and y belong to the left part of the visibility region and x is to the left with respect to y; – x, y ∈V and y1 = x1 −1 > (L + 1)/2, namely, x and y belong to the right part of the visibility region and x is to the right with respect to y. 2. The model We consider two diﬀerent species of particles, i.e., active and passive, moving inside Λ (we shall sometimes use in the notation the symbols A and P to respectively refer to them). Note that the sites of the external boundary of the room, that is to say the sites x ∈Z2 \ Λ such that there exists y ∈Λ nearest neighbor of x, cannot be accessed by the particles. The state of the system will be a conﬁguration η ∈Ω= {−1, 0, 1}Λ and we shall say that the site x is empty if ηx = 0, occupied by an active particle Volume 17, Issue 1, 460–477. Mathematical Biosciences and Engineering 463 if ηx = 1, and occupied by a passive particle if ηx = −1. The number of active (respectively, passive) particles in the conﬁguration η is given by nA(η) = P x∈Λ δ1,ηx (resp. nP(η) = P x∈Λ δ−1,ηx), where δ·,· is Kronecker’s symbol. Their sum is the total number of particles in the conﬁguration η. The dynamics in the room is modeled via a simple exclusion random walk with the two species of particles undergoing two diﬀerent microscopic dynamics: the passive particles perform a symmetric simple exclusion dynamics on the whole lattice, while the active particles, on the other hand, perform a symmetric simple exclusion walk outside the visibility region, whereas inside such a region they experience a drift pushing them towards the exit. In other words, the whole room is obscure for the passive particles, while, for the active ones, only the region outside the visibility region is obscure. Mathematical Biosciences and Engineering 2. The model he rate c(η, η′) be equal Next, we let the rate c(η, η′) be equal Next, we let the rate c(η, η′) be equal Next, we let the rate c(η, η′) be equal – to 1 if η′ can be obtained by η by replacing with 0 a −1 or a 1 at the exit (particles leave the room); can be obtained by η by replacing with 0 a −1 or a 1 at the exit (particles leave the room); – to 1 if η′ can be obtained by η by replacing with 0 a −1 or a 1 at the exit (particles leave the room); – to 1 if η′ can be obtained by η by exchanging a −1 with a 0 between two neighboring sites of Λ – to 1 if η′ can be obtained by η by replacing with 0 a −1 or a 1 at the exit (particles leave the room); – to 1 if η′ can be obtained by η by exchanging a −1 with a 0 between two neighboring sites of Λ (motion of passive particles inside Λ); – to 1 if η′ can be obtained by η by exchanging a −1 with a 0 between two neighboring sites of Λ (motion of passive particles inside Λ); – to 1 + ϵ(x, y) if η′ can be obtained by η by exchanging a +1 at site x with a 0 at site y, with x and y nearest neighbor sites of Λ (motion of active particles inside Λ); – to 1 + ϵ(x, y) if η′ can be obtained by η by exchanging a +1 at site x with a 0 at site y, with x and y nearest neighbor sites of Λ (motion of active particles inside Λ); g p – to 0 in all the other cases. The inﬁnitesimal generator L acts on continuous bounded functions f : Ω→R as L(η) = X η′∈Ω c(η, η′)[ f(η′) −f(η)]. (2.1) (2.1) The probability measure induced by the Markov chain started at η is denoted by Pη and the related expectation is denoted by Eη. We refer to [19, 20] where similarly–in–spirit models are described mathematically in a rigorous fashion. The probability measure induced by the Markov chain started at η is denoted by Pη and the related expectation is denoted by Eη. Mathematical Biosciences and Engineering 2. The model We refer to [19, 20] where similarly–in–spirit models are described mathematically in a rigorous fashion. The initial number of active (respectively, passive) particles is denoted by NA = nA(ηx(0)) (respec- tively, NP = nP(ηx(0))). We also let N = NA + NP be the initial total number of particles. For any choice of the initial conﬁguration η(0) in Ω, the process will eventually reach the empty conﬁguration 0 corresponding to zero particles in the room which is an absorbing point of the chain. As alternative working scenario, we will study the dynamics described above also in the presence of a solid obstacle hindering the pedestrian ﬂow in the room. The obstacle is made of an array of sites permanently occupied by ﬁctitious particles. In such a way these sites are never accessible to the actual interacting particles within our system. The dynamics in both the presence and in the absence of the obstacle is the same and all the param- eters have the same meaning and take similar values. In other words, we are implicitly assuming that Volume 17, Issue 1, 460–477. 464 Figure 2. Conﬁgurations of the model sampled at diﬀerent times (increasing in lexicographic order). Parameters: L = 15, wex = 7, Lv = 5, and ε = 0.3. Red pixels represent active parti- cles, blue pixels denote passive particles, and gray sites are empty. In the initial conﬁguration (top left panel) there are 70 active and 70 passive particles. Figure 2. Conﬁgurations of the model sampled at diﬀerent times (increasing in lexicographic order). Parameters: L = 15, wex = 7, Lv = 5, and ε = 0.3. Red pixels represent active parti- cles, blue pixels denote passive particles, and gray sites are empty. In the initial conﬁguration (top left panel) there are 70 active and 70 passive particles. Figure 3. As in Figure 2, the obstacle is a centered 5 × 5 square. This ﬁxed obstacle is depicted with white pixels. Figure 3. As in Figure 2, the obstacle is a centered 5 × 5 square. This ﬁxed obstacle is depicted with white pixels. Volume 17, Issue 1, 460–477. Mathematical Biosciences and Engineering 465 the presence of the obstacle does not aﬀect the pedestrian behavior: the obstacle is simply a barrier that must be avoided by the walking pedestrian. 2. The model Although, in principle, there is no restriction on the choice of the obstacle geometry, in this frame- work we always consider centered squared obstacles. A thorough investigation of the eﬀect of the choice of obstacle geometry on the evacuation time for mixed pedestrian populations moving thorough partially obscure rooms deserves special attention and will be done in a forthcoming work. We simulate this process using the following scheme: at time t we extract an exponential random time τ with parameter the total rate P ζ∈Ωc(η(t), ζ) and set the time equal to t + τ. We then select a conﬁguration using the probability distribution c(η(t), η)/ P ζ∈Ωc(η(t), ζ) and set η(t + τ) = η. As we have already pointed out in Section 1, the main goal of the paper is to detect drafting in pedestrian ﬂows, namely, identify situations when the evacuation of passive particles is favored by the presence of the active ones, even if no leadership or other kind of information exchange is allowed. We expect that this phenomenon will be eﬀective, provided the active particles will spend a suﬃciently long time in the room. This seems to help eﬃciently passive particles to escape. This eﬀect is illustrated in the Figures 2 and 3, where we show the conﬁguration of the system at diﬀerent times both in the absence, and respectively, in the presence of an obstacle. Indeed, the sequences of conﬁgurations show that, though the evacuation of active particles is faster than that of the passive ones, even at late times the fraction of active particles is still reasonably high. 3. The evacuation time Consider the dynamics deﬁned in Section 2, given a conﬁguration η ∈Ω. We let τη be the ﬁrst hitting time to the empty conﬁguration, i.e. τη = inf{t > 0 : η(t) = 0}, (3.1) (3.1) for the chain started at η. Given a conﬁguration η ∈Ω, we deﬁne the evacuation time starting from η as for the chain started at η. Given a conﬁguration η ∈Ω, we deﬁne the evacuation time starting from η as as Tη = Eη[τη] . (3.2) (3.2) Tη = Eη[τη] . We have deﬁned the evacuation time as the time needed to evacuate all the particles initially in the system, that is to say the evacuation time is the time at which the last particle leaves the room. In this section as well as in the next one, we study numerically the evacuation time for a ﬁxed initial random condition and then produce various realizations of the process for speciﬁc values of the initial drift ε and of the visibility depth Lv. To this end, we consider two geometrically diﬀerent situations: (i) the empty roomand (ii) the room with a squared obstacle positioned at the center. Mathematical Biosciences and Engineering 3.1. The empty room case We consider the system deﬁned in Section 2 for L = 15 (side length of the room), wex = 7 (exit width), NP = 70 (initial number of passive particles) NA = 0, 70 (initial number of active particles) Lv = 2, 5, 7, 15 (visibility depth), and ε = 0.1, 0.3, 0.5 (drift). More details are provided in the ﬁgure captions. All the simulations are done starting the system from the same initial conﬁguration chosen once for all by distributing the particle at random with uniform probability. More precisely, two initial Volume 17, Issue 1, 460–477. 466 (a) (b) Figure 4. Two initial conﬁgurations for the lattice gas dynamics. Blue and red pixels rep- resent, respectively, passive and active particles. The thick dashed line surrounding a large fraction of the grid denotes the presence of reﬂecting boundary conditions. The exit door is located in presence of the missing segment of dashed line. In (a) only NP passive particles are present. In (b), the passive particles occupy the same initial positions as in (a), and NA active particles are also included (we ﬁx NA = NP). We shall compare the evacuation time relative to the two conﬁgurations in (a) and (b). (a) (b) (b) (a) Figure 4. Two initial conﬁgurations for the lattice gas dynamics. Blue and red pixels rep- resent, respectively, passive and active particles. The thick dashed line surrounding a large fraction of the grid denotes the presence of reﬂecting boundary conditions. The exit door is located in presence of the missing segment of dashed line. In (a) only NP passive particles are present. In (b), the passive particles occupy the same initial positions as in (a), and NA active particles are also included (we ﬁx NA = NP). We shall compare the evacuation time relative to the two conﬁgurations in (a) and (b). conﬁgurations are considered, one for the case NP = 70 and NA = 0 and one for the case NP = 70 and NA = 70, chosen in such a way that in the two cases the initial positions of the passive particle is the same, see Figure 4 for a schematic illustration. We then compute the time needed to evacuate all the particles initially in the systems and, by aver- aging over 105 diﬀerent realizations of the process, we compute a numerical estimate of the evacuation time (3.2) for the chosen initial condition. Mathematical Biosciences and Engineering 3.1. The empty room case 620 640 660 680 700 720 740 760 0 0.2 0.4 0.6 0.8 1 evacuation time ε 620 640 660 680 700 720 740 760 2 4 6 8 10 12 14 evacuation time Lv Figure 5. Evacuation time in an empty room for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). dynamics of the passive particles does not depend on ε. We observe that the small ﬂuctuations visible in the data represented by solid disks Figure 5 come from considering averages over a ﬁnite number of diﬀerent realizations of the process (all starting from the given initial conﬁguration). Since the number particles in the initial conﬁguration in the experiments with the presence of active particles is double with respect to that considered in the case of only passive particles, one would expect a larger evacuation time. This is indeed the case for a small visibility depth, i.e. for Lv = 2. In such a case, it is worth noting that the evacuation time decreases when the drift is increased as it is in fact reasonable since a larger drift favors the fast evacuation of active particles, but it remains larger than the evacuation time in absence of active particles for all the values of ε that we considered. On the other hand, for larger values of the visibility depth, as long as the drift is large enough, the evacuation time in the presence of active particles becomes smaller than the one measured in the presence of only passive particles. This eﬀect is rather surprising. It can interpreted by saying that the presence of active particles, which have some information about the location of the exit, helps passive particles to evacuate the room even if no information exchange is allowed, and, mostly, even in presence of the exclusion con- straint of the lattice gas dynamics. Indeed, passive particles continue their blind symmetric dynamics, nevertheless their evacuation time is reduced. 3.1. The empty room case Results are reported in Figure 5. The main result of our investigation is the following: the evacuation time Tη corresponding to the initial conﬁguration with active particles (see the illustration (b) in Figure 4) is less than that corre- sponding to the initial conﬁguration with sole passive particles (see the illustration (a) in Figure 4). This result is non–trivial since our lattice dynamics is based on a hard core exclusion principle [20] – the motion of particles towards the exit is hindered by the presence of nearby particles. In the context of this work, we refer to this phenomenon as drafting, marking this way the analogy with the drafting or aerodynamic drag eﬀect encountered by pelotons of cyclists racing towards the goal; we refer the reader, for instance, to [21, 22] and references cited therein, for wind tunnel and computational evi- dence on drafting. It is crucial to note that the presence of active particles is essential for the onset of this phenomenon: if all active particles in the conﬁguration (b) in Figure 4 (represented by the red pixels) were replaced by passive ones (blue pixels), the evacuation time would clearly become larger than with the conﬁguration (a). This is essentially due to the exclusion constraint of the lattice gas dynamics. In the left panel in Figure 5, the dependence of the evacuation time on the drift ε is shown. Open symbols refer to the evacuation time for NP = 70 and NA = 70; for each value of ϵ we repeat the measure of the evacuation time also for a system in which only passive particles are present. We then obtain the sequence of solid disks reported in the ﬁgure which is approximatively constant, since the Volume 17, Issue 1, 460–477. 467 620 640 660 680 700 720 740 760 0 0.2 0.4 0.6 0.8 1 evacuation time ε 620 640 660 680 700 720 740 760 2 4 6 8 10 12 14 evacuation time Lv Figure 5. Evacuation time in an empty room for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). Mathematical Biosciences and Engineering 3.1. The empty room case sole passive particles though the total number of particles to be evacuated is doubled. As before we interpret these data as an evidence of the presence of the drafting eﬀect. Remarkably, for suﬃciently large drift ε, the evacuation time is not monotonic with respect to the visibility depth. In other words, there is an optimal value of Lv which minimizes the evacuation time. The fact that for Lv too large, i.e., comparable with the side length of the room, the evacuation time increases with Lv can be explained remarking that if active particles exit the system too quickly then passive particles, left alone in the room, evacuate it with their standard time. Hence, the drafting eﬀect is visible as long as the parameters ε and Lv make the motion of the active particles towards the exit enough faster than that of passive particles, but not too fast. Indeed, if the active particles move too slowly, they behave as passive ones: this would make the evacuation time larger, due to the standard exclusion constraint of our lattice gas dynamics. On the other hand, if the active particles move too fast, passive particles remain soon alone on the lattice, therefore the evacuation time relative to the conﬁguration of type (b) in Figure 4 reduces to that relative to the conﬁguration of type (a). Before concluding this Section, we shall also highlight the eﬀect of varying the relative amount of active and passive particles in the initial conﬁguration. In Figure 6 we also present the average evacuation times for the cases with NP = 70, NA = 35 and NP = 140, NA = 0, for two diﬀerent values of Lv. One readily notices that when the number of passive particles is doubled (case NP = 140 and NA = 0) the evacuation time increases and it obviously results to be independent of ε and Lv. For small visibility depth (Lv = 2 in the left panel in Figure 6) the evacuation time for the case NA = 35 and NP = 70 is smaller than the one mesured in the case NA = 70 and NP = 70 for any ε and shows a monotonic decrease. 3.1. The empty room case The sole interaction among passive and active particles is the exclusion rules, hence one possible interpretation of this eﬀect is that active particles, while walking toward the exit, leave behind a sort of empty sites wake. Passive particles, on the other hand, can beneﬁt of such an empty path and be thus blindly driven towards the exit. A diﬀerent interpretation is that passive particles, due to the exclusion rule, are pushed by active particles towards the exit. In the right panel of Figure 5, for the same choices of parameters and initial conditions, we show the evacuation time as a function of the visibility depth Lv for several values of the drift ε. Data can be discussed similarly as we did for those plotted in the left panel of the same ﬁgure: for small drift, and for any choice of the visibility length, the evacuation time in presence of active particles is larger than the one measured with sole passive particles. But, if the drift is increased, for a suﬃciently large visibility depth, the evacuation time in presence of active particles becomes smaller than the one for Volume 17, Issue 1, 460–477. 468 640 650 660 670 680 690 700 710 720 730 740 750 0 0.2 0.4 0.6 0.8 1 evacuation time ε 620 640 660 680 700 720 740 760 0 0.2 0.4 0.6 0.8 1 evacuation time ε Figure 6. Evacuation time in an empty room for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks), NA = 35 and NP = 70 (open triangles), NA = 70 and NP = 70 (open circles) and NA = 0 and NP = 140 (open squares). Left panel: Lv = 2. Right panel: Lv = 7. 620 640 660 680 700 720 740 760 0 0.2 0.4 0.6 0.8 1 evacuation time ε 640 650 660 670 680 690 700 710 720 730 740 750 0 0.2 0.4 0.6 0.8 1 evacuation time ε Figure 6. Evacuation time in an empty room for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks), NA = 35 and NP = 70 (open triangles), NA = 70 and NP = 70 (open circles) and NA = 0 and NP = 140 (open squares). Left panel: Lv = 2. Right panel: Lv = 7. Mathematical Biosciences and Engineering 3.2. The room with an obstacle Simulations similar with those described in Section 3.1 have been run in presence of a centered square obstacle made of 5 × 5 sites of the room not accessible by both active and passive particles. As before, we have computed the evacuation time in such a case and results are reported in Figure 7. Simulations similar with those described in Section 3.1 have been run in presence of a centered square obstacle made of 5 × 5 sites of the room not accessible by both active and passive particles. As before, we have computed the evacuation time in such a case and results are reported in Figure 7. It is immediate to remark that plots in Figure 7 are very similar to those shown in Figure 5. Our interpretation of the results is then the same. We just mention that the vertical scale is slightly diﬀerent and we notice that, in presence of an obstacle, the drafting eﬀect is slightly increased. The fact that the presence of an obstacle with suitable geometry can favor the evacuation of a room is a fact already established in the literature, see, e.g., [6,14–16] and references therein. 3.1. The empty room case Evacuation time in a room with a 5 × 5 squared centered obstacle for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). Mathematical Biosciences and Engineering 3.1. The empty room case The results are more interesting for larger visibility depth (Lv = 7 in the right panel in Figure 6): if the drift ε is large enough, namely, larger than about 0.2, the total evacuation time in presence of active particles becomes smaller than the one measured for sole passive particles both for the case NA = 35 and NP = 70 and NA = 70 and NP = 70, that is to say, in both cases the drafting eﬀect shows up. More interestingly, the evacuation time is smaller in the case in which more active particles are present (open circles in the picture): this is a sort of signature of the drafting eﬀect. Volume 17, Issue 1, 460–477. 469 680 700 720 740 760 780 800 820 0 0.2 0.4 0.6 0.8 1 evacuation time ε 680 700 720 740 760 780 800 820 2 4 6 8 10 12 14 evacuation time Lv Figure 7. Evacuation time in a room with a 5 × 5 squared centered obstacle for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). 680 700 720 740 760 780 800 820 0 0.2 0.4 0.6 0.8 1 evacuation time ε 680 700 720 740 760 780 800 820 2 4 6 8 10 12 14 evacuation time Lv Figure 7. Evacuation time in a room with a 5 × 5 squared centered obstacle for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). ure 7. Evacuation time in a room with a 5 × 5 squared centered obstacle for L = 15 Figure 7. 4.1. The empty room case We consider the system deﬁned in Section 4 for L = 15 (side length of the room), wex = 7 (exit width), NP = 70 (number of passive particles) NA = 0, 70 (number of active particles) Lv = 2, 5, 7, 15 (visibility depth), and ε = 0.1, 0.3, 0.5 (drift). More details on the selected parameters regimes are provided in the ﬁgure captions. As in Section 3.1, all the simulations share the same initial conﬁguration obtained by distributing the particle at random with a uniform probability. More precisely, two initial conﬁgurations are considered, one for the case NP = 70 and NA = 0 and one for the case NP = 70 and NA = 70, chosen in such a way that in the two cases the initial positions of the passive particle is the same, see Figure 4. We then let the system evolve and compute the ratio of the number of particles jumping from the exit to the reservoir to time. We consider, in particular, the ﬂux of passive particles, in absence and in presence of the active ones. This observable ﬂuctuates until it approaches a roughly constant value after about k = 6.36 × 107 MC steps (corresponding, approximately, to time 328342) is reached. Our results are reported in Figure 8. We show the dependence of the stationary ﬂux of passive particles on the drift ε in the left panel of Figure 8. Open symbols refer to the ﬂux for NP = 70 and NA = 70; for each value of ϵ we repeat the measure of the ﬂux time also for a system in which only passive particles are present. We then obtain the sequence of solid disks reported in the ﬁgure which is approximatively constant, since the dynamics of the passive particles does not depend on ε. In the right panel of Figure 8, for the same choices of parameters and initial conditions, we show the stationary ﬂux as a function of the visibility depth Lv for several values of the drift ε. Both ﬁgures exhibit ﬁrstly an increase of the ﬂux in presence of active particles at zero drift or zero visibility depth with respect to the case in which only passive particles are present (ﬁlled disks in Figure 8. 4. The stationary ﬂux 470 following modiﬁcation: following modiﬁcation: – if η′ can be obtained by η by adding a +1 at an empty site x then c(η, η′) = [NA −nA(η)]/(L2 − ( ) ( )) ( i i i l f h i i i h ) – if η′ can be obtained by η by adding a +1 at an empty site x then c(η, η′) = [NA −nA(η)]/(L2 − nA(η) −nP(η)) (moving an active particle from the reservoir to an empty site in the room); – if η′ can be obtained by η by adding a +1 at an empty site x then c(η, η′) = [NA −nA(η)]/(L2 − nA(η) −nP(η)) (moving an active particle from the reservoir to an empty site in the room); nA(η) −nP(η)) (moving an active particle from the reservoir to an empty site in the nA(η) nP(η)) (moving an active particle from the reservoir to an empty site in the room); – if η′ can be obtained by η by adding a −1 at an empty site x then c(η, η′) = [NP −nP(η)]/(L2 − nA(η) −nP(η)) (moving a passive particle from the reservoir to an empty site in the room). – if η′ can be obtained by η by adding a −1 at an empty site x then c(η, η′) = [NP −nP – if η′ can be obtained by η by adding a −1 at an empty site x then c(η, η′) = [NP −nP(η)]/(L2 − nA(η) −nP(η)) (moving a passive particle from the reservoir to an empty site in the room). At time t, the quantities NA −nA(η(t)) and NP −nP(η(t)) represent the number of active, and re- spectively, passive particles in the reservoir at time t, whereas L2 −nA(η(t)) −nP(η(t)) is the number of empty sites of the room at time t. With these changes in the deﬁnition of the rate, the total number of particles in the system (consid- ering the room and the reservoir) is conserved. The number of particles in the room, on the other hand, will ﬂuctuate due to the fact that particles can accumulate in the reservoir. 4. The stationary ﬂux In the study of this dynamics, the main quantity of interest is the stationary outgoing ﬂux or current which is the value approached in the inﬁnite time limit by the ratio between the total number of particle that in the interval (0, t) jumped from the exit to the reservoir and the time t. Mathematical Biosciences and Engineering 4. The stationary ﬂux To detect non–trivial behaviors as time elapses beyond a characteristic walking timescale, we con- sider now a modiﬁed version of the model proposed in Section 2. Essentially, the current situation is as follows: Particles exiting the system are introduced back in one site, randomly chosen among the empty sites of the room so that the total number of active and passive particles is approximatively kept constant during the evolution. This way, the system reaches a ﬁnal stationary state and in such a state we shall measure the ﬂux of exiting active and passive particles. The main idea is to add a reservoir in which particles exiting the room are collected. Particles in the reservoir are then introduced inside Λ with rates depending on the number of particles in such a reservoir and on the number of empty sites in the room. More precisely, recall the deﬁnition of the number of active and passive particles nA(η) and nP(η) in the conﬁguration η and ﬁx two non–negative integer numbers NA and NP. Consider the Markov process deﬁned in Section 2 with an initial conﬁguration with total number of active and passive particles respectively equal to NA and NP and rates c(η, η′), for η, η′ ∈Ω, deﬁned as in Section 2 with the Volume 17, Issue 1, 460–477. 4.1. The empty room case This situation can be understood by considering that, despite the exclusion constraint of the lattice gas dynamics, doubling the number of particles can justify the increase of the ﬂux at zero drift, if no complete clogging is reached. It is instructive to follow the sequence of empty symbols in Figure 8 for increasing values of ε or Lv. Note that an increase of the drift yields a monotonic increase Volume 17, Issue 1, 460–477. 471 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0 0.2 0.4 0.6 0.8 1 outgoing flux ε 0.56 0.58 0.6 0.62 0.64 0.66 0.68 0.7 0.72 0.74 2 4 6 8 10 12 14 outgoing flux Lv Figure 8. Stationary ﬂux of passive particles in an empty room for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0 0.2 0.4 0.6 0.8 1 outgoing flux ε 0.56 0.58 0.6 0.62 0.64 0.66 0.68 0.7 0.72 0.74 2 4 6 8 10 12 14 outgoing flux Lv Figure 8. Stationary ﬂux of passive particles in an empty room for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). of the stationary ﬂux as long as the visibility depth is not too large. In fact, the monotonic increase of the ﬂux is not observed with Lv = 15 (see the open squares in the left panel). This means that in the presence of such a large visibility depth the evacuation of the passive particles can be hindered by the presence of active particles if the drift is not large enough. Mathematical Biosciences and Engineering 4.1. The empty room case A quite interesting and a priori unexpected fact is the non–monotonicity of the stationary ﬂux with respect to the visibility depth: this can be seen by looking at the diﬀerent curves in the left panel and it is also put in evidence in the plots of the right panel. It is also worth looking at the behavior of the transient ﬂuxes as functions of time in the same evacuation set-up discussed earlier in Section 3, in which no external particle reservoir is included. To this aim, for a given initial condition, we averaged the ﬂux of passive particles over 103 diﬀerent realizations of the process. Coherently with the behavior observed for the stationary ﬂuxes in Figure 8, we notice once more that the presence of active particle enhances the outgoing ﬂux of passive particles, for all the considered values of ε and Lv, this being again a trace of the underlying drafting phenomenon. To get a deeper insight in this interesting eﬀect, we show in Figure 10 the stationary occupation number proﬁle. To obtain these results, we have run the dynamics for a suﬃciently long time (order of 6.36 × 107 MC steps) so that the system reaches the stationary state. Starting oﬀfrom that time, we have averaged the occupation number \|ηx(t)\| over time at each site of the room. The resulting function takes values between zero and one; see Figure 10 for an illustration. The plots indicate that for large drift and large visibility depth that clogging along the median ver- tical line can take place. the occurrence of such clogging situations partly explain the not–monotonic behavior of the stationary ﬂux with varying the visibility depth. The emergence of the central clogging is related to the large value of the drift pointing towards the central direction. This phenomenon reminds the faster–is–slower behavior already pointed out in the literature [23,24], even if in this case the origin of the phenomenon can be traced back to the intensity of the drift rather than to the pedestrian speed. Volume 17, Issue 1, 460–477. 4.1. The empty room case 472 0 0.2 0.4 0.6 0.8 1 1.2 0 10000 20000 30000 40000 50000 outgoing flux time 0 0.2 0.4 0.6 0.8 1 1.2 0 10000 20000 30000 40000 50000 outgoing flux time 0 0.2 0.4 0.6 0.8 1 1.2 0 10000 20000 30000 40000 50000 outgoing flux time 0 0.2 0.4 0.6 0.8 1 1.2 0 10000 20000 30000 40000 50000 outgoing flux time Figure 9. Transient ﬂux of passive particles in an empty room for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). In the various panels, shown are the cases with ε = 0.1 (empty triangles), ε = 0.3 (empty circles) and ε = 0.5 (empty squares). Diﬀerent values of Lv are considered: Lv = 2 (top left panel), Lv = 5 (top right panel), Lv = 7 (bottom left panel), and Lv = 15 (bottom right panel). 0 0.2 0.4 0.6 0.8 1 1.2 0 10000 20000 30000 40000 50000 outgoing flux time 0 0.2 0.4 0.6 0.8 1 1.2 0 10000 20000 30000 40000 50000 outgoing flux time 0 0.2 0.4 0.6 0.8 1 1.2 0 10000 20000 30000 40000 50000 outgoing flux time 0 0.2 0.4 0.6 0.8 1 1.2 0 10000 20000 30000 40000 50000 outgoing flux time time time Figure 9. Transient ﬂux of passive particles in an empty room for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). In the various panels, shown are the cases with ε = 0.1 (empty triangles), ε = 0.3 (empty circles) and ε = 0.5 (empty squares). Diﬀerent values of Lv are considered: Lv = 2 (top left panel), Lv = 5 (top right panel), Lv = 7 (bottom left panel), and Lv = 15 (bottom right panel). 4.2. The room with an obstacle Simulations similar with those described in Section 4.1 have been run in presence of a centered square obstacle made of 5 × 5 sites of the room not accessible by both active and passive particles. As before, we have computed the stationary ﬂux and our results are reported in Figure 11. The results plotted in Figures 11 and 12 are very similar to those shown in Figure 8 and 10. Our interpretation of the results is essentially the same. Mind though that, in order to reach the stationary state, we had to run the dynamics for a larger time than in the case of the empty room model (i.e., of order of 9.0×107 MC steps). Also from the point of view of the computed stationary ﬂux measures, our results suggest that the presence of the obstacle slightly favors the exit of particles from the room. This was noted in Section 3.2 in connection with the evacuation time measurements; see also [6,14–16]. Comparing Figures 8 and 11 one realizes that the dependence of the stationary ﬂux on the drift and on the visibility depth is milder. This fact can be explained remarking that the phenomenon of accumulation of particles along the median vertical line of the room discussed in Section 4.1 is less evident. Again, the obstacle seems to be keeping particles far apart so that clogging is reduced. Mathematical Biosciences and Engineering Mathematical Biosciences and Engineering 5. Conclusions We studied the problem of the evacuation of a crowd of pedestrians from an obscure region. We start from the assumption that the crowd is made of both active and passive pedestrians. The hazardous motion of pedestrians due to lack of light and, possibly, combined also to a high level of stress is Volume 17, Issue 1, 460–477. 473 Figure 10. Occupation number proﬁle at stationarity for L = 15, wex = 7, xex = 5, NA = NP = 70, ε = 0.1, 0.3, 0.5 (from the top to the bottom), Lv = 2, 5, 7, 15 (from the left to the right). Figure 10. Occupation number proﬁle at stationarity for L = 15, wex = 7, xex = 5, NA = NP = 70, ε = 0.1, 0.3, 0.5 (from the top to the bottom), Lv = 2, 5, 7, 15 (from the left to the right). 0.5 0.6 0.7 0.8 0.9 1 0 0.2 0.4 0.6 0.8 1 outgoing flux ε 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 2 4 6 8 10 12 14 outgoing flux Lv Figure 11. Stationary ﬂux in a room with a 5 × 5 squared centered obstacle for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). 0.5 0.6 0.7 0.8 0.9 1 0 0.2 0.4 0.6 0.8 1 outgoing flux ε 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 2 4 6 8 10 12 14 outgoing flux Lv Figure 11. Stationary ﬂux in a room with a 5 × 5 squared centered obstacle for L = 15, wex = 7, NA = 0 and NP = 70 (solid disks) and NA = NP = 70 (open symbols). Left panel: Lv = 2 (open triangles), Lv = 5 (open circles), Lv = 7 (open pentagons), Lv = 15 (open squares). Right panel: ε = 0.1 (open triangles), ε = 0.3 (open circles), ε = 0.5 (open squares). Volume 17, Issue 1, 460–477. Mathematical Biosciences and Engineering 474 Figure 12. Mathematical Biosciences and Engineering 5. Conclusions Occupation number proﬁle at stationarity in presence of a 5 × 5 centered obstacle for L = 15, wex = 7, xex = 5, NA = NP = 70, ε = 0.1, 0.3, 0.5 (from the top to the bottom), Lv = 2, 5, 7, 15 (from the left to the right). Figure 12. Occupation number proﬁle at stationarity in presence of a 5 × 5 centered obstacle for L = 15, wex = 7, xex = 5, NA = NP = 70, ε = 0.1, 0.3, 0.5 (from the top to the bottom), Lv = 2, 5, 7, 15 (from the left to the right). Volume 17, Issue 1, 460–477. Mathematical Biosciences and Engineering Mathematical Biosciences and Engineering 475 modeled via a simple random walk with exclusion. The active (smart, informed, aware, ...) pedestrians, which are aware of the location of the exit, are supposed to be subject to a given drift towards the exit, while the passive (unaware, uninformed) pedestrians are performing a random walk within the walking geometry and eventually evacuate if they accidentally ﬁnd the exit. The particle system is strongly interacting via the site exclusion principle – each site can be occupied by only a single particle. The main observable is the evacuation time as a function of the parameters caracterizing the mo- tion of the aware pedestrians. We have found that the presence of the active pedestrians favors the evacuation of the passive ones. This is rather surprising since we explicitly do not allow for any com- munication among the pedestrians. This seems to be due to some sort of drafting eﬀect. A drag seems to arise due to the empty spaces left behind by the active pedestrians moving towards the exit and naturally ﬁlled by the completely random moving unaware pedestrians. We have also remarked that too smart active pedestrians can limit the drafting eﬀect: indeed, if they exit the room too quickly the unaware pedestrian do not have the time to take proﬁt of the wakes of empty side that they left during their motion towards the exit. A promising research line concerns the investigation of evacuation times when diﬀerent species of particles are assumed to choose among diﬀerent exit doors. Such topic is relevant not only for urban situations but also for tunnel ﬁres or for forrest ﬁres expanding towards the neighborhood of inhabited regions. 5. Conclusions The main open question in this context is the model validation. A suitable experiment design is needed to make any progress in this sense. This will be our target in forthcoming work. With regards to the building up of the aerodynamic drag, it would also be interesting to verify the onset of the drafting phenomenon in lattice gas models in the presence of non–standard transport regimes leading to uphill diﬀusion of particles; see [25] (and references cited therein) for the study of such transport mechanisms. Acknowledgments ENMC and MC thank FFABR 2017 for ﬁnancial support. We thank our collaborators Omar Richardson (Karlstad, Sweden) and Errico Presutti (Gran Sasso Science Institute, Italy) for very useful discussions on this and related topics, and Finn Nilson (Karlstad) for providing us with infos about the 2001 Roskilde Festival incident and the 1989 Hillsborough Disaster, mentioned in the footnote of p. 2. Conﬂict of interest There is no conﬂicts of interest. Mathematical Biosciences and Engineering 1. G. Albi, M. Bongini, E. Cristiani, et al., Invisible control of self–organizing agents leaving un- known environments, SIAM J. Appl. Math., 76 (2016), 1683–1710. References 1. G. Albi, M. Bongini, E. Cristiani, et al., Invisible control of self–organizing agents leaving un- known environments, SIAM J. Appl. Math., 76 (2016), 1683–1710. 2. K. Fridolf, E. Ronchi, D. Nilsson, et al., Movement speed and exit choice in smoke-ﬁlled rail tunnels, Fire Safety J., 59 (2013), 8–21. Volume 17, Issue 1, 460–477. Mathematical Biosciences and Engineering 476 3. F. M¨uller, O. Wohak and A. Schadschneider, Study of inﬂuence of groups on evacuation dynamics using a cellular automaton model, Transport. Res. Procedia, 2 (2014), 168–176. 4. J.–H. Wang and J.–H. Sun. Principal aspects regarding to the emergency evacuation of large–scale crowds: A brief review of literatures until 2010, Procedia Eng., 71 (2014), 1–6. 5. S. Xue, B. Jia, R. Jiang, et al., Pedestrian evacuation in view and hearing limited condition: The impact of communication and memory, Phys. Lett. A, 380 (2016), 3029–3035. 6. A. Ciallella, E. N. M. Cirillo, P. Curseu, et al., Free to move or trapped in your group: Mathematical modeling of information overload and coordination in crowded populations, Math. Mod. Meth. Appl. Sci., 28 (2018), 1831–1856. 7. C. von Kr¨uchten and A. Schadschneider, Empirical study on social groups in pedestrian evacuation dynamics, Physica A, 475 (2017), 129–141. 8. H. Oh and J. Park, Main factor causing “faster-is-slower” phenomenon during evacuation: rodent experiment and simulation, Sci. Rep., 7 (2017), 13724. 9. M. Colangeli, A. Muntean, O. Richardson, et al., Modelling interactions between active and pas- sive agents moving through heterogeneous environments, in G. Libelli, N. Bellomo (Eds), Crowd Dynamics, vol. 1: Theory, Models and Safety Problems (pp. 211– 254). Modeling and Simulation in Science, Engineering and Technology, Boston, Birkh¨auser, 2019. 10. O. Richardson, A. Jalba and A. Muntean, The eﬀect of environment knowledge in evacuation sce- narios involving ﬁre and smoke – a multiscale modelling and simulation approach, Fire Technol., 55 (2019), 415–436. 11. J. P. Agnelli, F. Colasuonno and D. Knopoﬀ, A kinetic theory approach to the dynamics of crowd evacuation from bounded domains, Math. Mod. Meth. Appl. S., 25 (2015), 109–129. 12. N. Bellomo, D. Clarke, L. Gibelli, et al., Human behaviours in evacuation crowd dynamics: From modelling to “big data” toward crisis management, Phys. Life Rev., 18 (2016), 1–21. 13. E. Cristiani, B. Piccoli and A. Tosin, Multiscale Modeling of Pedestrian Dynamics, Series in Modeling, Simulation and Applications, vol. 12, Springer, 2014. 14. A. Ciallella and E. N. M. Mathematical Biosciences and Engineering Volume 17, Issue 1, 460–477. 20. H. Spohn, Large Scale Dynamics of Interacting Particles, Springer-Verlag Berlin Heidelberg, 1991. References Cirillo, Linear Boltzmann dynamics in a strip with large reﬂective ob- stacles: stationary state and residence time, Kinetic Relat. Mod., 11 (2018), 1475–1501. 15. E. N. M. Cirillo, O. Krehel, A. Muntean, et al., Lattice model of reduced jamming by a barrier, Phys. Rev. E, 94 (2016), 042115. 16. E. Cristiani and D. Peri. Handling obstacles in pedestrian simulations: Models and optimization, Appl. Math. Model., 45 (2017), 285–302. 17. I. D. Couzin, J. Krause, N. R. Franks, et al., Eﬀective leadership and decision–making in animal groups on the move. Nature, 433 (2005), 513–516. 18. D. Andreucci, D. Bellaveglia, E. N. M. Cirillo, et al., Monte Carlo study of gating and selection in potassium channels. Phys. Rev. E, 84 (2011), 021920. 19. G. A. Pavliotis, Stochastic Processes and Applications: Diﬀusion Processes, the Fokker-Planck and Langevin Equations. Texts in Applied Mathematics, vol. 60, Springer Verlag, Berlin, 2014. Volume 17, Issue 1, 460–477. Mathematical Biosciences and Engineering 477 20. H. Spohn, Large Scale Dynamics of Interacting Particles, Springer-Verlag Berlin Heidelberg, 1991. 21. B. Blocken, T. van Druenen, Y. Toparlar, et al., Aerodynamic drag in cycling pelotons: new in- sights by CFD simulation and wind tunnel testing, J. Wind Eng. Ind. Aerod., 179 (2018), 319–337. 22. B. Blocken, Y. Toparlar, T. van Druenen, et al., Aerodynamic drag in cycling team time trials, J. Wind Eng. Ind. Aerod., 182 (2018), 128–145. 23. D. Helbing, I. Farkas and T. Vicsek, Simulating dynamical features of escape panic, Nature, 407 (2000), 487–490. 24. A. Kirchner and A. Schadschneider, Simulation of evacuation processes using a bionics–inspired cellular automaton model for pedestrian dynamics, Physica A, 312 (2002), 260–276. 25. M. Colangeli, A. De Masi and E. Presutti, Microscopic models for uphill diﬀusion, J. Phys. A: Math. Theor., 50 (2017), 435002. c⃝2020 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) c⃝2020 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) Volume 17, Issue 1, 460–477. Mathematical Biosciences and Engineering
https://openalex.org/W3174365683	https://digital.csic.es/bitstream/10261/280953/1/Targeting%20cytoskeletal_Llorente_Art2021.pdf	English	null	Targeting cytoskeletal phosphorylation in cancer	Exploration of targeted anti-tumor therapy	2,021	cc-by	9,636	Keywords Cytoskeleton, phosphorylation, cancer, actin, tubulin, vimentin, myosin Clara Llorente-González , Marta González-Rodríƴguez, Miguel Vicente-Manzanares* Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biologíƴa Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Cientíƴficas (CSIC)-University of Salamanca, 37007 Salamanca, Spain Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biologíƴa Molecular y Ce Consejo Superior de Investigaciones Cientíƴficas (CSIC)-University of Salamanca, 37007 Salamanca, Spain *Correspondence: Miguel Vicente-Manzanares, Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biologíƴa Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Cientíƴficas (CSIC)-University of Salamanca, 37007 Salamanca, Spain. miguel.vicente@csic.es Academic Editor: Jianxun Ding, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, China Received: February 26, 2021 Accepted: June 01, 2021 Published: June 28, 2021 Cite this article: Llorente-González C, González-Rodríƴguez M, Vicente-Manzanares M. Targeting cytoskeletal phosphorylation in cancer. Explor Target Antitumor Ther. 2021;2:292-308. https://doi.org/10.37349/etat.2021.00047 Abstract Phosphorylation of cytoskeletal proteins regulates the dynamics of polymerization, stability, and disassembly of the different types of cytoskeletal polymers. These control the ability of cells to migrate and divide. Mutations and alterations of the expression levels of multiple protein kinases are hallmarks of most forms of cancer. Thus, altered phosphorylation of cytoskeletal proteins is observed in most cancer cells. These alterations potentially control the ability of cancer cells to divide, invade and form distal metastasis. This review highlights the emergent role of phosphorylation in the control of the function of the different cytoskeletal polymers in cancer cells. It also addresses the potential effect of targeted inhibitors in the normalization of cytoskeletal function. Exploration of Targeted Anti-tumor Therapy Exploration of Targeted Anti-tumor Therapy © The Author(s) 2021. This is an Open Access article licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Exploration of Targeted Anti-tumor Therapy Open Access Review Introduction Cytoskeletal proteins form the backbone of the different types of structural polymers found in every eukaryotic cell. Such polymers include microfilaments (MF), mini-filaments, microtubules (MT) and intermediate filaments (IF). Each polymer has a relatively homogeneous composition. Monomeric cytoskeletal proteins bind in a head-to-tail manner to form long chains of different geometries and biophysical properties. These monomers include actin (which forms MF), myosin (mini-filaments), tubulin (MT), and various families of IF proteins, including keratins, desmins, glial fibrillary acidic protein (GFAP), peripherin, vimentin, internexins, nestins and others (reviewed in [1]). MFs and mini-filaments enable cells to adapt to their surroundings. They exert several roles in cell division and support cell migration in physiological and pathological contexts, for example during invasion and metastasis. MTs are essential as they form the physical scaffold that mediates an even separation of genetic material during cell division, but they play limited roles in cell migration. IFs confer mechanical resistance to the cells. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 292 © The Author(s) 2021. This is an Open Access article licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 292 Like every protein in the eukaryotic proteome, cytoskeletal proteins are substrates of diverse protein kinases. Phosphorylation changes their interactive and dynamic properties with respect to their non- phosphorylated forms. In the specific case of cytoskeletal proteins, phosphorylation controls their assembly and disassembly affinity and dynamics, as well as the biochemical and biophysical properties of the polymers themselves. Phosphorylation also modulates their interactome, which also affects the stability and function of the polymers. Genetic modifications affecting protein kinases are very frequent in cancer [2]. The most typical are mutations or deletions that cause loss of function or increased catalysis [3]. These two outcomes also emerge when portions of kinases become fused with incorrect pieces of DNA as part of genomic cancer recombination, leading to abnormal activation. One example is the Philadelphia translocation, which is typical of chronic myeloid leukemia (CML) cells. Introduction It consists of a reciprocal translocation between parts of human chromosomes 9 and 22 that leads to the production of a constitutively active Abelson kinase (BCR- ABL), which triggers uncontrolled proliferation by phosphorylating multiple substrates [4]. On the other hand, activating mutations may lead to unexpected effects on the different cytoskeletal systems. For example, mutations to the small GTPase RhoA may lead to increased activation of proteins that control mini-filament formation [5]. These events disturb the delicate balance of MFs and mini-filament dynamics that enable cells to maintain their form and function in the context of the host tissue. These and other examples found in the latter sections of the present work highlight the fact that alterations of the phosphorylation of cytoskeletal proteins caused by cancer-related mutations may change the dynamics, architecture, and function of the different cytoskeletal polymers. These events cause aberrant molecular behaviors that may confer specific properties to cancer cells, e.g., increased cell migration, invasion, division, mechanosensing, etc. The study of these modifications is complicated by the existence of multiple isoforms of these proteins, some of which have non-overlapping functions. There are many isoforms of actin, tubulin and myosin, and multiple forms and variants of IF. How phosphorylation impacts every isoform of every cytoskeletal protein is not only impossible to describe, but mostly unknown. Because of this, we describe only the current state of the art regarding the phosphorylation of selected isoforms of actin, myosin II, tubulin and vimentin. We focus on the functional effect of these phosphorylations, and what the consequences would be if the extent of these phosphorylations was altered in the context of cancer progression. While phosphorylation has proven crucial for the function of some of these filament-forming polymers, e.g., myosin II, the function of many of the phosphorylations described here is still unexplored. Thus, a major goal of this work is to provide a wide, yet incomplete, perspective of the field, identifying potential hotspots that may be amenable to specific targeting to treat diverse forms of cancer. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Actin Main human β-actin phosphorylation sites Gene Site Putative kinase Discovered by/inhibitor Effect References ACTB Ser33 PLK1 BI_4834 Probably mitosis [12] Tyr53 Src Biochemical assays, targeted mutation MF disassembly [16, 17] Tyr91 EGFR erlotinib not known [27] EGFR: epidermal growth factor receptor; Tyr: Tyrosine Figure 1. Homology between human actin isoforms Table 1. Main human β-actin phosphorylation sites Gene Site Putative kinase Discovered by/inhibitor Effect References ACTB Ser33 PLK1 BI_4834 Probably mitosis [12] Tyr53 Src Biochemical assays, targeted mutation MF disassembly [16, 17] Tyr91 EGFR erlotinib not known [27] EGFR: epidermal growth factor receptor; Tyr: Tyrosine Figure 1. Homology between human actin isoforms Figure 1. Homology between human actin isoforms Table 1. Main human β-actin phosphorylation sites Gene Site Putative kinase Discovered by/inhibitor Effect References ACTB Ser33 PLK1 BI_4834 Probably mitosis [12] Tyr53 Src Biochemical assays, targeted mutation MF disassembly [16, 17] Tyr91 EGFR erlotinib not known [27] EGFR: epidermal growth factor receptor; Tyr: Tyrosine Table 1. Main human β-actin phosphorylation sites Another potentially important residue is Tyr53. It is conserved from Dictyostelium discoideum to humans. It resides in the D-loop of actin [14], which is essential for actin polymerization [15]. Tyr53 phosphorylation decreases the affinity of actin monomers for each other, causing filament shortening [16]. This mechanism is critically important in the central nervous system. During synaptogenesis, Tyr53 phosphorylation increased actin turnover [17]. Although the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 inhibits its phosphorylation (and that of Tyr362, of unknown function), it is unlikely that this kinase, which phosphorylates Ser/threonine 18 (Thr), directly phosphorylates Tyr53 (or Tyr362). However, MEK control, directly or indirectly, some potential Tyr kinases that may phosphorylate Tyr53 (and/or Tyr362). For example, extracellular signal-regulated kinase (ERK), the canonical target of MEK [18], can phosphorylate and activate RhoA [19], which increases focal adhesion maturation [20]. In this manner, ERK would increase Src activation by recruiting it to focal adhesions. However, active Src does not remain in focal adhesions, but propagates rapidly [21]. Based on these data, a possible model emerges in which Src is activated at focal adhesions in a MEK/ERK-dependent manner. Active Src would diffuse from focal adhesions and phosphorylate filamentous actin in Tyr53. This could destabilize MFs, thereby decreasing its assembly in contractile actomyosin bundles associated to focal adhesions. Actin Actin forms MFs by adenosine triphosphate (ATP)-dependent polymerization. Together with myosin II mini- filaments, MFs constitute the contractile apparatus of animal cells. A vast array of nucleators, cross-linkers and other binding partners regulate multiple aspects of its ability to form filaments and the multiple cellular functions they enable [6, 7]. There are multiple isoforms of actin, including muscle-specific and non-muscle [8]. Due to its abundance, ubiquity and high degree of homology among isoforms (Figure 1), we focus on cytoplasmic, β-actin. Human β-actin (ACTB gene; Uniprot #P60709) is located in chromosome 7p22.1 in humans. β-actin forms MFs in every non-muscle cell lineage, mediating protrusion, assembly of contractile structures and other motility- related structures, e.g., podosomes [9]. Somatic mutations of the ACTB gene associated to cancer have not been reported. However, the filamentous state of actin is a checkpoint for cell proliferation that is deregulated in several types of cancer [10]. Although the effect of actin phosphorylation in MF dynamics has yet to be studied in detail, some phosphorylation events have been described that potentially affect actin filamentation and/or disassembly (see Table 1 for a list). For example, serine (Ser)33 appears phosphorylated in multiple phospho-proteomic analyses, including diverse types of cancer cells that include human epidermal growth factor receptor 2 (HER2)-positive, luminal A and triple negative breast cancer [2, 11] and lung cancer Page 293 Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 (http://phosphosite.org, search term = ACTB, Ser33). Its phosphorylation lies downstream of polo-like kinase 1 (PLK1) since the specific inhibitor BI 4834 abrogates it [12]. Although it is unclear whether PLK1 directly phosphorylates β-actin in Ser33, the crucial role of this kinase in cancer cell division [13] suggests that PLK1-dependent actin phosphorylation may control actin function (by controlling actin polymerization and/or cross-linking) in cancer cell proliferation. (http://phosphosite.org, search term = ACTB, Ser33). Its phosphorylation lies downstream of polo-like kinase 1 (PLK1) since the specific inhibitor BI 4834 abrogates it [12]. Although it is unclear whether PLK1 directly phosphorylates β-actin in Ser33, the crucial role of this kinase in cancer cell division [13] suggests that PLK1-dependent actin phosphorylation may control actin function (by controlling actin polymerization and/or cross-linking) in cancer cell proliferation. Figure 1. Homology between human actin isoforms Table 1. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Non-muscle myosin II Functionally, non-muscle myosin II (NMII) is a hexameric molecular motor made of different combinations of genes. It always comprises two heavy chains [myosin heavy chain II (MHCII)] and four light chains, two regulatory (RLC) and two structural (essential, ELC). There are three genes that encode MHCII isoforms: MYH9 (Uniprot #P35579, human chromosome 22q12.3), MYH10 (Uniprot #P35580, human chromosome 17p13.1) and MYH14 (Uniprot #Q7Z406, human chromosome 19q13.33); three genes that encode RLC: MYL9 (Uniprot #P24844, human chromosome 20q11.23), MYL12A (Uniprot #P19105, human chromosome 18p11.31) and MYL12B (Uniprot #O14950, human chromosome 18p11.31); and one gene that encodes ELC, MYL6 (Uniprot #P60660, human chromosome 12q13.2). The typical structure of NMII involves MHCII from the same gene forming a central homodimer (they do not heterodimerize). Each heavy chain contains two tandem IQ motifs that bind to ELC and RLC. These binding sites define the “neck” of the hexamer, which is flexible and enables the conformational movement that generates mechanical work upon ATP hydrolysis when the hexamer is bound to actin [28]. Actin binding and ATPase activities lie upstream of the neck in a ≈ 800 amino acid long globular head domain. Downstream of the neck, both heavy chains display a ≈ 1,000 amino acid long coiled-coil domain that supports dimerization. The C-terminus of the heavy chains ends in a non-helical domain of variable length that controls the oligomerization of the hexamer into larger order units termed mini-filaments (the name has a historic connotation based on electron microscopy (EM) visualization of thick and thin bands in muscle sarcomeres, which are made of myosin II and actin, respectively). Whereas binding to calcium-sensitive proteins controls muscle myosin II function, NMII is largely controlled by phosphorylation. In fact, RLC phosphorylation is essential for the conformational extension that is required for NMII hexamers to form mini-filaments [29]. Likewise, phosphorylations in the coiled coil domain controls dimerization; and those in the non-helical tailpiece (NHT) domain regulate oligomerization [30]. Phosphorylations of the globular domain of the heavy chain are less characterized. In Table 2, it summarizes the phosphorylations affecting the different chains of the NMII hexamer, including phosphorylations of RLC (MYL9/12) that modulate its function as well as that of the entire NMII hexamer; those of ELC (MYL6); as well as of the three genes of MHCII (MYH9/10/14). Table 2. Actin According to the actin treadmilling model [7], monomeric actin would eventually undergo Tyr53 dephosphorylation to be reused by the cell to form other structures, e.g., lamellipodia or podosomes/invadopodia in less contractile/more protrusive regions. In this regard, Src overexpression promotes invadopodia [22] which, similar to podosomes, appear in regions in which actomyosin bundles are scarce. Importantly, Tyr53 also appears nitrated [23], which accelerated filament elongation, promoting the formation of disorganized F-actin aggregates, which may be very important for actin dynamics in highly oxidative contexts, e.g., in lung cancer [24]. Finally, actin phosphorylation in Tyr91 has been observed in multiple types of cancer, including diverse subtypes of breast cancer [2], colorectal carcinoma [25], lung cancer [26] and diverse types of leukemia (http://phosphosite.org, search term = ACTB, Tyr91). Tyr91 phosphorylation was modestly affected by treatment of non-small cell lung cancer cells with the EGFR inhibitor erlotinib [27]. However, the effect of this phosphorylation in the regulation of the actin cytoskeleton in cancer cells has yet to be addressed, although it could be related to its ability to polymerize and/or form filaments. Similar to Tyr53, Tyr91 also appears nitrated in vivo [23], with potential implications in the regulation of actin dynamics by the oxidative state of the cell. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 294 Non-muscle myosin II Several kinases induce these phosphorylations, including ROCK, MLCK, CITK, MRCK and ZIPK/death-associated protein kinase 3 (DAPK3) [30]. Very recently, we have identified Tyr155 phosphorylation downstream of EGFR. However, this phosphorylation only occurs when RLC is not bound to NMII. Tyr155 phosphorylation prevents the association of RLC with NMII, thus de facto decreasing the amount of NMII available to form filaments [37]. On the other hand, the role of ELC phosphorylation in cellular physiology or NMII function remains practically unexplored. Tyr29 appears more phosphorylated in many types of cancer, but the kinase remains unidentified [39]. Conversely, Tyr89 phosphorylation is dependent of EGFR III [40] and its phosphorylation is inhibited in cells treated with the EGFR inhibitor gefitinib [41]. However, whether this phosphorylation decreases NMII assembly is unknown, and currently under investigation in our lab. Regarding the heavy chains, there are isoform-specific differences regarding heavy chain phosphorylation that have potential effects on diverse types of filaments depending on their molecular composition. In general, head domain phosphorylations have the potential to control actin binding and ATPase activity of myosin II. However, this has been poorly explored. Conversely, phosphorylations of the coiled coil domains of MHCII-A/B/C are better characterized. These regions are important for dimerization, and phosphorylation has been shown to decrease the stability of the hexamers and hinders their lateral association with other hexamers to form filaments. This revealed that lateral interactions are highly dependent on the net charge of the interacting regions [42]. Phosphorylation of Thr1800, Ser1803 and Ser1808 in MHCII-A, Ser1810 and Thr1815 in MHCII-B and Thr1832/Ser1838 in MHCII-C decrease the formation of filaments of the corresponding isoform [43]. The kinases that regulate most of these phosphorylations are α-kinases, for example TRPM6/7 [43]. However, how theses kinases distinguish between isoforms is unclear. Finally, phosphorylations at the end of the coiled-coil or into the NHT domain of MHCII decrease mini- filament formation. One such residue is Ser1916 in MHCII-A. Its phosphorylation by PKCβ reduces filament stability [44, 45] as it increases NMII-A interaction with Mts1/S100A4, which forces NMII-A to stay in an assembly-incompetent conformation [46]. Phosphorylation of Ser1943 in MHCII-A has this effect. Casein kinase (CK)-II phosphorylates MHCII-A in Ser1943, promoting NMII-A filament disassembly [47]. In MHCII-B, Ser1935 and Ser1937 (MHCII-B) are phosphorylated by PKCζ [48, 49], but they have a similar effect. Non-muscle myosin II Main human NMII (RLC, ELC and MHCII-A, B, C) phosphorylation sites Gene Site Putative kinase Discovered by/inhibitor Effect References MYL9/ MYL12 Ser1/2 PKCα Targeted mutation Inhibits ATPase activity [32, 33] Thr18 CITK, ZIPK, ROCK1/2 Targeted mutation, biochemical assays Synergizes with pSer19 to stabilize conformation and boost ATPase activity [38, 109] Ser19 MLCK, MRCK, CITK, ZIPK, ROCK1/2 ML-7, dominant negatives, Y-27632, siRNA Conformational extension and increased ATPase activity [35, 110] Tyr155 EGFR Targeted mutation, cetuximab Inhibited NMII assembly [37] MYL6 Tyr29 not known not known Carcinoma progression [111] Tyr89 EGFR? Genfitinib not known [41] MYH9 Tyr158 Src siRNA Decreases listeria infection [112] Thr1800, Ser1803, Ser1808 TRPM6/7 Biochemical assays Decreases filament stability [43] Ser1916 PKCβ Go6976 Decreases filament stability, increases Mts1 binding [45] Ser1943 CK-II Targeted mutation Decreases filament formation [47] MYH10 Ser1810, Thr1815 TRPM6/7 Biochemical assays Decreases filament stability [43] Ser1935 PKCζ Targeted mutation, PKCζ pseudosubstrate Impairs filament stability and cell polarity [49] Ser1937 PKCζ Biochemical assays, siRNA Impairs filament stability [48] MYH14 Thr1832/1838 TRPM6/7 Biochemical assays Decreases filament stability [43] PKCs: protein kinase C; ROCK: RhoA-coiled coil kinase; MLCK: myosin light chain kinase; CITK: citron kinase; MRCK: myotonic dystrophy kinase-related; ZIPK: zipper-interacting protein kinase; siRNA: small interfering RNA; TRPM6/7: transient receptor potential melastatin 6/7 Table 2. Main human NMII (RLC, ELC and MHCII-A, B, C) phosphorylation sites Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 295 Page 295 RLC is the most important regulatory hotspot of myosin II by phosphorylation. Several residues have been described, including Ser1/2. Their phosphorylation inhibits NMII function, as they decrease ATP catalysis on the NMII hexamer head [31]. Although they control NMII function in response to growth factors downstream of conventional PKCs [32, 33], their mutation to a non-phosphorylatable version does not prevent cell division [34]. Conversely, Ser19 phosphorylation mediates the conversion of folded, assembly incompetent into extended, assembly competent NMII hexamers. Extended hexamers whose RLC is phosphorylated in Ser19 immediately form bipolar filaments that grow by lateral association, as outlined below [29]. Ser19 phosphorylation also increases ATP catalysis in the associated MHCII [35, 36]. On the other hand, Thr18 only appears phosphorylated if Ser19 is also phosphorylated [37]. Based on its in vitro effect boosting ATP catalysis of the bound MHCII, Thr18 is considered a synergy site with Ser19. It also has increases the half-life of NMII mini-filaments, which is essential during cell migration [38]. Non-muscle myosin II Importantly, and because NMII-B filaments are more stable than those made of NMII-A, these phosphorylations impair cell polarity and migration [49]. Tubulin There are three isoforms of tubulin, each one including several variants. For the sake of brevity, and due to the grouped homology among them (Figure 2 and Table S1), we focus on α1-tubulin, encoded by the gene TUBA1A (Uniprot #Q71U36, human chromosome 12q13.12), β1-tubulin, encoded by the gene TUBB1 (Uniprot #Q9H4B7, human chromosome 20q13.32) and γ1-tubulin, encoded by the gene TUBG1 (Uniprot #P23258, human chromosome 17q21.2). α- and β-tubulins are the major components of polymeric MTs. γ-tubulin is mainly present at the centrosome [also known as MT-organizing center, (MTOC)], nucleating polymerization by forming the γ-tubulin ring complex (γTuRC). This complex acts as a template for α/β Page 296 Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 monomer incorporation [50]. Phosphorylations affecting these subunits are summarized in Table 3, but their effects in tubulin dynamics are very poorly characterized, particularly in cancer. A few significant residues include Ser48 (and Ser75 of β-tubulin). These are likely Aurora kinase sites, as two independent Aurora kinase (AURK) inhibitors reduce their phosphorylation levels [51]. Due to the key role of AURK in cancer [52], it will be extremely interesting to study whether these sites are involved in the potential regulation of MT dynamics. In this regard, Aurora kinase A (AURKA) inhibition inhibits osteosarcoma cell division by preventing MT stabilization to form the mitotic spindle [53]. Figure 2. Grouped homology among human tubulin isoforms. Range shown in the homology of an isoform with itself refers to the minimal and maximal homology among sub-isoforms (see Table S1 for full details) Figure 2. Grouped homology among human tubulin isoforms. Range shown in the homology of an isoform with itself refers to the minimal and maximal homology among sub-isoforms (see Table S1 for full details) Table 3. Main human tubulin phosphorylation sites Gene Site Putative kinase Discovered by/inhibitor Effect Reference TUBA Ser48 AURK AZD1152, ZM447439 Not known [51] Ser165 PKCα bisindolylmaleimide EMT [54] TUBB Ser75 AURK AZD1152, ZM447439 Not known [51] TUBG Ser131 SadB Targeted mutation Centrosome duplication [55] Ser385 SadB Targeted mutation Promotes γ-tubulin interaction with the chromatin [56] SadB: synapses of amphids defective kinase Table 3. Main human tubulin phosphorylation sites SadB: synapses of amphids defective kinase On the other hand, tubulin phosphorylation in Ser165 by PKCα increases MT dynamics, cell motility and acquisition of a mesenchymal phenotype characterized by expression of neural (N)-cadherin [54]. Tubulin This strongly suggests that this phosphorylation could be a key event in the acquisition of mesenchymal phenotypes, which is a typical event during the transition of tumors from non-invasive to invasive states. Finally, phosphorylation of centrosomal γ-tubulin in Ser131/385 is mediated by SadB. This phosphorylation is involved in centrosome duplication during mitosis, a key event during cell division. A phospho-mimetic form induces spontaneous centrosome duplication, whereas a non-phosphorylatable mutant impairs centrosome duplication [55]. SadB also phosphorylates γ-tubulin in Ser385, regulating S-phase progression by moderating the activities of E2 promoter-binding factors. When phosphorylated by SadB in this amino acid, γ-tubulin increases its nuclear localization [56]. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Vimentin Vimentin (UniProt # P08670) is located on human chromosome 10p13. Vimentin forms very stable IF. Unlike actin, myosin and tubulin, IFs are not very dynamic, but they endow cells with structural stability. Importantly, vimentin is an IF typical of mesenchymal and hematopoietic cells, and a signature gene of the epithelial-mesenchymal transition (EMT) [57]. As such, it is a marker of cells that evolve into mesenchymal phenotypes, acquiring migratory capability as tumors become invasive. The development of cancer affects this protein, including epigenomic alterations [58, 59] and somatic mutations in squamous lung cancer [60], gastric adenocarcinoma [61], and other types of cancer. Vimentin undergoes extensive phosphorylation, which potentially controls its cellular function. The best characterized phospho-residues are grouped in the non-helical N-terminus domain [62] (see Table 4). Some crucial residues include Ser5, Ser7, Ser8, Ser9 and Ser10, which are phosphorylated by PKCα [63]. Since phosphorylation of these residues control leukocyte transmigration downstream of phosphatidyl inositol 3-kinase, isoform γ (PI3Kγ) [64], it is possible that they also control CTC extravasation, which tends to mimic Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 297 leukocyte diapedesis [65]. In this regard, vimentin localizes to the trailing edge of migrating leukocytes and controls cortex rigidity [66]. This may be important to reduce cancer cell attrition in the bloodstream [67]. The ras-related C3 botulinum toxin substrate 1 (Rac1)/p21-activated kinase 1 (PAK1) pathway controls the phosphorylation of Ser26, Ser51 and Ser66, impairing IF assembly. Whether this is related to the weak oncogenic ability of Rac [68] is currently unknown. Table 4. Main human vimentin phosphorylation sites Gene Site Putative kinase Discovered by/inhibitor Effect Reference VIM Ser5/7/8/9/10 PKCα Calyculin A (phosphatase) Cell polarity [63] Ser26/51/66 PAK1 Biochemical assays, selumetinib, vemurafenib Abnormal assembly [113, 114] Ser39 AKT/PKB A-674563 Proteolytic protection, slowed polymerization? [69] Ser56 AKT/PKB ROCK A-674563 siRNA Filament disassembly [69] [72] Ser72 ROCK1 Targeted mutation, Y27632 Increased cell migration [75, 76, 115] Ser73 AURKB Biochemical assays Mitosis? [114, 116] Ser83 PLK-1, CaMKII Biochemical assays, KN-93 (CaMKII inhibitor) Filament disassembly and pathogen interaction [77, 117, 118] CaMKII: calmodulin-dependent protein kinase II Table 4. Main human vimentin phosphorylation sites Key residues include Ser39, Ser56, Ser72 and Ser83. Is it feasible to target cytoskeletal phosphorylation to treat cancer? A possible role for MT phosphorylation in cancer cell proliferation and migration Is it feasible to target cytoskeletal phosphorylation to treat cancer? A ibl l f MT h h l ti i ll lif ti d i ti Vimentin In cancer cells, Ser39 phosphorylation by protein kinase B (AKT/PKB) protects vimentin from proteolysis and enhances tumor growth and metastasis [69] by altering filament assembly [63], which regulates cortex plasticity and could underlie the fact that cancer cells are overall softer than non-cancer cells [70]. On the other hand, Ser56 is phosphorylated by the cyclin dependent kinase (CDK) 1. This phosphorylation recruits PLK1, which enhances phosphorylation in Ser82 [71]. Consequently, cell arrest in G2/M induced by taxanes lead to an accumulation of phospho-Ser56 vimentin in a CDK1-dependent manner. Ser56 is also phosphorylated downstream of ROCK and PAK1 in hypoxia [72]. Ser56 phosphorylation promotes disassembly of perinuclear vimentin, controlling filament stability [73] and promoting cancer cell invasiveness [74]. Likewise, two independent studies indicated that Ser72 phosphorylation downstream of ROCK1 is important for cancer cell migration. A phospho-mimetic mutation of Ser72 increased cancer cell speed, whereas a non-phosphorylatable form impaired sphingolipid-triggered cell migration, respectively [75, 76]. Finally, Ser83 phosphorylation by calcium/CaMKII or PLK1 controls β1 integrin expression at the plasma membrane, controlling cell adhesiveness during invasion [77]. Importantly, phosphorylation of Ser39, Ser72 and Ser83 is preserved when vimentin is processed and peptides presented associated to major histocompatibility complexes. CD4+ T cells distinguish between non-phosphorylated vs. phosphorylated vimentin peptides [78]. Since these phosphorylations are elevated in metastatic cells [78], they could be useful as immunotherapeutic targets. Is it feasible to target cytoskeletal phosphorylation to treat cancer? A possible role for MT phosphorylation in cancer cell proliferation and migration A possible role for MT phosphorylation in cancer cell proliferation and migration Tubulin phosphorylation is arguably the least understood of cytoskeletal phosphorylations. The apparent lack of interest in the field may be due to the fact that early use of anti-tubulin polymerization drugs such as colchicine or vincas; or MT turnover inhibitors, e.g., taxanes, was very successful to inhibit mitosis in cancer cells [79]. The scant information available emerges from global phospho-proteomics approaches. However, tubulin appears heavily phosphorylated in cancer cells, which strongly suggests that targeting phosphorylation could be of therapeutic interest. Two of the most prominent kinases that target MTs are AURKA and AURKB, and clinical approaches are underway to address the viability of their inhibition to treat different types of cancer [80]. However, the rationale behind their use is that AURK inhibition impairs mitosis, which is the same as targeting MT dynamics via vincas or taxanes. It is likely that AURK-dependent inhibition of cell division is Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 298 independent of MT phosphorylation. Definitive proof would emerge from experiments aimed at interrogating whether phospho-mimetic forms of Ser48 (α-tubulin) and/or Ser75 (β-tubulin) confer resistance to AURK inhibitors. Recent work has highlighted that AURKA and PLK1 are essential for MT dynamics and centrosome positioning during T cell activation [81], hence their inhibition can play a role in curbing T-cell lymphomas and other T-cell-dependent malignancies. However, it will be important to determine whether the effect of AURK inhibitors is due to direct phosphorylation of tubulin. An intriguing possibility is to target tubulin phosphorylation to complement vinca or taxane treatments, particularly to counteract the development of resistance, which is observed in many forms of advanced cancer. The assumption is that taxane-resistant cells develop mechanisms to undergo mitosis in the presence of these inhibitors, overcoming G2/M arrest, e.g., in the presence of hypoxia [82]. An earlier study showed that phosphorylation impairs tubulin polymerization promoting protein (TPPP)-dependent tubulin polymerization in the brain [83], confirming that targeting tubulin phosphorylation could affect its dynamics through different mechanisms than those of vinca or taxanes. In this manner, targeting tubulin phosphorylation could be a potentially useful approach to complement current therapies aimed at blocking cancer cell division. Compared to their central role in cell division, the role of MTs in cell migration is more controversial. Thus, whether inhibiting their phosphorylation could have an effect on invasion is uncertain. MTs control cell polarity [84] and preserve the integrity of the cell as it migrates [85]. A possible role for MT phosphorylation in cancer cell proliferation and migration Since MTs control vesicle traffic, targeting MT dynamics through phosphorylation in this context could impair cancer cell secretion, decreasing the ability of cancer cells to exert modifications on the tumor microenvironment. Targeting myosin and actin phosphorylation: a gateway to curb metastasis Actin and myosin are also important for mitosis. Different studies have shown that inhibiting MYH10 expression promotes multinucleation due to cytokinesis failure [86, 87]. Since NMII activation relies on RLC phosphorylation, it is theoretically possible to inhibit NMII function by targeting the kinases that mediate RLC Ser19 phosphorylation, which is critical for NMII filamentation. Multinucleation due to failed cytokinesis is also observed when myosin-specific kinases, e.g., CITK are inhibited or deleted [88]. CITK depletion, which reduces NMII phosphorylation and activation, reduces tumor growth in multiple myeloma [89] and medulloblastoma [90]. Interestingly, this does not happen when other myosin kinases, e.g., ROCK, are targeted (in fact, ROCK inhibitors are routinely used to culture stem cell in vitro to favor growth and prevent differentiation [91-93]). This highlights the central role of myosin regulation in many different processes, which may render targeting NMII phosphorylation impractical to inhibit proliferation. On the other hand, targeting NMII (and actin) phosphorylation could prevent tumor cell dissemination. Different lines of evidence have suggested that elevated NMII phosphorylation and activity in the cortex changes the cellular phenotype from epithelial, or mesenchymal, into amoeboid [94, 95]. Amoeboid shape is characteristic of rapidly migrating cells, e.g., leukocytes, and is mainly integrin-independent [96]. In addition, elevated levels of phosphorylated NMII correlate with more aggressive tumors, e.g., gliosis-to-glioblastoma progression [97]. A recent study has highlighted that melanoma cells that undergo a mesenchymal-to- amoeboid transition display elevated levels of phosphorylated NMII, while also producing immunosuppressive signals [98]. The same group has shown that targeting ROCK alters the sensitivity of cancer cells to mitogen- activated protein kinase (MAPK) inhibitors [99], indicating that abnormalities in NMII phosphorylation downstream of ROCK may be targeted to confer cellular sensitivity to other families of inhibitors. However, the different levels of regulation dependent on NMII phosphorylation are only beginning to be understood. This is a very active line of investigation in our lab. Possible effects of targeting vimentin phosphorylation in cancer cell division and tumor mechanics Vimentin knockout mice are viable and fertile, displaying only minor developmental defects [100]. A possible role for MT phosphorylation in cancer cell proliferation and migration Other types of IFs are likely to compensate for the loss of vimentin in a tissue-specific manner, for example epidermal keratins in the skin or GFAP in the central nervous system [101]. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 299 Vimentin is prominently expressed in cells that are (or become) motile, including fibroblasts, mesenchymal cells, leukocytes and invasive cancer cells. This has suggested that vimentin could be targeted to prevent cancer cell motility. Indeed, vimentin is upregulated during EMT, which is a common occurrence in many carcinomas, and its repression in these cells decrease breast and colon tumor cell migration, as shown by siRNA depletion of vimentin in migrating mesenchymal cells and overexpression of vimentin in epithelioid, non-migrating tumor cells [102, 103]. In addition, higher expression of vimentin correlates with decreased survival in colorectal cancer, which could be related to a decreased metastatic ability [104]. Regarding vimentin phosphorylation, some sites favor IF assembly, whereas others promote disassembly, hence it is difficult to make general statements regarding the effect of its phosphorylation in cancer progression. An interesting fact is that many mitotic kinases, e.g., CDKs, PLK1, AURK, induce vimentin phosphorylation, hence its phosphorylation is likely to favor cell division. A model emerges in which elevated vimentin phosphorylation promotes IF disassembly, favoring cancer cell division. Likewise, recent evidence indicates that IFs may promote tumor cell migration [105], hence it is possible that similar mechanisms favor tumor cell dissemination. An intriguing possibility is that the preservation of these post-translational modifications during antigenic presentation [78] could be therapeutically useful to design novel immunotherapy-based strategies. Phospho-vimentin peptides could bear higher specificity for highly transformed cells, improving the cytotoxic T lymphocyte (CTL) response against them. Bystander inhibition of cytoskeletal phosphorylation in current therapies While a few cytoskeletal components, e.g., myosin II, have well-defined kinomes, most of them lack specific kinases. However, most of the reported effects on actin, tubulin and vimentin phosphorylation in cancer cells emerge from studies using kinase-specific drugs. Hence, we cannot rule out that some specific phenotypes caused by current treatments are related to cytoskeletal phosphorylations. A key example is that of paclitaxel, which induces mitotic arrest at G2/M, promoting vimentin phosphorylation in Ser56 via CDK1 [71]. A possible role for MT phosphorylation in cancer cell proliferation and migration A few years ago, we demonstrated that dasatinib, a Tyr kinase inhibitor used to treat CML and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) with several potential targets [106] induced myosin II phosphorylation, leading to increased contractility and vascular leakage [107]. This indicated that one of dasatinib substrates phosphorylated and inactivated a contractility inhibitor, perhaps ROCK. Likewise, we have recently shown that myosin light chain is a target of EGFR, which is a therapeutic target for the treatment of breast cancer, among others [37]. It is also possible that therapies aimed at inhibiting kinases in cancer cells have unexpected effects on non-cancer cells associated to the tumor microenvironment. For example, cancer-associated fibroblasts (CAFs) are very contractile, likely displaying elevated levels of phosphorylated NMII. NMII phosphorylation in these cells induces the formation of stress fibers, which predicts contact guidance for surrounding breast cancer cells [108]. Therapies designed to alter cytoskeletal phosphorylations in tumor cells could also have a dramatic impact on the tumor microenvironment, which will be a fascinating field of research in years to come. Conclusion Targeting cytoskeletal phosphorylation has the potential to dramatically alter the mechano-chemical properties of tumor cells, inhibiting their ability to develop the cancer program, at least at a preclinical level. However, these approaches may also have potentially severe side effects, as every cell, normal or cancerous, requires the cytoskeleton. MT inhibitors, discovered over 60 years ago, offered early promise as MT-targeted therapies that improved the outcome of many types of cancer by inhibiting tumor cell division. These therapies are still the first line of chemotherapeutic treatment in many types of cancer. Hence, it is evident that phosphorylation of cytoskeletal components is altered when patients are treated with kinase-targeted therapies. We have only scratched the surface in characterizing these effects. Many of them will be unavoidable consequences of treatments aimed at other fundamental processes, potentially causing side effects that will have to be dealt with. But the potential exists to discover that some cytoskeletal phosphorylation-specific effects underlie unexpected and important effects of current and future targeted therapies. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 300 Declarations CLG: writing and revision; MGR: writing; MVM: conceptualization, writing, revision and securing funding. C fli f i Conflicts of interest The authors declare that they have no conflict of interest. Ethical approval Supplementary materials The supplementary materials for this article are available at: https://www.explorationpub.com/uploads/ Article/file/100247_sup_1.pdf. Funding This work was funded by the Spanish Ministry of Science and Innovation (SAF2017-87408), AECC Seed award (2018-IDEAS18018VICE) and ECRIN-M3 from AECC/AIRC/CRUK. IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Ministry of Education of the Castilla-León Government (CLC-2017-01). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Copyright © The Author(s) 2021. Availability of data and materials Availability of data and materials Data are publicly available and reproduced from public and private, but fully and freely searchable, databases. Funding Ethical approval Not applicable. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 301 Page 301 Page 301 Consent to participate Not applicable. Consent to publication Not applicable. Consent to participate Not applicable. Consent to participate Not applicable. Not applicable. Consent to publication Not applicable. Consent to publication Not applicable. Not applicable. References 1. Herrmann H, Bar H, Kreplak L, Strelkov SV, Aebi U. Intermediate filaments: from cell architecture to nanomechanics. Nat Rev Mol Cell Biol. 2007;8:562-73. 1. Herrmann H, Bar H, Kreplak L, Strelkov SV, Aebi U. Intermediate filaments: from cell architecture to nanomechanics. Nat Rev Mol Cell Biol. 2007;8:562-73. 2. Mertins P, Mani DR, Ruggles KV, Gillette MA, Clauser KR, Wang P, et al. Proteogenomics connects somatic mutations to signalling in breast cancer. Nature. 2016;534:55-62. 2. Mertins P, Mani DR, Ruggles KV, Gillette MA, Clauser KR, Wang P, et al. Proteogenomics connects somatic mutations to signalling in breast cancer. Nature. 2016;534:55-62. 3. Giamas G, Stebbing J, Vorgias CE, Knippschild U. Protein kinases as targets for cancer treatment. Pharmacogenomics. 2007;8:1005-16. 4. Cilloni D, Saglio G. Molecular pathways: BCR-ABL. Clin Cancer Res. 2012;18:930-7. 5. Kataoka K, Ogawa S. Variegated RHOA mutations in human cancers. Exp Hematol. 2016;44:1123-9. 6. Campellone KG, Welch MD. A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol. 2010;11:237-51. 7. Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments. Cell. 2003;112:453-65. 8. Ampe C, Van Troys M. Mammalian actins: isoform-specific functions and diseases. Handb Exp Pharmacol. 2017;235:1-37. 9. Hans Georg Mannherz, editor. The actin cytoskeleton and bacterial infection. Cham, Switzerland: Springer; 2017. 10. Molinie N, Gautreau A. The Arp2/3 regulatory system and its deregulation in cancer. Physiol Rev. 2018;98:215-38. 11. Mertins P, Yang F, Liu T, Mani DR, Petyuk VA, Gillette MA, et al. Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels. Mol Cell Proteomics. 2014;13:1690-704. 12. Grosstessner-Hain K, Hegemann B, Novatchkova M, Rameseder J, Joughin BA, Hudecz O, et al. Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome. Mol Cell Proteomics. 2011;10:M111.008540. 13. Liu X, Lei M, Erikson RL. Normal cells, but not cancer cells, survive severe Plk1 depletion. Mol Cell Biol. 2006;26:2093-108. 14. Baek K, Liu X, Ferron F, Shu S, Korn ED, Dominguez R. Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding. Proc Natl Acad Sci U S A. 2008;105:11748-53. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 302 Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 302 15. Chou SZ, Pollard TD. Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides. References Proc Natl Acad Sci U S A. 2019;116:4265-74. 16. Liu X, Shu S, Hong MS, Levine RL, Korn ED. Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments. Proc Natl Acad Sci U S A. 2006;103:13694-9. 17. Bertling E, Englund J, Minkeviciene R, Koskinen M, Segerstrale M, Castren E, et al. Actin tyrosine-53- phosphorylation in neuronal maturation and synaptic plasticity. J Neurosci. 2016;36:5299-313. 18. Shaul YD, Seger R. The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta. 2007;1773:1213-26. 19. Tong J, Li L, Ballermann B, Wang Z. Phosphorylation and activation of RhoA by ERK in response to epidermal growth factor stimulation. PLoS One. 2016;11:e0147103. 20. Chrzanowska-Wodnicka M, Burridge K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol. 1996;133:1403-15. 21. Wang Y, Botvinick EL, Zhao Y, Berns MW, Usami S, Tsien RY, et al. Visualizing the mechanical activation of Src. Nature. 2005;434:1040-5. 22. Kelley LC, Ammer AG, Hayes KE, Martin KH, Machida K, Jia L, et al. Oncogenic Src requires a wild-type counterpart to regulate invadopodia maturation. J Cell Sci. 2010;123:3923-32. 23. Aslan M, Ryan TM, Townes TM, Coward L, Kirk MC, Barnes S, et al. Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization. J Biol Chem. 2003;278:4194-204. 24. Zhan X, Huang Y, Qian S. Protein tyrosine nitration in lung cancer: current research status and future perspectives. Curr Med Chem. 2018;25:3435-54. 25. Bai Y, Li J, Fang B, Edwards A, Zhang G, Bui M, et al. Phosphoproteomics identifies driver tyrosine kinases in sarcoma cell lines and tumors. Cancer Res. 2012;72:2501-11. 26. Tsai CF, Wang YT, Yen HY, Tsou CC, Ku WC, Lin PY, et al. Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics. Nat Commun. 2015;6:6622. 27. Yoshida T, Zhang G, Smith MA, Lopez AS, Bai Y, Li J, et al. Tyrosine phosphoproteomics identifies both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer. Clin Cancer Res. 2014;20:4059-74. 28. Juanes-Garcia A, Llorente-Gonzalez C, Vicente-Manzanares M. Non muscle myosin II. In: Choi S, editor. Encyclopedia of Signaling Molecules. New York: Springer; 2018. 29. Craig R, Smith R, Kendrick-Jones J. Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules. Nature. 1983;302:436-9. 30. Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR. References Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol. 2009;10:778-90. 31. Nishikawa M, Sellers JR, Adelstein RS, Hidaka H. Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase. J Biol Chem. 1984;259:8808-14. 32. Asokan SB, Johnson HE, Rahman A, King SJ, Rotty JD, Lebedeva IP, et al. Mesenchymal chemotaxis requires selective inactivation of myosin II at the leading edge via a noncanonical PLCgamma/PKCalpha pathway. Dev Cell. 2014;31:747-60. 33. Komatsu S, Ikebe M. The phosphorylation of myosin II at the Ser1 and Ser2 is critical for normal platelet- derived growth factor induced reorganization of myosin filaments. Mol Biol Cell. 2007;18:5081-90. 34. Beach JR, Licate LS, Crish JF, Egelhoff TT. Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells. BMC Cell Biol. 2011;12:52. 34. Beach JR, Licate LS, Crish JF, Egelhoff TT. Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells. BMC Cell Biol. 2011;12:52. 35. Adelstein RS, Conti MA. Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature. 1975;256:597-8. 35. Adelstein RS, Conti MA. Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature. 1975;256:597-8. 35. Adelstein RS, Conti MA. Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature. 1975;256:597-8. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 303 Page 303 36. Trybus KM, Lowey S. Conformational states of smooth muscle myosin. Effects of light chain phosphorylation and ionic strength. J Biol Chem. 1984;259:8564-71. 37. Aguilar-Cuenca R, Llorente-González C, Chapman JR, Talayero VC, Garrido-Casado M, Delgado-Arévalo C, et al. Tyrosine phosphorylation of the myosin regulatory light chain controls non-muscle myosin II assembly and function in migrating cells. Curr Biol. 2020;30:2446-58.e6. 38. Vicente-Manzanares M, Horwitz AR. Myosin light chain mono- and di-phosphorylation differentially regulate adhesion and polarity in migrating cells. Biochem Biophys Res Commun. 2010;402:537-42. 39. Gu TL, Deng X, Huang F, Tucker M, Crosby K, Rimkunas V, et al. Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma. PLoS One. 2011;6:e15640. 40. Huang PH, Mukasa A, Bonavia R, Flynn RA, Brewer ZE, Cavenee WK, et al. Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proc Natl Acad Sci U S A. 2007;104:12867-72. 41. Moritz A, Li Y, Guo A, Villen J, Wang Y, MacNeill J, et al. References Alvarado-Kristensson M, Rodriguez MJ, Silio V, Valpuesta JM, Carrera AC. SADB phosphorylation of gamma-tubulin regulates centrosome duplication. Nat Cell Biol. 2009;11:1081-92. 56. Eklund G, Lang S, Glindre J, Ehlen A, Alvarado-Kristensson M. The nuclear localization of gamma-tubulin is regulated by SadB-mediated phosphorylation. J Biol Chem. 2014;289:21360-73. 57. Scheel C, Weinberg RA. Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links. Semin Cancer Biol. 2012;22:396-403. 58. Moinova H, Leidner RS, Ravi L, Lutterbaugh J, Barnholtz-Sloan JS, Chen Y, et al. Aberrant vimentin methylation is characteristic of upper gastrointestinal pathologies. Cancer Epidemiol Biomarkers Prev. 2012;21:594-600. 59. Song BP, Jain S, Lin SY, Chen Q, Block TM, Song W, et al. Detection of hypermethylated vimentin in urine of patients with colorectal cancer. J Mol Diagn. 2012;14:112-9. 60. Cancer Genome Atlas Research Network. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012;489:519-25. 61. Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014;513:202-9. 62. Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. 1994;63:345-82. 63. Eriksson JE, He T, Trejo-Skalli AV, Harmala-Brasken AS, Hellman J, Chou YH, et al. Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments. J Cell Sci. 2004;117:919-32. 64. Barberis L, Pasquali C, Bertschy-Meier D, Cuccurullo A, Costa C, Ambrogio C, et al. Leukocyte transmigration is modulated by chemokine-mediated PI3Kgamma-dependent phosphorylation of vimentin. Eur J Immunol. 2009;39:1136-46. 65. Reymond N, d'Agua BB, Ridley AJ. Crossing the endothelial barrier during metastasis. Nat Rev Cancer. 2013;13:858-70. 66. Brown MJ, Hallam JA, Colucci-Guyon E, Shaw S. Rigidity of circulating lymphocytes is primarily conferred by vimentin intermediate filaments. J Immunol. 2001;166:6640-6. 67. Paterlini-Brechot P, Benali NL. Circulating tumor cells (CTC) detection: clinical impact and future directions. Cancer Lett. 2007;253:180-204. 68. Maldonado MDM, Dharmawardhane S. Targeting Rac and Cdc42 GTPases in cancer. Cancer Res. 2018;78:3101-11. 69. Zhu QS, Rosenblatt K, Huang KL, Lahat G, Brobey R, Bolshakov S, et al. Vimentin is a novel AKT1 target mediating motility and invasion. Oncogene. 2011;30:457-70. 70. Ramos JR, Pabijan J, Garcia R, Lekka M. The softening of human bladder cancer cells happens at an early stage of the malignancy process. Beilstein J Nanotechnol. 2014;5:447-57. 71. Yamaguchi T, Goto H, Yokoyama T, Sillje H, Hanisch A, Uldschmid A, et al. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis. J Cell Biol. 2005;171:431-6. 72. References Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases. Sci Signal. 2010;3:ra64. 42. Ricketson D, Johnston CA, Prehoda KE. Multiple tail domain interactions stabilize nonmuscle myosin II bipolar filaments. Proc Natl Acad Sci U S A. 2010;107:20964-9. 43. Clark K, Middelbeek J, Dorovkov MV, Figdor CG, Ryazanov AG, Lasonder E, et al. The alpha-kinases TRPM6 and TRPM7, but not eEF-2 kinase, phosphorylate the assembly domain of myosin IIA, IIB and IIC. FEBS Lett. 2008;582:2993-7. 44. Beach JR, Hussey GS, Miller TE, Chaudhury A, Patel P, Monslow J, et al. Myosin II isoform switching mediates invasiveness after TGF-beta-induced epithelial-mesenchymal transition. Proc Natl Acad Sci U S A. 2011;108:17991-6. 45. Ludowyke RI, Elgundi Z, Kranenburg T, Stehn JR, Schmitz-Peiffer C, Hughes WE, et al. Phosphorylation of nonmuscle myosin heavy chain IIA on Ser1917 is mediated by protein kinase C beta II and coincides with the onset of stimulated degranulation of RBL-2H3 mast cells. J Immunol. 2006;177:1492-9. 46. Li ZH, Spektor A, Varlamova O, Bresnick AR. Mts1 regulates the assembly of nonmuscle myosin-IIA. Biochemistry. 2003;42:14258-66. 47. Dulyaninova NG, Malashkevich VN, Almo SC, Bresnick AR. Regulation of myosin-IIA assembly and Mts1 binding by heavy chain phosphorylation. Biochemistry. 2005;44:6867-76. 48. Even-Faitelson L, Ravid S. PAK1 and aPKCzeta regulate myosin II-B phosphorylation: a novel signaling pathway regulating filament assembly. Mol Biol Cell. 2006;17:2869-81. 49. Juanes-Garcia A, Chapman JR, Aguilar-Cuenca R, Delgado-Arevalo C, Hodges J, Whitmore LA, et al. A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front-back polarity. J Cell Biol. 2015;209:23-32. 50. Kollman JM, Merdes A, Mourey L, Agard DA. Microtubule nucleation by gamma-tubulin complexes. Nat Rev Mol Cell Biol. 2011;12:709-21. 51. Kettenbach AN, Schweppe DK, Faherty BK, Pechenick D, Pletnev AA, Gerber SA. Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Sci Signal. 2011;4:rs5. 52. Tang A, Gao K, Chu L, Zhang R, Yang J, Zheng J. Aurora kinases: novel therapy targets in cancers. Oncotarget. 2017;8:23937-54. 53. Asteriti IA, Giubettini M, Lavia P, Guarguaglini G. Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces. Mol Cancer. 2011;10:131. 54. Markovsky E, de Stanchina E, Itzkowitz A, Haimovitz-Friedman A, Rotenberg SA. Phosphorylation state of Ser165 in α-tubulin is a toggle switch that controls proliferating human breast tumors. Cell Signal. 2018;52:74-82. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 304 Page 304 Page 304 55. References Zhang JG, Zhou HM, Zhang X, Mu W, Hu JN, Liu GL, et al. Hypoxic induction of vasculogenic mimicry in hepatocellular carcinoma: role of HIF-1 alpha, RhoA/ROCK and Rac1/PAK signaling. BMC Cancer. 2020;20:32. 73. Li QF, Spinelli AM, Wang R, Anfinogenova Y, Singer HA, Tang DD. Critical role of vimentin phosphorylation at Ser-56 by p21-activated kinase in vimentin cytoskeleton signaling. J Biol Chem. 2006;281:34716-24. 74. Thaiparambil JT, Bender L, Ganesh T, Kline E, Patel P, Liu Y, et al. Withaferin A inhibits breast cancer invasion and metastasis at sub-cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation. Int J Cancer. 2011;129:2744-55. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 305 Page 305 75. Terriac E, Coceano G, Mavajian Z, Hageman TA, Christ AF, Testa I, et al. Vimentin levels and serine 71 phosphorylation in the control of cell-matrix adhesions, migration speed, and shape of transformed human fibroblasts. Cells. 2017;6:2. 76. Hyder CL, Kemppainen K, Isoniemi KO, Imanishi SY, Goto H, Inagaki M, et al. Sphingolipids inhibit vimentin-dependent cell migration. J Cell Sci. 2015;128:2057-69. 77. Rizki A, Mott JD, Bissell MJ. Polo-like kinase 1 is involved in invasion through extracellular matrix. Cancer Res. 2007;67:11106-10. 78. Ohara M, Ohara K, Kumai T, Ohkuri T, Nagato T, Hirata-Nozaki Y, et al. Phosphorylated vimentin as an immunotherapeutic target against metastatic colorectal cancer. Cancer Immunol Immunother. 2020;69:989-99. 79. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer. 2004;4:253-65. 80. Du R, Huang C, Liu K, Li X, Dong Z. Targeting AURKA in Cancer: molecular mechanisms and opportunities for Cancer therapy. Mol Cancer. 2021;20:15. 81. Blas-Rus N, Bustos-Moran E, Perez de Castro I, de Carcer G, Borroto A, Camafeita E, et al. Aurora A drives early signalling and vesicle dynamics during T-cell activation. Nat Commun. 2016;7:11389. 82. Guo Q, Lu L, Liao Y, Wang X, Zhang Y, Liu Y, et al. Influence of c-Src on hypoxic resistance to paclitaxel in human ovarian cancer cells and reversal of FV-429. Cell Death Dis. 2018;8:e3178. 83. Hlavanda E, Klement E, Kokai E, Kovacs J, Vincze O, Tokesi N, et al. Phosphorylation blocks the activity of tubulin polymerization-promoting protein (TPPP): identification of sites targeted by different kinases. J Biol Chem. 2007;282:29531-9. 84. Gomes ER, Jani S, Gundersen GG. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell. 2005;121:451-63. 85. References Vicente-Manzanares M, Newell-Litwa K, Bachir AI, Whitmore LA, Horwitz AR. Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells. J Cell Biol. 2011;193:381-96. 86. Bao J, Jana SS, Adelstein RS. Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA- induced defects in COS-7 cell cytokinesis. J Biol Chem. 2005;280:19594-9. 87. Vicente-Manzanares M, Zareno J, Whitmore L, Choi CK, Horwitz AF. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. J Cell Biol. 2007;176:573-80. 88. D'Avino PP. Citron kinase-renaissance of a neglected mitotic kinase. J Cell Sci. 2017;130:1701-8. 89. Sahin I, Kawano Y, Sklavenitis-Pistofidis R, Moschetta M, Mishima Y, Manier S, et al. Citron Rho-interacting kinase silencing causes cytokinesis failure and reduces tumor growth in multiple myeloma. Blood Adv. 2019;3:995-1002. 90. Pallavicini G, Sgro F, Garello F, Falcone M, Bitonto V, Berto GE, et al. Inactivation of citron kinase inhibits medulloblastoma progression by inducing apoptosis and cell senescence. Cancer Res. 2018;78:4599-612. 91. Sun CC, Chiu HT, Lin YF, Lee KY, Pang JH. Y-27632, a ROCK inhibitor, promoted limbal epithelial cell proliferation and corneal wound healing. PLoS One. 2015;10:e0144571. 92. Gauthaman K, Fong CY, Bongso A. Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion. Stem Cell Rev Rep. 2010;6:86-95. 93. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007;25:681-6. 94. Liu YJ, Le Berre M, Lautenschlaeger F, Maiuri P, Callan-Jones A, Heuze M, et al. Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell. 2015;160:659-72. 95. Ruprecht V, Wieser S, Callan-Jones A, Smutny M, Morita H, Sako K, et al. Cortical contractility triggers a stochastic switch to fast amoeboid cell motility. Cell. 2015;160:673-85. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 306 Page 306 Page 306 96. Lammermann T, Bader BL, Monkley SJ, Worbs T, Wedlich-Soldner R, Hirsch K, et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature. 2008;453:51-5. 97. Miroshnikova YA, Mouw JK, Barnes JM, Pickup MW, Lakins JN, Kim Y, et al. Tissue mechanics promote IDH1-dependent HIF1alpha-tenascin C feedback to regulate glioblastoma aggression. Nat Cell Biol. 2016;18:1336-45. 98. Georgouli M, Herraiz C, Crosas-Molist E, Fanshawe B, Maiques O, Perdrix A, et al. References Regional activation of myosin II in cancer cells drives tumor progression via a secretory cross-talk with the immune microenvironment. Cell. 2019;176:757-74 e23. 99. Orgaz JL, Crosas-Molist E, Sadok A, Perdrix-Rosell A, Maiques O, Rodriguez-Hernandez I, et al. Myosin II reactivation and cytoskeletal remodeling as a hallmark and a vulnerability in melanoma therapy resistance. Cancer Cell. 2020;37:85-103.e9. 100. Colucci-Guyon E, Portier MM, Dunia I, Paulin D, Pournin S, Babinet C. Mice lacking vimentin develop and reproduce without an obvious phenotype. Cell. 1994;79:679-94. 101. Evans RM. Vimentin: the conundrum of the intermediate filament gene family. Bioessays. 1998;20:79-86. 102. Mendez MG, Kojima S, Goldman RD. Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J. 2010;24:1838-51. 103. McInroy L, Maatta A. Down-regulation of vimentin expression inhibits carcinoma cell migration and adhesion. Biochem Biophys Res Commun. 2007;360:109-14. 104. Al-Maghrabi J. Vimentin immunoexpression is associated with higher tumor grade, metastasis, and shorter survival in colorectal cancer. Int J Clin Exp Pathol. 2020;13:493-500. 105. Lavenus SB, Tudor SM, Ullo MF, Vosatka KW, Logue JS. A flexible network of vimentin intermediate filaments promotes migration of amoeboid cancer cells through confined environments. J Biol Chem. 2020;295:6700-9. n H, Jabbour E, Grimley J, Kirkpatrick P. Dasatinib. Nat Rev Drug Discov. 2006;5:717-8. 106. Kantarjian H, Jabbour E, Grimley J, Kirkpatrick P. Dasatinib. Nat Rev Drug Discov. 2006;5: 107. Kreutzman A, Colom-Fernandez B, Jimenez AM, Ilander M, Cuesta-Mateos C, Perez-Garcia Y, et al. Dasatinib reversibly disrupts endothelial vascular integrity by increasing non-muscle myosin II contractility in a ROCK-dependent manner. Clin Cancer Res. 2017;23:6697-707. 108. Wang J, Schneider IC. Myosin phosphorylation on stress fibers predicts contact guidance behavior across diverse breast cancer cells. Biomaterials. 2017;120:81-93. 109. Ikebe M, Koretz J, Hartshorne DJ. Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin. J Biol Chem. 1988;263:6432-7. 110. Feng Y, LoGrasso PV, Defert O, Li R. Rho kinase (ROCK) inhibitors and their therapeutic potential. J Med Chem. 2016;59:2269-300. 111. Sharma K, D'Souza RC, Tyanova S, Schaab C, Wisniewski JR, Cox J, et al. Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling. Cell Rep. 2014;8:1583-94. 112. Almeida MT, Mesquita FS, Cruz R, Osorio H, Custodio R, Brito C, et al. Src-dependent tyrosine phosphorylation of non-muscle myosin heavy chain-IIA restricts Listeria monocytogenes cellular infection. J Biol Chem. 2015;290:8383-95. 113. 118. Stefanovic S, Windsor M, Nagata KI, Inagaki M, Wileman T. Vimentin rearrangement during African swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase II. J Virol. 2005;79:11766-75. 117. Oguri T, Inoko A, Shima H, Izawa I, Arimura N, Yamaguchi T, et al. Vimentin-Ser82 as a memory phosphorylation site in astrocytes. Genes Cells. 2006;11:531-40. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 116. Yokoyama T, Goto H, Izawa I, Mizutani H, Inagaki M. Aurora-B and Rho-kinase/ROCK, the two cleavage furrow kinases, independently regulate the progression of cytokinesis: possible existence of a novel cleavage furrow kinase phosphorylates ezrin/radixin/moesin (ERM). Genes Cells. 2005;10:127-37. References Stuart SA, Houel S, Lee T, Wang N, Old WM, Ahn NG. A phosphoproteomic comparison of B-RAFV600E and MKK1/2 inhibitors in melanoma cells. Mol Cell Proteomics. 2015;14:1599-615. 114. Goto H, Tanabe K, Manser E, Lim L, Yasui Y, Inagaki M. Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK). Genes Cells. 2002;7:91-7. 115. Goto H, Kosako H, Tanabe K, Yanagida M, Sakurai M, Amano M, et al. Phosphorylation of vimentin by Rho- associated kinase at a unique amino-terminal site that is specifically phosphorylated during cytokinesis. J Biol Chem. 1998;273:11728-36. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 307 Page 307 116. Yokoyama T, Goto H, Izawa I, Mizutani H, Inagaki M. Aurora-B and Rho-kinase/ROCK, the two cleavage furrow kinases, independently regulate the progression of cytokinesis: possible existence of a novel cleavage furrow kinase phosphorylates ezrin/radixin/moesin (ERM). Genes Cells. 2005;10:127-37. 117. Oguri T, Inoko A, Shima H, Izawa I, Arimura N, Yamaguchi T, et al. Vimentin-Ser82 as a memory phosphorylation site in astrocytes. Genes Cells. 2006;11:531-40. 118. Stefanovic S, Windsor M, Nagata KI, Inagaki M, Wileman T. Vimentin rearrangement during African swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase II. J Virol. 2005;79:11766-75. 118. Stefanovic S, Windsor M, Nagata KI, Inagaki M, Wileman T. Vimentin rearrangement during African swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase II. J Virol. 2005;79:11766-75. Explor Target Antitumor Ther. 2021;2:292-308 \| https://doi.org/10.37349/etat.2021.00047 Page 308
https://openalex.org/W3042123066	https://f1000research.com/articles/9-687/v2/pdf	English	null	Case Report: Cryptococcal meningitis in Hodgkin’s Lymphoma patient receiving brentuximab-vedotin therapy	F1000Research	2,020	cc-by	4,826	F1000Research 2020, 9:687 Last updated: 06 SEP 2024 Open Peer Review Approval Status 1 2 version 2 (revision) 12 Aug 2020 view version 1 08 Jul 2020 view view First published: 08 Jul 2020, 9:687 https://doi.org/10.12688/f1000research.24816.1 Latest published: 12 Aug 2020, 9:687 https://doi.org/10.12688/f1000research.24816.2 v2 CASE REPORT Case Report: Cryptococcal meningitis in Hodgkin’s Lymphoma patient receiving brentuximab-vedotin therapy [version 2; peer review: 2 approved] Tatiana Cunha Pereira 1, Rita Rb-Silva 2, Rita Félix Soares1, Nelson Domingues2, José Mariz2 1Medical Oncology Department, Instituto Português Oncologia de Coimbra Francisco Gentil E. P. E., Coimbra, Portugal 2Onco-Hematology Department, Instituto Português de Oncologia do Porto, Porto, Portugal First published: 08 Jul 2020, 9:687 https://doi.org/10.12688/f1000research.24816.1 Latest published: 12 Aug 2020, 9:687 https://doi.org/10.12688/f1000research.24816.2 v2 Abstract Cryptococcus neoformans infections occur mostly in immunodeficient individuals, being the most common opportunistic fungal central nervous system (CNS) infection in HIV seropositive patients. Moreover, other conditions affecting host immunity, such as hematologic malignancies, organ transplantation and immunosuppressive drugs are implicated as risk factors. The authors present a case of a 48-year-old male with Hodgkin Lymphoma for 26 years and submitted to several lines of treatment, diagnosed with cryptococcal meningitis while on therapy with brentuximab. The patient presented with positive cerebral spinal fluid (CSF) cryptococcal antigen plus positive blood cultures. He was put under induction antifungal treatment with liposomal amphotericin B and flucytosine, as well as corticosteroid therapy with dexamethasone with headache improvement and a favorable clinical evolution. There are no reported cases of cryptococcal meningoencephalitis under CD30-directed monoclonal antibody. Furthermore, this case illustrates the risk of Cryptococcus neoformans infection in immunocompromising conditions other than HIV, underlining the need of considering this differential diagnosis when physicians face an opportunistic neuroinfection. Keywords Open Peer Review Approval Status 1 2 version 2 (revision) 12 Aug 2020 view version 1 08 Jul 2020 view view Adriana Roque , Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal Coimbra Institute for Clinical and Biomedical Research (iCBR), Coimbra, Portugal 1. Sarah A. Schmalzle, University of Maryland School of Medicine, Baltimore, USA 2. Any reports and responses or comments on the article can be found at the end of the article. F1000Research 2020, 9:687 Last updated: 06 SEP 2024 CASE REPORT Case Report: Cryptococcal meningitis in Hodgkin’s Lymphoma patient receiving brentuximab-vedotin therapy [version 2; peer review: 2 approved] Tatiana Cunha Pereira 1, Rita Rb-Silva 2, Rita Félix Soares1, Nelson Domingues2, José Mariz2 1Medical Oncology Department, Instituto Português Oncologia de Coimbra Francisco Gentil E. P. E., Coimbra, Portugal 2Onco-Hematology Department, Instituto Português de Oncologia do Porto, Porto, Portugal First published: 08 Jul 2020, 9:687 https://doi.org/10.12688/f1000research.24816.1 Latest published: 12 Aug 2020, 9:687 https://doi.org/10.12688/f1000research.24816.2 v2 Abstract Cryptococcus neoformans infections occur mostly in immunodeficient individuals, being the most common opportunistic fungal central nervous system (CNS) infection in HIV seropositive patients. Moreover, other conditions affecting host immunity, such as hematologic malignancies, organ transplantation and immunosuppressive drugs are implicated as risk factors. The authors present a case of a 48-year-old male with Hodgkin Lymphoma for 26 years and submitted to several lines of treatment, diagnosed with cryptococcal meningitis while on therapy with brentuximab. The patient presented with positive cerebral spinal fluid (CSF) cryptococcal antigen plus positive blood cultures. He was put under induction antifungal treatment with liposomal amphotericin B and flucytosine, as well as corticosteroid therapy with dexamethasone with headache improvement and a favorable clinical evolution. There are no reported cases of cryptococcal meningoencephalitis under CD30-directed monoclonal antibody. Furthermore, this case illustrates the risk of Cryptococcus neoformans infection in immunocompromising conditions other than HIV, underlining the need of considering this differential diagnosis when physicians face an opportunistic neuroinfection. Keywords Open Peer Review Approval Status 1 2 version 2 (revision) 12 Aug 2020 view version 1 08 Jul 2020 view view Adriana Roque , Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal Coimbra Institute for Clinical and Biomedical Research (iCBR), Coimbra, Portugal 1. Sarah A. Schmalzle, University of Maryland School of Medicine, Baltimore, USA 2. Any reports and responses or comments on the article can be found at the end of the article. F1000Research 2020, 9:687 Last updated: 06 SEP 2024 Abstract Cryptococcus neoformans infections occur mostly in immunodeficient individuals, being the most common opportunistic fungal central nervous system (CNS) infection in HIV seropositive patients. Moreover, other conditions affecting host immunity, such as hematologic malignancies, organ transplantation and immunosuppressive drugs are implicated as risk factors. view view The authors present a case of a 48-year-old male with Hodgkin Lymphoma for 26 years and submitted to several lines of treatment, diagnosed with cryptococcal meningitis while on therapy with brentuximab. The patient presented with positive cerebral spinal fluid (CSF) cryptococcal antigen plus positive blood cultures. He was put under induction antifungal treatment with liposomal amphotericin B and flucytosine, as well as corticosteroid therapy with dexamethasone with headache improvement and a favorable clinical evolution. There are no reported cases of cryptococcal meningoencephalitis under CD30-directed monoclonal antibody. Furthermore, this case illustrates the risk of Cryptococcus neoformans infection in immunocompromising conditions other than HIV, underlining the need of considering this differential diagnosis when physicians face an opportunistic neuroinfection. Adriana Roque , Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal Coimbra Institute for Clinical and Biomedical Research (iCBR), Coimbra, Portugal 1. Keywords C. neoformans, Brentuximab-vedotin, Hodgkin Lymphoma, Meningitis Keywords Keywords C. neoformans, Brentuximab-vedotin, Hodgkin Lymphoma, Meningitis Page 1 of 9 F1000Research 2020, 9:687 Last updated: 06 SEP 2024 Corresponding author: Tatiana Cunha Pereira (tatiana.cunhapereira@gmail.com) Author roles: Cunha Pereira T: Conceptualization, Writing – Original Draft Preparation; Rb-Silva R: Writing – Review & Editing; Félix Soares R: Writing – Review & Editing; Domingues N: Supervision, Writing – Review & Editing; Mariz J: Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2020 Cunha Pereira T et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Cunha Pereira T, Rb-Silva R, Félix Soares R et al. Case Report: Cryptococcal meningitis in Hodgkin’s Lymphoma patient receiving brentuximab-vedotin therapy [version 2; peer review: 2 approved] F1000Research 2020, 9:687 https://doi.org/10.12688/f1000research.24816.2 First published: 08 Jul 2020, 9:687 https://doi.org/10.12688/f1000research.24816.1 Corresponding author: Tatiana Cunha Pereira (tatiana.cunhapereira@gmail.com) Author roles: Cunha Pereira T: Conceptualization, Writing – Original Draft Preparation; Rb-Silva R: Writing – Review & Editing; Félix Soares R: Writing – Review & Editing; Domingues N: Supervision, Writing – Review & Editing; Mariz J: Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2020 Cunha Pereira T et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Cunha Pereira T, Rb-Silva R, Félix Soares R et al. Case Report: Cryptococcal meningitis in Hodgkin’s Lymphoma patient receiving brentuximab-vedotin therapy [version 2; peer review: 2 approved] F1000Research 2020, 9:687 https://doi.org/10.12688/f1000research.24816.2 First published: 08 Jul 2020, 9:687 https://doi.org/10.12688/f1000research.24816.1 Amendments from Version 1 REVISED As suggested by the peer review reports, grammar corrections have been made in this new version. As suggested by the peer review reports, grammar corrections have been made in this new version. As suggested by the peer review reports, gra have been made in this new version. Any further responses from the reviewers can be found at the end of the article Any further responses from the reviewers can be found at the end of the article At first evaluation, the patient was conscious and aware, hemodynamic stable and subfebrile, presenting general tremors and limited cervical mobility. Learning points Learning points g p • Cryptococcal meningitis is a common opportunistic central nervous system (CNS) infection among HIV- positive patients. However, it also affects HIV seronegative patients. Blood workup revealed elevated C-reactive protein with 73.2 mg/L (normal range under 5 mg/L), without other abnormalities. Blood workup revealed elevated C-reactive protein with 73.2 mg/L (normal range under 5 mg/L), without other abnormalities. • Every immunocompromising condition must be assessed and considered a risk factor for an opportun- istic fungal meningoencephalitis. A therapeutic agent affecting host immunity, such as with CD30-directed monoclonal antibody, may predispose to opportunistic infections. A head computed tomography (CT) scan showed the pre-existing cystic lesion in the left cerebellopontine angle with a slight right brainstem deviation, without associated edema (Figure 2A), confirmed by magnetic resonance imaging (Figure 2B). The case was discussed with the Neurosurgery Department and a lumbar puncture was postponed as it was considered a high-risk procedure. The patient started antibiotics with ceftriaxone (2 g q12h) and ampicillin. (2g q4h) At day 4, blood cultures came back positive for Cryptococcus neoformans sensitive to Posaconazole, Amphotericin B and Itraconazole, so that patient started Liposomal Amphotericin B (3mg/kg id) and Flucytosine (100 mg/kg per day orally in four divided doses) for 14 days and low dose corticosteroid therapy (4 mg per day). There was a progressive improvement of the symptoms and patient was discharged after 19 days with prescription of Fluconazole (400mg per day). • Cryptococcal meningitis diagnosis may be challenging in cases presenting negative cerebral spinal fluid (CSF) cultures, but cryptococcal polysaccharide antigen titers in CSF correlate with fungal burden. Background Cryptococcus species have a major predilection for the lungs with potential to spread further, mainly through continuity or through hematogenic and lymphoid pathways, with possible penetration through the blood-brain barrier and CNS involvement1–4. Cryptococcus neoformans infections occur mostly in immu- nodeficient individuals, being the most common opportunistic CNS infection in HIV-positive patients, counting up to 1 million new infections annually worldwide3,4. It also occurs in transplant recipients, patients with hematological malig- nancies, as well as patients receiving immunosuppressive medications1,2,4. After one month of treatment, a ventricular puncture was per- formed and normal pressure cerebrospinal fluid (CSF) revealed glucose consumption and elevated levels of proteins (Table 1), as well as positivity for cryptococcal polysaccharide capsular antigen. Follow-up lumbar punctures were performed to assess Figure 1. Head computed tomography (CT) scan revealing a large left extra-axial cystic lesion that was being monitored before current symptomatology. This case reports an opportunistic CNS infection in a patient with Hodgkin Lymphoma under brentuximab after multiple lines of treatment for over 20 years, including an allogenic stem cell transplantation. Despite being reported as a common fungal infection in HIV-patients, neuroinfections in patients under CD30-directed monoclonal antibody therapy or other drugs besides immunosuppressants are a rare occurrence. rresponding author: Tatiana Cunha Pereira (tatiana.cunhapereira@gmail.com) Corresponding author: Tatiana Cunha Pereira (tatiana.cunhapereira@gmail.com) Author roles: Cunha Pereira T: Conceptualization, Writing – Original Draft Preparation; Rb-Silva R: Writing – Review & Editing; Félix Soares R: Writing – Review & Editing; Domingues N: Supervision, Writing – Review & Editing; Mariz J: Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2020 Cunha Pereira T et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Cunha Pereira T, Rb-Silva R, Félix Soares R et al. Case Report: Cryptococcal meningitis in Hodgkin’s Lymphoma patient receiving brentuximab-vedotin therapy [version 2; peer review: 2 approved] F1000Research 2020, 9:687 https://doi.org/10.12688/f1000research.24816.2 First published: 08 Jul 2020, 9:687 https://doi.org/10.12688/f1000research.24816.1 p g st published: 08 Jul 2020, 9:687 https://doi.org/10.12688/f1000research.24816.1 Page 2 of 9 F1000Research 2020, 9:687 Last updated: 06 SEP 2024 one autologous bone marrow transplant in 1998, as well as an allogenic stem cell transplant in 2001, followed by several lines of chemotherapy. From October 2018 to this episode, the patient was taking brentuximab due to a hepatic hilar lesion. Sequencial imaging assessments showed a large left infratentorial arachnoid cystic lesion that was being monitored. (Figure 1). Amendments from Version 1 As suggested by the peer review reports, grammar corrections have been made in this new version. Any further responses from the reviewers can be found at the end of the article REVISED Case presentation A 48-year-old Caucasian male presented at the outpatient clinic in May 2019 with holocranial headache, more intense at occipital level, lasting for 6 days, with increasing intensity over the last couple of hours, associated with photophobia and vomiting. The patient was diagnosed in 1993 with Classic Hodgkin Lymphoma, nodular sclerosis subtype, stage IVB, achieving complete remission after first line chemotherapy. Since then, the patient suffered several relapses and underwent radiotherapy, Figure 1. Head computed tomography (CT) scan revealing a large left extra-axial cystic lesion that was being monitored before current symptomatology. Page 3 of 9 Page 3 of 9 F1000Research 2020, 9:687 Last updated: 06 SEP 2024 Figure 2. Head computed tomography (CT) scan showed the pre-existing cystic lesion in the left cerebellopontine angle with a slight right brainstem deviation, without associated edema (2A), as confirmed by magnetic resonance imagining (MRI) (2B). Figure 2. Head computed tomography (CT) scan showed the pre-existing cystic lesion in the left cerebellop brainstem deviation, without associated edema (2A), as confirmed by magnetic resonance imagining (MRI) (2 Figure 2. Head computed tomography (CT) scan showed the pre-existing cystic lesion in the left cerebellopontine angle with a slight right brainstem deviation, without associated edema (2A), as confirmed by magnetic resonance imagining (MRI) (2B). Table 1. Cerebrospinal fluid profile evolution throughout treatment. CSF – Cerebrospinal fluid. LP – Lumbar puncture. NV – Normal value. LP date Characteristic 27-06-2019 17-07-2019 31-07-2019 16-09-2019 Appearance Clear Clear Clear Clear Nucleated cells count 104/μL 43/μL 35/μL 5/μL Glucose (NV: 2.8 – 4.4 mmol/L) 2,3 mmol/L 3.1 mmol/L 3,4 mmol/L 3,3 mmol/L Protein level (NV: 150 – 450 mg/L) 838 mg/L 583 mg/L 529 mg/L 544 mg/L CSF culture Negative Negative Negative Negative Cryptococcus neoformans antigen Positive Positive Positive Positive Table 1. Cerebrospinal fluid profile evolution throughout treatment. CSF – Cerebrospinal fluid. LP – Lumbar puncture. NV – Normal value. LP date Characteristic 27-06-2019 17-07-2019 31-07-2019 16-09-2019 Appearance Clear Clear Clear Clear Nucleated cells count 104/μL 43/μL 35/μL 5/μL Glucose (NV: 2.8 – 4.4 mmol/L) 2,3 mmol/L 3.1 mmol/L 3,4 mmol/L 3,3 mmol/L Protein level (NV: 150 – 450 mg/L) 838 mg/L 583 mg/L 529 mg/L 544 mg/L CSF culture Negative Negative Negative Negative Cryptococcus neoformans antigen Positive Positive Positive Positive Table 1. Cerebrospinal fluid profile evolution throughout treatment. CSF – Cerebrospinal fluid. LP – Lumbar puncture. NV – Normal value. Case presentation In a recent review of Cryptococcus neoformans infections in patients with cancer, 82% corresponded to patients with haematological malignancies and from these patients, approxi- mately 54% had lymphoma5. CSF characteristics and cryptococcal antigen assessment. Patient was kept under consolidation therapy with Flucona- zole for 10 weeks with a favorable clinical evolution, as well as decreasing levels of protein and nucleated cells count as seen in Table 1. Patient maintains close surveillance under regular appointments at the Onco-Haematology Clinic. However, headache complaints increased in intensity shortly after dexam- ethasone discontinuation with an intermittent pattern. Patient died in another hospital about 8 months after the meningitis diagnosis due to a cardiovascular event. CSF characteristics and cryptococcal antigen assessment. Patient was kept under consolidation therapy with Flucona- zole for 10 weeks with a favorable clinical evolution, as well as decreasing levels of protein and nucleated cells count as seen in Table 1. Patient maintains close surveillance under regular appointments at the Onco-Haematology Clinic. However, headache complaints increased in intensity shortly after dexam- ethasone discontinuation with an intermittent pattern. Patient died in another hospital about 8 months after the meningitis diagnosis due to a cardiovascular event. The patient presented several conditions affecting host immunity due to several previous lines of treatment for over 25 years. However, Cryptococcus species were not considered the etiological agent for a possible opportunistic neuroinfection, emphasizing the need for an initial lumbar puncture to exclude fungal agents. This procedure was not possible at first evaluation and it delayed the start of antifungal therapy. Consent Data availability Underlying data All data underlying the results are available as part of the article and no additional source data are required. Written informed consent for publication of their clinical details and clinical images was obtained from the patient prior to their death. All data underlying the results are available as part of the article and no additional source data are required. 1. Li SS, Mody CH: Cryptococcus. Proc Am Thorac Soc. 2010; 7(3): 186–96. PubMed Abstract \| Publisher Full Text 2. Beardsley J, Sorrell TC, Chen SCA: Central nervous system cryptococcal infections in non-HIV infected patients. J Fungi (Basel). 2019; 5(3): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Góralska K, Blaszkowska J, Dzikowiec M: Neuroinfections caused by fungi. Discussion Cryptococcal meningitis accounts for up to 1 million new infections annually, mainly affecting HIV-positive patients. Other immunocompromising conditions such as organ transplanta- tion, hematologic malignancies and immunosuppressive drugs constitutes other relevant risk factors to these opportunistic fungi CNS infections1–4. Although there are many published case reports of Crypto- coccosis in patients with lymphoma, this is the first reported case of Cryptococcal neuroinfection in a patient with Hodgkin’s Lymphoma treated with CD-30-directed monoclonal antibody. Page 4 of 9 Page 4 of 9 Page 4 of 9 F1000Research 2020, 9:687 Last updated: 06 SEP 2024 Sarah A. Schmalzle No new comments. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Infectious disease, HIV, cryptococcosis, Group A Strep I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. https://doi.org/10.5256/f1000research.27380.r68025 © 2020 Schmalzle S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. References 1. Li SS, Mody CH: Cryptococcus. Proc Am Thorac Soc. 2010; 7(3): 186–96. PubMed Abstract \| Publisher Full Text 2. Beardsley J, Sorrell TC, Chen SCA: Central nervous system cryptococcal infections in non-HIV infected patients. J Fungi (Basel). 2019; 5(3): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Góralska K, Blaszkowska J, Dzikowiec M: Neuroinfections caused by fungi. Infection. 2018; 46(4): 443–59. PubMed Abstract \| Publisher Full Text \| Free Full Text 4. Mazlarz EK, Perfect JR: Cryptococcosis. Infect Dis Clin North Am. 2016; 30(1): 179–206. PubMed Abstract \| Publisher Full Text \| Free Full Text 5. Schmalzle SA, Buchwald UK, Gilliam BL, et al.: Cryptococcus neoformans infection in malignancy. Mycoses. 2016; 59(9): 542–52. PubMed Abstract \| Publisher Full Text Infection. 2018; 46(4): 443–59. PubMed Abstract \| Publisher Full Text \| Free Full Text 4. Mazlarz EK, Perfect JR: Cryptococcosis. Infect Dis Clin North Am. 2016; 30(1): 179–206. PubMed Abstract \| Publisher Full Text \| Free Full Text 5. Schmalzle SA, Buchwald UK, Gilliam BL, et al.: Cryptococcus neoformans infection in malignancy. Mycoses. 2016; 59(9): 542–52. PubMed Abstract \| Publisher Full Text 1. Li SS, Mody CH: Cryptococcus. Proc Am Thorac Soc. 2010; 7(3): 186–96. PubMed Abstract \| Publisher Full Text 2. Beardsley J, Sorrell TC, Chen SCA: Central nervous system cryptococcal infections in non-HIV infected patients. J Fungi (Basel). 2019; 5(3): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Góralska K, Blaszkowska J, Dzikowiec M: Neuroinfections caused by fungi. Page 5 of 9 Open Peer Review Open Peer Review Current Peer Review Status: F1000Research 2020, 9:687 Last updated: 06 SEP 2024 Sarah A. Schmalzle There is not known resistance and generally all therapy is empiric. 12. Corticotherapy should be corticosteroid therapy. 13. This needs to be reworded for clarity: "However, Cryptococcus species were not considered the etiological agent for a CNS infection, emphasizing the need for an initial lumbar puncture to exclude fungal agents when approaching opportunist neuroinfection. This was not possible at first evaluation in this case which delayed antifungal therapy." 14. Is the background of the case’s history and progression described in sufficient detail? Yes Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? The use of 'under' to describe being prescribed a treatment is not commonly how this is stated in the US. Try "treated with" or "on treatment for ___ with ___". 9. Also not common to say patients are admitted at the outpatient clinic. Patients are seen or evaluated at a clinic, but admitted to a hospital. 10. This sentence also needs to be revised for proper grammar/syntax. "Throughout image assessments, a large left infratentorial arachnoid cystic lesion was being monitored.". 11. This sentence also needs to be revised for proper grammar/syntax. "Throughout image assessments, a large left infratentorial arachnoid cystic lesion was being monitored.". 11. Susceptibilities are not commonly reported in Crypto case reports. There is not known resistance and generally all therapy is empiric. 12. This needs to be reworded for clarity: "However, Cryptococcus species were not considered the etiological agent for a CNS infection, emphasizing the need for an initial lumbar puncture to exclude fungal agents when approaching opportunist neuroinfection. This was not possible at first evaluation in this case which delayed antifungal therapy." 14. Sarah A. Schmalzle Institute of Human Virology, University of Maryland School of Medicine, Baltimore This is a clinically relevant case report as it is purportedly the first report in a patient being treated with a particular immunosuppressive monoclonal Ab. This is a clinically relevant case report as it is purportedly the first report in a patient being treated with a particular immunosuppressive monoclonal Ab. There are several minor language and grammatical improvements to be made that will strengthen There are several minor language and grammatical improvements to be made that will strengthen Page 6 of 9 F1000Research 2020, 9:687 Last updated: 06 SEP 2024 and clarify the report. It should have an additional round of editing by a native English speaker. Examples: and clarify the report. It should have an additional round of editing by a native English speaker. Examples: p HIV-positive should be 'people living with HIV'. 1. Abstract: 'fungi central nervous system..' should be fungal. 2. Brentuximab should be in lower case throughout. It is not explained with it is 'brentuximab- vedotin' in keywords but 'brentuximab' elsewhere. 3. Brentuximab should be in lower case throughout. It is not explained with it is 'brentuximab- vedotin' in keywords but 'brentuximab' elsewhere. 3. This sentence needs an English language revision: "However, these fungi neuroinfection affects HIV seronegative patients." 5. Background - it is not accurate to say crypto has a predilection for the lungs. The lungs are the route of entry, but this is a neurotropic pathogen. 6. Background - it is not accurate to say crypto has a predilection for the lungs. The lungs are the route of entry, but this is a neurotropic pathogen. 6. 'penetration into' should be 'penetration through'. 7. Case presentation: "over the last couple hours" should be "over the last couple of hours" or "over the last several hours". 8. p p p "over the last several hours". The use of 'under' to describe being prescribed a treatment is not commonly how this is stated in the US. Try "treated with" or "on treatment for ___ with ___". 9. Also not common to say patients are admitted at the outpatient clinic. Patients are seen or evaluated at a clinic, but admitted to a hospital. 10. This sentence also needs to be revised for proper grammar/syntax. "Throughout image assessments, a large left infratentorial arachnoid cystic lesion was being monitored.". 11. Susceptibilities are not commonly reported in Crypto case reports. https://doi.org/10.5256/f1000research.27380.r66704 © 2020 Roque A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Is the case presented with sufficient detail to be useful for other practitioners? Yes Reviewer Report 20 July 2020 https://doi.org/10.5256/f1000research.27380.r66704 https://doi.org/10.5256/f1000research.27380.r66704 Adriana Roque 1 Clinical Hematology Department, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal 2 Faculty of Medicine, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra Institute for Clinical and Biomedical Research (iCBR), Coimbra, Portugal I think that this is an interesting report that claims for attention to rare infections in patients under immunotherapy and other novel therapies, especially when they are more difficult to diagnose (due to the location and the infectious agent). g g This report provides a concise and informative history of the case, mainly focus on the difficulties of the process. I consider that it would be important to provide information about the previous therapeutics that the patient had received, including the transplantation conditioning regimen, as well as the development of graft versus host disease (GvHD) and GvHD therapeutics, because it can help to explain the subjacent immunosuppression state. Another helpful information is to understand if Hodgkin lymphoma was under control at the time of CNS infection presentation. Is the background of the case’s history and progression described in sufficient detail? Partly Is the background of the case’s history and progression described in sufficient detail? Yes Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Page 7 of 9 F1000Research 2020, 9:687 Last updated: 06 SEP 2024 Yes Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes Is the case presented with sufficient detail to be useful for other practitioners? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Infectious disease, HIV, cryptococcosis, Group A Strep I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Yes given and outcomes? Yes Is the background of the case’s history and progression described in sufficient detail? Partly Reviewer Expertise: Hematology, Hematology-oncology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Is the background of the case’s history and progression described in sufficient detail? Partly Partly Are enough details provided of any physical examination and diagnostic tests, treatment Page 8 of 9 given and outcomes? Yes Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes Is the case presented with sufficient detail to be useful for other practitioners? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Hematology, Hematology-oncology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com F1000Research 2020, 9:687 Last updated: 06 SEP 2024 given and outcomes? Yes Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes Is the case presented with sufficient detail to be useful for other practitioners? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Hematology, Hematology-oncology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com F1000Research 2020, 9:687 Last updated: 06 SEP 2024 F1000Research 2020, 9:687 Last updated: 06 SEP 2024 given and outcomes? Yes Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes Is the case presented with sufficient detail to be useful for other practitioners? Yes Competing Interests: No competing interests were disclosed. Is the case presented with sufficient detail to be useful for other practitioners? Yes The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com Page 9 of 9
https://openalex.org/W1763675400	http://repo.lib.semmelweis.hu//bitstream/123456789/2217/1/Gyorffy_Cell_Reports_2015_u.pdf	English	null	APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer	Cell reports	2,015	cc-by	12,366	Article Article APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast C APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer Graphical Abstract Highlights d APOBEC3B is associated with poor survival in ER+ breast cancer patients d APOBEC3B controls breast cancer cell growth by promoting ER transcriptional activity d APOBEC3B can cause C-to-U mutations at ER target genes, to activate DNA repair d Repair of APOBEC3B-induced lesions allows chromatin remodelling that stimulates gene expression Authors Manikandan Periyasamy, Hetal Patel, Chun-Fui Lai, ..., Luca Magnani, Laki Buluwela, Simak Ali Correspondence simak.ali@imperial.ac.uk In Brief Periyasamy et al. show that APOBEC3B required for the regulation of gene expression by the estrogen receptor in breast cancer cells. They report APOBEC3B can promote cytidine deamination at gene regulatory regions with consequent repair providing a mechanism for chromatin remodelling that facilitates gene expression. Accession Numbers GSE56979 GSE57426 Periyasamy et al., 2015, Cell Reports 13, 108–121 October 6, 2015 ª2015 The Authors http://dx.doi.org/10.1016/j.celrep.2015.08.066 Authors Manikandan Periyasamy, Hetal Patel, Chun-Fui Lai, ..., Luca Magnani, Laki Buluwela, Simak Ali Authors Manikandan Periyasamy, Hetal Patel, Chun-Fui Lai, ..., Luca Magnani, Laki Buluwela, Simak Ali Correspondence simak.ali@imperial.ac.uk Periyasamy et al., 2015, Cell Reports 13, 108–121 October 6, 2015 ª2015 The Authors http://dx.doi.org/10.1016/j.celrep.2015.08.066 APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer Manikandan Periyasamy,1 Hetal Patel,1 Chun-Fui Lai,1 Van T.M. Nguyen,1 Ekaterina Nevedomskaya,2 Alison Harrod,1 Roslin Russell,3 Judit Remenyi,4 Anna Maria Ochocka,1 Ross S. Thomas,1 Frances Fuller-Pace,4 Bala´ zs Gy}orffy,5 Carlos Caldas,3 Naveenan Navaratnam,6 Jason S. Carroll,3 Wilbert Zwart,2 R. Charles Coombes,1 Luca Magnani,1 Laki Buluwela 1 and Simak Ali1,* Manikandan Periyasamy,1 Hetal Patel,1 Chun-Fui Lai,1 Van T.M. Nguyen,1 Ekaterina Nevedomskaya,2 Alison Harrod,1 Roslin Russell,3 Judit Remenyi,4 Anna Maria Ochocka,1 Ross S. Thomas,1 Frances Fuller-Pace,4 Bala´ zs Gy}orffy,5 Carlos Caldas,3 Naveenan Navaratnam,6 Jason S. Carroll,3 Wilbert Zwart,2 R. Charles Coombes,1 Luca Magnani,1 Laki Buluwela 1 and Simak Ali1,* 1Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK 2Department of Molecular Pathology, The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands 3Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK 4Division of Cancer Research, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK 5MTA TTK Lendu¨ let Cancer Biomarker Research Group, Second Department of Pediatrics, Semmelweis University and MTA-SE Pediatrics and Nephrology Research Group, Budapest 1085, Hungary 6MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, Lon Correspondence: simak.ali@imperial.ac.uk 6MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK Correspondence: simak.ali@imperial.ac.uk htt //d d i /10 1016/j l 2015 08 066 http://dx.doi.org/10.1016/j.celrep.2015.08.066 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). http://dx.doi.org/10.1016/j.celrep.2015.08.066 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0 SUMMARY importance of pioneer factors, particularly FOXA1 and GATA3 in directing ER by promoting chromatin accessibility and long- range chromatin interactions (Magnani et al., 2011; Ross-Innes et al., 2012). Critical for transcription regulation by ER is the or- dered recruitment of a multitude of transcriptional co-regulator complexes with enzymatic activities for histone modiﬁcation and chromatin remodeling (Me´ tivier et al., 2006), which promote short- and long-range protein-DNA and protein-protein interac- tions between enhancer regions and gene promoters, to facili- tate expression of ER target genes that drive breast cancer cell proliferation. The processes of transcription and DNA repair are intimately linked, as deﬁned most obviously for the basal transcription factor TFIIH, which is essential for transcription initiation by RNA polymerase II (PolII), but is also required for the transcription-coupled nucleotide excision repair (Compe and Egly, 2012; Kamileri et al., 2012). Other DNA repair pathways also aid transcription (Fong et al., 2013) by promoting active DNA demethylation (Bhutani et al., 2011; Nabel and Kohli, 2011), enhancer RNA (eRNA) synthesis (Puc et al., 2015), and chromatin remodeling (Ju et al., 2006; Perillo et al., 2008), through pro- cesses that can involve the generation of single- and double- strand DNA breaks at enhancer regions. Estrogen receptor a (ERa) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regula- tion of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the genera- tion of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homolo- gous end-joining (NHEJ) pathways. We provide evi- dence that transient cytidine deamination by A3B aids chromatin modiﬁcation and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, rein- forcing the physiological signiﬁcance of A3B for ER action. In Brief Periyasamy et al. show that APOBEC3B is required for the regulation of gene expression by the estrogen receptor in breast cancer cells. They report APOBEC3B can promote cytidine deamination at gene regulatory regions, with consequent repair providing a mechanism for chromatin remodelling that facilitates gene expression. Accession Numbers GSE56979 GSE57426 d APOBEC3B is associated with poor survival in ER+ breast cancer patients d APOBEC3B controls breast cancer cell growth by promoting ER transcriptional activity d APOBEC3B can cause C-to-U mutations at ER target genes, to activate DNA repair d Repair of APOBEC3B-induced lesions allows chromatin remodelling that stimulates gene expression Cell Reports Article Cell Reports Article 108 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors INTRODUCTION Real-time RT-PCR for 151 breast cancers again conﬁrmed that A3B is expressed in ER+ and ER breast cancer, as well as in the majority of breast cancer cell lines examined (Figures 1C and S1G–S1R), in agreement with previous ﬁndings (Burns et al., 2013a). (Figure S1C). There was no association between expression of other APOBECs and poor outcome in ER+ or ER breast cancer in the METABRIC dataset (data not shown). We extended this analysis to other gene expression microarray datasets. Kaplan- Meier plot analysis of Affymetrix microarray datasets similarly showed that high A3B expression is associated with poor outcome for ER+, but not ER, breast cancer (Figures S1D– S1F). Forest-plot analysis for relapse-free survival further conﬁrmed the importance of A3B in ER+ breast cancer (Fig- ure 1B). Real-time RT-PCR for 151 breast cancers again conﬁrmed that A3B is expressed in ER+ and ER breast cancer, as well as in the majority of breast cancer cell lines examined (Figures 1C and S1G–S1R), in agreement with previous ﬁndings (Burns et al., 2013a). been ascribed to APOBEC3G (Nowarski et al., 2012). Interest- ingly, ectopic expression of APOBEC3A (A3A) and A3B can pro- mote mutagenesis in cancer cells (Burns et al., 2013a; Landry et al., 2011; Taylor et al., 2013). been ascribed to APOBEC3G (Nowarski et al., 2012). Interest- ingly, ectopic expression of APOBEC3A (A3A) and A3B can pro- mote mutagenesis in cancer cells (Burns et al., 2013a; Landry et al., 2011; Taylor et al., 2013). Cancer genomes are marked by an accretion of somatic mu- tations. Recent whole-genome sequencing of breast cancer has yielded genome-wide mutational signatures, one of which is consistent with the DNA mutation proﬁles associated with cyti- dine deamination by APOBEC3 genes (Alexandrov et al., 2013; Nik-Zainal et al., 2012). Similar mutational signatures have been described in ovarian, bladder, cervical, head and neck, and lung cancer (Burns et al., 2013a, 2013b; de Bruin et al., 2014; Leonard et al., 2013; Roberts et al., 2013). A3B expression is frequently elevated in breast and other cancers that feature mutational landscapes consistent with cytidine deaminase activ- ity (Burns et al., 2013a, 2013b). This, together with the demon- stration that ectopic A3B expression can promote C-to-T muta- tions in breast cancer cells, has led to a proposed model in which A3B overexpression in breast cancer could aid tumor initiation and progression by driving somatic mutations in cancer. INTRODUCTION How- ever, breast cancers from patients featuring a germline copy- number polymorphism involving A3A and A3B, in which A3B is effectively deleted, carry a greater burden of mutations associ- ated with the APOBEC-dependent signature than those in which the A3A and A3B genes are intact (Nik-Zainal et al., 2014), bringing into question the importance of A3B in this process. A3B Regulates the Growth of ER+ Breast Cancer Cells Given the association between A3B expression and poor patient survival in ER+ breast cancer, we wondered whether A3B regu- lates the growth of ER+ breast cancer cells. To investigate this, we treated tumor xenografts of the ER+ and estrogen-regulated MCF7 breast cancer cell line, which expresses moderate levels of A3B, with A3B small interfering RNA (siRNA). A3B knockdown markedly inhibited MCF7 tumor growth (Figure 1D), demon- strating that A3B is required for MCF7 tumor growth in vivo. Remarkably, ER target gene expression was greatly reduced in these tumors (Figures 1E and 1F), suggesting that A3B impacts directly on ER function. Indeed, transfection of MCF7 cells in culture with two independent A3B siRNAs inhibited estrogen- stimulated growth, accompanied by reduced expression of ER regulated genes (Figures S2A–S2C). Inhibition of estrogen-regu- lated growth and estrogen-responsive gene expression was conﬁrmed in a second ER+ cell line (T47D) (Figures 1G and 1H). SkBr3 cells, which are null for A3B (Komatsu et al., 2008), were not affected by A3B siRNA (Figures S1I and S2D). Growth of the A3B+/ER MDA-MB-231 cells was also unaffected by A3B siRNA (Figure S2E). Furthermore, inhibition of ER target gene expression by a siRNA targeting the A3B 30-UTR was rescued by transfection of an A3B expression plasmid that lacks the 30 UTR. However, siRNAs targeting the A3B coding region knocked down endogenous and ectopic A3B and inhibited ER- regulated genes (Figures S2F and S2G). Taken together, these results demonstrate A3B speciﬁcity of the siRNAs and show that A3B regulates growth and expression of estrogen-respon- sive genes in breast cancer cells. To investigate the potential role of A3B in breast cancer, we analyzed its expression in breast cancer. Surprisingly, A3B expression was associated with poor patient survival in ER+ breast cancer, but not in ER breast cancer, despite the fact that A3B expression was higher in ER breast cancer than in ER+ breast cancer, thus implicating A3B in ER action. Here, we provide evidence for a molecular mechanism in which A3B causes local and transient C-to-U transitions at ER enhancers, leading to activation of base excision repair (BER) and non-ho- mologous end-joining (NHEJ) pathways, which in turn promote chromatin modiﬁcation and remodeling, to drive expression of ER target genes. INTRODUCTION The AID/APOBEC genes comprise a family of enzymes that mutate RNA or DNA by deaminating cytidine to uridine. Among their functions are RNA editing of the apolipoprotein B pre- mRNA by APOBEC1 and generation of antibody diversity by class-switch recombination and somatic hypermutation through DNA editing by AID (Conticello, 2008). In primates, there are seven closely related APOBEC3 genes, some of which function in retroviral restriction by promoting ‘‘hypermutation’’ in viral ge- nomes. These functions do not readily explain the potential roles of APOBEC3 genes in non-immune system tissues, including breast, lung, cervix, bladder, and ovary, or the overexpression of some members of the family in cancers from these tissues (Burns et al., 2013a, 2013b; Leonard et al., 2013), although a po- tential role in the repair of DNA double-strand breaks (DSBs), re- sulting in resistance of lymphoma cells to ionizing radiation, has Estrogens play a central role in promoting breast cancer devel- opment (Ali and Coombes, 2002) and are important in uterine and ovarian cancer (O’Donnell et al., 2005; Shang, 2006). Two closely related nuclear receptors, estrogen receptor a (ERa, herein referred to as ER) and ERb, mediate estrogen actions (Dahlman-Wright et al., 2006). ERa is dominant in breast cancer; 70% of breast cancers express ERa, and therapies to inhibit its activity have transformed breast cancer treatment. However, many patients develop resistance, with few treatment options being available for endocrine-therapy-resistant breast cancer (Osborne and Schiff, 2011). Gene expression proﬁling and approaches for genome-wide identiﬁcation of ER binding regions have allowed the identiﬁca- tion of direct ER targets in breast cancer cells and highlight the 108 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors (Figure S1C). There was no association between expression of other APOBECs and poor outcome in ER+ or ER breast cancer in the METABRIC dataset (data not shown). We extended this analysis to other gene expression microarray datasets. Kaplan- Meier plot analysis of Affymetrix microarray datasets similarly showed that high A3B expression is associated with poor outcome for ER+, but not ER, breast cancer (Figures S1D– S1F). Forest-plot analysis for relapse-free survival further conﬁrmed the importance of A3B in ER+ breast cancer (Fig- ure 1B). A3B Expression Is Associated with Poor Prognosis in ER+ Breast Cancer AID/APOBECs function in retroviral restriction and in the case of AID, in class-switch recombination and somatic hypermutation to generate antibody diversity by causing C-to-T mutations (Conticello, 2008), functions that do not explain the high-level expression of A3B in many cancers (Burns et al., 2013a, 2013b). Toward deﬁning the role of A3B in breast cancer sub- types, we determined the relationship between A3B levels and patient outcome. Analysis of the METABRIC (Curtis et al., 2012) series of 2,000 breast cancer patients revealed that high A3B expression is associated with poor survival (hazard ratio [HR] = 1.5, p = 1 3 1011) (Figures S1A and S1B). Interestingly, A3B expression was associated with poor outcome in ER+ (HR = 1.9; p = 4.5 3 1011) (Figure 1A), but not in ER (p = 0.18), breast cancer. A3B retained its signiﬁcance (HR = 1.59, p = 1.27 3 106) in multivariate analysis of ER+ breast cancer A3B Is Recruited Globally to ER Binding Regions, and This Requires Its Interaction with the ER Next, we sought to determine whether A3B is required for the ER transcriptional response. In a reporter gene assay, A3B stimu- lated ER activity (Figures 2A and 2B). A3B contains two zinc coordinating cytidine deaminase activity (CDA) domains (Conti- cello, 2008). Substitution of glutamic acid residues at positions 68 or 255 to glutamine, which inhibits the cytidine deaminase ac- tivity in the N- and C-terminal domains of A3B, respectively, reduced stimulation of ER activity. Hence, both CDA domains are required for modulation of ER activity by A3B, echoing a previous report, which showed that both CDA domains are Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 109 gure 1. A3B Regulates ER Activity to Promote Breast Cancer Cell Growth Kaplan-Meier plots of breast cancer survival for ER+ patients from METABRIC, according to A3B expression. Forest plot analysis of ER+ breast cancers for A3B expression. Affymetrix microarray datasets that included >100 ER+ breast cancers were used alysis: E-MTAB-365 (Guedj et al., 2012), GSE7390 (Desmedt et al., 2007), GSE3494 (Miller et al., 2005), GSE21653 (Sabatier et al., 2011), GSE2034 (Wang 05), and GSE12093 (Zhang et al., 2009). Hazard ratios and 95% conﬁdence intervals are plotted on the x axis. Real-time RT-PCR was carried out using RNA prepared from ER+ (n = 92) and ER (n = 49) breast cancers. Also shown are the expression proﬁles fo east cancer cell lines, with MCF7 and T47D expression highlighted. MCF7 tumors were treated weekly with vehicle (n = 6), control siRNA (n = 6), or A3B siRNA (n = 6). Mean tumor volumes are plotted ± SEM. RNA and p re prepared from tumors at the end of the experiment. Real-time RT-PCR relative to GAPDH levels (n = 3; p < 0.0001) for tumors. Immunoblotting of protein lysates (20 g) tumors is shown Figure 1. A3B Regulates ER Activity to Promote Breast Cancer Cell Growth g g (A) Kaplan-Meier plots of breast cancer survival for ER+ patients from METABRIC, according to A3B expression. (B) Forest plot analysis of ER+ breast cancers for A3B expression. Affymetrix microarray datasets that included >100 ER+ breast cancers were used in the analysis: E-MTAB-365 (Guedj et al., 2012), GSE7390 (Desmedt et al., 2007), GSE3494 (Miller et al., 2005), GSE21653 (Sabatier et al., 2011), GSE2034 (Wang et al., 2005), and GSE12093 (Zhang et al., 2009). Hazard ratios and 95% conﬁdence intervals are plotted on the x axis. A3B Is Recruited Globally to ER Binding Regions, and This Requires Its Interaction with the ER (C) Real-time RT-PCR was carried out using RNA prepared from ER+ (n = 92) and ER (n = 49) breast cancers. Also shown are the expression proﬁles for ER+ breast cancer cell lines, with MCF7 and T47D expression highlighted. (D) MCF7 tumors were treated weekly with vehicle (n = 6), control siRNA (n = 6), or A3B siRNA (n = 6). Mean tumor volumes are plotted ± SEM. RNA and protein d f t t th d f th i t ( ) g p y ( mg) (G) Hormone-depleted T47D cells transfected with A3B siRNAs were assessed for growth (n = 4). (H) mRNA levels were determined following transfection of hormone-depleted T47D cells with A3B siRNAs. mRNA expression is shown relative to the expression for the siControl samples (n = 3). Statistical signiﬁcance within each treatment group for each A3B siRNA relative to siControl is denoted by asterisks (p < 0.001). 110 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors Figure 2. A3B Interacts with ER and Is Recruited to ER Binding Regions (A) Schematic representation of A3B. Highlighted are the catalytic site mutations used here. (B) COS-1 cells cultured in hormone-free medium were transfected with an estrogen-responsive luciferase reporter gene, ER and A3B. Estrogen (10 nM added for 20 hr. Data are shown as fold activation, relative to reporter activity for vehicle treatment, following co-transfection of ER without A3B (vector co (n = 3; * = p < 0.05). Immunoblotting for ER and A3B following transfection of COS-1 cells is also shown. (C) Hormone-depleted MCF7 cells treated with estrogen were immunoprecipitated with an ER antibody. Input represents 10% of lysate used in the imm precipitations. (legend continued on next p Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors Figure 2. A3B Interacts with ER and Is Recruited to ER Binding Regions g g g (A) Schematic representation of A3B. Highlighted are the catalytic site mutations used here. (A) Schematic representation of A3B. Highlighted are the catalytic site mutations used here. (B) COS-1 cells cultured in hormone-free medium were transfected with an estrogen-responsive luciferase reporter gene, ER and A3B. Estrogen (10 nM) was added for 20 hr. Data are shown as fold activation, relative to reporter activity for vehicle treatment, following co-transfection of ER without A3B (vector control) (n = 3; * = p < 0.05). A3B Is Recruited Globally to ER Binding Regions, and This Requires Its Interaction with the ER Immunoblotting for ER and A3B following transfection of COS-1 cells is also shown. (C) Hormone-depleted MCF7 cells treated with estrogen were immunoprecipitated with an ER antibody. Input represents 10% of lysate used in the immuno- precipitations. (A) Schematic representation of A3B. Highlighted are the catalytic site mutations used here. (B) COS-1 cells cultured in hormone-free medium were transfected with an estrogen-responsive luciferase reporter gene, E added for 20 hr. Data are shown as fold activation, relative to reporter activity for vehicle treatment, following co-transfection (n = 3; * = p < 0.05). Immunoblotting for ER and A3B following transfection of COS-1 cells is also shown. 3 g g ed a e e ca a y c s e u a o s used e e mone-free medium were transfected with an estrogen-responsive luciferase reporter gene, ER and A3B. Estrogen (10 nM) was as fold activation, relative to reporter activity for vehicle treatment, following co-transfection of ER without A3B (vector control) tting for ER and A3B following transfection of COS-1 cells is also shown. (legend continued on next page) Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 111 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 111 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 111 nevertheless prevented. By contrast, siRNA-mediated A3B knockdown did not affect ER recruitment (Figure S3E). Thus, although A3B interacts with ER in a ligand-independent manner, its recruitment to chromatin is estrogen dependent by virtue of estrogen-stimulated recruitment of ER to chromatin. Further- more, given that A3B is not required for ER recruitment to DNA, A3B is unlikely to act as a pioneer factor. enzymatically active and contribute to C-to-T editing (Bogerd et al., 2007). A3B was co-immunoprecipitated with ER in MCF7 cells (Figure 2C), and chromatin immunoprecipitation (ChIP) as- says showed that A3B is recruited, in an estrogen-dependent manner, to the ER binding regions in the TFF1 and GREB1 genes (Figures 2D and S3A). AIB1, which interacts with ER in an estro- gen-dependent manner (Anzick et al., 1997), acts as a control. g p ( , ), ChIP sequencing (ChIP-seq), for A3B to deﬁne the global dis- tribution of A3B on chromatin, identiﬁed 24,486 binding sites in MCF7 cells treated with estrogen (Figures 2E and S3B). A3B binding was primarily observed at intronic and gene-distal re- gions (Figure S3C), suggesting that A3B binding occurs in gene regulatory regions. (D) MCF7 cells were treated with estrogen (10 nM, 45 min), followed by ChIP. Shown are the results of real-time PCR of ChIP DNA for the TFF1 gene promoter proximal ERE or a control region in the TFF1 gene to which ER binding is not observed (n = 3). (E) A3B ChIP-seq was carried out following treatment of MCF7 cells with 10 nM estrogen for 45 min. The Venn diagram shows A3B binding regions from the peak- calling analysis. 2 A3B Is Recruited Globally to ER Binding Regions, and This Requires Its Interaction with the ER ChIP-seq for ER has established that the great majority of ER binding events also map to sites within introns and at considerable distances upstream and down- stream of transcribed regions (Welboren et al., 2009). Analysis of the raw reads demonstrated a remarkably close genomic co-localization of the binding sites for the two proteins (r2 = 0.96) (Figure 2F). A3B binding sites were signiﬁcantly enriched in the vicinity of estrogen-responsive genes (Figure 2G) and were enriched in binding motifs for ER (Figure 2H; Table S1). Alignment of all ER binding sites showed that the majority of ER binding sites in MCF7 cells are bound by A3B and that estro- gen treatment results in global stimulation of A3B recruitment to ER binding regions (Figures 2I and 2J), as evident from the genome browser snapshots for the ER target genes TFF1, GREB1, FOS, and CTSD (Figure 2K). These results provide a compelling argument for a mechanism of chromatin-based collaboration between A3B and ER, toward the global regulation of ER target genes in breast cancer. A3B Promotes Cytidine Deamination to Generate C-to-U Transitions at ER Binding Regions in Breast Cancer Cells Mutational inactivation of the A3B catalytic domains inhibited ER stimulation in reporter gene assays (Figure 2A), implying that deamination of deoxycytidine to deoxyuridine (C to U) is required for the regulation of estrogen-responsive gene expression by A3B. We used differential DNA denaturation PCR (3D-PCR), which identiﬁes C-to-T changes, based on detecting PCR ampli- cons at lower denaturation temperatures arising from an increase in A/T content (Burns et al., 2013a; Suspe` ne et al., 2005), to deter- mine if A3B causes C-to-U changes at ER binding regions. Estro- gen treatment of MCF7 cells generated lower-temperature amplicons in the TFF1 promoter region (Figure 3A). Cloning and sequencing of the PCR products from three independent exper- iments identiﬁed C-to-T changes in a total of 12/109 (11%) clones from vehicle-treated cells, increasing to 43/114 (38%) following estrogen treatment (Figure 3B). Importantly, the overwhelming majority (37/43 [86%]) of these changes mapped to the A3B bind- ing region (Figure 3C). Sequencing of 3D-PCR products following A3B knockdown identiﬁed C-to-T changes in a total of 58/155 (37%) clones in siControl-transfected, compared with 13/163 (8%) clones in siA3B-transfected, MCF7 cells (Figures 3D and 3E), demonstrating that A3B is necessary for the C-to-U transi- tions as the TFF1 ER/A3B binding region. Similar results were obtained for the PDZK1 ER/A3B binding region, if A3B was knocked down in T47D cells (Figures S4A–S4E). Recovering the chromatin following ChIP for ER and perform- ing ChIP with A3B antibody (ChIP/reChIP) showed that ER and A3B are present concurrently at the TFF1 ERE (Figure 2L). ChIP for A3B followed by reChIP for ER provided similar results. Treatment of MCF7 cells with the anti-estrogen fulvestrant (aka ICI182,780), which speciﬁcally promotes the downregulation of ER protein (McClelland et al., 1996), resulted in ER loss and lack of ER binding to chromatin in ChIP assays (Figures 2L and S3D). Fulvestrant did not affect A3B protein levels, but A3B recruitment to the TFF1 and PDZK1 ER binding regions was Failure to repair cytidine deamination would result in the accu- mulation of deleterious mutations at gene enhancers. As dU is excised by uracil DNA glycosylase (UNG) (Stavnezer, 2011), we determined if UNG is required for the A3B-dependent cytidine deamintion at ER binding regions. UNG knockdown (F) Genome-wide enrichment correlation analysis for A3B and ER raw signals demonstrates correlation (r2 = 0.96) between A3B and ER binding sites. A3B Promotes Cytidine Deamination to Generate C-to-U Transitions at ER Binding Regions in Breast Cancer Cells A3B and ER ChIP-seq reads were normalized (wig ﬁle) and binned in windows of 100 kb where the average score was calculated. The data were then used to calculate genome-wide correlation using a Spearman’s correlation score. The score for each window is plotted and an interpolation line added to ease interpretation. (G) Analysis of A3B binding regions identiﬁed by ChIP-seq are signiﬁcantly enriched in the proximity of estrogen responsive genes. (H) Analysis of the relative enrichment of transcription factor binding sites was used to generate a Z score, which is represented by the size of the motif in the word cloud. (I) Average signal intensity of A3B ChIP-seq binding events, centered on ER binding regions, shows increased recruitment of A3B to ER binding regions globally, upon estrogen treatment. Signal intensity is a normalized count of individual, non-redundant ChIP fragments at single ER binding sites identiﬁed by the peak- calling algorithm (MACS 1.4) (J) Heatmap showing clustered binding signal for A3B ± estrogen. The window represents ±2.5-kb regions from the center of the ER binding events. The color scale represents relative enrichment based on raw signal. (K) Representative genome browser snapshots show A3B binding regions and overlap with ER binding regions, shown on the same scale. (L) ChIP with ER or A3B antibodies (denoted by ) was followed by recovery of the chromatin complexes and reChIP with antibodies for A3B, ER, or mouse immunoglobulins (IgG control) (n = 3). g ( g ) ( ) (M) MCF7 cells were treated with fulvestrant for 24 hr, followed by addition of estrogen for 45 min (n = 3). 112 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors igure 3. Estrogen Treatment Induces A3B-Dependent C-to-U Transitions at the ER and A3B Binding Region in the TFF1 Gene A) Genomic DNA was prepared from MCF7 cells treated with 10 nM estrogen for 45 min. Agarose gel analysis of 3D-PCR for the TFF1 promoter region is show B) Mutation analysis of 3D-PCR amplicons from vehicle- and estrogen-treated cells is represented as a percentage of clones harboring C-to-T changes. Clone om three independent experiments were sequenced, for a total of >100 clones per treatment. C) Each identiﬁed C-to-T transition is shown (blue dots), mapped to the region of the TFF1 gene (520 to +80) ampliﬁed by 3D-PCR. D) MCF7 cells transfected with A3B or control siRNA were treated with estrogen as described above. A3B Promotes Cytidine Deamination to Generate C-to-U Transitions at ER Binding Regions in Breast Cancer Cells E) 3D-PCR amplicons were cloned. Shown are the percentage of clones harboring C-to-T transitions for a total of >150 clones generated from three experiment en Treatment Induces A3B-Dependent C-to-U Transitions at the ER and A3B Binding Region in the TFF1 Gene (E) 3D-PCR amplicons were cloned. Shown are the percentage of clones harboring C-to-T transitions for a total of >150 clones generated from three experiments. (F) 3D-PCR of genomic DNA from MCF7 cells transfected with UNG siRNA were treated with estrogen. g cells transfected with myc-UGI were treated with estrogen for 12 hr, and RNA and protein were isolated. Real-time PCR and im es are shown. myc-UGI were treated with estrogen for 12 hr, and RNA and protein were isolated. Real-time PCR and immunoblotting for ER (K) Hormone-depleted MCF7 and T47D cells transfected with myc-UGI were grown in the presence or absence of estrogen for 5 days. Growth was measured using the sulforhodamine B (SRB) assay (p < 0.001; n = 4). (K) Hormone-depleted MCF7 and T47D cells transfected with myc-UGI were grown in the presence or absence of estrogen for 5 days. Growth was measured using the sulforhodamine B (SRB) assay (p < 0.001; n = 4). 3J, 3K, and S4G). Finally, ChIP showed that UNG was recruited to ER binding regions in the TFF1 and PDZK1 genes, its recruit- ment being prevented by A3B knockdown (Figures 4A–4C). Thus, UNG is required for ER function and is recruited to ER binding sites in an A3B-dependent manner. Importantly, these results indicate that repair of A3B driven cytidine deamination in- volves the action of UNG. resulted in lower-temperature amplicons at ER/A3B binding re- gions (Figure 3F), suggesting that lack of UNG ‘‘ﬁxes’’ the A3B directed C-to-U changes in DNA. The consequence of UNG knockdown was inhibition of ER target gene expression and MCF7 and T47D cell growth (Figures 3G, 3H, and S4F). We used the bacteriophage PBS2 uracil DNA glycosylase in- hibitor (UGI), which represses UNG activity in mammalian cells (Burns et al., 2013a), to conﬁrm the involvement of UNG. In a re- porter gene assay, ectopic expression of UGI prevented the stimulation of ER activity by A3B (Figure 3I). UGI transfection also repressed endogenous ER target gene expression and estrogen-stimulated growth of MCF7 and T47D cells (Figures Estrogen Binding to ER Promotes DNA Strand Breaks at ER Binding Regions The mechanism of immunoglobulin gene class switch recombi- nation involves cytidine deamination by AID and subsequent Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 113 Figure 4. Estrogen Treatment of MCF7 Cells Induces DSBs at ER Binding Regions Hormone-depleted MCF7 cells were used in all experiments. Figure 4. Estrogen Treatment of MCF7 Cells Induces DSBs at ER Binding Regions Hormone-depleted MCF7 cells were used in all experiments. p (A and B) ChIP for A3B, UNG, DNA-PKcs, and Ku70 was performed following estrogen addition to MCF7 cells transfected with siA3B or siControl. Real-time PCR was performed on recovered DNA, using primers ﬂanking the ER binding regions in TFF1 and PDZK1 genes (n = 3, p < 0.001). (C) A3B mRNA levels by real-time PCR for samples used above. (C) A3B mRNA levels by real-time PCR for samples used above. (D) Estrogen was added and cells were immuno- stained for gH2AX. Nuclei were visualized with the TOPRO DNA stain. (E) gH2AX in 100 cells from ﬁve replicates (total n = 500) was quantiﬁed using Cell Proﬁler 2.0. (F) OHT or FUL was added for 1 hr, followed by addition of estrogen (E2) for 10 min. Boxplots show the mean gH2AX foci number in 100 cells (n = 5) (E) gH2AX in 100 cells from ﬁve replicates (total n = 500) was quantiﬁed using Cell Proﬁler 2.0. (F) OHT or FUL was added for 1 hr, followed by addition of estrogen (E2) for 10 min. Boxplots show the mean gH2AX foci number in 100 cells (n = 5). (G) gH2AX ChIP (MCF7) and real-time PCR for the TFF1 ER binding site or promoter regions of the SCN2A1 and RPL13A genes (n = 3). (G) gH2AX ChIP (MCF7) and real-time PCR for the TFF1 ER binding site or promoter regions of the SCN2A1 and RPL13A genes (n = 3). (H) Venn diagram of gH2AX, A3B, and ER binding events from ChIP-seq experiments for estrogen- treated cells. Individual peaks were identiﬁed us- ing the same peak-calling algorithm (MACS1.4) using identical settings. (I) Analysis of the relative enrichment of tran- scription factor binding sites was used to generate a Z score, which is represented by the size of the motif in the word cloud. (J) Genome browser snapshots of gH2AX ChIP- seq in MCF7 cells treated with estrogen, H2O2, or vehicle. Estrogen Binding to ER Promotes DNA Strand Breaks at ER Binding Regions Similarly, UNG was required, as its knockdown also inhibited estrogen induction of gH2AX (Figure S5I). Moreover, estrogen induction of gH2AX in MDA-MB-231 cells expressing ER required A3B (Figures S6B and S6C). A3B was also necessary for optimal expression of ER-regulated genes in the ER-expressing MDA-MB-231 lines (Figure S6D). ( g ) Next, we used biotin-16-deoxyuridine triphosphate (dUTP) labeling of DSBs with terminal deoxynucleotide transferase (TdT) (Ju et al., 2006) followed by biotin ChIP and real-time PCR to directly determine if DSBs are formed at ER/A3B binding regions. Real-time PCR using primers located 30 to the region in the TFF1 gene to which the great majority of C-to-U transitions mapped showed 6-fold enrichment over the vehicle control within 10 min of estrogen addition (Figure 5D). DSBs in this re- gion were reduced to basal levels by 60 min, in general agree- ment with the reduction in gH2AX over this time frame. A similar rapid but transient induction of DSBs was observed for other ER target genes, but there were no detectable DSBs at the non-expressed SCN2A1 gene promoter, or at a region 2 kb 50 to the ER binding region in TFF1 (TFF1 control). PCR using primers A/C, which amplify across the region containing the C-to-U transitions failed to show estrogen stimulation of DSBs, indicating that estrogen induces DSBs within the region of the TFF1 gene that is characterized by A3B-mediated C-to-U transitions (Figure 5E). Estrogen induction of DSBs was prevented if the cells were transfected with A3B siRNAs (Figures 5E and 5F). Aligning the gH2AX peaks showed very little enrichment for gH2AX in vehicle-treated cells at ER binding regions (Figures 4K and 4L). H2O2 treatment, which induced gH2AX, also did not result in much enrichment of gH2AX at ER binding regions. Indeed, co-treatment with H2O2 and estrogen reduced gH2AX at ER regions, compared with estrogen alone. Indeed, the major- ity of gH2AX regions induced by estrogen were enriched for ER and A3B binding (Figure S7C). There was some enrichment for ER and A3B sites in gH2AX regions that were common to all treatments. However, there was very little overlap with ER or A3B sites for gH2AX regions present in vehicle treated, or following H2O2 treatment, indicating that estrogen/ER induced gH2AX occurs at sites that are quite distinct from those caused by DNA-damaging agents. Estrogen Binding to ER Promotes DNA Strand Breaks at ER Binding Regions Estrogen treatment stimulated gH2AX at the ER/A3B binding region in the TFF1 gene, but no gH2AX enrichment was observed at the promoter of a gene that is not expressed in MCF7 cells (SCN2A) (Ju et al., 2006), nor was gH2AX enrichment observed at the non-ER target gene RPL13A, which is expressed in MCF7 cells (Figure 4G). We un- dertook gH2AX ChIP-seq to determine the global distribution of gH2AX following estrogen treatment (Figure S7A). Peak calling identiﬁed 17,892 gH2AX binding events in the estrogen-treated samples. Using the deﬁnition that a binding region must overlap by at least one base pair, 54% (9,637/17,892) of gH2AX regions co-localized with A3B and/or ER binding events, with two-thirds (64%, 6,173/9,637) of these regions co-localizing to A3B and ER co-incident binding events (Figure 4H). Motif enrichment anal- ysis conﬁrmed that the gH2AX regions are highly enriched for ER (ESR1) binding motifs (Figure 4I). Correlation coefﬁcient values for the raw sequencing data conﬁrmed the co-localization of gH2AX regions with A3B (r2 = 0.69) and ER (r2 = 0.70) binding events (Figure S7B). Genomic loci exemplifying gH2AX at A3B and ER binding regions are shown in Figure 4J. and A3B binding regions. Estrogen treatment stimulated gH2AX at the ER/A3B binding region in the TFF1 gene, but no gH2AX enrichment was observed at the promoter of a gene that is not expressed in MCF7 cells (SCN2A) (Ju et al., 2006), nor was gH2AX enrichment observed at the non-ER target gene RPL13A, which is expressed in MCF7 cells (Figure 4G). We un- dertook gH2AX ChIP-seq to determine the global distribution of gH2AX following estrogen treatment (Figure S7A). Peak calling identiﬁed 17,892 gH2AX binding events in the estrogen-treated samples. Using the deﬁnition that a binding region must overlap by at least one base pair, 54% (9,637/17,892) of gH2AX regions co-localized with A3B and/or ER binding events, with two-thirds (64%, 6,173/9,637) of these regions co-localizing to A3B and ER co-incident binding events (Figure 4H). Motif enrichment anal- ysis conﬁrmed that the gH2AX regions are highly enriched for ER (ESR1) binding motifs (Figure 4I). Correlation coefﬁcient values for the raw sequencing data conﬁrmed the co-localization of gH2AX regions with A3B (r2 = 0.69) and ER (r2 = 0.70) binding events (Figure S7B). Genomic loci exemplifying gH2AX at A3B and ER binding regions are shown in Figure 4J. TFF1 and PDZK1 genes (Figure 5C). Estrogen Binding to ER Promotes DNA Strand Breaks at ER Binding Regions (J) Genome browser snapshots of gH2AX ChIP- seq in MCF7 cells treated with estrogen, H2O2, or vehicle. (K) Heatmap showing clustered binding signals for regions enriched in gH2AX for all treatment con- ditions. The windows represent ±5.0-kb regions from the center of the ER binding events. The color scale shows relative enrichment based on raw signal. (L) Average signal intensities of gH2AX ChIP-seq binding events centered on ER binding regions are shown for the different treatments. Signal in- tensity is a normalized count of individual, non- redundant ChIP fragments at single ER binding sites identiﬁed with the MACS1.4 peak-calling algorithm. dU excision by UNG results in the generation of DNA strand breaks that can be repaired by the NHEJ pathway (Kotnis et al., 2009; Nowarski et al., 2012). Moreover, high-level A3G expression in leukemia cells promotes DNA strand breaks (Kot- nis et al., 2009; Nowarski et al., 2012). Finally, A3B overexpres- sion promotes gH2AX (Burns et al., 2013a), which is activated at DNA strand breaks (DSB) and is thus a marker for DSB. We reasoned, therefore, that targeted A3B-mediated cytidine deam- ination and dU excision by UNG could promote DSB generation at ER/A3B enhancers. If so, then estrogen treatment of breast cancer cells should be sufﬁcient to cause DSBs in breast cancer cells. Indeed, treatment of MCF7 cells with estrogen induced gH2AX within 10 min (Figures 4D and 4E). gH2AX induction required ER, since synthetic ER ligands also induced gH2AX (Figures S5A and S5B), while treatment with anti-estrogens 4-hy- droxytamoxifen (OHT) or fulvestrant (FUL) prevented estrogen induction of gH2AX (Figures 4F and S5C), as did transfection with ER siRNA (Figure S5D). Estrogen similarly induced gH2AX in an ER-dependent manner in T47D cells (Figures S5E and S5F), but not in the ER- MDA-MB-231 cells (Figure S6A). How- ever, estrogen stimulation of gH2AX was possible in MDA-MB- 231 cells ectopically expressing ER. Estrogen induced gH2AX required DNA-PK and ATM activities and the gH2AX foci co-localized with 53BP1 (Figures S5G and S5H), verifying that estrogen treatment promotes DSBs. We used ChIP for gH2AX to determine if the DSBs localize to ER 114 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors and A3B binding regions. A3B Is Required for Histone Modiﬁcation and Recruitment of Chromatin Remodeling Factors at ER Binding Regions Transcription factors regulate gene expression by promoting the ordered recruitment of diverse complexes that modify and remodel chromatin, leading to transcription initiation. Recent studies show that DNA repair factors regulate gene expression by aiding chromatin remodeling (Fong et al., 2013). A3B knock- down prevented estrogen stimulation of histone modiﬁcations associated with transcription at ER target genes (Figures 6A and 6B). Importantly, H2AX phosphorylation at serine-139 (gH2AX) promotes chromatin recruitment of BRG1, the catalytic subunit of the SWI/SNF ATPase-dependent chromatin remodel- ing complex (Lee et al., 2010). In agreement with a model in which A3B-mediated cytidine deamination leads to H2AX activa- tion, A3B knockdown prevented BRG1 recruitment (Figures 6C and 6D). A3B knockdown also inhibited PolII recruitment to the TFF1 and GREB1 genes. Interestingly, the estrogen-stimulated gH2AX co-localized with activated (phosphorylated) PolII, further evidence that the estrogen/ER and A3B regulated DSB forma- tion occurs of transcriptionally active chromatin. In addition to regulating histone modiﬁcation and recruitment of chromatin re- modelers, A3B was required for PolII recruitment to ER/A3B binding regions (Figures 6E and 6F). The importance of H2AX activation was underscored by the fact that treatment with the DNA-PKcs inhibitor NU7441 or the ATM inhibitor KU55933 inhibited histone modiﬁcation, as well as BRG1 and PolII recruit- ment at ER target genes (Figures 6G–6M). Note that these treat- ments did not affect A3B and ER recruitment. As described above, the greater part of ER and A3B binding occurs at distal regions. Active regulatory regions such as en- hancers and promoters carry speciﬁc epigenetic modiﬁcations including H3K27ac (enhancers and promoters), H3K4me1 (enhancer speciﬁc), and H3K4me3 (promoter speciﬁc) (ENCODE Project Consortium, 2012). Interestingly, A3B was found at 93% of active enhancers and 7% of active promoters (Figure S7D). This was conﬁrmed by strong enrichment for BRD4 and p300, two ubiquitous co-activators found at active regulatory elements (Hnisz et al., 2013). In addition, using recently published GRO- seq data (Hah et al., 2013), we could identify bi-directional tran- scription at a subset of distally bound A3B sites, indicating the possibility of eRNA synthesis at these elements. APOBEC3B Action at ER Target Genes Causes Transient DNA Strand Breaks That Are Repaired by NHEJ APOBEC3B Action at ER Target Genes Causes Transient DNA Strand Breaks That Are Repaired by NHEJ Our results demonstrate that A3B induces C-to-U transitions and promotes UNG, DNA-PK, and Ku70 recruitment to ER binding regions (Figures 4A and 4B). We have also shown that estro- gen/ER rapidly induces gH2AX globally at ER and A3B binding regions. These ﬁndings imply that A3B action is required for DSB generation at ER binding regions. Indeed, A3B knockdown blocked estrogen-induced gH2AX in MCF7 cells (Figures 5A, 5B, and S5D) and prevented gH2AX at ER binding sites in the Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 115 ure 5. Estrogen-Induced DNA Strand Breaks Are Dependent on A3B Hormone-depleted MCF7 cells were used for all experiments. Cells transfected with siA3B or siControl were treated with estrogen. Shown are represen ges for gH2AX staining. The average gH2AX foci number per cell in 100 cells from ﬁve replicates ± SEM re 5. Estrogen-Induced DNA Strand Breaks Are Dependent on A3B ormone-depleted MCF7 cells were used for all experiments. Cells transfected with siA3B or siControl were treated with estrogen. Shown are represen es for gH2AX staining. h H2AX f i b ll i 100 ll f ﬁ li SEM Figure 5. Estrogen-Induced DNA Strand Breaks Are Dependent on A3B (A) Hormone-depleted MCF7 cells were used for all experiments. Cells transfected with siA3B or siControl were treated with estrogen. Shown are representative images for gH2AX staining Figure 5. Estrogen-Induced DNA Strand Breaks Are Dependent on A3B (B) The average gH2AX foci number per cell in 100 cells from ﬁve replicates ± SEM. ( ) y (D) Estrogen was added and cells end labeled by incubation with biotin-16-dUTP in the presence of terminal deoxynucleot performed with a biotin antibody. (E and F) Estrogen was added following siRNA transfections for 10 min. Biotin end labeling was performed as in (D). (C–F) n = 3; p < 0.001; NS, not signiﬁcant. DISCUSSION facilitate the program of estrogen-responsive gene expression (Me´ tivier et al., 2006). There is also gathering evidence that many protein complexes that sense and repair DNA damage are important for regulation of gene expression by ER and other transcription factors, their recruitment aiding chromatin modiﬁ- cation/remodeling to promote gene activation (Fong et al., 2013). For example, the BER protein thymine DNA glycosylase The molecular mechanisms by which ER drives breast cancer has identiﬁed transcription factors that direct ER to active en- hancers (Magnani et al., 2011) and revealed that ER controls the coordinated recruitment of chromatin remodeling and modi- ﬁcation proteins and the transcription machinery that together 116 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors Figure 6. A3B Is Required for Chromatin Remodeling and Activating Histone Modiﬁ- cations at ER Enhancers Figure 6. A3B Is Required for Chromatin Remodeling and Activating Histone Modiﬁ- cations at ER Enhancers All experiments were undertaken with hormone- depleted MCF7 cells. (A and B) ChIP for histone H3 and H3 modiﬁcations associated with gene activation, was performed following estrogen addition to cells transfected with siA3B or control siRNA (n = 3, p < 0.001). (C–E) ChIP for BRG1, ER, and PolII followed by real-time for A3B/ER binding sites in TFF1, PDZK1, and GREB1. acts as a co-activator for ER and promotes recruitment of the p160 co-activators and the CBP/p300 histone acetyltransferase (Lucey et al., 2005; Tini et al., 2002). One reason advanced for the function of DNA repair proteins in gene regulation is that they may aid in relieving torsional stress generated by transcription- induced DNA supercoiling (Ma and Wang, 2014). Movement of RNA polymerase (RNAP) along the DNA template during tran- scription generates over-winding (positive DNA supercoiling) in front and negative DNA supercoiling behind it. Failure to resolve DNA supercoiling will ultimately affect transcription. Additionally, generation of DNA supercoiling at one promoter can affect tran- moted 8-oxoguanine 8-oxoguanine-DNA g changes that facilitat enhancers and promo tion and their resolutio lation of gene express Here, we report an ER-regulated genes i initiated by estrogen importance of A3B in of the exceptionally (F) Cells were treated with estrogen for 10 min and immunostained for gH2AX (green) and PolII (red). (G–M) 5 mM NU7441 (DNA-PKcs inhibitor) or KU55933 (ATM inhibitor) was added for 1 hr fol- lowed by estrogen addition. ChIP was performed as above (n = 3, p < 0.001). DISCUSSION (N) Cells were treated with NU7441 or KU55933 for 1 hr, at which point estrogen was added. Real- time RT-PCR was performed with RNA prepared after 4 hr (n = 3; *p < 0.001). acts as a co-activator for ER and promotes recruitment of the moted 8-oxoguanin scription from a distal promoter, so- called topological promoter coupling (Ma and Wang, 2014). Given the recent identiﬁcation of active transcription at enhancer regions, which generates eRNAs, RNAP procession is also likely to create torsional stress at enhancer regions, which might contribute to inhibition of transcription from coupled gene promoters. Furthermore, regulation of transcription by enhancer regions entails communication between distal enhancers and regulated promoters through chromatin looping, a process that is also inﬂuenced by DNA supercoil- ing (Kulaeva et al., 2012). Thus, DNA topoisomerases, which relax negative and positive DNA supercoils, are impor- tant in transcription regulation. Indeed, ER- and androgen receptor (AR)-induced transcription in breast and prostate can- cer involves transient DSBs generated by TOP1 and TOP2 (Ju et al., 2006; Puc et al., 2015). Activation of the LSD1 his- tone demethylase by ER binding pro- e modiﬁcation of DNA and recruitment of glycosylase and BER to stimulate chromatin te interaction between ER-regulated gene oters (Perillo et al., 2008). Thus, DSB forma- ion is an important component of the regu- ssion by of ER. scription from a distal promoter, so- called topological promoter coupling (Ma and Wang, 2014). Given the recent identiﬁcation of active transcription at enhancer regions, which generates eRNAs, RNAP procession is also likely to create torsional stress at enhancer regions, which might contribute to inhibition of transcription from coupled gene promoters. Furthermore, regulation of transcription by enhancer regions entails communication between distal enhancers and regulated promoters through chromatin looping, a process that is also inﬂuenced by DNA supercoil- ing (Kulaeva et al., 2012). Thus, DNA topoisomerases, which relax negative and positive DNA supercoils, are impor- tant in transcription regulation. Indeed, ER- and androgen receptor (AR)-induced transcription in breast and prostate can- cer involves transient DSBs generated by TOP1 and TOP2 (Ju et al., 2006; Puc et al., 2015). Activation of the LSD1 his- tone demethylase by ER binding pro- moted 8-oxoguanine modiﬁcation of DNA and recruitment of 8-oxoguanine-DNA glycosylase and BER to stimulate chromatin changes that facilitate interaction between ER-regulated gene enhancers and promoters (Perillo et al., 2008). DISCUSSION Importantly, proteins that bind to other CpG modiﬁcations, including 5-hydroxymethlycytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC) are now being identiﬁed and include not only DNA repair factors but also chromatin regulators and transcription factors (Iurlaro et al., 2013; Spruijt et al., 2013). Our ﬁndings allow that there may be transcription regulatory proteins that interact with DNA containing dU and that are therefore recruited upon A3B-medi- ated cytidine deamination at gene enhancers, an intriguing pos- sibility that may deserve further investigation. Recent studies show that cytidine deamination is an important feature of the mutational landscape in breast (Alexandrov et al., 2013; Burns et al., 2013a; Nik-Zainal et al., 2012), ovarian (Leo- nard et al., 2013), lung (de Bruin et al., 2014), and other cancers (Burns et al., 2013b; Roberts et al., 2013), with high-level expres- sion of A3B, and experimental studies indicate that A3B may be a key driver of such mutational signatures (Burns et al., 2013a; Taylor et al., 2013). It is tempting to speculate that the cytidine- deaminase-associated mutational landscapes in breast cancer might be enriched at ER/A3B binding regions. In support of this possibility, AR was shown to promote DNA DSBs to aid intra- and inter-chromosomal translocations in prostate cancer cells, one mechanism for which involved the hormone-depen- dent recruitment of AID (Lin et al., 2009). Determination of the global ER binding proﬁles by ChIP-seq analysis of breast tumors has shown that there is a high level of plasticity in ER binding in breast cancer (Ross-Innes et al., 2012), such that investigation of any association between A3B/ER binding regions and somatic mutations may entail whole-genome sequencing, coupled with ER and A3B ChIP-seq in the same tumor. Interestingly, however, gene regulatory regions can be characterized by low levels of somatic mutations in cancer in the absence of additional defects in DNA repair (Polak et al., 2014). Our results suggest that activation of DNA repair pathways may protect enhancer regions from A3B-dependent mutagenesis. It would be important there- fore to investigate the involvement of defects in BER and/or NHEJ in cancer mutational landscapes that have been ascribed to A3B. Figure 7. Model for A3B-Mediated Activation of ER Enhancers toward Regulation of ER Target Gene Expression Estrogen binding to ER promotes its recruitment to ER enhancers. A3B, re- cruited to these regions through interaction with ER, provides enzymatic conversion of C to U. DISCUSSION Generation of U:G mismatches promotes DNA nicks through the action of UNG and AP endonuclease, resulting in DNA cleavage and repair by the non-homologous end-joining DNA repair pathway. Induction of transient C-to-U changes stimulates chromatin modiﬁcation and remodel- ing and PolII recruitment to facilitate expression of ER target genes. and A3B binding regions. Moreover, we show that estrogen treatment causes rapid induction of DSBs, as demonstrated by gH2AX activation. Importantly, the majority of estrogen-induced gH2AX occurs at ER and A3B binding regions, and we have shown that gH2AX induction is dependent on A3B. Our work demonstrates that A3B directs cytidine deamination at ER bind- ing regions to facilitate DSBs through activation of BER and sub- sequent repair of these lesions by the NHEJ pathway. The critical role of A3B action in the regulation of gene expression by ER is established by its requirement for breast cancer cell growth in vitro and in vivo. Based on these ﬁndings, we propose that the A3B-mediated generation of C-to-U changes and activation of DNA repair pathways facilitates chromatin remodeling and enhancer/promoter interaction. In support of this, A3B is required for SWI/SNF recruitment, activating histone modiﬁca- tions and PolII recruitment to ER binding regions (a schematic model is shown in Figure 7). and A3B binding regions. Moreover, we show that estrogen treatment causes rapid induction of DSBs, as demonstrated by gH2AX activation. Importantly, the majority of estrogen-induced gH2AX occurs at ER and A3B binding regions, and we have shown that gH2AX induction is dependent on A3B. Our work demonstrates that A3B directs cytidine deamination at ER bind- ing regions to facilitate DSBs through activation of BER and sub- sequent repair of these lesions by the NHEJ pathway. The critical role of A3B action in the regulation of gene expression by ER is established by its requirement for breast cancer cell growth in vitro and in vivo. Based on these ﬁndings, we propose that the A3B-mediated generation of C-to-U changes and activation of DNA repair pathways facilitates chromatin remodeling and enhancer/promoter interaction. In support of this, A3B is required for SWI/SNF recruitment, activating histone modiﬁca- tions and PolII recruitment to ER binding regions (a schematic model is shown in Figure 7). DISCUSSION Thus, DSB forma- tion and their resolution is an important component of the regu- lation of gene expression by of ER. acts as a co-activator for ER and promotes recruitment of the p160 co-activators and the CBP/p300 histone acetyltransferase (Lucey et al., 2005; Tini et al., 2002). One reason advanced for the function of DNA repair proteins in gene regulation is that they may aid in relieving torsional stress generated by transcription- induced DNA supercoiling (Ma and Wang, 2014). Movement of RNA polymerase (RNAP) along the DNA template during tran- scription generates over-winding (positive DNA supercoiling) in front and negative DNA supercoiling behind it. Failure to resolve DNA supercoiling will ultimately affect transcription. Additionally, generation of DNA supercoiling at one promoter can affect tran- Here, we report an alternative mechanism for transcription of ER-regulated genes in which DSBs are generated in a process initiated by estrogen-ER-dependent recruitment of A3B. The importance of A3B in ER action is implied by our observation of the exceptionally high genome-wide co-localization of ER Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 117 Figure 7. Model for A3B-Mediated Activation of ER Enhancers toward Regulation of ER Target Gene Expression Estrogen binding to ER promotes its recruitment to ER enhancers. A3B, re- cruited to these regions through interaction with ER, provides enzymatic conversion of C to U. Generation of U:G mismatches promotes DNA nicks through the action of UNG and AP endonuclease, resulting in DNA cleavage and repair by the non-homologous end-joining DNA repair pathway. Induction of transient C-to-U changes stimulates chromatin modiﬁcation and remodel- ing and PolII recruitment to facilitate expression of ER target genes. scription factors may limit gH2AX accumulation and spreading to check retention of DNA repair factors and thus control the extent of the DDR response. In agreement with this model, DNA DSB-induced H2AX phosphorylation is spread over large domains around the DSB (Iacovoni et al., 2010). This contrasts with the restricted gH2AX distribution induced by A3B recruit- ment to ER binding regions observed here. Thus, the mode of ATM and DNA-PKcs recruitment appears to determine the effect of DSBs on transcription. Proteins that preferentially bind to DNA sequences containing methylcytosine (mC) to regulate chromatin and control gene expression are well described. 118 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors Cell Lines, Plasmids, Antibodies, and Real-Time RT-PCR Assays Cell Lines, Plasmids, Antibodies, and Real-Time RT-PCR Assays Cell lines were obtained from and cultured in media recommended by ATCC. MCF7, T47D, SkBr3, COS-1 and HeLa cells were grown in DMEM containing 10% fetal calf serum (FCS). MDA-MB-231 cells stably expressing ER have been described (Bhat-Nakshatri et al., 2004). Hormone depletion was achieved by culturing cells for 72 hr in DMEM lacking phenol red and containing 5% dextran-coated charcoal-stripped FCS. Plasmids, antibodies, and real-time PCR primers are detailed in the Supplemental Experimental Procedures. DISCUSSION Interestingly, DNA damage by irradiation or with the use of models in which DNA DSBs are locally induced with restriction enzymes such as I-SceI has demonstrated that ATM promotes dynamic chromatin condensation and transcriptional silencing at DSBs (Khurana et al., 2014; Shanbhag et al., 2010). DNA- PKcs can repress transcription (Pankotai et al., 2012), but its recruitment to transcription-factor-promoted DSBs stimulates transcription (Ju et al., 2006). As proposed by Tjian and col- leagues (Fong et al., 2013), transient DSB generation by tran- In summary, our results identify an important role for A3B as a regulator of ER-mediated gene expression in breast cancer, with potential as a therapeutic target in ER+ breast cancer. To advance this possibility, it will be important to extend our ﬁndings to studies that identify global A3B regulated genes in breast can- cer cell lines, as well as in tumor samples. Moreover, as A3B is widely expressed in other cancers, it is likely that A3B inhibition represents an important therapeutic approach to inhibit regu- lated transcription in other cancer types. 118 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors Reporter Gene Assays Hormone-depleted COS-1 cells were transfected with ERE3-TATA-luc, pRL- TK, together with ER and hemagglutinin (HA)-tagged A3B. Estrogen (10 nM) or an equal volume of ethanol (vehicle) was added 5 hr following transfection. Transfection methodology and luciferase measurements were performed as described previously (Lai et al., 2013). Reporter gene assays in HeLa cells following transfection with ER, A3B, and myc-UGI-NLS were undertaken as above. Biotin Labeling of DSB Biotin labeling was performed as described previously (Ju et al., 2006) and detailed in the Supplemental Experimental Procedures. REFERENCES Immunoprecipitations were performed as described previously (Lopez-Garcia et al., 2006). Alexandrov, L.B., Nik-Zainal, S., Wedge, D.C., Aparicio, S.A., Behjati, S., Bian- kin, A.V., Bignell, G.R., Bolli, N., Borg, A., Børresen-Dale, A.L., et al.; Australian Pancreatic Cancer Genome Initiative; ICGC Breast Cancer Consortium; ICGC MMML-Seq Consortium; ICGC PedBrain (2013). Signatures of mutational pro- cesses in human cancer. Nature 500, 415–421. MCF7 Human Tumor Xenografts We are grateful to M.-A. Langlois and the late M. Neuberger for helpful discus- sions and plasmids. We thank B. Cullen and H. Weingard for APOBEC plas- mids and J. Cohen for UGI. We also thank A. Ashworth and P. Edwards for breast cancer cell lines and H. Nakshatri for MDA-MB-231-ER cells. For their help, we thank R. Kerkhoven and L. Game (Solexa sequencing) and K.I. Hng (microscopy). This work was funded by CRUK and Breast Cancer Now. N.N. is funded by the MRC. 10 mM siRNA prepared with the atelogene in vivo siRNA transfection kit (Koken, Japan) was injected weekly directly into tumors. Tumor volumes were determined twice weekly. At the end of the experiment, protein lysates were prepared from half of each tumor by homogenization in RIPA buffer. RNA was prepared from the remaining halves of each tumor using the RNAeasy kit (QIAGEN). The study was undertaken under the auspices of a UK Home Ofﬁce project license, using approved procedures. Received: May 14, 2015 Revised: July 16, 2015 Accepted: August 24, 2015 Published: September 24, 2015 Received: May 14, 2015 Revised: July 16, 2015 Accepted: August 24, 2015 Published: September 24, 2015 Received: May 14, 2015 Revised: July 16, 2015 ACCESSION NUMBERS The accession numbers for the ChIP-seq data reported in this paper are GEO: GSE56979 (A3B ChIP-seq) and GEO: GSE57426 (gH2AX ChIP-seq). ChIP ChIP was performed as described previously (Lai et al., 2013), using 10 mg of antibody and 100 ml of Protein A Dynalbeads (10002D; Invitrogen). Control ChIP was performed by the addition of mouse immunoglobulins (IgG). Bhat-Nakshatri, P., Campbell, R.A., Patel, N.M., Newton, T.R., King, A.J., Marshall, M.S., Ali, S., and Nakshatri, H. (2004). Tumour necrosis factor and PI3-kinase control oestrogen receptor alpha protein level and its transrepres- sion function. Br. J. Cancer 90, 853–859. 3D-PCR, Cloning, and Sequencing 3D-PCR was carried out as described elsewhere (Suspe` ne et al., 2005). Genomic DNA was prepared using the Invitrogen genomic extraction kit. Products from the second round PCR were puriﬁed using the QIAGEN PCR puriﬁcation kit, cloned into the TOPO-TA cloning vector (Invitrogen), and sequenced with the T7 sequencing primer. SUPPLEMENTAL INFORMATION Cells were transfected with siRNA using Lipofectamine RNAiMax (Invitrogen). For collecting RNA and protein, 10 nM estrogen was added after 48 hr; RNA and protein lysates were prepared after a further 12 hr. Cell growth was deter- mined using the sulforhodamine B (SRB) assay, as described previously (Lai et al., 2013). Details of siRNAs are provided in the Supplemental Experimental Procedures. Supplemental information includes Supplemental Experimental Procedures, seven ﬁgures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2015.08.066. Gene Expression Total RNA was prepared and real-time RT-PCR was performed as described previously (Ngan et al., 2009), using TaqMan gene expression assays from ABI. Accepted: August 24, 2015 Accepted: August 24, 2015 Published: September 24, 2015 Published: September 24, 2015 Immunoﬂuorescence Cells were cultured on glass coverslips in phenol red-free DMEM containing 5% double charcoal-stripped FCS for 3 days before the addition of ligands. Cells were ﬁxed and incubated with antibodies, as described in the Supple- mental Experimental Procedures. Images were acquired using a Zeiss LSM510 confocal microscope. Images were analyzed using Fuji Image J (NIH) and CellProﬁler (Broad Institute) for quantiﬁcation of staining. Ali, S., and Coombes, R.C. (2002). Endocrine-responsive breast cancer and strategies for combating resistance. Nat. Rev. Cancer 2, 101–112. Anzick, S.L., Kononen, J., Walker, R.L., Azorsa, D.O., Tanner, M.M., Guan, X.Y., Sauter, G., Kallioniemi, O.P., Trent, J.M., and Meltzer, P.S. (1997). AIB1, a steroid receptor coactivator ampliﬁed in breast and ovarian cancer. Science 277, 965–968. EXPERIMENTAL PROCEDURES number of reads obtained, percentage of reads aligned, peaks called, and additional analysis methods are detailed in the Supplemental Experimental Procedures. ChIP and Solexa Sequencing Transcription 5, e28636. Magnani, L., Eeckhoute, J., and Lupien, M. (2011). Pioneer factors: directing transcriptional regulators within the chromatin environment. Trends Genet. 27, 465–474. Hah, N., Murakami, S., Nagari, A., Danko, C.G., and Kraus, W.L. (2013). Enhancer transcripts mark active estrogen receptor binding sites. Genome Res. 23, 1210–1223. McClelland, R.A., Gee, J.M., Francis, A.B., Robertson, J.F., Blamey, R.W., Wakeling, A.E., and Nicholson, R.I. (1996). Short-term effects of pure anti- oestrogen ICI 182780 treatment on oestrogen receptor, epidermal growth factor receptor and transforming growth factor-alpha protein expression in human breast cancer. Eur. J. Cancer 32A, 413–416. Hnisz, D., Abraham, B.J., Lee, T.I., Lau, A., Saint-Andre´ , V., Sigova, A.A., Hoke, H.A., and Young, R.A. (2013). Super-enhancers in the control of cell identity and disease. Cell 155, 934–947. Iacovoni, J.S., Caron, P., Lassadi, I., Nicolas, E., Massip, L., Trouche, D., and Legube, G. (2010). High-resolution proﬁling of gammaH2AX around DNA dou- ble strand breaks in the mammalian genome. EMBO J. 29, 1446–1457. Me´ tivier, R., Reid, G., and Gannon, F. (2006). Transcription in four dimensions: nuclear receptor-directed initiation of gene expression. EMBO Rep. 7, 161–167. Iurlaro, M., Ficz, G., Oxley, D., Raiber, E.A., Bachman, M., Booth, M.J., Andrews, S., Balasubramanian, S., and Reik, W. (2013). A screen for hydrox- ymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 14, R119. Miller, L.D., Smeds, J., George, J., Vega, V.B., Vergara, L., Ploner, A., Pawitan, Y., Hall, P., Klaar, S., Liu, E.T., and Bergh, J. (2005). An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival. Proc. Natl. Acad. Sci. USA 102, 13550–13555. Ju, B.G., Lunyak, V.V., Perissi, V., Garcia-Bassets, I., Rose, D.W., Glass, C.K., and Rosenfeld, M.G. (2006). A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription. Science 312, 1798–1802. Nabel, C.S., and Kohli, R.M. (2011). Molecular biology. Demystifying DNA de- methylation. Science 333, 1229–1230. Ngan, S., Stronach, E.A., Photiou, A., Waxman, J., Ali, S., and Buluwela, L. (2009). Microarray coupled to quantitative RT-PCR analysis of androgen-regu- lated genes in human LNCaP prostate cancer cells. Oncogene 28, 2051–2063. Kamileri, I., Karakasilioti, I., and Garinis, G.A. (2012). Nucleotide excision repair: new tricks with old bricks. Trends Genet. 28, 566–573. Khurana, S., Kruhlak, M.J., Kim, J., Tran, A.D., Liu, J., Nyswaner, K., Shi, L., Jailwala, P., Sung, M.H., Hakim, O., and Oberdoerffer, P. (2014). ChIP and Solexa Sequencing ChIP DNA was ampliﬁed as described (Schmidt et al., 2009). Sequences were generated by the Illumina Hiseq 2000 genome analyzer (using 50 bp reads) and aligned to the Human Reference Genome (assembly hg19, February 2009) using Bowtie 1.0. The model-based analysis for ChIP-seq (MACS) peak caller version 1.4 (Zhang et al., 2008) was used to identify enriched regions of the genome by comparison to an input sample. MACS was used in the default setting with a p value threshold of 105. To call stimuli speciﬁc peaks, we used bedtool to subtract or concatenate BED ﬁles generated by MACS. The Bhutani, N., Burns, D.M., and Blau, H.M. (2011). DNA demethylation dynamics. Cell 146, 866–872. Bogerd, H.P., Wiegand, H.L., Doehle, B.P., and Cullen, B.R. (2007). The intrinsic antiretroviral factor APOBEC3B contains two enzymatically active cytidine deaminase domains. Virology 364, 486–493. Burns, M.B., Lackey, L., Carpenter, M.A., Rathore, A., Land, A.M., Leonard, B., Refsland, E.W., Kotandeniya, D., Tretyakova, N., Nikas, J.B., et al. (2013a). Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 119 APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494, 366–370. Kulaeva, O.I., Nizovtseva, E.V., Polikanov, Y.S., Ulianov, S.V., and Studitsky, V.M. (2012). Distant activation of transcription: mechanisms of enhancer ac- tion. Mol. Cell. Biol. 32, 4892–4897. Burns, M.B., Temiz, N.A., and Harris, R.S. (2013b). Evidence for APOBEC3B mutagenesis in multiple human cancers. Nat. Genet. 45, 977–983. Lai, C.F., Flach, K.D., Alexi, X., Fox, S.P., Ottaviani, S., Thiruchelvam, P.T., Kyle, F.J., Thomas, R.S., Launchbury, R., Hua, H., et al. (2013). Co-regulated gene expression by oestrogen receptor a and liver receptor homolog-1 is a feature of the oestrogen response in breast cancer cells. Nucleic Acids Res. 41, 10228–10240. Compe, E., and Egly, J.M. (2012). TFIIH: when transcription met DNA repair. Nat. Rev. Mol. Cell Biol. 13, 343–354. Conticello, S.G. (2008). The AID/APOBEC family of nucleic acid mutators. Genome Biol. 9, 229. Landry, S., Narvaiza, I., Linfesty, D.C., and Weitzman, M.D. (2011). APOBEC3A can activate the DNA damage response and cause cell-cycle arrest. EMBO Rep. 12, 444–450. Curtis, C., Shah, S.P., Chin, S.F., Turashvili, G., Rueda, O.M., Dunning, M.J., Speed, D., Lynch, A.G., Samarajiwa, S., Yuan, Y., et al.; METABRIC Group (2012). The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346–352. Lee, H.S., Park, J.H., Kim, S.J., Kwon, S.J., and Kwon, J. (2010). ChIP and Solexa Sequencing A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double-strand break repair. EMBO J. 29, 1434–1445. Dahlman-Wright, K., Cavailles, V., Fuqua, S.A., Jordan, V.C., Katzenellenbo- gen, J.A., Korach, K.S., Maggi, A., Muramatsu, M., Parker, M.G., and Gustafs- son, J.A. (2006). International Union of Pharmacology. LXIV. Estrogen receptors. Pharmacol. Rev. 58, 773–781. Leonard, B., Hart, S.N., Burns, M.B., Carpenter, M.A., Temiz, N.A., Rathore, A., Vogel, R.I., Nikas, J.B., Law, E.K., Brown, W.L., et al. (2013). APOBEC3B upregulation and genomic mutation patterns in serous ovarian carcinoma. Cancer Res. 73, 7222–7231. de Bruin, E.C., McGranahan, N., Mitter, R., Salm, M., Wedge, D.C., Yates, L., Jamal-Hanjani, M., Shaﬁ, S., Murugaesu, N., Rowan, A.J., et al. (2014). Spatial and temporal diversity in genomic instability processes deﬁnes lung cancer evolution. Science 346, 251–256. Lin, C., Yang, L., Tanasa, B., Hutt, K., Ju, B.G., Ohgi, K., Zhang, J., Rose, D.W., Fu, X.D., Glass, C.K., and Rosenfeld, M.G. (2009). Nuclear receptor-induced chromosomal proximity and DNA breaks underlie speciﬁc translocations in cancer. Cell 139, 1069–1083. Desmedt, C., Piette, F., Loi, S., Wang, Y., Lallemand, F., Haibe-Kains, B., Viale, G., Delorenzi, M., Zhang, Y., d’Assignies, M.S., et al.; TRANSBIG Consortium (2007). Strong time dependence of the 76-gene prognostic signature for node- negative breast cancer patients in the TRANSBIG multicenter independent validation series. Clin. Cancer Res. 13, 3207–3214. Lopez-Garcia, J., Periyasamy, M., Thomas, R.S., Christian, M., Leao, M., Jat, P., Kindle, K.B., Heery, D.M., Parker, M.G., Buluwela, L., et al. (2006). ZNF366 is an estrogen receptor corepressor that acts through CtBP and histone de- acetylases. Nucleic Acids Res. 34, 6126–6136. Lucey, M.J., Chen, D., Lopez-Garcia, J., Hart, S.M., Phoenix, F., Al-Jehani, R., Alao, J.P., White, R., Kindle, K.B., Losson, R., et al. (2005). T:G mismatch-spe- ciﬁc thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif. Nucleic Acids Res. 33, 6393–6404. ENCODE Project Consortium (2012). An integrated encyclopedia of DNA ele- ments in the human genome. Nature 489, 57–74. Fong, Y.W., Cattoglio, C., and Tjian, R. (2013). The intertwined roles of tran- scription and repair proteins. Mol. Cell 52, 291–302. Guedj, M., Marisa, L., de Reynies, A., Orsetti, B., Schiappa, R., Bibeau, F., MacGrogan, G., Lerebours, F., Finetti, P., Longy, M., et al. (2012). A reﬁned molecular taxonomy of breast cancer. Oncogene 31, 1196–1206. Ma, J., and Wang, M. (2014). Interplay between DNA supercoiling and tran- scription elongation. ChIP and Solexa Sequencing A macrohi- stone variant links dynamic chromatin compaction to BRCA1-dependent genome maintenance. Cell Rep. 8, 1049–1062. Nik-Zainal, S., Alexandrov, L.B., Wedge, D.C., Van Loo, P., Greenman, C.D., Raine, K., Jones, D., Hinton, J., Marshall, J., Stebbings, L.A., et al.; Breast Can- cer Working Group of the International Cancer Genome Consortium (2012). Mutational processes molding the genomes of 21 breast cancers. Cell 149, 979–993. Komatsu, A., Nagasaki, K., Fujimori, M., Amano, J., and Miki, Y. (2008). Iden- tiﬁcation of novel deletion polymorphisms in breast cancer. Int. J. Oncol. 33, 261–270. Nik-Zainal, S., Wedge, D.C., Alexandrov, L.B., Petljak, M., Butler, A.P., Bolli, N., Davies, H.R., Knappskog, S., Martin, S., Papaemmanuil, E., et al. (2014). Association of a germline copy number polymorphism of APOBEC3A and APOBEC3B with burden of putative APOBEC-dependent mutations in breast cancer. Nat. Genet. 46, 487–491. Kotnis, A., Du, L., Liu, C., Popov, S.W., and Pan-Hammarstro¨ m, Q. (2009). Non-homologous end joining in class switch recombination: the beginning of the end. Philos. Trans. R. Soc. Lond. B Biol. Sci. 364, 653–665. 120 120 Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors Nowarski, R., Wilner, O.I., Cheshin, O., Shahar, O.D., Kenig, E., Baraz, L., Bri- tan-Rosich, E., Nagler, A., Harris, R.S., Goldberg, M., et al. (2012). APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase- dependent DNA repair. Blood 120, 366–375. Shanbhag, N.M., Rafalska-Metcalf, I.U., Balane-Bolivar, C., Janicki, S.M., and Greenberg, R.A. (2010). ATM-dependent chromatin changes silence transcrip- tion in cis to DNA double-strand breaks. Cell 141, 970–981. Shang, Y. (2006). Molecular mechanisms of oestrogen and SERMs in endome- trial carcinogenesis. Nat. Rev. Cancer 6, 360–368. O’Donnell, A.J., Macleod, K.G., Burns, D.J., Smyth, J.F., and Langdon, S.P. (2005). Estrogen receptor-alpha mediates gene expression changes and growth response in ovarian cancer cells exposed to estrogen. Endocr. Relat. Cancer 12, 851–866. Spruijt, C.G., Gnerlich, F., Smits, A.H., Pfaffeneder, T., Jansen, P.W., Bauer, C., Mu¨ nzel, M., Wagner, M., Mu¨ ller, M., Khan, F., et al. (2013). Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146– 1159. Osborne, C.K., and Schiff, R. (2011). Mechanisms of endocrine resistance in breast cancer. Annu. Rev. Med. 62, 233–247. Stavnezer, J. (2011). Complex regulation and function of activation-induced cytidine deaminase. Trends Immunol. 32, 194–201. Pankotai, T., Bonhomme, C., Chen, D., and Soutoglou, E. (2012). DNAPKcs- dependent arrest of RNA polymerase II transcription in the presence of DNA breaks. Nat. Struct. Mol. Biol. 19, 276–282. ChIP and Solexa Sequencing Suspe` ne, R., Henry, M., Guillot, S., Wain-Hobson, S., and Vartanian, J.P. (2005). Recovery of APOBEC3-edited human immunodeﬁciency virus G->A hypermutants by differential DNA denaturation PCR. J. Gen. Virol. 86, 125–129. Perillo, B., Ombra, M.N., Bertoni, A., Cuozzo, C., Sacchetti, S., Sasso, A., Chiariotti, L., Malorni, A., Abbondanza, C., and Avvedimento, E.V. (2008). DNA oxidation as triggered by H3K9me2 demethylation drives estrogen- induced gene expression. Science 319, 202–206. Taylor, B.J., Nik-Zainal, S., Wu, Y.L., Stebbings, L.A., Raine, K., Campbell, P.J., Rada, C., Stratton, M.R., and Neuberger, M.S. (2013). DNA deaminases induce break-associated mutation showers with implication of APOBEC3B and 3A in breast cancer kataegis. eLife 2, e00534. Polak, P., Lawrence, M.S., Haugen, E., Stoletzki, N., Stojanov, P., Thurman, R.E., Garraway, L.A., Mirkin, S., Getz, G., Stamatoyannopoulos, J.A., and Sunyaev, S.R. (2014). Reduced local mutation density in regulatory DNA of cancer genomes is linked to DNA repair. Nat. Biotechnol. 32, 71–75. Tini, M., Benecke, A., Um, S.J., Torchia, J., Evans, R.M., and Chambon, P. (2002). Association of CBP/p300 acetylase and thymine DNA glycosylase links DNA repair and transcription. Mol. Cell 9, 265–277. Puc, J., Kozbial, P., Li, W., Tan, Y., Liu, Z., Suter, T., Ohgi, K.A., Zhang, J., Aggarwal, A.K., and Rosenfeld, M.G. (2015). Ligand-dependent enhancer acti- vation regulated by topoisomerase-I activity. Cell 160, 367–380. Wang, Y., Klijn, J.G., Zhang, Y., Sieuwerts, A.M., Look, M.P., Yang, F., Talan- tov, D., Timmermans, M., Meijer-van Gelder, M.E., Yu, J., et al. (2005). Gene- expression proﬁles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 365, 671–679. Roberts, S.A., Lawrence, M.S., Klimczak, L.J., Grimm, S.A., Fargo, D., Stoja- nov, P., Kiezun, A., Kryukov, G.V., Carter, S.L., Saksena, G., et al. (2013). An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat. Genet. 45, 970–976. Welboren, W.J., van Driel, M.A., Janssen-Megens, E.M., van Heeringen, S.J., Sweep, F.C., Span, P.N., and Stunnenberg, H.G. (2009). ChIP-Seq of ERalpha and RNA polymerase II deﬁnes genes differentially responding to ligands. EMBO J. 28, 1418–1428. Ross-Innes, C.S., Stark, R., Teschendorff, A.E., Holmes, K.A., Ali, H.R., Dunning, M.J., Brown, G.D., Gojis, O., Ellis, I.O., Green, A.R., et al. (2012). Dif- ferential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature 481, 389–393. Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nusbaum, C., Myers, R.M., Brown, M., Li, W., and Liu, X.S. (2008). Model- based analysis of ChIP-Seq (MACS). Genome Biol. Cell Reports 13, 108–121, October 6, 2015 ª2015 The Authors 121 ChIP and Solexa Sequencing 9, R137. Sabatier, R., Finetti, P., Cervera, N., Lambaudie, E., Esterni, B., Mamessier, E., Tallet, A., Chabannon, C., Extra, J.M., Jacquemier, J., et al. (2011). A gene expression signature identiﬁes two prognostic subgroups of basal breast can- cer. Breast Cancer Res. Treat. 126, 407–420. Zhang, Y., Sieuwerts, A.M., McGreevy, M., Casey, G., Cufer, T., Paradiso, A., Harbeck, N., Span, P.N., Hicks, D.G., Crowe, J., et al. (2009). The 76-gene signature deﬁnes high-risk patients that beneﬁt from adjuvant tamoxifen ther- apy. Breast Cancer Res. Treat. 116, 303–309. Schmidt, D., Wilson, M.D., Spyrou, C., Brown, G.D., Hadﬁeld, J., and Odom, D.T. (2009). ChIP-seq: using high-throughput sequencing to discover pro- tein-DNA interactions. Methods 48, 240–248.
https://openalex.org/W2039568446	https://europepmc.org/articles/pmc3797749?pdf=render	English	null	Functional Characterisation of the Maturation of the Blood-Brain Barrier in Larval Zebrafish	PloS one	2,013	cc-by	9,677	Abstract ming et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits ution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors are all former employees of DanioLabs Ltd (a biotechnology company) and a large part of this work was carried out during this period of employment. Work performed at DanioLabs Ltd was financed by DanioLabs Ltd and not by grant funding. The company no longer exists and there are no restrictions on publishing this data. Dr A Fleming continued working on this project after leaving the company to work at the University of Cambridge and this part of the work was funded by an MRC Skills Gap Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: We have the following interests: The majority of the work in the original manuscript was performed whilst all 3 authors were employees of DanioLabs Ltd (a biotechnology company). The company no longer exists and there are no shareholders or employees. Some of the work in this manuscript was used to support a patent application (Screening methods employing zebrafish and the blood brain barrier; WO2005080974) which has now lapsed. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials. E-mail: af425@cam.ac.uk ¤a Current address: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom ¤b Current address: Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom ¤c Current address: Department of Experimental Neurology, Heinrich Heine University, Duesseldorf, Germany ¤d Current address: Department of Neurology, Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom ¤a Current address: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom ¤b Current address: Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom ¤c Current address: Department of Experimental Neurology, Heinrich Heine University, Duesseldorf, Germany ¤d Current address: Department of Neurology, Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom next level of barrier function is provided by capillary pericytes, which wrap around the endothelial cells of the capillary walls. Lastly, the outermost layer comprises astrocyte end feet which surround the endothelium and pericytes. Angeleen Fleming¤a¤b*, Heike Diekmann¤c, Paul Goldsmith¤d DanioLabs Ltd., Cambridge Research Park, Cambridge, United Kingdom Abstract In addition to providing a physical barrier, these three cell types express a variety of enzymes, such as aminopeptidases, carboxypeptidases, endopeptidases and cholinesterases, which inactivate many drugs [2], and in some cases, may also activate pro-drugs. In spite of these physical and enzymatic barriers, certain molecules are able to freely diffuse across the BBB, but are prevented from accumulating in the brain as they Abstract Zebrafish are becoming increasingly popular as an organism in which to model human disease and to study the effects of small molecules on complex physiological and pathological processes. Since larvae are no more than a few millimetres in length, and can live in volumes as small as 100 microliters, they are particularly amenable to high- throughput and high content compound screening in 96 well plate format. There is a growing literature providing evidence that many compounds show similar pharmacological effects in zebrafish as they do in mammals, and in particular humans. However, a major question regarding their utility for small molecule screening for neurological conditions is whether a molecule will reach its target site within the central nervous system. Studies have shown that Claudin-5 and ZO-1, tight-junction proteins which are essential for blood-brain barrier (BBB) integrity in mammals, can be detected in some cerebral vessels in zebrafish from 3 days post-fertilisation (d.p.f.) onwards and this timing coincides with the retention of dyes, immunoreactive tracers and fluorescent markers within some but not all cerebral vessels. Whilst these findings demonstrate that features of a BBB are first present at 3 d.p.f., it is not clear how quickly the zebrafish BBB matures or how closely the barrier resembles that of mammals. Here, we have combined anatomical analysis by transmission electron microscopy, functional investigation using fluorescent markers and compound uptake using liquid chromatography/tandem mass spectrometry to demonstrate that maturation of the zebrafish BBB occurs between 3 d.p.f. and 10 d.p.f. and that this barrier shares both structural and functional similarities with that of mammals. Citation: Fleming A, Diekmann H, Goldsmith P (2013) Functional Characterisation of the Maturation of the Blood-Brain Barrier in Larval Zebrafish. PLoS ONE 8(10): e77548. doi:10.1371/journal.pone.0077548 Editor: Filippo Del Bene, Institut Curie, France Received April 11 2013; Accepted September 5 2013; Published October 16 2013 Citation: Fleming A, Diekmann H, Goldsmith P (2013) Functional Characterisation of the Maturation of the Blood-Brain Barrier in Larval Zebrafish. PLoS ONE 8(10): e77548. doi:10.1371/journal.pone.0077548 Edit Fili D l B I tit t C i F Received April 11, 2013; Accepted September 5, 2013; Published October 16, 2013 Received April 11, 2013; Accepted September 5, 2013; Published October 16, 2013 Copyright: © 2013 Fleming et al. This is an open-access article distributed under the terms of the Creative Commons Attributio unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Dye exclusion experiments Stocks of 2% Evans blue (961 Da) and 10% sodium fluorescein (376 Da) in 0.9% saline were prepared and stored at 4 °C. Larvae were anaesthetised by immersion in 0.2 mg/ml 3-amino benzoic acid (MS222) prior to being immobilised and positioned laterally in 3% methyl cellulose in embryo medium such that the heart was accessible. Larvae and juveniles from 2 days post-fertilisation (d.p.f.) to 30 d.p.f. were injected with 0.5 to 2 nl (depending on age) of dye or saline control into the pericardial region, using a standard zebrafish microinjection apparatus [15]. Fish were transferred to fresh embryo medium to recover from anaesthesia, then viewed using a fluorescence dissecting microscope at 1 hour intervals to determine the time of uptake of the dye into the circulation and the time at which dye had permeated from the circulation into non-vascular body tissue. Representative images of dye distribution in vivo were taken on a Zeiss AxioZoom V16 microscope with Apotome. Dye and saline injected larvae were anaesthetised at 4 or 6 hours after injection and fixed in 4% paraformaldehyde in PBS. Ten larvae per age group and injection treatment were embedded in Sakura Tissue-Tek OCT (Bayer, Newbury, UK) and frozen parasagittal sections were cut at 10 µm thickness. Expression of claudin-5 and ZO-1 have been detected in cerebral microvessels of larval zebrafish from as early as 2 and 3 days post-fertilisation (d.p.f.) respectively [7,9]. Furthermore, several studies have used fluorescent dyes or transgenically- encoded fluorescently-tagged plasma protein to demonstrate size dependent exclusion occurs from around 3 d.p.f. [7,9–11]. However, size exclusion seems to occur only in certain cerebral vessels at this age, while others are still “leaky”. In mammals, the BBB gradually matures during development, with permeability to small molecules decreasing with age (i.e. substances excluded from the adult rat brain do permeate embryonic brain capillaries) [12]. Similarly, the developing mouse BBB shows decreasing compound permeability as it matures [13]. In this study, we set out to investigate how size-dependent exclusion matures during larval development, to determine the degree of similarity in exclusion properties to those of mammalian BBB and to study the cellular components surrounding the vascular endothelium in the brain. We show that zebrafish do indeed gradually develop a sophisticated BBB with active transport systems. Methods are actively effluxed by specific transporters, the most notable of which is P-glycoprotein (Pgp) [1]. Conversely, inwardly directed transporter systems, such as GLUT1/Slc2a1 (glucose transport), Slc7a1 and Slc7a5 (amino acids), low-density lipoprotein receptors (LRPs) and ion pumps, permit the entry of a variety of molecules that would otherwise be unable to enter the brain [1]. Introduction In mammals, the blood-brain barrier (BBB) provides a complex obstacle to the penetration of drugs into the central nervous system (CNS). The first level of barrier is presented by the tight junctions between endothelial cells of the vasculature. These high resistance tight junctions, made up by proteins such as claudin-5 and ZO-1, render brain capillary endothelia tightly sealed, in contrast to “leaky” endothelial capillaries in the periphery. Thus, there is no paracellular movement of fluid and only minimal pinocytosis from capillaries into the CNS [1]. The 1 October 2013 \| Volume 8 \| Issue 10 \| e77548 PLOS ONE \| www.plosone.org 1 Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Husbandry and experimental procedures Embryos were collected from matings of adults of wildtype (TL, AB and WIK strains) and Tg(fli1a:EGFP)y1 transgenic fish [14] kept under standard conditions [15]. Embryos were reared in embryo medium (5 mM NaCl, 0.17 mM KCl,0.33 mMCaCl2, 0.33 mM Mg2SO4, 10-5% Methylene Blue) and staged using standard criteria [16]. All experiments were carried out in accordance with the Animals (Scientific Procedures) Act, 1986. Ethics statement This work was licenced by the Home Office under the Animals (Scientific Procedures) Act (Home Office licence number PPL80/2074 for work performed at DanioLabs Ltd and PPL 80/2322 for work performed at the University of Cambridge). Protocols were approved by the local Animal Welfare Ethical Review Committee (AWERC); namely, DanioLabs AWERC and University of Cambridge AWERC. Appropriate steps were taken to ameliorate suffering in all work. Experiments were designed with the minimum number of animals necessary to produce meaningful results. Zebrafish are popular as a vertebrate model with which to perform compound screens [3,4] and increasingly used to model human neurological disease processes, such as epilepsy and neurodegeneration [5,6]. Therefore, it is important to understand whether and when the zebrafish BBB forms and compare its characteristics and function with that of mammals. Adult zebrafish express the tight junction proteins ZO-1 and claudin-5 in the endothelial vascular cells within the brain [7]. In addition, size dependent exclusion of immuno-reactive compounds occurs in the adult zebrafish brain. For example, analysis of the distribution of enzymatically active compounds injected into the heart demonstrated that HRP (44kDa) was retained in cerebral vessels, whilst sulfo-NHS-Biotin (0.443kDa) diffused into the brain, indicating a size dependent exclusion mechanism [7]. Tight junctions play a major role in size-dependent exclusion in mammals and loosening of the size exclusion limit is observed in claudin-5 knockout mice [8]. Therefore, the presence of claudin-5 and ZO-1 in the adult zebrafish brain could account for these observed effects. These findings demonstrate that adult zebrafish possess a BBB but it is not known when this barrier becomes functional during zebrafish development. Dye exclusion experiments This allows for the design of rational strategies for screening compounds for neurological indications and interpretation of results, as well as potentially providing a system to predict whether a novel compound might penetrate the human brain. Transmission Electron Microscopy body tissue samples were collected in separate microfuge tubes on ice and excess liquid removed. 100 µl ice cold PBS was added and samples were briefly centrifuged and all excess liquid removed. The weight of the tissue sample was measured and the samples were frozen at -20 °C prior to extraction for LC/MS/MS. Larvae were anaesthetised at 3, 5, 8 and 10 d.p.f. by immersion in 0.2 mg/ml MS222, fixed in 4% glutaraldehyde in cacodylate buffer containing 0.006% hydrogen peroxide for 3 hours and washed in cacodylate buffer before post-fixing in osmium tetroxide. Samples were bulk stained with uranyl acetate, dehydrated in ethanol and embedded in Spurr’s resin. Thin parasagittal sections (5 nm) were prepared with a Leica Ultracut UCT, stained with uranyl acetate and lead citrate and viewed in a Philips CM100 electron microscope at 80 KV. For studies investigating the effect of Abcb1/4/5 efflux on drug distribution, larvae were exposed to the ABCB1/4/5 efflux inhibitor verapamil at 50 µg/ml, a concentration previously demonstrated to be non-toxic, for 2 hours prior to exposure to the test compound. Test compound was added to the well containing 50 µg/ml verapamil and larvae were incubated for a further hour at room temperature prior to tissue collection as described. For subsequent LC/MS/MS analysis, tissue samples were thawed and after addition of an internal standard (clozapine) extracted into an organic solvent (tertiary-butyl methyl ether, t-bme). The solvent extract was then evaporated and reconstituted in methanol/acetic acid, except for scopolamine exposed samples, which were reconstituted in acetonitrile/ammonia. Liquid chromatography for haloperidol, desloratadine and diphenhydramine (100 µl injections of extracted samples) was carried out using a Phenomenex Luna C18(2), 50 x 2 mm, 5 µm analytical column with a mobile phase gradient given in Table S1. Chromatography for scopolamine and scopolamine N-butyl bromide was performed using a Restek Ultra IBD, 150 x 3.2 mm, 5 µm analytical column with a mobile phase gradient given in Table S2. The analytes and internal standard were ionised under atmospheric pressure chemical ionisation (APCI) conditions operating in positive ion mode. Detection was via tandem mass spectrometry (MS/MS) using selected ion monitoring (SIM). MS/MS scan parameters under APCI conditions are given in Table S3. All analyses were performed using a Finnigan LCQ quadrupole ion trap mass spectrometer. The ratio of the amount of drug found in the trunk relative to that in the head was calculated. Drug exposure and distribution experiments Initial drug exposure assays were performed in larval zebrafish to identify a sufficiently high concentration of drug that could be detected by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis without causing toxicity. Maximum tolerated concentration (MTC) was determined using n=6 larvae per group. A non-toxic drug concentration of 15 µg/ml was selected for all subsequent compound exposure experiments except for scopolamine, which was used at 5 µg/ml. 60 larvae (equivalent to approximately 20 mg wet tissue) were transferred into a single well of a 12 well plate (Corning) and the final volume of embryo medium per well was adjusted to 1.5 ml. Compounds of interest were prepared as 2 mg/ml stocks in dimethyl sulfoxide (DMSO). 11.25 µl of stock was added to each well, except for scopolamine (3.75 µl). Larvae were incubated in the drug for 1 hour at room temperature then anaesthetised on ice. For whole uptake analysis, anaesthetised larvae were transferred to 1.5 ml microfuge tubes and all excess liquid was removed. 100 µl ice cold PBS was added, samples were briefly centrifuged and all excess liquid was removed. For brain versus body uptake analysis, larvae were anaesthetised by chilling prior to decapitation using fine iridectomy scissors and No. 5 watchmaker forceps. Head and The effect of Pgp inhibitors on the distribution of rhodamine 123 Rhodamine 123 (R123) was dissolved in 10% ethanol/0.9% saline to make a 0.5 mg/ml stock. Dye exclusion experiments on larvae at 3, 5, 8 and 10 d.p.f. were performed as described above using 0.2 nl R123. Some larvae were exposed to verapamil at 50 µg/ml for 2 hours prior to and during intrapericardial injection of R123 and for 3 hours after injection. At 3 hours after injection, larvae were anaesthetised and fixed in 4% PFA prior to processing for frozen sections. Quantification of fluorescence in the brain was performed as described above. Zebrafish abcb1/4/5 expression Larvae were anaesthetised at 3, 5, 8 and 10 d.p.f. by immersion in 0.2 mg/ml MS222, fixed in 4% PFA and processed for wholemount antibody staining as previously described [17]. Immunohistochemistry was performed with an antibody raised against mouse ABCB1 (Covance) at 1:100 dilution and the staining detected using Alexa568 fluorescent secondary antibody (Molecular Probes). Larvae were mounted in chamber slides in Prolong Gold antifade mounting medium (Invitrogen) and viewed using a Zeiss StereoZoom V16 microscope under fluorescence illumination. Optical sectioning was performed using an Apotome.2 (Zeiss) and representative images were captured using and AxioCam MR digital camera (Zeiss) and Zen 2012 software (Zeiss). In silico sequence analysis Protein sequences of mammalian and zebrafish ABCB gene family members were identified from ENSEMBL (human GRCh37; mouse GRCm38; zebrafish Zv9). BLAST searches were performed to identify whether un-annotated paralogues could be identified from the zebrafish genome. Sequence alignments were performed using ClustalW. Transmission Electron Microscopy Changes in trunk/head drug ratios of greater than 20% were considered to be indicative of altered distribution. Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Quantification of dye fluorescence Sections were visualised without mounting medium by fluorescence microscopy on an Olympus BX51 microscope. Three midline sagittal sections from each larva were identified and images were captured using a ColorView camera (Olympus) and AnalySis software (Soft Imaging System). The fluorescence intensity within the brain was quantified for each midline section of each larva in each treatment group using colour threshold and area measurements in AnalySis. Mean values, standard deviations and Student’s t-tests were calculated using Excel software (Microsoft Office). PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77548 2 Expression of the zebrafish homologue of mammalian P-glycoprotein is developmentally regulated Previous studies have reported that zebrafish, like rodents, have a duplicated abcb1 (P-glycoprotein, Pgp, or multi-drug resistance) gene [22]. However, in the recent release of the zebrafish genome sequence (Genome assembly: Zv9), ENSDARG00000021787 is now annotated as abcb5 (reported as abcb1a by [22]) and ENSDARG00000010936 as abcb4 (reported as abcb1b by [22]). In man, the ABCB gene family comprises 11 members (ABCB1, 4, 5, 6, 7, 8, 9, 10, 11, TAP1 and TAP2) whereas in zebrafish, abcb1, abcb6 and tap2 are missing, abcb11 is duplicated (abcb11a and abcb11b) and, in addition, zebrafish possess abcb3l1. Phylogenetic analysis of the mammalian and zebrafish abcb family suggests that the zebrafish abcb5 (ENSDARG00000021787) and abcb4 genes (ENSDARG00000010936) are the closest homologues of mammalian ABCB1/4/5 (Figure 2) (see also Figure S1A and S1B for alignment of human, mouse and zebrafish Abcb4 and Abcb5 protein sequences). Peptide alignment analysis shows Results Since no specific orthologue of mammalian ABCB1 could be identified in the zebrafish genome (zv9), and since mammalian ABCB4 and ABCB5 are known to be expressed in the vascular endothelial cells of the CNS [1], we hypothesised that zebrafish Abcb4 or Abcb5 might function as efflux transporters in the vasculature surrounding the CNS and therefore investigated the expression of these proteins. Expression of zebrafish Abcb1/4/5 correlates with the onset of active transport across the blood brain barrier Dye exclusion experiments were performed with Rhodamine 123 (R123), a fluorescent substrate for ABCB1 [24], ABCB4 [25] and ABCB5 [26] in mammals, to investigate the function of Abcd1/4/5 during zebrafish development. R123 was excluded from the brain at 8 d.p.f., but not at 3 and 5 d.p.f (Figure 4). This time line coincides well with the expression of Abcb1/4/5 in the vascular endothelium of the CNS. To investigate whether the exclusion of R123 was indeed due to efflux mediated by a Abcb1/4/5 transporter, dye exclusion experiments were repeated in the presence of verapamil, a known inhibitor of mammalian ABCB1 [24], ABCB4 [25] and ABCB5 [27]. Larvae were incubated in 50 µg/ml verapamil prior to, during and after injection of R123 and analysed 3 hours after injection for distribution of R123 fluorescence. In the presence of verapamil, R123 penetrated into the brain also at 8 d.p.f. and 10 d.p.f. (Figure 4). Since R123 was excluded from the brain at these time points when incubated without verapamil, these results suggests that R123 is actively transported out of the brain by zebrafish Abcb1/4/5 from 8 d.p.f. Developmental changes in the size of fluorescent molecules excluded from the CNS Dye exclusion experiments have long been used in rodent investigations into the integrity of the BBB [1,18–21]. The absence of dyes within the CNS following injection into peripheral vessels can be used as a marker of BBB integrity. We therefore used dyes of varying molecular weights to determine the developmental timecourse of size dependent exclusion from the zebrafish CNS. Dye exclusion experiments were initially performed on larvae from 2 to 30 d.p.f. and after initial analysis had narrowed down the relevant age range, studies were limited to larvae at 3, 4, 5, 8 and 10 d.p.f. Larvae were observed at 1 hour intervals after injection of the fluorescent dye into the pericardial cavity to determine when the fluorescent compound had diffused from the circulation into peripheral tissues (Figure 1A & B). Larvae were then anaethestised and fixed for cryosectioning. The fluorescent intensity of frozen sections of brain tissue was quantified for dye injected larvae and compared to saline injected siblings fixed at the same time point after injection. An example of the distribution and quantification process is shown in Figure 1C-E. In Evans blue (961 Da) injected zebrafish, fluorescence was observed in the brain at 3 d.p.f. but not at 5 d.p.f or subsequent stages (Figure 1F), suggesting that molecules of high molecular weight become excluded between 3 and 5 d.p.f. In contrast, exclusion of sodium fluorescein, a lower molecular weight compound (376 Da), was not observed until 10 d.p.f. (Figure 1G), suggesting a time dependent maturation relating to the size limit for molecules excluded from the CNS. Using an antibody against a highly conserved amino acid sequence found in all mammalian Pgp isoforms [23] and also present in zebrafish Abcb4 and Abcb5 (Figure 3A), optical sections of wholemount Tg(fli1a:EGFP)y1 larvae at 10 d.p.f. revealed specific staining in the vascular endothelium of the CNS. A range of larval ages were then examined to determine the earliest time point with specific vascular staining. Staining was observed in the vascular endothelium of the CNS at 8 d.p.f. (Figure 3B & C) but not earlier (Figure S2). Based on phylogenetic analysis, peptide homology and protein expression, we assume that either the zebrafish abcb5 (ENSDARG00000021787) or abcb4 gene (ENSDARG00000010936) is the functional homologue of mammalian ABCB1 and is hereafter referred to as abcb1/4/5. Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Gold antifade mounting medium (Invitrogen) and viewed using a Zeiss StereoZoom V16 microscope under fluorescence illumination. Optical sectioning was performed using an Apotome.2 (Zeiss) and representative images were captured using and AxioCam MR digital camera (Zeiss) and Zen 2012 software (Zeiss). that zebrafish Abcb4 has 63% similarity at the amino acid level to human ABCB1 and 63% similarity to human ABCB4 (Table 1). The zebrafish abcb5 protein has 57% similarity at the amino acid level to human ABCB1 and 50% to human ABCB5 (Table 1). BLAST searches of the latest zebrafish genome release (Ensembl Zv9) with both human ABCB1 and mouse ABCB1A and ABCB1B identify zebrafish Abcb4 and Abcb5 as the closest zebrafish homologues to mammalian ABCB1. Analysis of GFAP expression Tg(fli1a:EGFP)y1 larvae at 3, 5, 7 and 10 d.p.f. were fixed in 4 % PFA, then processed for antibody staining as previously described [17]. The GFAP antibody (zrf-1, ZIRC) was used at 1:25 dilution and detected with Alexa568 fluorescent secondary antibody. Larvae were mounted in chamber slides in Prolong PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77548 3 The formation of a blood brain barrier correlates with developmental changes in drug distribution A panel of 5 drugs known to either cross or not cross the BBB in mammals were selected to test in zebrafish larvae. Larvae of different ages were exposed to these compounds at non-toxic concentrations for 1 hour prior to collection of head October 2013 \| Volume 8 \| Issue 10 \| e77548 PLOS ONE \| www.plosone.org 4 Formation of the Zebrafish Blood-Brain Barrier Figure 1. Investigation of blood-brain barrier maturation using fluorescent dyes. Ai-iv) One hour after pericardial injection of Evans blue into 8 d.p.f. Tg(fli1a:EGFP)y1 larvae, strong Evan’s blue fluorescence could be observed in the dorsal aorta (arrows) and vena cava (arrowheads) and weak fluorescence could be observed in individual segmental vessels of the trunk. Bi-iv) 4 hours after injection of Evan’s blue, strong fluorescence is still observed in dorsal aorta and vena cava and additionally in segmental vessels. In addition, fluorescence is also observed in trunk muscles between the segmental vessels and in the fin mesenchyme demonstrating that the dye has penetrated into surrounding tissue. Ci) Parasagittal section of a 3 d.p.f. zebrafish larva 3 hours after pericardial injection with saline control. Cii) High magnification fluorescent imaging of the region of marked in Ci). Di) Parasagittal section of a 3 d.p.f. zebrafish larva 3 hours after pericardial injection with Evan’s blue. Dii) High magnification fluorescent imaging of the region of marked in Di). Ei) Parasagittal section of a 5 d.p.f. zebrafish larva 3 hours after pericardial injection with Evan’s Blue. Cii) High magnification fluorescent imaging of the region of marked in Ci). Cii – Eii) The fluorescent intensity of dye within the brain was quantified using image thesholding (pseudo-coloured green) and area over threshold was measured using AnalySis software. F & G) The fluorescence intensity of injected dyes was measured in the brain of zebrafish following peripheral injection at various time points of zebrafish development. Graphs show mean fluorescent intensity (± std dev.) for each treatment. F) Evans blue, (961 Da) a large molecule known to form multimers with serum proteins, is excluded from the brain from day 5. G) Sodium fluorescein (376 Da) permeates into the brain until 8 d.p.f. but is excluded at 10 d.p.f. Scale bar represents 250 µm in A and B and 50 µm in C - E. doi: 10.1371/journal.pone.0077548.g001 Figure 1. Investigation of blood-brain barrier maturation using fluorescent dyes. Ai-iv) One hour after pericardial injection of Evans blue into 8 d.p.f. The formation of a blood brain barrier correlates with developmental changes in drug distribution Tg(fli1a:EGFP)y1 larvae, strong Evan’s blue fluorescence could be observed in the dorsal aorta (arrows) and vena cava (arrowheads) and weak fluorescence could be observed in individual segmental vessels of the trunk. Bi-iv) 4 hours after injection of Evan’s blue, strong fluorescence is still observed in dorsal aorta and vena cava and additionally in segmental vessels. In addition, fluorescence is also observed in trunk muscles between the segmental vessels and in the fin mesenchyme demonstrating that the dye has penetrated into surrounding tissue. Ci) Parasagittal section of a 3 d.p.f. zebrafish larva 3 hours after pericardial injection with saline control. Cii) High magnification fluorescent imaging of the region of marked in Ci). Di) Parasagittal section of a 3 d.p.f. zebrafish larva 3 hours after pericardial injection with Evan’s blue. Dii) High magnification fluorescent imaging of the region of marked in Di). Ei) Parasagittal section of a 5 d.p.f. zebrafish larva 3 hours after pericardial injection with Evan’s Blue. Cii) High magnification fluorescent imaging of the region of marked in Ci). Cii – Eii) The fluorescent intensity of dye within the brain was quantified using image thesholding (pseudo-coloured green) and area over threshold was measured using AnalySis software. F & G) The fluorescence intensity of injected dyes was measured in the brain of zebrafish following peripheral injection at various time points of zebrafish development. Graphs show mean fluorescent intensity (± std dev.) for each treatment. F) Evans blue, (961 Da) a large molecule known to form multimers with serum proteins, is excluded from the brain from day 5. G) Sodium fluorescein (376 Da) permeates into the brain until 8 d.p.f. but is excluded at 10 d.p.f. Scale bar represents 250 µm in A and B and 50 µm in C - E. doi: 10.1371/journal.pone.0077548.g001 of different absorption routes, such as gut and gills and/or onset of expression of metabolising enzymes. and trunk tissue for analysis of drug concentration. A generic extraction method was developed and LC/MS/MS was performed to analyse the concentration of drug in head and trunk tissue in larvae of different ages. Drug/tissue concentrations were normalised against a standard and the relative amount of drug in head and trunk samples was compared to determine whether the drug distribution altered with larval age (Table 2). Anatomical features of a blood brain barrier Figure 2. Phylogenetic analysis of mammalian and zebrafish ABCB genes. Phylogenetic analysis of the ABCB gene family in human, mouse and zebrafish. The zebrafish genome does not contain an annotated orthologue of mammalian ABCB1. Zebrafish abcb4 and abcb5 are identified as the closest homologues to mammalian ABCB1, 4 and 5. doi: 10.1371/journal.pone.0077548.g002 Having established that exclusion of small molecules from the zebrafish brain occurs between 3 and 10 d.p.f. (depending on the size of the molecule and the method of exclusion), we undertook an anatomical timecourse analysis using transmission electron microscopy (TEM) to determine whether there was also anatomical evidence for a BBB and if so, to characterise the maturation of these features. At 3 d.p.f., no evidence of tight junctions in the vessels around the brain and spinal cord was observed (Figure 5A & B). At 5 d.p.f., however, tight junctions were identified in some, but not all vascular endothelium (data not shown). By 10 d.p.f., double membranes were observed in the vascular endothelium at all sites investigated, suggesting that tight junctions are present by this age (Figure 5C & D). In addition, pericytes and astrocyte feet processes were observed in some sections at 10 d.p.f. (Figure 5E) and evidence for the latter was further supported by the co- localisation of glial fibrillary associated protein (GFAP) with GFP-positive vessels in the brains of Tg(fli1a:EGFP)y1 larvae at 10 d.p.f. but not earlier (Figure S3). The zebrafish BBB matures during larval development Co-incubation with verapamil resulted in equalisation of the distribution of desloratadine in head and trunk samples at 8 d.p.f and an accumulation in the head versus the body at 10 d.p.f. Therefore, the exclusion of desloratadine from 8 d.p.f is dependent on the function of Abcb1/4/5 in zebrafish. The zebrafish BBB matures during larval development The zebrafish BBB matures during larval development Several studies have demonstrated that dyes or enzymatically active compounds are retained within cerebral microvessels of the larval zebrafish brain from as early as 3 d.p.f. [7,9–11], co-incident with the expression of the tight junction proteins ZO-1 and claudin5. However, these studies showed exclusion from only some, but not all cerebral vessels suggesting that there is not a fully functional BBB at this point. Here, we have combined anatomical analysis by TEM, functional investigation using fluorescent markers and compound uptake using LC/MS/MS to investigate the formation of the functional BBB in zebrafish and to demonstrate how this barrier matures during larval development. By administrating dyes of different molecular weights, we have shown that large molecular weight compounds are indeed excluded at 3 d.p.f. Although Evans blue is a fairly small molecule (961 Da), most of the dye becomes bound to serum albumin and has a size of >69kDa, therefore exclusion at 3 d.p.f. likely demonstrates exclusion of serum proteins and this is in keeping with the size exclusion findings from other studies [7,9–11]. By contrast, exclusion of sodium fluorescein (molecular weight of 376Da) does not take place until 10 d.p.f. indicating that the size dependent exclusion of molecules from the brain matures during larval development. Table 1. Similarity between human, mouse and zebrafish ABCB1, ABCB4 and ABCB5 peptide sequences. Table 1. Similarity between human, mouse and zebrafish ABCB1, ABCB4 and ABCB5 peptide sequences. ABCB1, ABCB4 and ABCB5 peptide sequences. Zebrafish Abcb4 Zebrafish Abcb5 Human ABCB1 63% 57% Human ABCB4 63% 55% Human ABCB5 54% 50% Mouse ABCB1A 63% 56% Mouse ABCB1B 61% 56% Mouse ABCB4 63% 54% Mouse ABCB5 52% 48% Peptide sequences were aligned using ClustalW. doi: 10.1371/journal.pone.0077548.t001 known that this drug is actively exported by Pgp (ABCB1) in mammals, we investigated whether the distribution of this drug would be altered by co-incubation with the ABCB1, ABCB4 and ABCB5 inhibitor, verapamil. Co-incubation with verapamil resulted in equalisation of the distribution of desloratadine in head and trunk samples at 8 d.p.f and an accumulation in the head versus the body at 10 d.p.f. Therefore, the exclusion of desloratadine from 8 d.p.f is dependent on the function of Abcb1/4/5 in zebrafish. known that this drug is actively exported by Pgp (ABCB1) in mammals, we investigated whether the distribution of this drug would be altered by co-incubation with the ABCB1, ABCB4 and ABCB5 inhibitor, verapamil. The formation of a blood brain barrier correlates with developmental changes in drug distribution The amount of drug absorbed over the 1 hour exposure period varied greatly with some larval tissue containing as little as 5.9 % of the medium concentration for scopolamine N-butyl bromide and as much as 949% of the medium concentration for haloperidol, a variation that may reflect the hydrophobic/hydrophilic properties of the drugs tested. Indeed, there is a loose correlation between the partition coefficient (logP) value for each compound and the percentage of the drug absorbed from the medium. In addition, the absorption of each drug was found to vary between the different larval ages examined. This may reflect the maturation All drugs were equally distributed in head and trunk samples at 5 d.p.f., indicating the absence of a BBB at this age. Furthermore, diphenhydramine and haloperidol, which do cross the BBB in mammals, showed an equal distribution in head and trunk samples at all ages examined, indicating that these drugs also cross the zebrafish BBB. In scopolamine treated larvae, the drug level was below the detection limits at 5 and 8 d.p.f. However, scopolamine was detected at equal concentrations in head and trunk samples in 10 d.p.f. larvae, suggesting that scopolamine also crossed the BBB, as in mammals. In contrast, scopolamine N-butyl bromide and desloratadine, two drugs that do not cross the mammalian BBB, showed lower concentrations in the head versus the trunk at 10 d.p.f. and from 8 d.p.f. respectively, suggesting that these compounds are also excluded from the brain in zebrafish larvae. Exclusion of desloratadine at 8 d.p.f. correlates with the earliest expression of Abcb1/4/5 in the vascular endothelium. Since it is PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77548 5 Formation of the Zebrafish Blood-Brain Barrier Discussion Figure 2. Phylogenetic analysis of mammalian and zebrafish ABCB genes. Phylogenetic analysis of the ABCB gene family in human, mouse and zebrafish. The zebrafish genome does not contain an annotated orthologue of mammalian ABCB1. Zebrafish abcb4 and abcb5 are identified as the closest homologues to mammalian ABCB1, 4 and 5. doi: 10.1371/journal.pone.0077548.g002 The zebrafish BBB possesses active transport systems Active transport systems play a critical role in the function of the BBB in mammals. The most significant transporter in mammals is ABCB1 protein (Pgp), although other ABCB family transporters and organic anion transporters are also expressed by brain endothelial cells (reviewed in 1). These transporters have diverse but overlapping substrate specificities that help determine the partitioning of small molecules between the blood and the brain. A clinical example of the role of Pgp efflux is the non-sedating effects of second-generation PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77548 6 Formation of the Zebrafish Blood-Brain Barrier Figure 3. Sequence comparison and expression of zebrafish ABCB1/4/5 homologues. A) Sequence alignment of human and mouse ABCB1 protein sequences with zebrafish Abcb4 and Abcb5. The zebrafish proteins show a high level of identity with the mouse and human ABCB1, ABCB4 and ABCB5 proteins. An antibody recognising the peptide sequences VQAALD (yellow) and VQEALD (blue) (Covance) was selected for use in zebrafish as the similarity of these peptides was conserved. B (low magnification) and C) (high magnification) 3D projections of optically sectioned wholemount Tg(fli1a:EGFP)y1 larvae stained with the anti-VQAALD antibody. Positive staining is observed in the vascular endothelium of the CNS at 8 d.p.f , but not at earlier timepoints (see Figure S2). High magnification images (Ci-iii) demonstrate the co- localisation of ABCB1/4/5 antibody staining (red) on cerebral vessels (green). Bi and Ci – GFP channel – maximum intensity projection of the cerebral vasculature of Tg(fli1a:EGFP)y1 transgenic larvae; Bii and Cii – Alexa 568 labelled antibody staining with ABCB1/4/5 antibody; Biii and Ciii – overlay. doi: 10.1371/journal.pone.0077548.g003 Figure 3. Sequence comparison and expression of zebrafish ABCB1/4/5 homologues. A) Sequence alignment of human and mouse ABCB1 protein sequences with zebrafish Abcb4 and Abcb5. The zebrafish proteins show a high level of identity with the mouse and human ABCB1, ABCB4 and ABCB5 proteins. An antibody recognising the peptide sequences VQAALD (yellow) and VQEALD (blue) (Covance) was selected for use in zebrafish as the similarity of these peptides was conserved. B (low magnification) and C) (high magnification) 3D projections of optically sectioned wholemount Tg(fli1a:EGFP)y1 larvae stained with the anti-VQAALD antibody. Positive staining is observed in the vascular endothelium of the CNS at 8 d.p.f , but not at earlier timepoints (see Figure S2). High magnification images (Ci-iii) demonstrate the co- localisation of ABCB1/4/5 antibody staining (red) on cerebral vessels (green). The zebrafish BBB possesses active transport systems Bi and Ci – GFP channel – maximum intensity projection of the cerebral vasculature of Tg(fli1a:EGFP)y1 transgenic larvae; Bii and Cii – Alexa 568 labelled antibody staining with ABCB1/4/5 antibody; Biii and Ciii – overlay. doi: 10.1371/journal.pone.0077548.g003 In mammals, ABCB1 is present in epithelial cells specialised in the secretion and excretion of undesired molecules, particularly in the gut, liver, kidney and BBB. Several different variants of ABCB5 are expressed in different tissues in humans; ABCB5.ts encodes the longest protein (1257 aa), believed to be a full transporter (i.e. the single protein functions antihistamines. Unlike the first generation counterparts (e.g. hydroxyzine, diphenhydramine and triprolidine), loratadine, desloratadine (the active metabolite of loratadine), and cetirizine (the active metabolite of hydroxyzine) are actively effluxed from the brain by Pgp and therefore do not have a sedative effect [28]. PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 7 October 2013 \| Volume 8 \| Issue 10 \| e77548 Formation of the Zebrafish Blood-Brain Barrier Figure 4. Rhodamine 123 distribution is altered in the presence of verapamil. A) Dye distribution and quantifica experiments using rhodamine 123 (R123) were performed as described for Figure 1. Graph shows mean fluorescent intensity (± dev.) for each treatment. Rhodamine 123 (grey bars), a substrate for mammalian ABCB1, ABCB4 and ABCB5, was excluded the brain by 8 d.p.f. which, coincides with the onset of zebrafish Abcb/4/5 staining in the vasculature of the CNS. When 8 d.p.f. 10 d.p.f. larvae were incubated with verapamil (white bars), an inhibitor of ABCB1, ABCB4 and ABCB5 in mammals, R123 faile be excluded, consistent with blocked Abcb1/4/5 function. B and C) Representative images of parasagittal section of 5 d.p.f. la following pericardial injection of saline (B) or Rhodamine 123 (C). Fluorescence in the brain is observed following injectio Rhodamine 123. Scale bar represents 50 µm. doi: 10.1371/journal.pone.0077548.g004 Figure 4. Rhodamine 123 distribution is altered in the presence of verapamil. A) Dye distribution and quantification experiments using rhodamine 123 (R123) were performed as described for Figure 1. Graph shows mean fluorescent intensity (± std dev.) for each treatment. Rhodamine 123 (grey bars), a substrate for mammalian ABCB1, ABCB4 and ABCB5, was excluded from the brain by 8 d.p.f. which, coincides with the onset of zebrafish Abcb/4/5 staining in the vasculature of the CNS. When 8 d.p.f. and 10 d.p.f. The zebrafish BBB possesses active transport systems larvae were incubated with verapamil (white bars), an inhibitor of ABCB1, ABCB4 and ABCB5 in mammals, R123 failed to be excluded, consistent with blocked Abcb1/4/5 function. B and C) Representative images of parasagittal section of 5 d.p.f. larvae following pericardial injection of saline (B) or Rhodamine 123 (C). Fluorescence in the brain is observed following injection of Rhodamine 123. Scale bar represents 50 µm. doi: 10.1371/journal.pone.0077548.g004 zebrafish Abcb1/4/5 in the vasculature suggests that these proteins may fulfil the role of mammalian Pgp. By immunohistochemical and dye exclusion experiments, we have shown here both anatomical and functional evidence for active transport of molecules across the BBB in zebrafish. Abcb1/4/5 is first detected in the vascular endothelium of the CNS at 8 d.p.f. Thus whilst non-Pgp/ABCB5 substrate small molecules (e.g. sodium fluorescein) can penetrate the BBB and remain in as an efflux pump) with expression restricted to the testis; ABCB5 beta (812 aa) encodes a half-transporter which is only active as a dimer and expression is restricted to pigment cells. Evolutionary analysis of ABCB5 has shown that the ancestral gene is a full transporter and that ABCB5, 1, 4 and 11 share a common ancestor which began duplicating early in the evolution of the chordate lineage. Zebrafish have homologues of ABCB4 and ABCB5, but not ABCB1. The expression of October 2013 \| Volume 8 \| Issue 10 \| e77548 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 Formation of the Zebrafish Blood-Brain Barrier Table 2. Age-dependent changes in distribution of drugs in zebrafish larvae. The zebrafish BBB possesses active transport systems Drug (BBB penetration in mammals)LogP1 Age (d.p.f.) Absorption (whole body) (% uptake) Head concentration (µg/g)Trunk concentration (µg/g)Trunk/Head ratio Scopolamine 0.98 5 - - - - (penetrant) 8 - - - - 10 12.8 .65 .63 1 Scopolamine N-butyl bromide -1.11 5 7.2 .89 1.26 1.4 (excluded) 8 5.9 .88 .87 1 10 11.3 1.47 1.93 1.3 Diphenhydramine 3.27 5 142.4 21.63 21.08 1 (penetrant) 8 184.1 31.21 24.03 0.8 10 358.7 52.8 54.8 1 Haloperidol 4.3 5 732.6 115.5 104.29 0.9 (penetrant) 8 949.5 136.35 148.51 1.09 10 713.5 112.7 101.35 0.9 Desloratadine (excluded) 3.2 (XlogP) 5 14.8 2.31 2.14 0.9 8 39.1 2.57 3.29 1.3 10 28.5 3.38 5.17 1.5 Desloratadine + verapamil 5 18.4 2.89 2.64 0.9 8 7.9 1.17 1.2 1 10 48.9 8.57 6.12 0.7 1 LogP values were obtained from ChemIDplus (http://chem.sis.nlm.nih.gov/chemidplus/); where LogP values were not available, XLogP values were obtained from PubChem (http://pubchem.ncbi.nlm.nih.gov/). Zebrafish larvae were exposed to 15 µg/ml of the test compound (5 µg/ml for scopolamine) for 1 hour and then collected for LC/MS/MS analysis. Absorption in whole larvae was calculated as percentage of drug concentration in the tissue compared to the medium. Drug concentrations from head and trunk tissue were calculated from duplicate samples and trunk/head ratio was used to highlight unequal tissue distribution as an indicator of BBB effects. Differences of greater than 20% (i.e. trunk/head ratio > 1.2) were considered to indicate lack of BBB penetration. doi: 10.1371/journal.pone.0077548.t002 1 LogP values were obtained from ChemIDplus (http://chem.sis.nlm.nih.gov/chemidplus/); where LogP values were not available, XLogP values were obtained from PubChem (http://pubchem.ncbi.nlm.nih.gov/). Zebrafish larvae were exposed to 15 µg/ml of the test compound (5 µg/ml for scopolamine) for 1 hour and then collected for LC/MS/MS analysis. Absorption in whole larvae was calculated as percentage of drug concentration in the tissue compared to the medium. Drug concentrations from head and trunk tissue were calculated from duplicate samples and trunk/head ratio was used to highlight unequal tissue distribution as an indicator of BBB effects. Differences of greater than 20% (i.e. trunk/head ratio > 1.2) were considered to indicate lack of BBB penetration. doi: 10.1371/journal.pone.0077548.t002 the brain until day 10, Pgp/ABCB1/4/5 substrates (e.g. R123 and desloratidine) are actively effluxed from day 8. showed highest accumulation in larvae and the compounds with low logP (scopolamine and n-butyl scopolamine) showed least absorption. The zebrafish BBB possesses active transport systems However, 2 compounds with logP of approximately 3.2 (diphenhydramine and desloratidine) did not show a consistent pattern in the percentage of compound absorbed. This suggests that hydrophobic properties of a compound are not the sole determinant of their uptake into zebrafish and is in keeping with findings of previous studies [33]. Nevertheless, our study confirmed that all 5 compounds tested showed the same distribution in older zebrafish larvae as in mammals. An equal ratio of trunk/head drug distribution (i.e. trunk/head= 1.0) was observed at all ages for scopolamine, diphenhyramine and haloperidol, compounds known to cross the BBB in mammals. By contrast, ratios of greater than 1.2 were observed for scopolamine N-butyl bromide (at 10 d.p.f.) and desloratidine (at 8 and 10 d.p.f.), compounds that do not cross the mammalian BBB. In mammals, drug distribution is calculated as brain:plasma ratios. These studies show higher exclusion ratios than observed in zebrafish e.g. 1:8 for desloratadine and 1:20 for fexofenadine [34]. While this may suggest that the BBB may not yet be fully formed in larval zebrafish, our findings with dye exclusion assays would suggest otherwise. It is more likely that the lack of a more dramatic exclusion ratio is due to the exposure route (i.e. via immersion), or collection and analysis of whole head rather than brain tissue in the current study (in contrast to analysis on isolated brain and plasma samples in mammals). A more definitive analysis of compound distribution October 2013 \| Volume 8 \| Issue 10 \| e77548 A zebrafish model for BBB compound penetration The presence of the BBB has been one of the greatest obstacles in the development of drugs to treat neurological conditions, with less than 1% of small molecules penetrating this barrier. Various models have been developed to predict drug permeation through membranes. For example, measurements of drug partitioning in solvents is widely employed, although perfusion through solvents is not identical to diffusion across biological membranes and provides no information about active transport systems [29]. Indeed, as a result of transport proteins such as P-glycoproteins, the net distribution in the CNS of a variety of hydrophobic drugs, such as digoxin, cyclosporin and loperamide, is relatively low [30]. Models of BBB involving cell culture systems play some role in assessing whether drugs may cross the BBB, but are limited in that transport systems and blood-brain barrier enzymes may be severely down regulated or not present at all [31]. Furthermore, cells in such culture systems do not form rigid tight junctions. Here, we have demonstrated that zebrafish larva may be used as an in vivo tool for the prediction of compound distribution. In accordance with other studies on compound uptake in larval zebrafish [32,33], we observed a loose correlation between the amount of compound absorbed and its logP value. The compound with the highest logP, haloperidol, PLOS ONE \| www.plosone.org October 2013 \| Volume 8 \| Issue 10 \| e77548 9 Formation of the Zebrafish Blood-Brain Barrier ure 5. Transmission electron microscopy analysis of blood-brain barrier maturation. Parasaggital sections through the n of zebrafish larvae at 3 d.p.f. and 10 d.p.f. A) At 3 d.p.f., blood vessels (bv) surrounding the brain are simple in structure. B) At h resolution, only a single membrane is observed (arrows) no evidence of double membranes was observed at any location mined. C) At 10 d.p.f., blood vessels surrounding the CNS are more complex in structure. D) At high resolution, a double layer mbrane is apparent (arrows), indicative of the presence of tight junctions. E) In some vessels at 10 d.p.f., pericytes (pc) could be erved surrounding endothelial cells (ec). In addition, astrocyte endfeet (ae) were observed around some vessels at 10 d.p.f. but at earlier ages. Magnification: A & C 5.2K; B, D & E 15.5K. 10.1371/journal.pone.0077548.g005 Figure 5. Transmission electron microscopy analysis of blood-brain barrier maturation. Parasaggital sections through the brain of zebrafish larvae at 3 d.p.f. and 10 d.p.f. A zebrafish model for BBB compound penetration A) At 3 d.p.f., blood vessels (bv) surrounding the brain are simple in structure. B) At high resolution, only a single membrane is observed (arrows) no evidence of double membranes was observed at any location examined. C) At 10 d.p.f., blood vessels surrounding the CNS are more complex in structure. D) At high resolution, a double layer membrane is apparent (arrows), indicative of the presence of tight junctions. E) In some vessels at 10 d.p.f., pericytes (pc) could be observed surrounding endothelial cells (ec). In addition, astrocyte endfeet (ae) were observed around some vessels at 10 d.p.f. but not at earlier ages. Magnification: A & C 5.2K; B, D & E 15.5K. doi: 10.1371/journal.pone.0077548.g005 October 2013 \| Volume 8 \| Issue 10 \| e77548 10 PLOS ONE \| www.plosone.org Formation of the Zebrafish Blood-Brain Barrier before and after BBB formation might be achieved by injection of compounds directly into the circulation. However, in our study, we sought to replicate the conditions used in standard zebrafish chemical screens, i.e. compound exposure by immersion, in order to determine whether the formation of the BBB limits the penetration of certain compounds into CNS tissue. These findings will therefore be of more relevance to groups performing compound screens with neurological and neurobehavioural endpoints and suggest that even following immersion (where absorption is thought to occur through the skin, gut and gills), compounds may be (partially) excluded from the CNS in older larvae. Since the 5 d.p.f. zebrafish larva is only 2-3 mm in length, brain dissection was not possible, and head samples therefore also contained non-CNS tissue such as skin, bone, muscle and gills. Nevertheless, our results suggest that changes from the 1:1 head:trunk concentration ratio can be used as an indicator of exclusion from the brain. These data would therefore suggest that zebrafish may be useful in determining whether a compound would penetrate the BBB in vivo, but not for accurate quantification. For example, analysis of compound uptake in head versus trunk at 5 d.p.f. and 10 d.p.f. could be used as a method for predicting BBB penetration. In addition, analysis of drug distribution at different larval ages (i.e. before and after the onset of Abcb1/4/5 expression) allows analysis of whether a particular drug is actively effluxed. Supporting Information Figure S1. Alignment of human, mouse and zebrafish ABCB4 and ABCB5 protein sequences. A) Alignment of human, mouse and zebrafish ABCB4 protein sequences. B) Alignment of human, mouse and zebrafish ABCB5 protein sequences. Peptide sequences were aligned using ClustalW. (DOCX) A zebrafish model for BBB compound penetration Since the use of zebrafish screens to identify novel therapeutics is gaining popularity [35,36], these results allow for the rational screening of small molecules for CNS endpoints in zebrafish by appropriate age selection. Figure S2. Expression of zebrafish ABCB1/4/5 homologue at 3 d.p.f. Maximum intensity projection of the cerebral vasculature of Tg(fli1a:EGFP)y1 transgenic embryos at 3 d.p.f. A) GFP labels the vasculature; B) Alexa 568 labelled antibody staining with ABCB1/4/5 antibody; C) overlay. ABCB1/4/5 antibody does not co-localise with the vascular endothelium at 3 d.p.f. although positive antibody staining is observed in the liver primordium (arrowhead). Figure S3. Expression of GFAP around cerebral vessels. Maximum intensity projections of Tg(fli1a:EGFP)y1 transgenic larvae stained with GFAP antibody. A) At 7 d.p.f., GFAP staining is observed in the glia but does not co-localise with GFAP in the vasculature. B (low magnification) and C (high magnification) At 10 d.p.f., GFAP staining is observed in some cerebral vessels (arrowheads). Scale bar represents 50 µm. (TIF) Acknowledgements We are grateful to all the former DanioLabs aquarium staff for care and maintenance of the fish facility, Janet Powell and Jeremy Skepper for help with transmission electron microscopy, Ben Shaw for help with fluorescence microscopy and image processing and Anne Ferguson-Smith for allowing us to use the Zeiss StereoZoom and Apotome system. Table S3. MS Scan Parameters under APCI conditions. (DOCX) Table S3. MS Scan Parameters under APCI conditions. (DOCX) In summary, the demonstration of a BBB in zebrafish larvae lends further weight to their importance as an experimental species for drug discovery, particularly for neurological indications. Furthermore, since the barrier is formed in larvae by 10 d.p.f., zebrafish may provide a rapid and accurate in vivo model for the prediction of BBB permeability for novel compounds. Author Contributions Conceived and designed the experiments: AF HD PG. Performed the experiments: AF HD. Analyzed the data: AF HD PG. Contributed reagents/materials/analysis tools: AF HD. Wrote the manuscript: AF HD PG. Table S1. LC conditions for haloperidol, desloratadine and diphenhydramine. (DOCX) Table S1. LC conditions for haloperidol, desloratadine and diphenhydramine. (DOCX) 6. Xi Y, Noble S, Ekker M (2011) Modeling neurodegeneration in zebrafish. Curr Neurol Neurosci Rep 11: 274-282. doi:10.1007/ s11910-011-0182-2. PubMed: 21271309. 7. Jeong JY, Kwon HB, Ahn JC, Kang D, Kwon SH et al. (2008) Functional and developmental analysis of the blood-brain barrier in zebrafish. Brain. Res Bull 75: 619-628. doi:10.1016/j.brainresbull. 2007.10.043. 5. Stewart AM, Desmond D, Kyzar E, Gaikwad S, Roth A et al. (2012) Perspectives of zebrafish models of epilepsy: what, how and where next? Brain. Res Bull 87: 135-143. doi:10.1016/j.brainresbull. 2011.11.020. Formation of the Zebrafish Blood-Brain Barrier Levin VA (1980) Relationship of octanol/water partition coefficient and molecular weight to rat brain capillary permeability. J Med Chem 23: 682-684. doi:10.1021/jm00180a022. PubMed: 7392035. 15. Westerfield M, Doerry E, Kirkpatrick AE, Driever W, Douglas SA (1997) An on-line database for zebrafish development and genetics research. Seminars in Cell and Developmental Biology 8: 477-488. doi:10.1006/ scdb.1997.0173. PubMed: 9441953. 30. Schinkel AH, Wagenaar E, Mol CA, van Deemter L (1996) P- glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Invest 97: 2517-2524. doi:10.1172/JCI118699. PubMed: 8647944. 16. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203: 253-310. doi:10.1002/aja.1002030302. PubMed: 8589427. 31. Terasaki T, Ohtsuki S, Hori S, Takanaga H, Nakashima E et al. (2003) New approaches to in vitro models of blood-brain barrier drug transport. Drug Discov Today 8: 944-954. doi:10.1016/S1359-6446(03)02858-7. PubMed: 14554158. 17. Kaslin J, Panula P (2001) Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio). J Comp Neurol 440: 342-377. doi:10.1002/cne.1390. PubMed: 11745628. 18. Petito CK, Schaefer JA, Plum F (1977) Ultrastructural characteristics of the brain and blood-brain barrier in experimental seizures. Brain Res 127: 251-267. doi:10.1016/0006-8993(77)90539-X. PubMed: 861758. 32. Berghmans S, Butler P, Goldsmith P, Waldron G, Gardner I et al. (2008) Zebrafish based assays for the assessment of cardiac, visual and gut function--potential safety screens for early drug discovery. J Pharmacol Toxicol Methods 58: 59-68. doi:10.1016/j.vascn. 2008.05.130. PubMed: 18585469. 19. Johansson BB, Lund S (1978) Effect of sympathetic stimulation on the blood brain barrier dysfunction induced by amphetamine and by epileptic seizures. Acta Physiol Scand 104: 281-286. doi:10.1111/j. 1748-1716.1978.tb06280.x. PubMed: 716982. 33. Alderton W, Berghmans S, Butler P, Chassaing H, Fleming A et al. (2010) Accumulation and metabolism of drugs and CYP probe substrates in zebrafish larvae. Xenobiotica 40: 547-557. doi: 10.3109/00498254.2010.493960. PubMed: 20528625. 20. Hawkins BT, Egleton RD (2006) Fluorescence imaging of blood-brain barrier disruption. J Neurosci Methods 151: 262-267. doi:10.1016/ j.jneumeth.2005.08.006. PubMed: 16181683. 34. Kalvass JC, Maurer TS, Pollack GM (2007) Use of plasma and brain unbound fractions to assess the extent of brain distribution of thirty-four drugs: Comparison of unbound concentration ratios to in vivo P- glycoprotein efflux ratios. Drug Metab Dispos. 21. Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Formation of the Zebrafish Blood-Brain Barrier Natl Acad Sci U S A 87: 152-156. doi:10.1073/pnas.87.1.152. PubMed: 1688652. 8. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H et al. (2003) Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 161: 653-660. doi:10.1083/jcb.200302070. PubMed: 12743111. Natl Acad Sci U S A 87: 152-156. doi:10.1073/pnas.87.1.152. PubMed: 1688652. 24. Hung CC, Chen CC, Lin CJ, Liou HH (2008) Functional evaluation of polymorphisms in the human ABCB1 gene and the impact on clinical responses of antiepileptic drugs. Pharmacogenet Genomics 18: 390-402. doi:10.1097/FPC.0b013e3282f85e36. PubMed: 18408562. 9. Xie J, Farage E, Sugimoto M, Anand-Apte B (2010) A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development. BMC Dev Biol 10: 76. doi:10.1186/1471-213X-10-76. PubMed: 20653957. 25. Morita SY, Tsuda T, Horikami M, Teraoka R, Kitagawa S et al. (2013) Bile salt-stimulated phospholipid efflux mediated by ABCB4 localized in nonraft membranes. J Lipid Res 54: 1221-1230. doi:10.1194/ jlr.M032425. PubMed: 23468132. 10. Zheng PP, Romme E, van der Spek PJ, Dirven CM, Willemsen R et al. (2010) Glut1/SLC2A1 is crucial for the development of the blood-brain barrier in vivo. Ann Neurol 68: 835-844. doi:10.1002/ana.22318. PubMed: 21194153. 26. Moitra K, Scally M, McGee K, Lancaster G, Gold B et al. (2011) Molecular evolutionary analysis of ABCB5: the ancestral gene is a full transporter with potentially deleterious single nucleotide polymorphisms. PLOS ONE 6: e16318. doi:10.1371/journal.pone. 0016318. PubMed: 21298007. 11. Tam SJ, Richmond DL, Kaminker JS, Modrusan Z, Martin-McNulty B et al. (2012) Death receptors DR6 and TROY regulate brain vascular development. Dev Cell 22: 403-417. doi:10.1016/j.devcel.2011.11.018. PubMed: 22340501. 12. Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vasc Pharmacol 38: 323-337. doi:10.1016/S1537-1891(02)00200-8. PubMed: 12529927. 27. Fukunaga-Kalabis M, Martinez G, Nguyen TK, Kim D, Santiago-Walker A et al. (2010) Tenascin-C promotes melanoma progression by maintaining the ABCB5-positive side population. Oncogene 29: 6115-6124. doi:10.1038/onc.2010.350. PubMed: 20729912. 13. Stewart PA, Hayakawa EM (1987) Interendothelial junctional changes underlie the developmental 'tightening' of the blood-brain barrier. Brain Res 429: 271-281. PubMed: 3567665. 28. Chen C, Hanson E, Watson JW, Lee JS (2003) P-glycoprotein limits the brain penetration of nonsedating but not sedating H1-antagonists. Drug Metab Dispos 31: 312-318. doi:10.1124/dmd.31.3.312. PubMed: 12584158. 14. Lawson ND, Weinstein BM (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 248: 307-318. doi:10.1006/dbio.2002.0711. PubMed: 12167406. 29. References 1. Daneman R (2012) The blood-brain barrier in health and disease. Ann Neurol 72: 648-672. doi:10.1002/ana.23648. PubMed: 23280789. 2. Pardridge WM (2002) Drug and gene delivery to the brain: the vascular route. Neuron 36: 555-558. doi:10.1016/S0896-6273(02)01054-1. PubMed: 12441045. 3. Kokel D, Rennekamp AJ, Shah AH, Liebel U, Peterson RT (2012) Behavioral barcoding in the cloud: embracing data-intensive digital phenotyping in neuropharmacology. Trends Biotechnol 30: 421-425. doi:10.1016/j.tibtech.2012.05.001. PubMed: 22652049. 4. Sager JJ, Bai Q, Burton EA (2010) Transgenic zebrafish models of neurodegenerative diseases. Brain Struct Funct 214: 285-302. doi: 10.1007/s00429-009-0237-1. PubMed: 20162303. October 2013 \| Volume 8 \| Issue 10 \| e77548 11 PLOS ONE \| www.plosone.org Formation of the Zebrafish Blood-Brain Barrier Kozler P, Pokorný J (2003) Altered blood-brain barrier permeability and its effect on the distribution of Evans blue and sodium fluorescein in the rat brain applied by intracarotid injection. Physiol Res 52: 607-614. PubMed: 14535837. 35. Delvecchio C, Tiefenbach J, Krause HM (2011) The zebrafish: a powerful platform for in vivo, HTS drug discovery. Assay Drug Dev Technol 9: 354-361. doi:10.1089/adt.2010.0346. PubMed: 21309713. 22. Annilo T, Chen ZQ, Shulenin S, Costantino J, Thomas L et al. (2006) Evolution of the vertebrate ABC gene family: analysis of gene birth and death. Genomics 88: 1-11. doi:10.1016/j.ygeno.2006.03.001. PubMed: 16631343. 36. MacRae CA, Peterson RT Drug screening in the zebrafish: an overview. Drug Discovery Today: Disease Models. 23. Georges E, Bradley G, Gariepy J, Ling V (1990) Detection of P- glycoprotein isoforms by gene-specific monoclonal antibodies. Proc 12 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 12 October 2013 \| Volume 8 \| Issue 10 \| e77548
https://openalex.org/W2608093180	https://repository.ubn.ru.nl/bitstream/handle/2066/182726/1/182726.pdf	English	null	Current MitraClip experience, safety and feasibility in the Netherlands	Netherlands heart journal	2,017	cc-by	5,211	Current MitraClip experience, safety and feasibility in the Netherlands Rahhab, Z.; Kortlandt, F.A.; Velu, J.F.; Schurer, R.A.J.; Delgado, V.; Tonino, P.; Boven, A.J. van; Branden, B.J.L. Van den; Kraaijeveld, A.O.; Voskuil, M.; Hoorntje, J.; Wely, M.H. van; Houwelingen, K. van; Bleeker, G.B.; Rensing, B.; Kardys, I.; Baan, J., Jr.; Heyden, J.A.S. Van der; Mieghem, N.M. van Current MitraClip experience, safety and feasibility in the Netherlands Rahhab, Z.; Kortlandt, F.A.; Velu, J.F.; Schurer, R.A.J.; Delgado, V.; Tonino, P.; Boven, A.J. van; Branden, B.J.L. Van den; Kraaijeveld, A.O.; Voskuil, M.; Hoorntje, J.; Wely, M.H. van; Houwelingen, K. van; Bleeker, G.B.; Rensing, B.; Kardys, I.; Baan, J., Jr.; Heyden, J.A.S. Van der; Mieghem, N.M. van 2017, Article / Letter to editor (Netherlands Heart Journal, 25, 6, (2017), pp. 394-400) Doi link to publisher: https://doi.org/10.1007/s12471-017-0992-1 Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/182726 Download date: 2024-10-24 2017, Article / Letter to editor (Netherlands Heart Journal, 25, 6, (2017), pp. 394-400) Doi link to publisher: https://doi.org/10.1007/s12471-017-0992-1 Note: To cite this publication please use the final published version (if applicable). DOI 10.1007/s12471-017-0992-1 Neth Heart J (2017) 25:394–400 ORIGINAL ARTICLE - DESIGN STUDY ARTICLE Current MitraClip experience, safety and feasibility in the Netherlands Z. Rahhab1 · F. A. Kortlandt2 · J. F. Velu3 · R. A. J. Schurer4 · V. Delgado5 · P. Tonino6 · A. J. Boven7 · B. J. L. Van den Branden8 · A. O. Kraaijeveld9 · M. Voskuil9 · J. Hoorntje10 · M. van Wely11 · K. van Houwelingen12 · G. B. Bleeker13 · B. Rensing2 · I. Kardys1 · J. Baan jr.3 · J. A. S. Van der Heyden2 · N. M. Van Mieghem1 Published online: 25 April 2017 © The Author(s) 2017. This article is an open access publication. Published online: 25 April 2017 © The Author(s) 2017. This article is an open access publication. Published online: 25 April 2017 © The Author(s) 2017. This article is an open access publication. Device success and technical success were 91 and 95%, respectively, and were consistent over the years. Signif- icant reduction of mitral regurgitation by MitraClip was achieved in 94% of patients and was observed more often in patients with functional mitral regurgitation (95% vs. 91%, p = 0.025). Device time declined from 145 min in 2009 to 55 min in 2016. N. M. Van Mieghem n.vanmieghem@erasmusmc.nl Abstract Purpose Data on MitraClip procedural safety and efﬁ- cacy in the Netherlands are scarce. We aim to provide an overview of the Dutch MitraClip experience. Methods We pooled anonymised demographic and pro- cedural data of 1151 consecutive MitraClip patients, from 13 Dutch hospitals. Data was collected by product spe- cialists in collaboration with local operators. Effect on mi- tral regurgitation was intra-procedurally assessed by trans- oesophageal echocardiography. Technical success and de- vice success were deﬁned according to modiﬁed deﬁni- tions of the Mitral Valve Academic Research Consortium (MVARC). Conclusion MitraClip experience in the Netherlands is growing with excellent technical success and device suc- cess. Over the years, device time decreased and more pa- tients were treated with ≥2 Clips. Keywords Valvular heart disease · Mitral valve · Mitral valve therapies Results Median age was 76 (interquartile range 69–82) years and 59% were males. Patients presented with ≥mod- erate mitral regurgitation and a predominance of functional mitral regurgitation (72%). Overall, 611 (53%) patients were treated with one Clip, 486 (42%) with ≥2 Clips and 54 (5%) received no Clip. The number of patients with ≥2 Clips increased from 22% in 2009 to 52% in 2016. N. M. Van Mieghem n.vanmieghem@erasmusmc.nl 1 Department of Cardiology, Thorax center, Erasmus Medical Center, Rotterdam, The Netherlands 2 Department of Cardiology, St. 1 Department of Cardiology, Thorax center, Erasmus Medical Center, Rotterdam, The Netherlands N. M. Van Mieghem n.vanmieghem@erasmusmc.nl 1 Department of Cardiology, Thorax center, Erasmus Medical Center, Rotterdam, The Netherlands 2 Department of Cardiology, St. Antonius Hospital, Nieuwegein, The Netherlands 3 Department of Cardiology, Academic Medical Center, Amsterdam, The Netherlands 4 Department of Cardiology, University Medical Center Groningen, Groningen, The Netherlands 5 Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands 6 Department of Cardiology, Catharina Hospital, Eindhoven, The Netherlands N. M. Van Mieghem n.vanmieghem@erasmusmc.nl Abstract Antonius Hospital, Nieuwegein, The Netherlands 3 Department of Cardiology, Academic Medical Center, Amsterdam, The Netherlands 4 Department of Cardiology, University Medical Center Groningen, Groningen, The Netherlands 5 Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands 6 Department of Cardiology, Catharina Hospital, Eindhoven, The Netherlands 7 Department of Cardiology, Zorggroep Noorderbreedte, Leeuwarden, The Netherlands 7 Department of Cardiology, Zorggroep Noorderbreedte, Leeuwarden, The Netherlands 1 Department of Cardiology, Thorax center, Erasmus Medical Center, Rotterdam, The Netherlands 1 Department of Cardiology, Thorax center, Erasmus Medical Center, Rotterdam, The Netherlands 8 Department of Cardiology, Amphia Hospital, Breda, The Netherlands 9 Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands 10 Department of Cardiology, University Medical Center Maastricht, Maastricht, The Netherlands 11 Department of Cardiology, Radboud University Medical Center, Nijmegen, The Netherlands 4 Department of Cardiology, University Medical Center Groningen, Groningen, The Netherlands 12 Department of Cardiology, Medisch Spectrum Twente, Twente, The Netherlands 12 Department of Cardiology, Medisch Spectrum Twente, Twente, The Netherlands 5 Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands 13 Department of Cardiology, Haga Hospital, The Hague, The Netherlands 6 Department of Cardiology, Catharina Hospital, Eindhoven, The Netherlands 13 Department of Cardiology, Haga Hospital, The Hague, The Netherlands Neth Heart J (2017) 25:394–400 395 Introduction conforms to the principles outlined in the Declaration of Helsinki. conforms to the principles outlined in the Declaration of Helsinki. Mitral regurgitation (MR) has a 2% prevalence in the gen- eral population and is more frequent in the elderly [1, 2]. Surgical treatment is considered the ‘gold standard’ for patients with symptomatic severe mitral regurgitation [3]. However, a signiﬁcant proportion (49%) of eligible patients are denied for surgery because of age, comorbidities or poor left ventricular function [4]. Study endpoints and deﬁnitions The primary endpoints were procedural safety expressed in ‘technical success’ and procedural efﬁcacy expressed in ‘device success’, both were modiﬁed from the Mitral Valve Research Consortium (MVARC) criteria [10]. Methods This multicentre observational retrospective study collected all patients (n = 1151) from 13 Dutch hospitals treated with MitraClip between January 2009 and June 2016. All patients were discussed in local multi-disciplinary heart teams including interventional cardiologists, imaging spe- cialist and cardiac surgeons, and were considered symp- tomatic and at high operative risk. All patients provided written informed consent for the MitraClip procedure. ●Technical success is deﬁned as successful deployment of the device with absence of procedural mortality and free- dom from emergency surgery. ●Device success is deﬁned as proper placement of the de- vice without procedural mortality and with reduction in post-procedural MR by ≥1 grade from baseline and to an absolute level of moderate MR. ●Signiﬁcant MR reduction: reduction in post-procedural MR by ≥1 grade from baseline. Procedural data were prospectively and anonymously collected by product specialists in collaboration with local operators and, after approval of the participating centres, retrospectively analysed. Effect on MR was intra-procedu- rally (onsite) assessed by transoesophageal echocardiogra- phy. The Medical Ethics Committee of the Erasmus Medi- cal Center reviewed the study protocol and waived the need for additional informed consent because of the non-inter- ventional character of this retrospective study (MEC-2016- 423) using anonymous data collection. The investigation ●Device time is deﬁned as the time from guide catheter insertion to guide catheter removal. MitraClip procedure The MitraClip device is a 4 mm wide, polyester-covered cobalt chromium V-shaped clip with two movable arms and grippers (Fig. 1a). All procedures are performed under gen- eral anaesthesia, using ﬂuoroscopic and transoesophageal echocardiographic guidance. A 24-French guide catheter is introduced in a femoral vein and delivered into the left atrium after transseptal puncture (Fig. 1b). The clip de- livery system is advanced through the guide catheter into the left atrium and positioned above the origin of the re- gurgitation jet, perpendicular to the mitral coaptation line (Fig. 1c). The arms of the Clip are opened and advanced into the left ventricle. The Clip is then gradually pulled back towards the left atrium in order to grasp both mitral valve leaﬂets (Fig. 1d). The grippers are lowered, the clip is closed (Fig. 1e) and the leaﬂets are approximated result- ing in a double mitral oriﬁce (Fig. 1f). Before releasing the Clip, the severity of MR is assessed and the transmitral gra- dient is measured. If the result is satisfactory, the Clip can be released. In case of inadequate MR reduction or a high transmitral gradient, the Clip can be opened and reposi- tioned or removed. More than 1 Clip may be necessary for signiﬁcant MR reduction. The MitraClip (Abbott Vascular, Menlo Park, CA) of- fers a completely percutaneous mitral valve edge-to-edge repair. The efﬁcacy and safety of the MitraClip device have been demonstrated in the EVEREST I (Endovascular Valve Edge-to-Edge Repair Study) trial [5]. Subsequently, the EVEREST II randomised controlled trial compared con- ventional surgery with MitraClip in operable patients with moderate-to-severe or severe, predominantly degenerative MR [6]. MitraClip was associated with superior safety and similar improvements in clinical outcomes. However, it was less effective in reducing MR [6]. Based on these results, the Food and Drug Administration approved MitraClip for high-risk patients with symptomatic degenerative MR. In European practice, the majority of patients treated with Mi- traClip have functional MR [7, 8]. In this clinical setting, MitraClip may improve survival and hospital readmissions [9]. Data on the Dutch MitraClip experience are scarce. We therefore aim to provide an informative overview of the cur- rent MitraClip procedural safety and efﬁcacy in the Nether- lands. Results A total of 1151 patients underwent percutaneous mitral valve edge to-edge repair with the MitraClip device. Rel- ative contributions of the participating centres are sum- marised in Fig. 2a. The overall cohort had a median age of 76 (IQR 69–82) years and 59% were males. All patients presented with ≥moderate MR at baseline, with a clear Statistical analysis Categorical variables are presented as frequencies and per- centages, and compared with the use of the Pearson Chi Square Test or the Fisher’s exact test, as appropriate. Con- tinuous variables are presented as means (± standard devi- Neth Heart J (2017) 25:394–400 396 Fig. 1 a MitraClip device with two movable arms and grippers; b Guide catheter advanced into the left atrium after transseptal puncture; c Posi- tioning of the MitraClip above the regurgitation jet perpendicular to mitral coaptation line; d The MitraClip is pulled back in order to capture both leaﬂets; e The grippers are lowered and the arms are closed approximating the leaﬂets; and f creating a double mitral oriﬁce. Image courtesy of Abbott Fig. 1 a MitraClip device with two movable arms and grippers; b Guide catheter advanced into the left atrium after transseptal puncture; c Posi- tioning of the MitraClip above the regurgitation jet perpendicular to mitral coaptation line; d The MitraClip is pulled back in order to capture both leaﬂets; e The grippers are lowered and the arms are closed approximating the leaﬂets; and f creating a double mitral oriﬁce. Image courtesy of Abbott dominance of functional MR (72%) (Table 1). Overall, 611 (53%) patients were treated with one Clip, 486 (42%) with ≥2 Clips and 54 (5%) received no Clip (Table 2). The num- ber of patients treated with ≥2 Clips increased from 22% in 2009 to 52% in 2016 (Fig. 2). Signiﬁcant MR reduction (≥1 grade) was achieved in 94% of patients. ation – SD), in case of normal distribution, or medians (in- terquartile range – IQR), in case of skewed distribution, and compared with the use of the Student’s t-test or the Mann- Whitney U test. Normality of the distributions was assessed using the Shapiro-Wilk test. We used a two-sided alpha level of 0.05 to indicate signiﬁcance. Statistical analyses were performed using SPSS software version 21.0 (SPSS Inc., Chicago, Illinois, USA). The overall device and technical success were 91 and 95%, respectively, and were consistent over the years (Fig. 2b). Intra-procedural death and need for emergency surgery occurred in 3 (0.3%) and 6 (0.5%) patients, respec- tively (Table 2). The median device time declined from 145 (IQR 108–177) minutes in 2009 to 55 (IQR 34–86) minutes in 2016 (Fig. 2b). One vs. ≥two MitraClips Patients treated with ≥2 Clips were more often males (68% vs. 53%, p < 0.001) with degenerative MR (33% vs. 23%, p < 0.001) and severe MR at baseline (81% vs. 53%, p < 0.001). Signiﬁcant MR reduction was similar in both groups (98% vs. 98%, p = 0.59) (Fig. 4) while median device time was higher in ≥2 Clips group (86 [IQR 58–120] vs. 51 [IQR 35–75] minutes, p < 0.001). Degenerative vs. functional MR Patients with degenerative MR were older (median age 82 [IQR 76–85] vs. 74 [IQR 67–79] years, p < 0.001), had 397 Neth Heart J (2017) 25:394–400 Table 1 Baseline characteristics of patients undergoing MitraClip im- plantation Total population 2009–2016 (n = 1151) Male, n (%) 684 (59) Age, median (IQR) 76 (69–82) Etiology MR Degenerative, n (%) 198 (17) Functional, n (%) 832 (72) Mixed, n (%) 118 (10) Unknown, n (%) 3 (0.3) Severity of MR at baseline Moderate, n (%) 19 (2) Moderate-to-severe, n (%) 388 (34) Severe, n (%) 744 (65) LVEF <30%, n (%) 500 (43) IQR interquartile range, MR mitral regurgitation, LVEF left ventricular ejection fraction more often severe MR at baseline (73% vs. 61%, p < 0.001) and were more often treated with ≥2 Clips (50% vs. 39%, p = 0.001) when compared to patients with functional MR. Patients in the latter group had more often signiﬁcant MR reduction (95% vs. 91%, p = 0.025) (Fig. 3) and a shorter device time (62 [IQR 40–99] minutes vs. 75 [IQR 49–110] minutes, p < 0.001). more often severe MR at baseline (73% vs. 61%, p < 0.001) and were more often treated with ≥2 Clips (50% vs. 39%, p = 0.001) when compared to patients with functional MR. Patients in the latter group had more often signiﬁcant MR reduction (95% vs. 91%, p = 0.025) (Fig. 3) and a shorter device time (62 [IQR 40–99] minutes vs. 75 [IQR 49–110] minutes, p < 0.001). Table 1 Baseline characteristics of patients undergoing MitraClip im- plantation Discussion To date, more than 1250 patients have undergone MitraClip treatment in the Netherlands. We present the largest Dutch multi-centre MitraClip study including 1151 patients. Key ﬁndings are: 1) MitraClip was predominantly used to treat functional MR; 2) MitraClip was successful in reducing MR in 94% of patients; 3) MitraClip was slightly more effective in patients with functional MR; 4) Over the years, implan- tation of ≥2 Clips became more frequent; 5) With growing experience, procedure time decreased with preserved device success and technical success. Table 2 Procedural characteristics of patients undergoing MitraClip implantation Total population 2009–2016 (n = 1151) Clips 0 Clips, n (%) 54 (5) 1 Clip, n (%) 611 (53) ≥2 Clips, n (%) 486 (42) Device Time (min)a, median (IQR) 66 (42–103) MR reduction 0, n (%) 75 (7) 1, n (%) 108 (9) 2, n (%) 587 (51) 3, n (%) 381 (33) ≥1, n (%) 1076 (94) Device successb, n (%) 1049 (91) Technical successc, n (%) 1097 (95) Intra-procedural death, n (%) 3 (0.3) Emergency surgery, n (%) 6 (0.5) IQR i il MR i l i i Table 2 Procedural characteristics of patients undergoing MitraClip implantation Patient demographics in our study were comparable with large European registries (i. e. ACCESS-Europe A Two- Phase Observational Study of the MitraClip System in Europe (ACCESS-EU) and German Transcatheter Mi- tral Valve Interventions Registry [TRAMI]) but different from the EVEREST-II trial. The EVEREST trial was con- ducted in the USA and included younger patients (67.3 ± 12.8 years) with preserved left ventricular ejection fraction (60 ± 10.1). In Europe, MitraClip is more often applied in functional MR, which contrasts with the clear dominance (73%) of degenerative MR in the USA (Table 3; [6–8]). IQR interquartile range, MR mitral regurgitation In our study, MitraClip seemed slightly more effective in functional MR than in degenerative MR (95% vs. 91%, p = 0.025). Intra-procedural death and moderate MR after Clip implantation were comparable with the ACCESS-EU and TRAMI registry (0.3% vs. 0% vs. 0% and 92% vs. 91% vs. 97%, respectively), conﬁrming the safety and efﬁcacy of MitraClip (Table 3). Over the years, practice changed with a higher frequency of implanting ≥2 Clips. Patients treated with ≥2 Clips were more often males with degenerative MR and severe MR at baseline. Discussion Patients with degenerative MR may have thicker Neth Heart J (2017) 25:394–400 398 0 50 100 150 200 250 300 350 St. Antonius Hospital Nieuwegein Amsterdam MC UMC Groningen Leiden UMC Erasmus MC MC Leeuwarden Catharina Hospital Amphia Hospital MUC Maastricht Radboud Nijmegen UMC Utrecht MS Twente Haga The Hague Paents treated > 250 200 - 250 100 - 200 < 100 0 20 40 60 80 100 120 140 160 0 10 20 30 40 50 60 70 80 90 100 M i n u t e s (%) Median device me Device success Technical Succes ≥ 2 Clips implanted a b Fig. 2 Overview of a the relative contributions of the participating centres and b procedural characteristics and the primary endpoints over the years 0 50 100 150 200 250 300 350 St. Antonius Hospital Nieuwegein Amsterdam MC UMC Groningen Leiden UMC Erasmus MC MC Leeuwarden Catharina Hospital Amphia Hospital MUC Maastricht Radboud Nijmegen UMC Utrecht MS Twente Haga The Hague P d a 0 20 40 60 80 100 120 140 160 0 10 20 30 40 50 60 70 80 90 100 M i n u t e s (%) b b a Fig. 2 Overview of a the relative contributions of the participating centres and b procedural characteristics and the primary endpoints over the years Fig. 2 Overview of a the relative contributions of the participating centres and b procedural characteristics and years years 0 10 20 30 40 50 60 70 80 90 100 0 grade 1 grade 2 grades 3 grades ≥1 grades (%) MR reduction Overall DMR FMR p = 0.025 p = 0.002 p < 0.001 p = 0.12 p = 0.025 Overall p < 0.001 Fig. 3 Comparison of reduction of mitral regurgitation in patients with degenerative mitral regurgitation versus functional mitral regurgi- tation. MR mitral regurgitation, DMR degenerative mitral regurgitation, FMR functional mitral regurgitation 0 10 20 30 40 50 60 70 80 90 100 0 grade 1 grade 2 grades 3 grades ≥1 grades (%) MR reduction Overall DMR FMR p = 0.025 p = 0.002 p < 0.001 p = 0.12 p = 0.025 Overall p < 0.001 Fig. Discussion MR mitral regurgitation, LVEF left ventricular ejection fraction, SD standard deviation aDevice success: deﬁned as proper placement of the device without procedural mortality and with reduction in post-procedural MR by ≥1 grade from baseline and to an absolute level of moderate MR bTechnical success: deﬁned as successful deployment of the device with absence of procedural mortality and freedom from emergency surgery ne and procedural characteristics of patients undergoing MitraClip implantation in different cohorts g g , j , aDevice success: deﬁned as proper placement of the device without procedural mortality and with reduction in post-procedural MR by ≥1 grade from baseline and to an absolute level of moderate MR bTechnical success: deﬁned as successful deployment of the device with absence of procedural mortality and freedom from emergency surgery risk patients, resulting in positive left ventricular remod- elling and improvement of functional capacity [14, 15]. tion therapy, who fulﬁl the echo criteria of eligibility, are judged inoperable or at high surgical risk by a heart team, and have a life expectancy greater than 1 year (recommen- dation Class IIb, level of evidence C) [3]. The American guidelines consider transcatheter mitral valve repair only for severely symptomatic patients with chronic severe pri- mary MR who have favourable anatomy for the repair pro- cedure and a reasonable life expectancy, but who have a pro- hibitive surgical risk because of severe comorbidities and remain severely symptomatic despite optimal guideline-di- rected medical therapy for heart failure (recommendation Class IIb, level of evidence B) [13]. Yet, a wealth of recent clinical data underscores procedural safety and efﬁcacy of MitraClip and a favourable longer-term outcome in selected patients. MitraClip seems an excellent treatment strategy in patients who are deemed at very high or prohibitive op- erative risk by heart team consensus. Several studies have shown signiﬁcant MR reduction in the vast majority of high- Also, heart failure patients who do not respond effec- tively to cardiac resynchronisation therapy and have at least moderate MR can improve with MitraClip. Auricchio et al. showed that 73% of cardiac resynchronization therapy non- responders (with functional MR) improved in functional class, and had increased left ventricular ejection fraction and reduced ventricular volumes after MitraClip treatment [16]. Ongoing randomised trials further elaborate on the value of MitraClip in functional MR. Discussion 3 Comparison of reduction of mitral regurgitation in patients with degenerative mitral regurgitation versus functional mitral regurgi 0 10 20 30 40 50 60 70 80 90 100 0 grade 1 grade 2 grades 3 grades ≥1 grades (%) MR reduction Overall 1 MitraClip ≥2 MitraClips Overall p = 0.018 p = 0.59 p = 0.29 p = 0.001 p = 0.012 p = 0.59 Fig. 4 Comparison of mitral regurgitation reduction in patients treated with 1 versus ≥2 MitraClips. MR mitral regurgitation Fig. 4 Comparison of mitral regurgitation reduction in patients treated with 1 versus ≥2 MitraClips. MR mitral regurgitation Fig. 3 Comparison of reduction of mitral regurgitation in patients with degenerative mitral regurgitation versus functional mitral regurgi- tation. MR mitral regurgitation, DMR degenerative mitral regurgitation, FMR functional mitral regurgitation [95% CI; 1.2–5.3], p = 0.013) with 83% sensitivity and 90% speciﬁcity for a cut-off value of ≥7.5 mm [12]. The increased device time in degenerative MR may also be ex- plained by thicker and more mobile leaﬂets since this may aggravate the grasping process. Another reason is simply because of implantation of more Clips. and more mobile leaﬂets and had (in our cohort) more often severe MR at baseline, which may explain why these patients in particular are treated with ≥2 Clips. A previous study identiﬁed anterior leaﬂet thickness (OR 1.7 per mm [95% CI; 1.16–2.57], p = 0.007) and a greater regurgitation volume at baseline (OR 1.21 per 10 ml [95% CI; 1.0–1.3], p = 0.01) as echocardiographic predictors for the need for more than 1 Clip [11]. Another study showed that the vena contracta (jet width) predicted need for >1 Clip (OR 2.5 and more mobile leaﬂets and had (in our cohort) more often severe MR at baseline, which may explain why these patients in particular are treated with ≥2 Clips. A previous study identiﬁed anterior leaﬂet thickness (OR 1.7 per mm [95% CI; 1.16–2.57], p = 0.007) and a greater regurgitation volume at baseline (OR 1.21 per 10 ml [95% CI; 1.0–1.3], p = 0.01) as echocardiographic predictors for the need for more than 1 Clip [11]. Discussion Another study showed that the vena contracta (jet width) predicted need for >1 Clip (OR 2.5 According to the latest European guidelines on valvular heart disease, MitraClip may be considered in patients with symptomatic severe primary and secondary MR, despite optimal medical therapy, including cardiac resynchroniza- Neth Heart J (2017) 25:394–400 399 Table 3 Baseline and procedural characteristics of patients undergoing MitraClip implantation in different cohorts MitraClip Netherlands ACCESS-EU Phase I German TRAMI Registry EVEREST-II (n = 1151) (n = 567) n = 1064 n = 184 Male, n (%) 684 (59) 362 (64) 658 (62) 115 (63) Age (years) 76 (69–82) 73.7 ± 9.6 75 (70–81) 67.3 ± 12.8 Etiology MR Degenerative, n (%) 198 (17) 117 (23) 246 (29) 135 (73) Functional, n (%) 832 (72) 393 (77) 590 (71) 49 (27) Mixed, n (%) 118 (10) – – – Unknown, n (%) 3 (0.3) – – – Severity of MR at baseline Moderate, n (%) 19 (2) 13 (2) 42 (5) 8 (4) Moderate-to-severe, n (%) 388 (34) 230 (41) – 130 (71) Severe, n (%) 744 (65) 324 (57) 827 (95) 46 (25) LVEF <30%, n (%) 500 (43) 193 (34) 294 (33) N. A. LVEF, mean ± SD N. A. N. A. N. A. 60 ± 10.1 Procedural 0 Clips, n (%) 54 (5) 2 (0.4) N. A. N. A. 1 Clip, n (%) 611 (53) (60) N. A. N. A. ≥2 Clips 486 (42) (40) N. A. N. A. Severity of MR after Clip moderate, n (%) 1057 (92) 475 (91) 417 (97) (77) Moderate-to-severe, n (%) 57 (5) 39 (8) – 41 (23) Severe, n (%) 37 (3) 7 (1) 17 (3) – Device successa, n (%) 1049 (91) N. A. N. A. N. A. Technical successb, n (%) 1097 (95) N. A. N. A. N. A. Intra-procedural death, n (%) 3 (0.3) 0 (0) 0 (0) N. A. Emergency surgery, n (%) 6 (0.5) N. A. N. A. N. A. Limitations 7. Maisano F, Franzen O, Baldus S, et al. Percutaneous mitral valve interventions in the real world: early and 1-year results from the ACCESS-EU, a prospective, multicenter, nonrandomized post-ap- proval study of the MitraClip therapy in Europe. J Am Coll Cardiol. 2013;62:1052–61. Given the retrospective observational character of this study and the onsite assessment of MR (i. e. absence of echo core lab), potential self-reporting bias may be introduced. Spe- ciﬁc echocardiographic (quantitative) parameters such as regurgitation volume and jet width were not available. In addition, data were limited to procedural outcome. Follow- up data are needed to evaluate the durability of device suc- cess. 8. Schillinger W, Hünlich M, Baldus S, et al. Acute outcomes after MitraClip therapy in highly aged patients: results from the German TRAnscatheter Mitral valve Interventions (TRAMI) Registry. Eu- roIntervention. 2013;9:84–90. 9. Giannini C, Fiorelli F, De Carlo M, et al. Comparison of percuta- neous mitral valve repair versus conservative treatment in severe functional mitral regurgitation. Am J Cardiol. 2016;117:271–7. Long-term efﬁcacy may reveal recurrence of MR (grade 3 or 4) as shown by the EVEREST-II trial and AC- CESS-EU study with more than moderate MR recurrence rates of 21% at 12 months in both studies. Furthermore, we also acknowledge that complications such as stroke, bleeding and vascular complications, although rare, may occur during follow-up. 10. Stone GW, Adams DH, Abraham WT, Mitral Valve Academic Re- search C, et al. Clinical trial design principles and endpoint deﬁni- tions for transcatheter mitral valve repair and replacement: part 2: endpoint deﬁnitions: a consensus document from the Mitral Valve Academic Research Consortium. Eur Heart J. 2015;36:1878–91. 11. Armstrong EJ, Rogers JH, Swan CH, et al. Echocardiographic predictors of single versus dual MitraClip device implantation and long-term reduction of mitral regurgitation after percutaneous repair. Catheter Cardiovasc Interv. 2013;82:673–9. 12. Alegria-Barrero E, Chan PH, Foin N, et al. Concept of the cen- tral clip: when to use one or two MitraClips(R). EuroIntervention. 2014;9:1217–24. References 1. Nkomo VT, Gardin JM, Skelton TN, Gottdiener JS, Scott CG, En- riquez-Sarano M. Burden of valvular heart diseases: a population- based study. Lancet. 2006;368:1005–11. The Multicentre Study of Percutaneous Mitral Valve Re- pair MitraClip Device in Patients With Severe Secondary Mitral Regurgitation (MITRA-FR) is comparing the safety, efﬁcacy and the cost-effectiveness of OMT versus OMT plus MitraClip in patients with severe secondary mitral re- gurgitation. 2. Iung B, Vahanian A. Epidemiology of valvular heart disease in the adult. Nat Rev Cardiol. 2011;8:162–72. 3. Joint Task Force on the Management of Valvular Heart Disease of the European Society of C, European Association for Cardio-Tho- racic S, Vahanian A, Alﬁeri O, Andreotti F, et al. Guidelines on the management of valvular heart disease (version 2012). Eur Heart J. 2012;33:2451–96. 4. Mirabel M, Iung B, Baron G, et al. What are the characteristics of patients with severe, symptomatic, mitral regurgitation who are denied surgery? Eur Heart J. 2007;28:1358–65. Expectedly, focused guidelines on valvular heart dis- ease will be updated in the foreseeable future and include stronger recommendations for MitraClip. For now, our study demonstrated substantial MitraClip experience in the Netherlands with excellent procedural safety and efﬁcacy. 5. Feldman T, Wasserman HS, Herrmann HC, et al. Percutaneous mi- tral valve repair using the edge-to-edge technique: six-month re- sults of the EVEREST Phase I Clinical Trial. J Am Coll Cardiol. 2005;46:2134–40. 6. Feldman T, Foster E, Glower DD, EVEREST II Investigators, et al. Percutaneous repair or surgery for mitral regurgitation. N Engl J Med. 2011;364:1395–406. Discussion The MATTERHORN (Mi- tral vAlve reconsTrucTion for advancEd Insufﬁciency of Functional or iscHaemic ORigiN) trial, is comparing Mi- traClip with reconstructive mitral valve surgery in high- risk patients with moderate-to-severe functional MR. The Cardiovascular Outcomes Assessment of the MitraClip Per- cutaneous Therapy for Heart Failure Patients with Func- tional Mitral Regurgitation (COAPT) trial is investigating Neth Heart J (2017) 25:394–400 400 the safety and efﬁcacy of MitraClip versus optimal medical treatment (OMT) in patients with moderate-to-severe or se- vere functional MR who have been assessed as not eligible for mitral valve surgery. References Conclusion 13. Nishimura RA, Otto CM, Bonow RO, American College of Cardi- ology, American Heart Association, et al. 2014 AHA/ACC guide- line for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Thorac Cardio- vasc Surg. 2014;148:e1–e132. MitraClip experience in the Netherlands is growing with excellent technical success and device success. Over the years, the device time decreased and more patients were treated with ≥2 Clips. g 14. Van den Branden BJ, Swaans MJ, Post MC, et al. Percutaneous edge-to-edge mitral valve repair in high-surgical-risk patients: do we hit the target? JACC Cardiovasc Interv. 2012;5:105–11. Conﬂict of interest J. Baan jr, J.A.S. Van der Heyden, N.M. Van Mieghem have received unrestricted research grants from Abbott Vas- cular. Z. Rahhab, F.A. Kortlandt, J.F. Velu, R.A.J. Schurer, V. Del- gado, P. Tonino, A.J. Boven, B.J.L. van den Branden, A.O. Kraai- jeveld, M. Voskuil, J. Hoorntje, M. van Wely, K. van Houwelingen, G.B. Bleeker, B. Rensing and I. Kardys have no competing interests. 15. Whitlow PL, Feldman T, Pedersen WR, Investigators EI, et al. Acute and 12-month results with catheter-based mitral valve leaﬂet repair: the EVEREST II (Endovascular Valve Edge-to-Edge Re- pair) High Risk Study. J Am Coll Cardiol. 2012;59:130–9. 16. Auricchio A, Schillinger W, Meyer S, Investigators P-C, et al. Cor- rection of mitral regurgitation in nonresponders to cardiac resyn- chronization therapy by MitraClip improves symptoms and pro- motes reverse remodeling. J Am Coll Cardiol. 2011;58:2183–9. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
https://openalex.org/W2965045703	https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=150584&dn=	English	null	Characterization of Biomarker Levels in Crimean–Congo Hemorrhagic Fever and Hantavirus Fever with Renal Syndrome	Viruses	2,019	cc-by	11,276	Received: 14 June 2019; Accepted: 25 July 2019; Published: 26 July 2019 Abstract: Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are important viral hemorrhagic fevers (VHF), especially in the Balkan region. Infections with Dobrava or Puumala orthohantavirus and Crimean-Congo hemorrhagic fever orthonairovirus can vary from a mild, nonspeciﬁc febrile illness, to a severe disease with a fatal outcome. The pathogenesis of both diseases is poorly understood, but it has been suggested that a host’s immune mechanism might inﬂuence the pathogenesis of the diseases and survival. The aim of our study is to characterize cytokine response in patients with VHF in association with the disease progression and viral load. Forty soluble mediators of the immune response, coagulation, and endothelial dysfunction were measured in acute serum samples in 100 HFRS patients and 70 CCHF patients. HFRS and CCHF patients had signiﬁcantly increased levels of IL-6, IL-12p70, IP-10, INF-γ, TNF-α, GM-CSF, MCP-3, and MIP-1b in comparison to the control group. Interestingly, HFRS patients had higher concentrations of serum MIP-1α, MIP-1β, which promote activation of macrophages and NK cells. HFRS patients had increased concentrations of IFN-γ and TNF-α, while CCHF patients had signiﬁcantly higher concentrations of IFN-α and IL-8. In both, CCHF and HFRS patients’ viral load signiﬁcantly correlated with IP-10. Patients with fatal outcome had signiﬁcantly elevated concentrations of IL-6, IFN-α2 and MIP-1α, while GRO-α, chemokine related to activation of neutrophils and basophils, was downregulated. Our study provided a comprehensive characterization of biomarkers released in the acute stages of CCHF and HFRS. Keywords: VHF; CCHF; HFRS; cytokines; biomarkers Keywords: VHF; CCHF; HFRS; cytokines; biomarkers Characterization of Biomarker Levels in Crimean–Congo Hemorrhagic Fever and Hantavirus Fever with Renal Syndrome Miša Korva 1, Katarina Resman Rus 1, Miša Pavletiˇc 1, Ana Saksida 1, Nataša Knap 1, Mateja Jelovšek 1, Katja Strašek Smrdel 1, Xhevat Jakupi 2, Isme Humolli 2, Jusuf Dedushaj 2, Miroslav Petrovec 1 and Tatjana Avšiˇc-Županc 1,* Miša Korva 1, Katarina Resman Rus 1, Miša Pavletiˇc 1, Ana Saksida 1, Nataša Knap 1, Mateja Jelovšek 1, Katja Strašek Smrdel 1, Xhevat Jakupi 2, Isme Humolli 2, Jusuf Dedushaj 2, Miroslav Petrovec 1 and Tatjana Avšiˇc-Županc 1,* 1 Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia 2 National Institute of Public Health of Kosovo, 10000 Pristina, Kosovo * Correspondence: tatjana.avsic@mf.uni-lj.si; Tel.: +38-615-437-450 2 National Institute of Public Health of Kosovo, 10000 Pristina, Kosovo * Correspondence: tatjana.avsic@mf.uni-lj.si; Tel.: +38-615-437-450 * Correspondence: tatjana.avsic@mf.uni-lj.si; Tel.: +38-615-437-450 viruses viruses viruses Article Characterization of Biomarker Levels in Crimean–Congo Hemorrhagic Fever and Hantavirus Fever with Renal Syndrome Miša Korva 1, Katarina Resman Rus 1, Miša Pavletiˇc 1, Ana Saksida 1, Nataša Knap 1, Mateja Jelovšek 1, Katja Strašek Smrdel 1, Xhevat Jakupi 2, Isme Humolli 2, Jusuf Dedushaj 2, Miroslav Petrovec 1 and Tatjana Avšiˇc-Županc 1,* 1 Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia 2 National Institute of Public Health of Kosovo, 10000 Pristina, Kosovo * Correspondence: tatjana.avsic@mf.uni-lj.si; Tel.: +38-615-437-450 Received: 14 June 2019; Accepted: 25 July 2019; Published: 26 July 2019 viruses Article Characterization of Biomarker Levels in Crimean–Congo Hemorrhagic Fever and Hantavirus Fever with Renal Syndrome Miša Korva 1, Katarina Resman Rus 1, Miša Pavletiˇc 1, Ana Saksida 1, Nataša Knap 1, Mateja Jelovšek 1, Katja Strašek Smrdel 1, Xhevat Jakupi 2, Isme Humolli 2, Jusuf Dedushaj 2, Miroslav Petrovec 1 and Tatjana Avšiˇc-Županc 1,* 1 Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia 2 National Institute of Public Health of Kosovo, 10000 Pristina, Kosovo * Correspondence: tatjana.avsic@mf.uni-lj.si; Tel.: +38-615-437-450 Received: 14 June 2019; Accepted: 25 July 2019; Published: 26 July 2019 www.mdpi.com/journal/viruses 1. Introduction Viral hemorrhagic fevers (VHF) are an etiologically diverse group of zoonoses with common pathophysiology. Clinical manifestations of infections vary from asymptomatic or nonspeciﬁc febrile illness that can progress to hypovolemic shock and multi-organ failure and death. Two important causative agents of VHF are present in the Balkan area: Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) and orthohantavirus Dobrava (DOBV) and Puumala (PUUV). Crimean-Congo hemorrhagic fever (CCHF) is a potentially fatal disease with reported a fatality rate of up to 30%. Cases of CCHF are reported in Kosovo, Eastern Europe, Africa, Asia, and the Middle East [1–4]. The causative agent, CCHFV, is a negative-stranded RNA virus that belongs to the Nairoviridae family of the order Bunyavirales. The virus is transmitted through bites of infected Viruses 2019, 11, 686; doi:10.3390/v11080686 www.mdpi.com/journal/viruses Viruses 2019, 11, 686 2 of 19 Ixodid ticks, mainly Hyalomma spp., or via direct contact with blood or tissues of viraemic hosts [1,2,5]. Infection in humans is characterized by a febrile illness with headache, myalgia, and petechial rash, frequently followed by a hemorrhagic state with necrotic hepatitis. The acute stage of the disease in survivors usually lasts from 15 to 20 days and is followed by a convalescent period, characterized by prolonged weakness and confusion [1,2]. p g Pathogenic orthohantaviruses are geographically widespread zoonotic agents from the Hantaviridae family of the order Bunyavirales. They are genetically and evolutionally closely linked to their small mammal natural hosts [6]. The main transmission route of infection from rodents to humans occurs via inhalation of virus-contaminated aerosols from rodent excreta (urine, saliva or feces). Infection in humans can manifest in two primary forms, Hemorrhagic fever with renal syndrome (HFRS), endemic in Europe and Asia, or Hantavirus pulmonary syndrome (HPS), endemic in the Americas. The clinical spectrum of human infections ranges from asymptomatic infection to severe disease with fatal outcome, partly dependent on the causative virus and partly on host genetic and immune factors [6,7]. The onset of the disease symptoms is abrupt, with fever accompanied by myalgia, headache, transient myopia, nausea, vomiting, diarrhea, abdominal pain, back pain, ﬂushed face, and dizziness [8–10]. Although acute kidney injury is a distinctive feature of HFRS, various diﬀerent clinical manifestations can develop, with pulmonary, hemorrhagic, pancreatobiliary, central nervous system, endocrine, and cardiovascular events [6,11]. Despite many research attempts, the pathogenesis of VHF remains largely unknown. 1. Introduction There are several reasons for that: in vitro and in vivo research requires high containment facilities. First, there are no easily accessible animal models, and there is a limited number of patients, most often hospitalized in a remote area. Two VHF, present in the Balkan region, share common pathophysiological and clinical manifestations such as coagulopathy, thrombocytopenia, vascular permeability and hemorrhages. It is thought that the course and outcome of the disease depends on the viral load, host genetic factors and host immune response [12–16]. Despite the knowledge gap in the pathogenesis of VHF, the cytokine storm (i.e., uncontrolled release of cytokines and chemokines) is largely accepted [17,18]. In the present study, we enrolled 170 patients with CCHF or HFRS and measured the levels of cytokines and chemokines associated with innate, adaptive Th1, Th2 responses, regulatory T immune response and those involved in endothelial dysfunction and coagulopathy as the major clinical signs of the VHF. The aim of the study was to characterize biomarkers involved in the pathogenesis of both VHF. 2.1. Ethics Statement The study was performed retrospectively and no additional sample was taken for the purpose of the study. The use of HFRS samples was approved by the Republic of Slovenia National Medical Ethics Committee (no. 69/03/12; 30/04/15). Collection of CCHF serum samples was part of the CCH Fever network (Collaborative Project) supported by the European Commission under the Health Cooperation Work Programme of the 7th Framework Programme (grant agreement no. 260427). Research was approved by the National Medical Ethics Committee of the Republic of Kosovo (no. 05-3193/2). The study was conducted according to the Declaration of Helsinki, we followed the Oviedo Convention on Human Rights and Biomedicine and Slovenian Code of Medical Ethics. 2.3. Cytokines and Chemokines Concentrations of 40 cytokines/chemokines were measured in acute serum samples (ﬁrst seven days after onset of symptoms) with seven diﬀerent Human Cytokine/Chemokine Panels (HCYTOMAG-60K, HCYP3MAG-63K, HCVD2MAG-67K, HCVD3MAG-67K, HCVD4MAG-67K, HSP1MAG-63K and HAGP1MAG-12K; Milliplex, Merck Millipore, Burlington, MN, USA) on a MagPix instrument (Luminex, Austin, TX, USA). To minimize inter-assay variation, all measurements in a single panel were performed on the same day in one complete experiment according to the manufacturer’s instructions. All samples were previously aliquoted and diluted to a ﬁnal concentration 1:5. For all plates in a single panel, simultaneous analysis was done with Milliplex Analyst 5.1 software. In the study, we have investigated cytokines/chemokines associated with innate (granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-regulated oncogene-alpha (GRO-α/CXCL-1), interferon alpha 2 (IFN-α2), interleukin 1-alpha (IL-1α), IL-1β, interleukin-1 receptor antagonist (IL-1RA), IL-6, IL-8, IL-29, monocyte chemoattractant protein 1 (MCP-1/CCL2), MCP-3/CCL7, macrophage colony-stimulating factor (M-CSF), macrophage inﬂammatory protein 1-alpha (MIP-1α), MIP-1β/CCL4, tumor necrosis factor alpha (TNF-α)), adaptive Th1 (IFN-γ, IL-12p70, IL-12p40, IP-10), adaptive Th2 (IL-4, IL-5), regulatory T immune response (IL-10) and those involved in endothelial dysfunction and coagulopathy (Angiopoietin-2, Fibrinogen, d-dimer, plasminogen activator inhibitor (PAI-1), platelet factor 4 (PF4), soluble CD40 ligand (sCD40L), sE-Selectin, sL-Selectin sP-Selectin, soluble intracellular adhesion molecule sICAM-1, soluble vascular adhesion molecule sVCAM-1, soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1), Thrombomodulin (TM), Tissue factor (TF), VEGF A, von Willebrand factor (vWF), von Willebrand factor-cleaving protease (ADAMTS13)). 2.2. Patients A total of 100 HFRS (23 female and 77 male) and 70 CCHF patients (24 female and 45 male) were included in the study. All HFRS patients were hospitalized in Slovenian hospitals between the years 2000 and 2014. The diagnosis of HFRS was based on clinical ﬁndings (at least two out of three: fever >38 ◦C, acute kidney injury, thrombocytopenia) and was conﬁrmed serologically and molecularly as described in detail previously [8,19]. Patients’ blood samples were obtained at the time 3 of 19 Viruses 2019, 11, 686 of hospitalization (median 2 days; Table 1), when the clinical diagnosis was conﬁrmed with laboratory tests. According to the collected anamnestic data none of the patients received any treatment before blood collection. The serum was separated from blood cells and serum aliquots were stored at −80 ◦C until further use. Among HFRS patients, 50 were infected with DOBV (genotype Dobrava) and 50 infected with PUUV. Based on the disease severity and outcome, patients were distributed into three groups: patients with fatal outcome (n = 3), patients with severe disease (n = 51) and patients with mild disease (n = 49). All 70 CCHF patients were treated at the Infectious Disease Clinic, University Clinical Center of Kosova, Pristina, Kosovo between the years 2001 and 2011. Clinical diagnosis of CCHF was conﬁrmed with serological and molecular tests as described previously in detail [13,14]. Patients’ blood samples were obtained at the time of hospitalization for laboratory diagnostics (median two days; Table 1). According to the collected anamnestic data none of the patients received any treatment before blood collection. Serum was separated from blood cells and aliquots were stored at −80 ◦C until further use. Detailed medical charts were obtained for 57 CCHF patients. Based on disease progression and the outcome patients were distributed into three groups: patients with fatal outcome (n = 14), patients with severe disease (n = 18) and patients with moderate disease (n = 25). Additionally, 30 healthy age- and gender-matched controls were also enrolled in our study. Their blood samples were processed and stored as described for patients’ samples. The study was done retrospectively. All enrolled subject have signed inform consent for the studies. 3. Results Study population included 170 VHF patients infected with orthohantaviruses or CCHFV. Among 100 HFRS patients, 50 were infected with DOBV (genotype Dobrava) and 50 infected with PUUV. Serum samples were collected during the acute phase of infection, median on the 2nd day of hospitalization, which is between 3–11 days after self-reported onset of symptoms (Table 1). Medical charts were collected for each patient and based on selected clinical and laboratory criteria [19,20] they were categorized into mild or severe groups. Three patients had fatal outcome of the disease, and they were all infected with DOBV. The criteria for severe HFRS were: 1. the need for dialysis, or 2. the lowest systolic blood pressure <90 mm Hg and/or clinical signs of shock, or 3. thrombocytopenia <50 × 109/L and 4. the presence of a) bleeding and/or b) renal failure manifested with oliguria (diuresis <500 mL/day) and/or >4× higher than the upper normal level of urea or creatinine. Out of 100 HFRS patients, 48 patients (23 infected with DOBV, 25 infected with PUUV) fulﬁlled the criteria for severe disease and 49 patients were found to have mild disease (24 infected with DOBV, 25 infected with PUUV). In all HFRS patients, viral RNA was detected in the acute sample. The viral load was ranging from 0.2–7.6 log RNA copies/mL for PUUV infected patient and from 1.3–8.3 log RNA copies/mL for DOBV infected patients. On the contrary, IgM and IgG antibodies were detected in 98% and 46% of HFRS patients, respectively (Table 1). Among 70 patients with acute CCHF infection, 14 patients had fatal outcome of the disease. We were able to collect detailed medical charts for 43/56 surviving CCHF patients and based on selected clinical and laboratory criteria patients were categorized into moderate or severe groups. Surviving patients fulﬁlling at least three of the following criteria were deﬁned as having severe CCHF: The presence of profound hemorrhagic manifestations (blood transfusion required), increased serum creatinine values, increased serum transaminase values, and hypotension (blood pressure <100/60 mm Hg) [14]. None of the patients included in the study received ribavirin treatment. Out of 43 CCHF patients, 25 patients had moderate and 18 patients had severe disease. Also, all CCHF patients enrolled in our study were found to be PCR positive, with viral load ranging from 2.0–10.0 log RNA copies/mL (Table 1). As expected, the highest viral loads were detected in CCHF patients with fatal outcome. 2.4. Statistical Analyses Statistical calculations and analysis were performed in GraphPad Prism 8 (GraphPad Software, La Jolla, CA). Statistical analysis values above and below the upper and lower end of the standard cure, were considered as maximum and minimum values, respectively. Values above the maximum were measured only in CCHF fatal cases in two cytokines: M-CSF (n = 9) and Angiopoietin-2 (n = 5). Biomarkers with >50% of measurements out of range were excluded from the analysis. To analyze the normal distribution of data the D’Agostino-Pearson normality test was performed. 4 of 19 Viruses 2019, 11, 686 The identiﬁcation of outliers was performed using Dixon’s Q test. Statistically signiﬁcant diﬀerences in the serum concentrations of cytokines between severe and mild DOBV or PUUV infection were determined using the Mann–Whitney test (P). The Kruskal–Wallis test was used to determine diﬀerences among groups in comparison between moderate, severe and fatal cases of CCHF and HFRS and for comparison of cytokine levels among DOBV and PUUV infected patients and control groups. Correlations between biomarkers and viral loads of HFRS patients and CCHF patients were done with a nonparametric two-tailed Spearman correlation test with a 95% conﬁdence interval. To control for the false discovery rate, P values were adjusted for multiple comparisons. Diﬀerences with P < 0.05 were considered signiﬁcant. 3. Results Antibody response was measured only in the minority of CCHF patients, IgM antibodies were found in 33% patients and IgG in only 7.1% of patients (Table 1). Serum samples were collected during the acute phase of infection, median on the 2nd day of hospitalization, which is between 2–10 days after self-reported onset of symptoms (Table 1). Bleeding and profound endothelial dysfunction with capillary leakages, the hallmark of VHF pathology, have been reported in 10% of HFRS patients included in the study (six infected with DOBV and four infected with PUUV) and in 45% of CCHF patients. 5 of 19 Viruses 2019, 11, 686 Table 1. Antibody response, viral RNA load, self-reported onset of symptoms and day of hospitalization, by disease course and causative agent. Virus Disease Course No. of Enrolled Patients Self-Reported Onset of Symptoms (Median; Min/Max) Day of Hospitalization (Median; Min/Max) Viral load (Median ; Min/Max) IgG Pos/Neg (Min; Max Titer) IgM Pos/Neg (Min; Max Titer) CCHF moderate 25 7 (2–10) 2 (1–7) 5.6 (2.2–7.9) 2/25(800; 1600) 7/25 (800; >6400) severe 18 5 (2–8) 2 (1–4) 6.9 (2.7–8.9) 1/18 (100) 6/18 (1600; 6400) fatal 14 4 (2–7) 2 (1–2) 8.9 (2.7–10.0) 0/14 9/14 (400; 3200) P 70 6 (2–10) 2 (1–7) 6.6 (2.0–10.0) 5/70 (100; 1600) 23/70 (400; >6400) PUU mild 25 6 (3–10) 2 (1–6) 3.7 (0.2–7.0) 15/25 (400; >6400) 25/25 (100; >6400) severe 25 6 (3–9) 2 (1–8) 4.6 (2.6–7.6) 13/25 (100; >6400) 24/25 (400; >6400) P 50 6 (3–10) 2 (1–8) 4.4 (0.2–7.6) 27/50 (100; >6400) 49/50 (100; >6400) DOB mild 25 7 (3–11) 2 (1–8) 4.6 (1.3–8.3) 9/25 (800; 3200) 24/25 (800; >6400) severe 22 7 (3–11) 2 (1–6) 5.7 (2.1–7.7) 9/22 (100; >6400) 22/22 (1600; >6400) fatal 3 4 (3–4) 2 (1–4) 5.2 (4.3–6.8) 0/3 3/3 (1600; 6400) P 50 7 (3–11) 2 (1–8) 4.8 (1.3–8.3) 18/50 (100; >6400) 49/50 (800; >6400) determined with quantitative real-time RT-PCR (log RNA copies/mL); text in bold represents the values for the CCHF, PUU, DOB groups regardless of the disease progress. onset of symptoms and day of hospitalization, by disease course and causative agent. Viruses 2019, 11, 686 6 of 19 3.1. Comparison of Cytokine and Chemokine Levels in HFRS Patients 3.1. Comparison of Cytokine and Chemokine Levels in HFRS Patients In acute serum samples of HFRS patients 26/40 cytokines and chemokines diﬀer signiﬁcantly in comparison to healthy controls. Six cytokines (IL-1α, IL-1β, IL-5, IL-12p40, IL-29 and M-CSF) were below the lower limit of detection in >60% of patients and thus were not evaluated further. Signiﬁcantly increased cytokines and chemokines in HFRS patients were those predominantly associated with innate and adaptive Th1 immune response (IFN-γ, IL-1RA, IL-6, IL-8, IL-12p70, IP-10 and TNF-α) in comparison to the control group (Figure 1). In addition, HFRS patients had higher concentrations of serum MCP-1, MCP-3, MIP-1α and MIP-1β, which regulate migration of monocytes and activation of macrophages, and NK cells at the site of infection. Also, GM-CSF, an immune modulatory cytokine that has the ability to induce proliferation of granulocyte and macrophage populations from precursor cells and to activate T-cell immune response, was signiﬁcantly higher in HFRS patients in comparison to the control group (Figure 1). Patients with PUUV had higher levels of anti-inﬂammatory IL-10 and GRO-α than those infected with DOBV (Figure 1). On the other hand, DOBV infected patients with severe disease had signiﬁcantly elevated levels of IL-6, IL-8, IL1-RA, TNF-α and MCP-1, while PUUV infected patients with severe disease also had higher levels of IP-10 in comparison to those with mild disease (Figure 2). Additionally, we have investigated factors indicating endothelial dysfunction in patients with HFRS. HFRS patients had signiﬁcantly increased serum concentrations of soluble fractions of adhesion molecules (sE-Selectin, sL-Selectin and sP-Selectin) and sVCAM-1, PECAM-1 which facilitate the entry of leukocytes into inﬂamed tissues, in comparison to the control group. In addition to that, patients with HFRS, especially severe cases, also had signiﬁcantly higher concentrations of Angiopoietin-2, vWF, Fibrinogen, TM and TF in comparison to control group, suggesting changes in hemostasis (Figure 2). On the contrary, sCD40 ligand, which is considered to contribute to the promotion of prothrombotic responses and production of angiogenesis-associated factor, was signiﬁcantly downregulated in HFRS patients in comparison to controls or to mild disease, regardless of the virus in question (Figure 2). 7 of 19 9 7 of 19 9 Viruses 2019, 11, 686 Viruses 2019 11 686 Figure 1. The cytokine and chemokine concentrations with significant differences in HFRS patients (DOBV, n = 50; PUUV, n = 50) in comparison to control group (n = 30). 3.1. Comparison of Cytokine and Chemokine Levels in HFRS Patients The samples were obtained median 6 days after the onset of symptoms. The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identified outliers are not included in the figures above: IFN-γ (DOBV n = 4; PUUV n = 3), MCP-3 (DOBV n = 6; PUUV n = 5), IL-4 (DOBV n = 5; Figure 1. The cytokine and chemokine concentrations with signiﬁcant diﬀerences in HFRS patients (DOBV, n = 50; PUUV, n = 50) in comparison to control group (n = 30). The samples were obtained median 6 days after the onset of symptoms. The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identiﬁed outliers are not included in the ﬁgures above: IFN-γ (DOBV n = 4; PUUV n = 3), MCP-3 (DOBV n = 6; PUUV n = 5), IL-4 (DOBV n = 5; PUUV n = 5), MIP-1α (DOBV n = 3; PUUV n = 8), IL-8 (DOBV n = 7; PUUV n = 4), VEGF-A (PUUV n = 6) and Fibrinogen (DOBV n = 8; PUUV n = 2). Figure 1. The cytokine and chemokine concentrations with significant differences in HFRS patients (DOBV, n = 50; PUUV, n = 50) in comparison to control group (n = 30). The samples were obtained median 6 days after the onset of symptoms. The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identified outliers are not included in the figures above: IFN-γ (DOBV n = 4; PUUV n = 3), MCP-3 (DOBV n = 6; PUUV n = 5), IL-4 (DOBV n = 5; Figure 1. The cytokine and chemokine concentrations with signiﬁcant diﬀerences in HFRS patients (DOBV, n = 50; PUUV, n = 50) in comparison to control group (n = 30). The samples were obtained median 6 days after the onset of symptoms. The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). 3.1. Comparison of Cytokine and Chemokine Levels in HFRS Patients Identiﬁed outliers are not included in the ﬁgures above: IFN-γ (DOBV n = 4; PUUV n = 3), MCP-3 (DOBV n = 6; PUUV n = 5), IL-4 (DOBV n = 5; PUUV n = 5), MIP-1α (DOBV n = 3; PUUV n = 8), IL-8 (DOBV n = 7; PUUV n = 4), VEGF-A (PUUV n = 6) and Fibrinogen (DOBV n = 8; PUUV n = 2). 8 of 19 Viruses 2019, 11, 686 6) and Fibr Figure 2. The cytokine and chemokine concentrations with significant differences in patients infected with DOBV (severe n = 25; mild n = 25) or PUUV (severe n = 25; mild n = 25), with regard to the disease progression and outcome. The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identified outliers are not included in the figures above: IL- Figure 2. The cytokine and chemokine concentrations with signiﬁcant diﬀerences in patients infected with DOBV (severe n = 25; mild n = 25) or PUUV (severe n = 25; mild n = 25), with regard to th disease progression and outcome. The statistically signiﬁcant diﬀerences are marked with * (p < 0.05 ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whisker depict minimum and maximum values (range). Identiﬁed outliers are not included in the ﬁgure above: IL-6 (DOBV severe n = 4; DOBV mild n = 1; PUUV severe n = 1; PUUV mild n = 4), MCP- (DOBV severe n = 1; DOBV mild n = 4; PUUV severe n = 5; PUUV mild n = 4), IL-8 (DOBV severe n = 5 Figure 2. The cytokine and chemokine concentrations with significant differences in patients infected with DOBV (severe n = 25; mild n = 25) or PUUV (severe n = 25; mild n = 25), with regard to the disease progression and outcome. The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identified outliers are not included in the figures above: IL- Figure 2. 3.1. Comparison of Cytokine and Chemokine Levels in HFRS Patients The cytokine and chemokine concentrations with signiﬁcant diﬀerences in patients infected with DOBV (severe n = 25; mild n = 25) or PUUV (severe n = 25; mild n = 25), with regard to the disease progression and outcome. The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identiﬁed outliers are not included in the ﬁgures above: IL-6 (DOBV severe n = 4; DOBV mild n = 1; PUUV severe n = 1; PUUV mild n = 4), MCP-1 (DOBV severe n = 1; DOBV mild n = 4; PUUV severe n = 5; PUUV mild n = 4), IL-8 (DOBV severe n = 5; DOBV mild n = 3), D-dimer (PUUV mild n = 3), Fibrinogen (DOBV severe n = 5; DOBV mild n = 1; PUUV mild n = 2). Viruses 2019, 11, 686 9 of 19 3.2. Comparison of Cytokine and Chemokine Levels in CCHF Patients Identified outliers are not included in the figures above: GM- CSF (CCHF n = 5), IFN-α (CCHF n = 5), IFNγ (CCHF n = 1), GRO-α (CCHF, n = 6), IL-10 (CCHF n = 5), MCP-3 (CCHF n = 8), IL-4 (CCHF n = 8), IL-6 (CCHF n = 1), MCP-1 (CCHF n = 1), MIP-1β (CCHF n = 7), M-CSF (CCHF n = 1), VEGF-A (CCHF n = 6), TM (CCHF n = 5). Figure 3. The cytokine and chemokine concentrations with signiﬁcant diﬀerences in CCHF patients (n = 70) in comparison to control group (n = 30). The samples were obtained median six days after the onset of symptoms. The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identiﬁed outliers are not included in the ﬁgures above: GM-CSF (CCHF n = 5), IFN-α (CCHF n = 5), IFNγ (CCHF n = 1), GRO-α (CCHF, n = 6), IL-10 (CCHF n = 5), MCP-3 (CCHF n = 8), IL-4 (CCHF n = 8), IL-6 (CCHF n = 1), MCP-1 (CCHF n = 1), MIP-1β (CCHF n = 7), M-CSF (CCHF n = 1), VEGF-A (CCHF n = 6), TM (CCHF n = 5). 10 of 19 of 19 Viruses 2019, 11, 686 Viruses 2019 11 68 10 of 19 of 19 Figure 3. The cytokine and chemokine concentrations with significant differences in CCHF patients (n = 70) in comparison to control group (n = 30). The samples were obtained median six days after the onset of symptoms. The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identified outliers are not included in the figures above: GM- CSF (CCHF n = 5), IFN-α (CCHF n = 5), IFNγ (CCHF n = 1), GRO-α (CCHF, n = 6), IL-10 (CCHF n = 5), MCP-3 (CCHF n = 8), IL-4 (CCHF n = 8), IL-6 (CCHF n = 1), MCP-1 (CCHF n = 1), MIP-1β (CCHF n = 7), M-CSF (CCHF n = 1), VEGF-A (CCHF n = 6), TM (CCHF n = 5). Figure 3. The cytokine and chemokine concentrations with signiﬁcant diﬀerences in CCHF patients (n = 70) in comparison to control group (n = 30). 3.2. Comparison of Cytokine and Chemokine Levels in CCHF Patients 3.2. Comparison of Cytokine and Chemokine Levels in CCHF Patients In acute serum samples of CCHF patients, 25/40 cytokines and chemokines signiﬁcantly diﬀer in comparison to healthy control. The levels of ﬁve cytokines (IL-1α, IL-1β, IL-5, IL-12p40 and IL-29) were below the lower limit of detection in >60% of patients and thus were not evaluated further. Predominantly CCHF patients had signiﬁcantly elevated levels of cytokines and chemokines associated with innate and adaptive Th1 immune response (GM-CSF, IFN-α2, IFN-γ, IL-1RA, IL-6, IL-8, MCP-1, MCP-3, M-CSF, MIP-1β, TNF-α, IL-12p70, IP-10) in comparison to the control group (Figure 3). Regardless of the disease course and outcome most of the patients had signiﬁcantly elevated levels of IL-12p70, IFN-γ and IP-10, an IFN-γ inducible chemokine that attracts monocytes, macrophages, T cells, NK cells and dendritic cells. Among fatal cases, signiﬁcantly elevated concentrations of IL-6 and IL-10 were detected in comparison to the survivors (Figure 4). Also, fatal cases have higher levels of cytokines related to macrophage and granulocyte proliferation (MIP-1α, GM-CSF, M-CSF), while GRO-α, chemokine related to activation of neutrophils and basophils, was higher in patients with moderate disease progression (Figure 4). Since most of the patients with CCHF have hemorrhage related disorders (petechia, ecchymosis and melena), markers of endothelial dysfunction and coagulopathy were also investigated. CCHF patients had signiﬁcantly elevated levels of d-dimer, ﬁbrinogen, vWF, PECAM-1 and TM compared to control group. Fatal CCHF patients had signiﬁcantly higher levels of ﬁbrinogen and TM, which are involved in disseminated intravascular coagulation (DIC). On the contrary, both markers of platelet activation, soluble eﬀector molecule sCD40L and PF4, were higher in patients with moderate diseases progression (Figure 4). uses 2019, 11, 686 10 Viruses 2019, 11, 686 10 of 19 Figure 3. The cytokine and chemokine concentrations with significant differences in CCHF patients (n = 70) in comparison to control group (n = 30). The samples were obtained median six days after the onset of symptoms. The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). 3.2. Comparison of Cytokine and Chemokine Levels in CCHF Patients The samples were obtained median six days after the onset of symptoms. The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identiﬁed outliers are not included in the ﬁgures above: GM-CSF (CCHF n = 5), IFN-α (CCHF n = 5), IFNγ (CCHF n = 1), GRO-α (CCHF, n = 6), IL-10 (CCHF n = 5), MCP-3 (CCHF n = 8), IL-4 (CCHF n = 8), IL-6 (CCHF n = 1), MCP-1 (CCHF n = 1), MIP-1β (CCHF n = 7), M-CSF (CCHF n = 1), VEGF-A (CCHF n = 6), TM (CCHF n = 5). 11 of 19 11 of 19 Viruses 2019, 11, 686 Viruses 2019, 11, 686 , , Figure 4. The cytokine and chemokine concentrations with significant differences in CCHF patients, with regard to the disease progression and outcome (moderate n = 24; severe n = 18; fatal n = 14). The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identified outliers are not included in the figures above: GM-CSF (moderate n = 3; severe n = 1), IFN-α (moderate n = 2; fatal n = 1), GRO-α (moderate n = 2; severe n = 3), IL-10 (moderate n = 4; fatal n = 1), IL-4 (moderate n = 3; severe n = 2), IL-6 (moderate n = 7; severe n = 3; fatal n = 1), MIP-1α (moderate n = 6; severe n = 4; fatal n = 2). Figure 4. The cytokine and chemokine concentrations with signiﬁcant diﬀerences in CCHF patients, with regard to the disease progression and outcome (moderate n = 24; severe n = 18; fatal n = 14). The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). 3.3. Soluble Biomarkers Involved in the Pathogenesis of both VHF Viruses 2019, 11, 686 As both, CCHFV and orthohantaviruses can cause a wide spectrum of the disease, from asymptomatic disease to a fatal outcome, we wanted to compare cytokines and chemokines levels response in both groups. Interestingly, in our study HFRS patients had a more diverse and higher concentrations of most soluble biomarkers in comparison to the CCHF patients. HFRS patients had higher concentrations of TNF-α and IFN-γ, where CCHF patients had signiﬁcantly higher concentrations of IFN-α2 and IL-8 (Figure 5). Also, HFRS patients had higher concentrations of serum MIP-1α, MIP-1β, which promote activation of macrophages and NK cells. What is more, patients with HFRS had higher markers of endothelial dysfunction (Angiopoetin-2, sVCAM-1, PAI-1, ADAMTS13, d-dimer, sP-Selectin, ﬁbrinogen, vWF, sE-Selectin, sPECAM-1, TF and TM) than CCHF patients. 3.3. Soluble Biomarkers Involved in the Pathogenesis of both VHF As both, CCHFV and orthohantaviruses can cause a wide spectrum of the disease, from asymptomatic disease to a fatal outcome, we wanted to compare cytokines and chemokines levels response in both groups. Interestingly, in our study HFRS patients had a more diverse and higher concentrations of most soluble biomarkers in comparison to the CCHF patients. HFRS patients had higher concentrations of TNF-α and IFN-γ, where CCHF patients had significantly higher concentrations of IFN-α2 and IL-8 (Figure 5). Also, HFRS patients had higher concentrations of serum MIP-1α, MIP-1β, which promote activation of macrophages and NK cells. What is more, patients with HFRS had higher markers of endothelial dysfunction (Angiopoetin-2, sVCAM-1, PAI-1, ADAMTS13, d-dimer, sP-Selectin, fibrinogen, vWF, sE-Selectin, sPECAM-1, TF and TM) than CCHF patients. g p d-dimer, sP-Selectin, fibrinogen, vWF, sE-Selectin, sPECAM-1, TF and TM) than CCHF patients. Figure 5. The cytokine and chemokine concentrations with significant differences between in HFRS (n = 100) and CCHF (n = 70) patients. The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the Figure 5. The cytokine and chemokine concentrations with signiﬁcant diﬀerences between in HFRS (n = 100) and CCHF (n = 70) patients. The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). 3.2. Comparison of Cytokine and Chemokine Levels in CCHF Patients Identiﬁed outliers are not included in the ﬁgures above: GM-CSF (moderate n = 3; severe n = 1), IFN-α (moderate n = 2; fatal n = 1), GRO-α (moderate n = 2; severe n = 3), IL-10 (moderate n = 4; fatal n = 1), IL-4 (moderate n = 3; severe n = 2), IL-6 (moderate n = 7; severe n = 3; fatal n = 1), MIP-1α (moderate n = 6; severe n = 4; fatal n = 2). Figure 4. The cytokine and chemokine concentrations with significant differences in CCHF patients, with regard to the disease progression and outcome (moderate n = 24; severe n = 18; fatal n = 14). The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identified outliers are not included in the figures above: GM-CSF (moderate n = 3; severe n = 1), IFN-α (moderate n = 2; fatal n = 1), GRO-α (moderate n = 2; severe n = 3), IL-10 (moderate n = 4; fatal n = 1), IL-4 (moderate n = 3; severe n = 2), IL-6 (moderate n = 7; severe n = 3; fatal n = 1), Figure 4. The cytokine and chemokine concentrations with signiﬁcant diﬀerences in CCHF patients, with regard to the disease progression and outcome (moderate n = 24; severe n = 18; fatal n = 14). The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identiﬁed outliers are not included in the ﬁgures above: GM-CSF (moderate n = 3; severe n = 1), IFN-α (moderate n = 2; fatal n = 1), GRO-α (moderate n = 2; severe n = 3), IL-10 (moderate n = 4; fatal n = 1), IL-4 (moderate n = 3; severe n = 2), IL-6 (moderate n = 7; severe n = 3; fatal n = 1), MIP-1α (moderate n = 6; severe n = 4; fatal n = 2). Viruses 2019, 11, 686 12 of 19 12 of 19 3.3. Soluble Biomarkers Involved in the Pathogenesis of both VHF Viruses 2019, 11, 686 3.3. Soluble Biomarkers Involved in the Pathogenesis of both VHF Viruses 2019, 11, 686 3.3. Soluble Biomarkers Involved in the Pathogenesis of both VHF Viruses 2019, 11, 686 Identiﬁed outliers are not included in the ﬁgures above: GM-CSF (HFRS n = 8; CCHF n = 5), IFN-α2 (HFRS n = 4; CCHF n = 5), IFN-γ (HFRS n = 8; CCHF n = 1), IL-10 (HFRS n = 8; CCHF n = 5), TNF-α (HFRS n = 6; CCHF n = 3), Angiopoetin-2 (HFRS n = 9; CCHF n = 1), D-dimer (HFRS n = 1; CCHF n = 4), P-selectin (HFRS n = 4; CCHF n = 7), pCAM-1 (HFRS n = 4; CCHF n = 2), TF (HFRS n = 6; CCHF n = 8), TM (HFRS n = 2; CCHF n = 5). Figure 5. The cytokine and chemokine concentrations with significant differences between in HFRS (n = 100) and CCHF (n = 70) patients. The statistically significant differences are marked with * (p < 0.05). ns= not statistically significant. The boxes represents medians with interquartile ranges; the Figure 5. The cytokine and chemokine concentrations with signiﬁcant diﬀerences between in HFRS (n = 100) and CCHF (n = 70) patients. The statistically signiﬁcant diﬀerences are marked with * (p < 0.05). ns= not statistically signiﬁcant. The boxes represents medians with interquartile ranges; the whiskers depict minimum and maximum values (range). Identiﬁed outliers are not included in the ﬁgures above: GM-CSF (HFRS n = 8; CCHF n = 5), IFN-α2 (HFRS n = 4; CCHF n = 5), IFN-γ (HFRS n = 8; CCHF n = 1), IL-10 (HFRS n = 8; CCHF n = 5), TNF-α (HFRS n = 6; CCHF n = 3), Angiopoetin-2 (HFRS n = 9; CCHF n = 1), D-dimer (HFRS n = 1; CCHF n = 4), P-selectin (HFRS n = 4; CCHF n = 7), pCAM-1 (HFRS n = 4; CCHF n = 2), TF (HFRS n = 6; CCHF n = 8), TM (HFRS n = 2; CCHF n = 5). Viruses 2019, 11, 686 (HFRS n = 9; C pCAM 1 (HFR 13 of 19 7), In addition, we have explored a possible correlation between viral load and diﬀerent measured biomarkers. The strongest correlation was found between viral load and IP-10 in both CCHF (r = 0.62, p < 0.0001) and HFRS patients (r = 0.34, p = 0.0007) (Figure 6). Positive correlation between viral load and IFN-α2 (r = 0.25 p = 0.0118) was found in HFRS patients, but not in CCHF patients. 3.3. Soluble Biomarkers Involved in the Pathogenesis of both VHF Viruses 2019, 11, 686 Moreover, positive correlations between viral load and endothelial dysfunction markers (Angiopoietin-2 (r = 0.45 p = 0.0001), PAI-1 (r = 0.43 p = 0.0002), ADAMTS3 (r = 0.27 p = 0.0266), d-dimer (r = 0.31 p = 0.01), sPECAM-1 (r = 0.41 p = 0.0007), TF (r = 0.34 p = 0.0047) and TM (r = 0.43 p = 0.0002)) were found in CCHF patients. In HFRS patients, viral load was only associated with Angiopoietin-2 (r = 0.21 p = 0.0394). In addition, we have explored a possible correlation between viral load and different measured biomarkers. The strongest correlation was found between viral load and IP-10 in both CCHF (r = 0.62, p < 0.0001) and HFRS patients (r = 0.34, p = 0.0007) (Figure 6). Positive correlation between viral load and IFN-α2 (r = 0.25 p = 0.0118) was found in HFRS patients, but not in CCHF patients. Moreover, positive correlations between viral load and endothelial dysfunction markers (Angiopoietin-2 (r = 0.45 p = 0.0001), PAI-1 (r = 0.43 p = 0.0002), ADAMTS3 (r = 0.27 p = 0.0266), d-dimer (r = 0.31 p = 0.01), sPECAM-1 (r = 0.41 p = 0.0007), TF (r = 0.34 p = 0.0047) and TM (r = 0.43 p = 0.0002)) were found in CCHF patients. In HFRS patients, viral load was only associated with Angiopoietin-2 (r = 0.21 p = 0.0394). Figure 6. Correlation between biomarker IP-10 and viral loads of HFRS patients (A), CCHF patients (B), patients infected with DOBV (C) and patients infected with PUUV (D). Nonparametric two-tailed Spearman correlation test with 95% confidence interval was used Figure 6. Correlation between biomarker IP-10 and viral loads of HFRS patients (A), CCHF patients (B), patients infected with DOBV (C) and patients infected with PUUV (D). Nonparametric two-tailed Spearman correlation test with 95% conﬁdence interval was used. Figure 6. Correlation between biomarker IP-10 and viral loads of HFRS patients (A), CCHF patients (B), patients infected with DOBV (C) and patients infected with PUUV (D). Nonparametric two-tailed S ea a o elatio te t ith 95% o fide e i te al a u ed Figure 6. Correlation between biomarker IP-10 and viral loads of HFRS patients (A), CCHF patients (B), patients infected with DOBV (C) and patients infected with PUUV (D). Nonparametric two-tailed Spearman correlation test with 95% conﬁdence interval was used. Spearman c 4. Discussion Studies have shown that TNF-α contributes to hematophagocytosis trough macrophage activation, has an antiﬁbrinolytic action and may disrupt endothelial permeability and integrity [28,29]. In addition, IL-8 is known as the major chemotactic factor for neutrophil granulocytes and was shown to positively correlate with kidney dysfunction in PUUV infected HFRS patients and with fatal cases of CCHF [30,31]. Elevated levels of diﬀerent pro-inﬂammatory cytokines, especially in severe and fatal CCHF patients and in severe HFRS patients have already been reported previously [12,14,15,18,19,30,32–38]. Similar cytokine patterns observed in our study in CCHF and HFRS patients, were seen also in patients with Ebola virus disease (EVD) where elevated levels of IL-1β, IL-1RA, IL-6, IL-8, IL-15, MIP-1α, MIP-1β, MCP-1, MCP-3, IP-10 and eotaxin were associated with fatal outcome [39,40]. Interleukine-6 and TNFα were shown to cause endothelial barrier dysfunction via the protein kinase C pathway resulting in ultrastructural changes in distribution of tight junctions, intracellular actin disorganization and cellular morphological changes [41,42]. We have measured signiﬁcantly increased concentrations of IL-6 in fatal CCHF patients in comparison to the survivors. Increased serum levels of IL-6 were reported in other cases of VHF, such as EVD and Lassa fever [43–45]. Besides, our results support the previous ﬁndings [18,30], that higher levels of cytokines MIP-1α, GM-CSF, M-CSF, which are all related to macrophage and granulocyte activation, are signiﬁcantly increased in fatal CCHF patients. However, higher levels of MIP-1β, chemoattractant and activator of NK cells, have been reported in CCHF patients with milder disease [30], while in our study there was no correlation with disease progression. A recent study showed, that high concentrations of IP-10 and MCP-1 strongly correlate with high viral load and severity of the disease in CCHF patients [18,46,47]. Most CCHF patients included in our study had signiﬁcantly elevated levels of IP-10 regardless of the disease course or outcome. In HFRS patients, signiﬁcantly higher levels of IP-10 were observed in PUUV infected patients with severe disease in comparison to those with mild disease. Similar results were seen in DOBV infected patients, but the diﬀerence between severe and mild disease was not signiﬁcant. Elevated levels of IP-10 were also previously reported in Hantaan infected patients with severe disease [48]. Above that, we have discovered a strong positive correlation between viral load and IP-10 in both CCHF and HFRS patients. Positive correlation between viral load and IFN-α2 was established in HFRS patients, but not in CCHF patients. Spearman c 4. Discussion 4. Discussion In VHF a severe clinical manifestation with a fatal outcome is proposed to be a result of uncontrolled release of cytokines and chemokines (i.e., a cytokine storm) [17,18]. The innate immune response represents the first line of defense against various viral infections, including CCHF and HFRS. Secretion of type I cytokines, mainly IFN-α and IFN-β, lead to upregulation of interferon stimulated genes (ISGs) such as MxA, which was shown to inhibit viral replication of CCHF and orthohantaviruses via interaction with the viral nucleocapsid protein [21–25]. It was reported that In VHF a severe clinical manifestation with a fatal outcome is proposed to be a result of uncontrolled release of cytokines and chemokines (i.e., a cytokine storm) [17,18]. The innate immune response represents the ﬁrst line of defense against various viral infections, including CCHF and HFRS. Secretion of type I cytokines, mainly IFN-α and IFN-β, lead to upregulation of interferon stimulated genes (ISGs) such as MxA, which was shown to inhibit viral replication of CCHF and orthohantaviruses via interaction with the viral nucleocapsid protein [21–25]. It was reported that early activation of IFN I response can be delayed by antagonistic action of CCHFV and orthohantaviruses, thus enabling early replication of viruses and their systemic spread [26,27]. In our study, both CCHF and HFRS, patients had signiﬁcantly increased cytokines and chemokines associated with strong innate and adaptive Th1 immune response in comparison to the control group. HFRS patients had higher concentrations of TNF-α and IFN-γ, where CCHF patients had signiﬁcantly higher concentrations of IFN-α2 and IL-8, proposing activation of diﬀerent immune cells. 14 of 19 Viruses 2019, 11, 686 Studies have shown that TNF-α contributes to hematophagocytosis trough macrophage activation, has an antiﬁbrinolytic action and may disrupt endothelial permeability and integrity [28,29]. In addition, IL-8 is known as the major chemotactic factor for neutrophil granulocytes and was shown to positively correlate with kidney dysfunction in PUUV infected HFRS patients and with fatal cases of CCHF [30,31]. Elevated levels of diﬀerent pro-inﬂammatory cytokines, especially in severe and fatal CCHF patients and in severe HFRS patients have already been reported previously [12,14,15,18,19,30,32–38]. Similar cytokine patterns observed in our study in CCHF and HFRS patients, were seen also in patients with Ebola virus disease (EVD) where elevated levels of IL-1β, IL-1RA, IL-6, IL-8, IL-15, MIP-1α, MIP-1β, MCP-1, MCP-3, IP-10 and eotaxin were associated with fatal outcome [39,40]. Spearman c 4. Discussion This is conferred with our results where, in general, patients with HFRS had higher levels of markers indicating endothelial dysfunction than CCHF patients, despite the fact that CCHF has a fatality rate of up to 30% and that most severe or fatal patients present with hemorrhages. In our study, HFRS patients had signiﬁcantly increased serum concentrations of sE-Selectin, sL-Selectin and sP-Selectin and sVCAM-1, PECAM-1, which facilitate the entry of leukocytes into inﬂamed tissues. Increased expression of ICAM-1 and sVCAM-1 molecules by endothelial cells of blood vessels aﬀects rolling, adhesion and transmigration of leucocytes and can result in local inﬂammatory response that can lead to endothelial cell damage and bleeding [54,55]. In addition to that, patients with HFRS, especially severe cases, also had signiﬁcantly higher concentrations of Angiopoietin-2, vWF, Fibrinogen, TM and TF in comparison to control group, suggesting changes in hemostasis. Angiopoietin-2 is a proangiogenic factor that has recently been implicated in the direct control of inﬂammation-related signaling pathways [56]. In our study, a positive correlation between viral load and Angiopoietin-2 was observed for HFRS and CCHF patients. On the contrary, Angiopoetin-1 was reported to inhibit hantavirus directed endothelial cell permeability [57]. Besides, Angiopoietin-2 several other endothelial dysfunction markers, like PAI-1, ADAMTS3, d-dimer, sPECAM-1, TF and TM were found to correlate with viral load, but only in CCHF patients. CCHF patients had signiﬁcantly elevated levels of d-dimer, ﬁbrinogen, vWF, PECAM-1 and TM compared to control group. Fatal CCHF patients had signiﬁcantly higher levels of ﬁbrinogen and TM, which are involved in DIC. Endothelial dysfunction and disseminated intravascular coagulation, accompanied with increased expression of sICAM-1, sVCAM-1 and TM has also been reported in fatal EVD cases [40,58]. On the other hand, higher concentrations of two inﬂammatory factors, GRO-α and sCD40L, seem to play a protective role in vascular damage and hemorrhaging in VHF. Increased levels of GRO-α were reported in milder cases of Dengue infections without blood leakage [59,60]. In our study, patients with PUUV had higher levels of GRO-α than those infected with DOBV, thus they were able to more eﬃciently regulate the immune response resulting in milder disease. Even more, fatal CCHF patients and severe DOBV patients had signiﬁcantly lower concentrations of GRO-α in comparison to patients with milder disease. Similar ﬁndings were reported concerning sCD40L, an important proinﬂammatory factor released by activated platelets. HFRS and CCHF patients had signiﬁcantly lower levels of sCD40 ligand in comparison to controls. Spearman c 4. Discussion We have shown that DOBV infected patients with severe disease had signiﬁcantly higher levels of MCP-1, MCP-3 and MIP-1α, which regulate migration of monocytes, macrophages and NK cells at the site of infection. Also, MCP-1, induced by VEGF, is known to cause vascular leakage in vivo due to reduced tight junctions between vascular endothelium cells [49,50]. Increased levels of MCP-1 were observed in Dengue hemorrhagic fever patients with increased vascular permeability [50]. It is thought that elevated levels of anti-inﬂammatory IL-10 in early stages of infection impairs Th1 cell response and enables virus replication and spread. It was shown that elevated concentration of IL-10 correlate with viral load and severity of the disease in HFRS patients [19,35]. Similar ﬁndings have also been conveyed for fatal CCHF patients [14], but not for survived severe cases [2,15]. Elevated levels of IL-10 were reported in Dengue hemorrhagic fever and in fatal cases of EVD too [43,51]. In our study, HFRS and CCHF patients had higher levels of IL-10 in comparison to the control group. When comparing both VHF, surprisingly higher IL-10 concentrations were measured in HFRS patients in comparison to CCHF patients. Another anti-inﬂammatory cytokine, IL-4, an important activation marker of Th2 immune response, was shown to be upregulated in early phase of HFRS [46]. Some studies reported that IL-4 was signiﬁcantly upregulated also in the late phase of severe HFRS, while others claimed diﬀerent results [16,36]. IL-4, was shown to be also increased in patients with Dengue hemorrhagic fever [52,53]. In our study, signiﬁcantly higher concentrations of IL-4 were measured in HFRS infected patients in comparison to the control group, but there was no diﬀerence between the causative agents or disease severity. Concentrations of IL-4 were also signiﬁcantly higher in CCHF patients, where highest levels were measured in patients with a fatal outcome. g In both investigated VHF, endothelial dysfunction with temporary capillary leakage, which can result in tissue edema and organ failure is the major hallmark of the disease. In HFRS, the pathogenesis is 15 of 19 15 of 19 Viruses 2019, 11, 686 induced without any obvious cytopathology in the capillary endothelium. Thus, it implies that vascular leakage is more inﬂuenced by host immune response than virus characteristics. Author Contributions: Conceptualization, M.K., A.S., K.R.R. and T.A.-Ž.; formal analysis, M.P., M.J., K.S.S., N.K., K.R.R. and M.K.; investigation, M.K., K.R.R., M.J., K.S.S., N.K., M.P. and A.S.; resources, T.A.-Ž., M.P.P., J.D., X.J. and I.H.; writing—original draft preparation, M.K., K.R.R., M.P. and T.A.-Ž.; writing—review and editing, N.K., T.A.-Ž., M.P.P. and M.K.; funding acquisition, M.P.P. and T.A.-Ž. Funding: The study was supported by the Slovenian Research Agency (grant no. P3-0083) and by the European Virus Archive goes Global (EVAg) project that received funding from the European Union Horizon 2020 research and innovation program under grant agreement no. 653316. The funders had no role in the design of the study; Spearman c 4. Discussion The lowest concentrations of sCD40L were measured in CCHF patients with fatal outcome, who also had the most pronounced thrombocytopenia. Signiﬁcantly, increased levels of sCD40L were reported in milder cases of Dengue hemorrhagic fever and EVD, suggesting a potential protective role of sCD40L in pathogenesis of VHF. However, low sCD40 levels also correlated with low platelet counts that could result in observed decreased concentration of sCD40L in these patients [40,59]. VHF are a group of clinically similar diseases, but the intensity of clinical symptoms can vary from asymptomatic to lethal. The diﬀerences in disease progression can only partly be explained by the direct inﬂuence of the virus and are severely inﬂuenced by the host’s immune response. The majority of the studies in this ﬁeld have shown that both, low and massive immune response can lead to lethal disease, and only if controlled release of cytokines is produced the viral infection results in a milder disease [61]. The key question now is how to control and modify the release of cytokines in order to limit the impact of the disease; however, the answer to this question is yet to be discovered. Author Contributions: Conceptualization, M.K., A.S., K.R.R. and T.A.-Ž.; formal analysis, M.P., M.J., K.S.S., N.K., K.R.R. and M.K.; investigation, M.K., K.R.R., M.J., K.S.S., N.K., M.P. and A.S.; resources, T.A.-Ž., M.P.P., J.D., X.J. and I.H.; writing—original draft preparation, M.K., K.R.R., M.P. and T.A.-Ž.; writing—review and editing, N.K., T.A.-Ž., M.P.P. and M.K.; funding acquisition, M.P.P. and T.A.-Ž. Funding: The study was supported by the Slovenian Research Agency (grant no. P3-0083) and by the European Virus Archive goes Global (EVAg) project that received funding from the European Union Horizon 2020 research and innovation program under grant agreement no. 653316. The funders had no role in the design of the study; Viruses 2019, 11, 686 16 of 19 16 of 19 in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Acknowledgments: Without dedicated infectious disease specialists and specialists of internal medicine from diﬀerent Slovenian hospitals, no clinical research would be possible. References Whitehouse, C.A. Crimean-Congo hemorrhagic fever. Antivir. Res. 2004, 64, 145–160. [CrossRef] [PubMed 1. Whitehouse, C.A. Crimean-Congo hemorrhagic fever. Antivir. Res. 2004, 64, 145–160. [CrossRef] [PubMed] 2. Ergonul, O. Crimean-Congo haemorrhagic fever. Lancet Infect. Dis. 2006, 6, 203–214. [CrossRef] 2. Ergonul, O. Crimean-Congo haemorrhagic fever. Lancet Infect. Dis. 2006, 6, 203–214. [CrossRef] 2. Ergonul, O. Crimean-Congo haemorrhagic fever. Lancet Infect. Dis. 2006, 6, 203–214. [CrossRef] 3. Weber, F.; Mirazimi, A. Interferon and cytokine responses to Crimean Congo hemorrhagic fever virus; an emerging and neglected viral zonoosis. Cytokine Growth Factor Rev. 2008, 19, 395–404. [CrossRef] [PubMed] 3. Weber, F.; Mirazimi, A. Interferon and cytokine responses to Crimean Congo hemorrhagic fever virus; an emerging and neglected viral zonoosis. Cytokine Growth Factor Rev. 2008, 19, 395–404. [CrossRef] [PubMed] 4. Thomas, S.; Thomson, G.; Dowall, S.; Bruce, C.; Cook, N.; Easterbrook, L.; O’Donoghue, L.; Summers, S.; Ajazaj, L.; Hewson, R.; et al. Review of Crimean Congo hemorrhagic fever infection in Kosova in 2008 and 2009: Prolonged viremias and virus detected in urine by PCR. Vector Borne Zoonotic Dis. 2012, 12, 800–804. [CrossRef] [PubMed] 5. Bente, D.A.; Forrester, N.L.; Watts, D.M.; McAuley, A.J.; Whitehouse, C.A.; Bray, M. Crimean-Congo hemorrhagic fever: History, epidemiology, pathogenesis, clinical syndrome and genetic diversity. Antivir. Res. 2013, 100, 159–189. [CrossRef] [PubMed] 6. Vaheri, A.; Strandin, T.; Hepojoki, J.; Sironen, T.; Henttonen, H.; Makela, S.; Mustonen, J. Uncovering the mysteries of hantavirus infections. Nat. Rev. Microbiol. 2013, 11, 539–550. [CrossRef] [PubMed] 7. Krautkramer, E.; Grouls, S.; Hettwer, D.; Rafat, N.; Tonshoﬀ, B.; Zeier, M. Mobilization of circulating endothelial progenitor cells correlates with the clinical course of hantavirus disease. J. Virol. 2014, 88, 483–489. [CrossRef] 8. Avsic-Zupanc, T.; Petrovec, M.; Furlan, P.; Kaps, R.; Elgh, F.; Lundkvist, A. Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia—A 10-year survey. Clin. Infect. Dis. 1999, 28, 860–865. [CrossRef] 9. Pal, E.; Strle, F.; Avsic-Zupanc, T. Hemorrhagic fever with renal syndrome in the Pomurje region of Slovenia—An 18-year survey. Wien. Klin. Wochenschr. 2005, 117, 398–405. [CrossRef] 10. Pal, E.; Korva, M.; Rus, K.R.; Kejzar, N.; Bogovic, P.; Kurent, A.; Avsic-Zupanc, T.; Strle, F. Sequential assessment of clinical and laboratory parameters in patients with hemorrhagic fever with renal syndrome. PLoS ONE 2018, 13, e0197661. [CrossRef] 11. Krautkramer, E.; Grouls, S.; Stein, N.; Reiser, J.; Zeier, M. Pathogenic old world hantaviruses infect renal glomerular and tubular cells and induce disassembling of cell-to-cell contacts. J. Virol. Spearman c 4. Discussion Thus, we would like to thank all of them for their help in collecting HFRS samples and retrieving patients’ case records: Franc Strle, Stanka Lotriˇc Furlan, Petra Bogoviˇc (University Medical Centre Ljubljana); Božena Kotnik Kevorkijan, Zvonko Baklan, Sibila Unuk, Rajko Saletinger (University Medical Centre Maribor); Emil Pal (General Hospital Murska Sobota); Anica Kurent (General Hospital Novo mesto); Gorazd Lešniˇcar, Irena Milotiˇc, Branko Šibanc (General Hospital Celje); Zala Plešivˇcnik, Davorin Benko (General Hospital Slovenj Gradec); Matjaž Klemenc, Peter Viˇciˇc (General Hospital Nova Gorica). Conﬂicts of Interest: The authors declare no conﬂict of interest. References [PubMed] 22. Kanerva, M.; Melen, K.; Vaheri, A.; Julkunen, I. Inhibition of puumala and tula hantaviruses in Vero cells by 2. Kanerva, M.; Melen, K.; Vaheri, A.; Julkunen, I. Inhibition of puumala and tula hantaviruses in Vero cell MxA protein. Virology 1996, 224, 55–62. [CrossRef] [PubMed] 23. Andersson, I.; Bladh, L.; Mousavi-Jazi, M.; Magnusson, K.E.; Lundkvist, A.; Haller, O.; Mirazimi, A. Human MxA protein inhibits the replication of Crimean-Congo hemorrhagic fever virus. J. Virol. 2004, 78, 4323–4329. [CrossRef] [PubMed] 24. Levine, J.R.; Prescott, J.; Brown, K.S.; Best, S.M.; Ebihara, H.; Feldmann, H. Antagonism of type I interferon responses by new world hantaviruses. J. Virol. 2010, 84, 11790–11801. [CrossRef] [PubMed] 25. Resman Rus, K.; Korva, M.; Bogovic, P.; Pal, E.; Strle, F.; Avsic-Zupanc, T. Delayed Interferon Type 1-Induced Antiviral State Is a Potential Factor for Hemorrhagic Fever With Renal Syndrome Severity. J. Infect. Dis. 2018, 217, 926–932. [CrossRef] 26. Andersson, I.; Karlberg, H.; Mousavi-Jazi, M.; Martinez-Sobrido, L.; Weber, F.; Mirazimi, A. Crimean-Congo hemorrhagic fever virus delays activation of the innate immune response. J. Med. Virol. 2008, 80, 1397–1404. [CrossRef] [PubMed] 7. Fajs, L.; Resman, K.; Avsic-Zupanc, T. Crimean-Congo hemorrhagic fever virus nucleoprotein suppre IFN-beta-promoter-mediated gene expression. Arch. Virol. 2014, 159, 345–348. [CrossRef] 28. Fisman, D.N. Hemophagocytic syndromes and infection. Emerg. Infect. Dis. 2000, 6, 601–608. [CrossRef] g y y 29. Fernandez-Mestre, M.T.; Gendzekhadze, K.; Rivas-Vetencourt, P.; Layrisse, Z. TNF-alpha-308A allele, a possible severity risk factor of hemorrhagic manifestation in dengue fever patients. Tissue Antigens 2004, 64, 469–472. [CrossRef] 30. Ergonul, O.; Seref, C.; Eren, S.; Celikbas, A.; Baykam, N.; Dokuzoguz, B.; Gonen, M.; Can, F. Cytokine response in crimean-congo hemorrhagic fever virus infection. J. Med Virol. 2017, 89, 1707–1713. [CrossRef] 31. Strandin, T.; Makela, S.; Mustonen, J.; Vaheri, A. Neutrophil Activation in Acute Hemorrhagic Fever With Renal Syndrome Is Mediated by Hantavirus-Infected Microvascular Endothelial Cells. Front. Immunol. 2018, 9, 2098. [CrossRef] 32. Linderholm, M.; Ahlm, C.; Settergren, B.; Waage, A.; Tarnvik, A. Elevated plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-6, and IL-10 in patients with hemorrhagic fever with renal syndrome. J. Infect. Dis. 1996, 173, 38–43. [CrossRef] 33. Muranyi, W.; Bahr, U.; Zeier, M.; Van Der Woude, F.J. Hantavirus infection. J. Am. Soc. Nephrol. 2005, 16, 3669–3679. [CrossRef] 34. Sadeghi, M.; Eckerle, I.; Daniel, V.; Burkhardt, U.; Opelz, G.; Schnitzler, P. Cytokine expression during early and late phase of acute Puumala hantavirus infection. BMC Immunol. References 2011, 85, 9811–9823. [CrossRef] [PubMed] 12. Papa, A.; Bino, S.; Velo, E.; Harxhi, A.; Kota, M.; Antoniadis, A. Cytokine levels in Crimean-Congo hemorrhagic fever. J. Clin. Virol. 2006, 36, 272–276. [CrossRef] [PubMed] 13. Duh, D.; Saksida, A.; Petrovec, M.; Ahmeti, S.; Dedushaj, I.; Panning, M.; Drosten, C.; Avsic-Zupanc, T. Viral load as predictor of Crimean-Congo hemorrhagic fever outcome. Emerg. Infect. Dis. 2007, 13, 1769–1772. [CrossRef] [PubMed] 14. Saksida, A.; Duh, D.; Wraber, B.; Dedushaj, I.; Ahmeti, S.; Avsic-Zupanc, T. Interacting roles of immune mechanisms and viral load in the pathogenesis of crimean-congo hemorrhagic fever. Clin. Vaccine Immunol. 2010, 17, 1086–1093. [CrossRef] [PubMed] 15. Kaya, S.; Elaldi, N.; Kubar, A.; Gursoy, N.; Yilmaz, M.; Karakus, G.; Gunes, T.; Polat, Z.; Gozel, M.G.; Engin, A.; et al. Sequential determination of serum viral titers, virus-speciﬁc IgG antibodies, and TNF-alpha, IL-6, IL-10, and IFN-gamma levels in patients with Crimean-Congo hemorrhagic fever. BMC Infect. Dis. 2014, 14, 416. [CrossRef] 17 of 19 17 of 19 Viruses 2019, 11, 686 16. Ozsurekci, Y.; Arasli, M.; Karadag Oncel, E.; Caglayik, D.Y.; Kaya, A.; Icagasioglu, F.D.; Engin, A.; Korukluoglu, G.; Elaldi, N.; Ceyhan, M. Can the mild clinical course of crimean-congo hemorrhagic fever in children be explained by cytokine responses? J. Med. Virol. 2013, 85, 1955–1959. [CrossRef] [PubMed] 17. Garanina, E.; Martynova, E.; Davidyuk, Y.; Kabwe, E.; Ivanov, K.; Titova, A.; Markelova, M.; Zhuravleva, M.; Cherepnev, G.; Shakirova, V.G.; et al. Cytokine Storm Combined with Humoral Immune Response Defect in Fatal Hemorrhagic Fever with Renal Syndrome Case, Tatarstan, Russia. Viruses 2019, 11, 601. [CrossRef] [PubMed] 18. Papa, A.; Tsergouli, K.; Caglayik, D.Y.; Bino, S.; Como, N.; Uyar, Y.; Korukluoglu, G. Cytokines as biomarkers of Crimean-Congo hemorrhagic fever. J. Med. Virol. 2016, 88, 21–27. [CrossRef] 19. Korva, M.; Saksida, A.; Kejzar, N.; Schmaljohn, C.; Avsic-Zupanc, T. Viral load and immune response dynamics in patients with haemorrhagic fever with renal syndrome. Clin. Microbiol. Infect. 2013, 19, e358–e366. [CrossRef] 20. Saksida, A.; Duh, D.; Korva, M.; Avsic-Zupanc, T. Dobrava virus RNA load in patients who have hemorrhagic fever with renal syndrome. J. Infect. Dis. 2008, 197, 681–685. [CrossRef] 1. Frese, M.; Kochs, G.; Feldmann, H.; Hertkorn, C.; Haller, O. Inhibition of bunyaviruses, phleboviru and hantaviruses by human MxA protein. J. Virol. 1996, 70, 915–923. [PubMed] 21. Frese, M.; Kochs, G.; Feldmann, H.; Hertkorn, C.; Haller, O. Inhibition of bunyaviruses, phleboviruses, and hantaviruses by human MxA protein. J. Virol. 1996, 70, 915–923. References 2011, 12, 65. [CrossRef] 35. Saksida, A.; Wraber, B.; Avsic-Zupanc, T. Serum levels of inﬂammatory and regulatory cytokines in patients with hemorrhagic fever with renal syndrome. BMC Infect. Dis. 2011, 11, 142. [CrossRef] 36. Wang, P.Z.; Li, Z.D.; Yu, H.T.; Zhang, Y.; Wang, W.; Jiang, W.; Bai, X.F. Elevated serum concentrations of inﬂammatory cytokines and chemokines in patients with haemorrhagic fever with renal syndrome. J. Int. Med. Res. 2012, 40, 648–656. [CrossRef] Viruses 2019, 11, 686 18 of 19 18 of 19 37. Tsergouli, K.; Papa, A. Immune response in Dobrava-Belgrade virus infections. Arch. Virol. 2016, 161, 3413–3420. [CrossRef] 38. Guo, J.; Guo, X.; Wang, Y.; Tian, F.; Luo, W.; Zou, Y. Cytokine response to Hantaan virus infection in patients with hemorrhagic fever with renal syndrome. J. Med. Virol. 2017, 89, 1139–1145. [CrossRef] 39. Wauquier, N.; Becquart, P.; Padilla, C.; Baize, S.; Leroy, E.M. Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis. PLoS Negl. Trop. Dis. 2010, 4, e837. [CrossRef] 40. McElroy, A.K.; Erickson, B.R.; Flietstra, T.D.; Rollin, P.E.; Nichol, S.T.; Towner, J.S.; Spiropoulou, C.F. Ebola hemorrhagic Fever: Novel biomarker correlates of clinical outcome. J. Infect. Dis. 2014, 210, 558–566. [CrossRef] 41. Ferro, T.; Neumann, P.; Gertzberg, N.; Clements, R.; Johnson, A. Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000, 278, L1107–L1117. [CrossRef] 42. Desai, T.R.; Leeper, N.J.; Hynes, K.L.; Gewertz, B.L. Interleukin-6 causes endothelial barrier dysfunction via the protein kinase C pathway. J. Surg. Res. 2002, 104, 118–123. [CrossRef] 43. Baize, S.; Leroy, E.M.; Georges, A.J.; Georges-Courbot, M.C.; Capron, M.; Bedjabaga, I.; Lansoud-Soukate, J.; Mavoungou, E. Inﬂammatory responses in Ebola virus-infected patients. Clin. Exp. Immunol. 2002, 128, 163–168. [CrossRef] 44. Lukashevich, I.S.; Tikhonov, I.; Rodas, J.D.; Zapata, J.C.; Yang, Y.; Djavani, M.; Salvato, M.S. Arenavirus-mediated liver pathology: Acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation. J. Virol. 2003, 77, 1727–1737. [CrossRef] 45. Vernet, M.A.; Reynard, S.; Fizet, A.; Schaeﬀer, J.; Pannetier, D.; Guedj, J.; Rives, M.; Georges, N.; Garcia-Bonnet, N.; Sylla, A.I.; et al. Clinical, virological, and biological parameters associated with outcomes of Ebola virus infection in Macenta, Guinea. JCI Insight 2017, 2, e88864. [CrossRef] 46. Baigildina, A.A.; Khaiboullina, S.F.; Martynova, E.V.; Anokhin, V.A.; Lombardi, V.C.; Rizvanov, A.A. Inﬂammatory cytokines kinetics deﬁne the severity and phase of nephropathia epidemica. Biomark. References Med. 2015, 9, 99–107. [CrossRef] 47. Papa, A.; Yagci Caglayik, D.; Christova, I.; Tsergouli, K.; Korukluoglu, G.; Uyar, Y. Crimean-Congo hemorrhagic fever: CXCL10 correlates with the viral load. J. Med. Virol. 2015, 87, 899–903. [CrossRef] 48. Zhang, Y.; Liu, B.; Ma, Y.; Yi, J.; Zhang, C.; Zhang, Y.; Xu, Z.; Wang, J.; Yang, K.; Yang, A.; et al. Hantaan virus infection induces CXCL10 expression through TLR3, RIG-I, and MDA-5 pathways correlated with the disease severity. Mediat. Inﬂamm. 2014, 2014, 697837. [CrossRef] 49. Yamada, M.; Kim, S.; Egashira, K.; Takeya, M.; Ikeda, T.; Mimura, O.; Iwao, H. Molecular mechanism and role of endothelial monocyte chemoattractant protein-1 induction by vascular endothelial growth factor. Arterioscler. Thromb. Vasc. Biol. 2003, 23, 1996–2001. [CrossRef] 50. Lee, Y.R.; Liu, M.T.; Lei, H.Y.; Liu, C.C.; Wu, J.M.; Tung, Y.C.; Lin, Y.S.; Yeh, T.M.; Chen, S.H.; Liu, H.S. MCP-1, a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells. J. Gen. Virol. 2006, 87, 3623–3630. [CrossRef] 51. Perez, A.B.; Garcia, G.; Sierra, B.; Alvarez, M.; Vazquez, S.; Cabrera, M.V.; Rodriguez, R.; Rosario, D.; Martinez, E.; Denny, T.; et al. IL-10 levels in Dengue patients: Some ﬁndings from the exceptional epidemiological conditions in Cuba. J. Med. Virol. 2004, 73, 230–234. [CrossRef] 52. Abhishek, K.S.; Chakravarti, A.; Baveja, C.P.; Kumar, N.; Siddiqui, O.; Kumar, S. Association of interleukin-2, -4 and-10 with dengue severity. Indian J. Pathol. Microbiol. 2017, 60, 66–69. [CrossRef] 53. Butthep, P.; Chunhakan, S.; Yoksan, S.; Tangnararatchakit, K.; Chuansumrit, A. Alteration of cytokines and chemokines during febrile episodes associated with endothelial cell damage and plasma leakage in dengue hemorrhagic fever. Pediatr. Infect. Dis. J. 2012, 31, e232–e238. [CrossRef] 54. Rice, G.E.; Munro, J.M.; Corless, C.; Bevilacqua, M.P. Vascular and nonvascular expression of INCAM-110. A target for mononuclear leukocyte adhesion in normal and inﬂamed human tissues. Am. J. Pathol. 1991, 138, 385–393. 55. Van Gorp, E.C.; Suharti, C.; Ten Cate, H.; Dolmans, W.M.; Van Der Meer, J.W.; Ten Cate, J.W.; Brandjes, D.P. Review: Infectious diseases and coagulation disorders. J. Infect. Dis. 1999, 180, 176–186. [CrossRef] Viruses 2019, 11, 686 19 of 19 19 of 19 56. Scholz, A.; Plate, K.H.; Reiss, Y. Angiopoietin-2: A multifaceted cytokine that functions in both angiogenesis and inﬂammation. Ann. N. Y. Acad. Sci. 2015, 1347, 45–51. [CrossRef] 57. Gavrilovskaya, I.N.; Gorbunova, E.E.; Mackow, N.A.; Mackow, E.R. References Hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor VEGF, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability. J. Virol. 2008, 82, 5797–5806. [CrossRef] 58. Kerber, R.; Krumkamp, R.; Korva, M.; Rieger, T.; Wurr, S.; Duraﬀour, S.; Oestereich, L.; Gabriel, M.; Sissoko, D.; Anglaret, X.; et al. Kinetics of Soluble Mediators of the Host Response in Ebola Virus Disease. J. Infect. Dis. 2018, 218, S496–S503. [CrossRef] 59. De Sousa Cardozo, F.T.G.; Baimukanova, G.; Lanteri, M.C.; Keating, S.M.; Ferreira, F.M.; Heitman, J.; Pannuti, C.S.; Pati, S.; Romano, C.M.; Sabino, E.C. Serum from dengue virus-infected patients with and without plasma leakage diﬀerentially aﬀects endothelial cells barrier function in vitro. PLoS ONE 2017, 12, e0178820. [CrossRef] 60. Soe, H.J.; Khan, A.M.; Manikam, R.; Samudi Raju, C.; Vanhoutte, P.; Sekaran, S.D. High dengue virus load diﬀerentially modulates human microvascular endothelial barrier function during early infection. J. Gen. Virol. 2017, 98, 2993–3007. [CrossRef] 61. Perdomo-Celis, F.; Salvato, M.S.; Medina-Moreno, S.; Zapata, J.C. T-Cell Response to Viral Hemorrhagic Fevers. Vaccines 2019, 7, 11. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W1796070797	https://europepmc.org/articles/pmc4584051?pdf=render	English	null	Overexpression of<i>GPC6</i>and<i>TMEM132D</i>in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival	BioMed research international	2,015	cc-by	7,491	Athanasios Karapetsas,1 Antonis Giannakakis,1,2 Denarda Dangaj,1,3 Evripidis Lanitis,1,3 Spyridon Kynigopoulos,4 Maria Lambropoulou,4 Janos L. Tanyi,5 Alex Galanis,1 Stylianos Kakolyris,6 Gregorios Trypsianis,7 George Coukos,3 and Raphael Sandaltzopoulos1 Athanasios Karapetsas,1 Antonis Giannakakis,1,2 Denarda Dangaj,1,3 Evripidis Lanitis,1,3 Spyridon Kynigopoulos,4 Maria Lambropoulou,4 Janos L. Tanyi,5 Alex Galanis,1 Stylianos Kakolyris,6 Gregorios Trypsianis,7 George Coukos,3 and Raphael Sandaltzopoulos1 1Laboratory of Gene Expression, Molecular Diagnosis and Modern Therapeutics, Department of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece 2 h 2Division of Genome and Gene Expression Data Analysis, Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671 gy g p 3Department of Oncology, University Hospital of Lausanne (CHUV), Ludwig Cancer Research Center, University of Lausanne, 1011 Lausanne, Switzerland 3Department of Oncology, University Hospital of Lausanne (CHUV), Ludwig Cancer Research Center, University of Lausanne, 1011 Lausanne, Switzerland y f 4Laboratory of Histology and Embryology, School of Medicine, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece 4Laboratory of Histology and Embryology, School of Medicine, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece 4Laboratory of Histology and Embryology, School of Medicine, Faculty of Health Sciences, Democritus University of Thrace, 68100 Al d p li G p 5Division of Gynecologic Oncology, University of Pennsylvania, Philadelphia, PA 19014, USA rtment of Clinical Oncology, School of Medicine, Faculty of Health Sciences, Democritus University of Thrace, 0 Alexandroupolis, Greeceh 6Department of Clinical Oncology, School of Medicine, Faculty of Health Sciences, Democritus University of Thra 68100 Alexandroupolis, Greeceh Medical Statistics Lab, School of Medicine, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexand 7Medical Statistics Lab, School of Medicine, Faculty of Health Sciences, Democritus University of 7Medical Statistics Lab, School of Medicine, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece 7Medical Statistics Lab, School of Medicine, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece 7Medical Statistics Lab, School of Medicine, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greec Correspondence should be addressed to Raphael Sandaltzopoulos; rmsandal@mbg.duth.gr Received 12 January 2015; Revised 27 May 2015; Accepted 28 May 2015 Received 12 January 2015; Revised 27 May 2015; Accepted 28 May 2015 Academic Editor: Beric Henderson Copyright © 2015 Athanasios Karapetsas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 712438, 9 pages http://dx.doi.org/10.1155/2015/712438 Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 712438, 9 pages http://dx.doi.org/10.1155/2015/712438 Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 712438, 9 pages http://dx.doi.org/10.1155/2015/712438 Athanasios Karapetsas,1 Antonis Giannakakis,1,2 Denarda Dangaj,1,3 Evripidis Lanitis,1,3 Spyridon Kynigopoulos,4 Maria Lambropoulou,4 Janos L. Tanyi,5 Alex Galanis,1 Stylianos Kakolyris,6 Gregorios Trypsianis,7 George Coukos,3 and Raphael Sandaltzopoulos1 Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (𝑝= 0.0067) and 0.861 (𝑝< 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (𝑝= 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling. 1. Introduction About 22,000 new cases and 14,000 deaths are expected in the United States alone in 2014 [1]. Because of the lack of obvious and specific symptoms at the onset of the disease, the majority of the cases are diagnosed at a late stage. The five-year overall survival rate of ovarian cancer patients is Despite the slight decrease in the incidence and death rates of ovarian cancer over the recent years, it remains the major cause of death due to gynecological malignancies. 2 BioMed Research International 2 approximately 44%. On the contrary, patients with early stage ovarian cancer exhibit significantly higher survival rates [2]. The age at the time of diagnosis, the stage of the disease, the histological subtype, and tumor grade are common prognostic factors used to predict clinical outcome [3]. Similarly, the expression levels of several genes have been found to correlate with patients’ survival [4–7]. For instance, patients with low/intermediate levels of BRCA1 mRNA exhibit higher overall survival following treatment with platinum-based chemotherapy compared to patients with high levels of BRCA1 mRNA [8]. Thus, characterization of molecular markers with prognostic value is of great impor- tance in order to stratify high-risk ovarian cancer patients and implement the most appropriate therapeutic scheme. involved in intercellular signalling and cell-cell recognition, aspects critical for the mechanisms of cell attraction and recruitment. We validated with qPCR the expression of GPC6 and TMEM132D in a cohort of stage I-II ovarian cancer patients. The expression of both genes correlated positively with the CD8A marker and thus with T-cell infiltration. Furthermore, the expression of both genes was monitored in a variety of ovarian cancer cell lines. Ultimately, we performed a retrospective survival analysis of the early stage ovarian cancer patients and correlated the mRNA levels of GPC6 and TMEM132D with the overall survival. Patients with high mRNA levels of GPC6 and/or TMEM132D exhibited survival benefit compared to patients with low mRNA levels of both genes. p pp p p Similar to several other types of solid cancers, ovarian tumors are immunogenic with various immune cell popula- tions infiltrating the tumor sites. Zhang et al. demonstrated that intratumoral infiltration of CD3+ T-lymphocytes cor- relates significantly with high progression-free and overall survival of ovarian cancer patients [9]. Since then, a number of studies have highlighted the prognostic significance of T- cell infiltration in ovarian cancer [10–13]. 2. Materials and Methods 2.1. Patients and Specimens. Ovarian cancer tumor speci- mens were obtained from patients undergoing primary debu- lking surgery by surgeons within the Gynecologic Oncology Division at the University of Pennsylvania. The stage of the disease was determined by the gynecologic oncologists. The histology and grade of the tumor samples were established by the surgical pathologist. Specimens were immediately snap-frozen and stored at −80∘C. The tissue collection was approved by the IRB committee of the University of Pennsylvania. The analysis of the samples took place at the Department of Molecular Biology and Genetics, abiding to the guidelines of the Ethics Commission of the Democritus University of Thrace. 2.2. Cell Lines and Culture. Human epithelial ovarian cancer cell lines SKOV3, OVCAR3, OVCAR5, A1847, A2780, and C30 were acquired from ATCC and cultured in RPMI 1640 with stable glutamine, supplemented with 10% FBS, 100 U/mL penicillin, and 100 𝜇g/mL streptomycin (all from Biosera). Mouse ovarian cancer cell line ID8 was originally donated by Drs. Kathy Robby and Paul Terranova and cultured in DMEM high glucose with stable glutamine and sodium pyruvate (Biosera), supplemented with 10% FBS, 100 U/mL penicillin, and 100 𝜇g/mL streptomycin. Cell lines were cultured at 37∘C, 5% CO2, in a humidified atmosphere and passaged for fewer than six months since receipt and stock thawing. p Recently, utilizing a fluorescent version of the ADDER (Amplification of Double-Stranded cDNA End Restriction Fragments) Differential Display methodology, we identified, for the first time, genes overexpressed in early stage ovar- ian cancer which are associated with CD8+ T-lymphocyte infiltration [19]. For instance, the mRNA levels of one of the identified genes, SMARCE1, correlated significantly with the expression of CD8A, a marker of T-cell infiltration. Importantly, forced overexpression of SMARCE1 in ovarian cancer cells induced the expression and secretion of certain chemokines and consequently triggered the chemotaxis of CD8+ T-lymphocytes in vitro [19]. In the present study, we evaluated the expression of two other overexpressed genes, GPC6 and TMEM132D, and investigated whether they could represent novel prognostic markers of survival in early stage ovarian cancer. We selected to study these two genes as they are both surface antigens. TMEM132D is a transmembrane protein, while GPC6 is a GPI-anchored protein on the outer surface of the cell which may be cleaved off and released in the extracellular space. Through the heparan sulfate glycosaminoglycan chains GPC6 can interact with other molecules and receptors of the membrane. 1. Introduction For instance, it has been well documented that infiltration with high numbers of CD8+ T-lymphocytes associates positively with survival benefit and favorable clinical outcome [14, 15]. So far, gene expression profiling by microarrays has been employed by three independent research groups in order to elucidate the genes and the underlying molecular mechanisms that govern T-cell infiltration in ovarian cancer [16–18]. All of these studies focused on advanced stage ovarian cancer and each identified a number of differentially expressed genes that were associated with CD8+ T-lymphocyte infiltration and immune responses and even with survival [16–18].l 3. Results For relative quantification, the formula RQ = 2−(ΔΔCt) was used. Prior to using the ΔΔCt method for relative quanti- fication of the transcripts, validation experiments were per- formed by applying the relative standard curve method in order to demonstrate that the PCR efficiencies of the targets CD8A, GPC6, and TMEM132D and of the housekeeping gene b-actin were approximately equal. In general, each reaction was run in triplicate and each PCR experiment included two nontemplate controls. By employing a fluorescent version of ADDER Differential Display, we have recently reported the identification of 128 genes overexpressed in early stage ovarian tumors enriched with CD8+ T-lymphocytes (TIL+ tumors) [19]. GPC6 and TMEM132D, encoding for the heparan sulfate proteoglycan Glypican-6 and the transmembrane protein 132D, respec- tively, were included in the identified genes (Figures 1(a) and 2(a), resp.). Here, we further evaluated the expression of these two genes in early stage ovarian cancer. 2.5. ADDER Fluorescent Differential Display. ADDER was adapted from [20] for fluorescent detection as previously described [19]. 3.1. GPC6 and TMEM132D Are Differentially Expressed in TIL+ Early Stage Ovarian Cancer and Their Expression Levels Correlate with CD8+ T-Cell Infiltration. In order to validate the expression of GPC6 and TMEM132D in early stage ovarian cancer, we measured by qPCR their mRNA levels in tumor samples from 38 stage I-II ovarian cancer patients (Figures 1(b) and 2(b), resp.). The expression of both genes was detectable in all patient samples analyzed, at various levels. To investigate whether GPC6 and TMEM132D expression correlates with CD8+ T-lymphocyte infiltration in early stage ovarian cancer, we used qPCR to quantify the mRNA levels of the CD8A marker in the same cohort of patients. Interestingly, a statistically significant positive correlation between the mRNA levels of GPC6 or TMEM132D and CD8A accordingly was observed (Figures 1(c) and 2(c), resp.). Thus, the mRNA expression levels of GPC6 and TMEM132D correlate with CD8+ T-lymphocyte infiltration in early stage ovarian cancer. Moreover, we quantified by qPCR the relative expression of GPC6 and TMEM132D in a variety of ovarian cancer cell lines and demonstrated that the expression of both genes is not restricted to the infiltrating immune cells and may actually originate from the tumor cells. As shown in Figure 1(d), expression of GPC6 was documented in all cell lines tested except one (i.e., OVCAR5). Similarly, all ovarian cancer cell lines expressed TMEM132D (Figure 2(c)). To further validate the expression 2.6. Immunohistochemistry. 2. Materials and Methods Overall survival was defined as the time interval from diagnosis/first surgery to death or last follow-up. Multivariate Cox proportional hazards regression analysis was performed in order to evaluate the independent effect of GPC6 and TMEM132D mRNA levels on overall sur- vival. Multivariate regression models included stage, grade, histotype, and CD8A mRNA levels. All tests were two-tailed and statistical significance was considered for 𝑝values < 0.05. conditions: 95∘C/3 min and then 40 cycles at 95∘C/15 sec- onds and at 59∘C/1 min. The housekeeping gene b-actin was employed as internal control for normalization. The primers for CD8A, GPC6, TMEM132D, and b-actin were as follows: CD8A forward 5󸀠-CCCTGAGCAACTCCATCA- TGT-3󸀠and reverse 5󸀠-GGCTTCGCTGGCAGGAA-3󸀠, GPC6 forward 5󸀠-GGGCACAGCAAAGCCAGATA- 3󸀠and reverse 5󸀠-TGGTTGGTGAGCCCATCAT-3󸀠, TMEM132D forward 5󸀠-CACTGGTCGCCGGTA- CTCAT-3󸀠and reverse 5󸀠-GACCTTCCGTCACTTTGGAAAA-3󸀠, b-actin forward 5󸀠-GCGCGGCTACAGCTTCA-3󸀠 and 2. Materials and Methods Moreover, glypicans may regulate Hedgehog, Wnt, BMP, and FGF signaling pathways. On the contrary, the exact biological role of TMEM132D still remains quite elusive. The localization of these proteins suggests that they may be 2.3. RNA Isolation and Reverse Transcription. Total RNA was isolated from tissue or from 1 × 106 cells with TRIzol reagent (Life Technologies). The quality, integrity, and quantity of the isolated RNA were assessed spectrophotometrically and by gel electrophoresis. Following treatment with RNase-free DNase, total RNA was reverse-transcribed using Superscript First-Strand Synthesis Kit for RT-PCR (Life Technologies) according to manufacturer’s instructions. 2.4. Quantitative PCR. qPCR was performed on a StepOne real-time PCR system (Applied Biosystems) using the KAPA SYBR Fast qPCR kit (KAPA Biosystems) under the following BioMed Research International 3 2.7. Statistical Analysis. Graphs and statistical analysis of the data were performed with GraphPad Prism 5 and SPSS ver- sion 19.0. Correlation of GPC6 and TMEM132 mRNA levels with CD8A expression levels was examined by Pearson coef- ficient correlation. The chi-square test was used to assess the association of the expression levels of GPC6 and TMEM132D with patients’ clinicopathologic characteristics (stage, grade, and histotype). To study whether mRNA levels of GPC6 and TMEM132D were predictive for overall survival, survival rates were calculated with the Kaplan-Meier method and the statistical difference between survival curves was determined with the log-rank test. Overall survival was defined as the time interval from diagnosis/first surgery to death or last follow-up. Multivariate Cox proportional hazards regression analysis was performed in order to evaluate the independent effect of GPC6 and TMEM132D mRNA levels on overall sur- vival. Multivariate regression models included stage, grade, histotype, and CD8A mRNA levels. All tests were two-tailed and statistical significance was considered for 𝑝values < 0.05. 2.7. Statistical Analysis. Graphs and statistical analysis of the data were performed with GraphPad Prism 5 and SPSS ver- sion 19.0. Correlation of GPC6 and TMEM132 mRNA levels with CD8A expression levels was examined by Pearson coef- ficient correlation. The chi-square test was used to assess the association of the expression levels of GPC6 and TMEM132D with patients’ clinicopathologic characteristics (stage, grade, and histotype). To study whether mRNA levels of GPC6 and TMEM132D were predictive for overall survival, survival rates were calculated with the Kaplan-Meier method and the statistical difference between survival curves was determined with the log-rank test. 3. Results The relative expression of GPC6 was plotted along with the res on levels after normalization to b-actin. A pooled mix of equal amounts of all samples served as calibrator (Cal). (c) P fficient of CD8A-GPC6 expression levels. The correlation is significant (𝑝= 0.0067). (d) The relative expression of GP cell lines was estimated by qPCR (normalized to b-actin). (e-f) Immunohistochemistry of GPC6 expression in repres rian tumor samples. (e) Negative expression of GPC6 protein in the tumor sample 314 with low mRNA levels of the res (f) Strong expression of the GPC6 protein in the 408 sample with also high mRNA levels (see 1(b)). Magnification (e-f n, bars: SEM. (a) GPC6 (b) Pearson r 95% C.I. 0.427 38 GPC6 p value p = 0.0067 Number of XY pairs 0.129 to 0.654 (c) mRNA relative expression Cell lines 45 40 10 8 6 4 2 0 OVCAR3 OVCAR5 SKOV3 ID8 A2780 A1847 C30 GPC6 (d) Low GPC6 High GPC6 (d) 50𝜇m High GPC6 (f) 50𝜇m Low GPC6 (e) Low GPC6 (f) (e) Figure 1: GPC6 is overexpressed in TIL+ early stage ovarian cancer and its expression correlates positively with CD8+ T-lymphocyte infiltration. (a) GPC6 was overexpressed in the TIL+ ovarian cancer sample as visualized by fluorescent ADDER (band indicated by an arrow). Lane (+) displays the gene expression profile of the TIL+ sample generated by the corresponding Differential Display primer set; lane (−) displays the expression profile of the TIL−sample and lane (C) displays the combined profile of equal amount of PCR products of both samples. Genes expressed equally in both samples appear as yellow bands in lane (C). (b) The expression of GPC6 was quantified by qPCR in samples derived from 38 stage I-II ovarian cancer patients. The relative expression of GPC6 was plotted along with the respective CD8A expression levels after normalization to b-actin. A pooled mix of equal amounts of all samples served as calibrator (Cal). (c) Pearson correlation coefficient of CD8A-GPC6 expression levels. The correlation is significant (𝑝= 0.0067). (d) The relative expression of GPC6 in 7 ovarian cancer cell lines was estimated by qPCR (normalized to b-actin). (e-f) Immunohistochemistry of GPC6 expression in representative early stage ovarian tumor samples. (e) Negative expression of GPC6 protein in the tumor sample 314 with low mRNA levels of the respective gene (see 1(b)). 3. Results Paraffin embedded tissue sam- ples from ovarian tumors were available from patients who underwent surgery. Four-micron sections (4 𝜇m) of representative blocks from each case were deparaffinized, rehydrated, and treated with 0.3% H2O2 for 5 min in methanol to prevent endogenous peroxidase activity and were immunostained by the peroxidase method (Envision System, DAKO, Carpinteria, California, USA) according to the manufacturer’s recommendations. After antigen retrieval and endogenous peroxidase blockade, the sections were incubated overnight at 4∘C with polyclonal antibodies against Glypican-6 (Acris, Germany) and TMEM132D (Novus Bio, USA) in 1 : 100 and 1 : 80 dilutions, respectively. Then, the sec- tions were incubated with secondary antibody at room tem- perature for 60 min. Finally, bound antibody complexes were stained for 10 min with 0.05% diaminobenzidine. Sections were then briefly counterstained with Mayer’s haematoxylin, mounted, and examined under a Nikon Eclipse 50i micro- scope. Control slides were incubated for the same period with nonimmunized rabbit serum (negative control). A positive control was always run in the assay. The staining results were evaluated by a pathologist based on the percentage of staining in tumor cells. 4 BioMed Research International C − + (a) CD8A GPC6 mRNA relative expression 40 30 20 10 0 658 630 107 537 197 305 290 547 661 565 x2 568 402 409 573 269 230 261 314 254 555 288 395 522 458 608 328 98 334 526 108 x3 206 241 x1 350 418 408 Cal Patient (b) Pearson r 95% C.I. 0.427 38 GPC6 p value p = 0.0067 Number of XY pairs 0.129 to 0.654 (c) mRNA relative expression Cell lines 45 40 10 8 6 4 2 0 OVCAR3 OVCAR5 SKOV3 ID8 A2780 A1847 C30 GPC6 (d) 50𝜇m Low GPC6 (e) 50𝜇m High GPC6 (f) C6 is overexpressed in TIL+ early stage ovarian cancer and its expression correlates positively with CD8+ T-lymp GPC6 was overexpressed in the TIL+ ovarian cancer sample as visualized by fluorescent ADDER (band indicate +) displays the gene expression profile of the TIL+ sample generated by the corresponding Differential Display prim ys the expression profile of the TIL−sample and lane (C) displays the combined profile of equal amount of PCR prod Genes expressed equally in both samples appear as yellow bands in lane (C). (b) The expression of GPC6 was quant les derived from 38 stage I-II ovarian cancer patients. 3. Results (f) Strong expression of the GPC6 protein in the 408 sample with also high mRNA levels (see 1(b)). Magnification (e-f): ×400. Columns: mean, bars: SEM. 5 BioMed Research International C − + (a) 658 630 107 537 197 305 290 547 661 565 x2 568 402 409 573 269 230 261 314 254 555 288 395 522 458 608 328 98 334 526 108 x3 206 241 x1 350 418 408 Cal Patient mRNA relative expression CD8A TMEM132D 120 80 25 20 15 10 5 0 (b) Number of XY pairs Pearson r 95% C.I. 0.861 38 TMEM132D p value p < 0.0001 0.747 to 0.923 (c) TMEM132D mRNA relative expression Cell lines OVCAR3 OVCAR5 SKOV3 ID8 A2780 A1847 C30 15 10 5 0 (d) 50𝜇m Low TMEM132D (e) 50𝜇m High TMEM132D (f) M132D is overexpressed in TIL+ early stage ovarian cancer and its expression correlates positively with CD8+ TMEM132D was overexpressed in the TIL+ ovarian cancer sample as visualized by fluorescent ADDER (ban The expression of GPC6 was quantified by qPCR in samples derived from 38 stage I-II ovarian cancer patien MEM132D was plotted along with the respective CD8A expression levels after normalization to b-actin. A pool amples served as calibrator (Cal). (c) Pearson correlation coefficient of CD8A-TMEM132D expression levels.h = 0.0067). (d) The relative expression of TMEM132D in 7 ovarian cancer cell lines was estimated by qPCR mmunohistochemistry of TMEM132D expression in representative early stage ovarian tumor samples. (e) Nega protein in the tumor sample 408 with low mRNA levels of the respective gene (see 2(b)). (f) Very strong ex tein in the 314 sample with also high mRNA levels (see 2(b)). Magnification (e-f): ×400. Columns: mean, bar (a) 658 630 107 537 197 305 290 547 661 565 x2 568 402 409 573 269 230 261 314 254 555 288 395 522 458 608 328 98 334 526 108 x3 206 241 x1 350 418 408 Cal Patient m CD8A TMEM132D 5 0 (b) Number of XY pairs Pearson r 95% C.I. 0.861 38 TMEM132D p value p < 0.0001 0.747 to 0.923 (c) TMEM132D mRNA relative expression Cell lines OVCAR3 OVCAR5 SKOV3 ID8 A2780 A1847 C30 15 10 5 0 (d) 50𝜇m Low TMEM132D (e) 50𝜇m High TMEM132D (f) Figure 2: TMEM132D is overexpressed in TIL+ early stage ovarian cancer and its expression correlates positively with CD8+ T-lymphocyte infiltration. 3. Results (a) TMEM132D was overexpressed in the TIL+ ovarian cancer sample as visualized by fluorescent ADDER (band indicated by an arrow). (b) The expression of GPC6 was quantified by qPCR in samples derived from 38 stage I-II ovarian cancer patients. The relative expression of TMEM132D was plotted along with the respective CD8A expression levels after normalization to b-actin. A pooled mix of equal amounts of all samples served as calibrator (Cal). (c) Pearson correlation coefficient of CD8A-TMEM132D expression levels. The correlation is significant (𝑝= 0.0067). (d) The relative expression of TMEM132D in 7 ovarian cancer cell lines was estimated by qPCR (normalized to b-actin). (e-f) Immunohistochemistry of TMEM132D expression in representative early stage ovarian tumor samples. (e) Negative expression of TMEM132D protein in the tumor sample 408 with low mRNA levels of the respective gene (see 2(b)). (f) Very strong expression of the TMEM132D protein in the 314 sample with also high mRNA levels (see 2(b)). Magnification (e-f): ×400. Columns: mean, bars: SEM. ( ) 50𝜇m High TMEM132D (f) 50𝜇m Low TMEM132D (e) (e) (f) Figure 2: TMEM132D is overexpressed in TIL+ early stage ovarian cancer and its expression correlates positively with CD8+ T-lymphocyte infiltration. (a) TMEM132D was overexpressed in the TIL+ ovarian cancer sample as visualized by fluorescent ADDER (band indicated by an arrow). (b) The expression of GPC6 was quantified by qPCR in samples derived from 38 stage I-II ovarian cancer patients. The relative expression of TMEM132D was plotted along with the respective CD8A expression levels after normalization to b-actin. A pooled mix of equal amounts of all samples served as calibrator (Cal). (c) Pearson correlation coefficient of CD8A-TMEM132D expression levels. The correlation is significant (𝑝= 0.0067). (d) The relative expression of TMEM132D in 7 ovarian cancer cell lines was estimated by qPCR (normalized to b-actin). (e-f) Immunohistochemistry of TMEM132D expression in representative early stage ovarian tumor samples. (e) Negative expression of TMEM132D protein in the tumor sample 408 with low mRNA levels of the respective gene (see 2(b)). (f) Very strong expression of the TMEM132D protein in the 314 sample with also high mRNA levels (see 2(b)). Magnification (e-f): ×400. Columns: mean, bars: SEM. 6 BioMed Research International 6 Table 1: Clinicopathologic characteristics of the stage I/II ovarian cancer patients. Table 1: Clinicopathologic characteristics of the stage I/II ovarian cancer patients. Table 1: Clinicopathologic characteristics of the stage I/II ovarian cancer patients. 3. Results Consistently, the tumors with low mRNA levels of TMEM132D showed little expression at protein level, whereas the tumors with high levels of TMEM132D mRNA also showed strong expression at protein level. In conclusion, GPC6 and TMEM132D could serve as potent markers of CD8+ T-cell infiltration in early stage ovarian cancer. of GPC6 and TMEM132D at protein level in the cancer cells, we performed immunohistochemistry in sections from representative tumor samples with either high or low mRNA levels of GPC6 and TMEM132D as categorized by qPCR (Figures 1(e)-1(f) and 2(e)-2(f)). As depicted in Figure 1(e), the tumors with low mRNA levels of GPC6 also showed negative expression of the gene at protein level. On the contrary, high expression of the GPC6 protein was observed in tumors with high mRNA levels of the gene. Consistently, the tumors with low mRNA levels of TMEM132D showed little expression at protein level, whereas the tumors with high levels of TMEM132D mRNA also showed strong expression at protein level. In conclusion, GPC6 and TMEM132D could serve as potent markers of CD8+ T-cell infiltration in early stage ovarian cancer. As about 25% of the tumors exhibited abundant T- cell infiltration and both GPC6 and TMEM132D correlated with infiltration, the 75th percentile median mRNA levels of GPC6 (1.8 relative expression units) or TMEM132D (5.0 relative expression units) were selected as the cut-off values to subdivide the patient cohort into two groups: patients with high and patients with low mRNA levels of the relevant gene. There was no significant age difference among the two groups of patients, neither in the case of GPC6 subdivision nor in the case of TMEM132D subdivision. The presence of high levels of GPC6 and TMEM132D was also analysed in relation to stage, grade, and type of the disease. Statistically significant differences were not observed for the mRNA levels of the two genes among different stages, grades, or types of tumors (Tables S1 and S2 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/712438). Kaplan-Meir curves were calculated for the mRNA levels in order to assess whether GPC6 or TMEM132D levels were predictive of 3.2. The mRNA Levels of GPC6 and TMEM132D Correlate with Patients’ Overall Survival in Early Stage Ovarian Cancer. In order to evaluate the survival predictive value of GPC6 and TMEM132D mRNA levels in early stage ovarian cancer, we performed a retrospective clinical analysis of the above- studied stage I-II ovarian cancer patients. 3. Results Characteristic 𝑁(%) All stages, 𝑁= 35 Stage I, 𝑁= 28 (80%) Stage II, 𝑁= 7 (20%) Age at diagnosis Median/mean 59/57.63 59/56.86 59/60.71 Range (34–83) (34–78) (51–83) Grade 0 2 (5.7) 2 (7.1) 1 12 (34.3) 10 (35.7) 2 (28.6) 2 8 (22.9) 5 (17.9) 3 (42.9) 3 13 (37.1) 11 (39.3) 2 (28.6) Histological subtype Serous 11 (31.4) 9 (32.1) 2 (28.5) Endometrioid 20 (57.2) 16 (57.2) 4 (57.2) Clear-cell 4 (11.4) 3 (10.7) 1 (14.3) Debulking status Optimal 34 (97.1) 28 (100) 6 (85.7) Suboptimal 1 (2.9) 1 (14.3) Response to therapy CRa 33 (94.3) 27 (96.4) 6 (85.7) PDb 2 (5.7) 1 (3.6) 1 (14.3) Survival Ovarian cancer deaths 6 (17.1) 4 (14.3) 2 (28.5) Total number of deaths 6 (17.1) 4 (14.3) 2 (28.5) aComplete response. bProgressive disease. (Table 1). All of the clear-cell and more than half of the endometrioid tumors (55%) were high grade. Similarly, about 54% of the serous tumours were also high grade. The median age at diagnosis for all patients was 59 years, while the mean overall survival among the entire cohort was 189 months (95% C.I. 164–213, Table 2). Six patients (17.14%) deceased due to the disease during the study period. There were no statistically significant differences in survival among the two stages or the histological subtypes of the disease (data not shown). (Table 1). All of the clear-cell and more than half of the endometrioid tumors (55%) were high grade. Similarly, about 54% of the serous tumours were also high grade. The median age at diagnosis for all patients was 59 years, while the mean overall survival among the entire cohort was 189 months (95% C.I. 164–213, Table 2). Six patients (17.14%) deceased due to the disease during the study period. There were no statistically significant differences in survival among the two stages or the histological subtypes of the disease (data not shown). of GPC6 and TMEM132D at protein level in the cancer cells, we performed immunohistochemistry in sections from representative tumor samples with either high or low mRNA levels of GPC6 and TMEM132D as categorized by qPCR (Figures 1(e)-1(f) and 2(e)-2(f)). As depicted in Figure 1(e), the tumors with low mRNA levels of GPC6 also showed negative expression of the gene at protein level. On the contrary, high expression of the GPC6 protein was observed in tumors with high mRNA levels of the gene. 3. Results Over twenty percent (21.4%) of the patients with low levels deceased due to the disease. Similar results were obtained for TMEM132D. Interestingly, all patients with high mRNA levels of GPC6 or TMEM132D were still alive at the end of the study. We then asked whether the expression of both GPC6 and TMEM132D genes in combination correlated with survival. We divided the patients into 2 groups based on the combined expression of GPC6 and TMEM132D: (i) patients with low levels of both genes (𝑛= 22, 62.9%) and (ii) patients with high mRNA levels of GPC6 and/or TMEM132D (𝑛= 13, 37.1%). No significant difference in age was observed among the two groups. Patients with low mRNA levels of both GPC6 and TMEM132D had a mean overall survival of 144 months (95% C.I. 114–174). Furthermore, 27.3% of the patients with low mRNA levels deceased due to the disease (Table 2). On the contrary, all the patients with high mRNA levels of GPC6 and/or TMEM132D were alive until the last follow-up (or the end of the study). In conclusion, as shown in Figure 3, high mRNA levels of GPC6 and/or TMEM132D correlate significantly with increased overall survival (𝑝= 0.032) in early stage ovarian cancer. Figure 3: High mRNA levels of GPC6 and/or TMEM132D corre- late with increased overall survival of early stage ovarian cancer patients. Kaplan-Meier survival curves calculated for both GPC6 and TMEM132D mRNA levels. Patients were divided into two groups: (i) patients with low mRNA levels of both GPC6 and TMEM132D (cut-off points 1.8 and 5, resp.) and (ii) patients with GPC6 and/or TMEM132D high levels. Patients with high mRNA levels of GPC6 and/or TMEM132D exhibit a favorable overall survival (𝑝= 0.032). Although CD8A is a classic established marker of T-cell infiltration in ovarian cancer, in our cohort of patients, the prognostic value of CD8A mRNA levels was statistically weak (𝑝= 0.805) possibly due to the small size of the sample and the low number of deaths. However, in the same cohort of patients, high mRNA levels of GPC6 and/or TMEM132 associated significantly with increased survival. and TMEM132D with CD8+ T-lymphocyte infiltration and favorable prognosis in early stage ovarian cancer. Both genes were found to be differentially expressed in TIL+ versus the TIL−early stage ovarian cancer [19]. 3. Results Patients x1, x2, and x3 were excluded from this analysis due to unavailability of complete data. The clinicopathologic characteristics of the 35 early stage ovarian cancer patients are shown in Table 1. Twenty-eight patients (80%) presented stage I ovarian cancer and seven patients (20%) were stage II. The majority of the patients had endometrioid and clear-cell ovarian tumors BioMed Research International BioMed Research International 7 Table 2: Survival characteristics of patients related to their tumoral GPC6 and TMEM132D mRNA levels. All GPC6 mRNA levels TMEM132D mRNA levels GPC6/TMEM132D mRNA levels Low High Low High Both low High GPC6 and/or TMEM132D N (%) 35 28 (80) 7 (20) 26 (74.3) 9 (25.7) 22 (62.9) 13 (37.1) Survival 5-year survival (%) 91.34 ± 4.78 88.73 ± 6.13 100 88.29 ± 6.36 100 85.45 ± 7.78 100 10-year survival (%) 84.69 ± 6.34 80.06 ± 8.04 100 78.73 ± 8.55 100 73.62 ± 10.27 100 Overall survival (months) Mean ± SE 189 ± 12 179 ± 15 Undefineda 152 ± 13 Undefineda 144 ± 15 Undefineda 95% C.I. 164–213 149–209 — 126–177 — 114–174 — Death cases (%) 6 (17.14%) 6 (21.4) 0 (0) 6 (23.1) 0 (0) 6 (27.3) 0 (0) 𝑝value (log-rank test) 0.214 0.099 0.032 aAll subjects alive. Table 2: Survival characteristics of patients related to their tumoral GPC6 and TMEM132D mRNA levels. High GPC6 and/or TMEM132D Low GPC6 and TMEM132D Log-rank p = 0.032 Time (months) Overall survival 1.0 0.8 0.6 0.4 0.2 0.0 0 50 100 150 200 250 Figure 3: High mRNA levels of GPC6 and/or TMEM132D corre- late with increased overall survival of early stage ovarian cancer patients. Kaplan-Meier survival curves calculated for both GPC6 and TMEM132D mRNA levels. Patients were divided into two groups: (i) patients with low mRNA levels of both GPC6 and TMEM132D (cut-off points 1.8 and 5, resp.) and (ii) patients with GPC6 and/or TMEM132D high levels. Patients with high mRNA levels of GPC6 and/or TMEM132D exhibit a favorable overall survival (𝑝= 0.032). High GPC6 and/or TMEM132D Low GPC6 and TMEM132D Log-rank p = 0.032 Time (months) Overall survival 1.0 0.8 0.6 0.4 0.2 0.0 0 50 100 150 200 250 survival (Figures S1(a) and (b), resp.). As shown in Table 2, the analysis suggested a strong trend towards increased survival for patients with high mRNA levels of GPC6. 3. Results Here we validated by qPCR analysis in a larger group of stage I/II patients (𝑛= 38) that the mRNA expression levels of the two genes significantly correlated with the mRNA levels of the T-cell infiltration marker CD8A. More importantly, we showed that early stage ovarian cancer patients with high mRNA levels of at least one of the two genes, GPC6 and TMEM132D, exhibited increased overall survival compared to patients with low levels of both genes. i Taken together, our data indicate GPC6 and TMEM132D as potential markers for CD8+ T-lymphocyte infiltration, favorable prognosis, and survival benefit in early stage ovar- ian cancer. References [1] R. Siegel, J. Ma, Z. Zou, and A. Jemal, “Cancer statistics, 2014,” CA: A Cancer Journal for Clinicians, vol. 64, no. 1, pp. 9–29, 2014. [2] American Cancer Society, Cancer Facts & Figures 2013, Ameri- can Cancer Society, Atlanta, Ga, USA, 2013. [3] C. H. Holschneider and J. S. Berek, “Ovarian cancer: epidemi- ology, biology, and prognostic factors,” Seminars in Surgical Oncology, vol. 19, no. 1, pp. 3–10, 2000. [4] D. Spentzos, D. A. Levine, M. F. Ramoni et al., “Gene expression signature with independent prognostic significance in epithelial ovarian cancer,” Journal of Clinical Oncology, vol. 22, pp. 4700– 4710, 2004. [5] M. J. Birrer, M. E. Johnson, K. Hao et al., “Whole genome oligonucleotide-based array comparative genomic hybridiza- tion analysis identified fibroblast growth factor 1 as a prognostic marker for advanced-stage serous ovarian adenocarcinomas,” Journal of Clinical Oncology, vol. 25, no. 16, pp. 2281–2287, 2007. On the other hand, little is known about the biolog- ical role of TMEM132D. Polymorphisms in TMEM132D gene have been associated with panic disorder [27, 28]. TMEM132D encodes a single-pass type I transmembrane protein initially discovered in mature oligodendrocytes. TMEM132D expression is detectable in brain, lung, pancreas, and testis, but intriguingly not in normal ovaries [29]. Thus, it is possible that the expression of TMEM132D is induced in ovarian cancer and by an unknown mechanism becomes implicated in CD8+ T-lymphocyte infiltration. Based on the presence of TMEM132D on the plasma membrane surface, one could hypothesize an involvement in cell-cell interactions or intercellular signaling mechanisms that could be impli- cated with T-cell recruitment. [6] Y. Tanaka, H. Kobayashi, M. Suzuki et al., “Reduced bikunin gene expression as a factor of poor prognosis in ovarian carcinoma,” Cancer, vol. 98, no. 2, pp. 424–430, 2003. [7] A. M. Wolf, H. Rumpold, D. Reimer, C. Marth, A. G. Zeimet, and D. Wolf, “High IL-12 p35 and IL-23 p19 mRNA expression is associated with superior outcome in ovarian cancer,” Gyneco- logic Oncology, vol. 118, no. 3, pp. 244–250, 2010. [8] J. E. Quinn, C. R. James, G. E. Stewart et al., “BRCA1 mRNA expression levels predict for overall survival in ovarian cancer after chemotherapy,” Clinical Cancer Research, vol. 13, no. 24, pp. 7413–7420, 2007. [9] L. Zhang, J. R. Conejo-Garcia, D. Katsaros et al., “Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer,” The New England Journal of Medicine, vol. 348, no. 3, pp. 203– 213, 2003. 4. Discussion It is well established that the expression of CD8A at the transcriptional level is a reliable indicator of CD8+ T-cell infiltration in ovarian cancer [9, 19]. In this study, we documented an association between the expression of GPC6 Our results suggest an involvement of GPC6 in the biology of lymphocyte infiltration in early stage ovarian BioMed Research International 8 8 Conflict of Interests In particular, NFAT induces the expression of GPC6 which in turn inhibits canonical Wnt and b-catenin signaling and activates Wnt5A signaling that results in activation of JNK and p38𝛼kinases [26]. Further studies are required in order to shed light on the role of GPC6 in ovarian cancer and the mechanism of CD8+ T-lymphocyte infiltration. It is reasonable to anticipate that GPC6 may mediate intercellular interactions with immune cells or that it may be involved in shaping a chemokine gradient, necessary for the CD8+ T- lymphocyte infiltration. The authors declare that there is no conflict of interests regarding the publication of this paper. Authors’ Contribution Denarda Dangaj and Evripidis Lanitis contributed equally to this work. Acknowledgment Athanasios Karapetsas was supported by a Ph.D. scholarship from the State Scholarships Foundation of Greece. Conflict of Interests tumors. GPC6 encodes for Glypican-6, a 62.7 kDa heparan sulfate proteoglycan [21, 22]. Glypicans are proteins bearing glycosaminoglycan chains. There are six members of the family (GPC1–GPC6) in mammals, with GPC6 being a close homologue to GPC4 (64% identity). Glypicans are attached to the outer surface of the membrane through a glycosylphosphatidylinositol (GPI) anchor but can also be released to the extracellular space [23, 24]. Their heparan sulfate glycosaminoglycan chains are at the C-terminus of the protein, close to the membrane, and are thought to facilitate the interaction of glypicans with other molecules and receptors of the membrane. It has been shown that glypicans may regulate Hedgehog, Wnt, BMP, and FGF sig- naling pathways [24]. Interestingly, in Drosophila the released glypicans are involved in the transport of Wnts, Hhs, and BMPs by creating a morphogen gradient [25]. Furthermore, GPC6 promotes invasive migration of breast cancer cells through a noncanonical Wnt5A signaling pathway [26]. In particular, NFAT induces the expression of GPC6 which in turn inhibits canonical Wnt and b-catenin signaling and activates Wnt5A signaling that results in activation of JNK and p38𝛼kinases [26]. Further studies are required in order to shed light on the role of GPC6 in ovarian cancer and the mechanism of CD8+ T-lymphocyte infiltration. It is reasonable to anticipate that GPC6 may mediate intercellular interactions with immune cells or that it may be involved in shaping a chemokine gradient, necessary for the CD8+ T- lymphocyte infiltration. tumors. GPC6 encodes for Glypican-6, a 62.7 kDa heparan sulfate proteoglycan [21, 22]. Glypicans are proteins bearing glycosaminoglycan chains. There are six members of the family (GPC1–GPC6) in mammals, with GPC6 being a close homologue to GPC4 (64% identity). Glypicans are attached to the outer surface of the membrane through a glycosylphosphatidylinositol (GPI) anchor but can also be released to the extracellular space [23, 24]. Their heparan sulfate glycosaminoglycan chains are at the C-terminus of the protein, close to the membrane, and are thought to facilitate the interaction of glypicans with other molecules and receptors of the membrane. It has been shown that glypicans may regulate Hedgehog, Wnt, BMP, and FGF sig- naling pathways [24]. Interestingly, in Drosophila the released glypicans are involved in the transport of Wnts, Hhs, and BMPs by creating a morphogen gradient [25]. Furthermore, GPC6 promotes invasive migration of breast cancer cells through a noncanonical Wnt5A signaling pathway [26]. References According to our results, early stage ovarian cancer patients with low mRNA levels of GPC6 and TMEM132D exhibit significantly reduced overall survival compared to patients with high levels of GPC6 and/or TMEM132D. Therefore, we suggest that GPC6 and TMEM132D mRNA levels could serve as markers of CD8+ T-cell infiltration and survival prognosis in early stage ovarian cancer. For example, monitoring tumoral GPC6 and TMEM132D mRNA levels could facilitate the identification of early stage ovarian cancer patients at high risk. Our conclusions underscore the necessity to elucidate the molecular mechanism of GPC6 and TMEM132D involvement in T-cell infiltration and their impact on cancer progression and also highlight their possi- ble importance as putative diagnostic/therapeutic targets. [10] M. Tomˇsov´a, B. Melichar, I. Sedl´akov´a, and I. ˇSteiner, “Prog- nostic significance of CD3+ tumor-infiltrating lymphocytes in ovarian carcinoma,” Gynecologic Oncology, vol. 108, no. 2, pp. 415–420, 2008. [11] N. Leffers, M. J. M. Gooden, R. A. De Jong et al., “Prognostic significance of tumor-infiltrating T-lymphocytes in primary and metastatic lesions of advanced stage ovarian cancer,” Cancer Immunology, Immunotherapy, vol. 58, no. 3, pp. 449–459, 2009. [12] L. Han, M. S. Fletcher, D. L. Urbauer et al., “HLA class I antigen processing machinery component expression and intratumoral T-cell infiltrate as independent prognostic markers in ovarian carcinoma,” Clinical Cancer Research, vol. 14, no. 11, pp. 3372– 3379, 2008. BioMed Research International 9 [28] A. Erhardt, N. Akula, J. Schumacher et al., “Replication and meta-analysis of TMEM132D gene variants in panic disorder,” Translational Psychiatry, vol. 2, article e156, 2012. [13] W.-T. Hwang, S. F. Adams, E. Tahirovic, I. S. Hagemann, and G. Coukos, “Prognostic significance of tumor-infiltrating T cells in ovarian cancer: a meta-analysis,” Gynecologic Oncology, vol. 124, no. 2, pp. 192–198, 2012. [29] H. Nomoto, T. Yonezawa, K. Itoh et al., “Molecular cloning of a novel transmembrane protein MOLT expressed by mature oligodendrocytes,” The Journal of Biochemistry, vol. 134, no. 2, pp. 231–238, 2003. [14] E. Sato, S. H. Olson, J. Ahn et al., “Intraepithelial CD8+ tumor- infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favourable prognosis in ovarian cancer,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, pp. 18538–18543, 2005. [15] M. Stumpf, A. Hasenburg, M.-O. References Riener et al., “Intraepithelial CD8-positive T lymphocytes predict survival for patients with serous stage III ovarian carcinomas: relevance of clonal selec- tion of T lymphocytes,” British Journal of Cancer, vol. 101, no. 9, pp. 1513–1521, 2009. [16] M. J. Callahan, Z. Nagymanyoki, T. Bonome et al., “Increased HLA-DMB expression in the tumor epithelium is associ- ated with increased CTL infiltration and improved progno- sis in advanced-stage serous ovarian cancer,” Clinical Cancer Research, vol. 14, no. 23, pp. 7667–7673, 2008. [17] B. Clarke, A. V. Tinker, C.-H. Lee et al., “Intraepithelial T cells and prognosis in ovarian carcinoma: novel associations with stage, tumor type, and BRCA1 loss,” Modern Pathology, vol. 22, no. 3, pp. 393–402, 2009. [18] N. Leffers, R. S. N. Fehrmann, M. J. M. Gooden et al., “Iden- tification of genes and pathways associated with cytotoxic T lymphocyte infiltration of serous ovarian cancer,” British Journal of Cancer, vol. 103, no. 5, pp. 685–692, 2010. [19] A. Giannakakis, A. Karapetsas, D. Dangaj et al., “Overexpres- sion of SMARCE1 is associated with CD8+ T-cell infiltration in early stage ovarian cancer,” The International Journal of Biochemistry & Cell Biology, vol. 53, pp. 389–398, 2014. [20] B. Kornmann, N. Preitner, D. Rifat, F. Fleury-Olela, and U. Schi- bler, “Analysis of circadian liver gene expression by ADDER, a highly sensitive method for the display of differentially expressed mRNAs,” Nucleic acids research, vol. 29, no. 11, article E51, 2001. [21] S. Paine-Saunders, B. L. Viviano, and S. Saunders, “GPC6, a novel member of the glypican gene family, encodes a product structurally related to GPC4 and is colocalized with GPC5 on human chromosome 13,” Genomics, vol. 57, no. 3, pp. 455–458, 1999. [22] M. Veugelers, B. De Cat, H. Ceulemans et al., “Glypican-6, a new member of the glypican family of cell surface heparan sulfate proteoglycans,” Journal of Biological Chemistry, vol. 274, no. 38, pp. 26968–26977, 1999. [23] J. Filmus, M. Capurro, and J. Rast, “Glypicans,” Genome Biology, vol. 9, article 224, 2008. [24] J. Filmus and M. Capurro, “The role of glypicans in Hedgehog signaling,” Matrix Biology, vol. 35, pp. 248–252, 2014. [25] T. Y. Belenkaya, C. Han, D. Yan et al., “Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans,” Cell, vol. 119, no. 2, pp. 231–244, 2004. [26] G. K. Yiu, A. Kaunisto, Y. R. Chin, and A. References Toker, “NFAT promotes carcinoma invasive migration through glypican-6,” Biochemical Journal, vol. 440, no. 1, pp. 157–166, 2011. [27] A. Erhardt, L. Czibere, D. Roeske et al., “TMEM132D, a new candidate for anxiety phenotypes: evidence from human and mouse studies,” Molecular Psychiatry, vol. 16, no. 6, pp. 647–663, 2011.
https://openalex.org/W2147395479	https://europepmc.org/articles/pmc4431510?pdf=render	English	null	Understanding why people participate in HIV surveillance	Bulletin of the World Health Organization	2,015	cc-by	2,728	Understanding why people participate in HIV surveillance Rather than excluding those who decline post-test coun selling, we have the obligation to understand the reasons for research participation – and non-participation – better, including attitudes to learning or confirming one’s HIV status. Longitudinal studies with repeated HIV testing are particularly well placed to investigate decision-making around HIV testing. The results of such studies could demonstrate whether refusal to participate or receive test results lead to biased estimates of HIV prevalence that weaken the public health utility of the data.6,7 They could also teach us about why and how people be come motivated to be tested8 and that knowledge could lead to improvements in the design of programmes of HIV testing. ■ People have argued that the benefits of human immunodefi ciency virus (HIV) testing and counselling are so important that participants in HIV surveys must be given their HIV test results and that individuals who decline to receive their test re sults should be excluded from participation in such surveys.1–3 In early attempts at HIV surveillance, there were many logistical issues that complicated the return of test results and few advantages to infected individuals in receiving their test results.4 Now the situation has changed radically with the widespread roll-out of HIV treatment and care – which not only prolongs life but also reduces sexual and vertical transmission of HIV.2 We agree that researchers now have an obligation to offer and encourage post-test counselling as part of a research encounter but argue that there are both practical and ethical reasons to allow study participants to opt out of post-test counselling. Acknowledgements Janet Seeley (London School of Hygiene & Tropical Medicine, London, England), Moffat Nyirenda (Karonga Prevention Study, Chilumba, Malawi), Xavier Gomez-Olive (School of Public Health, University of Witwatersrand, Johannesburg, South Africa), Constance Nyamukapa (Biomedical Research and Training Institute, Harare, Zimbabwe), Eveline Geubbels (Ifakara Health Institute, Dar es Salaam, United Republic of Tanzania), Sally Mtenga (Ifakara Health Institute, Dar es Sa laam, United Republic of Tanzania), Amek Nyaguara (KEMRI/ CDC Research and Public Health Collaboration, Kisumu, Ke nya), Jessica Nakiying-Miiro (MRC/UVRI Research Program on AIDS, Entebbe, Uganda) and Robert Newton (MRC/UVRI Research Program on AIDS, Entebbe, Uganda) support the viewpoint presented here. In African populations with HIV prevalence above 4%, between 30% and 81% of infected men and women have ever been tested for HIV.5 Those who know they are HIV-positive may be willing to donate blood samples for research purposes but may not want to repeat pre- and post-test counselling. The same may be true of those who feel certain that they are uninfected. Sexually active men and women are encouraged to be tested regularly. Research studies should provide the option for those who arrange to be frequently retested – so-called repeat testers – to be given their test results in a short format and be allowed to opt out of post-test counselling. They should also be prepared for participants who do not wish to collect their test results in any format. All participants – including those who provide blood samples for research but opt out of post-test counselling – can be asked to report their testing histories and testing motives. Understanding why people participate in HIV surveillance Basia Zaba,a Georges Reniers,a Emma Slaymaker,a Jim Todd,a Judith Glynn,a Amelia Crampin,b Mark Urassa,c Tom Lutalo,d Marie-Louise Newell,e Vic toria Hosegood,f Samuel Clarkg & Simon Gregsonh Understanding why people participate in HIV surveillance Basia Zaba,a Georges Reniers,a Emma Slaymaker,a Jim Todd,a Judith Glynn,a Amelia Crampin,b Mark Urassa,c Tom Lutalo,d Marie-Louise Newell,e Vic toria Hosegood,f Samuel Clarkg & Simon Gregsonh Understanding why people participate in HIV surveillance Basia Zaba,a Georges Reniers,a Emma Slaymaker,a Jim Todd,a Judith Glynn,a Amelia Crampin,b Mark Urassa,c Tom Lutalo,d Marie-Louise Newell,e Vic toria Hosegood,f Samuel Clarkg & Simon Gregsonh 1. Baggaley R, Johnson C, Garcia Calleja JM, Sabin K, Obermeyer C, Taegtmeyer M, et al. Routine feedback of test results to participants in clinic- and survey- based surveillance of HIV. Bull World Health Organ. 2015;93(5):352–5. doi: http://dx.doi.org/10.2471/BLT.15.153031 a London School of Hygiene & Tropical Medicine, Keppel Street, London, WC1E 7HT, England. b Karonga Prevention Study, Chilumba, Malawi. c National Institute of Medical Research, Mwanza, United Republic of Tanzania. d Rakai Health Sciences Program, Rakai, Uganda. e Southampton University, Southampton, England. f Africa Centre for Health and Population Studies, Mtubatuba, South Africa. g University of Washington, Seattle, United States of America. h Imperial College, London, England. Correspondence to Basia Zaba (email: basia.zaba@lshtm.ac.uk). Routine feedback of HIV test results Round table Routine feedback of HIV test results Round table Rachel Baggaley et al. respect for individual autonomy. Informed consent procedures typically tell willing subjects that they have a right not to an swer questions that make them uncomfortable and a right to withdraw from the study at any time without completing all the activities and procedures. Declining to receive a test result is an example of the right of participants to opt out of part of a study. Whether in research or clinical practice, public health utility would be better served through understanding peoples’ reasons for not wanting to receive an HIV test result – while informing individuals about all of the available testing and counselling options. 1. Baggaley R, Johnson C, Garcia Calleja JM, Sabin K, Obermeyer C, Taegtmeyer M, et al. Routine feedback of test results to participants in clinic- and survey- based surveillance of HIV. Bull World Health Organ. 2015;93(5):352–5. doi: http://dx.doi.org/10.2471/BLT.15.153031 2. Maher D. The ethics of feedback of HIV test results in population-based surveys of HIV infection. Bull World Health Organ. 2013 Dec 1;91(12):950–6. doi: http://dx.doi.org/10.2471/BLT.13.117309 PMID: 24347734 Round table discussion Round table discussion 2. Maher D. The ethics of feedback of HIV test results in population-based surveys of HIV infection. Bull World Health Organ. 2013 Dec 1;91(12):950–6. doi: http://dx.doi.org/10.2471/BLT.13.117309 PMID: 24347734 1. Baggaley R, Johnson C, Garcia Calleja JM, Sabin K, Obermeyer C, Taegtmeyer M, et al. Routine feedback of test results to participants in clinic- and survey- based surveillance of HIV. Bull World Health Organ. 2015;93(5):352–5. doi: http://dx.doi.org/10.2471/BLT.15.153031 2. Maher D. The ethics of feedback of HIV test results in population-based surveys of HIV infection. Bull World Health Organ. 2013 Dec 1;91(12):950–6. doi: http://dx.doi.org/10.2471/BLT.13.117309 PMID: 24347734 3. Baggaley R, Calleja JM, Marum L, Marum E. Knowledge is power; information is liberation. Bull World Health Organ. 2013 Dec 1;91(12):898– 898A. doi: http://dx.doi.org/10.2471/BLT.13.132464 PMID: 24347724 Accurate information as a tool to decrease HIV test refusals in research studies y y g That so many are convinced, wrongly, that an HIV test will inevitably produce a positive diagnosis is the consequence of rural Malawians’ incorrect understanding of the probabilities of HIV transmission. For example, most of our survey respon dents believed that an uninfected individual was certainly or highly likely to be infected with HIV during a single act of unprotected intercourse with an infected person.8 Would it not be preferable to treat those living amidst the HIV epidemic as having an ethical right to accurate information on the prob abilities of transmission? Efforts should be made to evaluate the potential benefits of disseminating accurate information on the probabilities of transmission, the approximate prevalence of HIV infection and the probability of a positive result in an HIV test – such that consent for HIV testing in surveys is fully, rather than incompletely, informed. We need to know whether such health education would be a liberation, lead to fewer test refusals in research studies and, importantly, increase the number of people who are willing to know their HIV status. ■ Susan C Watkins,a Philip Anglewicz,b Nicole Angotti,c Amy Kalerd & Ann Swidlere Susan C Watkins,a Philip Anglewicz,b Nicole Angotti,c Amy Kalerd & Ann Swidlere It has been argued that researchers conducting surveys that include testing for human immunodeficiency virus (HIV) have a duty to tell potential subjects that they do not have the right to participate if they refuse to receive their HIV test results.1,2 Furthermore, promotion of the routine feedback of such test results has been based on the grounds that knowl edge is power and information is liberation.3 However, other researchers argue that, although it is desirable to offer study participants post-test counselling, for practical and ethical reasons some study participants should be given the right to refuse such counselling.4 Although we support the right of participants to opt out of post-test counselling and thus not to receive their test results, we also propose that subjects who are – or may be – tested for HIV should be given informa tion that may decrease their resistance to learning their test results. We draw on data, collected between 1998 and 2013, on rural Malawians’ experience with – and perceptions of – HIV testing. Rachel Baggaley et al. Reynolds L, Cousins T, Newell ML, Imrie J. The social dynamics of consent and refusal in HIV surveillance in rural South Africa. Soc Sci Med. 2013 Jan;77:118–25. doi: http://dx.doi.org/10.1016/j.socscimed.2012.11.015 PMID: 23219165 8. Reynolds L, Cousins T, Newell ML, Imrie J. The social dynamics of consent and refusal in HIV surveillance in rural South Africa. Soc Sci Med. 2013 Jan;77:118–25. doi: http://dx.doi.org/10.1016/j.socscimed.2012.11.015 PMID: 23219165 a California Center for Population Studies at UCLA, 4284 Public Affairs Building, MC 723603, PO Box 957236, Los Angeles, CA 90405, United States of America (USA). b Tulane University School of Public Health and Tropical Medicine, New Orleans, USA. c Department of Sociology and Center on Health, Risk and Society, American University, Washington, USA. d Department of Sociology, University of Alberta, Edmonton, Canada. e Department of Sociology, University of California, Berkeley, USA. Correspondence to Susan C Watkins (swatkins@ccpr.ucla.edu). a California Center for Population Studies at UCLA, 4284 Public Affairs Building, MC 723603, PO Box 957236, Lo b Tulane University School of Public Health and Tropical Medicine, New Orleans, USA. c Department of Sociology and Center on Health, Risk and Society, American University, Washington, USA. d Department of Sociology, University of Alberta, Edmonton, Canada. e Department of Sociology, University of California, Berkeley, USA. Correspondence to Susan C Watkins (swatkins@ccpr.ucla.edu). References 1. Baggaley R, Johnson C, Garcia Calleja JM, Sabin K, Obermeyer C, Taegtmeyer M, et al. Routine feedback of test results to participants in clinic- and survey- based surveillance of HIV. Bull World Health Organ. 2015;93(5):352–5. doi: http://dx.doi.org/10.2471/BLT.15.153031 Importantly, the opt-out provisions we discuss here align with the general principles of research ethics. We agree that there is public health utility in informing people of their HIV status but we think that this should not override the ethics of 2. Maher D. The ethics of feedback of HIV test results in population-based surveys of HIV infection. Bull World Health Organ. 2013 Dec 1;91(12):950–6. doi: http://dx.doi.org/10.2471/BLT.13.117309 PMID: 24347734 a London School of Hygiene & Tropical Medicine, Keppel Street, London, WC1E 7HT, England. b Karonga Prevention Study, Chilumba, Malawi. c National Institute of Medical Research, Mwanza, United Republic of Tanzania. d Rakai Health Sciences Program, Rakai, Uganda. e Southampton University, Southampton, England. f Africa Centre for Health and Population Studies, Mtubatuba, South Africa. g University of Washington, Seattle, United States of America. h Imperial College, London, England. Correspondence to Basia Zaba (email: basia.zaba@lshtm.ac.uk). Bull World Health Organ 2015;93:356 \| doi: http://dx.doi.org/10.2471/BLT.14.135756 356 Routine feedback of HIV test results Round table Round table Routine feedback of HIV test results Round table Rachel Baggaley et al. Rachel Baggaley et al. 3. Baggaley R, Calleja JMG, Marum L, Marum E. Knowledge is power; information is liberation. Bull World Health Organ. 2013 Dec 1;91(12):898. doi: http://dx.doi.org/10.2471/BLT.13.132464 PMID: 24347724 for antenatal HIV testing in Malawi includes an opt-out pro vision, accounts from pregnant women attending antenatal clinics show that HIV testing is compulsory if the women are to receive antenatal care.5 Moreover, in population-based HIV surveys, fieldworkers are always under pressure to minimize the numbers of test refusals and may exert undue pressure on individuals who do not want to receive their test results. While exclusion from antenatal services is, presumably, much more serious than exclusion from survey participation, in both of these examples people are sanctioned for not giving consent – which is a clear ethical violation. 4. Temmerman M, Ndinya-Achola J, Ambani J, Piot P. The right not to know HIV-test results. Lancet. 1995 Apr 15;345(8955):969–70. doi: http://dx.doi. org/10.1016/S0140-6736(95)90707-6 PMID: 7619122 g 5. Staveteig S, Wang S, Head SK, Bradley SEK, Nybro E. Demographic patterns of HIV testing uptake in sub-Saharan Africa. DHS Comparative Reports No. 30. Calverton: ICF International; 2013. 6. Obare F. Nonresponse in repeat population-based voluntary counseling and testing for HIV in rural Malawi. Demography. 2010 Aug;47(3):651–65. doi: http://dx.doi.org/10.1353/dem.0.0115 PMID: 20879682 p g 7. Floyd S, Molesworth A, Dube A, Crampin AC, Houben R, Chihana M, et al. Underestimation of HIV prevalence in surveys when some people already know their status, and ways to reduce the bias. AIDS. 2013 Jan 14;27(2):233–42. doi: http://dx.doi.org/10.1097/QAD.0b013e32835848ab PMID: 22842993 7. Floyd S, Molesworth A, Dube A, Crampin AC, Houben R, Chihana M, et al. Underestimation of HIV prevalence in surveys when some people already know their status, and ways to reduce the bias. AIDS. 2013 Jan 14;27(2):233–42. doi: http://dx.doi.org/10.1097/QAD.0b013e32835848ab PMID: 22842993 Second, our ethnographic data depict the anguish that many suffer as they anticipate the future receipt of their test results – an issue that has rarely been discussed in the public health and social science literature.6 Two common misper ceptions among rural Malawian adults are that the result of an HIV test will almost always be positive and that a positive result will inevitably be followed by hastening psychological deterioration, suicidal thoughts and death. Yet survey data from people living in rural Malawi show that between 80% and 90% of respondents who believed that they were HIV-positive before they were tested learned that they were HIV-negative.7,8h 8. 1. Baggaley R, Johnson C, Garcia Calleja JM, Sabin K, Obermeyer C, Taegtmeyer M, et al. Routine feedback of test results to participants in clinic- and survey- based surveillance of HIV. Bull World Health Organ. 2015;93(5):352–5. doi: http://dx.doi.org/10.2471/BLT.15.153031 4. Zaba B, Reniers G, Slaymaker E, Todd J, Glynn J, Crampin A, et al. Understanding why people participate in HIV surveillance. Bull World Health Organ. 2015;93(5):356. doi: http://dx.doi.org/10.2471/BLT.14.135756 5. Angotti N, Dionne KY, Gaydosh L. An offer you can’t refuse? Provider- initiated HIV testing in antenatal clinics in rural Malawi. Health Policy Plan. 2011 Jul;26(4):307–15. doi: http://dx.doi.org/10.1093/heapol/czq066 PMID: 21047809 6. Kaler A, Watkins S. Asking God about the date you will die: HIV testing as a zone of uncertainty in rural Malawi. Demogr Res. 2010 Nov 9;23(32):905–32. doi: http://dx.doi.org/10.4054/DemRes.2010.23.32 PMID: 21562614 7. Kohler HP, Watkins SC, Behrman JR, Anglewicz P, Kohler I, Fleming P, et al. Cohort profile: the Malawi Longitudinal Study of Families and Health. Philadelphia: University of Pennsylvania; 2013. Available from http:// repository.upenn.edu/cgi/viewcontent.cgi?article=1045&context=psc_ working_papers [cited 2015 Feb 2]. 8. Anglewicz P, Kohler HP. Overestimating HIV infection: the construction and accuracy of subjective probabilities of HIV infection in rural Malawi. Demogr Res. 2009;20(6):65–96. doi: http://dx.doi.org/10.4054/DemRes.2009.20.6 PMID: 19672478 4. Zaba B, Reniers G, Slaymaker E, Todd J, Glynn J, Crampin A, et al. Understanding why people participate in HIV surveillance. Bull World Health Organ. 2015;93(5):356. doi: http://dx.doi.org/10.2471/BLT.14.135756 5. Angotti N, Dionne KY, Gaydosh L. An offer you can’t refuse? Provider- initiated HIV testing in antenatal clinics in rural Malawi. Health Policy Plan. 2011 Jul;26(4):307–15. doi: http://dx.doi.org/10.1093/heapol/czq066 PMID: 21047809 6. Kaler A, Watkins S. Asking God about the date you will die: HIV testing as a zone of uncertainty in rural Malawi. Demogr Res. 2010 Nov 9;23(32):905–32. doi: http://dx.doi.org/10.4054/DemRes.2010.23.32 PMID: 21562614 7. Kohler HP, Watkins SC, Behrman JR, Anglewicz P, Kohler I, Fleming P, et al. Cohort profile: the Malawi Longitudinal Study of Families and Health. Philadelphia: University of Pennsylvania; 2013. Available from http:// repository.upenn.edu/cgi/viewcontent.cgi?article=1045&context=psc_ working_papers [cited 2015 Feb 2]. References From the perspective of the organizations that promote HIV testing, it is axiomatic that people will benefit from knowing their HIV status. In Maher’s view, such benefit justifies sanctioning those who refuse to receive their test results.2 We disagree, for two reasons. First, the experience of many Malawians is that refusal to consent to testing may have serious consequences. For example, although the policy Bull World Health Organ 2015;93:357–358 \| doi: http://dx.doi.org/10.2471/BLT.14.136515 Bull World Health Organ 2015;93:357–358 \| doi: http://dx.doi.org/10.2471/BLT.14.136515 357 Discussion 8. Anglewicz P, Kohler HP. Overestimating HIV infection: the construction and accuracy of subjective probabilities of HIV infection in rural Malawi. Demogr Res. 2009;20(6):65–96. doi: http://dx.doi.org/10.4054/DemRes.2009.20.6 PMID: 19672478 Bull World Health Organ 2015;93:357–358 \| doi: http://dx.doi.org/10.2471/BLT.14.136515 Bull World Health Organ 2015;93:357–358 \| doi: http://dx.doi.org/10.2471/BLT.14.136515 358
https://openalex.org/W2165058735	https://www.frontiersin.org/articles/10.3389/fncel.2012.00017/pdf	English	null	Neural stem/progenitor cells as a promising candidate for regenerative therapy of the central nervous system	Frontiers in cellular neuroscience	2,012	cc-by	9,435	THE CENTRAL NERVOUS SYSTEM, AN ORGAN WITH AN “IMMUNOLOGICALLY SPECIAL” STATUS the brain, like growth factors, cytokines, or immunoglobulins (Goldstein and Betz, 1983; Joó, 1993). In addition, there is no proof of the existence of professional antigen presenting cells like dendritic cells, B cells, and macrophages in the unlesioned CNS (Wekerle et al., 1987) preventing the initiation and propagation of antigen-speciﬁc immune responses in the brain. In physiological conditions, the expression of antigens from the major histocom- patibility complex (MHC) by neural cells is very weak and even in some cases undetectable (Barker and Billingham, 1977; Mauer- hoff et al., 1988) allowing these cells to escape the recognition by antigen-speciﬁc T cells. However, only the normal CNS displays such an absence of immune response. Inﬂammation is the primary response of the immune system that occurs to defend the organism against danger signals pro- voked by an infection or irritation consecutive to the intrusion of pathogens. The speciﬁc property of the immune system is to discriminate the self from the non-self and thus to identify and eliminate the infectious agents. From this same mechanism originates the phenomenon of rejection in transplantation. How- ever, since Van Dooremal work in 1873 on tumor cell graft in the eye anterior chamber, it is known that speciﬁc sites in the organism display limited immune reactions. In 1948, Sir Peter Medawar, Nobel Prize of Medicine and pioneer in the ﬁeld of the immunology of transplantation, proposed the term of “immune privilege” to describe a reduced inﬂammation after inoculation of allogeneic tissues in some organs like the brain or the eye anterior chamber (Medawar, 1948). Later, this deﬁnition was extended to the particular property of speciﬁc organs or tissues to display a prolonged, and sometimes inﬁnite, survival when grafted in con- ventional sites of the organism. Thus, throughout the years, the cornea, the placenta, the eye anterior chamber (Billingham and Boswell, 1953; Hori et al., 2000), or the testis (Head et al., 1983) were examples of well-studied immune-privileged tissues. In this context, the central nervous system (CNS) and the immune system were traditionally perceived as separate morphological and func- tional entities, preventing the disturbance of the CNS homeostasis which is crucial to neuronal functioning. Edited by: Edited by: Afsaneh Gaillard, INSERM, University of Poitiers, France Reviewed by: Corette Wierenga, Utrecht University, Netherlands Mohamed Jaber, INSERM, University of Poitiers, France Correspondence: Philippe Naveilhan, INSERM U643, 30 Boulevard Jean Monnet, 44093 Nantes Cedex 01, France. e-mail: philippe.naveilhan@univ-nantes.fr Afsaneh Gaillard, INSERM, University of Poitiers, France Reviewed by: Corette Wierenga, Utrecht University, Netherlands Reviewed by: Corette Wierenga, Utrecht University, Netherlands Mohamed Jaber, INSERM, University of Poitiers, France Correspondence: Philippe Naveilhan, INSERM U643, 30 Boulevard Jean Monnet, 44093 Nantes Cedex 01, France. e-mail: philippe.naveilhan@univ-nantes.fr Keywords: transplantation, immunomodulation, immune reactions, stem cells, regenerative medicine CELLULAR NEUROSCIENCE CELLULAR NEUROSCIENCE REVIEW ARTICLE published: 11 April 2012 doi: 10.3389/fncel.2012.00017 REVIEW ARTICLE Virginie Bonnamain1,2,3, Isabelle Neveu1,2,3 and Philippe Naveilhan1,2,3* 1 INSERM U643, Nantes, France 2 Institut de Transplantation et de Recherche en Transplantation, Centre Hospitalier Universitaire de Nantes, Nantes, Fra 3 Faculté de Médecine, Université de Nantes, Nantes, France 2 Institut de Transplantation et de Recherche en Transplantation, Centre Hospitalier Universitaire de Nantes, Nantes, France 3 Faculté de Médecine, Université de Nantes, Nantes, France Neural transplantation is a promising therapeutic strategy for neurodegenerative diseases and other disorders of the central nervous system (CNS) such as Parkinson and Hunting- ton diseases, multiple sclerosis or stroke. Although cell replacement therapy already went through clinical trials for some of these diseases using fetal human neuroblasts, several signiﬁcant limitations led to the search for alternative cell sources that would be more suit- able for intracerebral transplantation.Taking into account logistical and ethical issues linked to the use of tissue derived from human fetuses, and the immunologically special status of the CNS allowing the occurrence of deleterious immune reactions, neural stem/progenitor cells (NSPCs) appear to be an interesting cell source candidate. In addition to their ability for replacing cell populations lost during the pathological events, NSPCs also display surprising therapeutic effects of neuroprotection and immunomodulation. A better knowledge of the mechanisms involved in these speciﬁc characteristics will hopefully lead in the future to a successful use of NSPCs in regenerative medicine for CNS disorders. Frontiers in Cellular Neuroscience Neural stem/progenitor cells as promising candidates for regenerative therapy of the central nervous system Virginie Bonnamain1,2,3, Isabelle Neveu1,2,3 and Philippe Naveilhan1,2,3* 1 INSERM U643, Nantes, France 2 Institut de Transplantation et de Recherche en Transplantation, Centre Hospitalier Universitaire de Nantes, Nantes, France 3 Faculté de Médecine, Université de Nantes, Nantes, France THE CENTRAL NERVOUS SYSTEM, AN ORGAN WITH AN “IMMUNOLOGICALLY SPECIAL” STATUS Encouraging results obtained from animal models of Parkinson disease led to small-scale clinical trials. Isacson’s group performed a study where 12 Parkinson disease patients received a unilateral striatal graft constituted of a porcine ventral mesen- cephalon (25–28 days of gestation) cell suspension (Schumacher et al., 2000). Even though a signiﬁcant decrease in Uniﬁed Parkin- son’s Disease Rating Scale (UPDRS) scores was observed, positron emission tomography (PET) analyses did not show any increase in the [18F]ﬂuorodopa uptake, underlining a lack of improve- ment in axon termination density of dopaminergic neurons from the graft and no increase in dopa-decarboxylase activity. More- over, a postmortem study revealed that only 638 TH-positive cells out of the 12 millions of porcine neuroblasts transplanted had survived at that stage, with a lymphocytic inﬁltration at the bor- der and within the graft, despite the fact that the patient was under cyclosporine A treatment (Deacon et al., 1997). If some work has shown that neural xenografts are able to survive for a long time in the CNS without the use of immunosuppressors (Björklund et al., 1982; Daniloff et al., 1985), most of the recent studies, including those from our group, indicate that intracere- bral xenografts trigger a strong immune reaction leading to the fast destruction of the graft through the invasion of microglial cells/macrophages, T lymphocytes, and dendritic cells within 5– 7 weeks post-transplantation (Rémy et al., 2001; Melchior et al., 2002; Michel et al., 2006; Figure 1). Xenograft rejection is more aggressive than that observed with allotransplantation and mainly occurs through the involvement of T cells. A genetically modi- ﬁed swine has been engineered to express CTLA4-Ig, a human molecule inhibiting T cells, under the neuron-speciﬁc enolase pro- moter. This construction allowed porcine transgenic neurons to locally deliver an immunosuppressive molecule after transplan- tation in the brain (Martin et al., 2005). In the CNS, xenograft survival has been signiﬁcantly prolonged by administration of immunosuppressive drugs targeting T cells like cyclosporine A. However, this immunosuppressor at doses required to inhibit the j ti h t id ff t (R i 2006) d i across the BBB on a regular basis (Hickey et al., 1991; Cayrol et al., 2008) were convincing proofs of the strong bidirectional com- munication between the immune and the nervous systems. THE CENTRAL NERVOUS SYSTEM, AN ORGAN WITH AN “IMMUNOLOGICALLY SPECIAL” STATUS This vision that the CNS could escape the immune surveillance was supported by the dis- covery of the blood–brain barrier (BBB) preventing the exchange between a wide range of soluble molecules from the blood and During certain pathological processes, speciﬁc genes are acti- vated leading to the change of this immunologically non-reactive tissue into an environment favorable to the development of inﬂammatory reactions. In these conditions, macrophages are recruited from the pool of blood monocytes and inﬁltrate the perivascular spaces. In addition, microglial cells from the CNS are activated and acquire phagocytosis and antigen presentation abilities (Aloisi, 2001). Microglia can induce the production of pro-inﬂammatory cytokines like TNF and IL-1β (Sivakumar et al., 2011) and, along with reactive astrocytes, become able to present antigens through class I and II MHC molecules (Höftberger et al., 2004), allowing CNS-inﬁltrated T cells to recognize the antigenic peptides and behave as potent immune effectors (Cornet et al., 2000). The last two decades have thus witnessed the questioning of the concept of immune privilege. Discovery of the perme- ability of the BBB under pathological circumstances (Kebir et al., 2007), the existence of an unconventional lymphatic drainage in the CNS (Hatterer et al., 2006), and the migration of leucocytes April 2012 \| Volume 6 \| Article 17 \| 1 Frontiers in Cellular Neuroscience www.frontiersin.org NSPC for CNS cell therapy Bonnamain et al. proven to be an efﬁcient source for cell therapy in animal models of neurodegenerative diseases. Interest in these cells started to grow after ﬁrst studies revealed that xenografts derived from the ventral mesencephalon of pigs (21–26 days of gestation) could survive in the brain of a rat model of Parkinson disease under immunosup- pression (Freeman et al., 1988; Huffaker et al., 1989). Beyond the long-term survival, xenotransplanted neural cells also displayed a long-distance axonal outgrowth (Wictorin et al., 1990; Deacon et al., 1994). Isacson et al. (1995) have shown that neurons iso- lated from several areas of the porcine fetal brain and transplanted in homotypic or ectopic lesioned cerebral regions of an adult rat could project axons in the deafferented zones, thus rebuild- ing the brain parenchyma cytoarchitecture. This inter-species and targeted axonal growth of porcine dopaminergic neuroblasts sig- niﬁcantly restored in several months an efﬁcient dopaminergic innervation. A signiﬁcant functional recovery in transplanted rats was also observed and was correlated to the number of tyrosine hydroxylase(TH)-positivecellsandtothesizeof thegraft(Galpern et al., 1996). CELL REPLACEMENT THERAPY FOR THE CNS CELL REPLACEMENT THERAPY FOR THE CNS Neuronal cell death is the main characteristic of CNS acute disor- ders like stroke or trauma but also of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington diseases. As the adult CNS displays very weak capacities of endogenous cell replacement and repair, both being necessary to attain a signiﬁcant functional recovery, cell replacement therapy for the CNS repre- sents a promising avenue for the treatment of these pathological conditions. In fact, clinical impact of cell replacement strategy has already been assessed in Parkinson disease patients by allo- transplantation of fetal mesencephalic cells. The results of the clinical trials showed, in some patients, a long-term symptomatic improvement associated with a survival of the grafted cells and a partial reinnervation of the striatum (Björklund and Lindvall, 2000). A functional recovery has also been noticed in a small cohort of patients suffering from Huntington disease and grafted with human fetal striatal neuroblasts (Bachoud-Lévi et al., 2000, 2006), with one study even showing integration of cells from the donor into the host tissue (Freeman et al., 2000). Despite these promising outcomes, several issues remain to be assessed (Björk- lund et al.,2003). Tissue availability, ethical and logistical concerns linked to the use of human material, cell viability and purity, or appearance of deleterious side-effects like dyskinesia (Olanow et al., 2003) represent important obstacles that need to be over- come before resuming clinical trials on neural allotransplantation. Moreover, the discovery of biological signs of alloimmunization to donor’s antigens like the appearance of anti-HLA antibodies in Huntington disease patients following fetal neural grafts (Krys- tkowiak et al., 2007) underlined the importance of considering immune responses in the CNS as a crucial parameter for future cell transplantation strategies. In order to at least circumvent some of the problems mentioned above, alternative cell sources for intracerebral transplantation like porcine neuroblasts have been considered (Pakzaban and Isacson, 1994). THE CENTRAL NERVOUS SYSTEM, AN ORGAN WITH AN “IMMUNOLOGICALLY SPECIAL” STATUS The brain is now more readily considered as an organ with special immune characteristics and subject to immunological surveil- lance than a strictly immune-privileged tissue (Hickey,2001). This dual status of the CNS, being both more favorable to the trans- plantation than the periphery but also a propitious environment for deleterious inﬂammatory reactions in response to pathologi- cal conditions (Kerschensteiner et al., 2009) could in part explain why fetal neuron allografts in the brain are usually well tolerated under moderate immunosuppressive treatment (Björklund and Lindvall, 2000; Bachoud-Lévi et al., 2006; Krystkowiak et al., 2007) while intracerebral xenografts are systematically rejected (Larsson et al., 2000). Frontiers in Cellular Neuroscience INTRACEREBRAL XENOTRANSPLANTATION Porcine fetal neural tissue has been for a long time considered as the more adequate cell source for xenotransplantation in the human brain. Indeed, swine offer the advantage of a relatively easy breeding allowing an excellent access to fetal material and can be genetically modiﬁed. In fact, porcine fetal cells have been April 2012 \| Volume 6 \| Article 17 \| 2 Frontiers in Cellular Neuroscience Frontiers in Cellular Neuroscience www.frontiersin.org NSPC for CNS cell therapy Bonnamain et al. depletion (Honey et al., 1990; Okura et al., 1997). The admin- istration of two successive high doses of anti-CD4 monoclonal antibodies resulted in a longer survival of discordant xenografts (between species differing from the expression of the gal epitope, like pig and human), although not prolonged, suggesting that other immune components than T cells are implicated (Wood et al., 1996). Indeed, B cells and immunoglobulins have been shown to intervene in the survival and function of the graft. The importance of immunoglobulins has been highlighted by compar- ing the survival of a cell suspension derived from porcine ventral mesencephalon xenografted in the brain of wild-type (WT) or immunoglobulin-deﬁcient (Ig-KO) mice. Most of the Ig-KO mice displayed for more than 4 weeks a functional graft with very little immune cell inﬁltration while xenograft survival beyond 4 days remained exceptional in WT mice. After a prolonged survival of about 4 weeks, an immune cellular response with a high pro- portion of CD8-positive T cells occurred and led to the rejection of the graft in Ig-KO mice (Larsson et al., 1999). These observa- tions indicate that immunoglobulins play an important part in the fast initiation of discordant neural xenografts rejection, a phe- nomenon that appears to be unavoidable despite all the efforts made to control it. Overall, these pre-clinical and clinical stud- ies led to disappointing results and limited the enthusiasm and hope initially raised by the use of fetal xenogeneic neuroblasts in regenerative medicine. It is now important to ﬁnd more adequate cell sources for intracerebral transplantation. In theory, the ideal candidate should be tolerogenic and safe and have the proper- ties to be easily ampliﬁed, resist to long-term storage, be efﬁcient for cell replacement and allow manipulation to adjust to a spe- ciﬁc pathology. INTRACEREBRAL XENOTRANSPLANTATION Until these days, no cell type possesses all those characteristics and several cells at different stages of the neuronal lineage have been tested in animal models and clinical trials (Guil- laume and Zhang, 2008). Among them, neural stem/progenitor cells (NSPCs) seem to represent a good candidate. FIGURE 1 \| Characterization of xenograft inﬁltration by activated immune cells. Kinetics (A). In the absence of immunosuppressive FIGURE 1 \| Characterization of xenograft inﬁltration by activated immune cells. Kinetics (A). In the absence of immunosuppressive treatment, the rejection process of fetal neurons xenografted in the rat striatum involves cells from the macrophagic lineage. After a peak of microglial activation and dendritic cell inﬁltration in the ﬁrst days post-transplantation, probably consecutive to the surgery, a latency phase is observed. Around 5 weeks post-transplantation, rejection process is initiated and strong microglial cell activation at the border but also within the graft is observed. This phenomenon is strongly correlated with a massive T cell and dendritic cell inﬁltration. Immunostaining (B). Speciﬁc porcine neuroﬁlament is revealed by NF70 antibody and allows visualization of the graft. Immune reaction is assessed by Ox62 (dendritic cells), Ox42 (microglial cells/macrophages), and R73 (T cells) antibodies. Scale bar: 200 μm. FIGURE 1 \| Characterization of xenograft inﬁltration by activated immune cells. Kinetics (A). In the absence of immunosuppressive treatment, the rejection process of fetal neurons xenografted in the rat striatum involves cells from the macrophagic lineage. After a peak of microglial activation and dendritic cell inﬁltration in the ﬁrst days post-transplantation, probably consecutive to the surgery, a latency phase is observed. Around 5 weeks post-transplantation, rejection process is initiated and strong microglial cell activation at the border but also within the graft is observed. This phenomenon is strongly correlated with a massive T cell and dendritic cell inﬁltration. Immunostaining (B). Speciﬁc porcine neuroﬁlament is revealed by NF70 antibody and allows visualization of the graft. Immune reaction is assessed by Ox62 (dendritic cells), Ox42 (microglial cells/macrophages), and R73 (T cells) antibodies. Scale bar: 200 μm. Frontiers in Cellular Neuroscience NSPCs, THE FUTURE OF INTRACEREBRAL TRANSPLANTATION? Stem cells are deﬁned by their ability to self renew to maintain the pool of undifferentiated cells, to proliferate to give rise to lineage-restricted progenitors, and to differentiate into a range of mature cell types. Among them, multipotent neural stem cells (NSCs), derived from embryonic or adult nervous systems, can be cultivated in vitro as neurospheres, a mixture of neural stem cells and progenitors (NSPCs), in presence of growth factors such as bFGF (Vescovi et al., 1993; Gritti et al., 1996), and EGF (Reynolds and Weiss,1992). Withdrawal of growth factors induces their differentiation into neurons, astrocytes, and oligodendro- cytes (Reynolds and Weiss, 1992; Reynolds et al., 1992). These properties make them an interesting source of cells for neural repair after injury or disease. Besides their differentiation poten- tial, NSPCs seem to have a selective advantage for their survival when transplanted in the brain. In a xenotransplantation context, Armstrong and colleagues as well as our group demonstrated that porcine NSPCs were able to survive longer than porcine neurob- lasts when grafted into the striatum of non-immunosuppressed rats (Armstrong et al., 2001; Michel-Monigadon et al., 2011). As NSPCswerereportedtoexpressnoorlowlevelsof MHCmolecules (Klassen et al., 2001; Hori et al., 2003), this phenomenon was ﬁrstly linked to a lesser immunogenicity of these cells compared to more mature cells (Odeberg et al., 2005). However, immuno- genic properties of NSPCs cannot totally justify their delayed rejection, and recent studies conﬁrmed the expression of MHC class I and class II molecules by NSPCs, under normal or inﬂam- matory conditions (Sergent-Tanguy et al., 2006; Johansson et al., 2008; Yin et al., 2008; Laguna Goya et al., 2011). Beneﬁcial effects of NSPC transplantation have been shown in pre-clinical mod- els of several neurologic disorders such as Parkinson disease April 2012 \| Volume 6 \| Article 17 \| 3 Frontiers in Cellular Neuroscience www.frontiersin.org NSPC for CNS cell therapy Bonnamain et al. (Richardson et al., 2005), Huntington disease (McBride et al., 2004), or multiple sclerosis but also in other pathologies includ- ing renal ischemia-reperfusion (Wang et al., 2009b). However, the different mechanisms by which these cells exert their therapeutic effect remain unclear. The replacement of cells that have been lost or damaged was for a long time thought to be the main function of transplanted stem cells but it is now clear that somatic stem cells could also induce several beneﬁcial effects far beyond the cell replacement itself. NSPCs, THE FUTURE OF INTRACEREBRAL TRANSPLANTATION? For instance, it has recently been demon- strated that several stem cell types (embryonic, mesenchymal, or neural) display a strong immunosuppressive potential (Fändrich et al., 2002; Zappia et al., 2005; Einstein et al., 2007), favorable to their use in transplantation strategies for immune-related dis- eases like multiple sclerosis. It is also possible that NSPCs induce neural repair through intrinsic properties of neuroprotection and immunomodulation by releasing directly at the graft site a range of molecules (immunomodulatory molecules, growth factors, stem cell regulatory factors) spatially and temporally orchestrated by the microenvironment (Pluchino et al., 2009). region of the CNS, ampliﬁed in culture and reimplanted in the patient to repair cerebral or spinal damage without the need for immunosuppressive treatment (Pfeifer et al.,2006). To date, autol- ogous transplantation of differentiated NSPCs was performed in one Parkinson disease patient with encouraging results (Lévesque et al., 2009). If the invasive surgery to isolate NSPCs may have critical consequences like permanent damage in the taking area, this strategy presents the advantage to avoid immune compatibil- ity problems and could allow patient-speciﬁc gene modiﬁcations of the cells prior to transplantation (Stojkovic and Lako, 2011). Genetic engineering of autologous NSPCs could for example be used to repair potential genetic damage linked to the patient’s disease (like in Huntington disease), stimulate dopaminergic dif- ferentiation in the case of Parkinson disease (Wagner et al., 1999) or dedifferentiate NSPCs toward a pluripotent state using the Oct4 gene (Kim et al., 2009). Despite the immunologically special status of the CNS,long-term survival of neural allografts is compromised in the host without immunosuppression (Krystkowiak et al., 2007; Rota Nodari et al., 2010). Immunogenicity is therefore an impor- tant parameter to consider for the survival of transplanted cells. NSPCs express class I MHC and co-stimulatory molecules (Imi- tola et al., 2004; Sergent-Tanguy et al., 2006) but the expression of MHC class II molecules remain undetectable in physiological conditions (Hori et al.,2003). However in the case of an inﬂamma- tory process, in particular under IFNγ exposure, the expression of immunogenic molecules at NSPC’s surface is strongly increased (Johansson et al., 2008; Laguna Goya et al., 2011) which com- promises their survival and consequently their long-term effects. Moreover, NSPC allotransplantation studies have revealed that NSPCs derived from adult tissue could be less efﬁcient than those derived from the fetal CNS (Guillaume and Zhang, 2008). NSPCs, THE FUTURE OF INTRACEREBRAL TRANSPLANTATION? Indeed, the latter have demonstrated a greater capacity of proliferation in vitro and would more easily differentiate into neurons in vivo. NSPCs derived from the adult brain display weak propensity to integrate in the cerebral tissue, show limited synapse formation and their survival remains relatively short (Dziewczapolski et al., 2003), compromising a potential functional recovery. These obser- vations suggest that NSPCs should preferentially be isolated from the fetal CNS prior to transplantation. However, one must keep in mind that the use of human fetal NSPCs, like ES cells, is lim- ited by ethical and logistical issues. The use of NSPCs from animal species like swine could alleviate problems linked to the use of fetal human cells. In several studies, porcine NSPCs have been shown to have a reduced immunogenicity that could optimize their survival in vivo (Armstrong et al., 2001). Porcine NSPCs are also particu- larly interesting because of their multipotency. Barker’s group has demonstrated that porcine NSPCs xenografted in cyclosporine A- immunosuppressed rats presenting a 6-hydroxydopamine lesion to model Parkinson disease could differentiate into dopaminer- gic neurons (Armstrong et al., 2002). In addition, the xenografted cells have proven to signiﬁcantly integrate in the host tissue and partially reconstitute damaged neuronal circuitry (Harrower et al., 2006). In a recent study, our group has shown that NSPCs derived from the cerebral cortex of pig embryos at 28 days of gestation could survive signiﬁcantly longer than porcine neuroblasts iso- lated from the ventral mesencephalon after transplantation in the ADMINISTRATION ROUTE AND SOURCE OF TRANSPLANTED NSPCs Deﬁning the best route of administration of cells represents a con- straint for NSPC transplantation and is very dependent on the type of CNS lesions (focal or multifocal). Anatomic and patho- logic characteristics of CNS focal disorders like Parkinson disease, spinal cord lesions, Huntington disease, or stroke suggest that intracerebraltransplantationof cellsdirectlyatthesiteof thelesion would be the more appropriate strategy to facilitate tissue regener- ation. However, the presence of several lesion areas in diseases like multiple sclerosis or epilepsy represents a major limit for intrale- sional cellular transplantation approaches, and some groups were able to show a therapeutic effect of NSPC systemic transplan- tation by intravenous or intrathecal route (Pluchino et al., 2003; Einstein et al., 2007). Efﬁciency of restorative transplantation can also depend on the differentiation stage of the grafted cells. In some cases where a speciﬁc cell type is selectively lost during the pathogenic event, like the dopaminergic neurons in Parkinson disease, transplantation of pre-differentiated cells sharing similar properties in the affected region could allow a better functional recovery (Kim et al., 2002; Lévesque et al., 2009). However, the use of multipotent undifferentiated cells could be the best strat- egy in the case of a lesion or disease affecting several cell types in extended areas. Indeed these cells could spontaneously dif- ferentiate in vivo under the inﬂuence of the microenvironment in cells with desired phenotypes. It has recently been show that undifferentiated human NSPCs could survive and differentiate into neurons and glial cells after xenotransplantation into the rat spinal cord (Mothe et al., 2011). AUTO, ALLO, AND XENOTRANSPLANTATION OF NSPCs AUTO, ALLO, AND XENOTRANSPLANTATION OF NSPCs Autologous NSPCs derived from rodent adult nervous tissue and transplanted in the CNS can functionally integrate in the cerebral parenchyma, conﬁrming their potential use in therapy (Taupin and Gage, 2002). Isolation of NSPCs from the human adult brain offers the opportunity to perform autologous transplan- tation, in which NSPCs would be isolated from an unlesioned April 2012 \| Volume 6 \| Article 17 \| 4 Frontiers in Cellular Neuroscience www.frontiersin.org NSPC for CNS cell therapy Bonnamain et al. directly in the lesioned area using genetically engineered stem cells (Müller et al., 2006). This type of strategy would especially be appropriate in ischemic pathologies where altered blood ﬂow decreases the access to the lesion site by systemic administration route. NSPC’s neuroprotective effect often goes along with an increase in the bioavailability of the main neurotrophic factors like NGF, BDNF, CNTF, and GDNF (Carletti et al., 2011). This has been demonstrated in rodents suffering from primary cen- tral inﬂammatory diseases like multiple sclerosis (Pluchino et al., 2003), spinal cord lesions (Lu et al., 2003), or stroke, and also in rodent models of neurodegenerative disorders associated with an immune reaction such as Parkinson and Huntington diseases (McBride et al.,2004; Ryu et al.,2004; Richardson et al.,2005). Cel- lular and molecular mechanisms implicated in this phenomenon remain unclear but may reside in the intrinsic properties of neu- rospheres, cellular artifacts resulting from NSPC culture and from which most of transplanted NSPCs are derived. Typically, neu- rospheres are generated in vitro after about 10 days in culture in the absence of serum and in the presence of high concen- trations of growth factors (EGF/bFGF). This culture protocol allows the selection of NSPCs responding to those factors. Thus, after transplantation, neurosphere-derived NSPCs might be more sensitive to environmental signals (especially bFGF) in the host tissue triggering neurotrophin secretion by neighboring cells (Lu et al., 2003). was associated with an absence of T lymphocyte, dendritic and activated microglial cell inﬁltration within the NSPC healthy grafts and with a trophic effect of the grafted cells on the host dopaminergic system (Michel-Monigadon et al., 2011). However, we did not observe any dopaminergic differentiation suggesting that NSPCs could be beneﬁcial far beyond the cell replacement process itself. IMMUNOMODULATION Accumulating evidence suggest that stem cells, like mesenchymal stem cells (MSCs) or embryonic stem cells, could interact with components of the immune system, leading to the modulation of many effector functions (Fändrich et al., 2002; Zappia et al., 2005). For instance, MSCs are known to suppress T cell prolif- eration in vitro (Di Nicola et al., 2002) and to induce long-term graft survival in an allogeneic context (Bartholomew et al., 2002; Chabannes et al., 2007). Regarding NSPCs, a growing number of studies highlighted their immunomodulatory properties, in vivo and in vitro (Bonnamain et al., 2011). It was clearly demonstrated that NSPCs were able to attenuate experimental autoimmune encephalomyelitis (EAE) when injected centrally or peripherally (Einstein et al., 2003, 2007; Pluchino et al., 2005). If the site of action (i.e., CNS or lymph nodes) is not clearly deﬁned, NSPCs have the capacity to inhibit the proliferation of lymph nodes- derived T cells, in response to either concanavalin A (ConA) or to myelin oligodendrocyte glycoprotein, in vitro (Einstein et al., 2003). A recent study showed that syngenic NSPCs transplanted in a model of focal spinal cord injury were able to interact with activated-macrophages in situ to decrease their number and increase the proportion of regulatory T cells (Cusimano et al., 2012). This conﬁrms the importance of the interactions between NSPCs and immune cells to reconﬁgure the deleterious inﬂamma- tory environment and thus promote the healing or regeneration processes. As systemic transplantation of NSPCs was shown to be beneﬁcial in animal models of autoimmune disease like mul- tiple sclerosis (Einstein et al., 2007), it could be hypothesized that NSPCs exert their immunomodulatory effect through a strong paracrine mechanism. Previous studies revealed the expression of immune molecules like TGFβ-1 by NSPCs (Klassen et al., 2003), THERAPEUTIC EFFECTS OF TRANSPLANTED NSPCs CELL REPLACEMENT Human NSPCs transplanted in the brain of immunodeﬁcient mice can proliferate, migrate, and differentiate depending on the injection site (Tamaki et al., 2002). Allotransplantation of NSPCs has proven to be efﬁcient in animal models of stroke and spinal cord lesion. Murine NSPCs from C17.2 cell line implanted in an ischemic mouse brain could survive, integrate, and differentiate into neurons and glial cells (Snyder et al., 1997). Animal models of cerebral ischemia have also been used to demonstrate that NSPCs delivered by intraventricular injection could migrate toward and through the damaged tissue, inte- grate, and differentiate into the main neural cell types (Riess et al., 2002). Moreover, NSPCs genetically modiﬁed to overex- press NT-3 have shown a potent integration and a signiﬁcant regenerative capacity in a model of hypoxic and ischemic cere- bral lesion, associated with a decrease in the severity of brain parenchyma damage, an improvement of axonal outgrowth, and a diminution of the glial scar (Park et al., 2006). These studies suggest that, under certain conditions, the lesioned CNS could represent a permissive environment for the maintenance of trans- planted NSPCs. Functional integration and reconstruction of the neuronal circuitry is an important goal for restorative therapy. In this context, several groups have shown that NSPCs cultured in vitro could give rise to functional neurons connected and elec- trically active (Auerbach et al., 2000) that could integrate in the host cortical connections after transplantation (Snyder et al.,1997; Lundberg et al., 2002; Park et al., 2002). Despite these encourag- ing results, proofs of the ability of NSPCs to differentiate into a sufﬁcient number of functional neurons that could regener- ate lost functions by massive cell replacement remain relatively rare. Functional beneﬁts resulting from NSPC transplantation are hardly correlated to the number of fully differentiated neu- ral cells obtained from the grafted cells. This inefﬁciency to complete the differentiation process and the tendency to main- tain an undifferentiated phenotype within the host tissue suggest that transplanted NSPCs partially exert their therapeutic effect by alternative mechanisms. Frontiers in Cellular Neuroscience REFERENCES Aloisi, F. (2001). Immune function of microglia. Glia 36, 165–179. Cornet, A., Bettelli, E., Oukka, M., Cambouris, C., Avellana-Adalid, V., Cornet, A., Bettelli, E., Oukka, M., Cambouris, C., Avellana-Adalid, V., Kosmatopoulos, K., and Liblau, R. S. (2000). Role of astrocytes in anti- gen presentation and naive T-cell activation. J. Neuroimmunol. 106, 69–77. Armstrong, R. J., Harrower, T. P., Hurel- brink, C. B., McLaughin, M., Rat- cliffe, E. L., Tyers, P., Richards, A., Dunnett, S. B., Rosser, A. E., and Barker, R. A. (2001). Porcine neu- ral xenografts in the immunocompe- tent rat: immune response following grafting of expanded neural precur- sor cells. Neuroscience 106, 201–216. survival in vivo. Exp. Hematol. 30, 42–48. Billingham, R. E., and Boswell, T. (1953). Studies on the problem of corneal homografts. Proc. R. Soc. Lond. B Biol. Sci. 141, 392–406. Einstein, O., Karussis, D., Grigori- adis, N., Mizrachi-Kol, R., Reinhartz, E., Abramsky, O., and Ben-Hur, T. (2003). Intraventricular transplanta- tion of neural precursor cell spheres attenuatesacuteexperimentalallergic encephalomyelitis. Mol. Cell. Neu- rosci. 24, 1074–1082. Cusimano, M., Biziato, D., Brambilla, E., Donegà, M., Alfaro-Cervello, C., Snider, S., Salani, G., Pucci, F., Comi, G., Garcia-Verdugo, J. M., De Palma, M., Martino, G., and Pluchino, S. (2012). Transplanted neural stem/precursor cells instruct phagocytes and reduce secondary tis- sue damage in the injured spinal cord. Brain 35(Pt. 2), 447–460. Björklund, A., Dunnett, S. B., Brundin, P., Stoessl, A. J., Freed, C. R., Breeze, R. E., Levivier, M., Peschanski, M., Studer, L., and Barker, R. (2003). Neural transplantation for the treat- ment of Parkinson’s disease. Lancet Neurol. 2, 437–445. Armstrong, R. J. E., Hurelbrink, C. B., Tyers, P., Ratcliffe, E. L., Richards, A., Dunnett, S. B., Rosser, A. E., and Barker, R. A. (2002). The potential for circuit reconstruction by expanded neural precursor cells explored through porcine xenografts in a rat model of Parkinson’s disease. Exp. Neurol. 175, 98–111. Fändrich, F., Dresske, B., Bader, M., and Schulze, M. (2002). Embryonic stem cells share immune-privileged features relevant for tolerance induc- tion. J. Mol. Med. 80, 343–350. Björklund, A., and Lindvall, O. (2000). Cell replacement therapies for cen- tral nervous system disorders. Nat. Neurosci. 3, 537–544. Daniloff, J. K., Low, W. C., Bodony, R. P., and Wells, J. (1985). Cross-species neural transplants of embryonic sep- tal nuclei to the hippocampal forma- tion of adult rats. Exp. Brain Res. 59, 73–82. Freeman, T. B., Cicchetti, F., Hauser, R. A., Deacon, T. REFERENCES W., Li, X.-J., Her- sch, S. M., Nauert, G. M., Sanberg, P. R., Kordower, J. H., Saporta, S., and Isacson, O. (2000). Transplanted fetal striatum in Huntington’s dis- ease: phenotypic development and lack of pathology. Proc. Natl. Acad. Sci. U.S.A. 97, 13877–13882. Björklund, A., Stenevi, U., Dunnett, S. B., and Gage, F. H. (1982). Cross- species neural grafting in a rat model of Parkinson’s disease. Nature 298, 652–654. Auerbach, J. M., Eiden, M. V., and McKay, R. D. (2000). Transplanted CNS stem cells form functional synapses in vivo. Eur. J. Neurosci. 12, 1696–1704. Deacon, T., Schumacher, J., Dinsmore, J., Thomas, C., Palmer, P., Kott, S., Edge, A., Penney, D., Kassissieh, S., Dempsey, P., and Isacson, O. (1997). Histological evidence of fetal pig neu- ral cell survival after transplantation into a patient with Parkinson’s dis- ease. Nat. Med. 3, 350–353. Bonnamain, V., Neveu, I., and Naveil- han, P. (2011). In vitro analyses of the immunosuppressive proper- ties of neural stem/progenitor cells using anti-CD3/CD28-activated T cells. Methods Mol. Biol. 677, 233– 243. Bachoud-Lévi, A.-C., Gaura, V., Brugières, P., Lefaucheur, J.-P., Boissé, M.-F., Maison, P., Baudic, S., Ribeiro, M.-J., Bourdet, C., Remy, P., Cesaro, P., Hantraye, P., and Peschanski, M. (2006). Effect of fetal neural trans- plants in patients with Huntington’s disease 6 years after surgery: a long- term follow-up study. Lancet Neurol. 5, 303–309. Freeman, T. B., Wojak, J. C., Brandeis, L., Michel, J. P., Pearson, J., and Flamm, E. S. (1988). Cross-species intracerebral grafting of embryonic swine dopaminergic neurons. Prog. Brain Res. 78, 473–477. Deacon, T. W., Pakzaban, P., Burns, L. H., Dinsmore, J., and Isacson, O. (1994). Cytoarchitectonic devel- opment, axon–glia relationships, and long distance axon growth of porcine striatal xenografts in rats. Exp. Neu- rol. 130, 151–167. Carletti, B., Fpiemonte, F., and Rossi, F. (2011). Neuroprotection: the emerg- ing concept of restorative neural stem cell biology for the treatment of neu- rodegenerative diseases. Curr. Neu- ropharmacol. 9, 313–317. Galpern, W. R., Burns, L. H., Dea- con, T. W., Dinsmore, J., and Isac- son, O. (1996). Xenotransplanta- tion of porcine fetal ventral mesen- cephalon in a rat model of Parkin- son’s disease: functional recovery and graft morphology. Exp. Neurol. 140, 1–13. Bachoud-Lévi, A. C., Rémy, P., Nguyen, J. P., Brugières, P., Lefaucheur, J. P., Bourdet, C., Baudic, S., Gaura, V., Maison, P., Haddad, B., Boissé, M. F., Grandmougin, T., Jény, R., Bar- tolomeo, P., Dalla Barba, G., Degos, J. NEUROPROTECTION Transplanted NSPCs can signiﬁcantly improve the survival and the functions of endogenous glial and neuronal progenitor cells that have survived the pathologic event. NSPCs display a strong tropism for tissue lesions and seem to migrate toward these critical sites to release molecules preventing death and facilitat- ing regeneration of targeted cell populations (Ourednik et al., 2002). Several groups are interested in this particular tropism of NSPCs and stem cells in general for damaged zones. This property could indeed be used to deliver therapeutic molecules April 2012 \| Volume 6 \| Article 17 \| 5 Frontiers in Cellular Neuroscience www.frontiersin.org NSPC for CNS cell therapy Bonnamain et al. the common knowledge that the CNS was an immunologically privileged tissue deprived of any immune reaction and that transplanted NSPCs were only efﬁcient through mechanisms of neuronal replacement. It is now clear that neuroprotection and immunomodulation capacities of these cells play a major part in the beneﬁcial effects observed in pre-clinical models of neurode- generative diseases and other CNS affections. Elucidation of these mechanisms may be a crucial step in the control and improve- ment of NSPC transplantation as a major therapeutic strategy in regenerative medicine. or inducible nitric oxide synthase (iNOS) and prostaglandin E2 in NSPC cell line (Wang et al.,2009a). Even if the implication of some of these molecules in the immunosuppressive effect of NSPC cell line has already been suggested, the mechanism remains poorly understood. CONCLUSION Signiﬁcant progresses in stem cell biology have generated con- siderable enthusiasm for the use of these cells in therapeutic strategies for CNS disorders. Numerous studies have challenged adult rat and human mesenchymal stem cells. Blood 110, 3691–3694. Einstein, O., Fainstein, N., Vaknin, I., Mizrachi-Kol, R., Reihartz, E., Grigoriadis, N., Lavon, I., Baniyash, M., Lassmann, H., and Ben-Hur, T. (2007). Neural precursors atten- uate autoimmune encephalomyelitis by peripheral immunosuppression. Ann. Neurol. 61, 209–218. Deans, R., Moseley, A., and Hoff- man, R. (2002). Mesenchymal stem cells suppress lymphocyte prolifera- tion in vitro and prolong skin graft survival in vivo. Exp. Hematol. 30, 42–48. REFERENCES REFERENCES C., Nerrière-Daguin, V., Josien, R., Brachet, P., Naveilhan, P., and Neveu, I. (2006). Dendritic cell recruitment following xenograft- ing of pig fetal mesencephalic cells into the rat brain. Exp. Neurol. 202, 76–84. Head, J. R., Neaves, W. B., and Billing- ham, R. E. (1983). Immune privilege in the testis. I. Basic parameters of allograft survival. Transplantation 36, 423–431. Joó, F. (1993). The blood–brain barrier in vitro: the second decade. Neu- rochem. Int. 23, 499–521. Larsson, L. C., Czech, K. A., Widner, H., and Korsgren, O. (1999). Discor- dant neural tissue xenografts survive longer in immunoglobulin deﬁcient mice. Transplantation 68, 1153–1160. Kebir, H., Kreymborg, K., Ifergan, I., Dodelet-Devillers, A., Cayrol, R., Bernard, M., Giuliani, F., Arbour, N., Becher, B., and Prat, A. (2007). Human TH17 lymphocytes promote blood–brain barrier disruption and central nervous system inﬂamma- tion. Nat. Med. 13, 1173–1175. Hickey, W. F. (2001). Basic principles of immunological surveillance of the normal central nervous system. Glia 36, 118–124. Michel-Monigadon, D., Bonnamain, V., Nerrière-Daguin, V., Dugast, A.-S., Lévèque, X., Plat, M., Venturi, E., Brachet, P., Anegon, I., Vanhove, B., Neveu, I., and Naveilhan, P. (2011). Trophic and immunoregu- latory properties of neural precur- sor cells: beneﬁt for intracerebral transplantation. Exp. Neurol. 230, 35–47. Lévesque, M. F., Neuman, T., and Rezak, M. (2009). Therapeutic microinjec- tion of autologous adult human neu- ral stem cells and differentiated neu- rons for Parkinson’s disease: ﬁve-year post-operative outcome. Open Stem Cell J. 1, 20–29. Hickey,W. F., Hsu, B. L., and Kimura, H. (1991). T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28, 254–260. Kerschensteiner, M., Meinl, E., and Hohlfeld, R. (2009). Neuro-immune crosstalk in CNS diseases. Neuro- science 158, 1122–1132. Lu, P., Jones, L. L., Snyder, E. Y., and Tuszynski, M. H. (2003). Neu- ral stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp. Neurol. 181, 115–129. Höftberger, R., Aboul-Enein, F., Brueck, W., Lucchinetti, C., Rodriguez, M., Schmidbauer, M., Jellinger, K., and Lassmann, H. (2004). Expression of major histocompatibility complex class I molecules on the different cell types in multiple sclerosis lesions. Brain Pathol. 14, 43–50. Mothe, A. J., Zahir, T., Santaguida, C., Cook, D., and Tator, C. H. (2011). Neuralstem/progenitorcellsfromthe adult human spinal cord are mul- tipotent and self-renewing and dif- ferentiate after transplantation. PLoS ONE 6, e27079. doi: 10.1371/jour- nal.pone.0027079 Kim, J. REFERENCES H. (2004). Human neural stem cell transplants improve motor function in a rat model of Huntington’s disease. J. Comp. Neurol. 475, 211–219. Krystkowiak, P., Gaura, V., Labalette, M., Rialland, A., Remy, P., Peschan- ski, M., and Bachoud-Lévi, A.-C. (2007). Alloimmunisation to donor antigens and immune rejection fol- lowing foetal neural grafts to the brain in patients with Huntington’s disease. PLoS ONE 2, e166. doi: 10.1371/journal.pone.0000166 Guillaume, D. J., and Zhang, S.-C. (2008). Human embryonic stem cells: a potential source of transplantable neural progenitor cells. Neurosurg. Focus 24, E3. Isacson, O., Deacon, T. W., Pakz- aban, P., Galpern, W. R., Dins- more, J., and Burns, L. H. (1995). Transplanted xenogeneic neural cells in neurodegenerative disease mod- els exhibit remarkable axonal target speciﬁcity and distinct growth pat- terns of glial and axonal ﬁbres. Nat. Med. 1, 1189–1194. Medawar, P. B. (1948). Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br. J. Exp. Pathol. 29, 58–69. Harrower, T. P., Tyers, P., Hooks, Y., and Barker, R. A. (2006). Long-term survival and integration of porcine expanded neural precursor cell grafts in a rat model of Parkinson’s disease. Exp. Neurol. 197, 56–69. Laguna Goya, R., Busch, R., Mathur, R., Coles, A. J., and Barker, R. A. (2011). Human fetal neural precursor cells can up-regulate MHC class I and class II expression and elicit CD4 and CD8 T cell proliferation. Neurobiol. Dis. 41, 407–414. Melchior, B., Rémy, S., Nerrière- Daguin, V., Heslan, J.-M., Soulillou, J.-P., and Brachet, P. (2002). Tempo- ral analysis of cytokine gene expres- sion during inﬁltration of porcine neuronal grafts implanted into the rat brain. J. Neurosci. Res. 68, 284–292. Hatterer, E., Davoust, N., Didier-Bazes, ill l li Hatterer, E., Davoust, N., Didier-Bazes, M., Vuaillat, C., Malcus, C., Belin, M.-F., and Nataf, S. (2006). How to drain without lymphatics? Den- dritic cells migrate from the cere- brospinal ﬂuid to the B-cell follicles of cervical lymph nodes. Blood 107, 806–812. Johansson, S., Price, J., and Modo, M. (2008). Effect of inﬂamma- tory cytokines on major histocom- patibility complex expression and differentiation of human neural stem/progenitor cells. Stem Cells 26, 2444–2454. Larsson, L. C., Czech, K. A., Brundin, P., and Widner, H. (2000). Intrastri- atal ventral mesencephalic xenografts of porcine tissue in rats: immune responses and functional effects. Cell Transplant. 9, 261–272. Michel, D. REFERENCES B., Sebastiano, V., Wu, G., Araúzo-Bravo,M.J.,Sasse,P.,Gentile, L., Ko, K., Ruau, D., Ehrich, M., van den Boom, D., Meyer, J., Hübner, K., Bernemann, C., Ortmeier, C., Zenke, M., Fleischmann, B. K., Zaehres, H., and Schöler, H. R. (2009). Oct4- induced pluripotency in adult neural stem cells. Cell 136, 411–419. Lundberg, C., Englund, U., Trono, D., Björklund, A., and Wictorin, K. (2002). Differentiation of the RN33B cell line into forebrain projection neuronsaftertransplantationintothe neonatal rat brain. Exp. Neurol. 175, 370–387. Honey, C. R., Clarke, D. J., Dall- man, M. J., and Charlton, H. M. (1990). Human neural graft function in rats treated with anti-interleukin II receptor antibody. Neuroreport 1, 247–249. Müller, F.-J., Snyder, E. Y., and Loring, J. F. (2006). Gene therapy: can neural stem cells deliver? Nat. Rev. Neurosci. 7, 75–84. Kim, J.-H., Auerbach, J. M., Rodríguez- Gómez, J. A., Velasco, I., Gavin, D., Lumelsky, N., Lee, S.-H., Nguyen, J., Sánchez-Pernaute, R., Bankiewicz, K., and McKay, R. (2002). Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418, 50–56. Martin, C., Plat, M., Nerriére-Daguin, V., Coulon, F., Uzbekova, S., Ven- turi, E., Condé, F., Hermel, J.-M., Hantraye, P., Tesson, L., Anegon, I., Melchior, B., Peschanski, M., Le Mauff, B., Boeffard, F., Sergent- Tanguy, S., Neveu, I., Naveilhan, P., Soulillou, J. P., Terqui, M., Brachet, P., and Vanhove, B. (2005). Trans- genic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation. Transgenic Res. 14, 373–384. Hori, J., Joyce, N., and Streilein, J. W. (2000). Epithelium-deﬁcient corneal allografts display immune privilege beneath the kidney capsule. Invest. Ophthalmol. Vis. Sci. 41, 443–452. Odeberg, J., Piao, J.-H., Samuelsson, E.-B., Falci, S., and Akesson, E. (2005). Low immunogenicity of in vitro-expanded human neural cells despite high MHC expression. J. Neu- roimmunol. 161, 1–11. Hori, J., Ng, T. F., Shatos, M., Klassen, H., Streilein, J. W., and Young, M. J. (2003). Neural progenitor cells lack immunogenicity and resist destruc- tion as allografts. Stem Cells 21, 405–416. Klassen, H., Schwartz, M. R., Bai- ley, A. H., and Young, M. J. (2001). Surface markers expressed by mul- tipotent human and mouse neural progenitor cells include tetraspanins and non-protein epitopes. Neurosci. Lett. 312, 180–182. Okura, Y., Tanaka, R., Ono, K., Yoshida, S., Tanuma, N., and Matsumoto, Y. (1997). Treatment of rat hemiparkin- son model with xenogeneic neural transplantation: tolerance induction by anti-T-cell antibodies. J. Neurosci. Res. 48, 385–396. Huffaker, T. K., Boss, B. REFERENCES D., Lisovoski, F., Ergis, A. M., Pailhous, E., Cesaro, P., Hantraye, P., and Peschanski, M. (2000). Motor and cognitive improvements in patients with Huntington’s disease after neural transplantation. Lancet 356, 1975–1979. Cayrol, R., Wosik, K., Berard, J. L., Dodelet-Devillers, A., Ifergan, I., Kebir, H., Haqqani, A. S., Kreym- borg, K., Krug, S., Moumdjian, R., Bouthillier, A., Becher, B., Arbour, N., David, S., Stanimirovic, D., and Prat, A. (2008). Activated leuko- cyte cell adhesion molecule promotes leukocyte trafﬁcking into the central nervous system. Nat. Immunol. 9, 137–145. Di Nicola, M., Carlo-Stella, C., Magni, M., Milanesi, M., Longoni, P. D., Mat- teucci, P., Grisanti, S., and Gianni, A. M. (2002). Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspeciﬁc mitogenic stimuli. Blood 99, 3838–3843. Goldstein, G. W., and Betz, A. L. (1983). Recent advances in understanding brain capillary function. Ann. Neurol. 14, 389–395. Dziewczapolski, G., Lie, D. C., Ray, J., Gage, F. H., and Shults, C. W. (2003). Survival and differentiation of adultrat-derivedneuralprogenitor cells transplanted to the striatum of hemiparkinsonian rats. Exp. Neurol. 183, 653–664. Gritti, A., Parati, E. A., Cova, L., Frol- ichsthal, P., Galli, R., Wanke, E., Faravelli, L., Morassutti, D. J., Roisen, F., Nickel, D. D., and Vescovi, A. L. (1996). Multipotential stem cells from the adult mouse brain prolifer- ateandself-renewinresponsetobasic Barker, C. F., and Billingham, R. E. (1977). Immunologically privileged sites. Adv. Immunol. 25, 1–54. Chabannes, D., Hill, M., Merieau, E., Rossignol, J., Brion, R., Soulillou, J. Chabannes, D., Hill, M., Merieau, E., Rossignol, J., Brion, R., Soulillou, J. P., Anegon, I., and Cuturi, M. C. (2007). A role for heme oxygenase- 1 in the immunosuppressive effect of (1977). Immunologically privileged sites. Adv. Immunol. 25, 1–54. Bartholomew,A., Sturgeon, C., Siatskas, M., Ferrer, K., McIntosh, K., Patil, Bartholomew,A., Sturgeon, C., Siatskas, M., Ferrer, K., McIntosh, K., Patil, S., Hardy, W., Devine, S., Ucker, D., S., Hardy, W., Devine, S., Ucker, D., April 2012 \| Volume 6 \| Article 17 \| 6 Frontiers in Cellular Neuroscience www.frontiersin.org NSPC for CNS cell therapy Bonnamain et al. ﬁbroblast growth factor. J. Neurosci. 16, 1091–1100. ﬁbroblast growth factor. J. Neurosci. 16, 1091–1100. Snyder, E.Y., and Khoury, S. J. (2004). Neural stem/progenitor cells express costimulatory molecules that are dif- ferentially regulated by inﬂammatory and apoptotic stimuli. Am. J. Pathol. 164, 1615–1625. McBride, J. L., Behrstock, S. P., Chen, E.-Y., Jakel, R. J., Siegel, I., Svendsen, C. N., and Kordower, J. REFERENCES Neural xenotransplantation: recon- struction of neuronal circuitry across species barriers. Neuroscience 62, 989–1001. Wictorin, K., Clarke, D. J., Bolam, J. P., Brundin, P., Gustavii, B., Lindvall, O., and Björklund, A. (1990). Exten- sive efferent projections of intra- striatally transplanted striatal neu- rons as revealed by a species-speciﬁc neuroﬁlament marker and antero- grade axonal tracing. Prog. Brain Res. 82, 391–399. Reynolds, B. A., and Weiss, S. (1992). Generation of neurons and astro- cytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710. Snyder, E. Y., Yoon, C., Flax, J. D., and Macklis, J. D. (1997). Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apop- totic degeneration in adult mouse neocortex. Proc. Natl. Acad. Sci. U.S.A. 94, 11663–11668. Park, K. I., Himes, B. T., Stieg, P. E., Tessler, A., Fischer, I., and Sny- der, E. Y. (2006). Neural stem cells may be uniquely suited for com- bined gene therapy and cell replace- ment: evidence from engraftment of neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury. Exp. Neurol. 199, 179–190. Rezzani, R. (2006). Exploring cyclosporine A-side effects and the protective role-played by antiox- idants: the morphological and immunohistochemical studies. Histol. Histopathol. 21, 301–316. Wood, M. J., Sloan, D. J., Wood, K. J., and Charlton, H. M. (1996). Indef- inite survival of neural xenografts induced with anti-CD4 mono- clonal antibodies. Neuroscience 70, 775–789. Stojkovic, M., and Lako, M. (2011). Neural stem cells, a step closer to clinic? Stem Cells 29, 1477–1478. Richardson, R. M., Broaddus, W. C., Holloway, K. L., and Fillmore, H. L. (2005). Grafts of adult subependy- mal zone neuronal progenitor cells rescue hemiparkinsonian behav- ioral decline. Brain Res. 1032, 11–22. Park, K. I., Teng, Y. D., and Snyder, E. Y. (2002). The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconsti- tute lost tissue. Nat. Biotechnol. 20, 1111–1117. Yin, L., Fu, S.-L., Shi, G.-Y., Li, Y., Jin, J.- Q., Ma, Z.-W., and Lu, P.-H. (2008). Expression and regulation of major histocompatibility complex on neu- ral stem cells and their lineages. Stem Cells Dev. 17, 53–65. Tamaki, S., Eckert, K., He, D., Sutton, R., Doshe, M., Jain, G., Tushin- ski, R., Reitsma, M., Harris, B., Tsukamoto,A.,Gage,F.,Weissman,I., and Uchida, N. (2002). Engraftment of sorted/expanded human central nervous system stem cells from fetal brain. J. Neurosci. Res. 69, 976–986. Riess, P., Zhang, C., Saatman, K. E., Laurer, H. L., Longhi, L. REFERENCES D., Morgan, A. S., Neff, N. T., Strecker, R. E., Spence, M. S., and Miao, R. (1989). Xenografting of fetal pig ventral mes- encephalon corrects motor asymme- try in the rat model of Parkinson’s disease. Exp. Brain Res. 77, 329–336. Mauerhoff, T., Pujol-Borrell, R., Mirakian, R., and Bottazzo, G. F. (1988). Differential expression and regulation of major histocompati- bility complex (MHC) products in neural and glial cells of the human fetal brain. J. Neuroimmunol. 18, 271–289. Klassen, H. J., Imfeld, K. L., Kirov, I. I., Tai, L., Gage, F. H., Young, M. J., and Berman, M. A. (2003). Expression of cytokines by multipo- tent neural progenitor cells. Cytokine 22, 101–106. Olanow, C. W., Goetz, C. G., Kordower, J. H., Stoessl, A. J., Sossi, V., Brin, M. F., Shannon, K. M., Nauert, G. M., Perl, D. P., Godbold, J., and Freeman, T. B. (2003). A double- blind controlled trial of bilateral fetal Imitola, J., Comabella, M., Chandraker, A. K., Dangond, F., Sayegh, M. H., April 2012 \| Volume 6 \| Article 17 \| 7 Frontiers in Cellular Neuroscience www.frontiersin.org NSPC for CNS cell therapy Bonnamain et al. nigral transplantation in Parkinson’s disease. Ann. Neurol. 54, 403–414. neural precursor cells ameliorates renal ischemia-reperfusion injury. Nephron Exp. Nephrol. 112, e20–e28. nigral transplantation in Parkinson’s disease. Ann. Neurol. 54, 403–414. rejection of porcine neurons and endothelial cells transplanted into the rat brain. Xenotransplantation 8, 136–148. Sergent-Tanguy, S., Véziers, J., Bon- namain, V., Boudin, H., Neveu, I., and Naveilhan, P. (2006). Cell surface antigens on rat neural pro- genitors and characterization of the CD3 (+)/CD3 (−) cell populations. Differentiation 74, 530–541. Ourednik, J., Ourednik, V., Lynch, W. y P., Schachner, M., and Snyder, E. Y. (2002). Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat. Biotech- nol. 20, 1103–1110. Wekerle, H., Sun, D., Oropeza-Wekerle, R. L., and Meyermann, R. (1987). Immune reactivity in the nervous sys- tem: modulation of T-lymphocyte activation by glial cells. J. Exp. Biol. 132, 43–57. Reynolds, B. A., Tetzlaff, W., and Weiss, S. (1992). A multipotent EGF- responsive striatal embryonic pro- genitor cell produces neurons and astrocytes. J. Neurosci. 12, 4565– 4574. Sivakumar, V., Foulds, W. S., Luu, C. D., Ling, E., and Kaur, C. (2011). Reti- nal ganglion cell death is induced by microglia derived pro-inﬂammatory cytokines in the hypoxic neonatal retina. J. Pathol. 224, 245–260. Pakzaban, P., and Isacson, O. (1994). REFERENCES G., Raghu- Pfeifer, K., Vroemen, M., Caioni, M., Aigner, L., Bogdahn, U., and Wei- dner, N. (2006). Autologous adult rodent neural progenitor cell trans- plantation represents a feasible strat- egy to promote structural repair in the chronically injured spinal cord. Regen. Med. 1, 255–266. Riess, P., Zhang, C., Saatman, K. E., Laurer, H. L., Longhi, L. G., Raghu- pathi, R., Lenzlinger, P. M., Lif- shitz, J., Boockvar, J., Neugebauer, E., Snyder, E. Y., and McIntosh, T. K. (2002). Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury. Neurosurgery 51, 1043–1052; discussion 1052–1054. Zappia, E., Casazza, S., Pedemonte, E., Benvenuto, F., Bonanni, I., Gerdoni, Zappia, E., Casazza, S., Pedemonte, E., Benvenuto, F., Bonanni, I., Gerdoni, E., Giunti, D., Ceravolo, A., Caz- zanti, F., Frassoni, F., Mancardi, G., and Uccelli, A. (2005). Mesenchy- mal stem cells ameliorate experimen- tal autoimmune encephalomyelitis inducing T-cell anergy. Blood 106, 1755–1761. Taupin, P., and Gage, F. H. (2002). Adult neurogenesis and neural stem cells of the central nervous system in mammals. J. Neurosci. Res. 69, 745–749. Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani, G., Dina, G., Galli, R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi, A. L., and Martino, G. (2003). Injection of adult neuro- spheres induces recovery in a chronic model of multiple sclerosis. Nature 422, 688–694. Vescovi, A. L., Reynolds, B. A., Fraser, D. D., and Weiss, S. (1993). bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells. Neuron 11, 951–966. Rota Nodari, L., Ferrari, D., Giani, F., Bossi, M., Rodriguez-Menendez, Rota Nodari, L., Ferrari, D., Giani, F., Bossi, M., Rodriguez-Menendez, V., Tredici, G., Delia, D., Vescovi, A. L., and De Filippis, L. (2010). Long-term survival of human neural stem cells in the ischemic rat brain upon transient immunosuppression. PLoS ONE 5, e14035. doi: 10.1371/ journal.pone.0014035 V., Tredici, G., Delia, D., Vescovi, Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any com- mercial or ﬁnancial relationships that could be construed as a potential con- ﬂict of interest. Wagner, J., Akerud, P., Castro, D. S., Holm, P. C., Canals, J. M., Sny- der, E. Y., Perlmann, T., and Arenas, E. (1999). Induction of a midbrain dopaminergic phenotype in Nurr1- overexpressing neural stem cells by type 1 astrocytes. Nat. Biotechnol. 17, 653–659. REFERENCES Pluchino, S., Zanotti, L., Brini, E., Ferrari, S., and Martino, G. (2009). Regeneration and repair in multi- ple sclerosis: the role of cell trans- plantation. Neurosci. Lett. 456, 101–106. Ryu, J. K., Kim, J., Cho, S. J., Hatori, K., Nagai, A., Choi, H. B., Lee, M. C., McLarnon, J. G., and Kim, S. U. (2004). Proactive transplantation of human neural stem cells prevents degeneration of striatal neurons in a rat model of Huntington disease. Neurobiol. Dis. 16, 68–77. Received: 15 February 2012; paper pend- ing published: 06 March 2012; accepted: 26 March 2012; published online: 11 April 2012. Citation: Bonnamain V, Neveu I and Naveilhan P (2012) Neural stem/ progenitor cells as promising candidates for regenerative therapy of the central ner- vous system. Front. Cell. Neurosci. 6:17. doi: 10.3389/fncel.2012.00017 Received: 15 February 2012; paper pend- ing published: 06 March 2012; accepted: 26 March 2012; published online: 11 April 2012. Received: 15 February 2012; paper pend- ing published: 06 March 2012; accepted: 26 March 2012; published online: 11 April 2012. Pluchino, S., Zanotti, L., Rossi, B., Brambilla, E., Ottoboni, L., Salani, G., Martinello, M., Cattalini, A., Bergami, A., Furlan, R., Comi, G., Constantin, G., and Martino, G. (2005). Neurosphere-derived multi- potent precursors promote neu- roprotection by an immunomod- ulatory mechanism. Nature 436, 266–271. Citation: Bonnamain V, Neveu I and Naveilhan P (2012) Neural stem/ progenitor cells as promising candidates for regenerative therapy of the central ner- vous system. Front. Cell. Neurosci. 6:17. doi: 10.3389/fncel.2012.00017 Wang, L., Shi, J., van Ginkel, F. W., Lan, L., Niemeyer, G., Martin, D. R., Sny- der, E. Y., and Cox, N. R. (2009a). Neural stem/progenitor cells modu- late immune responses by suppress- ing T lymphocytes with nitric oxide and prostaglandin E2. Exp. Neurol. 216, 177–183. Schumacher, J. M., Ellias, S. A., Palmer, E. P., Kott, H. S., Dinsmore, J., Dempsey, P. K., Fischman, A. J., Thomas, C., Feldman, R. G., Kas- sissieh, S., Raineri, R., Manhart, C., Penney, D., Fink, J. S., and Isacson, O. (2000). Transplantation of embry- onic porcine mesencephalic tissue in patients with PD. Neurology 54, 1042–1050. vous system. Front. Cell. Neurosci. 6:17. doi: 10.3389/fncel.2012.00017 Copyright © 2012 Bonnamain, Neveu and Naveilhan. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non- commercial use, distribution, and repro- duction in other forums, provided the original authors and source are credited. Wang, P. H. REFERENCES M., Schwindt, T. T., Barn- abé, G. F., Motta, F. L. T., Semedo, P., Wang, P. H. M., Schwindt, T. T., Barn- abé, G. F., Motta, F. L. T., Semedo, P., Beraldo, F. C., Mazzali, M., Dos Reis, M. A., Teixeira Vde, P. A., Pacheco- Silva, A., Mello, L. E., and Câmara, N. O. (2009b). Administration of Rémy, S., Canova, C., Daguin- Nerrière, V., Martin, C., Melchior, B., Neveu, I., Charreau, B., Soulil- lou, J. P., and Brachet, P. (2001). Different mechanisms mediate the April 2012 \| Volume 6 \| Article 17 \| 8 Frontiers in Cellular Neuroscience www.frontiersin.org
https://openalex.org/W4232398544	https://literator.org.za/index.php/literator/article/download/593/763	Afrikaans	null	A collection of Litera for Volume 17, Issue 1	Literator	1,996	cc-by	17,489	Literator 17(1) April 1996:169-170 ISSN 0258-2279 Gedígte Bernard Odendaal Thabo ya Kreste 171 Henning Pieterse Latent 179 Sinestesie 172 Gevangenes 180 Com Henk Piemeef 173 Impasse 181 Hein Viljoen Roukonsert vir Uys 182 Pianis 175 Tony Ullyatt Huwelik 176 Strangers at the altar 183 Kantoormasjien 177 Your olive-green raincoat 184 Broodgewoon 178 • K ortverhaal Nelia Engelrecht En daar was lig • Paternoster Skryfskool Kortverhale 187 191 Die rabriek Litera word fmansieel gesteun deur/ The section Litera is financially supported by smnuiu mDit sKtmMCEKUNSiE FOUNDATION FOR THE CREATTVE ARTS Li 17(1) A il 1996 169 170 ISSN 0258 2279 169 Gedígte Bernard Odendaal Thabo ya Kreste 171 Henning Pieterse Latent 179 Sinestesie 172 Gevangenes 180 Com Henk Piemeef 173 Impasse 181 Hein Viljoen Roukonsert vir Uys 182 Pianis 175 Tony Ullyatt Huwelik 176 Strangers at the altar 183 Kantoormasjien 177 Your olive-green raincoat 184 Broodgewoon 178 • K ortverhaal Nelia Engelrecht En daar was lig • Paternoster Skryfskool Kortverhale 187 191 Die rabriek Litera word fmansieel gesteun deur/ The section Litera is financially supported by smnuiu mDit sKtmMCEKUNSiE FOUNDATION FOR THE CREATTVE ARTS Li 17(1) A il 1996 169 170 ISSN 0258 2279 169 Gedígte Bernard Odendaal Thabo ya Kreste 171 Henning Pieterse Latent 179 Sinestesie 172 Gevangenes 180 Com Henk Piemeef 173 Impasse 181 Hein Viljoen Roukonsert vir Uys 182 Pianis 175 Tony Ullyatt Huwelik 176 Strangers at the altar 183 Kantoormasjien 177 Your olive-green raincoat 184 Broodgewoon 178 • K ortverhaal Nelia Engelrecht En daar was lig • Paternoster Skryfskool Kortverhale 187 191 Die rabriek Litera word fmansieel gesteun deur/ The section Litera is financially supported by smnuiu mDit sKtmMCEKUNSiE FOUNDATION FOR THE CREATTVE ARTS Literator 17(1) April 1996:169-170 ISSN 0258-2279 169 Gedígte Bernard Odendaal Thabo ya Kreste 171 Henning Pieterse Latent 179 Sinestesie 172 Gevangenes 180 Com Henk Piemeef 173 Impasse 181 Hein Viljoen Roukonsert vir Uys 182 Pianis 175 Tony Ullyatt Huwelik 176 Strangers at the altar 183 Kantoormasjien 177 Your olive-green raincoat 184 Broodgewoon 178 • K ortverhaal Nelia Engelrecht En daar was lig • Paternoster Skryfskool Kortverhale 187 191 R ubriek vir skeppende w erk Met die afdeling Litera tree ons toe tot die mark vir skeppende werk in al die tale waar daar normaalweg in Literator geskryf word. Dit is die wens van die Redaksie dat die gedigte en kortverhale wat hierby verskyn, baie gedigte, kortverhale en prosa- en draraafragniente in ander inheemse tale sal roep, sodat hierdie blad één geïntegreerde beeld van die korter skryfwerk van jong Suid-Afrikaanse skrywers kan bied. Dit is met vreugde dat ons die uitnodiging aan veral digters en kortverhaalskrywers rig om skeppende werk voor te lê vir plasing in hierdie rubriek, met die voorbehoud dat ons nie oor die mannekrag beskik om daaroor te korrespondeer nie. Bydraes in viervoud moet in dubbelspasiëring getik wees, met die naam en adres van die insender regs bo aan die eerste blad van gekramde tekste en op elke losbladvel wat gestuur word. Stuur alle bydraes aan die Hoofredakteur, Literator (629), Buro vir Wetenskaplike Tydskrifle, Privaatsak X6001, Potchefstroom 2520. Gedígte Die rabriek Litera word fmansieel gesteun deur/ The section Litera is financially supported by smnuiu mDit sKtmMCEKUNSiE FOUNDATION FOR THE CREATTVE ARTS 169 Send all contributions to the Editor-in-Chief, Literator, (629), Bureau for Scholarly Journals, Private Bag X6001, Potchefstroom 2520. Section for creative w riting In introducing the section Litera we are entering the market for creative work in all the languages that Literator normally caters for. It is the wish of the Editorial Board that the poems and short stories printed in this issue will only be the precursor of many poems, short stories, prose and drama fragments in all the indigenous languages, so that the journal will be able to present one integrated image of the short pieces of writing which young South Africans can offer. It is with great joy that we extend this invitation to especially poets and short fiction writers to submit creative work with a view to publishing them in this section. The only reservation is that we do not have the manpower to enter into correspodence about the works submitted. Four copies of contributions have to be submitted. The submissions have to be typed in double spacing, with the name and address of the author at the top right o f the first page of stapled text, and on every loose sheet sent in. Send all contributions to the Editor-in-Chief, Literator, (629), Bureau for Scholarly Journals, Private Bag X6001, Potchefstroom 2520. Literator 17(1) April 1996:169-170 ISSN 0258-2279 170 Bernard Odendaal Literator 17(1) April 1996:171-173 Thabo ya Kreste a Kreste (Vader Claerhout) (Vader Claerhout) ’n Mens sou die maklike lag van God wou inkleur: magrietjies pienk, skemers groen, die slaghaan vloekrooi tipeer; die land met sonneblom, die lyf met neutmuskaat insmeer; die dagbreekson in soliede jaspis vas wou kleur. Sou krabbelend Gods primitiefste vreugdes raak wou vat: die infame kurwe van ’n tiet, skurfle van ’n voet; die lakse simmetrie van lokasiehuisies, oorhoeks; ’n kierieskraal Christus koersvas deur die grote Vrystaat. Tog rym dit eenmaal nié. Dit bars uitbundig oral uit in rooigrasgeel, nagblou, olyfgroenlyf, nuwehoedpienk - ’n skaterende palet wat elke kunsgreep ontglip en die vaste dans van lyne goedertieren oorspoel met die wonderlike ding wat hier by ons gebeur het; hoe God se onberekenbare vreugde Hom gewreek het. Augustus 1985 ISSN 0258-2279 171 Gedigte / Poems Bernard Odendaal Sinestesie (na ’n fiktiewe skildery) Die oggend glip die oop gordyne binne, blakend - ’n intense, blonde jongeling teen diep, dadelbruin skadu’s; stryk blosend oor die pluistapyt se pienk, oor syige hopies bra en broekieskous; aai oor blougrys plooiinge, pastel-lig gevleg in ’n afgevalle laken; betas ’n bedtafel se kristallyne vrag: smukdosies, ’n flessie iets, ’n snoer, ’n blatante pienk krisant gestingel in ’n waterkraf; ontdek, op die pêrelgrys bed, haar ledemate, loom uitgespoel aan die oewer van ’n droom - en wek, in ’n vemikking van sinne, sensasies van andermansekstase: bleek heupe maanlig beweeglik in die kantgordyn; naggeluide langgesliert soos parfiime, soos strelinge; langsame boleros van die lyf ... oorstelping uit alle oorde en hortende ejakulasies van woorde. Literator 17(1) April 1996:171-173 Sinestesie (na ’n fiktiewe skildery) Literator 17(1) April 1996:171-173 172 ISSN 0258-2279 Bernard Odendaal Literator 17(1) April 1996:171-173 Bernard Odendaal Bernard Odendaal Oom Henk Pierneef Later neem hy stif en passer, liniaal. Stip die fokuspunt. Haal tot vlakke deur. Versit die berge in balans. Bal saam die toringwolk. Golf die rflensveld, es ’n sloom rivier. Sambreel die borne oop. Laat tuimel hier, laat daar steil styg. Hutjies lyk op klippers uit die stapelkrans geval... Hy staan terug; blik besorg, verlig - oorstelp dat kuns die wildemis so klein kan kry. Maart 1995 Literator 17(1) April 1996:171-173 173 ISSN 0258-2279 Hein Viljoen Literator 17(1) April 1996:175-178 Pianis vir Hennie Coetzee innig buig hy oor sy instrument glimmende swart hout wit ivoor strooi kristaldrappels deur die saal die note plooi hul na sy hande snare neem sy arms oor infiltreer die adellike kop deur die jare witter aan ’t worde soos sy hemp die klawers eggo musiek vul sy kop en sypel in sy rug af hy haai skaars asem sylyfneem die steinway se sonorante aanwesigheid aan soos die swart poot bewegingloos tril sy been op ’n swaar blinkbrons roller Literator 17(1) April 1996:175-178 ISSN 0258-2279 175 Gedigte / Poems Hein Viljoen Literator 17(1) April 1996:175-178 Huwelik tydens die oorbekende moet julle hoor hoe heilig seremonie ’n vlaag wind met Sy eie hand lug blaas deur ’n orrelpyp neem jy onverwags en dis of die kerse helderder brand wat skuif voor die venster weg verklaar julle regtens ’nd u if ’n wolk plotseling weggetrek lig ligter as lig brandende beurende vlam Literator 17(1) April 1996:175-178 ISSN 0258-2279 176 Hein Viljoen Hein VUjoen Literator 17(1) April 1996; 175-178 Kantoormasjien miskien is dit die nek wat eerste ingee - skokbrekers afgeslyt deur te veel buig, draai, te veel knik of dalkies swig en die oë, ja, beelde word - selfs deur ’n lens - te dof geregistreer om nog effektief te kan flinksioneer ander bewegende dele raak ook kapot knieë, heupe, net steun-steun kom dit weer orent, kniel gaan eintlik nie smeermiddels help ook nie en dan die pomp wat aangepak raak van jare se gal en ander minder digterlike vloeistowwe laat sirkuleer versonke hoop frustrasie suur van verdriet en gesmoorde ideale vreet aan silinderwande prostaat gekneus gesit versper die afVoer van besoedelstowwe obstruksies in uitlaatkanale die hande knokkerig kan nie meer suiwer vat o f netjies aanteken van die gees is daar nouliks iets oor dalk ’n hort o f twee flou skaduwee van die vaart die vuur, die dinamiek toe hy nog nuutwas. Geskik vir skroot. Sy dienslewe is verby. Gee horn ’n pakket. Literator 17(1) April 1996; 175-178 ISSN 025^-2219 177 Gedigte /Poems Hein Viljoen Broodgewoon vandag ’n dag soos alle dae opstaan skeer werk toe gaan terugkom eet slaap vaal vandieniks nikswerd leeg voel my hart klop die son kom op pienkerig roos agter wolke my liggaam se prosesse loop kou asemhaal verteer ekskreseer goeie opelyf gehad vandag liefhê peins praat verbeel ’n ordínêre dag dit is die ergste ek word van a na b vervoer deur ’n meganiese wonder tik ’n gedig elektronies of korrespondeer met Gerda ver oor die water maak nouliks kontak met mense om my darem bewus van kinders wat irriteer ’n lang storie vir Hans lees Willemien wakker maak vir skool hulle word amper vanself groot en ek het ’n week laas in die Bybel gelees wat ’n onsamehangende geteem so ordinêr soos die kind in die arms bed toe dra lang wimpers op sy wang sproetjies oor sy neus uiteindelik wonderbaarlik aan die slaap toelaat dat Maretha my lecture oor entrepreneurskap vir Daan aan die gis hou oor my planne niks het eintlik gebeur ’n dag so gewoon soos volgraanbrood Literator 17(1) April 1996:175-178 178 ISSN 0258-2279 Henning Pieterse (1956) Literator 17(1) April 1996:179-182 Literator 17(1) April 1996:179-182 Latent Vader, dis nou al sewe jaar dat ons jou daar in die voorstad van manner en graniet alleen moes laat om tot niet te gaan tot iets soos stuifsand in die wind geskik vir boom en plant al is jou liggaam weg bly jou handewerk vervleg soos reënbooggare in die netwerk van ons erf: jou afdak vir die karavaan is waar my motor deesdae staan jou waterbessie is al hoog die rubbettooom gooi steeds soos altyd blare af die groot papajaboom het van ouderdom verdroog soggens sing die voëltjies nog die goggas leef van blarevog die opslagkosmos spog heuphoog met blomme in die agterplaas in my sien almal jou: die stem, die stap, die sit en staan geneties is jy vasgelê vervleg in die negatief wat in my wag om tot wasdom te ontvou 1986 Vader, dis nou al sewe jaar dat ons jou daar in die voorstad van manner en graniet alleen moes laat om tot niet te gaan tot iets soos stuifsand in die wind geskik vir boom en plant al is jou liggaam weg bly jou handewerk vervleg soos reënbooggare in die netwerk van ons erf: jou afdak vir die karavaan is waar my motor deesdae staan jou waterbessie is al hoog die rubbettooom gooi steeds soos altyd blare af die groot papajaboom het van ouderdom verdroog soggens sing die voëltjies nog die goggas leef van blarevog die opslagkosmos spog heuphoog met blomme in die agterplaas in my sien almal jou: die stem, die stap, die sit en staan geneties is jy vasgelê vervleg in die negatief wat in my wag om tot wasdom te ontvou 1986 1986 Literator 17(1) April 1996:179-182 179 ISSN 0258-2279 Gedigte /Poems Henning Pieterse (1956) Gevangenes vas vang God in sy geosfeer mens en dier om te vermeer vas teen die akwariumruit skiet visse parend kuit epigonies vasgevang vis ek beelde op wat lesers van my knittelverse hokus-pokus fop Gevangenes 180 ISSN 0258-2279 Henning Pieterse (1956) Henning Pieterse (1956) Impasse (assimileer (ww.): verwerk, opneem, inneem, gelykmaak, aanpas) Literator 17(1) April 1996:179-182 Literator 17(1) April 1996:179-182 Literator 17(1) April 1996:183-185 Impasse (assimileer (ww.): verwerk, opneem, inneem, gelykmaak, aanpas) jare terug het ons mekaar op die haamaalddraai in die koue bergpas reeds verleer nooit kon ons één keer eers hobbelloos glad impas bymekaar vandag voel ek onhartstogtelik: impotent vir leef beswerend tel ek reëls meet klanke greep-greep af pen my drome tot swart sandsuikerskadu’s vas op rimpellose reinwit waterpaspapier maak my mee-ster van die spel dat ek totaal kan impas as stippel in die poespas van die groot heelal Literator 17(1) April 1996:179-182 ISSN 0258-2279 181 Gedigte / Poems Henning Pieterse (1956) Beeld 14 Aug. 1987 182 ISSN 0258-2279 Tony Ullyatt Strangers at the altar Looking at altars through the light splintered by gorgeous windows I can almost see that man on his cross. Looking at altars through the light splintered by gorgeous windows I can almost see that man on his cross. Then the quietness and tlie light offer a momentary but elusive glimpse into tranquillity. ISSN 0258-2279 183 Gedigte /Poems Tony Ullyatt Literator 17(1) April 1996; 183-185 Literator 17(1) April 1996:183-185 Your olive-green raincoat: variations on a theme by Leonard Cohen Your olive-green raincoat can never be famous; it’s not even tattered or tom. Your olive-green raincoat can never be famous; it’s not even tattered or tom. Your olive-green raincoat can never be famous; it’s not even tattered or tom. You bought it that summer the rain was quite heavy; since then, it has scarcely been worn. And a man with a beard peers into train windows in search of his only true love. He waits at the station for every arrival; her heart will fit his like a glove. The widow is lonely, makes antimacassars; her cats are fed fat out o f tins. There’s a clock ticking loud in a portrait-hung hall but no one to laugh at her sins. 184 ISSN 0258-2279 Tony Ullyatt The man watching tv craves someone to love him but his only friend is old age. His sink’s ftill of dishes and all of them dirty; your vases are full of dead sage. And somewhere there’s someone who’s playing the piano; the music’s been dead for a while. She’s singing a song of an olive-green raincoat; the words are so sad tíiat I smile. The dark night has fallen: now everyone’s singing as stars peter out one by one. And no one is bothered about crucifixions or just where your raincoat has gone. Literator 17(1) April 1996:183-185 ISSN 0258-2279 185 186 En daar was lig Nelia Engelbrecht Nelia Engelbrecht Eers ná Christian het die dorpsmense verstaan. Van lig en van donker, maar veral van lig. Hy was maar tafelhoog toe hulle op die dorp aangekom het. Hy en sy ma, Bettina, het in die wit huis langs die spruit kom woon, sodat hy na die kloosterskool vir blindes kon gaan. Sy pa het nie van die platteland gehou nie; daarom het hy verkies om in die stad agter te bly. Buitendien het die mistieke band tussen moeder en seun horn stom en eenkant gelaat. Vir die terloopse verbyganger, die enkele vreemdeling op die dorp, wat dit nie altyd duidelik wie van die twee siende was nie. Met hul arms deur mekaar s’n gehaak, het die vrou en seun gestap: soggens en smiddae tussen huis en skool, en teen skemer met die voetpad af na die spruit. Daar, tussen die soet gras en die plons van visse, het die seun sy fluit opgelig en note sterlig oor hulle gesif. Met haar vingers omhoog, het sy ma verwonderd in die lug getas en met ’n glimlag die blink klanke teen haar gesig gedruk. Die dorp was trots op die seun. ’n Musiekonderwyser het spesiaal een maal per week vanaf ’n buurdorp gekom om vir hom musieklesse te gee. Die seun het selfs so nou en dan stad toe gegaan, waar hy die een na die ander musiek- kompetisie gewen het. Mettertyd was die eens vergete dorpie ’n flikkering op die landkaart. Die seun het veel meer as trots aan die dorpsmense gegee. In hom was hulle verlore jeug, hulle ou vreugdes en verwondering opgesluit. Deur sy musiek het hy dit alles aan hulle teruggegee. Met sy ontroerende klanke, sy onbevange lag en sy helder oë het hy rafels kleur in die jarelange vaalte wat oor die plekkie gehang het, geweef. Soos die son, kon hy onverwagte skakerings uit die kleurloosste skepsels kaats. Mettertyd het die inwoners, asof uit ’n diep, droomlose slaap, wakker geword. Meneer Veriioef het die stof van sy verfkwaste afgeblaas en die lendelam esel teen sy kombuismuur staangemaak. Jan Vermeer het weer lewe uit die 187 Literator 17(1) April 1996:187-189 ISSN 0258-2279 En daar was lig wilgerhout agter sy huis begin kerf en Marijke Bloem het aarselend die koel hout van haar viool onder haar ken gedruk. En bo-oor alias, soos ’n wakende engel, was die silwer lig uit Christian se fluit. Nelia Engelbrecht Wanneer hy gespeel het, het die dorpsmense met ontsag gefluister dat hy nie die aarde nie, maar die hemel sien. In die klooster het die nonne saans ’n spesiale kers vir die seun aangesteek. Hulle het hul hande saamgeslaan oor die enkele lig wat selfs die verste hoeke van die kapel geel en warm laat gloei het. In die skemeruur, wanneer die wind die seun se ligte fluitspel deur die dorpie gedra het, het die priester sy kop gebuig en gedink aan die Lig wat gekom het om in die duistemis te skyn. En hy het gesien dat die duistemis die Lig nie oorweldig het nie. In die kroeg het die drinkers en geselsers vir ’n paar oomblikke stil geraak. Terwyl hulle geluister het, het hul beplooide en bebaarde gesigte bo-aards in die roserige laatlig geblink. Daama het hulle geglimlag en hul gesprekke hervat, maar gedemp en murmelend, asof uit eerbied. Langs die swaar kastrolle, die muwwe wasbalies en die stryende kinders het die vroue orent gekom en dronierig geluister na die note wat skoon, soos witgewaste duiwe, deur hul vensters vlieg. Selfs die kinders het nie onaangeraak gebly nie. Want Christian se musiek het die bestaan van nimfe en onskuld bevestig en vertel van die heeriike misterie van toekomstige dinge. Dit was juis die kinders wat hulle gevind het. Vervleg in die dryfhout het hulle oop-oog na bo gestaar, hulle arms steeds ingehaak. Op die seun se gesig was ’n uitdrukking van intense verwondering, asof hy in die laaste moment plotseling oorkom is deur al die kleure en al die skoonheid waarvan hy nog altyd net moes droom. Betower deur sy fluitspel, het die seun en sy ma nie die onaardse gegrom van die stygende water gehoor nie. Ná die reëns hoër op teen die berg, het die donker spruit hulle dié middag onverwags en met ongekende brutaliteit gegryp en meegesleur. Daama het die wolke daelank swaar oor die dorp gehang en elke stippeltjie lig verdring. Op die derde dag het die droewige tou mense die twee kiste moeisaam teen die heuwel op gedra. By die graf het die priester sy kop gebuig en geroep na die Lig wat in die duistemis kom skyn het. Die nonne het vergeefs die flikkerende kerse met hul Literator 17(1) April 1996:187-189 ISSN 0258-2279 188 Nelia Engelbrecht bakhande teen die wind probeer beskerm. Litemtor 17(1) April 1996:187-189 En oral - rondom hulle en in hulle - was daar Lig. Nelia Engelbrecht Die dorpsmense het hul koppe skeef gehou en sender hoop geluister. Die seun se pa het met sy rug na die dorp gestaan en net die vreeshke donker tussen die grimmige wolke gesien. Daarom het die wonder by horn verby- gegaan. ’n Jong vrou het dit eerste gesien. Haar verbaasde uitroep het die stilte van bo na onder oopgeskeur. Die dorpsmense het opgekyk. Die lig het eers in ’n tenger streep deur die grys marmer gebeur. Toe, asof weggestoot deur ’n onsigbare hand, het die donker massas meegegee. ’n Magtige vloed lig het uit die liemel gestort en in goud oor die dorp gegolf. Hulle voete het asof vanself die pad terug gevind. Hoe nader hulle aan die dorp gekom het, hoe duidehker het hulle dit gehoor: fluitklanke wat lang, dun lyne tussen hemel en aarde span. En oral - rondom hulle en in hulle - was daar Lig. Litemtor 17(1) April 1996:187-189 ISSN 0258-2279 189 190 Literator 17(1) April 1996:191 Skryfkursus vir swart Afrikaanse skrywers Paternoster (1995) Die kortverhale wat in hierdie gedeelte van LITERA geplaas word, is almal die produkte van ’n skryfkursus wat na die tweede simposium vir swart Afrikaanse skrywers op Paternoster aangebied is in Oktober 1995. Omdat gepoog is om die verhale se eiesoortige egtheid en spontaneïteit te beiiou, is so min as moontlik deur my of die Redaksie verander aan die verhale se struktuur en verhaallyn. Tydens die driedagkursus kon die skrywers egter self hul werk redigeer. Die beperkte publikasiemoontlikhede vir jong Afrikaanse skrywers het die Redaksie laat besluit om met dié publikasie iets vir die jong Afrikaanse skrywer te doen. Met hierdie verhale word ook gefllustreer dat Afrikaans in baie stemme kan praat! 0 ns dank aan Afrikaans Stereo vir fmansiële ondersteuning om die publiseer van hierdie afdeling van LITERA te help moontlik maak. Hans du Plessis Hans du Plessis Direkteur, ATKV-skryfskool van die PU vir CHO Literator 17(1) April 1996:191 ISSN 0258-2279 191 Literator 17(1) April 1996:193 Inhoudsopgawe Sien, hoor, praat: geen kwaad Balman versus Balman ’n Bok vir ’n gaping Mis Marigoldstraat, die ‘main road’ vannie scheme Brandewyn en coke special Liefde le ef... boemelaar of te not Frans Mouton se tuiskoms Literator 17(1) April 1996:193 193 ISSN 015%-im 194 “Mammie’sie byrie hysie,” het ek gesê. Maar sy het nie regtig na sy ma kom soek nie, het sy my van buite af laat verstaan: sy het my kom haal, het sy gesê, en my skool toe gedra ... En dis omtrent al herimieringe wat oorgebly het van daai eerste skooldag, amper twintig jaar gelede. Daar was wel nog iets wat my opgeval het toe ek by die klas instap; wat dit was, kan ek egter nie nou oproep nie. Ek weet wel dat toe dit naweek word, het ek my ouers vir die eerste keer sien baklei. Op hoërskool het ek ’n opstel daaroor so begin, en goeie punte daarvoor gekry: “ ’n Dronk man en dronk vrou baklei een aand oor-weet-nie-wat-nie in hul kombuis. Dis my pa en my ma. Die hele dag het hulie saam met ander mense gedrink, gesing, geiag. Maar net toe die mense weg is, het die bakieiery begin. " Dit was ’n Saterdagaand in die middel van die winter, en buite was dit al donker. Ek het by my suster op die kooi in die kamer gesit en hartseer gehuil, terwyl ek aanhoudend by die kombuis ingeloer het. Dit het my suster geirriteer. “Hou jou mond, man,” het sy gesê en my dan teen haar vasgedruk. Die wekker op die spieëlkassie langs die kooi het net verby sewe-uur getik. Dié tyd van die aand begin die jongmense van Caledon se bruin buurt, Bergsig, gewoonlik regmaak om disko toe te gaan; die Apostolies hou biduur of ’n kolfiekroeg; ’n paar dronkies sit en tiep by die smokkelhuis. In die kombuis het my ma ’n bord kos in my pa se gesig gegooi. My pa het kwaad geword en het haar omgestoot dat haar kop teen die rand van die tafel kap. ’n Pierinkie het van die tafel geval, stukkend. “Ooo jere, my kop,” het my ma gehuil. “Djy wil mossie fokkienwil ophou nie,” het my pa gesê. Voordat hy by die huis uitgestap het om nog wyn te gaan soek. Sonder om my ma op te help. My suster het ’n waslap gevat en die bloed van my ma se gesig afgevee; sy’t ’n stuk ou laken as verband om my ma se kop gedraai. Daarna het sy die deur toegemaak en die kers in die groen blaker doodgeblaas. Die volgende oggend was my ma se gesig lelik opgeswel en het sy besluit om in die kooi te bly. Sien, hoor, praat: geen kwaad Heinrich Wyngaard Dit was ’n sonskyndag op Caledon, met ’n paar wolkies hier en daar in die lug - soos gister in die Kaap - toe ek daai Dinsdagoggend gereed maak vir my eerste skooldag. Vir die begin van ’n staptog wat twaalf lange jare sou duur. Omdat ek eers teen einde Julie verjaar, was ek at sewe jaar oud toe ek saam met my suster, Leona Susanna, by die huis uitstap Swartberg Primêr toe. Ek was nie baie opgewonde oor die vooruitsig van skoolgaan nie, want om by die huis te bly en heeldag te speel, was vanselfsprekend lekkerder. Maar omdat ek uitgevat was in my nuwe skooikiere het ek nie gemaaind ora enige plek te gaan nie. (Nuwe klere was in daai dae nie elke dag se ding nie.) By die skool aangekom, was daar ’n miemes van woelinge voor die kunsklas waar die nuwe sub-A’tjies - die meeste met snotneuse, vaal strepe oor hul skurwe wange - ingeskryf moes word. M a’s het sonder ophou met mekaar geklets oor huissake en die prys van skoolgoed wat wragtag al weer op is, terwyl die kiiiders verskrik rondgekyk het. En toe, enkele minute voor die kletsers en die verskriktes by die klas moes ingaan, los my suster my toe alleen om betyds by haar eie klas te wees. “Gan sam in assie anner mense ingan,” het sy gesê, voor sy weggestap het. “En as hulle jou naam sê - j y weet moes hoe’s jou naam? - gan jy vorentoe.” “Ja’k sil,” het ek kopskuddend geantwoord. En toe’s sy daar weg. Maar toe die instappery uiteindelik begin, is ek op die ou end alleen buite. Ek het so ’n rukkie gewag om te kyk of iemand my gaan kora roep, maar toe niks gebeur nie, besluit Leona Susanna se bybiebroer (die jongste van Karel en Kitriena) om terug huis toe te gaan. Net ná ek egter met my rooi brandweerwaentjie met die een stukkende wiel begin speel het, het ’n antie van my wie se kind ook dié dag begin te skoolgaan het, aan die deur geklop. “Kitriena!” het sy geroep. Literator 17(1) April 1996:195-197 ISSN 0258-2279 195 Sien, hoor, praat: geen kwaad Sien, hoor, praat: geen kwaad “Mammie’sie byrie hysie,” het ek gesê. Literator 17(1) April 1996:195-197 “Mammie’sie byrie hysie,” het ek gesê. Ek het buite in die Sondagsonnetjie met my rooi brandweer- waentjie met die een stukkende wiel gespeel. My suster, Leona Susaima, het rys op die stoof gesit, die kombuis uitgevee en die splinters van gisteraand se pierinkie opgetel. My pa was nog nie by die huis nie. Literator 17(1) April 1996:195-197 ISSN 0258-2279 196 Heinrich Wyngaard Op my eerste skooldag, onthou ek nou, is dit wat eerste my aandag getrek het toe ek by juffrou Daniëls se klas ingestap het: ’n Beeldjie met drie apies op die rak. Een van hulle het sy oë toegehou. Een sy ore. Die ander een sy mond. Die ander een sy mond. ISSN 0258-2279 197 198 “Natuurlik.” Sy knik, met een van die glimlagte wat sy vir haar eie raense wegbêre. Soos sy die badkamer binnestap, laat val sy haar cream satynjapon, wat ’n teekol op die voorkant het. Hy sien haar kaal rug en vol sitvlak. Hy sien haar kaal rug en vol sitvlak. As sy die badwater intap, loop hy vinnig na die japon toe, tel dit op en hang dit oor die stoel wat voor die dressing table staan. Sy eie klere hang hy in die klein, netjies afgewerkte hangkas - die gitsswart broek met sy gestrykte witstreepsakdoek in die een sak, die spierwit langmouhemp bo-oor. En die sagte, ou jersey. Die jersey wat haar so pla. Die tweede keer, dink sy, terwyl sy die spons water oor haar gesig spoel. En die laaste keer, belowe sy haarself plegtig. Sy staan op, klim uit die wit bad, en draai die groot handdoek om haar ofF-white liggaam. Hy het intussen onder die duvet ingekruip. Hy lê op sy rug met sy kop op sy hande. Sy oë is toe. Dis die tweede keer. Hy jaag haar kaal liggaam uit sy gedagtes uit en maak vinnig sy oë oop - sy’t die ligte afgesit, soos hy gevra het laaskeer. Balman versus Balman Enrico Parry “Gaan was jou eers, asseblief,” versoek hy haar beleefd, “en los die bad- kamerdeur oop. Ek sal myself tuismaak hier. Ek ken mos darem al die plek.” “Natuurlik.” Dis donker. Hy voel haar kaal liggaam warm langs hom inkruip. Sy oë is oop, en hy sien niks, niemand. Sy begin saggies, soos twee weke gelede, dieselfde weeksaand. Dieselfde skoon, agtermekaar kamer. “Na gelang van die kerkraadsvergadering, spyt dit my om u mee te deel dat u dienste as leraar van die gemeente hiermee beëindig word. U word versoek om “Na gelang van die kerkraadsvergadering, spyt dit my om u mee te deel dat u dienste as leraar van die gemeente hiermee beëindig word. U word versoek om Literator 17(1) April 1996:199-202 199 ISSN 0258-2279 Balman versus Balman die pastorie binne drie maande van bogenoemde datum te ontruim ...” Sy kry eers seer as sy long dringend oor haar borste speel, tot dit ferm word van ingehoue plesier. Hy is teer en lief met sy liggaam, sy pynlik netjies instandgehoude mansmenslyf. “Weet jy wat se redes gee liulle nogal aan?” het hy vir haar gevra ná die vorige keer, toe hulle agtema in pikdonkerte gelê en rook het aan haar Pall Mall pakkie. “Wat? Hoe het jy hulle kwaad gemaak?” het sy gevra. “Onhigienies - ek ruik na sweet; ek spoel nie die toilet af as ek klaar gepie het nie, ek was nie. ‘Dominee Balman, u trek nie die toilet af as u klaar is nie.’” Hy het mallerig gelag. “Spoel jou pis af, John Bahnan! Was jou heste, Jan Balie!” het hy hard geskree. “Spoel jou pis af, John Bahnan! Was jou heste, Jan Balie!” het hy hard geskree. “Spoel jou pis af, John Bahnan! Was jou heste, Jan Balie!” het hy hard geskree. “Sjj,” het sy gepaai, haar sigaret in die vol asbak doodgedruk, en haar arm oor hom gesit, soos met ’n verleë kind by die skool. “Sjj,” het sy gepaai, haar sigaret in die vol asbak doodgedruk, en haar arm oor hom gesit, soos met ’n verleë kind by die skool. “Sjj,” het sy gepaai, haar sigaret in die vol asbak doodgedruk, en haar arm oor hom gesit, soos met ’n verleë kind by die skool. Sy oë was souterig toe sy dit soen. Sy oë was souterig toe sy dit soen. “Sjj, ek is hier. Delia is by jou, John. Sjj.” Sy oë was souterig toe sy dit soen. “Sjj, ek is hier. Delia is by jou, John. Sjj.” Hy het teruggesak en bewerig gefluister. Hy het teruggesak en bewerig gefluister. Literator 17(1) April 1996:199-202 Hy moes uithaal en wys. “Special requests, special payment raise, Mister Balman,” het ou Miesies Pietersen gesê, “Deedee is a very special girl. And not stupid at all.” “Ja, ek stink nie nou meer nie.” Hy het ’n lang ruk stilgebly. “Beslis nie. Netjies en skoon,” het sy geantwoord. “Netjies en skoon,” het hy haar herhaal, “Netjies en skoon wou sy he; en toe sy dit uiteindelik nie kry nie, het sy geloop. Met altweetjies my meisiekinders ... Eerwaarde Dominee J.J. Balman hiermee gedagvaar om op 3 September in hof nommer twee van die Magistraatskantore by Hoogstraat 17 te verskyn in die saak A. Balman versus J. Balman.” “Netjies en skoon,” het hy haar herhaal, “Netjies en skoon wou sy he; en toe sy dit uiteindelik nie kry nie, het sy geloop. Met altweetjies my meisiekinders ... Eerwaarde Dominee J.J. Balman hiermee gedagvaar om op 3 September in hof nommer twee van die Magistraatskantore by Hoogstraat 17 te verskyn in die saak A. Balman versus J. Balman.” “En waar is A. Balman vandag?” Delia het met die rook kringe in die donker geblaas. “En waar is A. Balman vandag?” Delia het met die rook kringe in die donker geblaas. “George, Pacaltsdorp, Delville Park, Pumastraat drie-sewe-drie. Met ’n jong onderwyser twee jaar jonger as sy. Op die hof.” Daar het hy opgehou. Sy het later self begin vertel, maar net sóveel. “Ek doen dit nie vir die geld nie.” “Ek doen dit nie vir die geld nie.” “Nié?” het dominee gevra. “Nié?” het dominee gevra. “Kyk, jy leef nou maar eenmaal met ’n byl oor jou nek as jy onderwys. Maar jy kry dárem nog jou tjek elke end van die maand,” het sy geantwoord, sonder dat die toon in sy stem haar gepla het. “Kyk, jy leef nou maar eenmaal met ’n byl oor jou nek as jy onderwys. Maar jy kry dárem nog jou tjek elke end van die maand,” het sy geantwoord, sonder dat die toon in sy stem haar gepla het. “Klere, kos, ligte en water,” het hy haar uitgawes gelys. “Nou waarom dan? Hoekom dan dit?” “Nou waarom dan? Hoekom dan dit?” “Nou waarom dan? Hoekom dan dit?” “Ek geniet dit. Elke oomblik daarvan.” Vanaand het sy haar fmaal uitgeniet, dink sy as sy weer langs hom inkruip ná die was. Hulle lê stil langs mekaar. Hy rook nie. Haar pakkie lê toe op die bedkassie. Dis donker. “‘Jy moet jou was, assebheftog!’ het sy gesê. ‘Skandelik!’ is wat sy ges “‘Jy moet jou was, assebheftog!’ het sy gesê. ‘Skandelik!’ is wat sy gesê het.” Hy het stilgebly vir ’n oomblik, “Ja-nee, Mister Balman. Mense wil van ’n skoon mens ’n lewenspolis koop. Personell Manager se vereiste.” Hy het stilgebly vir ’n oomblik, “Ja-nee, Mister Balman. Mense wil van ’n skoon mens ’n lewenspolis koop. Personell Manager se vereiste.” Delia soen sy naeltjie. Vanaand, soos die eerste keer, is hulle altwee stil. Net hulle lywe wat sweet en spoeg en seks praat. Delia soen sy naeltjie. Vanaand, soos die eerste keer, is hulle altwee stil. Net hulle lywe wat sweet en spoeg en seks praat. Eers as sy ook haar tevredenheid bereik, dan sal hulle gaan was. Eers hy, dan sy. Eers as sy ook haar tevredenheid bereik, dan sal hulle gaan was. Eers hy, dan sy. Dan gaan hulle rook. Pall Mall, uit sy pakkie. Hy kreun as sy bo-op hom s it... Hy kreun as sy bo-op hom s it... “My vrou is vroeg uit my uit,” het hy daardie Woensdagaand sy storie vertel, “Die dominee het seker té veel gestink.” “My vrou is vroeg uit my uit,” het hy daardie Woensdagaand sy storie vertel, “Die dominee het seker té veel gestink.” “Jy stink nie nou meer nie,” het sy gesê. Literator 17(1) April 1996:199-202 ISSN 0258-2279 200 _Enrico Parry Hy het gesmeek om háár te sien, al sou dit net wees om met haar te praat - die mooiste meisie van die huis. Hy moes uithaal en wys. Hulle bly toegemaak in hulleself Swaarmoedig. ISSN 0258-2279 Literator 17(1) April 1996:199-202 201 Literator 17(1) April 1996:199-202 Na ’n ruk staan Delia op, trek haar aan in die donkerte. Panty, broek, bloes, skoene. Sy stap deur toe en hy kyk na haar soos sy in die deur staan, met die gang se lig wat haar omlyn. Hy wil dit nie hoor nie. Na ’n ruk staan Delia op, trek haar aan in die donkerte. Panty, broek, bloes, skoene. Batman versus Balman Na ’n ruk staan Delia op, trek haar aan in die donkerte. Panty, broek, bloes, skoene. Sy stap deur toe en hy kyk na haar soos sy in die deur staan, met die gang se lig wat haar omlyn. Hy wil dit nie hoor nie. “Jy vra nie weer vir my nie, Dadda.’ Literator 17(1) April 1996:199-202 ISSN 0258-2279 202 ’n Bok vír ’n gaping Willem Fransman Ons huis is naby ’n kroeg in die hartjie van die stad geleë. Die kroeg is ’n klein ou plekkie waar die manne tog te lekker kan kuier. Tot hul vroue se ontsteltenis! Dit is ’n lekker plek, want daar ontmoet jy allerhande karakters. En vang die manne goed gees! Ek gaan dikwels daarheen. Want my vrou weet: ek vrek oor ’n mooi storie. En dáár loop hulle op twee bene, my maat. Maar dis nie die enigste rede nie. Eintlik hou ek nie daarvan om my jenewertjie alleen te drink nie. En dit was dan ook daar dat ek met oom Sarel Grootlieg kennis gemaak het. Grootlieg was blykbaar nie sy van nie, maar in ’n kroeg vra niemand mos jou van nie! Oom Sarel was ’n karakter in eie reg. So asof die tyd vir hom stil gestaan het. Die mense weet te vertel dat hy net nooit sy hoed afhaal nie. Ook nie in die kerk nie. Dit was ’n vilthoed wat met ’n tiervelband afgerond is. Dit het al baie son en wind getrotseer. Ek het in my sondigheid gewonder of die oom die hoed afhaal as hy bed toe gaan of... Die dag toe ek oom Sarel ontmoet het, het hy nie bedsake op die brein gehad nie, maar sy geiiefde sport, rugby, op die hart. Ek verstaan dat hy almal altyd as swaer aangespreek het. Vir my was ’n swaer nog altyd my vrou se broer of my suster se man of die een met wie se vrou jy ’n skelm verhouding het! Ek weet nie waar hy my geplaas het nie. Ek was tog op geen manier familie van hom nie. En sy vrou was ook al jare lank onder die kluite! “Swaer” sê hy met ’n lekker dop brandewyn in die hand, “Hierdie oom van jou kan nie glo dat daar nou so ’n groot ophef van die Springbokspan met die blommetjies op die bors gemaak word nie. Lyk my aimal het gerieflik vergeet van die befaamde span van die Vyftigs.” “Kyk Swaerie, hierdie omie van jou is as’t ware met ’n rugbybol in sy hande gebore.” Hy maak homself gemaklik, bly vir ’n rukkie stil. Toe hy opmerk dat hy my onverdeelde aandag het, neem hy weer ’n teug aan sy brandewyn, en vervolg. ’n Bok vír ’n gaping Literator 17(1) April 1996:203-207 m ISSN 0258-2279 'n Bok vir ’n gaping “Die doktertjie daar by Putsonderolie se kraamhospitaaltjie, dis nou waar joii omie gebore is, kon sy oë nie glo toe ek uit die oond gehaal was nie.” Die oom vertel met ’n stem wat swel van trots hoe die dokter aan die verpleegster gesê het dat hy nog nooit so ’n baba met sulke groot hande gesien iiet nie. Ook die lenige armpies en die spekke agter die onderbene was vir hom ’n rariteit! Toe uiter oom Sarel Grootlieg die woorde wat my byna aan my jenewertjie laat stik. “Swaer, toe sê die doktertjie wragtag: ‘Hier is vandag ’n Superspringbok gebore’. Ek meen, Swaer, dit was eintlik profetiese woorde wat daai dag gespreek was!” “Oom Sarel, wil Oom vir my sê al die uiterlike tekens van Springbok wees was toe al teenwoordig? Ek meen, oom, ek praat nou van bol sense, side steps, spoed, drop, place and score, alles! Maar oom, dis mos ongelooflik!” Oom Sarel het my met sy swaar kop en deurpriemende oë aangekyk asof ek die eerste keer in my lewe die waarheid gepraat het. “Hoe dan anders jong. lemand van my portuur kom maar eenmaal in ’n leeftyd. Jou oom het sommer gou gewys talent staan regoor hom geskryf! Ek was bestem om gou roem te verwerf en Springbok te word!” Terwyl hy dit sê, bekyk ek hom deeglik. Die man oorkant my kon nog vir die dorpsspan uitgedraf het, maar vir die Springbokke! Dat hy groot is, is duidelik, dat hy hande soos twee kalkoene had, was ook duidelik. Die wynglas was dan skaars sigbaar daarin. Boonop het sy arms my eerder laat dink aan dié van ’n oerang-oetang. Maar ek kon vir geen oomblik die oom sien as ’n Springbokrugbyspeler van weleer nie. Die oom se rasperstem ruk my tot die werklikheid: “Kyk, Swaer, toe ou Brandy Cockroach en sy vlieënde All Blacks by die middel van die eeu hier kom toer, het almal gesê dis verby met Springbok se kind! Hulle was ’n formidabele span, met ’n manjifieke rekord. Hulle het vyftig wedstryde en sestien toetse gespeel en in almal koning gekraai.” “Hulle was nou vir jou,” borduur die oom voort, “’n klompie vlieënde geraamtes. Ongelooflike voetvlugtiges, ek bedoel vlugvoetiges! Reusagtig, biltongtaai en balhonger. ’n Bok vír ’n gaping Een koerant het selfs geskryf hulle was bal-orig, want hulle kon ons vroue eenvoudig net nie uitlos nie!” IVIy mond hang nou behoorlik oop. My dop skoon vergete! “Kyk, Swaer, hulle was nou ’n goed ge-oliede rugbymasjien wat al die provinsies vir brekfis opgeëet het.” Ek kon my indink watter vreesaanjaende gesig hulle moes wees. Verbeel jou, ’n rugbyspan wat uit vyftien Jonah Lomu’s bestaan! Ek sê dit ook vir oom Sarel. Literator 17(1) April 1996:203-207 204 ISSN 0258-2279 Willem Fransman “Kyk Boetie, almal, tot die nasionale keurders was met ’n soort van lamsiekte geslaan. Alle koerante het dit dan ook uitgebasuin; ‘Swart gevaartes gaan arme Bokke lewendig verslind!’ ‘Dis verby met die Groen en Goud.’ ‘Bokke se uur het geslaan!’” Ook maar goed dat ons nasionale keurders die Bokke destyds van koerante af weggehou het! Dit sou mos enige mens ontsenu het. “Oom Sarel, dit sou mos enige mens die ritteltit gegee het”, sê ek gemaak emstig. “Swaer, jou siddering is als puur vemiet! Dink so ’n bietjie man. Jou oom Sarel was dan al daar. Wat het daai dokter-profeet, wat jou oom gevang het, gesê?... Tjaag, jy is ook maar dom!” “Ek het nie vergeet nie, Oom, maar een swaeltjie maak mos nie ’n somer nie? Hy maak dan nie eens ’n nes nie!” Nou was die oom behoorlik kwaad! O f so het ek gedink! “Ek sien jy is nog nie so bekend met jou oom se talente nie, maar laat ek jou dan inlig! Op daai toer van ou Brandy Cockroach en sy lot, het één swael, ’n groot intelligente, almintige balhorige bliksemstraal (dis nou jou oom), op die rugby-horison verskyn en in die Bokspan nesgeskop.” Nou het die oom my voile aandag. Op die manier waarop hy hom regskuif, kon ek agterkom, hier kom ’n storie! “Kyk, Swaer, toe die Springbokkeurders daai nag om twaalf-uur hul span oor die radio aankondig, daar was toe mos nog nie televisie nie, was daar net nege spelers in die span. Agt vories en een besondere talentvolle reus in die agter- lyn. Dit was duidelik dat hulle lank oor die spansamestelling gedink het.” “En wie was die besondere agterspeler in die unieke Bokspan, Oom?” vra ek heel geamuseerd. “Kyk swaerie, daai negende man, was jou oom Sarel, die nimlike. Wie dan anders, of het jy al weer die dokter-profeet se woorde vergeet?” Toe voel ek eers ’n gek! ’n Bok vír ’n gaping Ek moes mos reken dit was die oom, dis dan sy storie! Gelukkig vervolg die oom. “Ou Brandy en sy spul het hulle natuurlik vrek gelag, maar die Volk het geweet: die keurders weet wat hulle doen! Kyk, Swaer, toe’t ons nog in die keurders geglo!” Hy gee my ’n deurpriemende kyk, gooi sy kop agteroor dat ek vrees sy hoed sal afval en slaan die laaste van sy brandewyntjie weg. Met ’n veeg van die rugkant van sy regterhand oor sy mond vervolg hy: “Van die afskop af was dit duidelik dat ons ’n anderste wedstryd sal hê. Met die eerste skrum het Pissie Piston die bol vinnig gehaak. Die slotte en die agsteman het gangetjie gemaak en toe skiet daai bol in jou omie se wagtende hande! Kyk, dit was nou ’n triek wat ek en ou Pissie lank geoefen het! Ek 205 ISSN 0258-2279 Literator 17(1) April 1996:203-207 Literator 17(1) April 1996:203-207 'n Bok vir 'n gaping swaai toe hierdie lang slangarms van my en skiet die ból so met ’n boog uit na die fly-halfposisie. In dieselfde beweging dra hierdie bene van my my lyf en ek vang die ból in ’n aíwaartse gang. Toe slinger ek die bol na die binnesenter en voor die Alls weet wat hulle getref het, is jou omie weer daar. Ek gryp die bol en gee ’n kort aangee na die buite-senter.” Ek is nou saam met die oom in die wedstryd! Myjenewertjie skoon vergete! “’n Swart hand”, vervolg die oom, “wil die bol intersep, maar jou oom se hand is eerste by en ek slinger die bol vinnig vleuel toe! Die All Blackvleuel won jou omie tackle, toe gryp hy in die lug! Ek systap binne en buite toe (sy kop maak die bewegings), gooi die verdediging heeltemal oorhoops, uiter my vrees- aanjaende strydkreet en gaan druk onder die pale. Die skare was rasend. So iets was nog nooit in ’n toetswedstryd gesien nie!” Sy oë blink van trots, of was dit die voggies wat die trane so vinnig na die oë laat skiet het? “Toe skop ek nog die bol so hard met die vervyfskop dat dit soos ’n vrot tamatie oopgebars het. Gelukkig was daar soveel krag in die skop dat dit tog oor die dwarslat geseil het.” “Oom was toe sommer skrumskakel, losskakel, binne- en buitesenter en vleuel. Hoef ek dan verder te twyfel? ’n Bok vír ’n gaping Ek het darem ’n uithaler oom!” sê ek gelate en drink die laaste van my jenewertjie. “Kyk, Swaer”. Hy beduie aan die kroegman om sy glas weer te vul sonder om van sy storielyn af te wyk. Dit lyk nou asof sy borskas so ’n paar millimeters verder uitstoot van trots. “Daai dag het ons Brandy Cockroach en sy lot cleaners toe gevat. Hulle doppies was behoorlik geklink. Jou omie het dwars- deur hulle voorspelers vore getrek, self losskrums afgedwing en gewen en vier drieë gedruk. Dis nou behalwe die drie ander wat ek vir die voorspelers help druk het! Daai dag het ons hulle met ’n rekordtelling geklop wat tot vandag toe op die boeke is!” sê oom Sarel Grootlieg en neem weer ’n sluk van sy dop. “Oom meen, die Bokke van vandag was vemiet so bang vir die Jonah Lomu- vent? Hulle moes net by Oom kom kers opsteek het! O f beter nog; hulle kon Oom as hul spesiale adviseur aangewys het. Ek meen, met so ’n lewendige legende tussen ons het ons mos niks te vrees nie!” “Ag Swaer, wat sil hulle nou so ’n verrimpelde ou bleeksiel soos ek naby hulle wil hê. As jy klaar vir jou volk en vaderland rugby gespeel het, dan vergeet almai van jou. Jy kry nie eens ’n ou gratis kaartjie vir ’n toets nie. Laat staan nog spesiale adviseur!” Daar sak ’n somberheid oor hom as hy lank na sy glas staar en met sy vinger oor die glas se rand trek. As hy opkyk, is die vonkel skielik weer terug. Literator 17(1) April 1996.-203-207 ISSN 0258-2279 206 Willem Fransman “Swaerie, ek moet darem sê, ons het daai Alls goed opgemoer. Hulle was nooit weer ’n force in intemasionale ragby nie.” Ek kyk horn met groot deemis aan, want ek weet dat hy pas die grootste klomp bog kwytgeraak het. Maar hoe laat ek hom verstaan dat ek niks glo nie. “Oom, ek glo nou dat dokters, omdat hulle babas gewoonlik eerste sien, wel profetiese woorde kan spreek. Oom het so wragtag ’n Superspringbok geword,” sê ek en klop hom op die skouer. Hy staan stadig op, raak met sy regterhand aan sy hoed in ’n groet-aksie en beweeg deurwaarts. Sê nou net vir een oomblik die oom se stone is tog waar! My oë volg hom soos hy uitstap. (Opregte woorde van dank aan my vriend, Nic Venter, wat die idee van die storie verskaf het.) Literator 17(1) April 1996:203-207 Kirby van der Menve Kirby van der Menve Kirby van der Menve Vanoggend bly ek op die paadjie, hoog teen die slaap van die berg. Die Kaapse mis hang nat en lood en pleister die berggrasse teen die walle. Die sig is nul en die lug is stil, net die mis wat diep en stug oor die klowe lê. En die bekende bergpaadjie is ’n stomp, newelagtige tonnel. Die landskap van see, berg en biesies en boom en bas lê dig in die mis se wintervag. Die geure van klam grond en saggeweekte blaar hang ge- suspendeerd, soos fyn druppels. Ek word mis, afgebreek in my elemente, die mis vloei deur die holtes van my oë en my ore en ek word deel van die massa wat strek oor die ganse bergreeks, oor die see, tot by die doodkoue suidpool. En ek hoor hom, eers die ligte trilling op die senuweedrade van die mis. Dan sy voetstappe, ferm en vinnig. Sy drif brand ’n tonnel deur die gestolde mis. Die oerinstink van die jagter suur in my maag en die adrenalien val amper uit my mond uit. Sy intensie golf voor hom uit en dit rol teen my rug op en kriewel oor my kopvel. Ek is reg vir hom, al my sintuie gekonsentreerd. Kom wat wil, hy sal hom ook teen my vasloop. Soos die jongmamie wat op mooi dae die berg opgejaag kom, aangehits deur testosteroon en die matelose arrogansie van die jeug. Sal ek hom ook voorlê, vooruit strompel soos ’n gekweste voël, hygend sweet en so nou-en-dan vooroor staan met my palms op my knieë, maar skelm, skelm onderlangs loer, hulle uitlok om my verby te steek. Maar ek hou hulle net op die fyn tergende afstand. Ooreis hulle en gee dan skielik in. Dan sit en wag ek vir die valse glimlag, die vriendelike groet en die bemoedigende woord. Maar dis die glinstering in die oog waama ek soek. Watter vermetel- heid om my te wil gebruik om hulle krag teen te meet! My te wil gebruik as maatstaf van hulle gewaande manlikheid asof ek net ’n klein trappie is waarop hulle met hulle ego’s kan khm. Ek laat my nie gebruik nie. Ek sal self die eerste fyn krakie slaan. Die kraak wat hulle uiteindelik sal afbreek tot brokstukke. Die pynlike besef van hulle mortaliteit, nietig, soos die rotsbrokkie waarop ek hier trap. ’n Bok vír ’n gaping Aanvanklik dink ek hy is behoorlik gekoring van twee dubbele brandewyntjies. Toe tref dit my tussen die oë. Die oom is nie SÓ dronk nie. Oom Sarel het ’n horrelvoet. (Opregte woorde van dank aan my vriend, Nic Venter, wat die idee van die storie verskaf het.) Literator 17(1) April 1996:203-207 ISSN 0258-2279 207 S Kirby van der Menve Literator 17(1) April 1996:209-212 209 ISSN 0258-2279 M s_ Dan slaan ek gevoelloos toe, sê vinnig in die verbygaan tot siens en hardloop katagtig onder hulle uit. Wanneer ek hulle later weer op die berg raakloop, ignoreer hulle my, maar ek voel niks, ek voel heeltemal niks. Dis dan juis my beloning; die dikbek en die geknakte stert. Dit wil tog gedoen wees, op my ouderdom. Ek is ’n gesonde 38, goeie longe en as ek self so moet sê, ek het nogal ’n paar sterk bene. Maar nou bewe hulle, van afwagting en my hart klop in ray nek. Die bloed ruis in my ore soos die see, onsigbaar, hier naby op die rotse. Dan is hy op my. sommer deur poeletjies, bars deur die erikas en heide wat donsig glim onder hul las van duisende druppels. Die sproei lawe my stomende gesig. Die koel water meng met my warm sweet en die pekel loop in my oë, in by my lippe en my long kwyl. Ek is deumat van sweet en sagte reën en ek droog my gesig met my hande. Teen ’n steilte op, soos die paadjie al hoër klim, gruis die sandsteenbrokke onder die trekkersoie van my harde bergstewels. Die grens van die gruis word die grens in my bors. Op ’n gelykte sien ek figure in die mis. “Jou broer is daardie kant toe”, sê ’n lang ou, ’n regte Mountain Club o f S.A. old boy tipe. Ek voel skaam dat hy die spel kon snap en kwaad - eintlik hoogs verontwaardig dat hy my teenstander as my broer kon aansien. Maar die breuk gee my die kans om my bril skoon te vryf, en toe sien ek waarom: die mis het intussen gelig en die groep kon die jagtog van die hoogte af aanskou. En opeens sien ek hom, soos ’n bobbejaan teen die steil paadjie op skarrel. Ek voel verlig, nou weet ek presies waarheen hy op pad is. Die steil paadjie hang soos ’n lang dun rafel teen die krans. Ek sien hom hang aan die punt van die rafel en ek word gevul met heerlikheid. Nou moet ek net kophou, nie oorhaastig word nie. My bene is geswel, maar dis net krag en spier. Ek is in beheer, en ek gee hom kans, laat hy homself aan die rafel ophang, homself laat uitrafel. Hy hoor my en hy kyk verdwaas om. Ek sien die verontwaardiging en die skok toe hy my doodluiters sien aankom. En nou klim hy met mening, maar dit word ’n spartel. Hy kyk weer om en die gaping is kleiner. Hy spartel en hy kruip. Hy kruip skaamteloos op sy knieë oor die rotsbanke. En ek lag, want ek het hom en ek tart hom soos ’n kat met sy prooi. En ek wag, want ek weet hy is baie naby die einde van sy draadjie. Toe hy knak, kom staan ek by hom. “Lyk my jy ’t intussen besluit om nie meer geld te gaan opgrawe nie.” “Ja,” sê hy gedwee, “dis te koud. Dan is hy op my. Heeltemal uit pas met die prentjie wat ek vir myself voorgehou. het. Uit pas met die berg en die teësinnige weer. Middeljarig en die tekens van die middeldertigs het reeds in sy stywe grys langbroek kom sit. Sy stywe bont t-hemp beaam sy minagting vir die winter en vir die nat mis wat soos toegerypte spinnerakke aan elke boomtak, fynbos en in ons hare koek. A1 wat hy met hom saamdra, is die eienaardige instrument wat my dwing om te vra: “Wat se snaakse ding is daai, my broer?” “Weet jy dan nie? Dis ’n metal detekter.” Dit sê hy ietwat geirriteerd, seker om my te intimideer, maar voeg dan tog by asof hy my gedagtes kan lees: “Ek het al baie geld opgetel, daar waar die Boere vroeër vir goud gegrawe het, maar verskoon my, ek het nog ver om te gaan.” “Wat het jy sover opgetel?” vra ek nog agter hom aan. “’n Klomp pennies en ’n tiekie.” Ek weet sy sarkasme was gemik om my uit te lok, of om my op my plek te sit. En die mis sluk hom in, self sy voetval versmoor. Ek lig my bene, ek moet hom inhaal, binne trefafstand bly, maar hy’s weg. Ek slaan oor in ’n draflfie om net sy voetstappe te hoor, dan weet ek ten minste waar hy is. Ek verbeel my dat ek hom voor my tussen die bosse sien vleg, maar hy bly net tergend buite die bereik van my sintuie. Maar, dit weet ek beslis, hy sal nie wegkom nie, al moet ek hom inhardloop. Van wegkom kan hy maar vergeet, oor my dooie liggaam miskien ja. Maar hy’s net weg, soos ’n gees wat hierdie skemer-oggend, deur voorafbepaalde tyd en noodlot homself vir ’n oombUk kom manifesteer, hier kom ambraal en jou voortaan kwel dat jou nagsweet mis word. Jou laat wonder of hierdie oggend se besondere digte mis nie ook maar net die kondensasie van die nagtelike gesweet van al die menigte gekwelde siele is nie. Nou gee ek nie meer ’n duiwel om nie. Hy sal nie wegkom nie, al verloor ek iets in die proses of al moet ek ook tromp-op in hom vas hardloop. Ek hardloop Literator 17(1) April 1996:209-212 210 ISSN 0258-2279 Kirby van der Merwe sommer deur poeletjies, bars deur die erikas en heide wat donsig glim onder hul las van duisende druppels. Literator 17(1) April 1996:209-212 Dan is hy op my. Ek gaan maar liewerste na my vriend toe wat hier bo-op die berg woon.” “So, dan sien ek jou weer later.” Met dié woorde los ek hom daar. En my oë glinster. Ek probeer so vinnig as moontlik groter afstand tussen ons kry, vir die finale steek. Ek weet hy kan my duidelik sien, maar ek kyk nie weer om nie. Op die kruin skuil ek agter ’n rots teen die ysige wind. Op die plato sien ek ’n kliphuisie ingekruip onder ’n paar denne en die rook uit die skoorsteen raak verstrengel in die takke. Uiteindelik kom hy swaar om ’n rots, natgesweet en uitasem, sy systeek in sy gesig gekerf Hy wil nie met my praat nie. Ek het gehoop hy sal my saamnooi Literator 17(1) April 1996:209-212 211 ISSN 025^-2219 Mis_ Mis_ na sy vriend toe, maar hy loop net dikbek verder. Hy klou nog vas aan sy splinternuwe rooi metaalverklikker. Langs die pad terug, verkyk ek my aan die landskap, vars en dromerig soos ’n pastelskets. Die proteas bloos met wolmondjies. Verder ondertoe, waar die bosbouspanne se veldtog teen die indringers fel was, lê ’n denneboom gedissekteer, haar ingewande oop en bloot vir almai ora te lees. Ek break ’n takkie van ’n bossie af en skeur ’n sagte groen basrepie daarvan af. A1 langs die pad maak ek vir my ’n bossie blommetjies bymekaar wat ek met die basrepie vasbind, een blommetjie van elke iynbosplant wat ek die oggend teëkom. Literator 17(1) April 1996:209-212 ISSN 0258-2279 212 Marigoldstraat, die 'main road ’ vannie scheme Oom Ransie het toe besluit om hom liewer te bepaal by die rede waarvoor Bankie ingeroep was. “Mevrou, djy word daarvan beskuldig dat djy onwettig handel dryf en as gevolg daarvan het djy jou huurkontrak verbreek.” Bankie het haar anns gevou en gesê: “Wie beskuldig vi’ my dat ek onwettig handel dryf?” Oom Ransie het gebewe van boosheid: “Ek, oom Ransie, wat ’n lewendige God dien beskuldig jou van onwettige handel dryf.” Om beter te skel het Bankie haar hande in die heupe gekap en gesê: “Ek is baie jammer, maar ek word nie deur toiletpapier verhoor nie. Daar is baie mense wat onwettig handel dryf met suckers, lekkers, pop-com en toffie-appels en hoekom is hulle dannie hie’ nie?” Bankie was baie boos toe sy gesê het: “As daai die geval is, dan ga’n ek na my lawyer om te ga’n hoor of ’n mens soe verhoo’ kan word.” Oom Ransie het horn nie gesteur daaraan nie en het bygevoeg “Ek dien die Here. My Here issie bang vi’ jou lawyer of die ANC nie. Djy’s soe vol politick; hoekom ga’n djy nie somme’ na die ANC nie.” Bankie het haar liggaam spoggerig rondgewaai toe sy gesê het: “Die ANC praat met die Nasionale Party en nie met huishondjies nie.” Oom Ransie het met sy hand op die tafel geklap engesê: “O ’s sal sien, o ’s sal sien; more is djy uit. Ek ko sit jou self uit as die witmense nie wil nie. Ek la’t nie met my mors nie. Hie’ innie kleurlingdorp is o’s baas. Ek wietie hoeko issie res van my komitee soe stil nie, is hulle ook dan bang vi’ djou.” Die res van die Bestuurskomitee was verstom en het nooit so iets verwag nie. “Djy kan maa’ sê wat djy wil, maa’ more is djy uit. Die polisie het vir jou net te veel al uitgedra en nou weet jy skielik niks van onwettige handeldryf nie. Djy dink sieke’ ek wietie dat djy oek ’n moneylender is nie. Dis behalwe die mense se disability- en pensionboekies wat djy agterhou as hulle jou skuld.” Bankie het gou gereageer; “Djy kan mos somme’ nou my huis kom omdol en kyk of djy ienage disability- of pensionboekie sal kry, maa’ as djy nie kry nie, hang jou moer nog vandag aan ’n draadjie sonne ’n pennetjie. Marigoldstraat, die ‘main road’ vannie scheme Ek sal vi’ jou wys wat kan die Boere se toiletpapier maak met goed wat in die toilet behoort.” Bankie het toe geantwoord: “Van goed wat in die toiletpot behoort te is?” Literator 17(1) April 1996:213-217 213 ISSN 0258-2279 Marigoldstraat, die 'main road ’ vannie scheme Marigoldstraat, die 'main road ’ vannie scheme Marigoldstraat, die ‘main road’ vannie scheme Leonard Koza Marigoldstraat is die hoofstraat van die behuisingskema. Die redes daarvoor is bale duidelik. Eerstens is dit die straat met die meeste winkels en smokkel- huise. Dan is dit ook die straat waarin die meeste gevegte plaasvind. There is never a dull moment in Marigoldstraat. Dit is ook die straat met die interessantste karakters en persoonlikhede. Toe Marigoldstraat nog vol plakkers was, het hulle dit Appelboord gedoop. Tot die SAP was vir die Appelboord skrikkerig. ’n Speurder van jare se ondervinding het op ’n keer genoem dat die skema baie stil sou gewees het as daar net beslag gelê kon word op al die wapens in Appelboord. Marigoldstraat het sekere karakters wat dit laat uitstaan bokant al die ander strate. Wat belangrik is van die karakters is dat hulle soms gereed is om hul bloed te stort vir sekere beginsels: of dit nou reg of verkeerd is. Ten spyte van alles bly hulle menslik en liefdevol in vergelyking met die ander bekeerde broers en susters van die ander strate. In die ander strate loop die mense met lang en suur gesigte rond omdat hulle te emstig is om hemel toe te gaan. Party wil nie eers groet nie, want hulle mag punte verloor en dan kan hulle nie die hemelpoort ingaan nie. Bankie is een van die karakters in Marigoldstraat wat geen bang haar op haar hoof het nie. Sy was al vele kere opgeroep om voor die Kleurlingbestuurs- komitee te verskyn: die voorsitter van die Kleurlingbestuurskomitee is oom Ransie. Die voorsitter het haar kwaai aangespreek en gesê: “Mevrou, is u getroud?” waarop sy geantwoord het: “Waarom sê djy dan Mevrou?” Die voorsitter het hom toe vererg en gesê: “Ek het gehoo’ van jou, maa’ ek issie bang vi’ jou nie. Ek dien die Here.” Bankie het toe geantwoord: “Ek het oek gehoo’ van Kleurlingbestuurskomitees.” Met ’n breë glimlag het sy gesê: “Ek het gehoo’ hulle is die Boere se toiletpapier.” Oom Ransie het so boos geraak dat hy byna ’n hartaanval gekry het. Hy het haar toe verseker: “Djy kan vi’ jou kiaa’ maak, more is djy uit! Marigoldstraat, die 'main road ’ vannie scheme Met djou sal ek nie speel nie, sowaar as my mama, ’n Kind slaan nie ’n grootmens nie, m aa’ die Here sal my nie straf as ek ’n member van die Management Commitee op sy moer gie nie.” Bankies het vorentoe gemik om vir oom Ransie te gryp, maar hy was te vinnig vir haar. Hy het ’n paar meter vanaf haar gestaan en gesê: “Met ray God spring ek oo’ enige muur.” Bankie het met haar vinger na hom gewys en gesê: “Djou geluk dat ek jou nie innie hande kry nie. Die Boere sou net djou stukke opgetel het.” Die res van die lede van die Bestuurskomitee het verstom gesit en kyk. Ouma Meelbol was een van die vroulike lede van die komitee. Sy kon nie die wanorde verdra nie. “Meneer Voorsit ek gaan nou die polisie roep. 0 n s kan nie so aangaan nie. Hulle sê alreeds ons is die Boere se toiletpapier. Voordat hulle ons opfrommel, roep ons liewer die polisie. Die mense toon geen respek vir die komitee nie. Die mense wil ons nie erken nie. Meneer Voorsitter, dink jy nie ons moet maar liewer bedank nie?” Bankie was in haar noppies toe sy dit hoor. “Ek stem saam met Literator 17(1) April 1996:213-217 214 ISSN 0258-2279 LeonardKoza Ouma Meelbol, djulle kan gerus bedank.” “Dame, ek is baie jammer, maar my ma het nie ’n Ouma Meelbol in die lewe gebring nie”. Bankie het net gelag terwyl oom Ransie gesê het: “Ek sal nooit bedank nie, want dan beteken dit dat ek moet swig voor die k on im u n i s me O o m Ransie was nog steeds besig om te praat toe die polisie opgedaag het. Die een konstabel het uitgeklim om te vemeem waarom hulle ontbied was. Alles was doodstil, maar toe die konstabel die komiteekamer se deur oopmaak, het die rumoer hom getref soos ’n warrelwind. Bankie is so gewoond aan polisiemanne wat gereeld by haar woning klopjagte uitvoer dat sy haar glad nie aan die polisie gesteur het nie. Oom Ransie was weer baie bly toe hy die polisie gewaar en het gesê: “Daa’ kó die polisie, hulle hoo’ hoe lelik praat djy.” Bankie het vorentoe gestap en gesê; “Dink julle ek is bang vi’ die moffies. Marigoldstraat, die 'main road ’ vannie scheme Die polisie weet, djy kan hulle nou vra; innie Appelboord se tyd het ek baie baklei dat ek kaalboude tussen hulle staan, maa’ vi’ my word nie in ’n vangwa gegooi nie. I’m very sorry,” het Bankie gespog. Die polisie het vir Bankie geken en hulle het geweet dat sy nie sou omgee om hulle in die verleentheid te bring nie. Dit was duidelik dat die polisie glad nie enige een in hegtenis wou neem nie, want dit sou net onsmaaklikheid veroorsaak het. Die komitee het weer verlang dat Bankie opgesiuit word. Oom Ransie en die een vroulike lid het geweldig geprotesteer. “Sy kannie vi’ o’s soe beledig nie,” het oom Ransie gesê. “Dan het sy nog die vemietelheid om vir my Ouma Meelbol te noem asof ek haar speeimaat is.” Bankie het vreeslik gelag en dit het vir oom Ransie vreeshk kwaad gemaak. “Ek ga’n na die minister met julle, want o’s staan die regering by deur dik en dun en nou wil julle nie vi’ haa’ opsluit nie. Ek ga’n ook na julle kommandant om vi’ hom te vertel van julle. Dit lyk my djulle drink daa’; daa’om kan julle haa’ niks maak nie.” Die konstabel het geglimlag toe hy se: “Meneer, kan jy bewys wat jy nou net gesê het?” Oom Ransie het gebewe van woede en gesê: “Onthou net dat ek sal twee keer dink voordat ek weer uitkom as jy die polisie moet ontbied.” Die konstabel het in die vangwa geklim en weggery sonder nog om te luister hoe oom Ransie agtema skree: “Ek is die baas van hierdie dorp. Julle sal net kom as ek julle nodig het”. Bankie, wat eenkant gestaan het, het nader gestap en gesê: “Ek loep oek nou, want ek wil more my lawyer ga’n sien.” Oom Ransie wat nie ver van haar af gestaan het nie het haar gehoor en toe gesê: “Djy kan maa’ na jou lawyer toe ga’n, maa’ onthou net ek is die een wat baklei het dat al die pondokkies afgegooi word en dat daa’ vi’ julle huise gebou moet word.” Daar was ’n groot sug van verligting toe Bankie die gebou verlaat het aangesien haar betoog ander mense wat ook moes verskyn, kon beinvloed. Oom Ransie het op sy horlosie gekyk en aan die ander komiteelede gesê: “Kan djulle nou sien hoe die smokkelaars die komitee se tyd mors. Marigoldstraat, die 'main road ’ vannie scheme Net so mors hulle o’s mense 215 Literator 17(1) April 1996:213-2] 7 ISSN 0258-2279 Marigoldstraat, die 'main road' vannie scheme se lewens ook op met die gifstowwe wat hulle verkoop en as jy praat, dan moet djy hoo’ hulle ga’n lawyer toe.” se lewens ook op met die gifstowwe wat hulle verkoop en as jy praat, dan moet djy hoo’ hulle ga’n lawyer toe.” Mevrou Sofie Kramer was die volgende karakter van Marigoldstraat wat voor die Bestuurskomitee moes verskyn. Oom Ransie het probeer om haar eers sag te maak voordat hy haar aangeval het. “Ma-Kramer,” het hy gesê, “ek het voor jou familie grootgeword en hoop net dat jy jou beter sal gedra as Bankie, jou buurvrou. Ek sou jou nie ingeroep het nie, maar die behuisingskantoor kry net te veel klagtes van jou.” Sofie Kramer het haar gesig skeef getrek soos ’n skut wat korrel. Die voorsitter het probeer paai deur te sê: “O’s wietie of dit waa’ issie, m aa’ die klagte se dat jy onwettig handel dryf, en anne raense se jong kinders by jou huis opgaar. Daar word ook gese dat jy een vadie Raad se kamers uitverhuur en dat jy anne vrouens se mans na jou huis lok. Die bure kla gedurig van geraas as gevolg van diskopartytjies by jou huis. Jy het oek gloe kinnes van verskillende mans maa’ daai’s jou eie biesagheid, wil jy enige iets se?” Sofie Kramer het eers die hele komitee deurgekyk voordat sy begin praat het. Haar oë het stil in haar kop gestaan en sy het diep gesluk. Dit was duidelik dat sy vinnig van iets onslae wou raak. Die voorsitter het weer vir haar gevra: “Is djy sieker daa’s niks wat djy wil sê nie?” Sofie Kramer het stadigies haar logge liggaam gelig en gesê: “Meneer die voorsitter, julle is mos ’n klomp moifies, djulle makeer net panties. Djulle call julleself leaders, m aa’ djulle skinne dan soes wasvrouens. Watse respek kan o’s ordinary mense vi’ julle het? Ek is haastig van die huis af sonder om my familie kos te gie. As ek gewiet het dis julle style sal ek mos nooit gekom het nie. Djulle praat van onwettige handel dryf asof ek die enigste een is in die scheme wat biesagheid maak. Ek het nog nooit ander mense se kinnes opgegaar nie. Marigoldstraat, die 'main road ’ vannie scheme Die kinnes kom vra altyd iets om te iet en dan gie ek vi’ hulle. Die blêrrie mense is te sleg om hulle kinnes kos te gie, maar hulle hardloop na julle om te kla ek gaar hulle kinnes op. Ek lok nie anne vrouens se mans na my huis toe nie. Saans speel die mans kaarte en dominoes. Moet ek nou die mans wegja? Waarom maak die vrouens nie hul mans vas met kettings nie. Is ’n mens nie toegelaat om ’n bietjie music te speel as iemand veijaar nie? Is dit hoe o’se rates gemors word om julle te betaal om my sulke nonsense te vra. Somme’ vanaand skop ek my neigbour in sy moer. Hy moenie van my by julle moffies kom skinne nie.” Die voorsitter het sy hamertjie gekap vir orde, maar Sofie het haar nie daaraan gesteur nie. “Ek skop julle somme; nou in julle ma se ....” Die voorsitter het besef dat hulle niks met haar kan uitrig nie. Sy was meer aggressief as die vorige huurder. “Djulle het ’n fine cheek om te skinne van my los kinnes. Wys my ’n straat innie scheme wat nie los kinnes het nie. Ek wiet van soveel kamstige kerklike mense innie scheme wat los kinnes het. Daa’ 216 Literator 17(1) April 1996:213-217 ISSN 0258-2279 LeonardKoza word nou net van myne gepraat omdat my kinnes se pa baie ko visit. Djulle gie my kinnes fokkol, soe hou julle bekke van my af.” word nou net van myne gepraat omdat my kinnes se pa baie ko visit. Djulle gie my kinnes fokkol, soe hou julle bekke van my af.” Die voorsitter het haar mooi gevra: “Mevrou Kramer, is djy nou klaar gepraat, want ek wil oek iets sê?” “Meneer voorsitter of oom Ransie, ek is nog nie klaa’nie. Wie het vi’ djulle gekies. Omtrent tien persent van Tiervlei se mense het vir julle gekies. Djulle represent mos nie al die mense nie. Djulle is kamstig soe big deal, maa’ hie’ innie Tiervlei kry mense huise wat uit Namakwaland, Mitchell’s Plain en anne surrounding areas uitkom. Mense wat innie Tiervlei gebore is, word vertel hulle moet geduldig wees, want die waglys is lank.” Oom Ransie se wange het gebewe van boosheid. Hy het gehakkei en gepraat met sy linkerhand op sy hart. Sy het haar handsak opgetel en is swaai-swaai daar uit. Literator 17(1) April 1996:213-217 Marigoldstraat, die 'main road ’ vannie scheme “Ek is ’n hartlyer en die dokter het gesê dat ek nie moet kwaad word nie, maa’ mense ek voel gegrief. Mevrou Kramer het vergeet dat ek hierdie gemeenskap meer as dertig jaar dien. Ek het by tye van stormreëns baie nagte kniediep in die water gestaan terwyl anne leiers slaap, maa’ vandag word ek so beledig. Hierdie komitee support nie Apartheid nie. Ek wiet baie min mense het kom stem, maa’ o’s kannie help as die bietjie wat kom stem het vi’ o’s gestem het nie. A ’mal innie Tiervlei is nou opgemaak met ’n ANC, maa’ waa’ was hulle toe Tiervlei nog bos was?” Mevrou Kramer het skerp gereageer en gesê: “Djulle moet glad nie praat vannie ANC nie, want hulle negotiate met die Nasionale Party terwyl julle skinne’ van my los kinnes. Djulle onderskat die mense van Tiervlei, maa’ ek sê vi’ julle; one day is one day, djulle sal nog wakke’ skrik dan is daa’ nie mee’ ’n Bestuurskomitee nie. Ek hoep julle vat note dat die mense innie Township is op van julle.” Literator 17(1) April 1996:213-217 217 ISSN 0258-2279 218 Brandewyn en coke special Die seuns sê nee, hulle hoort daar, hulle het dan vir die oom gesê. Die ooni sê issie en sy ghout beginne pyn, so kwaad raak hy. Dis nou waar die bewyse- storie inpas. Die oom sê hulle is besig cm te steel, kyk hoe lyk die plek. Nee, sê hulle, hulle het partytjie gehou, dis waarom dit so deurmekaar is. Sê die man van die gemeenskapspohsie: “Maar as meneer dan dink hulle steel, hoekom arresteer meneer hulle dan nie self nie? 0 n s het in elk geval nie nou tyd om ’n saak te maak nie. 0 n s moet nog na ’n ander inbraak hier anderkant gaan. Maar ons sal die manne se name vat just in case ...” En daar ry hulle, los die diewe net daar vir die oom om te arresteer. Toé pyn die oom se ghout. “Juffrou, ek sou so graag wou, maar die voet was nét so hoog opgeswel,” en hy beduie amper so ’n halwe meter van die grond af. “Maar ek dink ek gaan dit nie daar los nie, juffrou.” Hy bel sy seuns en hy skakel weer die polisie. ’n Ruk later kom die twee konstabels weer daar aan en die oom se twee seuns ry saam na die adresse van die twee jongmans. Dis nou nadat hulle oorkant was en gesien het daar is ingebreek. Die twee knape woon net ’n paar strate weg. Die een, ten voile gekleed, maak die deur oop en sê hy slaap lankal. Die ander een maak oop, das om die nek en dikdronk. “Seker die Klippies,” voeg die antie tussenin. Hy slaap al la..a..a..nkal. “Sien,” sê die man van Korrektiewe Dienste, “dit kan nie hulle wees nie. Hulle slaap al la..a..a..nkal.” ♦ ♦ ♦ ♦ ♦ Ek skrik vervaard wakker en sit regop om te sien waar ons is. “Ons is nou net by George verby,” se die bestuurder van die kombi. Ons is op pad terug vanaf Port Ehzabeth waar ons vir ’n paar dae gaan kerjakker het. Die res van die toergeselskap lê ook nog en slaap. “Kan ons nie in Mosselbaai stop sodat ek gou kan huis toe bel nie?” Pappie tel die telefoon hygend op en tussen stotterende asmatiese hygkrete leer ek dat alles nog o.k. was toe hy die vorige dag gaan kyk het of my huis nog o.k. is. Brandewyn en coke special Ingrid Geldenhuys “Weet jy wat vra daai blêddie polisieman my, juffrou? Hy wou van my weet watter bewyse ek het dat daai twee skarminkels besig was om by jou huis in te break en of ek kan bewys dat hulle nié daar woon nie. Magistraat! Van wanneer af het ’n mens bewyse nodig om jou bure te k en ...” Dis my buunnan. ’n Wonderlike ou omie met ghout in sy regtervoet, ’n be-gate swirlkous op sy weerbarstige rooikop en ’n mond wat breed glimlag om die goudsplitte te laat vonkel. Maar dierbaar. Die nie-ampte!ike brandwag- bobbejaan van die buurt as ons almai in die werk of met vakansie is. Dié dag van die inbraak gewaar einste buurman van oorkant dat die buurseun van langsaan die straat patrolleer. “Patrolleer” is buurman se woord, want dis geen geheim dat dié leeglêer, wil-nie-werker net vir kwaadgeld rondloop nie. Hy is betrokke by ’n bende en omdat hy alewig by die huis is, weet hy presies wie is wanneer nie tuis nie en by watter huis is dit veilig om in te breek. Uit dié eenmanpatrolhe is dit vir buurman duidelik dat iets aan’t broeie was en sy uitkykinstinkte staan op aandag! Teen 19h00 hoor hulle die kenmerkende sein- fluite van die bendes. Buurman stap uit en sien twee wildvreemde jongmans op my stoep. Hy stap nader, wetende dat ek met vakansie is, en vra wat hulle daar soek, want hulle hoort nie daar nie. “Wat weet oom, ons pas die huis op.” Maar buurman wis dis ’n hegstorie en sê hy gaan die polisie bel. Toe verdwyn hulle. Maar hy voel nie gems nie. Later die aand merk hy bewegende figure binne die huis op, en al die hgte is skielik aan. Hy skakel onmiddellik die polisie om te se ’n inbraak is besig om op daai oomblik plaas te vind. Hulle kom en buurman en vrouhef stap saam met hulle by die oop agterhek in om hulle onkant te vang. Daar kry hulle dieselfde twee jongmans lustig aan’t drinke in my voorhuis. Hulle is so tuis soos twee huisbrakke. “Dis die Here se waarheid, juflrou. Ek’t hulle met my eie oë gesien. Hulle’t daar gesit met ’n bottel Klipdrift en ’n halwe bottel Coke,” sê die antie. Literator 17(1) April 1996:219-223 219 ISSN 0258-2279 Ek draai cm, niks maak verder saak nie. Toe eers raak ek bewus van die res van die vakansiegeselskap wat stilswyend in die deuropening staan. “Is jy okay”, vra iemand. Verslae gaan sit ek midde-in die chaos en probeer inneem wat my getref het. Elke denkbare laai en kas is uitgegooi. My leefhiimte, my werkruimte gerelegeer tot ’n urienstinkende rommelhoop. Wat van my kosbare skoeneversameUng? Soos ’n wafferse Imelda Marcos het ek ’n maniese passie vir skoene - lelikes, grasgroenes, skokpienkes, beblomdes, beblinkertjiedes - ag, sommer net oor hulle vir my mooi is en elkeen ’n eie storie vertel. Ek onthou die grasgroen paar, die “patent leathers” wat ek gekoop het voor ek weg is. Ek maak my skoenekas oop ... daar’s niks in nie. Ek smyt die papiere eenkant toe. Assebhef, Here, net die groen paar. Dis ook weg. Wat wil hulle daarmee maak? Dis dan so lelik?! Dis net ek wat dink hulle’s pragtig. Trane biggel oor my wange. Die opname van wat weg is, is skrikwekkend. Ek is ’n alleenloper en het oor die jare my arsenaal van huisware en kantoor-ameublement goed aangevul. Maar dis nou alles weg: rekenaar, antwoordmasjien, faks, diktafoon, mikro- golf, twee leerbaadjies, ketel (dis so oud, dis moeilik om water daarin te kook), glastopstoof, lêers, handsakke, floppies/stiffies, koordlose telefoon ... Te veel om te noem. Net dit waarop ek my hoof moet neerlê, het oorgebly. Die lewe wat ek vir myself gebou en bymekaargemaak het, is in een orgie van waansin uitgetippex. Asof dit nooit bestaan het nie. Ook my onderklerelaai is gefynkam. En hulle het nie sommer enige ou bloemer gevat nie. Nee, die sexy goedjies wat in stelletjies kom. lewers loop iemand met my onderklere rond. Gr...r..r..il.. Ek staa! my vir die vraag op almal se lippe. “Het jy versekering?” NEE! gil my siel terug. Dit gebeur tog net met ander mense. Spottend lê die onin- gevulde versekeringsvorm bo-op die gemorshoop vir my en loer. Ek het nooit sover gekom om dit in te vul nie. My gedagte-rat kom krakend in beweging. Ek hoor die skamiere ... ek moet oproepe maak. Die polisie, my pa, my familie, my vriende ... Dit kan maar seker nog ’n nag wag tot more-oggend. Niemand kan tog nou enige iets doen nie. Die nuus het egter deur die nag soos ’n ondergrondse struggle-boodskap versprei. Elke keer as die telefoon lui, moet ek van vooraf begin. Brandewyn en coke special Maar daar’s knaende onrus wat maar nie wil gaan lê nie. Ons ry die Kaap omtrent 22h30 binne. Die reën val in triestige slierte oor die voorruit. Toe ons die draai vat, sien ek al die ligte van my huis brand. Ek het hulle beslis nie aan gelos nie. Ek klou my sleutel krampagtig vas terwyl ek na die voordeur stap. “Wil julle nie maar solank in die kombi wag nie.” Hulle het my eerste kom aflaai. Die deur swaai oop nog voordat ek die sleutel kan draai. Die stank van katte en urine sypel my neus binne. Die grootste chaos wat jy jou kan voorstel, begroet my. Oral lê goed rondgestrooi. Al wat deur my gedagtes flits, is my rekenaar. Laat hy tog daar staan waar ek hom gelos het. Soos altyd wanneer ek uit was, dryf die obsessie my om te gaan seker maak dat hy getrou vir my staan en wag. Die hy in my lewe met wie ek al my gedagtes d e e l... die Literator 17(1) April 1996:219-223 220 ISSN 0258-2279 Ingrid Geldenhuys niiddelpunt van my bestaan. My kamerdeur is oopgebreek. Die kat spring miaauende van die bed af en hardloop die kamer uit. Stadig dwaal my oë na my rekenaar se staanplek op die lessenaar. Hy’s nie daar nie, sy rooi oog sal my nooit weer hallo sê nie. niiddelpunt van my bestaan. My kamerdeur is oopgebreek. Die kat spring miaauende van die bed af en hardloop die kamer uit. Stadig dwaal my oë na my rekenaar se staanplek op die lessenaar. Hy’s nie daar nie, sy rooi oog sal my nooit weer hallo sê nie. Ek draai cm, niks maak verder saak nie. Ek draai cm, niks maak verder saak nie. “Jammer oor 221 Literator 17(1) April 1996:219-223 ISSN 0258-2279 Brandewyn en coke special wat met jou gebeur het. Het jy versekering? Ek” “Dankie. Nee. Alles.” Dit voel soos ’n begrafhis. Ek kan dit later nie meer verdra nie, vat my laaste geld en gaan koop ’n antwoordmasjien. Die boodskap? “You’ve reached the ransacked home of Miss X. If you want to have a soup kitchen at this address, please do so. They’ve even cleaned out the freezer. Instead of sympathy, please leave a donation after the beep.” Die gelui het nie opgehou nie. Nie dat ek ondankbaar is nie, inteendeel, dit het my eerder op my knieë gedwing (miskien was dit die Goeie Vader se toetsrede, waarom dié ding met my gebeur het) oni dankie te sê vir die wonderwerk van vriende. Nou vir die Korrektiewe Dienste wat gebuk gaan onder titels soos Gemeenskapsvriendelike polisiemag, Misdaadvoorkomingsforums, ens. Ek skakel hulle om te hoor of iemand die inbraak aangemeld het, want die predikant wat oorkant my bly se seun sê hy het die polisie ontbied, so kan ek asseblief met die persoon gemoeid met die inbraak by dié adres gesels. Hulle weet nie van ’n inbraak nie. Niemand het hulle ontbied nie. Huh? Ek moet dan maar ’n klag lê en dan sal hulle kom ondersoek instel. Ek lê toe ’n klag. Toe die polisie daar aankom, voorsien ek hulle van ’n telefoonnommer, wat die diewe blykbaar langs my telefoon vergeet het, om hulle met hul saak te help. Ewe behulpsaam is ek. Hulle sal op die saak ingaan; hulle stuur onmiddellik die speurders. Konstabels, speurders? Ek verstaan nie die “pecking order” nie. Twee jong knape kom volgende. Die een spring rats oor die agterste muur en Voila! Daar lê twee swartsakke en toe hulle dit oopmaak, is dit vol skoene. Dis net God se genade dat die vulliskanveiers dit nie weggeneem het nie, want dit was juis Maandagoggend en Maandag is vullisdag. “Is jy familie van Imelda Marcos, jufïrou?” Ek dink glad nie dis snaaks nie, maar gaan nogtans histeries aan die lag. Uit pure verligting of ’n wrang vorm van geregtigheid? A1 wat vir my op daardie oomblik sin gee aan my bestaan, is die paar groen skoene wat ek styf teen my bors vasdruk. Geen vingerafdrukke word geneem nie. Hulle is daar weg met ’n lys van wat teruggevind is. “Dis jou opinie, juflrou.” “Dis jou opinie, juflrou.” Ons spring in my vriendin se motor en ry na Bellville-Suid se pohsiestasie. Wat daar gebeur, is lagwekkend, om die minste te se. Nêrens in die K- watsenaam is so iets aangeteken nie. In elk geval, sê die luitenant op sy onbeskofste, hulle rekenaars was die naweek af. Die land is lamgelê deur misdaad, hulle rekenaar is af. Nou’t ek als gehoor. Die moer in. My termometer is na aan kookpunt. Nêrens sal dit dus op rekord wees dat daar iewers gesteelde goedere ingegee was nie. Ons moet maar wag tot hulle die geskrewe K-items vir die rekenaar gevoer het, dan sal hulle my skakei. Ek draai cm, niks maak verder saak nie. Die volgende oggend, 08h00, is daar ’n ander speurder. “Goeie more, het hulle hier ingebreek? Waar het hulle ingekom?” sê hy in een asem. “Daar is geen ruit gebreek of teken van geforseerde toegang nie,” antwoord ek. “Sjoe”, se hy. Binne een minuut stap hy dwarsdeur die huis en sê toe sonder enige verdere woord tot siens. My mond hang oop en ek moet my kaak met my hand toedruk. Nog steeds geen vingerafdrukke... tot op vandag nie. Intussen skakel my vriendin wie se nommer Pappie aan die polisie gegee het as kontakpersoon totdat ek terug is. “Het jy jou goed gekry?” vra sy. Kalm ... kalm ... “Watter goed?” waag ek versigtig. Die blitspatrollie het haar die nag van die inbraak geskakel en gesê sy moenie worry nie, sy moet vir haar Literator 17(1) April 1996:219-223 222 ISSN 0258-2279 Ingrid Geldenhuys vriendin sê hulle het die twee outjies gevang en die goed teruggekry. Ek moet dit kom uitken sodra ek terug is. Hy skakei van Maitland-polisiestasie. vriendin sê hulle het die twee outjies gevang en die goed teruggekry. Ek moet dit kom uitken sodra ek terug is. Hy skakei van Maitland-polisiestasie. My h a rt... sing. Ek gaan my rekenaar terugkry! Sy kom gou oor en ons twee gaan aan’t bel. Nee, sê die polisieman vies by die Maitland-polisiestasie, hulle weet van geen so ’n oproep nie. Geen regdenkende polisieman sal so ’n oproep maak nie. Hy sal gaan seker maak in die K-watsenaam, maar hy is seker dis iemand wat ’n grap wou maak? My vriendin raak rooi. Natuurlik het iemand van die polisie haar geskakel. Sy’s mos nie befok in haar kop nie. Iemand lieg en dis beslis nie sy nie. Hulle gee ons die telefoonnommers van ander polisiestasies om te skakei en verwys my terug na Bellville-polisiestasie waar die saak aangemeld is. Bid jou aan, ons moet die speurwerk doen. “Meneer, sersant, of wie jy ook al is, as ek ’n speurder wou gewees het, sou ek nie skoolgegaan het nie,” kap ek terug, want toe is ek moerig. Literator 17(1) April 1996:219-223 Ek wag nou nog. As die een sê hy weet nie, lyk dit die ander sê in ’n koor agtema hulle weet nie. Toe stap die kaptein in, hy is swart, en skielik loop daai onbeskofte wit polisieman oor van vriendelikheid en lek hy asof hy ’n seel lek. Ek weier egter oni verlei te word. Ek is al jarelank in die struggle. Maar nie eens die kaptein se tussentrede het hulle tot aksie opgekommandeer nie. Skakei ek hulle kantore, is die man gemoeid met my saak op verlof Hy het nou nog nie met my kontak gemaak nie. My rekenaar staan op iemand anders se lessenaar, iemand anders dra my onderklere, spog in my leerbaadjie, drink kofTie met water gekook in my ou ketel. Ek is uit my huis gedryf, my lewe is gestroop, my privaatheid is gewelddadig verkrag. Die misdaadafdeling se rekenaars is naweek-af Nie eens my groen skoene loop meer lekker as ek hulle aantrek nie. Literator 17(1) April 1996:219-223 ISSN 0258-2279 223 224 Liefde leef... boemelaar of te not boemelaar o f te not spottend, “is die lewe dam nog orraait of moet ek jou bietjie tnigsit innie hewe?” Katy Willemse vererg haar nie oor Jan se opmerking nie; sy ken horn veels te goed om sy woorde van flussies erastig op te neem. “So asof jy nooit die enigste mens was wat Jan Balie se diepste menswees geken het nie!” dink Jan as Katy uitdrukkingloos voor haar uitstaar. Twee siele was amper een totdat sy gaan trou het met ’n ryk man van die Kaap. Sy en haar man kom woon toe boonop in KalUesbaai, so naby aan die gebroke Jan BaUe. As Katy die Willemse se winkel binnegaan, kan Jan nie help om die lyf wat eens syne was, agtema te kyk nie. “Hoe mooi het sy nie geword nie. Die volheid, ja die volheid was maar altyd daar.” Hy wonder soms oor hoe sy lewe sou gewees het as hy vyftien jaar gelede met sy jeugliefde, Katy, getrou het. Sou sy nog altyd so pragtig gewees het. O f sou swaarkry haar ouer laat lyk h e t...? Hy was pas mondig, sy agtien en in die fleur van hul lewens. Sorgeloos en ongebonde soos net die jeug kan. Vry om die liefde van die jeug te ervaar. Hy was skipper van “Groot Katy” die boot van Katy se pa, mister Willemse, die man wat byna elke koninkryk op Kalliesbaai besit. Sy het pas begin skoolhou vir die Kindertuin en kan nog bloos soos geen ander meisie op Kalliesbaai nie. Die twee van hulle sou trou sodra Jan genoeg gespaar het vir ’n redelike huis om nie die Willemse se status te beledig nie. “Die Balies is arm mense” het die vrouens van Kalliesbaai altyd geskinder. “Mense wat ’n troue van hul seun met ’n ryk dogter sou goedkeur, terwyl die Willemse vals genoeg was om net hulle dogter se geluk so te wil verseker.” Tog kon nie die Balies of die Willemse keer dat Katy wegloop met ’n Kaapse man en later geboorte gee aan ’n pragtige seuntjie nie. Nie eens toe Katy en haar nuwe familie hul intrek neem by haar pa se strandhuis nie. Nog minder kon die Kalliesbaaiers Jan Balie keer toe hy ná tien jaar sy vissersmanslewe vir ’n stoepsitlewe by die Willemse se kafee verruil. Liefde leef... boemelaar of te not Abigail Smith “Middag! Middag, miesies Lea! Die Baai is mooi vemore, nê? Ek wou maar net vra, miesies Lea, hoe lyk dit m et...” Groot ou Lea Steenkamp gee Jan Balie net een hooghartige kyk vanuit haar effens té brilletjie vir háár aristokratiese neus. Met ’n vinnige tred stap sy weg van die man wat sy voor haar sien grootword het. Die man met die lang maer, maar tog stewige liggaamsbou wat vroeër jare deur talle bakvissies begeer was, ten spyte van die Bybelse skrywe oor begeerte. Hy gee een harde sug as hy miesies Lea sien wegstap, want ken hy wat Jan Balie is dan nie nou al ná jare se bedel die mense van Kalliesbaai nie. Die Samaritane wat hom laat besef dat God nie slaap nie én die suiniges wat altyd maak asof hulle Jan Balie van g ’n kant ken nie en haastig op pad na een of ander belangrike afspraak is. Ja, nee wat! Jan Balie ken sy mense asof hy hulle gemaak het. “Hêy! Ou Jannie, issit al weer een van daardie dae?” skree ’n man met ’n blinklyfsnoek in sy hand op pad na êrens. “Ja man. Is mossie elke dag Krismis op KalUesbaai nie, jong. So gaan dit maar met Jan Balie!” Die man stap weg nog voor Jan Balie ’n stukkie vis kon bedel, maar ’n ou gryskopvrou wat met haar vriendelike plooigesig en vlesige mond ’n tien- sentstuk in sy hand stop, verhinder hom om weer sinies of verbitterd te word. As hy gaan sit op sy blik op die stoep van Mister Willemse se kafee, stap ’n kort mollige vroutjie op hom a f ’n Hemelse prentjie! “More Jan,” sê sy as hy opkyk en sy sien die spot in sy oë of is dit vermaak? Tog, wat maak dit vandag nog saak? Haar en Jan se lewens het nou eenmaal vyftien jaar gelede elkeen sy eie koers ingeslaan. “More Katy,” sy stem is hard en die pyn kruip in sy oë al is sy stem ook half “More Katy,” sy stem is hard en die pyn kruip in sy oë al is sy stem ook half Literator 17(1) April 1996:225-227 225 ISSN 0258-2279 Liefde leef... Literalor 17(1) April 1996:225-227 Liefde leef... boemelaar of te not Die see bring te veel herinneringe terug van sy dae as jong klong saam met sy jeugliefde. Sy grootste geskenk, Katy Willemse! ’n Geskenk wat sommer so sonder waarskuwing weggeruk is uit sy lewe. ’n Geskenk wat gebly het in sy hart, sie! en oë wat onbewustehk oral tussen mense soek. Jan Balie ruk as hy die nou al bekende voetstappe van sy enigste liefde hoor nader kom. As sy by hom wou verbyskuur so asof hy nooit die “magic” in haar jongmeisiedae was nie, gryp hy haar vas. En as sy skrik, tree Jan nie weg nie, Literator 17(1) April 1996:225-227 226 ISSN 0258-2279 _Abigail Smith _Abigail Smith maar bring haar al nader aan hom. Hy trek haar vas, Katy sien die baard- stoppels, die bruingebrande gesig en ontmoet die oë van ’n nou ouer man. Sy sien die lig daarin, vermeng met ’n bietjie spot. Dan ... Twee paar honger lippe vind ná vyftien jaar weer die wonder van die vlees saam met die gees. Twee siele word weer een, soos destyds. Twee harte herleef weer die wonder van lig. As Jan die lieflike vrou uit sy arms laat gaan, steier sy amper agteruit en stap twee treë terug. “Here Jan!” Dis ’n uitroep van seer, van liefde! Die twee mense se oë ontmoet, hulle weet dit albei eens: “Liefde leef nog na al die hartseeijare!” “Here Jan!” Dis ’n uitroep van seer, van liefde! Die twee mense se oë ontmoet, hulle weet dit albei eens: “Liefde leef nog na al die hartseeijare!” ISSN 0258-2279 227 228 Frans Mouton se tuiskoms Edwina H. Fransman Annette, ’n aantreklike donkerkop, stap op ’n lieflike Saterdagoggend in Parow se hoofstraat. Sy is op soek na ’n apteek wat afslag op spesiale produkte bied. Skielik steek sy vas. Voor haar sien s y ’n bondeltjie vodde. Sy skreef haar oë om beter daama te kyk. “Maar dis dan ’n mens!” sê sy. Eers toe die bondeltjie-mens opkyk, besef Annette dat sy hardop gepraat het. Die grys oë wat na haar opstaar, is so vol leed dat sy daarvoor skrik. In haar hele negentienjarige bestaan het sy nog nie so ’n verwese skepsel teëgekom nie. Toe sy besef dat dit ’n bedelaar is, wil sy eers ’n paar muntstukke in sy hand stop, maar besluit dan daarteen. Sy buk af na die bedelaar en vra: “Wat is jou naam en waar kom jy vandaan?” “Dit maak nie saak nie, juflroutjie.” antwoord hy. “Ek soek net ’n geldjie vir kos en sigarette.” Annette, wat self baie hardkoppig kan wees, ignoreer sy pleidooi en herhaal haar vraag. “Frans Mouton.” antwoord die bedelaar baie onwillig. Annette deins ’n tree terug, verstom! “Het jy gesê, Frans Mouton?” vra sy hees. “Is jy seker dis jou regte naam? Waarvandaan kom jy dan?” wil sy nou dringend weet. Frans verstaan nie die meisie se getorring nie, maar hy begin nietemin sy storie aan haar te vertel. “Dit was sowat tien jaar gelede dat die ongeluk my getref het. Ek was ’n gesiene man in die gemeenskap. Ek was ouderling in die kerk en ’n liefdevolle man en vader.” Annette is meegevoer deur sy vertelling, tog bly sy nugter. “Jy was dit alles!” se sy verwonderd. “Jy was dit alles!” se sy verwonderd. “Jy was dit alles!” se sy verwonderd. “Jy was dit alles!” se sy verwonderd. “Ja. By die skool, waar ek Afnkaansonderwyser was, was ek baie gewild, veral onder die kinders. Tot op daardie noodlottige Vrydagmiddag ...” Frans bly opeens stil. “Ja. By die skool, waar ek Afnkaansonderwyser was, was ek baie gewild, veral onder die kinders. Tot op daardie noodlottige Vrydagmiddag ...” Frans bly opeens stil. “Ja. By die skool, waar ek Afnkaansonderwyser was, was ek baie gewild, veral onder die kinders. Tot op daardie noodlottige Vrydagmiddag ...” Frans bly opeens stil. 229 Literator 17(1) April 1996:229-232 ISSN 0258-2279 Annette kyk hom vraend aan. Annette kyk hom vraend aan. Annette kyk hom vraend aan. “Wat het dan daardie dag gebeur?” “Wat het dan daardie dag gebeur?” Frans Mouton kyk haar stip aan, haal diep aseni en vertel verder. “Ek was op pad huis toe na ’n vergadering by die skool daardie middag. Soos gewoonlik was die straat waarin ek gebly het, vol kinders en honde. Skielik het ’n hond oor die straat gehardloop. Terwyl ek nog na die hond gestaar en probeer vermy het, het ’n kind van nêrens voor my motor verskyn. Dit was te laat om te rem. Die volgende oomblik het ek net die slag gehoor...” Annette, wat so intens na die man staan en luister, kom skielik agter dat haar gesig nat is. Sy word ook bewus van die mense wat op en af verby hulle beweeg. Sy vee oor haar gesig met ’n snesie en vra Frans ora verder te vertel. Hy is self verwonderd oor die kind se vreenide reaksie. “Die dogtertjie was op slag dood. Soos te verstane was daar ’n hofsaak. Alhoewel ek onskuldig bevind is, het die gemeenskap my nie vrygespreek nie. Ek kon myself ook nie vergewe nie, al het ek geweet dat dit nie my skuld was nie.” Frans klink nou so pateties en gebroke dat Annette vinnig afbuk en sy vuil hand in hare neem. “Natuurlik was dit nie jou skuld nie, Pa.” sê sy sag. Frans se kop ruk so vinnig op dat sy nek eintUk seerkry. “Pa! H etjy my Pa, genoem? W ieisjyjuffrou?” vrahyoorbluf “Ek is jou dogter, Annette. Onthou jy my nie meer nie? 0 n s was dan sulke goeie maats. Die laaste keer dat ek jou gesien het, was ek byna tien jaar oud. Jy het net skielik verdwyn en ons agtergelaat. Waarom?” eis sy nou van hom. “Ja, my kind, ek het net verdwyn. Ek kon nie meer met my gewete saamleef nie. Jou ma het my ook kwalik geneem. Haar vriendinne wou haar skielik nie meer ken nie. Ek weet dat ek nie regverdig teenoor julle was nie, maar ... dit was al uitweg. Ek het op straat beland en begin bedel om aan die lewe te bly.” Annette, wat nog al die jare na haar pa verlang het, gooi haar arms om sy nek. Sy skrik toe sy voel hoe maer sy bolyf is. Sy besluit daar en dan om vandag nog haar mense bymekaar te bring. “Pa, sal jy assebhef saam met my huis toe kom?” vra sy dringend. “Wat het dan daardie dag gebeur?” “Pa, sal jy assebhef saam met my huis toe kom?” vra sy dringend. Frans skrik eflfens. Hy kan dit nog nie glo dat sy lieflingsdogter hom gevind het nie. Maar wat sy nóú wil doen, verstaan hy nog minder! Frans skrik eflfens. Hy kan dit nog nie glo dat sy lieflingsdogter hom gevind het nie. Maar wat sy nóú wil doen, verstaan hy nog minder! Literator 17(1) April J996:229-232 230 ISSN 0258-2279 Edwina H. Fransman “Kind, wil jy ’n ou vuil bedelaar huis toe neem? Wat gaan jou ma sê? En Heleen en Wiinpie? Ek kan dit eenvoudig net nie doen nie. Moet my asseblief ook nie dwing nie.” smeek hy. “Kind, wil jy ’n ou vuil bedelaar huis toe neem? Wat gaan jou ma sê? En Heleen en Wiinpie? Ek kan dit eenvoudig net nie doen nie. Moet my asseblief ook nie dwing nie.” smeek hy. Annette kry ’n vasberade trek op haar gesig. Sy kyk Frans Mouton vas in die oë. Annette kry ’n vasberade trek op haar gesig. Sy kyk Frans Mouton vas in die oë. “Jy is my pa. Jy het tien jaar gelede net verdwyn. Hoe dink jy het ons gevoel! Kyk wat het jy aan jouself gedoen. Dink jy nie dis nou tyd om op jou spoor terug te loop nie? Mammie en die ander twee sal jou met ope arms verwelkom.” Frans sien die smeking in sy dogter se gevoelvolle oë. Hy besef skielik dat hy sy gesin werklik die afgelope tien jaar gemis het. Frans neem Annette se hand. “Kom ons gaan, my kind. Ek weet nou waar my lojaliteit lê.” Annette, wat al weer vergeet het waar sy haar bevind, gooi haar arms om die bedelaar se nek en soen sy wang. “Kom, Pa, Mamma se motor staan ’n paar strate verder. En Pa, ek is baie trots op jou.” sê sy. Onbewus van die verbaasde voetgangers, stap die twee hand aan hand die straat af. Annette het heeltemal vergeet van die apteek waama sy op soek was. Die afstand na die huis word in rekordtyd afgelê. Sy is so opgewonde dat sy nie eens wonder oor haar ma se reaksie nie. Wat vir haar saak maak, is die feit dat sy haar pa na al die jare opgespoor het. Rika, Annette se ma, is besig om blomme te pluk toe die motor stilhou. “Kind, wil jy ’n ou vuil bedelaar huis toe neem? Wat gaan jou ma sê? En Heleen en Wiinpie? Ek kan dit eenvoudig net nie doen nie. Moet my asseblief ook nie dwing nie.” smeek hy. “Wat het dan daardie dag gebeur?” Sy kyk verbaas op toe sy sien dat ’n tweede persoon uit die motor klim. Toe sy sien hoe vuil en verflenter die wese daar uitsien, wil sy haar eers vererg. Sy besef dan dat haar dogter ’n baie goeie rede vir die man se teenwoordigheid moet hê. Die lang skraal vrou merk toe eers die stralende uitdrukking op Annette se gesig. Rika loop solank die huis binne en wag die twee in die sitkamer in. Sy skrik merkbaar toe sy die bedelaar van naderby sien. “Frans, is dit jy?” kom dit byna fluisterend van haar. “Ja, Riekie, dis ek. Ek is jammer dat ek jou so laat skrik, maar ons dogter het aangedring dat ek huis toe kom.” Rika staar net na Frans. Skielik bars sy in trane uit. Sy kan regtig nie glo dat sy hom na soveel jare weer sien nie. Hy neem gretig die goedversorgde hand wat sy na hom uithou. Litemtor 17(1) April 1996:229-232 231 ISSN 0258-2279 Fram Mouton se tuiskoms Fram Mouton se tuiskoms “Riekie, ek kan nie glo dat jy nog so pragtig lyk na al die jare nie. Kan my my vergewe vir wat ek julle aangedoen het?” Rika se gesig verhelder in ’n mooi glimlag. “Ek sal jou enigiets vergewe, my man. Jy is ook welkom om weer by ons te kom bly.” Rika se gesig verhelder in ’n mooi glimlag. “Ek sal jou enigiets vergewe, my man. Jy is ook welkom om weer by ons te kom bly.” Intussen het Annette die ander kinders gaan roep. Die vyftienjarige Heleen en die eiQarige Wimpie kyk Frans verwonderd aan. Hulle kan nie glo dat die vuil bedelaar hul pa is nie. Die bekkige Wimpie fluister aan Heleen; Intussen het Annette die ander kinders gaan roep. Die vyftienjarige Heleen en die eiQarige Wimpie kyk Frans verwonderd aan. Hulle kan nie glo dat die vuil bedelaar hul pa is nie. Die bekkige Wimpie fluister aan Heleen; “Die spreekwoord sê mos: Mens kan jou vriende kies, maar beslis nie jou familie nie!” “Die spreekwoord sê mos: Mens kan jou vriende kies, maar beslis nie jou familie nie!” “Ek hoop nie ons vriende sien hom terwyl hy so lyk nie!” fluister Heleen terug. “Ons sal so-iets mos nooit oorleef nie.” As Frans Mouton sy seun en dogter groet, krimp hy ineen. Sy eie kinders is vir hom vreemdelinge! Literator 17(1) April 1996:229-232 “Wat het dan daardie dag gebeur?” Hy sal beslis hard aan hul verhouding moet werk. As Frans Mouton sy seun en dogter groet, krimp hy ineen. Sy eie kinders is vir hom vreemdelinge! Hy sal beslis hard aan hul verhouding moet werk. Nadat Frans gewas is en skoon klere aanhet, lyk dinge aansienlik beter. Almal voel meer op hul gemak. Annette lyk baie in haar skik met haarself. Sy glimlag van oor tot oor en kan nie genoeg na haar pa kyk nie. Tien jaar is darem ’n lang tyd. Rika en Frans sit op die rusbank met hul hande inmekaar gevleg. Daar is baie dinge om uit te pluis. Ook besef hulle dat baie probleme nog sal opduik, maar hulle is nou bereid om dit saam die hoof te bied. Literator 17(1) April 1996:229-232 232 ISSN 0258-2279
https://openalex.org/W2040738271	https://zenodo.org/record/2300812/files/article.pdf	Javanese	null	Museums and the State	Nature	1,920	public-domain	10	© 1920 Nature Publishing Group © 1920 Nature Publishing Group
https://openalex.org/W2626899692	https://nottingham-repository.worktribe.com/preview/856553/WEMDCD2017-Modulated%20Field%20Current%20-%20Final%20Version%20for%20Submission.pdf	English	null	Power quality improvement by pre-computed modulated field current for synchronous generators	null	2,017	cc-by	3,405	Machine drawings and prototype on which this work is based were provided by the Cummins Innovation Centre. The authors are all with the PEMC group of the University of Nottingham, NG7 2RD Power Quality Improvement by Pre-Computed Modulated Field Current for Synchronous Generators Daniel Fallows, Stefano Nuzzo, Alessandro Costabeber, Michael Galea operation. This concept of modifying the typically DC field current, is the basic ‘philosophy’ on which the power quality improvement method described in this paper is built. Abstract – Although power quality aspects of electrical machines have been extensively studied and investigated for a large number of years, room for improvement still exists in the field of classic, wound-field, synchronous generators. This paper proposes an innovative method of power quality improvement for single-phase synchronous generators in which the usual DC field current is replaced by a calculated current waveform. The optimised field current waveform is designed in such a way that harmonics created by the machine geometry and the winding configuration are significantly reduced. A number of variants of the basic method mentioned above, where power quality improvements are addressed by generating ‘extra’ harmonics, have been proposed before. In [6], [7] harmonics are introduced onto the field current to generate related harmonics on the machine output that compensate the distribution system. In [8] the field reconstruction method (FRM) is used to calculate1an optimised field current waveform to reduce the total harmonic distortion (THD) of the output voltage of a small three-phase machine. A FE model of the SG under investigation is used to validate a FRM model which is then used to prove the concept. Although significant improvement was achieved, the method showed limitations due to the interaction of the three phases. Index Terms – Magnetic field modulation, power generation, power quality, synchronous generator I. INTRODUCTION S ynchronous generators (SGs), of the wound-field type, are one of the most common machines used for power generation across the power range from a few kVA to many MVA. Power quality is a key performance characteristic and although these machines represent a well-known and consolidated technology, recent development in adjacent technologies such as power electronics, and modelling and analysis tools, has renewed research interest in their design and improvement. S The above methods show positive results but fail to address the implementation of such techniques to single-phase machines. This paper will therefore focus on the improvement that can be achieved to the power quality of commercial single-phase SGs. As a vessel to do this, a modified SG optimised for maximum power output (at the cost of lower power quality figures) is used. In order to improve the generator output, various methodologies exist and have been investigated, including optimisation and modifications to the stator winding configuration, relative to the ‘classical’ layout [1-3], improvements to the lamination geometry [2] and the implementation of more disruptive techniques (such as skewing of the core packs or modulated damper cage) [1-4]. While these have all been shown to achieve significant improvements in terms of power quality, typically these design choices have a ‘negative’ effect in terms of reduced total output and of course more complex manufacturing requirements [1], [3]. A very important aspect to note, especially considering the ever-increasing stringent requirements in this area, is the fact that reduced power quality also affects the efficiency of SGs [5]. B. Voltage waveform at rated operating point B. Voltage waveform at rated operating point The general voltage waveform of the prototype machine model was captured, when operating at the DC field current so at to produce the rated voltage, as shown in Fig. 3. The harmonic components of this waveform, can be observed in Fig. 4, which shows that the 5th, 7th and slot (around 1.2 kHz) harmonics are significant, due to the combination of the full pitch layout, and the absence of skewing and damper cage effects. Fig. 3 - Output waveform at rated field current. Fig. 1 - FE model of studied machine. In order to highlight and emphasize the potential improvements possible by optimising the field current waveform, a downgrade of the above machine was implemented. The modified machine, henceforth referred to as the prototype machine, was developed with a full pitch winding, with no skewing of the core packs and with no rotor damper bars. A 2D model of the prototype machine was also built. The geometry and cross-sectional scheme of the modified machine are identical to that of the prototype machine as the only differences are those given above. Fig. 3 - Output waveform at rated field current. Fig. 3 - Output waveform at rated field current. Fig. 3 - Output waveform at rated field current. The two models (production and prototype machines) were then used to investigate the proposed methodologies as presented below. Fig. 4 – Amplitude harmonic spectrum of the no-load output voltage at rated field current. A. Open-circuit characteristic The FE model of the production machine was verified against experimental test data by open-circuit characterisation testing. Fig. 2 compares the open circuit characteristics of the production machine from both FE and experimental testing. Excellent similarity (between predicted and measured values) can be observed. The FE model of the production machine was modified to the specification of the prototype machine (full pitch winding, etc) and the open-circuit characterisation test carried out. From Fig. 2, the increased output of the prototype machine can also be observed. This is the result of the configuration changes described above (full pitch winding, etc). In particular the rated operating voltage (1 p.u.) was achieved with 13% less field current. Fig. 4 – Amplitude harmonic spectrum of the no-load output voltage at rated field current. II. FE MODEL VERIFICATION AND SIMULATION RESULTS A commercial 48.5kVA SG, whose main specifications are given in Table 1, is studied in this paper and used as a platform to prove the concept. The machine, henceforth referred to as the production machine, is produced with a two-thirds short pitch winding, a stator skew of one slot pitch and a damper winding characterised by four bars-per-pole. A detailed 2D model of the production machine was built, shown in Fig. 1, and verified against test data. Skewing was achieved through the use of the multislice method and the damper bars were modelled as solid conductors to account for skin effect. The verification results are shown in Fig. 2. TABLE 1 PARAMETERS OF STUDIED MACHINE Parameter Value Rated Power 48.5kVA Rated Voltage 240V Rated Frequency 50Hz Rotor Type Salient Pole Number of Poles 4 In SGs, the harmonic distortion of the output voltage is a result of non-sinusoidal flux linkage interaction with the armature coils, mainly caused by the inevitable limitations in the machine geometry. For a given machine it is well known that the flux linkage is a function of the rotor position, field flux and armature winding configuration. Of these, only the field flux can, to a certain extent, be actively controlled and adjusted (through the field current) while the machine is in TABLE 1 Stator Slots 48 Stator Outer Diameter 340mm Stack Length 240mm Fig. 1 - FE model of studied machine. Fig. 2 - Open circuit characteristic comparison. Fig. 2 - Open circuit characteristic comparison. 2) Creation of Improved Current Waveform The flux linkage values were calculated to give a sinusoidal output of 240V at 50Hz. These values were then input into the function to calculate the field current required at each angle of rotor rotation. Fig. 5 shows the field current waveform that was produced along with the nominal field current for the same operating point. ߣ= ݂(ܫ௙, ߠ) (1) (1) For a given machine it is possible to create a two- dimensional matrix that gives the instantaneous field current required to produce a specific flux linkage at a particular rotor position. Fig. 5 - Modulated field current waveform. In Fig. 5, it can be seen that there is a spike in the field current at each of the flux linkage zero crossing points due to the interpolation function assumption discussed in the previous section. This could be removed with further post- processing of the field current waveform. Fig. 6 shows the frequency components of the field current waveform. Apart from the DC component, the dominant peaks can be seen at every even harmonic decaying in steps as the frequency increases. Although the components are significant they represent less than 0.5% of the DC amplitude. Fig. 5 - Modulated field current waveform. The matrix can then be used as a lookup table to find the field current values for any chosen flux linkage waveform (and by extension voltage waveform). The resulting current waveform can be input to the field causing the machine to produce the chosen voltage waveform. However, when applied to a real, practical machine there will be limitations to this method, including:  The inductance of the field winding preventing large, rapid changes in current;  Slip-rings or a rotor mounted power electronic converter required to supply the excitation; Fig. 5 - Modulated field current waveform.  The damper cage action smoothing changes in the field flux thereby cancelling the effects of the modulated field current. In Fig. 5, it can be seen that there is a spike in the field current at each of the flux linkage zero crossing points due to the interpolation function assumption discussed in the previous section. This could be removed with further post- processing of the field current waveform. This paper represents work focused on proving the concept described rather than addressing the real-world implementation. 2) Creation of Improved Current Waveform Some discussion will be made in response to this but fully addressing these challenges will be left to future work. Fig. 6 shows the frequency components of the field current waveform. Apart from the DC component, the dominant peaks can be seen at every even harmonic decaying in steps as the frequency increases. Although the components are significant they represent less than 0.5% of the DC amplitude. C. Considerations on the preliminary investigation The results shown in Fig. 2 indicate and verify the accuracy of the developed FE models for the production machine. Considering that the model of the prototype machine is based and built on that of the production machine, then it can be safely assumed that the model of the prototype machine is also current value between flux linkage values for a given rotor position. (partially) validated. In Fig. 3 it can be observed that the prototype machine, as expected, achieves a significantly higher output for a given field current at the expense of power quality. The aim of this paper is therefore to achieve the same (or improved) power quality as the production machine whilst maintaining the increased output benefits. To populate the matrix a suite of transient simulations with motion representing the prototype machine operating at no- load condition have been carried out. The field current has been varied from 0A to 12A, in steps of 0.5A, while the time step has been set such that each step was equal to one third a degree of rotation. These procedures were done with the commercial package ANSYS Maxwell and the results post- processed with MATLAB. 1) Matrix Search and Interpolation Function A function was created that loops through the flux linkage values for a given rotor position and locates the closest points to a given flux linkage value. The assumption is made that the flux linkage increases with field current, which is always true, except for when the flux linkage is close to zero.  The instantaneous flux linkage values for any given voltage waveform can be calculated for each point of rotation of a machine. The function carries out a linear interpolation between the points to return the required field current. A linear interpolation is not a true match but is acceptable given the density of the field current simulations (every 0.5A).  The instantaneous flux linkage is a product of the current flowing in the field windings, the rotor position and the number of turns on both the armature and field windings. The number of turns can be considered constants, thus the field current and the rotor position, can be considered as inputs for the expression given in (1). A. Concept It is well known that the voltage produced by a machine is equal to the rate-of-change of flux linkage, as governed by Faraday’s law. Therefore, in order to achieve a sinusoidal voltage output, the flux must also be sinusoidal. The following considerations are also very important to note: C. Simulation Results of Improved Field Waveform In order to show the validity of the proposed method, the waveform shown in Fig. 5 was applied to the FE model of the prototype machine as a piecewise linear current source. The resulting output voltage waveform is shown in Fig. 7 with the original waveform for comparison. Fig. 8 shows a comparison between the harmonic contents of the two waveforms. Fig. 9 - Field voltage required to produce the modulated current waveform. An excellent improvement in terms of voltage waveform quality can be observed, particularly in the frequency components identified in Section II. A comparison in terms of THD is given in Table 2 showing that the modulated field prototype had a marked improvement over the production machine, which was the main objective of this work. Fig. 7 - Comparison of output waveform with and without field modulation. Fig. 9 - Field voltage required to produce the modulated current waveform. D. Performance of Field Modulation Under load The required modulated field current waveform will change when the machine is placed on load. To maintain rated voltage the field current will have to increase which will push the iron into a different region of the saturation curve. Similarly the current flow in the armature winding will modify the air-gap flux thereby changing the harmonic content of the output voltage. Fig. 7 - Comparison of output waveform with and without field modulation. Fig. 7 - Comparison of output waveform with and without field modulation. An in-depth study of field modulation as applied to a loaded machine is beyond the scope of this paper but will be investigated in future works. Fig. 8 - Comparison of output frequency content with and without field modulation. B. Production of Field Current Matrix FE analysis was used to create the lookup matrix described in the previous section, as a matrix of flux linkage for each rotor position and field current. The use of FE analysis ensures that non-linear effects such as saturation and eddy currents are also taken into account. Interpolation functions have been developed and implemented in MATLAB to estimate the field Fig. 6 - Frequency components of the modulated field current. TABLE 2 THD VALUES FOR EACH MACHINE Machine THD (%) Production (short pitch winding and skewed stator) 1.46 Prototype (using DC field current) 3.90 Prototype (using modulated field current) 0.828 Although the method showed significant room for improving the power quality of the 48.5kVA SG considered in this paper, a limitation of this method is the high field voltage required to produce a modulated field current. Fig. 9 shows a plot of the field voltage and confirms the levels required. The spikes present in the calculated field current waveform are behind the most significant voltage spikes. With further post- processing of this waveform it should be possible to limit the voltage requirements to 60V peak (the level at which the majority of the waveform already achieves). Fig. 6 - Frequency components of the modulated field current. IV. REFERENCES [1] Kostenko, M. and Piotrovsky, L., Electrical Machines. Vols. 1 and 2. Moscow: Mir, 1974. [2] I. Boldea, Synchronous generators: The electric generators handbook. Boca Raton, FL: Taylor and Francis (CRC Press), 2005. [2] I. Boldea, Synchronous generators: The electric generato Stefano Nuzzo received his B.Sc. and M.Sc. degrees in Electrical Engineering from the University of Pisa, Pisa, Italy, in 2011 and 2014, respectively. He is currently a Ph.D. student with the Power Electronics, Machines and Control Group, The University of Nottingham, Nottingham, U.K. He spent six months with The University of Nottingham as a Visiting Student in 2013, where he was involved in developing analytical and numerical models for the analysis of permanent magnet synchronous machines. His current research interests include the analysis and design of salient-pole synchronous generators and brushless excitation systems. [3] J. Pyrhonen, T. Jokinen, V. Hrabovcova, and H. Niemel, Design of rotating electrical machines. Chichester, United Kingdom: Wiley, John & Sons, 2008. [4] S. Nuzzo, M. Degano, M. Galea, C. Gerada, D. Gerada and N. Brown, "Improved Damper Cage Design for Salient-Pole Synchronous Generators," in IEEE Transactions on Industrial Electronics, vol. 64, no. 3, pp. 1958-1970, March 2017. [5] C. Debruyne, J. Desmet, J. Rens and J. De Kock, "The effect of a reduced power quality on the energy efficiency of stand-alone generator systems," 2015 IEEE International Electric Machines & Drives Conference (IEMDC), Coeur d'Alene, ID, 2015, pp. 1902-1909. Alessandro Costabeber received the M.S. degree with honours in electronic engineering from the University of Padova, Padova, Italy, in 2008 and the Ph.D. degree from the same university in 2012, on energy efficient architectures and control techniques for residential microgrids. In 2014 he joined the PEMC group, Department of Electrical and Electronic Engineering, University of Nottingham, Nottingham, UK as Lecturer in Power Electronics. His current research interests include HVDC converters topologies, high power density converters for aerospace applications, control solutions and stability analysis of AC and DC microgrids, control and modelling of power converters, power electronics and control for distributed and renewable energy sources. Dr. Costabeber received the IEEE Joseph John Suozzi INTELEC Fellowship Award in Power Electronics in 2011. [6] M. T. Abolhassani, H. A. Toliyat and P. Enjeti, "An electromechanical active harmonic filter," IEMDC 2001. IEEE International Electric Machines and Drives Conference (Cat. No.01EX485), Cambridge, MA, 2001, pp. 349-355. [7] M. T. Abolhassani, H. A. Toliyat and P. E. Conclusions and Next Steps This paper has demonstrated a simple and effective method to improve power quality in single-phase SGs. Use of this method in machines reduces manufacturing cost by easing the design and increases power density though the use of full pitched windings. The next step will be to verify the simulation results against a real machine under test. This will be achieved with a slip-ring excited machine and programmable signal generator. A future paper will detail the development of a rotating converter to replace the standard diode rectifier and provide the field modulation capability. Fig. 8 - Comparison of output frequency content with and without field modulation. Once verified at test the implementation through the use of a rotating power converter on a brushless exciter machine will be investigated. Generator Technologies where he was involved with engineering software development and synchronous machine analysis. His special fields of interest include synchronous generator excitation systems and embedded computing. Generator Technologies where he was involved with engineering software development and synchronous machine analysis. His special fields of interest include synchronous generator excitation systems and embedded computing. IV. REFERENCES Enjeti, "Harmonic compensation using advanced electric machines," Industrial Electronics Society, 2001. IECON '01. The 27th Annual Conference of the IEEE, Denver, CO, 2001, pp. 1388-1393 vol.2. Available: http://home.process.com/ Intranets/wp2.htp Available: http://home.process.com/ Intranets/wp2.htp [8] M. L. M. Kimpara, J. O. P. Pinto, B. Fahimi, P. E. M. J. Ribeiro, R. B. Godoy and L. E. B. Silva, "Field reconstruction method applied for harmonic voltage mitigation in salient pole synchronous generators," 2013 Brazilian Power Electronics Conference, Gramado, 2013, pp. 890- 895. Michael Galea received his PhD in electrical machines design from the University of Nottingham, UK, where he has also worked as a Research Fellow. He is currently a Lecturer in Electrical Machines and Drives within the PEMC research group of the University of Nottingham. He is the Deputy Director of the Institute for Aerospace Technology at the University of Nottingham, where he is also a Lecturer in Aerospace Systems Integration and where he manages a number of diverse projects related to the more / all electric aircraft and associated fields. His main research interests are design, analysis and thermal management of electrical machines and drives and the more electric aircraft. V. BIOGRAPHIES V. BIOGRAPHIES Daniel Fallows graduated in 2015 with a Masters in Engineering from the University of Nottingham, UK, and was awarded the IET Prize 2015. Currently he is a PhD student within the PEMC group at the University of Nottingham. His employment experience includes working at Cummins
https://openalex.org/W4382985050	https://link.springer.com/content/pdf/10.1007/s10592-023-01540-3.pdf	English	null	Genomics of founders for conservation breeding: the Jasper caribou case	Conservation genetics	2,023	cc-by	12,316	Conservation Genetics (2023) 24:855–867 https://doi.org/10.1007/s10592-023-01540-3 Conservation Genetics (2023) 24:855–867 https://doi.org/10.1007/s10592-023-01540-3 RESEARCH ARTICLE Abstract Conservation breeding programs are increasingly used as recovery actions for wild animals; bringing founders into captivity to rear captive populations for future reintroduction into the wild. The International Union for the Conservation of Nature recommends that founders should come from genetically close populations and should have sufficient genetic diversity to avoid mating among relatives. Genomic data are highly informative for evaluating founders due to their high resolution and ability to capture adaptive divergence, yet, their application in that context remains limited. Woodland caribou are federally listed as a Species at Risk in Canada, with several populations facing extirpation, such as those in the Rocky Mountains of Alberta and British Columbia (BC). To prevent local extirpation, Jasper National Park (JNP) is proposing a conservation breeding program. We examined single nucleotide polymorphisms for 144 caribou from 11 populations encompassing a 200,0002 km area surrounding JNP to provide information useful for identifying appropriate founders for this program. We found that this area likely hosts a caribou metapopulation historically characterized by high levels of gene flow, which indicates that multiple sources of founders would be appropriate for initiating a breeding program. However, population structure and adaptive divergence analyses indicate that JNP caribou are closest to populations in the BC Columbia range, which also have suitable genetic diversity for conservation breeding. We suggest that collaboration among jurisdictions would be beneficial to implement the program to promote recovery of JNP caribou and possibly other caribou populations in the surrounding area, which is strategically at the periphery of the distribution of this endangered species. 6 Ministry of Land, Water and Resource Stewardship Northeast Region, 400‑10003‑110Th Avenue, Fort St. Genomics of founders for conservation breeding: the Jasper caribou case Maria Cavedon1 · Lalenia Neufeld2 · Laura Finnegan3 · Dave Hervieux4 · Anita Michalak5 · Agnes Pelletier6 · Jean Polfus7 · Helen Schwantje8 · Geoff Skinner2 · Robin Steenweg7 · Caeley Thacker8 · Jocelyn Poissant5 · Marco Musiani9 Received: 5 December 2022 / Accepted: 7 June 2023 / Published online: 3 July 2023 © The Author(s) 2023 words Caribou · Genomics · Conservation breeding · Founders · National Parks · Recovery actions * Marco Musiani marco.musiani@unibo.it 9 Dipartimento Scienze Biologiche Geologiche Ambientali, Università Di Bologna, Via Zamboni, 33 ‑ 40126 Bologna, Italia 5 Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada Introduction As the rate of biodiversity loss caused by anthropogenic activities dramatically increases, numerous species and populations are now extirpated or face the threat of extir‑ pation (Pimm et al. 2014). Consequently, recovery actions like conservation breeding programs are sometimes used to reintroduce species to areas where they used to occur, or to augment populations (Seddon et al. 2007; Brichieri- Colombi and Moehrenschlager 2016; Bubac et al. 2019). Conservation breeding programs encompass the act of bringing rare or endangered animals into captivity with the aim of rearing captive populations for eventual rein‑ troduction of their progeny into the wild (Seddon et al. 2014). Examples of such programs for ungulates include those conducted for the European bison (Bison bonasus) in Poland (Tokarska et al. 2009), the Przewalski’s horse in Mongolia and China (Der Sarkissian et al. 2015), the American bison (Bison bison) in Oklahoma (Kleiman 1989), USA, the Arabian oryx (Oryx leucoryx) in Arabia (Arif et al. 2010), and the Pére David's deer (Elaphurus davidianu) in China (Dayuan et al. 2022). f Until recently, genetic studies aimed at identifying found‑ ers for conservation breeding programs have been based on small sets of putatively neutral molecular markers (i.e., regions of the genome that are non-coding) such as micros‑ atellites and/or mitochondrial DNA (mtDNA) (Russello and Jensen 2018). However, these markers have known limita‑ tions, including limited resolution and ability to capture adaptive differentiation. Single nucleotide polymorphisms (SNPs), have the potential to overcome such limitations and be more informative for the selection of founders (Allendorf et al. 2010; He et al. 2016). The higher resolution of SNPs can provide more precise estimates of genetic parameters (e.g., genetic diversity and inbreeding) of candidate found‑ ers when compared to microsatellites and mtDNA (Luikart et al. 2003; Ivy et al. 2009; Galla et al. 2020). In addition, as SNPs may be located in genes, they can be used to identify specific loci of interest in founders that might be desirable to maintain (e.g., loci under selection) in the captive and reintroduced populations (Laikre 1999; Luikart et al. 2003; Weeks et al. 2011; Flanagan et al. 2018; Wright et al. 2021). Despite multiple examples of successful conservation breeding programs, their execution is challenging (Fischer and Lindenmayer 2000; Armstrong and Seddon 2008). To improve their success, guidelines and best practices are continuously improved (Frankham 2010). Introduction For example, the International Union for the Conservation of Nature (IUCN) guidelines indicate that reintroductions and other conservation translocations should be based on plans that evaluate economic, social, and ecological aspects, such as a species’ current and historical distribution, and the removal or substantial reduction, of extinction threat(s) (IUCN/SSC 2013). The IUCN guidelines emphasize that selection of appropriate founders is a critical factor affect‑ ing reintroduction success (IUCN/SSC 2013; Forsman 2014). Ideally, founders should have appropriate demo‑ graphic characteristics (e.g., number, sex, and age), come from geographically close populations, and have character‑ istics (i.e., ecology, behaviour, etc.) similar to the popula‑ tions being restored (Robert 2009; Soorae 2018). Caribou (Rangifer tarandus), also known as reindeer, are a Holarctic species that is globally declining, and listed as a Species at Risk in Canada (Environment Canada 2014). Woodland caribou populations located at the southern edge of the species’ distribution, such as those in the study area in the Rocky Mountains of Alberta (AB) and British Colum‑ bia (BC) (Fig. 1), are particularly at risk of disappearing, also due to climate change (Vors and Boyce 2009). These caribou populations belong to one subspecies (Woodland Caribou) and two Designatable Units (DU; Central and Southern Mountain), which largely overlap in meaning with Evolutionary Significant Units (ESUs; Crandall et al. 2000; Green 2005; COSEWIC 2011). DUs are further divided into subpopulations, also known as “herds” (although herds of Woodland Caribou are not social groups; Bergerud 1988), delineated for management and conservation purposes (Environment Canada 2014). Considering the genetics of founders is crucial for the success of conservation breeding programs (Araki et al. 2007; Witzenberger and Hochkirch 2011). From a genetic perspective, the best source populations are those that are genetically similar to the populations that had been extirpated and have sufficient genetic diversity to reduce mating among relatives of captive individuals (Frankham 2010; Pelletier et al. 2009; Miller et al. 2010). However, recent work has also indicated that genetic diversity is important and matching genetic groups may be overstated if there is not a biological difference between the groups, In the southern Rocky Mountains of Canada, caribou herds occur on public lands available for multiple land uses including resource extraction, and in provincially and federally protected areas, however even in protected areas some have been extir‑ pated and others are declining (Environment Canada 2014; Serrouya et al. 2019). Abstract John, BC V1J 6M7, Canada * Marco Musiani marco.musiani@unibo.it 1 Deparment of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada 2 Jasper National Park of Canada, Parks Canada, Jasper, Canada 3 fRI Research, 1176 Switzer Drive, Hinton, AB T7V 1V3, Canada 4 Fish and Wildlife Stewardship Branch, Alberta Environment and Protected Areas, Grande Prairie, AB T8V 6J4, Canada 5 Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada 1 Deparment of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada 7 Canadian Wildlife Service – Pacific Region, Environment and Climate Change Canada, 1238 Discovery Ave, Kelowna, BC V1V 1V9, Canada 2 Jasper National Park of Canada, Parks Canada, Jasper, Canada 8 Wildlife and Habitat Branch, Ministry of Forests, Lands, Natural Resource Operations and Rural Development, Government of British Columbia, 2080 Labieux Road, Nanaimo, BC V9T 6J 9, Canada 8 Wildlife and Habitat Branch, Ministry of Forests, Lands, Natural Resource Operations and Rural Development, Government of British Columbia, 2080 Labieux Road, Nanaimo, BC V9T 6J 9, Canada 3 fRI Research, 1176 Switzer Drive, Hinton, AB T7V 1V3, Canada 4 Fish and Wildlife Stewardship Branch, Alberta Environment and Protected Areas, Grande Prairie, AB T8V 6J4, Canada 9 Dipartimento Scienze Biologiche Geologiche Ambientali, Università Di Bologna, Via Zamboni, 33 ‑ 40126 Bologna, Italia :(0123 1 3456789) 3 Conservation Genetics (2023) 24:855–867 856 i.e. (Frankham et al. 2017; Ralls et al. 2020). Accounting for proper genetic characteristics of founders can increase the survival probability of released individuals and improve the chance of having healthy and self-sustaining reintroduced populations in the long term (Robert 2009; Williams and Hoffman 2009). Introduction In the mountain national parks, the last 9 caribou were extirpated from Banff in 2009 (Hebblewhite et al. 2010), and caribou now only occur in Revelstoke and Jas‑ per (JNP) national parks. Caribou in JNP are either extirpated or facing near-extirpation, a status that calls for conservation 1 3 3 Conservation Genetics (2023) 24:855–867 857 Fig. 1 Caribou sampled in the southern Rocky Mountains of Canada for genomic analyses (n = 137; sampling locations in Fig. 3). Black lettered circles indicate sampled herds (i.e., a term used to indicate subpopulations, although herds of Woodland caribou are not social groups) with circle size proportional to sample size (mean = 12.45, SD = 7.03, range 2–20). Grey-scale polygons show the distribution of Designatable Units (DUs) encompassing multiple herds b). Following IUCN guidelines, JNP is taking actions to aid recovery by exploring conservation breeding of JNP caribou. As all caribou herds within JNP are either extirpated, near extirpated or declining and as the habitat for caribou has been considered either favourable or improving, conservation breed‑ ing was considered a viable option by conservation planners and stakeholders (Foundations of Success and Parks Canada 2021; Salafsky et al. 2022). This exploration includes assess‑ ment of genetic information to identify options for selection of founder animals (Parks Canada Agency 2022a, b). Caribou genetics studies conducted in the southern Rocky Mountains of AB and BC have primarily used neu‑ tral molecular markers (i.e., mitochondrial DNA haplotypes and autosomal microsatellites) (McDevitt et al. 2009; Ser‑ rouya et al. 2012; Weckworth et al. 2012 Yannic et al. 2014), although more recent work has used genomic data (Cave‑ don et al. 2019; Taylor et al. 2022). MtDNA indicated the presence of a hybrid zone where sympatric individuals have either Beringian/Northern or Southern mitochondrial line‑ age (McDevitt et al. 2009; Yannic et al. 2014). Studies based on microsatellites and SNPs also indicated the presence of shared genetic traits (McDevitt et al. 2009; Serrouya et al. 2012; Weckworth et al. 2012; Cavedon et al. 2019; Taylor et al. 2022). For example, population structure studies indi‑ cated that JNP caribou had genetic characteristics of both Central and Southern Mountain DU herds but that it was not possible to ascertain which characteristics predominated (McDevitt et al. 2009; Serrouya et al. 2012; Weckworth et al. 2012). Introduction However, past studies did not survey an adequate number of herds in the immediate JNP area to clarify local genetic structure and inform founder selection. Fig. 1 Caribou sampled in the southern Rocky Mountains of Canada for genomic analyses (n = 137; sampling locations in Fig. 3). Black lettered circles indicate sampled herds (i.e., a term used to indicate subpopulations, although herds of Woodland caribou are not social groups) with circle size proportional to sample size (mean = 12.45, SD = 7.03, range 2–20). Grey-scale polygons show the distribution of Designatable Units (DUs) encompassing multiple herds We conducted a genomic study to inform JNP’s planned conservation breeding program. We characterised genomic diversity in 144 individuals from 11 herds in a 200,000 km2 area using a newly developed caribou SNP array (Carrier et al. 2022), and sampling equally from the Central and Southern Mountain DUs. Our aims were to: (i) define levels of genetic differentiation, (ii) evaluate population structure, and (iii) detect signatures of adaptive divergence between population groups determined with population structure analyses. The goal of our study was to identify suitable founder populations for the establishment of a JNP conserva‑ tion breeding program, and to interpret the findings in view of genetic and ecological traits of caribou in the southern Rocky Mountains of AB and BC. actions (Parks Canada Agency 2022a, b). Caribou declines in JNP are due to multiple factors. Decades ago the JNP caribou lost migratory access to traditional forested foothills outside of the national park, due to high levels of habitat change and loss from industrial land uses. Loss of annual migration has been demonstrated to compromise the population viability of central mountain caribou (Williams et al. 2021). In addition, caribou remaining within JNP have been negatively affected by apparent competition with elk (Holt 1977) mediated by wolves (Bradley and Neufeld 2012). A rapid increase of elk density, which occurred after their reintroduction in 1960, resulted in higher wolf densities and an increase in predation rate on elk and caribou. Recently, lower wolf and elk densi‑ ties have resulted in more favourable ecological conditions for caribou, and potential for recovery (Parks Canada Agency 2022a, b). Despite these current more favourable conditions, caribou within JNP continue to struggle with low survival rates, and the population is now too small to recover with‑ out population augmentation (Parks Canada Agency 2022a, actions (Parks Canada Agency 2022a, b). Introduction Caribou declines in JNP are due to multiple factors. Decades ago the JNP caribou lost migratory access to traditional forested foothills outside of the national park, due to high levels of habitat change and loss from industrial land uses. Loss of annual migration has been demonstrated to compromise the population viability of central mountain caribou (Williams et al. 2021). In addition, caribou remaining within JNP have been negatively affected by apparent competition with elk (Holt 1977) mediated by wolves (Bradley and Neufeld 2012). A rapid increase of elk density, which occurred after their reintroduction in 1960, resulted in higher wolf densities and an increase in predation rate on elk and caribou. Recently, lower wolf and elk densi‑ ties have resulted in more favourable ecological conditions for caribou, and potential for recovery (Parks Canada Agency 2022a, b). Despite these current more favourable conditions, caribou within JNP continue to struggle with low survival rates, and the population is now too small to recover with‑ out population augmentation (Parks Canada Agency 2022a, Assessing and comparing diversity of caribou caribou monitoring activities between 2012 and 2020, corresponding to ≥ one generation for caribou (following McLoughlin et al. 2003).The collected samples spanned a 200,000 km2 study area located in the mountainous region along the southern AB and BC border in proximity to JNP, Canada (Fig. 1). Caribou in this area belong to either the Central Mountain DU or the Southern Moun‑ tain DU (COSEWIC 2011) and are from the montane cordillera ecozone, which includes habitats ranging from alpine tundra, to dense coniferous forests, to dry sagebrush and grasslands (Ecological Stratification Working Group 1995). We used the R package DARTR (Gruber et al. 2018) to esti‑ mate observed (Ho) and expected heterozygosity (He) and inbreeding coefficients (FIS, with 95% Confidence Intervals (CI) of bootstrap values) for each herd as well as pairwise fixation index values (FST) between herds. We then used FST values to derive the number of migrants per generation (Nm; i.e., an estimate of gene flow; Wright 1931). The applied metrics of FST are sensitive to a species’ heterozygosity, and FST as well as the population STRUCTURE analyses (below) also to sample sizes. Due to low sample size, we therefore combined individuals from the Banff herd (n = 3) with those from JNP (n = 15). We also combined individ‑ uals from the Purcell South herd (n = 4) with those from South Selkirk herd (n = 3). The pooling of these samples was warranted due to known genetic similarities (McDevitt et al. 2009; Serrouya et al. 2012; Weckworth et al. 2012) and geographic proximity. We estimated a kinship matrix between caribou individuals using the R package POPKIN (Ochoa and Storey 2021), and from this, we also obtained the inbreeding coefficients for each caribou. To avoid a bias and potential underestimation of kinship and inbreeding, we used a SNP dataset including all caribou individuals (i.e., also including relatives), and where SNPs were filtered by LD and HWE as is standard practice. ) We extracted DNA from samples using the Qiagen kits following manufacturer protocols for both single spin col‑ umns (DNeasy Blood & Tissue Kit) and 96-well plates (QIAamp 96 DNA QIAcube HT kit). DNA samples were then quantified using either the BioTek Synergy LX Mul‑ timode Reader or the Thermo Fisher Qubit 3 Fluorometer following Thermo Fisher with Qubit and Quant-iT 1X dsDNA high sensitivity (HS) or broad range (BR) Kits. Sampling and genomic data Blood and tissue samples were collected from both live and deceased animals as part of government agencies’ 1 3 858 Conservation Genetics (2023) 24:855–867 Assessing and comparing diversity of caribou One hundred and forty-four samples that yielded ≥ 400 ng of DNA were selected for analyses. Samples were normal‑ ized to a quantity of 400 ng, dried on a Thermo Scientific Savant SpeedVac DNA 130 Integrated Vacuum Concentra‑ tor System, and shipped at room temperature to Genome Québec Ltd (Montréal, Québec) where genotyping using the Illumina Caribou 60 K SNP array (Carrier et al. 2022) was outsourced. The array accounts for SNPs evenly dis‑ tributed across the entire genome (~ every 50 Kb) with known minor alleles across populations world-wide. In addition, a subset of SNPs was selected to represent rare and local alleles which could be used for ecotype and population assignments—information urgently needed for conservation planning. Population structure analyses (2005) was calculated using STRUCTURE HARVESTER to help determine the 1 3 3 Conservation Genetics (2023) 24:855–867 859 to 0.39 (mean 0.37 + SD 0.01). Inbreeding coefficients (FIS) ranged from 0.003 to 0.047 (mean 0.019 + SD 0.015). Pair‑ wise fixation index (FST) between herds ranged from 0.01 to 0.09 (mean 0.06 + SD 0.02). FST values between JNP and the other herds varied from 0.06 to 0.09 (mean 0.07 + SD 0.04; Table 2). The estimated number of migrants per gen‑ eration (Nm values) between herds assessed pairwise were all > 1 (mean 5.38 + SD 4.10). The estimated Kinship matrix indicated moderate relatedness among caribou individuals within the same herd (Fig. 2), with the exception of cari‑ bou within the Hart South and North Caribou herds. The matrix indicated lower relatedness among caribou individu‑ als belonging to different herds. The inbreeding coefficients for caribou individuals calculated from the kinship matrix followed a gaussian distribution (Fig. S1). most appropriate number of genetic clusters (Earl and von Holdt 2012). For comparison, we also assessed population structure with a maximum likelihood approach implemented in ADMIXTURE v1.3 and the most appropriate number of genetic clusters was detected by examining the cross-vali‑ dation errors for K varying from 1 to 10 (Alexander et al. 2009). Lastly, as is common in genetic studies examining population structure (see for example Cavedon et al. 2022a, b, c), we performed a principal component analysis (PCA) to determine population groups using the program SMART‑ PCA within EIGENSTRAT v3.0 (Price et al. 2006). Population structure analyses To assess isolation by distance (IBD), we performed a Man‑ tel test to detect the potential correlation between the geo‑ graphic and genetic distances calculated between individu‑ als pairwise. We calculated the geographic distances with the function “distGeo” within the R package GEOSPHERE (Karney 2013), whereas we calculated the genetic distances with the function “gl.dist.ind” within the R package DARTR (Gruber et al. 2018). Lastly, we performed the mantel test with the function “gl.ibd” also within the dartR package. We used PLINK v1.9 (Purcell et al. 2007) to perform data quality control, which included filtering out individu‑ als and SNPs with call rates < 0.98 and SNPs with a minor allele frequency (MAF) < 0.01. After filtering there were 44,112 SNPs remaining and 137 individuals from both the Central Mountain DU (nherds = 6, individuals = 77) and Southern Mountain DU (nherds = 5, individuals = 60) (Fig. 1). For population structure analyses (below), we used PLINK to further exclude SNPs exhibiting strong linkage dis‑ equilibrium (“–indep-pairwise 50 5 0.5”) and those not in Hardy–Weinberg equilibrium (“–hwe 0.001”), leaving 36,053 SNPs for each individual (Purcell et al. 2007). In addition, for population structure analyses, we removed 42 animals which had a close relative in the dataset (28 and 14 belonging to the Central Mountain DU and South‑ ern Mountain DUs, respectively) based on an identity by descent (IBD; –genome) degree of recent shared ancestry threshold of 0.25 (second-degree relatives) (see for exam‑ ple Kominakis et al. 2021). To visualize patterns of population structure, we calcu‑ lated pairwise Nei’s genetic distances (Nei 1972) between all individuals using the R package StAMPP v1.6.1 (Pemb‑ leton et al. 2013) and constructed a neighbour-joining tree based on the genetic distances using the R package APE 5.2 (Paradis et al. 2004). We also evaluated population structure using STRUCTURE v2.3.4, which uses a Bayesian iterative algorithm to place samples into clusters (K) whose mem‑ bers share similar patterns of genetic variation (Pritchard et al. 2000; Falush et al. 2003). We ran STRUCTURE with 20,000 burn-in iterations followed by 50,000 sampling itera‑ tions for K = 1 through 10 (Schweizer et al. 2016; Cave‑ don et al. 2022a, b, c). Each run was performed 10 times, and the ΔK statistic of Evanno et al. Population structure of caribou individuals We found a significant correlation between geographic dis‑ tance and genetic distance, across the 137-individual SNP data set (r = 0.499; Mantel test P = 0.001; Fig. S2). JNP and Banff individuals grouped together in the neighbor-joining tree and were most similar to caribou from Columbia North, Purcell South, and South Selkirk. JNP and Banff individuals then grouped together with caribou from À La Pêche, and then caribou from Redrock-Prairie Creek (a group that was admixed with some Hart South individuals) (Fig. 3). The remaining groups (Hart South, North Cariboo, Quintette, and Moberly) were admixed and branched together sepa‑ rately from JNP and Banff (Fig. 3). f STRUCTURE and ADMIXTURE analyses both sup‑ ported the presence of two genetic clusters (K = 2, Fig. 4). Results also indicated that JNP and Banff individuals had similar assignment to individuals belonging to the Columbia North, Purcell South, and South Selkirk populations, but were different from individuals from À La Pêche, Redrock- Prairie Creek, Quintette, Moberly, Hart South, and North Cariboo. Detection of adaptive divergence between caribou population groups We identified putatively adaptatively divergent SNPs between population groups determined with population structure analyses (see results) with BAYESCAN v.2.1 (Foll and Gaggiotti 2008). BAYESCAN tests whether population group-specific allele frequencies, measured by an FST coef‑ ficient, are significantly different from the allele frequency within the common gene pool. It also assigns a posterior probability (alpha) to a model in which selection explains the difference in allele frequencies better than a null model. A positive alpha indicates population group-specific direc‑ tional selection, while a negative alpha suggests balancing or purifying selection. We ran analyses using a prior odd of 10, where SNPs were considered to be under selection when below the false discovery rate (FDR) threshold of 0.05. f b Individuals from South Selkirk and Purcell South were combined (see “Methods” section) a Individuals from Banff and Jasper were combined Genetic diversity and gene flow Colors indicate the degree of relatedness between individuals (darker color indi‑ cate higher relatedness). Individuals were grouped by herd: MOB = Moberly; QUI = Quintette: RED = Redrock-Prairie Creek; ALP = À La Pêche: JNP = Jasper + Banff; HS = Hart South; NC = North Caribou; COL = Columbia North; PS = South Selkirk and Purcell South Fig. 3 Neighbor-joining tree of caribou individuals sampled southern Rocky Mountains of Canada. The tree was based o genetic distance between individuals calculated using genom SNPs data. Branches represent caribou individuals and colors sent herds Fig. 3 Neighbor-joining tree of caribou individuals sampled in the Fig. 2 Kinship matrix of caribou individuals. Colors indicate the degree of relatedness between individuals (darker color indi‑ cate higher relatedness). Individuals were grouped by herd: MOB = Moberly; QUI = Quintette: RED = Redrock-Prairie Creek; ALP = À La Pêche: JNP = Jasper + Banff; HS = Hart South; NC = North Caribou; COL = Columbia North; PS = South Selkirk and Purcell South Fig. 3 Neighbor-joining tree of caribou individuals sampled in the southern Rocky Mountains of Canada. The tree was based on Nei’s genetic distance between individuals calculated using genome-wide SNPs data. Branches represent caribou individuals and colors repre‑ sent herds Fig. 2 Kinship matrix of caribou individuals. Colors indicate the degree of relatedness between individuals (darker color indi‑ cate higher relatedness). Individuals were grouped by herd: MOB = Moberly; QUI = Quintette: RED = Redrock-Prairie Creek; ALP = À La Pêche: JNP = Jasper + Banff; HS = Hart South; NC = North Caribou; COL = Columbia North; PS = South Selkirk and Purcell South Columbia North, South Selkirk, Purcell South, JNP, and Banff individuals (Fig. 5b). JNP and Banff caribou could not be distinguished from Columbia North, Purcell South, and South Selkirk along the PC1 axis, whereas they could be distinguished from other caribou along the PC2 axis. The first and second axis accounted for 3.37 and 2.79% of the observed genetic variation, respectively (the 3rd axis accounted for 1.88%). Our analyses therefore determined four distinguishable groups (Fig. 5b), including caribou indi‑ viduals with varying proportions of assignment to the two major genetic clusters detected with STRUCTURE (Fig. 5c). A population group (called “JNP”) was formed by individ‑ uals from the JNP and Banff herds. Genetic diversity and gene flow Individuals were grouped by herd: Fig. 3 Neighbor-joining tree of caribou individuals sampled in the southern Rocky Mountains of Canada. The tree was based on Nei’s genetic distance between individuals calculated using genome-wide SNPs data. Branches represent caribou individuals and colors repre‑ sent herds Table 2 Values of pairwise fixation index (FST) (below diagonal) and number of migrants per generation (Nm) (above diagonal) calculated using SNPs between caribou herds belonging to Central Mountain or Southern Mountain DU, sampled in western Canada between 2012 and 2020 a Three individuals from Banff were added to the Jasper pool bTwo individuals from South Selkirk were added to the Purcell South pool (see “Methods” section) Pairwise FST (Nm) Jasper À La Pêche Moberly Quintette Redrock Columbia North Hart South North Cariboo Purcell South Jaspera – 2.88 2.53 3.32 3.32 2.88 3.92 3.92 2.53 À La Pêche 0.08 – 3.92 4.75 6 2.88 6 6 2.88 Moberly 0.09 0.06 – 0.04 0.05 0.09 0.04 6 2.88 Quintette 0.07 0.05 6 – 0.03 0.07 12.25 8.08 3.32 Redrock 0.07 0.04 4.75 8.08 – 3.32 12.25 8.08 3.32 Columbia North 0.08 0.08 2.53 3.32 0.07 – 3.92 3.92 3.92 Hart South 0.06 0.04 6 0.02 0.02 0.06 – 24.75 4.75 North Cariboo 0.06 0.04 0.04 0.03 0.03 0.06 0.01 – 4.75 Purcell Southb 0.09 0.08 0.08 0.07 0.07 0.06 0.05 0.05 – Table 2 Values of pairwise fixation index (FST) (below diagonal) and number of migrants per generation (Nm) (above diagonal) calculated using SNPs between caribou herds belonging to Central Mountain or Southern Mountain DU, sampled in western Canada between 2012 and 2020 Three individuals from Banff were added to the Jasper pool f b Two individuals from South Selkirk were added to the Purcell South pool (see “Methods” section) f b Two individuals from South Selkirk were added to the Purcell South pool (see “Methods” section) ( ) Fig. 3 Neighbor-joining tree of caribou individuals sampled in the southern Rocky Mountains of Canada. The tree was based on Nei’s genetic distance between individuals calculated using genome-wide SNPs data. Branches represent caribou individuals and colors repre‑ sent herds Columbia North, South Selkirk, Purcell South, JN Banff individuals (Fig. 5b). JNP and Banff caribou not be distinguished from Columbia North, Purcell and South Selkirk along the PC1 axis, whereas they be distinguished from other caribou along the PC2 Fig. 2 Kinship matrix of caribou individuals. f Two individuals from South Selkirk were added to the Purcell South pool (see “Methods” section) Genetic diversity and gene flow Observed heterozygosity (Ho) of caribou herds in the study area ranged from 0.33 to 0.39 (mean 0.37 + SD 0.02; Table 1) and expected heterozygosity (He) ranged from 0.35 Table 1 Values of observed and expected heterozygosity (Ho and He respectively), and inbreeding coefficient (FIS, with Confidence Inter‑ vals in parenthesis) calculated using SNPs for caribou herds belong‑ ing to the Central Mountain or Southern Mountain DU, sampled in western Canada between 2012 and 2020 1 p y), gfi ( IS,i vals in parenthesis) calculated using SNPs for caribou herds belong‑ a Individuals from Banff and Jasper were combined b Individuals from South Selkirk and Purcell South were combined (see “Methods” section) Central Mountain DU Southern Mountain DU Jaspera À La Pêche Moberly Quintette Redrock Columbia North Hart South North Cari‑ boo Purcell Southb Ho 0.35 0.38 0.39 0.38 0.39 0.36 0.39 0.39 0.33 He 0.36 0.38 0.38 0.39 0.38 0.35 0.39 0.39 0.36 FIS 0.028 (0.024, 0.032) 0.017 (0.014, 0.020) 0.029 (0.026, 0.032) 0.004 (0.002, 0.006) 0.021 (0.018, 0.024) 0.014 (0.012, 0.016) 0.004 (0.002, 0.006) 0.003 (0.001, 0.005) 0.047 (0.022, 0.072) 1 3 Conservation Genetics (2023) 24:855–867 860 Columbia North, South Selkirk, Purcell South, JNP, and Table 2 Values of pairwise fixation index (FST) (below diagonal) and number of migrants per generation (Nm) (above diagonal) calculated using SNPs between caribou herds belonging to Central Mountain or Southern Mountain DU, sampled in western Canada between 2012 and 2020 a Three individuals from Banff were added to the Jasper pool b Two individuals from South Selkirk were added to the Purcell South pool (see “Methods” section) Pairwise FST (Nm) Jasper À La Pêche Moberly Quintette Redrock Columbia North Hart South North Cariboo Purcell South Jaspera – 2.88 2.53 3.32 3.32 2.88 3.92 3.92 2.53 À La Pêche 0.08 – 3.92 4.75 6 2.88 6 6 2.88 Moberly 0.09 0.06 – 0.04 0.05 0.09 0.04 6 2.88 Quintette 0.07 0.05 6 – 0.03 0.07 12.25 8.08 3.32 Redrock 0.07 0.04 4.75 8.08 – 3.32 12.25 8.08 3.32 Columbia North 0.08 0.08 2.53 3.32 0.07 – 3.92 3.92 3.92 Hart South 0.06 0.04 6 0.02 0.02 0.06 – 24.75 4.75 North Cariboo 0.06 0.04 0.04 0.03 0.03 0.06 0.01 – 4.75 Purcell Southb 0.09 0.08 0.08 0.07 0.07 0.06 0.05 0.05 – Fig. 2 Kinship matrix of caribou individuals. Colors indicate the degree of relatedness between individuals (darker color indi‑ cate higher relatedness). Discussion We conducted a genomic study to identify candidate found‑ ers for a conservation breeding program currently planned for caribou, an approach suggested as a best practice for endangered species in general, but requiring substantial preparation and research to be successful (Russello and Jensen 2018). Our work relied on a newly developed SNP- array specifically produced for caribou (Carrier et al. 2022), which in this study produced around 40,000 SNPs that were successfully genotyped in 95% of the sampled individuals. Across the 200,000 km2 study area we found that, despite wide-spread recent declines (Serrouya et al. 2019), caribou subpopulations (herds) have retained levels of genetic diver‑ sity and natural connectivity (e.g. more than one migrant per generation [Wright 1931; Slatkin 1987; Weeks et al 2011] genetically estimated by this study between any pair of herds) which in other studies have been considered adequate for conservation interventions including translocations of individuals. However, we found two major genetic clusters and additional population groups, which should inform con‑ servation planning. If a conservation breeding program is established in JNP, founders could be selected from clusters or population groups most similar to JNP as a first priority, while also taking into consideration information on poten‑ tially-adaptive divergence (see discussion below on results of outlier analyses).i Fig. 4 Population structure of caribou sampled in the southern Rocky Mountains of Canada. a Bar plots from STRUCTURE and ADMIX‑ TURE analyses indicate individual proportions of assignment into two main genetic clusters (red or blue color). The most likely num‑ ber of clusters (K) obtained with STRUCTURE (higher values best) and ADMIXTURE (lower values best) is indicated with circles on the respective scatter plots. b Map showing distribution of sampled cari‑ bou (capture coordinates) with proportion of belonging to either clus‑ ter for each individual obtained with STRUCTURE (pie chart) The levels of heterozygosity we identified (including He and Ho) were aligned with other reported values for cari‑ bou and approaching levels considered as “high” in wild mammals (see for example Cavedon et al. 2019). The lev‑ els of inbreeding coefficients we also determined (FIS), which provide information on relatedness among individu‑ als (Crow and Kimura 1970; Caballero et al. 2021), were low to moderate (see Solmundson et al. 2020). Discussion Therefore, our findings indicate that the study area’s wild caribou have retained levels of genetic diversity, which if properly main‑ tained in captive populations could perhaps circumvent risk of inbreeding depression. Also valuable to avoid inbreeding depression, in this study, we identified close relatives (those with IBD > 0.25 corresponding to second-degree relatives), which should be discarded when selecting source founders. Kinship is an important tool used to identify breeders for captive populations and can be used to effectively evaluate one additional by individuals from Columbia North, Purcell South, and South Selkirk (“COL”). The fourth and final pop‑ ulation group (“CR”) included individuals from the remain‑ ing sampled herds, which were also close on the PCA plot (Hart South, North Cariboo, Redrock-Prairie Creek, Quin‑ tette, and Moberly). Genetic diversity and gene flow One more population group was formed by individuals from À La Pêche (“ALP”), A principal component analysis indicated that individuals belonging to Central Mountain or Southern Mountain DUs could not be obviously separated along the two principal axes (Fig. 5a). It also indicated that some Central and South‑ ern Mountain DU caribou were genetically close, includ‑ ing an assemblage formed by À La Pêche, Redrock-Prairie Creek, Quintette, Moberly, Hart South, and North Cariboo individuals, and including another assemblage formed by 3 Conservation Genetics (2023) 24:855–867 861 Fig. 4 Population structure of caribou sampled in the southern Rocky Mountains of Canada. a Bar plots from STRUCTURE and ADMIX‑ TURE analyses indicate individual proportions of assignment into two main genetic clusters (red or blue color). The most likely num‑ ber of clusters (K) obtained with STRUCTURE (higher values best) and ADMIXTURE (lower values best) is indicated with circles on the respective scatter plots. b Map showing distribution of sampled cari‑ bou (capture coordinates) with proportion of belonging to either clus‑ ter for each individual obtained with STRUCTURE (pie chart) identified 89 outlier SNPs divergent between JNP and ALP, with FST values ranging between 0.20 and 0.36 (average 0.25). We also detected 68 SNPs diverging between JNP and CR, with FST values ranging between 0.15 and 0.24 (average 0.19). Lastly, we found only 53 outlier SNPs between JNP and COL with FST values ranging between 0.21 and 0.29 (average 0.25; Fig. 6). identified 89 outlier SNPs divergent between JNP and ALP, with FST values ranging between 0.20 and 0.36 (average 0.25). We also detected 68 SNPs diverging between JNP and CR, with FST values ranging between 0.15 and 0.24 (average 0.19). Lastly, we found only 53 outlier SNPs between JNP and COL with FST values ranging between 0.21 and 0.29 (average 0.25; Fig. 6). Divergence between caribou population groups To assess putatively adaptive divergence, we ran pairwise BAYESCAN analyses between the four caribou popula‑ tion groups identified with PCA analyses (see above). We 1 3 Conservation Genetics (2023) 24:855–867 862 862 Conservation Genetics (2023) 24:855–867 the potential for future inbreeding in wild populations (Bal‑ lou and Lacy 1995; Frankham et al. 2017; Flesch et al. 2022). Our kinship findings indicated moderate relatedness only among caribou individuals within the same herd and suggested that inbreeding could be minimized by relying on founders from different source herds, should a captive popu‑ lation be established. Our results also indicated that herds known to overlap (example, Hart South and North Caribou with other neighbouring herds; Environment Canada 2014) naturally had lower relatedness. Future studies could exam‑ ine inbreeding and relatedness in more detail, such as by examining “runs of homozygosity” (see Broman and Weber 1999) across the genome, an approach that may show sig‑ natures of natural and/or human mediated selection (Kardos et al. 2015; Caballero et al. 2021; Solmundson et al. 2020). We estimated that the number of migrants per genera‑ tion (Nm) were all greater than one, indicating significant historical and/or recent gene flow among all herd pairs. One migrant per generation, as a minimum, is typically considered sufficient to offset genetic deterioration within subunits (Wright 1931; Slatkin 1987; Weeks et al 2011). It is therefore likely that caribou herds in the study area were connected until recently, with barriers to gene flow likely arising in the last decades. Consistent with this interpreta‑ tion, barriers to dispersal have been identified in studies of radio-collared southern mountain caribou (Van Oort et al. 2011), which likely reflect contemporary, non-historical pat‑ terns (i.e., those observed during the 2–3 years lifespan of a GPS collar), and which were deployed just on females (i.e., the least vagile sex in caribou, as discussed in Cavedon et al. 2022a, b, c). By contrast, presence of barriers between popu‑ lation ranges was not as consequential: habitat suitability followed by predation risk was associated with overall gene flow in a caribou study conducted previously (Gubili et al. 2017). 3 Divergence between caribou population groups Importantly,, levels of Nm exceeding one have been historically used to manage wildlife populations as one unit (Mills and Allendorf 1996; Vucetich and Waite 2000; Wang 2004), and perhaps the same threshold could be applied to caribou (manageable in the future as a unit, similar to other wildlife populations) The levels of Nm we detected could be Fig. 5 Principal Component Analysis (PCA) of caribou sampled in the southern Rocky Mountains of Canada. Circles represent caribou individuals, and colors represent DUs (in Panel a), herds (in Panel b) or proportion of assignment to the two major genetic clusters detected with STRUCTURE (in Panel c). Dashed circles in panel B indicate the caribou popula‑ tion groups we detected, which were then used in analyses of adaptive divergence: ALP = À La Pêche; COL = Columbia North, Purcell South, and South Selkirk; JNP = Jasper and Banff; CR = Moberly, Quintette, Redrock-Prairie Creek, Hart South, and North Cariboo Fig. 5 Principal Component Analysis (PCA) of caribou sampled in the southern Rocky Mountains of Canada. Circles represent caribou individuals, and colors represent DUs (in Panel a), herds (in Panel b) or proportion of assignment to the two major genetic clusters detected with STRUCTURE (in Panel c). Dashed circles in panel B indicate the caribou popula‑ tion groups we detected, which were then used in analyses of adaptive divergence: ALP = À La Pêche; COL = Columbia North, Purcell South, and South Selkirk; JNP = Jasper and Banff; CR = Moberly, Quintette, Redrock-Prairie Creek, Hart South, and North Cariboo the potential for future inbreeding in wild populations (Bal‑ lou and Lacy 1995; Frankham et al. 2017; Flesch et al. 2022). Our kinship findings indicated moderate relatedness only among caribou individuals within the same herd and suggested that inbreeding could be minimized by relying on founders from different source herds, should a captive popu‑ lation be established. Our results also indicated that herds known to overlap (example, Hart South and North Caribou with other neighbouring herds; Environment Canada 2014) naturally had lower relatedness. Future studies could exam‑ ine inbreeding and relatedness in more detail, such as by examining “runs of homozygosity” (see Broman and Weber 1999) across the genome, an approach that may show sig‑ natures of natural and/or human mediated selection (Kardos et al. 2015; Caballero et al. 2021; Solmundson et al. 2020). Divergence between caribou population groups the potential for future inbreeding in wild populations (Bal‑ lou and Lacy 1995; Frankham et al. 2017; Flesch et al. 2022). Our kinship findings indicated moderate relatedness only among caribou individuals within the same herd and suggested that inbreeding could be minimized by relying on founders from different source herds, should a captive popu‑ lation be established. Our results also indicated that herds known to overlap (example, Hart South and North Caribou with other neighbouring herds; Environment Canada 2014) naturally had lower relatedness. Future studies could exam‑ ine inbreeding and relatedness in more detail, such as by examining “runs of homozygosity” (see Broman and Weber 1999) across the genome, an approach that may show sig‑ natures of natural and/or human mediated selection (Kardos et al. 2015; Caballero et al. 2021; Solmundson et al. 2020). i d h h b f i subunits (Wright 1931; Slatkin 1987; Weeks et al 2011). It is therefore likely that caribou herds in the study area were connected until recently, with barriers to gene flow likely arising in the last decades. Consistent with this interpreta‑ tion, barriers to dispersal have been identified in studies of radio-collared southern mountain caribou (Van Oort et al. 2011), which likely reflect contemporary, non-historical pat‑ terns (i.e., those observed during the 2–3 years lifespan of a GPS collar), and which were deployed just on females (i.e., the least vagile sex in caribou, as discussed in Cavedon et al. 2022a, b, c). By contrast, presence of barriers between popu‑ lation ranges was not as consequential: habitat suitability followed by predation risk was associated with overall gene flow in a caribou study conducted previously (Gubili et al. 2017). Importantly,, levels of Nm exceeding one have been historically used to manage wildlife populations as one unit (Mills and Allendorf 1996; Vucetich and Waite 2000; Wang 2004), and perhaps the same threshold could be applied to caribou (manageable in the future as a unit, similar to other wildlife populations). The levels of Nm we detected could be We estimated that the number of migrants per genera‑ tion (Nm) were all greater than one, indicating significant historical and/or recent gene flow among all herd pairs. One migrant per generation, as a minimum, is typically considered sufficient to offset genetic deterioration within 1 3 Conservation Genetics (2023) 24:855–867 863 863 Conservation Genetics (2023) 24:855–867 suitable for conservation translocations among these popula‑ tions. Divergence between caribou population groups This study’s populations are also within natural disper‑ sal distances characteristic of the species, whereas dispersals themselves might be infrequent recently, likely due to habitat fragmentation (Van Oort et al. 2011). Nonetheless, further analyses on gene flow are recommended, as both FST and Nm are known to also depend on genetic diversity and on sample sizes (Holsinger and Weir 2009), which were limited in this study. Population structure results indicate that caribou individ‑ uals could be grouped in two major genetic clusters, which is consistent with both past and current studies conducted in western Canada (see McDevitt et al. 2009; Cavedon et al. 2022a, b, c). In this study, we targeted common variants (SNPs with MAF > 0.01), which are suited to examine the deep evolutionary history of species (Gibson 2012). Our findings may therefore have captured similar diversification patterns as those detected with mtDNA analyses, which indicated the presence of either a Beringian/Northern or a S th li i th t d (M D itt t l 2009 The two genetic clusters we identified did not overlap fully with currently recognized DUs. We found that although caribou from some Central Mountain DU herds were geneti‑ cally similar to one another, they were also similar to caribou from some Southern Mountain herds (note the mainly blue circles in Fig. 4b), and this information could also be used to plan future translocations. Our findings indicating the pres‑ ence of two genetic clusters and potentially two DUs in the area are consistent with other recent studies also relying on genomic data for caribou (Cavedon et al. 2022a, b, c; Taylor et al. 2020, 2021). Our findings, however, partially contrast with past studies that rely on neutral markers (autosomal microsatellites and mtDNA), which have also indicated the presence of two main clusters and DUs, but with slightly different boundaries (McDevitt et al. 2009; Serrouya et al. 2012; Weckworth et al. 2012). This can be explained by the fact that these neutral genetic markers cannot detect local adaptation, unlike genomic SNPs (Luikart et al. 2003; Allen‑ dorf et al. 2010). Th i i l t l i d t t d f di ti Fig. 6 Signatures of adaptive divergence between caribou population groups determined by outlier analyses. Divergence between caribou population groups Our findings indicating the pres‑ ence of two genetic clusters and potentially two DUs in the area are consistent with other recent studies also relying on genomic data for caribou (Cavedon et al. 2022a, b, c; Taylor et al. 2020, 2021). Our findings, however, partially contrast with past studies that rely on neutral markers (autosomal microsatellites and mtDNA), which have also indicated the presence of two main clusters and DUs, but with slightly different boundaries (McDevitt et al. 2009; Serrouya et al. 2012; Weckworth et al. 2012). This can be explained by the fact that these neutral genetic markers cannot detect local adaptation, unlike genomic SNPs (Luikart et al. 2003; Allen‑ dorf et al. 2010). The principal component analysis detected four distin‑ guishable groups, which we used for further analyses of suitable for conservation translocations among these popula‑ tions. This study’s populations are also within natural disper‑ sal distances characteristic of the species, whereas dispersals themselves might be infrequent recently, likely due to habitat fragmentation (Van Oort et al. 2011). Nonetheless, further analyses on gene flow are recommended, as both FST and Nm are known to also depend on genetic diversity and on sample sizes (Holsinger and Weir 2009), which were limited in this study. The two genetic clusters we identified did not overlap fully with currently recognized DUs. We found that although caribou from some Central Mountain DU herds were geneti‑ cally similar to one another, they were also similar to caribou from some Southern Mountain herds (note the mainly blue circles in Fig. 4b), and this information could also be used to plan future translocations. Our findings indicating the pres‑ ence of two genetic clusters and potentially two DUs in the area are consistent with other recent studies also relying on genomic data for caribou (Cavedon et al. 2022a, b, c; Taylor et al. 2020, 2021). Our findings, however, partially contrast with past studies that rely on neutral markers (autosomal microsatellites and mtDNA), which have also indicated the presence of two main clusters and DUs, but with slightly different boundaries (McDevitt et al. 2009; Serrouya et al. 2012; Weckworth et al. 2012). This can be explained by the fact that these neutral genetic markers cannot detect local adaptation, unlike genomic SNPs (Luikart et al. 2003; Allen‑ dorf et al. 2010). The two genetic clusters we identified did not overlap fully with currently recognized DUs. Divergence between caribou population groups The horizontal axis indicates the BAYESCAN-assessed log10 of the q value (the false discov‑ ery rate (FDR) analog to the p-value) and the vertical axis is the mean genetic differentiation (FST). Each point represents a SNP and significant outliers are visible right of the grey verti‑ cal line. Identifiers represent population groups: ALP = À La Pêche; COL = Columbia North, Purcell South, and South Selkirk; JNP = Jasper and Banff; CR = Moberly, Quintette, Redrock-Prairie Creek, Hart South, and North Cariboo suitable for conservation translocations among these popula‑ tions. This study’s populations are also within natural disper‑ sal distances characteristic of the species, whereas dispersals The two genetic clusters we identified did not overlap fully with currently recognized DUs. We found that although caribou from some Central Mountain DU herds were geneti‑ Fig. 6 Signatures of adaptive divergence between caribou population groups determined by outlier analyses. The horizontal axis indicates the BAYESCAN-assessed log10 of the q value (the false discov‑ ery rate (FDR) analog to the p-value) and the vertical axis is the mean genetic differentiation (FST). Each point represents a SNP and significant outliers are visible right of the grey verti‑ cal line. Identifiers represent population groups: ALP = À La Pêche; COL = Columbia North, Purcell South, and South Selkirk; JNP = Jasper and Banff; CR = Moberly, Quintette, Redrock-Prairie Creek, Hart South, and North Cariboo i Fig. 6 Signatures of adaptive divergence between caribou population groups determined by outlier analyses. The horizontal axis indicates the BAYESCAN-assessed log10 of the q value (the false discov‑ ery rate (FDR) analog to the p-value) and the vertical axis is the mean genetic differentiation (FST). Each point represents a SNP and significant outliers are visible right of the grey verti‑ cal line. Identifiers represent population groups: ALP = À La Pêche; COL = Columbia North, Purcell South, and South Selkirk; JNP = Jasper and Banff; CR = Moberly, Quintette, Redrock-Prairie Creek, Hart South, and North Cariboo The two genetic clusters we identified did not overlap fully with currently recognized DUs. We found that although caribou from some Central Mountain DU herds were geneti‑ cally similar to one another, they were also similar to caribou from some Southern Mountain herds (note the mainly blue circles in Fig. 4b), and this information could also be used to plan future translocations. Divergence between caribou population groups Outlier SNPs may indicate divergence due to demographic events or selection processes related to local adaptation (Luikart et al. 2003; Allendorf et al. 2010). Previous genomic studies conducted for caribou on a larger scale also identified candidate loci under selection that were associated with ecological, behav‑ ioral, and climatic factors (Cavedon et al. 2019, 2022a, b, c). Together, the results of our current and previous studies (Cavedon et al. 2019, 2022a, b, c) indicate that caribou pop‑ ulation groups in the study area may be differently adapted due to selective forces (e.g., environmental and climatic con‑ ditions), an important detail that should be accounted for in any conservation plan (Des Roches et al. 2021). step to identify suitable units or groups for sourcing founder animals beyond arbitrary, human-created boundaries. Pre‑ serving and restoring such intraspecies diversity, though the path we indicated for caribou, is key to maintaining a spe‑ cies’ evolutionary potential, and in turn its critical ecological functions (Des Roches et al. 2021). Supplementary Information The online version contains supplemen‑ tary material available at https://doi.org/10.1007/s10592-023-01540-3. Acknowledgements We would like to thank the Aseniwuche Winewak Caribou Patrol, Bevan Ernst, Leo DeGroot, Mike Klaczek, and Aaron Reid for their efforts for caribou monitoring and conservation manage‑ ment. We also thank the Italian PNRR project Spoke 4.4, Scenarios of Area-based conservation planning and management, for supporting MM’s line of research. y p Our study of caribou genomics and distribution should inform selection of founders for the proposed JNP conserva‑ tion breeding program. We found that the study area likely hosted a historic caribou metapopulation, characterized by high levels of gene flow which has led to high levels of genetic diversity. Our results showed that JNP could poten‑ tially acquire founders from any of the sampled herds, some of which, thanks to recovery actions, have recently started to increase and might be able to sustain the removal of some individuals (Government of Alberta 2017; Eacker et al. 2019; McNay et al. 2022). However, population structure analyses and adaptive divergence analyses indicate that JNP caribou are most genetically related to caribou in the Colum‑ bia range of BC. Selecting founders from the Columbia range would provide the best chance of maintaining genetic traits most similar to JNP caribou. Herds in the Columbia range are however numerically low compared to others (e.g., 184 individuals in Columbia North vs. Divergence between caribou population groups We found that although caribou from some Central Mountain DU herds were geneti‑ cally similar to one another, they were also similar to caribou from some Southern Mountain herds (note the mainly blue circles in Fig. 4b), and this information could also be used to plan future translocations. Our findings indicating the pres‑ ence of two genetic clusters and potentially two DUs in the area are consistent with other recent studies also relying on genomic data for caribou (Cavedon et al. 2022a, b, c; Taylor et al. 2020, 2021). Our findings, however, partially contrast with past studies that rely on neutral markers (autosomal microsatellites and mtDNA), which have also indicated the presence of two main clusters and DUs, but with slightly different boundaries (McDevitt et al. 2009; Serrouya et al. 2012; Weckworth et al. 2012). This can be explained by the fact that these neutral genetic markers cannot detect local adaptation, unlike genomic SNPs (Luikart et al. 2003; Allen‑ dorf et al. 2010). suitable for conservation translocations among these popula‑ tions. This study’s populations are also within natural disper‑ sal distances characteristic of the species, whereas dispersals themselves might be infrequent recently, likely due to habitat fragmentation (Van Oort et al. 2011). Nonetheless, further analyses on gene flow are recommended, as both FST and Nm are known to also depend on genetic diversity and on sample sizes (Holsinger and Weir 2009), which were limited in this study. Population structure results indicate that caribou individ‑ uals could be grouped in two major genetic clusters, which is consistent with both past and current studies conducted in western Canada (see McDevitt et al. 2009; Cavedon et al. 2022a, b, c). In this study, we targeted common variants (SNPs with MAF > 0.01), which are suited to examine the deep evolutionary history of species (Gibson 2012). Our findings may therefore have captured similar diversification patterns as those detected with mtDNA analyses, which indicated the presence of either a Beringian/Northern or a Southern lineage in the study area (McDevitt et al. 2009; Serrouya et al. 2012; Weckworth et al. 2012; Yannic et al. 2014; Taylor et al. 2021). The principal component analysis detected four distin‑ guishable groups, which we used for further analyses of potentially adaptive divergence. Overall, we found more 1 864 Conservation Genetics (2023) 24:855–867 outlier SNPs between JNP and ALP than between JNP and CR and especially between JNP and COL. Divergence between caribou population groups 405 in Hart; see also Serrouya et al. 2021; McNay et al. 2022) and might not tolerate the removal of individuals. However, admixture of two caribou genetic clusters has also been consistently documented throughout the study area (see McDevitt et al. 2009, this study), therefore indicating that any conservation program should aim at maintaining such diversity (sensu Frankham et al. 2017, Ralls et al. 2020) too. It should also be taken into consideration that caribou genetic diversity in the study area is correlated with ecological and behavioural diversity also including seasonal migration (McDevitt et al. 2009; Cavedon et al. 2022a, b, c), and any conservation pro‑ gram should aim at maintaining all diversities in these par‑ tially migrating populations (Cavedon et al. 2019). Author contributions Maria Cavedon designed the study (with Marco Musiani and Lalenia Neufeld), conducted lab analyses (with Anita Michalak), analyzed genomic data, and drafted the manuscript (with Marco Musiani, Anita Michalak, and Jocelyn Poissant). Laura Finne‑ gan, Dave Hervieux, Agnes Pelletier, Jean L. Polfus, Helen Schwantje, Geoff Skinner, Robin Steenweg, and Caeley Thacker gathered genetic and/or biological samples. All authors critically reviewed drafts and approved the final version of the manuscript. Funding This work was supported by the Alberta Conservation Asso‑ ciation, Alberta Innovates, Alberta Upstream Petroleum Research Fund, Canadian Association of Petroleum Producers, Canada's Oil Sands Innovation Alliance, Conoco-Phillips, Environment and Climate Change Canada's Canadian Wildlife Service, Exxon, fRI Research, Governments of Alberta and British Columbia, Natural Sciences and Engineering Research Council of Canada, Parks Canada Agency, and Teck Resources. Data Availability Caribou sample locations and population and DU range maps are available from the Government of British Columbia –see https://catalogue.data.gov.bc.ca/dataset/caribou-herd-locations- for-bc. Restrictions apply to the availability of this Species At Risk’s raw SNP data, which were used under license for this study’s academic personnel. 1 3 References https://doi.org/10.1002/wsb.950 Bradley M, Neufeld L (2012) Climate and management interact to explain the decline of woodland caribou (Rangifer tarandus cari‑ bou) in Jasper National Park. Rangifer 32:183–191. https://doi. org/10.7557/2.32.2.2268 Earl DA, VonHoldt BM (2012) STRUCTURE HARVESTER: a web‑ site and program for visualizing STRUCTURE output and imple‑ menting the Evanno method. Conserv Genet Resour 4(2):359– 361. https://doi.org/10.1007/s12686-011-9548-7i Brichieri-Colombi TA, Moehrenschlager A (2016) Alignment of threat, effort, and perceived success in North American conservation translocations. Conserv Biol 30(6):1159–1172. https://doi.org/ 10.1111/cobi.12743 Ecological Stratification Working Group (1995) A national ecologi‑ cal framework for Canada. Agriculture and Agri-Food Canada, Research Branch, Centre for Land and Biological Resources Research and Environment Canada, State of the Environment Directorate, Ecozone Analysis Branch, Ottawa Broman KW, Weber JL (1999) Long homozygous chromosomal seg‑ ments in reference families from the Centre d’Etude du Polymor‑ phisme Humain. Am J Hum Genet 65(6):1493–1500. https://doi. org/10.1086/302661 Bubac CM, Johnson AC, Fox JA, Cullingham CI (2019) Conservation translocations and post-release monitoring: identifying trends in failures, biases, and challenges from around the world. Biol Cons 238:108239 Environment Canada (2014) Recovery strategy for the Woodland Cari‑ bou, Southern Mountain population (Rangifer tarandus caribou) in Canada. Species at Risk Act Recovery Strategy Series, Envi‑ ronment Canada, Ottawa Caballero A, Villanueva B, Druet T (2021) On the estimation of inbreeding depression using different measures of inbreeding from molecular markers. Evol Appl 14(2):416–428. https://doi. org/10.1111/eva.13126 Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clus‑ ters of individuals using the software structure: a simulation study. Mol Ecol 14(8):2611–2620. https://doi.org/10.1111/j. 1365-294X.2005.02553.x Carrier A, Prunier J, Poisson W, Trottier-Lavoie M, Gilbert I, Cave‑ don M et al (2022) Design and validation of a 63K genome- wide SNP-genotyping platform for caribou/reindeer (Rangifer tarandus). BMC Genomics 23(1):1–14. https://doi.org/10.1186/ s12864-022-08899-6 Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and cor‑ related allele frequencies. Genetics 164(4):1567–1587. https:// doi.org/10.1093/genetics/164.4.1567 Fischer J, Lindenmayer DB (2000) An assessment of the published results of animal relocations. Biol Cons 96(1):1–11. https://doi. org/10.1016/S0006-3207(00)00048-3 Cavedon M, Gubili C, Heppenheimer E, vonHoldt B, Mariani S, Heb‑ blewhite M et al (2019) Genomics, environment and balancing selection in behaviourally bimodal populations: The caribou case. Mol Ecol 28(8):1946–1963. https://doi.org/10.1111/mec. 15039 Flanagan SP, Forester BR, Latch EK, Aitken SN, Hoban S (2018) Guidelines for planning genomic assessment and monitoring of locally adaptive variation to inform species conservation. Evol Appl 11(7):1035–1052. References Cavedon M, VonHoldt B, Hebblewhite M, Hegel T, Heppenheimer E, Hervieux D et al (2022c) Selection of both habitat and genes in specialized and endangered caribou. Conserv Biol. https://doi. org/10.1111/cobi.13900 Alexander DH, Novembre J, Lange K (2009) Fast model-based estimation of ancestry in unrelated individuals. Genome Res 19(9):1655–1664. https://doi.org/10.1101/gr.094052.109 Committee on the Status of Endangered Wildlife in Canada (COSE‑ WIC) (2011) Designatable units for caribou (Rangifer tarandus) in Canada. Committee on the Status of Endangered Wildlife in Canada, Ottawa Allendorf FW, Hohenlohe PA, Luikart G (2010) Genomics and the future of conservation genetics. Nat Rev Genet 11(10):697–709. https://doi.org/10.1038/nrg2844f Crandall KA, Bininda-Emonds OR, Mace GM, Wayne RK (2000) Considering evolutionary processes in conservation biology. Trends Ecol Evol 15(7):290–295. https://doi.org/10.1016/S0169- 5347(00)01876-0 Araki H, Cooper B, Blouin MS (2007) Genetic effects of captive breed‑ ing cause a rapid, cumulative fitness decline in the wild. Science 318(5847):100–103. https://doi.org/10.1126/science.1145621 Arif IA, Khan HA, Shobrak M, Al Homaidan AA, Al Sadoon M, Al Farhan AH (2010) Measuring the genetic diversity of Arabian Oryx using microsatellite markers: implication for captive breed‑ ing. Genes Genet Syst 85(2):141–145 Crow JF, Kimura M (1970) An introduction to population genetics theory. Harper & Row Publishers, New York Dayuan X, Yuanyuan Z, Zhibin C, Zhenyu Z, Ming C, Mengdi F et al (2022) Père David’s deer (Elaphurus davidianus) in China: pop‑ ulation dynamics and challenges. J Resour Ecol 13(1):41–50. https://doi.org/10.5814/j.issn.1674-764x.2022.01.005 g y Armstrong DP, Seddon PJ (2008) Directions in reintroduction biol‑ ogy. Trends Ecol Evol 23(1):20–25. https://doi.org/10.1016/j. tree.2007.10.003 Der Sarkissian C, Ermini L, Schubert M, Yang MA, Librado P, Fuma‑ galli M et al (2015) Evolutionary genomics and conservation of the endangered Przewalski’s horse. Curr Biol 25(19):2577–2583. https://doi.org/10.1016/j.cub.2015.08.032 Ballou JD, Lacy RC (1995) Identifying genetically important individu‑ als for management of genetic variation in pedigreed populations. In: Ballou JD, Gilpin M, Foose TJ (eds) Population management for survival and recovery: analytical methods and strategies in small population conservation. Columbia University Press, New York, pp 76–111 Des Roches S, Pendleton LH, Shapiro B, Palkovacs EP (2021) Con‑ serving intraspecific variation for nature’s contributions to people. Nat Ecol Evol 5(5):574–582. https://doi.org/10.1038/ s41559-021-01403-5 Bergerud AT (1988) Caribou, wolves and man. Trends Ecol Evol 3(3):68–72. https://doi.org/10.1016/0169-5347(88)90019-5 Eacker DR, Hebblewhite M, Steenweg R, Russell M, Flasko A, Her‑ vieux D (2019) Web-based application for threatened woodland caribou population modeling. Wildl Soc Bull 43(1):167–177. Declarations Conflict of interest The authors have no relevant financial or non-fi‑ nancial interests to disclose. Ethics Approval Research was conducted under research permits of Governments of British Columbia and Alberta, Parks Canada and Uni‑ versity of Calgary. Approval was granted by the University of Calgary Animal Care Committee Studies AC16-0195 and AC20-0110. Open Access This article is licensed under a Creative Commons Attri‑ bution 4.0 International License, which permits use, sharing, adapta‑ tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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In addition to genetic analyses, it will be important to conduct population viability analyses, or an equivalent assessment approach, to understand the conservation impli‑ cations of caribou removals for source populations of this species at risk (Hoban et al. 2012).i Similarly to caribou, other endangered species can benefit from the evaluation of genomic data applied to conserva‑ tion breeding programs (Russello and Jensen 2018), as a key 1 3 Conservation Genetics (2023) 24:855–867 865 References https://doi.org/10.1111/eva.12569fi Cavedon M, Poissant J, vonHoldt B, Michalak A, Hegel T, Heppenhe‑ imer E, Hervieux D, Schwantje H, Neufeld L, Polfus J, Schwantje H, Steenweg R, Musiani M (2022a) Population structure of threatened caribou in western Canada inferred from genome- wide SNP data. Conserv Genet 23:1089–1103 Flesch EP, Graves T, Thomson J, Proffitt K, Garrott R (2022) Average kinship within bighorn sheep populations is associated with con‑ nectivity, augmentation, and bottlenecks. Ecosphere 13:e3972 Foll M, Gaggiotti O (2008) A genome-scan method to identify selected loci appropriate for both dominant and codominant markers: a Bayesian perspective. Genetics 180(2):977–993. https://doi.org/ 10.1534/genetics.108.092221 Cavedon M, vonHoldt B, Hebblewhite M, Hegel T, Heppenheimer E, Hervieux D et al (2022b) Genomic legacy of migration in endangered caribou. PLoS Genetics 18(2):e1009974. https://doi. org/10.1371/journal.pgen.1009974 1 3 Conservation Genetics (2023) 24:855–867 866 Karney CFF (2013) Algorithms for geodesics. J Geod 87:43–55. https://doi.org/10.1007/s00190-012-0578-z Karney CFF (2013) Algorithms for geodesics. J Geod 87:43–55. https://doi.org/10.1007/s00190-012-0578-z Forsman A (2014) Effects of genotypic and phenotypic variation on establishment are important for conservation, invasion, and infec‑ tion biology. Proc Natl Acad Sci USA 111(1):302–307. https:// doi.org/10.1073/pnas.1317745111 p g Kleiman DG (1989) Reintroduction of captive mammals for conserva‑ tion. Bioscience 39(3):152–161. https://doi.org/10.2307/1311025 Kominakis A, Tarsani E, Hager-Theodorides AL, Mastranestasis I, Gkelia D, Hadjigeorgiou I (2021) Genetic differentiation of mainland-island sheep of Greece: implications for identify‑ ing candidate genes for long-term local adaptation. PLoS One 16(9):e0257461. https://doi.org/10.1371/journal.pone.0257461 Foundations of Success and Parks Canada (2021) Assessing the Evi‑ dence for Adoption of a Conservation Breeding Strategy to Enable Recovery of Southern Mountain Caribou Populations in Jasper National Park, Canada. Frankham R (2010) Where are we in conservation genetics and where do we need to go? Conserv Genet 11(2):661–663. https://doi.org/ 10.1007/s10592-009-0010-2 Laikre L (1999) Hereditary defects and conservation genetic manage‑ ment of captive populations. Zoo Biol 18(2):81–99. https://doi. org/10.1002/(SICI)1098-2361(1999)18:2%3c81::AID-ZOO1% 3e3.0.CO;2-2 Frankham R, Ballou JD, Ralls K, Eldridge M, Dudash MR, Fenster CB et al (2017) Genetic management of fragmented animal and plant populations. Oxford University Press, Oxford Luikart G, England PR, Tallmon D, Jordan S, Taberlet P (2003) The power and promise of population genomics: from genotyping to genome typing. Nat Rev Genet 4(12):981–994. https://doi.org/ 10.1038/nrg1226 Galla SJ, Moraga R, Brown L, Cleland S, Hoeppner MP, Maloney RF et al (2020) A comparison of pedigree, genetic and genomic estimates of relatedness for informing pairing decisions in two critically endangered birds: Implications for conservation breed‑ ing programmes worldwide. References Evol Appl 13(5):991–1008. https:// doi.org/10.1111/eva.12916 McDevitt AD, Mariani S, Hebblewhite M, Decesare NJ, Morgantini L, Seip D et al (2009) Survival in the Rockies of an endangered hybrid swarm from diverged caribou (Rangifer tarandus) line‑ ages. Mol Ecol 18(4):665–679. https://doi.org/10.1111/j.1365- 294X.2008.04050.x Gibson G (2012) Rare and common variants: twenty arguments. Nat Rev Genet 13(2):135–145. https://doi.org/10.1038/nrg3118 McLoughlin PD, Dzus E, Wynes BOB, Boutin S (2003) Declines in populations of woodland caribou. J Wildl Manag 67:755–761 Government of Alberta (2017) DRAFT: provincial woodland caribou range plan. https://open.alberta.ca/dataset/932d6c22-a32a-4b4e- a3f5-cb2703c53280/resource/3fc3f63a-0924-44d0-b178-82da3 4db1f37/download/draft-caribourangeplanandappendices-dec20 17.pdf. Accessed 25 May 2023 McNay RS, Lamb CT, Giguere L, Williams SH, Martin H, Sutherland GD, Hebblewhite M (2022) Demographic responses of nearly extirpated endangered mountain caribou to recovery actions in central British Columbia. Ecol Appl 32:e2580. https://doi.org/ 10.1002/eap.2580 Green DM (2005) Designatable units for status assessment of endan‑ gered species. Conserv Biol 19(6):1813–1820. https://doi.org/ 10.1111/j.1523-1739.2005.00284.x Miller W, Wright SJ, Zhang Y, Schuster SC, Hayes VM (2010) Opti‑ mization methods for selecting founder individuals for captive breeding or reintroduction of endangered species. Pac Symp Bio‑ comput 15:43–53. https://doi.org/10.1142/9789814295291_0006 Gruber B, Unmack PJ, Berry OF, Georges A (2018) dartr: An r package to facilitate analysis of SNP data generated from reduced repre‑ sentation genome sequencing. Mol Ecol Resour 18(3):691–699. https://doi.org/10.1111/1755-0998.12745 t 15:43–53. https://doi.org/10.1142/9789814295291_0006 Mills LS, Allendorf FW (1996) The one-migrant-per-generation rule in conservation and management. Conserv Biol 10(6):1509–1518. https://doi.org/10.1046/j.1523-1739.1996.10061509.x Gubili C, Mariani S, Weckworth BV, Galpern P, McDevitt AD, Hebble‑ white M et al (2017) Environmental and anthropogenic drivers of connectivity patterns: a basis for prioritizing conservation efforts for threatened populations. Evol Appl 10(2):199–211 Nei M (1972) Genetic distance between populations. Am Nat 106(949):283–292 He X, Johansson ML, Heath DD (2016) Role of genomics and tran‑ scriptomics in selection of reintroduction source populations. Conserv Biol 30(5):1010–1018. https://doi.org/10.1111/cobi. 12674 Ochoa A, Storey JD (2021) Estimating FST and kinship for arbitrary population structures. PLoS Genet 17(1):e1009241. https://doi. org/10.1371/journal.pgen.1009241 Paradis E, Claude J, Strimmer K (2004) APE: analyses of phylogenet‑ ics and evolution in R language. Bioinformatics 20(2):289–290. https://doi.org/10.1093/bioinformatics/btg412 Hebblewhite M, White C, Musiani M (2010) Revisiting extinction in national parks: mountain caribou in Banff. Conserv Biol 24(1):341–344 Parks Canada Agency (2022a) Jasper National Park Caribou Program Progress Report 2017–2020. Jasper National Park, Parks Canada Agency Hoban S, Bertorelle G, Gaggiotti OE (2012) Computer simulations: tools for population and evolutionary genetics. Nat Rev Genet 13(2):110–122. References https://doi.org/10.1038/nrg3130 Parks Canada Agency (2022b) Proposal for consultation: conservation breeding strategy to rebuild small caribou populationpopulations in Jasper National Park. Jasper National Park of Canada, Parks Canada Agency Holsinger KE, Weir BS (2009) Genetics in geographically structured populations: defining, estimating and interpreting FST. Nat Rev Genet 10(9):639–650. https://doi.org/10.1038/nrg2611 Pelletier F, Réale D, Watters J, Boakes EH, Garant D (2009) Value of captive populations for quantitative genetics research. Trends Ecol Evol 24(5):263–270. https://doi.org/10.1016/j.tree.2008. 11.013 Holt RD (1977) Predation, apparent competition, and the structure of prey communities. Theor Popul Biol 12(2):197–229. https://doi. org/10.1016/0040-5809(77)90042-9 IUCN, SSC (2013) Guidelines for reintroductions and other conserva‑ tion translocations. Version 1.0. IUCN Species Survival Com‑ mission, Gland Pembleton LW, Cogan NO, Forster JW (2013) St AMPP: An R pack‑ age for calculation of genetic differentiation and structure of mixed-ploidy level populations. Mol Ecol Resour 13(5):946–952. https://doi.org/10.1111/1755-0998.12129 Ivy JA, Miller A, Lacy RC, DeWoody JA (2009) Methods and pros‑ pects for using molecular data in captive breeding programs: an empirical example using parma wallabies (Macropus parma). J Hered 100(4):441–454. https://doi.org/10.1093/jhered/esp019 Pimm SL, Jenkins CN, Abell R, Brooks TM, Gittleman JL, Joppa LN et al (2014) The biodiversity of species and their rates of extinc‑ tion, distribution, and protection. Science 344(6187):1246752. https://doi.org/10.1126/science.1246752 Kardos M, Luikart G, Allendorf FW (2015) Measuring individual inbreeding in the age of genomics: marker-based measures are better than pedigrees. Heredity 115(1):63–72. https://doi.org/10. 1038/hdy.2015.17 Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D (2006) Principal components analysis corrects for stratification 1 3 Conservation Genetics (2023) 24:855–867 867 in genome-wide association studies. Nat Genet 38(8):904–909. https://doi.org/10.1038/ng1847 cycles before the Last Glacial Maximum. Mol Ecol 30(23):6121– 6143. https://doi.org/10.1111/mec.16166 p g g Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155(2):945– 959. https://doi.org/10.1093/genetics/155.2.945 Taylor RS, Manseau M, Redquest B, Keobouasone S, Gagné P, Martineau C, Wilson PJ (2022) Whole genome sequences from non-invasively collected caribou faecal samples. Con‑ serv Genet Resour 14(1):53–68. https://doi.org/10.1007/ s12686-021-01235-2 p g g Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D et al (2007) PLINK: a tool set for whole-genome associa‑ tion and population-based linkage analyses. Am J Hum Genet 81(3):559–575. https://doi.org/10.1086/519795 Tokarska M, Marshall T, Kowalczyk R, Wójcik JM, Pertoldi C, Kris‑ tensen TN et al (2009) Effectiveness of microsatellite and SNP markers for parentage and identity analysis in species with low genetic diversity: the case of European bison. References Heredity 103(4):326–332. https://doi.org/10.1038/hdy.2009.73 Ralls K, Sunnucks P, Lacy RC, Frankham R (2020) Genetic rescue: a critique of the evidence supports maximizing genetic diversity rather than minimizing the introduction of putatively harmful genetic variation. Biol Cons 251:108784 Van Oort H, McLellan BN, Serrouya R (2011) Fragmentation, dis‑ persal and metapopulation function in remnant populations of endangered mountain caribou. Anim Conserv 14(3):215–224. https://doi.org/10.1111/j.1469-1795.2010.00423.x g Robert A (2009) Captive breeding genetics and reintroduction success. Biol Cons 142(12):2915–2922. https://doi.org/10.1016/j.biocon. 2009.07.016 Vors LS, Boyce MS (2009) Global declines of caribou and reindeer. Glob Change Biol 15(11):2626–2633. https://doi.org/10.1111/j. 1365-2486.2009.01974.xfi Russello MA, Jensen EL (2018) Ex situ wildlife conservation in the age of population genomics. Population genomics: wildlife. Springer, Cham, pp 473–492 Vucetich JA, Waite TA (2000) Is one migrant per generation sufficient for the genetic management of fluctuating populations? Anim Conserv 3(3):261–266. https://doi.org/10.1111/j.1469-1795. 2000.tb00111.x Salafsky N, Irvine R, Boshoven J, Lucas J, Prior K, Bisaillon JF et al (2022) A practical approach to assessing existing evidence for specific conservation strategies. Conserv Sci Pract 4(4):e12654. https://doi.org/10.1111/csp2.12654 Wang J (2004) Application of the one-migrant-per-generation rule to conservation and management. Conserv Biol 18(2):332–343. https://doi.org/10.1111/j.1523-1739.2004.00440.x Schweizer RM, Vonholdt BM, Harrigan R, Knowles JC, Musiani M, Coltman D et al (2016) Genetic subdivision and candidate genes under selection in North American grey wolves. Mol Ecol 25(1):380–402. https://doi.org/10.1111/mec.13364 Weckworth BV, Musiani M, McDevitt AD, Hebblewhite M, Mariani S (2012) Reconstruction of caribou evolutionary history in Western North America and its implications for conservation. Mol Ecol 21(14):3610–3624. https://doi.org/10.1111/j.1365-294X.2012. 05621.x Seddon PJ, Armstrong DP, Maloney RF (2007) Developing the science of reintroduction biology. Conserv Biol 21(2):303–312. https:// doi.org/10.1111/j.1523-1739.2006.00627.xfi g j Seddon PJ, Griffiths CJ, Soorae PS, Armstrong DP (2014) Revers‑ ing defaunation: restoring species in a changing world. Science 345(6195):406–412. https://doi.org/10.1126/science.1251818 Weeks AR, Sgro CM, Young AG, Frankham R, Mitchell NJ, Miller KA et al (2011) Assessing the benefits and risks of transloca‑ tions in changing environments: a genetic perspective. Evolut Appl 4(6):709–725f Serrouya R, Paetkau D, McLellan BN, Boutin S, Campbell M, Jenkins DA (2012) Population size and major valleys explain microsatel‑ lite variation better than taxonomic units for caribou in western Canada. Mol Ecol 21(11):2588–2601. https://doi.org/10.1111/j. 1365-294X.2012.05570.x Williams SE, Hoffman EA (2009) Minimizing genetic adaptation in captive breeding programs: a review. Biol Cons 142(11):2388– 2400. https://doi.org/10.1016/j.biocon.2009.05.034 Serrouya R, Seip DR, Hervieux D, McLellan BN, McNay RS, Steen‑ weg R et al (2019) Saving endangered species using adaptive management. References Proc Natl Acad Sci USA 116(13):6181–6186. https://doi.org/10.1073/pnas.1816923116 Williams SH, Steenweg R, Hegel T, Russell M, Hervieux D, Heb‑ blewhite M (2021) Habitat loss on seasonal migratory range imperils an endangered ungulate. Ecol Solut Evid 2(1):e12039 Witzenberger KA, Hochkirch A (2011) Ex situ conservation genet‑ ics: a review of molecular studies on the genetic consequences of captive breeding programmes for endangered animal species. Biodivers Conserv 20(9):1843–1861. https://doi.org/10.1007/ s10531-011-0074-4 Serrouya R, Bollefer K, Cook R, Gilbert S, Gill R, Legebokow C, MacBeth B, Schwantje H, Thacker C (2021) Final Report for Revelstoke Caribou Rearing in the Wild. Nelson: British Colum‑ bia Ministry of Forests, Lands, Natural Resource Operations and Rural Developmentl Wright S (1931) Evolution in Mendelian populations. Genetics 16(2):97. https://doi.org/10.1093/genetics/16.2.97 Slatkin M (1987) Gene flow and the geographic structure of natural populations. Science 236(4803):787–792. https://doi.org/10. 1126/science.3576198 Wright BR, Hogg CJ, McLennan EA, Belov K, Grueber CE (2021) Assessing evolutionary processes over time in a conserva‑ tion breeding program: a combined approach using molecular data, simulations and pedigree analysis. Biodivers Conserv 30(4):1011–1029. https://doi.org/10.1007/s10531-021-02128-4 Solmundson K, Bowman J, Wilson PJ, Taylor RS, Horn RL, Keob‑ ouasone S, Manseau M (2020) Genomic islands of heterozygo‑ sity maintained across caribou populations despite inbreeding. bioRxiv. https://doi.org/10.1101/2020.12.29.424772 Yannic G, Pellissier L, Ortego J, Lecomte N, Couturier S, Cuyler C et al (2014) Genetic diversity in caribou linked to past and future climate change. Nat Clim Chang 4(2):132–137. https://doi.org/ 10.1038/nclimate2074 Soorae PS (ed) (2018) Global reintroduction perspectives, 2018: case studies from around the globe. IUCN-International Union for Conservation of Nature and Natural Resources, Gland Taylor RS, Manseau M, Horn RL, Keobouasone S, Golding GB, Wil‑ son PJ (2020) The role of introgression and ecotypic parallel‑ ism in delineating intraspecific conservation units. Mol Ecol 29(15):2793–2809. https://doi.org/10.1111/mec.15522 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. p g Taylor RS, Manseau M, Klütsch CF, Polfus JL, Steedman A, Hervieux D et al (2021) Population dynamics of caribou shaped by glacial 1 3
https://openalex.org/W2460308583	https://www.repository.cam.ac.uk/bitstream/1810/256810/1/Fuhr-2016-Qualitative_Sociology-VoR.pdf	English	null	Vicarious Group Trauma among British Jews	Qualitative sociology	2,016	cc-by	14,114	Qual Sociol DOI 10.1007/s11133-016-9337-4 Vicarious Group Trauma among British Jews Christina Fuhr1 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Given that literature on the intra- and inter-generational transmission of traumas is mainly based on secondary literature and focuses on the transmission of trauma memory in terms of the historical knowledge of group trauma, this article develops the theory of vicarious group trauma and tests this theory by exploring vicarious traumatization in the everyday lives of Jews in Britain through the methods of observation and in-depth interviewing. Vicarious group trauma is defined as a life or safety-threatening event or abuse that happened to some members of a social group but is felt by other members as their own experience because of their personal affiliation with the group. The article finds that the vicarious sensation of traumatic group experiences can create anxiety, elicit perceptions of threat and, by extension, hypervigilance among Jews. The findings demonstrate that group traumas of the past inter- penetrate and interweave with members’ current lives and in this way can also become constitutive of their group identity. An institutional focus on threats to Jews can inform the construction and reinforcement of traumatization symptoms and accordingly vicarious group trauma. This article suggests an association between the level of involvement of group members in the collective’s social structure and the prominence of vicarious group trauma among them. Keywords Britain . Emotions . Holocaust . Identity. Jews . Memory. Perception . Trauma . Traumatization Keywords Britain . Emotions . Holocaust . Identity. Jews . Memory. Perception . Trauma . Traumatization There is a focus in sociology on the intra- and inter-generational transmission of traumas (e.g. Alexander et al. 2004; Eyerman et al. 2011). Most of this research is based on secondary literature and focuses on the transmission of trauma memory in terms of the historical knowledge of group trauma and its impact on the identity of the collective. In this article, I extend this work by studying the symptoms of traumatization among members who have not gone through the group trauma themselves. I argue that in this way transmitted traumas can become constitutive of members’ group identity. To do so, I develop the theory of vicarious * Christina Fuhr christina.fuhr@gmail.com 1 Woolf Institute, 12-14 Grange Road, Cambridge, UK 1 Woolf Institute, 12-14 Grange Road, Cambridge, UK Qual Sociol group trauma. Vicarious group trauma is defined in this article as a life- or safety-threatening event or abuse that happened to some members of a social group but is felt by other members as their own experience because of their personal affiliation with the group. When many members of a group feel as if they experienced the group trauma themselves by having symptoms of traumatization or can be attested to have those symptoms, we speak of vicarious group trauma. This article will test the theory by exploring vicarious traumatization in the everyday lives of Jews in Britain through the methods of observation and in-depth interviewing. Therefore, the article presents a description of the actual traumas that are communicated to and remem- bered by different kinds of Jewish people. It finds that secular participants—meaning syna- gogue (affiliated) members who are less or not at all engaged with Judaism as a religion, culture and community as well as synagogue (unaffiliated) members—tend to remember only the Holocaust. This is proposed to be related to the communication of the event through mainstream culture and education in addition to, if applicable, personal family stories. Devout affiliated members have a propensity to also remember earlier Jewish traumas such as the enslavement in Egypt and their exodus over a hundred years later. This is suggested to be related to the observation of holy days associated with those traumas in Jewish schools, homes and synagogues, on top of, if applicable, family stories. Keywords Britain . Emotions . Holocaust . Identity. Jews . Memory. Perception . Trauma . Traumatization Due to a personal identification with the trauma on an emotional level because of a shared group membership, members of the community can feel the traumatic experience as their own. The article then illustrates that the vicarious sensation of such traumatic group experiences can create anxiety, elicit perceptions of threat and, by extension, hypervigilance among Jews. Vicarious traumatization symptoms, particularly perceptions of threat and hypersensitivity, are prominent among community-involved unaffiliated and particularly among engaged affiliated members. The narratives below demonstrate that group traumas of the past interpenetrate and interweave with members’ current lives and in this way can also become constitutive of their group identity. Lastly, the article shows how an institutional focus on past and present threats to Jews can inform the construction of traumatization symptoms and accordingly vicarious group trauma. Considering that vicarious trauma is present to a greater extent among community-involved Jews, especially affiliated engaged ones, this type of member is also found to be more exposed to constant and multiple traumatization mediators—communicated indicators of current threat and past trauma—through communal structures because of their physical and social proximity to them. Thus, this article indicates an association between the level of involvement of group members in their collective’s social structure and the prominence of vicarious trauma among them. Overall, the article illustrates how a group’s institutions, including the family, can create a siege mentality among members by emphasizing threats to them. The Jewish community presents an obvious and interesting case study as research has shown that trauma memory and particularly the Holocaust is prominent among members of this group and an essential part of their identity (e.g. Alexander 2004a; Freud 1932; Zerubavel 1996; Lazar et al. 2008; Kugelmass 1996); however, there is a lack of such research with regards to British Jews, highlighting the necessity for this present study. The evidence provided for the Jewish group is regarded as symptomatic of the impact conflict can have on any minority group. For example, the recent war in Syria can be expected to affect many Syrians around the world as much as the 1994 genocide for the Rwandan diaspora. Equally disturbing can be traumas that are less recent, such as the times of slavery for the Afro- American community in the United States or the partition of Punjab in 1947 for Sikhs. Vicarious Group Trauma There has been a focus in sociological research on the exploration of trauma transmission. Most of such research, however, uses secondary literature to examine how trauma memory in the form of historical facts and experiences of trauma is transmitted to members who have not personally experienced the trauma, and can become part of their association with the group and in this way binds them together rather than disconnecting them (Assmann 2006; Eyerman 2011; Eyerman et al. 2011). Zerubavel (1996), for instance, theorizes that Bbeing social presupposes the ability to experience events that happened to groups and communities to which we belong long before we joined them as if they were part of our own past.^ Alexander et al. (2004, 1) similarly assert that Ba cultural trauma occurs when members of a collectivity feel they have been subjected to a horrendous event that leaves indelible marks upon their group consciousness, marking their memories forever.^ To construct a cultural trauma, the memory of the traumatic event needs to be part of the cultural and public discourse and represented as being destructive and threatening to the existence of the group, its culture, and members’ personal identity. Contributors to the book apply the model in a series of case studies, including the Holocaust and slavery in the United States. There is also a small set of qualitative and quantitative literature that analyses the Holocaust as a cultural trauma specifically in the second and third-generation offspring of Holocaust survivors (Scharf 2007; Kellerman 2001; Litvak-Hirsch and Bar-On 2006), also in comparison to the offspring of non-survivors (Lazar et al. 2004). The survey findings by Lazar et al. (2008) suggest that there are sociocultural mechanisms at work in Israel—such as Holocaust education and the country’s perceived role as providing security for Jewish people—that influence the third-generation offspring of both Holocaust survivors and non-survivors to perceive the Holocaust as a cultural trauma.1 Indeed, there are various social mechanisms that can be responsible for the transmission of trauma memory. Research by scholars such as Alexander et al. (2004) and Eyerman (2011) demonstrates the importance of carrier groups (e.g. the mass media) in imparting trauma to members who have not experienced it directly. In this way the trauma becomes part of the group identity and can therefore also change a group’s Bfuture identity in fundamental and irrevocable ways^ (Alexander 2004b). 1 These recent quantitative findings resonate with analyses from earlier studies based on secondary literature such as Ben-Amos and Bet-El’s (1999) research about collective trauma remembrance in the form of memorial ceremonies in Israeli schools and its impact on Jewish national identity. Keywords Britain . Emotions . Holocaust . Identity. Jews . Memory. Perception . Trauma . Traumatization All of Qual Sociol those traumas have been communicated through social and, thus, institutional mechanisms such as museums, civic commemorations and the media. Hence, the perspectives of Jews in this study will provide a significant example of the real and long-term effects transmitted group traumas can have on their members and the identification with their collective as well as the powerful role institutions play in creating and maintaining a vicarious trauma experience. Vicarious Group Trauma Carrier groups Barticulate the significance of and represent the trauma for the collective…making it available for communication and shared understanding…They help transform emotional responses into words and images that can be dispersed and remembered^ (Eyerman 2011). Hirsch (2008), for instance, examines how the trauma of the Holocaust is transmitted through the arts, specifically photography, to the second generation. Hirsch (2008, 103) developed the concept of postmemory to describe Bthe rela- tionship of the second generation to powerful, often traumatic, experiences that preceded their Qual Sociol births but that were nevertheless transmitted to them so deeply as to seem to constitute memories in their own right.^ Landsberg (1997, 2004) emphasizes the importance of mass cultural technologies—producing a unique transmission of images and narratives about the past—in making it possible to take on memories of events through which people did not live. The sensuous engagement that experiential museums or technological developments (e.g. the mass dissemination of film) provide enables secondary memory formation. Landsberg (1997, 66) referred to these memories as prosthetic memories, arguingthatwhileBthe traumatic experiencesare notoriginallybased, they are nevertheless experiences with one’s own body…and as such, become part of one’s personal archive of experience, informing not only one’s subjectivity, but one’s relationship to the present and future tenses.^ g y j y, p p While the sociological literature has focused on the transmission of trauma memory on the group level, the psychiatric and psychology literature has concentrated on exploring trauma transmission at the individual level, understanding the term trauma simply Bas a wound inflicted not upon the body but upon the mind^ (Caruth 1996; Eyerman 2011). Accordingly, trauma is an emotional experience of the historical knowledge of trauma that is stored in a person’s long- term memory and is so strong as to bypass rationality and affect the person’s cognition and emotions. Such research has looked at how the trauma can be transmitted to another person. Studies such as by McCann and Pearlman (1990) found that trauma may be experienced by people who come into contact with trauma victims over a prolonged period of time, e.g. family members or therapists, as they are able to suffer signs and symptoms of traumatization similar to those of the victim. They termed the process vicarious traumatization. The outcome has also been labelled secondary trauma by others such as Figley (1983). Vicarious Group Trauma A personal association with the transmitted trauma knowledge on the emotional level as a result of group membership can be a common phenomenon among members because they are able to feel that the trauma could have happened to them as well. This emotional immediacy makes the memory powerful enough to elicit strong emotions and perceptions. Group membership is crucial in the construction of vicarious group trauma. For example, a non-Jewish person visiting the Holocaust Museum in Washington DC may start feeling a connection with the trauma and adopt the prosthetic memory as described by Landsberg (2004). It may, however, be unlikely that the memory will be strong enough to result in secondary traumatization – by having an emotional experience that allows them to feel as if they themselves have been subject to the traumatic experience and results in symptoms of trauma- tization – if they have no direct ethno-religious association with the group, or, as noted above, if they have not spent time with a survivor of that trauma. The historical fact of trauma, which may also involve an emotional response providing the memory with strength, can be taken on by anyone without any direct personal relation to the traumatic event, or the person(s) it happened to. However, the actual traumatization with its symptoms as a result of the communicated trauma knowledge and accordingly the viscerally felt traumatic experience may not be created. It follows, then, that vicarious group trauma can act within and across many generations and become part of members’ identity. The theory distinguishes itself clearly from other theories on trauma transmission, such as Hirsch’s (2008) concept of postmemory, as the focus of its conceptualization rests on the transmission of the actual trauma in terms of an emotional traumatic experience rather than the historical knowledge of trauma. Reviewing the literature, we find that there is a gap in sociological research studying secondary group traumatization on the micro-level. Ethnographic research is needed to study not only how vicarious group traumatization manifests itself among individual members, impacting their identification with the group, but also how the traumatization is informed and sustained. While there have been interesting analyses of how past Jewish traumas can be a part of group identity and can be effective in sustaining that identity (e.g. Freud 1932), there is little published research about group trauma among British Jews and its effect. Vicarious Group Trauma For example, there is evidence that children of Holocaust survivors (Danieli 1985; Prince 2009) and Vietnam combat veterans (Rosenheck and Nathan 1998) can experience social and psychological difficulties that are symptomatic of posttraumatic stress disorder. The psychiatric and psychology literature dem- onstrates on the individual level how the actual trauma can be transmitted to another person who has not lived through the traumatic experience themselves by analyzing the symptoms of traumatization that they display. Nevertheless, there is a gap in the sociological literature exploring empirically vicarious traumatization in everyday life on the group level. As a response, I outline a theory of vicarious group trauma. Vicarious group trauma is defined in this article as a life or safety-threatening event or abuse that happened to some members of a social group but is felt by other members as their own experience as they personally identify with the trauma on an emotional level because of their shared group membership. As mentioned above, sociological and psychological literature already suggests it is the resulting emotional experience of the life- or safety-threatening event or abuse, despite not having suffered the trauma themselves, which creates trauma memory. For example, Landsberg (2009) argues that people who have not lived through trauma can still adopt its memory through the personal emotional experience of empathy in response to the communicated trauma knowledge. I extend this theory by arguing that through individuals’ personal association with the trauma as a member of the group, the historical fact of trauma or the communicated knowledge of the life or safety-threatening event or abuse can become an emotional trauma experience – an emotional wound - for individuals, who have not directly experienced it. The historical knowledge is tagged with powerful emotions so that it becomes an experience for them, and in this way, the transmitted trauma can impact on their emotions and perceptions of the world. When many members can be attested, or they themselves attest, to have this emotional experience associated with the trauma, the personal visceral trauma becomes a shared trauma; in other words vicarious group trauma. Qual Sociol The actual trauma memory can differ between members who have gone through the trauma themselves and those who have not, but both types of members may, nonetheless, have an emotional experience of the remembered trauma, which can be felt in similar ways. Vicarious Group Trauma Cooke (2000) examines how debates over the perceived appropriateness of Holocaust memorial sites in London structure various discourses concerning Anglo-Jewish identity. Another study, by Berman (2004), looks at Holocaust commemorations in London and discusses the relationship between the commem- oration and unity formation, suggesting that associated rituals and collective mourning help to bind this community together. While both are helpful in shedding light on the importance of trauma remembrance in the creation and perpetuation of Jewish identity in Britain, neither of these or other studies engaged in primary research methods such as ethnography. This work uses in-depth interviewing and observations to contribute to the existing research by exploring vicarious traumatization among members of the British Jewish community. Methods This research is part of a larger ethnographic study on Jewish identity construction and perpetuation in contemporary Britain. The data are derived from observations of, and semi- structured interviews with, British Jews. The interviews first asked very open questions about Qual Sociol interviewees’ sense of Jewishness. This allowed me to follow up on what the respondents regarded as important in their identification with Judaism. The rest of the questions dealt with their upbringing, the impact being Jewish had on their current lives, the community, Jewish language, their relations to other Jews, as well as their views on particular topics, namely Christmas, partnership, Israel, and anti-Semitism past and present. Interview findings in this article are not based on answers to a specific question about the Holocaust. While I asked BDo you think that the Holocaust has an impact on your life?^ I only posed this question towards the end of the interview as part of a wider set of questions dealing with anti-Semitism. By then vicarious group trauma and symptoms of traumatization had often already come up in implicit and explicit ways. The data were collected between October 2008 and October 2009. My study focuses on Modern Orthodox as well as Progressive (Liberal and Reform) Jews and Jews without synagogue affiliation. These three groups are the largest ones in Britain. Among the affiliated Jewish groups, the Modern Orthodox is the biggest group (55 %), followed by the Progressive (19.4 % Reform, 8.7 % Liberal)2 and then the Ultra-Orthodox (11 %), Sephardim (3.5 %) and Masorti (2.7 %)3 (Graham and Vulkan 2010). The unaffiliated population size ranges between 25 and 50 % according to statistics (Schmool and Cohen 1998; Graham and Vulkan 2010) and predictions made by researchers from the Institute of Jewish Policy Research in London. Although most of the in-depth interviews were conducted with Jews from these three groups, I also interviewed a few Masorti and Ultra-Orthodox members in order to get a well- rounded overview of Jewish identification. Of the 105 interviewees I conducted, four inter- views were with Ultra-Orthodox members, 33 with Modern Orthodox Jews, four with Masorti, 19 with Progressive and 45 with unaffiliated Jews. All respondents were above 18. The majority of interviewees were below 50. The inclusion of a substantial number of interviewees below 50 allowed me to look carefully at whether younger Jews express significantly different answers to my interview questions. 2 Liberal Judaism in Britain is similar to the Reform Judaism in North America. The North American Reform movement and British Liberal Judaisms are located on the more radical end of the non-orthodox movements of Judaism, followed by the British Reform movement. 3 Masorti Judaism is synonymous to Conservative Judaism in the United States and Canada. 4 As part of a full year of observation in different Jewish communities, I participated in the marking of every major holy day in the Jewish calendar at homes and different synagogues. I also visited people’s houses, or met with them for food or drinks outside of the home at other times. I went to a bris (circumcision rite) and several birthdays, engagements and weddings. I did observations at the Jewish Learning Exchange, Jewish Book Week, the UK Jewish Film Festival, exhibitions about Jews and Judaism, Jewish theatre plays in London, youth group meetings such as Bnei Akiva gatherings, and at the Jewish cultural conference Limmud. I visited a Jewish primary school and went to talks, seminars, workshops and movies about Jewish topics at the Jewish Community Centre for London, London Jewish Cultural Centre, the Jewish Learning Exchange, synagogues, Universities, the Oxford University Chabad Society, and the Oxford University Jewish society. I spent time walking around Jewish areas, sitting in cafes and restaurants, and going to Jewish shops to observe members’ interactions with each other. In terms of political observations, I visited the office of the Chief Rabbi and the United Synagogue Beth Din (rabbinical court), Tribe (the youth organization of the United Synagogue), the Board of Deputies of British Jews, the Institute for Jewish Policy Research, United Jewish Israel Appeal, Birthright Israel and the Community Security trust (CST). Methods I refer to the expert interviewees by their title, which is their real title, and surname in the text, as it is important to distinguish the experts I interviewed in an official capacity from the respondents, who spoke to me as a private person. Regarding the observational part of the study, I have conducted most of the observations in densely Jewish-populated areas in North London, considering that the majority of Jews live in London and most of those London Jews live in the North-West London boroughs of Barnet, Hackney, Camden and Harrow (JPR 2000). Certain neighbourhoods in these boroughs have a Jewish population of 50–75 % (Graham et al. 2007). Conducting observations in such densely populated neighbourhoods provided me with a profound understanding of the social structure and texture of the Jewish community. I obtained access to observations such as synagogue services as well as social gatherings (e.g. Sabbath dinners and seminars) through gatekeepers such as rabbis and individuals met during observations and interviews.4 During my observa- tional fieldwork, my reason for attendance was either communicated when being introduced to participants, or I communicated my researcher status in social interactions with participants such as by talking with them about my study as well as the reasons for my attendance. I also conducted 16 expert interviews with employees, researchers and representatives of Jewish institutions as well as with academics active in Jewish-related fields of study. They were also allocated pseudonyms at random. I refer to the expert interviewees by their title, which is their real title, and surname in the text, as it is important to distinguish the experts I interviewed in an official capacity from the respondents, who spoke to me as a private person. Regarding the observational part of the study, I have conducted most of the observations in densely Jewish-populated areas in North London, considering that the majority of Jews live in London and most of those London Jews live in the North-West London boroughs of Barnet, Hackney, Camden and Harrow (JPR 2000). Certain neighbourhoods in these boroughs have a Jewish population of 50–75 % (Graham et al. 2007). Conducting observations in such densely populated neighbourhoods provided me with a profound understanding of the social structure and texture of the Jewish community. I obtained access to observations such as synagogue services as well as social gatherings (e.g. Methods Sabbath dinners and seminars) through gatekeepers such as rabbis and individuals met during observations and interviews.4 During my observa- tional fieldwork, my reason for attendance was either communicated when being introduced to participants, or I communicated my researcher status in social interactions with participants such as by talking with them about my study as well as the reasons for my attendance. Methods I expected to observe differences between younger and older Jews with respect to experiences of traumatization in response to transmitted trauma memory, given that the Holocaust may be a more distant memory for younger Jews. Such differences could not be observed. The interview sample has an approximately equal spread of gender (56 % male, 44 % female) and different levels of engagement with Judaism. Engagement with Judaism is measured in terms of the interviewees’ level of ethno-religious practice (e.g. observance of the major holy days and dietary restrictions) and their involvement with the community, including the culture. Regarding the former, I looked at the respondents’ involvement with other Jewish people (e.g. friends and acquaintances) and in community institutions. In consideration of the latter, I looked at their engagement in BJewish activities,^ such as reading about Jewish related issues or books by Jewish authors. The overall project was primarily interested in finding out how Jewish identity is sustained and perpetuated. Thus, while the study also included unaffiliated Jews who would not mention that they were Jewish unless they were asked and were completely removed from the Jewish community, religion and culture, its focus was on Jews who maintain an affiliation to Judaism. The sample does not reflect a wide demographic spread in terms of social class. The majority of the respondents Qual Sociol identified themselves as middle class (84 %). The statistic of middle-class respondents does, however, resonate with the results of the 2011 Census, suggesting that a majority of Jews in Britain are middle class (Graham et al. 2007). Most of the interviewees were from London, as over half of Britain’s enumerated Jews (171,960 of 269,568) live in the London region (Graham et al. 2012; Graham 2013). All interviewees gave informed consent. For reasons of confidentiality, the interviewees are identifiable by pseudonyms, which I selected on an arbitrary basis. Still, if respondents had Jewish names, I allocated them different but equally Jewish names. The article will also mention their age bracket, status of affiliation, and Holocaust association where applicable. Holocaust association where applicable. I also conducted 16 expert interviews with employees, researchers and representatives of Jewish institutions as well as with academics active in Jewish-related fields of study. They were also allocated pseudonyms at random. Remembered Traumas Participants often remembered traumas in which their family members died due to or survived anti-Semitism. The remembered traumas varied from discrimination and hostility against Jews to attacks and organized persecution throughout history. Over half of my interview sample mentioned close or distant family members enduring anti-Semitism in the past. These remem- bered traumas were mainly more recent ones, such as the Eastern European Pogroms, the Qual Sociol Holocaust, and the persecution of Jews in Argentina and Iraq. For example, Modern Orthodox Evan, in his 40s, whose parents fled from Baghdad, noted, ‘‘I have grown up in a Baghdadi family…you are conscious of the fact that there have been terrible times for Jews in Iraq.’’ A few respondents also remembered earlier family traumas, such as the persecution of relatives in Portugal and Spain. For example, when unaffiliated Brianne, who was in her 50s, defined her identity, she argued: It is about having some built-in understanding where Jews have come from, what’s happening to the Jewish populations over the centuries and identifying with that… knowing that one branch of my family were expelled from Spain in the fifteenth century for being Jewish. I have quite a lot of Sephardies in my family so I know that that part of my family suffered persecution as far as that. In narratives about their lives, however, such interviewees referred not only to the trauma experiences of their own family members but also to the trauma of the group as a whole. For instance, the unaffiliated Erica, who was in the same age bracket as Brianne, and whose parents survived the Holocaust and became refugees in England, felt Jewish because of her family’s history of persecution. Still, she referred to the impact the Holocaust trauma had on her in general terms: You’ll find that’s quite a common trait in the next generation of people who have suffered trauma that they become anxious…There’s a part of me that separates me from others that is the Jewish part of me that defines very much my reaction to situations…I suppose in any political situation, there’s always in the back of your mind, what is the Jewish angle to it. How does it affect being Jewish? It has been quite horrible with all the stuff going on in the Middle East. You do feel quite sort of anxious. Remembered Traumas The Reform interviewee Richard, who was in his 20s, mentioned anti-Semitism in associ- ation with his anxiety about his physical safety in Britain and argued that it reminded him of the history of anti-Semitism, noting the Holocaust, the Eastern European Pogroms as well as the following memory of his family trauma: BMy family had to leave Limerick in Ireland due to a pogrom in the early twentieth century, which was a religious-based pogrom, based on a local Catholic priest.^ Interviewees also remembered traumas even if their family did not directly experience them and most importantly they could also remember them as their own trauma. The young unaffiliated Jim, who lived removed from the Jewish community and did not have any family involved in Holocaust, stated: BThey [the Nazis] killed us.^ Modern Orthodox Ophra, who was in her 30s and whose in-laws went through the Holocaust and lost everything they owned, offered another example: BI am Jewish. The history of Jewish persecution is my history…The Nazis have tried to kill us. This is part of me.^ As the examples demonstrate many participants used subjective and personal pronouns such as I, us and we when talking about their group’s traumatic experiences, already indicating the vicariousness of the trauma for them. Interestingly, there was a subtle difference in trauma memory between secular and engaged affiliated participants. Secular Jews focused more exclusively on the memory of the Holocaust, whereas engaged affiliated participants tended to remember the Holocaust in association with earlier traumas, such as the enslavement in Egypt and their exodus over a hundred years later, the destruction of the First and Second Temple, the rededication of the holy Temple in Jerusalem at the time of the Maccabean Revolt of the second century BC, or the trauma of being nearly annihilated in the ancient Persian Empire. My findings suggest that engaged Qual Sociol affiliated members’ longer trauma memory can be related to their observation of holy days associated with those earlier traumas in the home, synagogues and Jewish (Sunday) schools. The enslavement in Egypt and the subsequent exodus of Jews is commemorated at Pesach, the first and second destruction of the Temple is remembered on the 9th of Av, the rededication of the Holy Temple in Jerusalem is commemorated in the festival of Chanukah, and the trauma of nearly being annihilated in the ancient Persian Empire is commemorated at Purim. Remembered Traumas In contrast, remembrance of the Holocaust among all types of participants is not unexpected considering the focus placed on the Holocaust in Jewish and non-Jewish education as well as mainstream culture such as movies, documentaries and exhibitions about the Holocaust. For instance, Jim, quoted above, who went to a non-Jewish school, said: BI did my A-level coursework on the persecution of Jews in the past 2000 years. Every country that I go to on our holiday, my mum used to take me to the Holocaust museum there, every single one. So I have been in Paris, Washington, New York, Rome, all over.^ The communication of past trauma can inform the personalization of the trauma memory in terms of creating awareness that one could have also suffered the trauma because of one’s group membership, assisting in creating a vicariously felt trauma experience as the following sections demonstrate. Vicarious Traumatization It makes me more cautious on the streets…I have an absolute terror of being hurt…it separates me from the non-Jewish world and strengthens and connects me more with the J i h l It l h t d J i h l d f This quote highlights how the mechanisms in response to this type of trauma memory can interrelate and the importance of group membership in experiencing vicarious traumatization and thus vicarious group trauma. Even the very unaffiliated Alonso, in his 50s, who grew up in Argentina and had family members suffering anti-Semitism there, claimed that his group shares an inherent trauma anxiety, which he argued is related to him being connected with other Jews in the past and present. There is a sense of national anxiety in Jews…If someone would say something very bad about Israel; I would react in a different manner than you are going to react, because obviously you feel immediately what is actually touched is something that…is more in the limbic system. That is where the emotions are located and associated with a much more primitive way of being. A comment like that is going to touch the trauma that has gone on for 2000 years, so I cannot react like you…The trauma is actually connected to a historical part of being part of a group. So, for instance, let’s say that Hitler would have actually won. I’m telling you today that I wouldn’t be here talking to you so it can actually not be traumatic for me, but that’s also why it is. This quote also illustrates that anxiety can be associated with other asserted mechanisms, in this case the perceived threat of a future trauma. The fact that this particular unaffiliated interviewee related his trauma anxiety to the continuous persecution of Jews over the years makes him an exception to a strong pattern; most secular Jews in my ethnographic sample related their anxiety to the Holocaust alone. This can be associated with the fact that secular members do not tend to follow any or many ethno-religious rituals related to the commem- oration of earlier traumas, but are exposed to diverse information about the Holocaust in their daily lives, e.g. through family, the media or schools. 5 There was an arson attempt on the Brondesbury Synagogue in Willesden, North-West London on the fourth of January 2009 in response to Israel’s operation on December 27 2008. Assailants attempted to set fire to its entrance door. The interviewee may have recalled the arson attempt as a firebomb attack as the Jewish Chronicle’s headline about the event was Bbid to firebomb synagogue as protests grow^ (Silver 2009). Vicarious Traumatization The mechanisms elicited in response to trauma memory will be analysed indepen- dently of whether participants have family members that survived or were killed in Jewish traumas. This is because, as the last section has illustrated, interviewees who had family members involved in traumas did not only refer to the trauma of their family in narratives about their lives, but also the traumas of the whole group. Moreover, I did not find a significant difference in the mechanisms elicited by vicarious group trauma between the participants with family histories of Jewish trauma and those without them. To illustrate the lack of disparity between these two samples, the article will provide details of whether the quoted respondents had close family members that survived or were killed in the Holocaust where applicable. Firstly, remembered traumas, especially more recent ones like the Holocaust and the Eastern European pogroms, elicited in many participants a feeling of anxiety; they felt anxious about having to undergo another trauma in the future because of their Jewishness. This trauma anxiety, in turn, informed their identification with the group. Anxiety is defined in this study as a deep-seated emotional state of discomfort and apprehension about future uncertainties. For the Modern Orthodox interviewee Johanna, who was in her 20s and did not have close family who died in or survived the Holocaust, the Holocaust in conjunction with earlier traumas evoked anxiety, which informed her sense of group belonging and accordingly identity: I can’t really disassociate the Holocaust from my images of anti-Semitism throughout Jewish history…it’s been up and down. Sometimes we’ve been absolutely great like now in Britain. Jews are at a height of doing fine, and then we’ll be the lowest of low, and there will be crusades or a pogrom or something…Anyone who is Jewish can be a victim of anti-Semitism. So it makes me feel bonded with them [Jews] in that way. This interviewee’s trauma anxiety was also connected with hypervigilance through the perceived threat of anti-Semitism: Qual Sociol It makes me more cautious on the streets…I have an absolute terror of being hurt…it separates me from the non-Jewish world and strengthens and connects me more with the Jewish people. It also pushes me more towards Jewish people and away from non- Jewish people. It makes me more suspicious and more distrustful. Vicarious Traumatization Whereas some participants, such as those quoted above, expressed anxiety in relation to vicarious group trauma very overtly, others expressed their anxiety more subtly in narratives about anti-Semitism or Israel. For instance, when I asked the community-engaged Modern Orthodox Steve whether it is still important to fight against anti-Semitism, he answered: ‘‘It’s imperative…because it’s based on forethoughts [of another trauma].’’ Similarly, Rudolph, in his 60s, who, unlike Steve, had close relatives going through the Holocaust, said when talking about anti-Semitism in Britain: You can never discount that [meaning another trauma happening to Jews]. One should never discount that. There have been problems for Jews in the last few years in England…Bad things do happen to Jews in England. Synagogues are being firebombed.5 People are being attacked in the street…Will it ever go back to twelfth century York? Qual Sociol Interestingly, a majority of respondents, mainly those who were affiliated, expressed their trauma anxiety in view of possible safety options: namely having Israel as their Bsafe haven^ (Zachariah, Modern Orthodox, 20s) or Binsurance policy^ (Hugh, Modern Orthodox, 50s); a place that would provide Jews with security in the event of another trauma. Even the mid- twenties Modern Orthodox Jonah, who did not have any direct Holocaust link and who was less involved in the community, felt comforted by the existence of Israel: I feel more secure with the fact that I have this insurance policy of Israel…Every Jew can go to Israel, no questions will be asked. They get citizenship immediately.6 They allowed in a million Russians in 1991 and these Russian Jews were very privileged...When people try to get rid of it, it makes me very uncomfortable. It makes me think actually when there were problems in Ethiopia or Yemen...they [Israelis] actually airlifted them in…The Israeli air force went in and got them out…I like having Israel for that reason. This particular finding should be read in light of the historical context in which political Zionism used the Holocaust and Jews’ existing trauma anxiety as a justification for the foundation of Israel (Novick 1999) and more importantly that there is support from Jewish institutions for the State of Israel. Although synagogues of the Progressive movement may not be as supportive of Israel as the Modern Orthodox movement and its leading institution, the United Synagogue, most synagogues will still be generally pro-Israel. For instance, the United Synagogue, to which most of the affiliated members in Britain belong (Graham and Vulkan 2010), Bstrongly believe[s] in the centrality of Israel in Jewish life^ (JLC 2016). Secondly, trauma can sensitize people to perceive potential dangers in situations or things in their environment. Accordingly, I found that many participants perceived threats from gentiles or non-Jewish entities based on the traumas their group had to endure in the past. Some participants perceived danger in the form of prospective actions from states such as Iran or terrorist groups such as Hamas. They drew on evidence from previous Jewish traumas to validate their perceptions, suggesting that threat perceptions are fuelled by vicariously expe- rienced group traumas. 6 The Law of Return is legislation enacted by the Israeli government in 1950 and amended in 1970 in response to Jewish persecution over the centuries, particularly the Holocaust. It gives all persons of Jewish ancestry (e.g. even grandchildren of Jews) and their spouses the right to move to Israel and get Israeli citizenship (Richmond 1993). For example, the young Modern Orthodox community-involved Alon, without close relatives experiencing the Holocaust, opined: I get worried about what’s happening in Iran...There’s going to be another threat to Jewish people and it is how Iran promises to wipe out Israel off the map…It probably just means that I am paranoid…X-million Jews would be killed for no reason like in the Holocaust...being Jewish and having that knowledge of history and knowledge of persecution…it does add an extra dimension of worries in the equation. The quote illustrates the importance of a personal association with the group in feeling perceptions of threats in the present because of trauma to Jews in the past. Several participants perceived anti-Semitism due to Israel’s domestic policy with regards to the occupied territories as threatening because it reminded them of traumas experienced by their group in the past. In particular affiliated participants’ expressed worries about a perceived increase in anti-Semitism following the airstrikes and ground invasion in Gaza in December 2008. This threat perception was often expressed very subtly in responses to the interview Qual Sociol question about whether it is important to fight against anti-Semitism. For example, the community engaged Reform interviewee Robin, in his 30s, who did not mention family relations involved in Jewish traumas, first distanced himself from the notion that it was still important to fight against anti-Semitism by asserting that he does not think the Holocaust could happen again: BI am not worried that I will be physically in danger in this country.^ However, later in the interview when he talked about the recent Gaza conflict, it suddenly appeared that Robin did have a threat perception, a threat that was for him connected to the Holocaust and to even more recent attacks on Jews, such as the attack on a Chabad centre in Mumbai in November 2008:7 It [anti-Semitism] doesn’t make me feel massively insecure as a Jew in Britain. It doesn’t feel it’s at that stage Byet.^ Only very, very recently have I become more personally worried because I think during the Gaza conflict some of the rhetoric became quite scary. 7 The Rabbi Gavriel and Rivkah Holtzberg, the directors of Chabad-Lubavitch of Mumbai, were killed during the terrorist attack by Islamist terrorists on their centre in Mumbai on November 26 2008. 8 The CST is the Community Security trust. It is a charitable organization funded by the Jewish community to provide representation and advice for the community on matters of security and anti-Semitism; e.g. provision of a yearly anti-Semitic discourse and incidents reports. Its aim is to protect it from “bigotry, anti-Semitism and terrorism” (CST 2011). This organization provides physical security, training and advice for the protection of British Jews. 9 Crime statistics show that physical and verbal attacks against Jews are relatively rare in Britain (CST 2010). From 2003 to 2013, there has been an average of 2 extreme cases of violence involving serious physical harm or a threat to life, 90 assaults, 66 damage and desecration incidents, 37 threats, 367 incidents of abusive behaviors against Jews and 24 anti-Semitic cases found in literature (CST 2014). Comparing the average number of anti- Semitic incidents from 2003 to 2013—586 incidents (CST 2014)—to the number of Jews by religion living in Great Britain—269,568 according to the 2011 Census (Graham 2013)—results in the statistic that 1 in 460 members experiences anti-Semitism in this country. The discrepancy between the relatively low number of anti- Semitic incidents and the prevalence of a threat perception among participants suggests that talking about and looking out for threats can reinforce a threat perception. 9 Crime statistics show that physical and verbal attacks against Jews are relatively rare in Britain (CST 2010). From 2003 to 2013, there has been an average of 2 extreme cases of violence involving serious physical harm or a threat to life, 90 assaults, 66 damage and desecration incidents, 37 threats, 367 incidents of abusive behaviors against Jews and 24 anti-Semitic cases found in literature (CST 2014). Comparing the average number of anti- Semitic incidents from 2003 to 2013—586 incidents (CST 2014)—to the number of Jews by religion living in Great Britain—269,568 according to the 2011 Census (Graham 2013)—results in the statistic that 1 in 460 members experiences anti-Semitism in this country. The discrepancy between the relatively low number of anti- Semitic incidents and the prevalence of a threat perception among participants suggests that talking about and looking out for threats can reinforce a threat perception. I do occasionally worry about security in Jewish buildings…At some point there probably will be a terrorist attack on a Jewish target in Britain…It will happen…In Mumbai, they attacked the Lubavitch home…We are not anywhere near that [the Holocaust]…I worry what’s gonna happen in Israel and how that’s gonna impact on Jews…I do support the CST financially.8 Robin’s attempt to distance himself from a perception of threat in association with the vicarious group trauma indicates that he consciously understands that such a mechanism can be elicited in response to this memory.9 His apparent inability to prevent himself from experiencing it points to the power of this personalized trauma memory. Moreover, in several conversations perceived threat of another trauma for Jews was expressed in a very offhand manner. After a Sabbath lunch in the beginning of 2009, a Modern Orthodox male in his early twenties talked about wanting to make Aliyah (i.e. move to Israel). He spoke mainly about the positive aspects of living in Israel, but, surprisingly, while getting ready to leave, he added: BIt’s getting dangerous here. Anti-Semitic attacks are increasing in Britain and all around the world. Look at France, all the Jews are leaving… They move to Israel.^ Without vicarious group trauma it would be unlikely for such a strong statement to be asserted. Considering that the Gaza conflict had erupted not long before and that members in the community talked about the increase in anti-Semitism due to Israel’s military actions, this particular threat response is understandable. When talking about previous traumas, some interviewees such as the young Modern Orthodox interview Josie, who did not have any direct Holocaust family connections, made 7 The Rabbi Gavriel and Rivkah Holtzberg, the directors of Chabad-Lubavitch of Mumbai, were killed during terrorist attack by Islamist terrorists on their centre in Mumbai on November 26 2008. 7 The Rabbi Gavriel and Rivkah Holtzberg, the directors of Chabad-Lubavitch of Mumbai, were killed during the terrorist attack by Islamist terrorists on their centre in Mumbai on November 26 2008. 8 The CST is the Community Security trust. It is a charitable organization funded by the Jewish community to provide representation and advice for the community on matters of security and anti-Semitism; e.g. provision of a yearly anti-Semitic discourse and incidents reports. Its aim is to protect it from “bigotry, anti-Semitism and terrorism” (CST 2011). This organization provides physical security, training and advice for the protection of British Jews. 9 Qual Sociol a link to talk about current threats to Jews, which demonstrates the association between the two even more clearly: We have that [the Holocaust] in common. Innately you feel threatened, we know the history of the Jewish people and it’s hard for the Jewish people; they’ve always had problems…In every generation it happens, we have had Hitler and the Nazis, now we think there’s a growing threat whether it’s the BNP [British National Party] or Islamic Extremism coming out publically saying they will kill the Jews, for example. There’s this feeling there and you can’t really ignore it. It also came to my attention that members often look out for signs of threat to their group— such as negative news about Israel or anti-Semitism—in the mainstream newspapers or in online media. For instance, the Liberal Anthony, in his 60s, whose family has been in the country for centuries, and subsequently would not be expected to have a threat perception, expressed his perception in this way: BIf you are Jewish, you look out for what they write about Jews. If they write something bad about Israel, you just see it. Such news stands out to you. It wouldn’t stand out to you [non-Jews].^ Anthony felt that Jews were, as he called it, Bhardwired^ to notice such reports or comments because of their personal and, hence, emotional association with the trauma memory. Some participants also noted that this threat perception also made them sensitive to the way gentiles talk about Jews or topics relating to them in their presence. Accordingly, a perception of threat can be expressed in sensitivity to possible threats and, as shown below, can also shade into hypervigilance. For instance, the unaffiliated Eric, in his 60s, who grew up with his mother and two aunts, who all survived concentration camps, noted: BYou just hear when people talk about Israel or Jews. It’s like you’re always on guard. You always want to know whether people are anti-Semitic; whether there’s a threat for us. It’s in us. History has taught us to be on the lookout.^ Negatively perceived comments about the group or Jewish issues, told by gentiles or written in the broadsheet press, as well as online, were often interpreted by participants as a sign of anti-Semitic sentiments or an increase of it amongst non-Jews as well as an indicator of threat to Jews. For example Adena, a Modern Orthodox in her 20s, said: Even though I was lucky enough not to have had my family—like my close family—directly affected by the Holocaust, I think that you can never lose sight of the fact that I think you’re living in a country at the mercy of that country….I live in a very benevolent country; I’m very lucky. Could that change in the future? Yes…Jews in Germany were very comfortable before the War, there was no one more German than the Jews, completely integrated into society, and I don’t know if we’ll ever lose sight of the feeling that times could change, and that we can’t depend on it. Later in her interview, she gave an example of how the perception of threat in response to her vicariously felt trauma experience can manifest: Jews are very conscious of anything negative or high profile about anybody who is Jewish. For example, Bernard Madoff…Immediately non-Jews will think Bhe’s Jewish^ [derogatory tone] and immediately all the stereotypes will come up: Jew, wealthy, fraud, thief, criminal, all Jews are rich and are thieves…With the current economic climate, people need to have a scapegoat and the fear is that Jews will become the scapegoat again because of people like that. Qual Sociol Thirdly, the perception of threat can even take the form of hypervigilance, a state of constant alertness. It can be expressed in hyperawareness and hypersensitivity to threat indicators. Threat indicators are cues that are perceived as being negatively related to one’s group; through them threat beliefs can be continually reinforced. Some participants directly referred to their own or others’ hypervigilance as paranoia or over- or hyper-sensitivity such as the middle-aged Masorti interviewee Johnda: BYou are suspicious [because of the Holocaust]; a constant fear that they [non-Jews] don’t like you…It’s the identity of Jews. I cannot believe that there are Jews who don’t feel this particular hypersensitivity.^ Adah, a Modern Orthodox woman in her 20s, who did not have immediate relations who died in or survived the Holocaust, gave a poignant example of such hypervigilance: We are all victims of anti-Semitism [ironically said]…Anti-Semitism won’t go away and it might be disguised by anti-Zionism or anti-this or anti-that…I think like for me the Holocaust is not prevalent, it doesn’t cut in my Jewish identity, but for a lot of people it is…A friend of mine got a ticket and he said that the officer was, for sure, anti-Semitic. And I was like BNo, you were driving at 50 miles an hour in a 30 zone. What do you expect?^ He was like BNo, no, he was anti-Semitic; he could have let me off.^ It’s victimization. People think that everything that happens to them is because they are Jewish such as BI didn’t get the job because they thought I was a Jew^…Everyone who is rude to them is so because they are Jewish. Another example of hypersensitivity surfaced one Sabbath afternoon in a group conversa- tion about anti-Semitism over the centuries. A Modern Orthodox in his mid-twenties recounted a story about how his hypervigilance due to vicariously experienced anti-Semitism manifested itself in his life. He had just started a new job as an investment banker in the city. Because he wore a kippah (a skullcap) and had requested to have certain working days off for major holy days, he was recognizably Jewish. He felt that he had to work extra hard and be better than others in order to prevent negative attitudes towards him from surfacing. His anticipation of anti-Semitism was informed by the history of discrimination against Jews. Right from the start of his new employment, he felt that one colleague in particular was anti-Semitic. He started, as he called it, Ban anti-Semitism tally,^ noting down every instance in which he perceived that the colleague was racist towards him. After having gathered enough evidence, he decided to talk to other colleagues about this employee’s behaviour towards him. Institutional Focus on Threat There is an institutional focus on threats to Jews that can assist in the construction of a sense of anxiety, threat perceptions and, by extension, hypervigilance for those exposed to it – that is, mainly community-involved Jews and particularly, affiliated community-involved Jews. Community-involved affiliated Jews are generally more engaged in Jewish religious and non-religious structures and thus more exposed to narratives about past and present threats to Jews, which, in turn, can explain why vicarious traumatization symptoms are more prominent and intense among them than they are among less engaged affiliated and unaffiliated Jews. Through observational research I found that both non-religious and religious Jewish organizations, such as schools, which affiliated, but also community-involved unaffiliated Jews tend to attend (Graham et al. 2014; Rocker 2014), place an emphasis on communicating past and present threats to their members. This cannot only make the trauma more immediate for them but can also inform the construction of traumatization symptoms and accordingly vicarious group trauma. For instance, unaffiliated Claire, who was in her 20s without any close family experiencing the Holocaust, noted in terms of schools: It [meaning Jewish trauma] started with like the blood libels, Pogroms in Eastern Europe and in a way like we are taught [in Jewish educational institutions] as if it’s always led up to the Holocaust…They [teachers] tell you Jewish persecution will never end and that others are always out to get you. Claire, who grew up in a middle-of-the-road Orthodox home, did not identify with this siege mentality and related her lack of vicarious trauma symptoms to the fact that she had religiously and physically distanced herself from the community since going to university and living away from home. Both types of organizations arrange many events to inform members about threats to them. This includes events about anti-Semitism in the present that often connect it to anti-Semitism in the past; for example events about anti-Semitism in regard to Israel, which has been coined as the new anti-Semitism (Klug 2003). There are various lectures, seminars, workshops and movie screenings that focus on threats to the group at places such as synagogues, university Jewish societies, Chabad society centers and other cultural and political group meetings. Holocaust survivors and now also their children give talks within the community about the experience. One reason often given for doing so is to raise awareness that another trauma like the Holocaust could be prevented. He realized that his perception was, as he labelled it Bparanoia,^ when he found out that this person was equally rude to others and thus Bjust a bad person.^ The unaffiliated Avery, 50s, who was not, at the moment of the interview, involved in the community and did not have a direct Holocaust link, but had a strong community background, referred to the inescapability of his Jewishness due to the Holocaust memory when explaining his sense of anxiety and hypervigilance: I’m full of Jewish blood. You are born into a club. It’s not a club that you can leave even if you choose to because the blood is in you…with Jews there’s this warmth, which isn’t with British people…There’s always that fear that…they might turn against you because you are Jewish. We are brought up in our childhood to expect it…There might be a point to persecute me in the future…As long as there are Jews there will be anti-Semitism. It [being Jewish] is something that you feel could be used against you…Images that come up: Germany in the 1930s, organized anti-Semitism. It’s about never having to drop your guard by believing that these people would never use this tool against you. Qual Sociol The quote illustrates, that even for secular members, threat perception can morph into the more intensified form of hypervigilance. Still, as this section has demonstrated, traumatization symptoms were more prominent and intense among community-involved Jews, especially among engaged affiliated participants. Institutional Focus on Threat The academic Dr. Goldstein contributed to this observation: BI was actually discussing a possible program of talks at one of the large United Synagogue Communities and I found it fascinating that they said they can only guarantee a good turnout when there’s a discussion on anti-Semitism.^ This quote, furthermore, indicates that institutions place importance on perpetuating traumatization Qual Sociol symptoms. The fact that there is an audience that wants to hear about threats suggests their success in this regard. symptoms. The fact that there is an audience that wants to hear about threats suggests their success in this regard. Synagogues, like schools, were found to be key places where threats are accentuated. Such institutions provide regular interaction platforms where members meet and in this way are particularly effective in supporting the construction of traumatization symptoms among its members. In the synagogues in which I did observations, the rabbis not only focused on the observation of past threats to Jews, such as through the marking of holy days associated with those earlier traumas, but also on possible present threats to the group in their communication with synagogue members. At the time of the European Parliament elections, for instance, a Modern Orthodox rabbi urged his congregation during the Sabbath morning service to vote so that the BNP would not get any seats, to prevent another trauma like the Holocaust. Such statements can reinforce the importance of the trauma and simultaneously strengthen percep- tions of threat among Jews, as illustrated in the following comment by Adah, who was involved, amongst other Jewish institutions, in this synagogue community, about a week after hearing the rabbi’s speech: BLook at the European Elections now, the BNP have got a strong chance of getting seats…and that’s worrying. It is worrying because that’s like how Hitler came into power. It’s happening before our very eyes.^ Adah was quoted earlier as saying that the Holocaust does not have an impact on her, but here we see that this memory elicits a threat perception in her. As with Robin, this demon- strates that although there can be a cognitive understanding of the mechanisms that can be elicited in response to the transmitted trauma, this does not necessarily inoculate people from experiencing them, particularly if they engage with community institutions. Such evidence also suggests that the impact of vicarious group trauma is strong enough to bypass a person’s rational thinking. Institutional Focus on Threat The fact that the CST is a charitable organization, funded primarily by Jews themselves, implies not only that there is a considerable level of perceived threat within the community in Britain, but also that this community assists in perpetuating this institutional focus on threat. The CST particularly focuses on threat prevention by offering security advice and training for Jewish schools, synagogues and other communal institutions, and to Jewish individuals such as pupils and university students. Some of the participants mentioned that they did not feel comfortable wearing Jewish symbols outside of the community because they did not want to fuel anti-Jewish sentiments and be a target of anti-Semitism. The following example shows how such threat perceptions can be reinforced among members by an institutional focus on threat: I didn’t feel secure wearing T-shirts with Israeli writing on them [at my university]. I felt like people would make offensive comments. The CST advised us against it or tried to facilitate us to do it. The CST always tries to allow the community to operate as much as possible as it wants to do. So rather than saying BDon’t wear that T-shirt,^ they say BWear that T-shirt and we’ll make sure that someone is here so that you can.^ (Aron, community-involved unaffiliated, 20s) The CST also advises Jewish institutions on appropriate security measures to prevent anti- Semitic incidents, such as using CCTV cameras, security alarms, and most importantly, security personnel on their premises. For instance, after the Gaza invasion in 2008, the CST advised Jewish institutions to increase their security measures. The impact this had on members can be seen in that the Modern Orthodox community-involved Dr. Goldstein – who argued that Bthe Holocaust is not just a shadow^ and that it Bmakes Jews want to battle against anti-Semitism^ – subsequently agreed with the increase of security in her daughter’s Jewish primary school as a response to the Gaza incursion: They have employed an extra security guard. They have already got two full-time security guards and they have changed the closing time of the school. It used to be that the younger children would finish at three thirty and the older children at four. Institutional Focus on Threat The interview with Adah was conducted in a Jewish café. While I was talking to her, a Board of Deputies of British Jews delegate came to our table to encourage us to vote in the upcoming election. The conversation between my interviewee and the delegate turned to how thankful she was for his efforts so that the BNP would not win any seats. This caught the attention of a male Jewish immigrant from Israel in his mid-thirties sitting next to us. He interrupted them saying: BNo one is here for us.^ Adah started a conversation with him in Hebrew and then turned to me to argue: BSee that’s the general Jewish attitude. He says, ‘They are all as bad as each other, they all want to kill us, what do I care? So if I vote Labour, Conservative or BNP, they all want to kill us, so what difference does it make?’^ Adah then said to me: BVictim mentality. Perfect example. They believe no matter who gets into the European parliament; they all want to kill us.^ This conversation illustrates how easy it is to be confronted with an institutional traumatization mediator—as in the form of this delegate— when in a Jewish area. The described situation shows that institutional agents can assist in the construction and reinforcement of members’ sense of perceived threat and, by extension, hypervigilance. This would be unlikely to occur, however, without a sense of group affiliation. Moreover, it highlights that non-religious institutions also focus on possible threats posed to the group. Regarding the focus on threat by non-religious institutions, the Jewish community in Britain even has its own security organization called the Community Security trust (CST). CST represents and advises the community on matters of security for Jews. CST also monitors hate crimes against members and possible terrorist threats (e.g. CST 2004). According to the CST representative I interviewed, Mr. Finkelstein, it is even important to monitor threats posed to the group in other countries, as those threats could spill over into Britain. The Holocaust was Qual Sociol referenced as an example of how threat can spread. This illustrates how vicarious group trauma can even affect organizations’ outlooks, in this case making it an institutional goal to inform the community about threats and to safeguard it through prevention work. Institutional Focus on Threat Because that meant that the school gates were opened until ten past four, so for a whole forty minutes, which was seen to be too big a security threat, they have now changed it, half that gap and made the infants finish not until ten to four so that the school gates would only be opened for twenty minutes, which I find to be an unbelievably radical move. One assumes that this is because of a real perceived threat because that requires an awful lot of commitment from parents and teachers to implement. The fact that the school and parents complied with the security advice and implemented additional security measures illustrates the community’s heightened level of perceived threat and, by extension, acute sensitivity to threat indicators, as well as institutions’ power to accentuate such threat—a power that can be justified because of the Holocaust. The strong influence of the CST’s focus on threat monitoring on Jews’ sense of threat perception is further illustrated in the following quote by Alon, who helps to secure the premise of his local synagogue during services: People are being trained by the CST. They are having emergency buzzers that alarm the police…People get nervous when people drive past [the synagogue] slowly and take Qual Sociol photos…so you have to be aware and have heightened security measures. I think there is a serious, serious threat. photos…so you have to be aware and have heightened security measures. I think there is a serious, serious threat. In regard to the impact of security advice by institutions such as the CST, the young Modern Orthodox – involved Saul, who shortly after the interview became quite detached from the religious community and moved abroad to experience a liberal non-Jewish life, gave a critical account: If you listen to the CSTyou’d probably get quite worried, and you’d be worried when you walk out on the street, although they’re trying to get people to feel safer about things…I don’t like what it does to you as a person. If you think that people are out there to get you, it makes you a really nervous wreck, and it just makes you not feel part of society. This suggests that while security measures (e.g. Institutional Focus on Threat CST patrols on holy days in Jewish neighborhoods) function mainly as deterrents against low-level, less organized attacks against the community and provide an image of safety, their existence can also reinforce the perception of being under constant threat and thus hypersensitivity as well as fuel anxiety. Furthermore, my observational evidence suggests that Jewish news organizations focus on threats to Jews as they frequently report about anti-Semitism in the past and present. This was also picked up by some of the participants, mainly secular members who have grown up in the community by living in Jewish neighborhoods and attending Jewish schools and/or syna- gogues. These participants argued that such intense focus on threats to members by the Jewish media can assist in bringing the trauma of the past into the present and accordingly can reinforce members’ existing anxiety, threat perceptions and hypervigilance: When I read the Jewish Chronicle, there are always tales about anti-Semitism, there is always something about anti-Semitism so one is reminded of these things and you don’t need the Holocaust [said ironically]. (Ruben, Modern Orthodox, 60s) If you read the Jewish Chronicle on a daily basis you start thinking another Holocaust is about to happen. (Josephine, Masorti, 40s) To sum up, the findings indicate a focus in the Jewish community and particularly by its institutions, on threats to the group, which stands in association with vicarious group trauma. This focus on threat can assist in the construction and reinforcement of anxiety, threat perceptions and, by extension, hypervigilance. It can accentuate the trauma memory by making the trauma more immediate for members. Considering that most of the less community- engaged Jews were not exposed to constant and multiple traumatization mediators because of their physical and social distance from the community, it can be inferred that the traumatization mediators provided by community institutions have a strong impact only on engaged Jews, particularly on affiliated involved Jews. This can explain why vicarious traumatization symp- toms were found to be less prominent and intense among less community-involved Jews. Conclusion This article has developed a sociological theory of vicarious group trauma. The theory purports that people can feel a threatening event and abuse that were inflicted on members of their social group as if this had happened to them. Individuals identify with the traumatic incident or abuse personally as members of their group so that it can be an emotional experience for them. Qual Sociol Through a personal association with the trauma as members of the group, a historical fact of group trauma can become an emotional trauma experience for them strong enough to bypass rationality and elicit strong emotions and perceptions, i.e. vicarious traumatization. We speak of vicarious group trauma when many members of a collectivity can be attested to have or they themselves feel as if they have a visceral identification with their group’s trauma; reflected in signs and signals of traumatization. In this way, vicarious group trauma can inform members’ sense of belonging to the group and, therefore, their identity. Using the method of ethnography, this article applied the theory by exploring how transmitted group traumas manifest themselves in the everyday experiences of Jews in Britain and, in this way, impacts their identification with the group. To do so, the article showed first which traumas Jews remembered and concluded that group traumas are remembered selectively, termed ‘‘chosen trauma’’ by Volkan (2001). Secular participants tended to remember only the Holocaust, which was proposed to be related not only to the communication of the event through personal family stories but also more generally through mainstream culture and education. For example, the Holocaust Education Trust reports that Holocaust education is generally part of the national curriculum in British schools even though it is not a formal requirement in Wales, Northern Ireland, Scotland or in independent schools in England (HET 2011). There are also many exhibitions (e.g. the Holocaust exhibition at the Imperial War Museum), renowned plays, blockbuster movies, and award-winning documentaries about the Holocaust. Devout affiliated members had a propensity to also remember earlier Jewish traumas, such as the enslavement in Egypt and their exodus over a hundred years later. I suggested that this was related to the observation of holy days associated with those traumas in synagogues, Jewish schools and homes in addition to personal family stories. For instance, the enslavement in Egypt and the subsequent exodus of Jews is commemorated at Passover. Conclusion At the Passover Seder, a ritualistic dinner, participants are required to view themselves as if they came out of Egypt. Passages like the following ones are read aloud: BWe were slaves to Pharaoh in Egypt, but the Lord our God brought us out^ (Deuteronomy: 6–21), and, BYou shall tell your son on that day, it is because of what God did for me when I went forth from Egypt^ (Zerubavel 1996: 290, Exodus 13:8). In this way, the article showed that there are not only Jewish institutions including the family but also non-Jewish ones that assist in the transmission of trauma memory and highlight members’ difference from gentiles, which in turn can inform identification with the group. g g g , g p The article then illustrated that the vicarious sensation of traumatic group experiences can create anxiety and elicit perceptions of threat and, by extension, hypervigilance, which can inform group identification. It is well-documented in the psychology literature that transmitted trauma can elicit these traumatization symptoms in people who come into contact with trauma victims over a prolonged period of times, such as family members or therapists (McCann and Pearlman 1990); case studies have been written about the traumatization symptoms found in second and third- generation offspring of Holocaust survivors (Danieli 1985; Prince 2009). My study adds to literature in this area as there is little research exploring secondary traumatization among Jews on the group- level, including Jews who have not been in close contact with trauma survivors. The findings resonate with research such as that by Hirsch and Chaitin (2010), which describes Jewish-Israeli young adults, with and without family connections to Holocaust survivors, as fearful because of the Holocaust. Similarly, Lazar et al. (2008) relate to trauma anxiety by asserting that it exists among Israeli Jews, including those without direct connection to Holocaust survivors, in the form of needing Israel as a place of safety. Their article connects Jews’ association of Israel as a safe haven with political Zionism’s focus on the need for a sovereign Jewish state by arguing that the lack of it Bwas one major contributing factor that Jews in Europe, prior to and during the Holocaust, were singled out for massive persecution^ (Lazar et al. 2008: 116). Qual Sociol Lastly, the article showed how group institutions can contribute to the construction and reinforcement of not only trauma memory but also of vicarious group trauma. Conclusion It demonstrated that there is an extant institutional focus on past and present threats to Jews, which can assist in the creation and reinforcement of traumatization symptoms among members. The article found that traumatization symptoms, particularly perceptions of threat and hypersensitivity, were prominent among community-involved Jews, and especially among affiliated engaged members. Their level of involvement in the social structure allowed for increased exposure to constant and multiple institutional traumatization agents such as security guards in front of synagogue. Thus, the findings indicate that institutional platforms of a group’s social structure provide a powerful instrument for informing and perpetuating trauma memory and, with it, vicarious group trauma. The article adds to sociological research that offers largely theoretical analyses regarding the transmission of the historical fact of memory and how the memory must be presented in public for it to become a social trauma (Assmann 2006; Eyerman 2011; Eyerman et al. 2011). My work has provided a systematic empirical account of the impact transmitted group trauma can have on the emotions and perceptions of members independent of any direct relation or contact with a survivor of the group’s trauma, and the role the social structure with its institutions plays in this regard. In this way, the findings can be applied more widely to other groups with a history of trauma. Scholars may seek to explore the extent to which vicarious group trauma is present in other minorities. Case studies could include the impact of the times of slavery on African- Americans in the United States or the partition of Punjab in 1947 and the 1984 attack on the Golden Temple for Sikhs and the effects of more recent traumatic experiences such as the conflict in Syria and Rwandan genocide on the respective diaspora communities. All of those traumas have been communicated through institutional mechanisms, such as the media and cultural organizations like museums. For example, the memories of the Sikh’s traumatic experiences are used in discourses by organizations and community elites in the diaspora to maintain and strengthen members’ group belonging (Barrier 2006; Tatla 2006; Jacobsen and Myrvold 2011). Thus, these group traumas would be worth an examination in view of how the social structure with its institutions assists in their perpetuation within and across generations. Conclusion Acknowledgments The author thanks Random House, St Catherine’s College and the Sociology Department of the University of Oxford for the financial support in order for me to conduct this study as well as Michael Biggs, Shana Cohen, Gabriella Elgenius, Miri Freud-Kandel, Thomas Grund, Anthony Heath, Brian Klug, Avner Offer and the journal’s reviewers for valuable comments. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and repro- duction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Alexander, Jeffrey C. 2004b. Towards a theory of cultural trauma. In Cultural trauma and collective identity, eds. Jeffrey Alexander, Ron Eyerman, Bernhard Giesen, Neil J. Smelser, and Piotr Sztompka, 1–30. Berkeley: University of California Press. Alexander, Jeffrey C., Ron Eyerman, Bernhard Giesen, Neil J. Smelser, and Piotr Sztompka. 2004. Cultural trauma and collective identity. Berkeley: University of California Press. Alexander, Jeffrey C. 2004a. On the social construction of moral universals. The Bholocaust^ from war crime to trauma. In Cultural trauma and collective identity, eds. Jeffrey C. Alexander, Ron Eyerman, Bernhard Giesen, Neil J. Smelser, and Piotr Sztompka, 196–263. Berkeley: University of California Press. References Alexander, Jeffrey C. 2004a. On the social construction of moral universals. The Bholocaust^ from war crime to trauma. In Cultural trauma and collective identity, eds. Jeffrey C. Alexander, Ron Eyerman, Bernhard Giesen, Neil J. Smelser, and Piotr Sztompka, 196–263. Berkeley: University of California Press. Alexander, Jeffrey C. 2004b. Towards a theory of cultural trauma. In Cultural trauma and collective identity, eds. Jeffrey Alexander, Ron Eyerman, Bernhard Giesen, Neil J. Smelser, and Piotr Sztompka, 1–30. Berkeley: University of California Press. Alexander, Jeffrey C., Ron Eyerman, Bernhard Giesen, Neil J. Smelser, and Piotr Sztompka. 2004. Cultural trauma and collective identity. Berkeley: University of California Press. Qual Sociol Assmann, Aleida. 2006. Der lange Schatten der Vergangenheit: Erinnerungskultur und Geschichtspolitik München: C. H. Beck Verlag. Barrier, Norman, Gerald 2006. Trauma and memory within the Sikh diaspora: Internet dialogue. Sikh Formations: Religion, Culture, Theory 2 (1): 33–56. Ben-Amos, Avner, and Ilana Bet-El. 1999. Holocaust day and memorial day in Israeli schools: Ceremonies, education and history. Israel Studies 4(1): 258–284. Berman, Judith E. 2004. Holocaust commemorations in London and Anglo-Jewish (dis-)Unity. Journal of Modern Jewish Studies 3(1): 51–71. Caruth, Cathy. 1996. Unclaimed experience: trauma, narrative, and history. Baltimore: Johns Hopkins University Press. oke, Steven. 2000. Negotiating memory and identity: The Hyde Park holocaust memorial, London. Journal of Historical Geography 26(3): 449–465. g p y ( ) CST. 2004. The community security trust: Terrorist incidents against Jewish communities and Israeli citizens abroad, 1968–2003. London: Community Security Trust. g p y ( ) T. 2004. The community security trust: Terrorist incidents against Jewish communities and Israeli citizens b d 1968 2003 L d C it S it T t abroad, 1968–2003. London: Community Security Trust. abroad, 1968–2003. London: Community Security Trust. y y CST. 2010. Antisemitic incidents report 2009. London: Community Security Trust. 2010. Antisemitic incidents report 2009. London: Co CST. 2014. Antisemitic Incidents Report 2013 London: Community Security Trust. Danieli, Yael. 1985. The treatment and prevention of long-term effects and intergenerational transmission of victimization. A lesson from holocaust survivors and their children. In Trauma and its wake, ed. Charles R. Figley, 278–294. New York: Brunner/Mazel. g y, Eyerman, Ron. 2011. The cultural sociology of political assassination: from MLK and RFK to Fortuyn and Van Gogh. Palgrave Macmillan: Cultural sociology. New York. rman, Ron, Jeffrey C. Alexander, and Elizabeth Butler Breese. 2011. Narrating trauma: on the impact of collective suffering. Boulder: Paradigm. Figley, Charles R. 1983. References Catastrophes: An overview of family reaction. In Compassion fatigue: Coping with secondary traumatic stress disorder in those who treat the traumatized, eds. C.R. Figley, and H.I. McCubbin, 1–20. New York: Brunner/Mazel. Freud, Sigmund. 1932. Moses and Monotheism. International psycho-analytical library, vol. no 33. Lond Hogarth Press. Freud, Sigmund. 1932. Moses and Monotheism. International psycho-analytical library, vol. no 33. London: Hogarth Press. G h D id 2013 2011 Thi i d hi k i L d I i f J i h P li R h g Graham, David. 2013. 2011 census: Thinning and thickening. London: Institute for Jewish Policy Research Graham, David, and Daniel Vulkan. 2010. Synagogue membership in the United Kingdom in 2010. London: Institute for Jewish Policy Research. Graham, David, Marlena Schmool, and Stanley Waterman. 2007. Jews in Britain: A snapshot from the 2001 census. London: Institute for Jewish Policy Research. y Graham, David, Jonathan Boyd, and Daniel Vulkan. 2012. 2011 census (England and Wales): Initial insig about the UK Jewish population. London: Institute for Jewish Policy Research. Graham, David , Daniel Staetsky, and Jonathan Boyd. 2014. Jews in the United Kingdom in 2013: Preliminary findings from the national Jewish community survey. London: Institute for Jewish Policy Research. g y y y HET. 2011. Holocaust education in the UK. London: Holocaust Educational Trust. Hirsch, Marianne. 2008. The generation of postmemory. Poetics Today 29(1): 103–128. Hirsch, Tal Litvak, and Julia Chaitin. 2010. BThe shoah runs through our veins^: the relevance of the holocaust for jewish-israeli young adults Idea 14 (1). Jacobsen, Knut A., and Kristina Myrvold. 2011. Sikhs in Europe: Migration, Identities and Representations. Farnham: Ashgate Publishing Limited. g g JLC. 2016. United Synagogue. http://www.thejlc.org/portfolio/united-synagogue/. Accessed 13 May 2016. JPR. 2000. A Community of Communities: Report of the commission on representation of the interests of the British Jewish community. London: Institute for Jewish Policy Research. JPR. 2000. A Community of Communities: Report of the commission on representation of the interests of JPR. 2000. A Community of Communities: Report of the commission on represe British Jewish community. London: Institute for Jewish Policy Research. Kellerman, Natan P.F. 2001. Psychopathology in children of holocaust survivors: A review of the research literature. Israel Journal of Psychiatry and Related Sciences 38(1): 36–46. P.F. 2001. Psychopathology in children of holocaust Klug, Brian. 2003. The collective Jew: Israel and the new antisemitism. Patterns of Prejudice 37(2): 117–138. Kugelmass, Jack. 1996. References Missions to the past: Poland in contemporary Jewish thought and deed. In In Tense Past: Cultural Essays in Trauma and Memory, eds. Paul Antze, and Michael Lambek Lambeck, 199–214. New York Routledge. Landsberg, Alison. 1997. America, the holocaust, and the mass culture of memory: Toward a radical politics of empathy. New German Critique 71: 63–86. Landsberg, Alison. 2004. Prosthetic memory: The transformation of American remembrance in the age of m Landsberg, Alison. 2004. Prosthetic memory: The tra culture. New York: Columbia University Press. Landsberg, Alison. 2004. Prosthetic memory: The tr Landsberg, Alison. 2004. Prosthetic memory: The transformation of American remembrance in the age of mass culture. New York: Columbia University Press. culture. New York: Columbia University Press. Landsberg, Alison. 2009. Memory, Empathy, and the Politics of Identification. International Journal of Polit Culture, and Society 22(2): 221–229. Qual Sociol Lazar, Alon, Julia Chaitin, Tamar Gross, and Dan Bar-on. 2004. Jewish Israeli teenagers, national identity, and the lessons of the holocaust. Holocaust and Genocide Studies 18: 188–204. Lazar, Alon, Tal Litvak-Hirsch, and Julia Chaitin. 2008. Between culture and family: Jewish-Israeli young adults relation to the holocaust as a cultural trauma. Traumatology 14: 93–102. Litvak-Hirsch, Tal, and Dan Bar-On. 2006. To rebuild life: A longitudinal study of the influences of the holocaust on the following generations. Family Process 45: 465–483. McCann, Lisa, and Laurie Anne Pearlman. 1990. Vicarious traumatization: A framework for understanding the psychological effects of working with victims. Journal of Traumatics Stress 3: 131–149. Novick, Peter. 1999. The holocaust and collective memory. London: Clays Ltd.. . 1999. The holocaust and collective memory. London Prince, Robert. 2009. Psychoanalysis traumatized: The legacy of the holocaust. The American Journal of Psychoanalysis 69: 179–194. y y Richmond, Nancy. 1993. Israel's law of return: Analysis of its evolution and present. Application. Dickinson Journal of International Law 12(1): 95–133. Rocker, Simon. 2014. Survey: Demand for schools has peaked. The Jewish Chronicle Online. Accessed 30 January 2015. Rosenheck, Robert, and Paul Nathan. 1998. Secondary traumatization in children of Vietnam veterans. Hospital and Community Psychiatry 36: 538–539. Scharf, Miri. 2007. Long-term effects of trauma: Psychosocial functioning of the second and third generation holocaust survivors. Development and Psychopathology 19: 603–622. Schmool, Marlena, and Frances Cohen. 1998. A profile of British Jewry: Patterns and trends at the turn of a century. London: Board of Deputies of British Jews. Silver, Craig. 2009. Bid to firebomb synagogue as protests grow. References London: The Jewish Chronicle. Tatla, Darshan S. 2006. The morning after: Trauma, memory and the Sikh predicament since 1984. S Formations: Religion, Culture, Theory 2(1): 57–88. Volkan, Vamik, D. 2001. Transgenerational transmission and chosen traumas: An aspect of large-group ident Group Analysis 34 (79): 1–20. Zerubavel, Yael. 1996. Social memories: Steps to a sociology of the past. Qualitative Sociology 19(3): 283–299. Zerubavel, Yael. 1996. Social memories: Steps to a sociology of the past. Qualitative Sociology 19(3): 283–299. Zerubavel, Yael. 1996. Social memories: Steps to a sociology of the past. Qualitative Sociology 19(3): 283–299. Christina Fuhr is a Research Fellow at St. Edmund’s College, University of Cambridge, and at the Woolf Institute in Cambridge.
https://openalex.org/W4362620652	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_SF2_from_Antibody_Binding_and_Neutralization_of_Live_SARS-CoV-2_Variants_Including_BA_4_5_Following_Booster_Vaccination_of_Patients_with_B-cell_Malignancies/22546038/1/files/40009506.pdf	English	null	Supplementary Figure SF2 from Antibody Binding and Neutralization of Live SARS-CoV-2 Variants Including BA.4/5 Following Booster Vaccination of Patients with B-cell Malignancies	null	2,023	cc-by	514	Supplemental Figure 2. Positive correlations in antibody binding titers between anti-Spike IgG and anti-RBD, anti-NTD IgG, and anti-spike IgA and IgM. A) IgG binding titers against full-length spike protein correlate with anti-RBD (orange) and anti-NTD (purple) binding IgG titers. B-C) Anti-spike IgG titers correlate better with anti-spike IgA (B) than IgM (C). Horizontal and vertical dotted lines = background 0.8427 0.8694 NTD IgG (AU/mL) RBD IgG (AU/mL) 102 103 104 105 106 101 R2= R2= 102 103 104 105 106 101 anti-Spike IgG (AU/mL) P < 0.0001 P < 0.0001 A B C 102 103 104 105 106 101 102 103 104 105 106 101 anti-Spike IgG (AU/mL) anti-Spike IgM (AU/mL) 0.3709 R2= P = 0.0019 102 103 104 105 106 101 102 103 104 105 106 101 anti-Spike IgG (AU/mL) anti-Spike IgA (AU/mL) 0.6222 R2= P < 0.0001 Supplemental Figure 2. Positive correlations in antibody binding titers between anti-Spike IgG and anti-RBD, anti-NTD IgG, and anti-spike IgA and IgM. A) IgG binding titers against full-length spike protein correlate with anti-RBD (orange) and anti-NTD (purple) binding IgG titers. B-C) Anti-spike IgG titers correlate better with anti-spike IgA (B) than IgM (C). Horizontal and vertical dotted lines = background 0.8427 0.8694 NTD IgG (AU/mL) RBD IgG (AU/mL) 102 103 104 105 106 101 R2= R2= 102 103 104 105 106 101 anti-Spike IgG (AU/mL) P < 0.0001 P < 0.0001 A B C 102 103 104 105 106 101 102 103 104 105 106 101 anti-Spike IgG (AU/mL) anti-Spike IgM (AU/mL) 0.3709 R2= P = 0.0019 102 103 104 105 106 101 102 103 104 105 106 101 anti-Spike IgG (AU/mL) anti-Spike IgA (AU/mL) 0.6222 R2= P < 0.0001 0.8427 0.8694 NTD IgG (AU/mL) RBD IgG (AU/mL) 102 103 104 105 106 101 R2= R2= 102 103 104 105 106 101 anti Spike IgG (AU/mL) P < 0.0001 P < 0.0001 A A Supplemental Figure 2 Positive correlations in antibody binding titers between anti Spike IgG and anti Spike IgG (AU/mL) B C 102 103 104 105 106 101 102 103 104 105 106 101 anti-Spike IgG (AU/mL) anti-Spike IgM (AU/mL) 0.3709 R2= P = 0.0019 102 103 104 105 106 101 102 103 104 105 106 101 anti-Spike IgG (AU/mL) anti-Spike IgA (AU/mL) 0.6222 R2= P < 0.0001 B 102 103 104 105 106 101 102 103 104 105 106 101 anti-Spike IgG (AU/mL) anti-Spike IgA (AU/mL) 0.6222 R2= P < 0.0001 C 102 103 104 105 106 101 102 103 104 105 106 101 anti-Spike IgG (AU/mL) anti-Spike IgM (AU/mL) 0.3709 R2= P = 0.0019 C B Supplemental Figure 2. Positive correlations in antibody binding titers between anti-Spike IgG and Supplemental Figure 2. Positive correlations in antibody binding titers between anti-Spike IgG and anti-RBD, anti-NTD IgG, and anti-spike IgA and IgM. A) IgG binding titers against full-length spike protein correlate with anti-RBD (orange) and anti-NTD (purple) binding IgG titers. B-C) Anti-spike IgG titers correlate better with anti-spike IgA (B) than IgM (C). Horizontal and vertical dotted lines = background antibody levels determined from pre-pandemic samples. Correlations were statistically significant using extra-sum-of-squares F test.
https://openalex.org/W2757128688	http://dspace.vutbr.cz/bitstream/11012/201700/1/14451Article%20Text5676211020170928.pdf	English	null	Location Accuracy of Commercial IP Address Geolocation Databases	Informacinės technologijos ir valdymas	2,017	cc-by	6,688	Corresponding author: komosny@feec.vutbr.cz Corresponding author: komosny@feec.vutbr.cz This paper deals with finding the geographical location of Internet nodes remotely with no need to communicate with the nodes located (client-independently). IP geolocation is used in a number of areas, such as content person alisation, on-line fraud prevention and detection, and digital media law enforcement. One of the main concerns when studying the accuracy of client-independent geolocation is the groundtruth dataset. As we show in the re lated work, the used groundtruth influences the results a lot. We construct an error-free groundtruth dataset con sisting of nodes with GPS-precise locations. We also record the country, region, city, and ISP for each groundtruth node. Using the created groundtruth, we study the accuracy of eight IP location databases in a number of scenari os, such as effect of city area and population, effect of ISP assignment, and number of not-returned locations. KEYWORDS: location, geolocation, IP address, groundtruth, accuracy, city, database, MaxMind, DB-IP, IP 2Location, ipinfo, Skyhook, Neustar, Eurek, GeoBytes. Miroslav Voznak VSB-TU Ostrava, Department of Telecommunications, 17 listopadu 15/2172, 708 33 Ostrava, Czech Republic e-mail: miroslav.voznak@vsb.cz Location Accuracy of Commercial IP Address Geolocation Databases Brno University of Technology, Department of Telecommunications, Technicka 12, 616 00 Brno, Czech Republic e-mail: komosny@feec.vutbr.cz ITC 3/46 Journal of Information Technology and Control Vol. 46 / No. 3 / 2017 pp. 333-344 DOI 10.5755/j01.itc.46.3.14451 © Kaunas University of Technology 333 333 Information Technology and Control 2017/3/46 Location Accuracy of Commercial IP Address Geolocation Databases Saeed Ur Rehman Auckland University of Technology, School of Engineering, Computer and Mathematical Sciences Private Bag 92006, Auckland 1142, New Zealand; e-mail: saeed.rehman@aut.ac.nz 2. Current problems of IP geolocation In this section, we discuss the current problems of finding the geographical location of the Internet de vices by their IP addresses. In this paper we deal with client-independent geo location that is used for a broad variety of Internet services and applications, including social networks (detection of virtual identity misuse or username/ password sharing), web-based services (detection of suspicious logins), e-shops (detection of on-line credit card frauds), banking (prevention of phishing attacks), and electronic content distribution (enforc ing territory restriction given by digital media laws). The use of the IP address space is controlled by IANA (Internet Assignment Numbers Authority). IANA allocates the major segments of IP addresses to five regional registers (RIRs) – AFRINIC (Africa), APNIC (Asia/Pacific), ARIN (North America), LACNIC (Lat in America), and RIPE NCC (Europe, the Middle East, and Central Asia). The regional registers further allo cate IP address segments ISPs. Such allocation can be direct or through two types of intermediary entities – national internet registry (NIR) and local internet registry (LIR). The records of the allocated IP address segments are stored in a database that is managed by a regional register, as shown in Figure 1. Along with the IP allocation records, the registers maintain contact information of the organizations with the assigned IP addresses. The stored contacts provide a way to lo cate IP devices in some extent. However, there are no official rules for filling the contact information by the organizations, and thus the provided location informa tion can lead to wrong results. Next major concern is that the IP addresses that fall into one allocation seg ment can be distributed on a large geographical area depending on the type and size of the organization. A good example are ISPs that operate at the national level or organizations with branches at different locations. The contribution of this paper is the following: 1 Based on an inconsistency of the location accuracy results presented in the related work (described in Section 4), we construct an error-free groundtruth dataset. It consists of Internet nodes with known GPS-precise locations. The dataset guarantees the avoidance of wrong location accuracy results. 2 The related work typically depends on the groundtruth nodes coming from large cities. Our groundtruth dataset covers all types of cities, from very small to very large. 1. Introduction (client-dependent) or without its assistance (cli ent-independent). Client-dependent methods use (client-dependent) or without its assistance (cli ent-independent). Client-dependent methods use Geographical location of Internet devices can be obtained with an assistance of the device located 334 2017/3/46 Information Technology and Control dent location accuracy. Section 5 describes the method for error-free construction of the groundtruth dataset. In Section 6 we present and discuss our location accu racy evaluation. In Section 7 we summarize the results. technologies such as GPS, accelerometers, and trian gulation in WiFi or cellular mobile networks. These methods rely on specific properties or features of the devices located. Typically, location accuracy is with in tens of meters. On the other hand, client-indepen dent methods are used to locate any Internet device and they do not require any additional properties or features in the devices located. Several methods are used, such as IP geolocation databases. Accuracy of client-independent geolocation is lower, typically at city level. 2. Current problems of IP geolocation This allows us to study more properties that influence the location accu racy, such as the effect of city area and population. 3 When locating nodes that belong to the same In ternet service provider (ISP), we observe that the results show the same or similar locations in spite of very different real (correct) positions. We par ticularly study the change of location performance from this ISP node assignment point of view. The domain names (DNS) can also indicate the lo cation of IP nodes, however, there are no rules for geographical naming despite some standardization Figure 2 Geolocation database filling schemes Network measurement is also used for IP geoloca tion [19, 11, 4]. Measurement-based geolocation works with a positive correlation between communication latency and geographical distance [23]. The latency is measured from a set of servers with known location to the IP address located. The results are converted to maximal geographical distances from the servers to the IP address. These geographical distances delim it the area where the IP address is located. There are several known methods that use this approach, such as Constraint-Based Geolocation [9], Octant [29], Spotter [20], or Topology-based Geolocation [13]. A disadvantage of measurement-based geolocation is the latency instability which leads to false location estimations [18, 28]. Other known problems are long location times [22, 21]. Figure 1 Figure 1 IP address allocation The paper is structured as follows: the next section defines the problem that we address. We describe why low accuracy is reached with client-independent geo location. We give an example of locations provided by several location databases and demonstrate the loca tion error. Section 3 presents the location accuracy as reported by the database vendors. In Section 4 we sur vey the related work that deals with client-indepen IP address allocation 335 2017/3/46 Information Technology and Control uses location information available through Internet resources, such as crawling web pages [7, 3, 1, 10] and measuring the network [17]. The bottom-up scheme uses locations that are collected by external resourc es, such as GPS or WiFi network scanning. efforts [2]. Some ISPs use internal geographical nam ing schemes for their networking devices and such in formation can be used for IP geolocation as described in [24]. The use of domain names for IP geolocation was particularly described in [6, 26, 5]. An enhance ment of DNS defines a new LOC record which stores latitude, longitude, and altitude for domain names. However, this enhancement gives a poor location ef ficiency (high number of not-returned locations) and large location errors [14]. 3. Claimed accuracy by location service vendors In this section, we study the claimed location accura cy by major geolocation database providers. We sum marize the found claimed accuracy in Table 1. The databases typically return geographical coordinates (latitude and longitude), country, region, and city. The Skyhook database ‘Hyperlocal IP Pro’ differs from others by returning region and city only when the es timation reaches a certain level of trustworthiness. Database-based IP geolocation defines blocks of con tinuous IP addresses and stores location information for them. These IP blocks may be smaller than the IANA allocation segments and thus provide a better location accuracy. There are two schemes to obtain the location information to be linked with the IP blocks: top-down and bottom-up. These sources of lo cations are shown in Figure 2. The top-down scheme Table 2 Cumulative percentage of estimated locations within maximum location error [km] – created from source [25] [%] Vendor <50 <100 <150 <250 MaxMind 68 73 76 78 IP2Location 62 65 66 68 IPligence 73 75 76 78 HostIP 37 39 42 45 Netaculity 45 49 50 54 Geobytes 33 35 40 45 Triukose et al. [27] focus on IP geolocation of mo bile devices. They discuss the use of network ad dress translation and how it affects IP geolocation. They study the accuracy of the public and private IP addresses separately. In some cases, they observe large location errors at the scale of inter-continental distances. We particularly study such large errors in section 6.6. We summarize the results of their work in Table 3. Two databases are used: MaxMind and Table 1 Shavitt and Zilberman [25] use a groundtruth data set that is based on an algorithm which groups IP addresses into virtual Points of Interest (PoPs). The algorithm discovers the sets of the routers at the same location. For this purpose, they use latency mea surements and topology discovery. The accuracy of the results depends on the PoPs identified locations. Six major geolocation databases are evaluated: Max Mind, IP2Location, IPligence, HostIP, Netaculity, and Geobytes. We summarize the results in Table 2. As the source of the accuracy data, we use the cumu lative probability function showing the database lo cation deviation from the locations of the identified groundtruth PoPs. Table 1 Claimed accuracy by vendors Vendor/database Country [%] City [%] IPv4 IPv6 MaxMind/GeoIP2 Precision 84 40 100 % YES – N/A DB-IP/IP address to location + ISP N/A N/A 7 mil. YES – 586,718 IP2Location/DB24 N/A 77 14 mil. YES – N/A Neustar/where 99.9 N/A 100 % YES – 100 % Eurek/professional edition N/A N/A 100 % NO Geobytes/Geo IP Location 97 75 98 % NO 336 2017/3/46 Information Technology and Control MaxMind publishes the accuracy data for 23 coun tries (www.maxmind.com/en/geoip2-city-data base-accuracy). For the purpose of comparison at the country level, we use the maximum location error of 250 km to evaluate the result as correct. Based on this range, 4 % of the location queries are reported to be resolved incorrectly, and 12 % of location queries are reported to be unresolved (not-returned) at the coun try level. For the city level, 48 % of the location que ries give an incorrect city, and 11 % the location que ries are unresolved. DB-IP does not publish any data on location accuracy, only the number of IP address space covered. IP2Location publishes a comprehen sive location accuracy data for 250 countries (www. ip2location.com/data-accuracy). The published data cover only the city coverage (the country level is not included). For the purpose of comparison at the city level, we use the maximum error of 50 miles. Neustar does not publish any data on accuracy of their geolo cation services. However, we found that the accuracy of the databases was evaluated by Pricewater- house Coopers (www.neustar.biz/‌resources/‌product-lit erature/‌neustar-ip-‌intelligence-pwc-audit). The result is 99.9 % accuracy for the country level. Neus tar claims to cover all of the IP address space. Eurek does not provide any location accuracy information about their products. The only information provided is that it covers the whole IPv4 address space. Geo Bytes provides some basic data about their accuracy (www.geobytes.com/‌faq/). It claims to resolve 98 % of IP addresses with accuracy of 97 % at the country level. Another information published is that 80 % of returned locations are within the maximum error distance of 100 km and 75 % of locations are within the maximum location error of 50 km. ipinfo does not publish any accuracy related information. The same holds for Skyhook. within a maximal error of 50, 100, 150, and 250 km. Table 4 4 and 20-96 % of the nodes can be located within range of 250 km. Cumulative percentage of estimated locations within maximum location error [km] – created from source [12] [%] The worst results were achieved when one ISP-based groundtruth dataset was used. The second worst re sults were achieved when mobile devices were lo cated. The best results were obtained when using a groundtruth dataset that was obtained from the cen troid-based algorithm based on location votes from the databases. Vendor <50 <100 <150 <250 MaxMind 81 85 89 92 Digital Envoy 88 91 93 96 IPligence 78 82 85 89 HostIP 76 82 83 87 Poese et al. [24] focus on the differences between the claimed accuracy by the database vendors and the real-case location accuracy. The accuracy is evaluat ed by using a groundtruth dataset that was obtained via a large European ISP. They create PoPs based on the network prefixes. The locations of the groundtruth nodes are obtained by using an internal naming scheme of the ISP. The databases studied are HostIP, IP2Location, InfoDB, MaxMind, and Software77. Ta ble 5 shows the results for IP address blocks that are smaller than the groundtruth ISP prefixes. Table 3 The locations of the ground nodes are obtained by using an internal n scheme of the ISP. The databases studied are H IP2Location, InfoDB, MaxMind, and Software7 ble 5 shows the results for IP address blocks th smaller than the groundtruth ISP prefixes. Table 5 IP2Location (DB11.LITE). The table shows the val ues for the public IP addresses studied. The private addresses studied give larger values up to a maximum error of 400 km. After this value, the results are sim ilar with no significant difference. We note that a cel lular network is used in this study and such networks give worse results compared to general Internet net works. IP2Location (DB11.LITE). The table shows the val ues for the public IP addresses studied. The private addresses studied give larger values up to a maximum error of 400 km. After this value, the results are sim ilar with no significant difference. We note that a cel lular network is used in this study and such networks give worse results compared to general Internet net works. Cumulative percentage of estimated locations within maximum location error [km] – created from source [24] [%] Vendor <50 <100 <150 <250 InfoDB 50 63 70 80 MaxMind 35 42 50 63 IP2Location 10 15 18 20 Huffaker et al. [12] evaluate eight databases. They point out the absence of a substantial groundtruth dataset. They propose a method to evaluate the ac curacy by a centroid-based algorithm working with majority of location votes from the databases. The location accuracy in a form of cumulative probabili ty is only provided for five databases: MaxMind Geo, MaxMind Lite, IPligence, Digital Envoy, and HostIP. For the data shown in Table 4, we use the location accuracy for the PlanetLab groundtruth dataset. The results show a better location accuracy most probably because of the use of the groundtruth dataset, which was created by using the location voting algorithm. Another reason is that the PlanetLab groundtruth dataset is strongly oriented towards the major cit ies [15, 16]. Other papers that deal with database-based IP geolo cation are [30, 8]. These papers do not give any results for the cumulative maximum location error probabil ities, and they focus on different related topics, such as the covered IP address space. By summarizing the related work, we show that there are very large differences in the results. The related work shows that: 1 from 10 to 88 % of the nodes can be located within 50 km range, 2 15-91 % of the nodes can be located within range of 100 km, 3 18-93 % of the nodes can be located within range of 150 km, Table 3 Comparing data from the related work is difficult due to the use of different evaluation techniques. An ex ample is the use of different distance thresholds for the city-level accuracy. In the related work it varies from 40 to 100 km. Therefore, we use a different mea sure to compare the related work. We work with the independent cumulative probabilities of locations Cumulative percentage of estimated locations within maximum location error [km] – created from source [27] Cumulative percentage of estimated locations within maximum location error [km] – created from source [27] [%] Vendor <50 <100 <150 <250 IP2Location 28 34 47 57 MaxMind 15 22 32 45 Vendor <50 <100 <150 <250 IP2Location 28 34 47 57 MaxMind 15 22 32 45 337 2017/3/46 Information Technology and Control Information Technology and Control IP2Location (DB11.LITE). The table shows th ues for the public IP addresses studied. The p addresses studied give larger values up to a max error of 400 km. After this value, the results ar ilar with no significant difference. We note that lular network is used in this study and such net give worse results compared to general Interne works. Huffaker et al. [12] evaluate eight databases. point out the absence of a substantial ground dataset. They propose a method to evaluate th curacy by a centroid-based algorithm working majority of location votes from the databases location accuracy in a form of cumulative prob ty is only provided for five databases: MaxMin MaxMind Lite, IPligence, Digital Envoy, and H For the data shown in Table 4, we use the lo accuracy for the PlanetLab groundtruth datase results show a better location accuracy most pro because of the use of the groundtruth dataset, was created by using the location voting algo Another reason is that the PlanetLab ground dataset is strongly oriented towards the majo ies [15, 16]. Table 4 Cumulative percentage of estimated locations within maximum location error [km] – created from source [ Vendor <50 <100 <150 MaxMind 81 85 89 Digital Envoy 88 91 93 IPligence 78 82 85 HostIP 76 82 83 Poese et al. [24] focus on the differences betwe claimed accuracy by the database vendors an real-case location accuracy. The accuracy is ev ed by using a groundtruth dataset that was obt via a large European ISP. They create PoPs bas the network prefixes. Figure 3 Example of same-ISP nodes used in groundtruth dataset Figure 5 g Groundtruth nodes in cities with different area 3 We aimed to cover a variation of cities in the groundtruth. The cities covered are very small to very large, i.e. we did not focus only on major cities. The city population and area of the groundtruth nodes is shown in Figures 4 and 5. The median value for the city population is 16 925. The median value for the city area is 50 km2. After applying the proposed method to the orig inal dataset, we obtained about 700 error-free groundtruth nodes. The trusted nodes were situated in 16 countries, 52 regions, and 270 cities. They were assigned to 319 ISPs. 5. Construction of error-free groundtruth dataset One of the main concerns when evaluating location accuracy is the groundtruth dataset. As we show in the related work, the used groundtruth influences the location accuracy results a lot. Being aware of this problem, we construct an error-free groundtruth dataset. We collected the original groundtruth public IP addresses by using a developed mobile application. 338 2017/3/46 Information Technology and Control Figure 4 Groundtruth nodes in cities with different population Figure 5 Groundtruth nodes in cities with different area Figure 4 Groundtruth nodes in cities with different population Figure 4 Groundtruth nodes in cities with different population Figure 4 The groundtruth geographical locations were ob tained by using in-built GPS in the used mobile devic es. We also recorded the country, region, city, and ISP for each groundtruth IP address. Figure 4 Groundtruth nodes in cities with different population Groundtruth nodes in cities with different population The groundtruth construction is as follows: 1 In order to keep the dataset free of the problems that described in the related work, we strictly fol lowed the rule to use only one node per ISP in a city (i.e. there is only one node which belong to an ISP in a city). This restricted the size of the origi nal dataset a lot. On the other hand, it assured the proper distribution of the nodes and the location accuracy results are not influenced by repeating the same or similar locations. 2 We additionally filtered the dataset to store only the nodes that belong to same ISPs and, at the same time, situated in at least 10 different cities for each ISP involved. The reason is that the location da tabases give the same or a small set of locations for the same-ISP nodes that are correctly located in many different places [27]. An example distri bution of the nodes that belong to such an ISP is shown in Figure 3. Absolute location error details [km] Database Mean Std Dev 1st q. Median 3rd q. MaxMind 64 86 3 18 113 DB-IP 142 775 5 50 181 IP2Location 91 290 4 26 146 ipinfo 95 98 5 47 187 Skyhook 50 76 1 10 70 Neustar 93 100 6 51 181 Eurek 69 112 3 17 119 Geobytes 657 1768 21 168 261 Figure 6 Relative location accuracy a city only when the estimation is believed to be cor rect, otherwise the city returned is null. The most significant differences in absolute location error between all the databases are around a maximal error of 150 km. The best database gives about 90 % of the locations within this error range (Skyhook) while the worst returns only 45 % of the results within this error range (GeoBytes). This difference becomes smaller for large-errors that we separately study in section 6.6. All the databases return almost all loca tions within maximum error range of 300 km except the database GeoBytes. The results show that the majority of the databases achieve nearly 100 % accuracy at the country level. The worst accuracy is achieved by the database Geo Bytes which is around 80 %. There are much worse results for the region and city estimations. The best databases return around half of the estimations cor rect at the region level. The worst database is again GeoBytes with about 20 % of the estimated correct regions. At the city level, the best databases give about 30 % of the estimations correct. We notice a signifi cant drop for the Skyhook database which returns only about 15 % of the cities correctly. We, however, note that this database returns a city only when there is a high probability of a correct match. The results show that the majority of the databases achieve nearly 100 % accuracy at the country level. The worst accuracy is achieved by the database Geo Bytes which is around 80 %. There are much worse results for the region and city estimations. The best databases return around half of the estimations cor rect at the region level. The worst database is again GeoBytes with about 20 % of the estimated correct regions. At the city level, the best databases give about 30 % of the estimations correct. We notice a signifi cant drop for the Skyhook database which returns only about 15 % of the cities correctly. We, however, note that this database returns a city only when there is a high probability of a correct match. Table 6 provides specific numbers on absolute loca tion accuracy. The standard deviation shows large variation of the location errors for the databases DB- IP and GeoBytes. Figure 7 local language. Some of the databases return the En glish form, but the others keep the local name. For a proper evaluation, we stored the English forms of place names in the groundtruth dataset. If a database returned a place name in the local language, we found its English form for a proper evaluation. Figure 7 Cumulative probability of absolute location error Cumulative probability of absolute location error Cumulative probability of absolute location error Figure 6 Relative location accuracy 6.1 Relative location accuracy By using the groundtruth dataset constructed, we evaluated relative location accuracy in terms of the correctly estimated countries, regions, and cities. The results are shown in Figure 6. During this evaluation, we faced a particular problem of the place names which are sometimes different in English and in the 339 Information Technology and Control 2017/3/46 Table 7 Location accuracy details for nodes belonging to same ISP [km] Location accuracy details for nodes belonging to same ISP [km] Database Mean Std Dev 1st q. Median 3rd q. MaxMind 118 86 35 112 201 DB-IP 144 78 78 158 206 IP2Location 118 88 38 107 205 ipinfo 145 91 44 171 215 Skyhook 87 81 21 61 138 Neustar 145 77 80 159 204 Eurek 117 88 35 102 201 Geobytes 483 1142 54 177 236 6.2 Absolute location accuracy 12 We evaluated absolute location accuracy as the geo graphical distance between the estimated and correct coordinates. We noticed great differences between absolute and relative location accuracy. A good exam ple is the Skyhook database. Figure 7 shows cumula tive probabilities of maximal location errors for each database. The most accurate database is Skyhook. However, regarding relative accuracy (Figure 6), Sky hook gives the worst result at the city level. The rea son behind this inconsistency is that Skyhook returns 340 2017/3/46 Information Technology and Control figure shows the cumulative probability for the same- ISP nodes located within a maximum location error. The gap between the location error probabilities for the databases is smaller compared to the general case shown in Figure 7. The graph also shows generally worse cumulative probabilities. The databases again locate all the nodes within a maximum error distance of 300 km, except the database GeoBytes. Figure 10 g Absolute location error of nodes belonging to same-ISP g Absolute location error of nodes belonging to same-ISP Figure 8 Location error change with different city area Figure 9 Location error change with different city population 6.4 Effect of ISP assignment The databases typically return the same or a small set oflocationsforthenodesassignedtothesameISP We The specific numbers of this evaluation are shown Table 7. The database with the lowest median locati error is again Skyhook. However, the median val for the database Skyhook is about 50 km worse, com pared to the general multi-ISP scenario. Table 7 Location accuracy details for nodes belonging to same IS [km] Database Mean Std Dev 1st q. Median 3rd q MaxMind 118 86 35 112 201 DB-IP 144 78 78 158 206 IP2Location 118 88 38 107 205 ipinfo 145 91 44 171 215 Skyhook 87 81 21 61 138 Neustar 145 77 80 159 204 Eurek 117 88 35 102 201 Figure 8 Location error change with different city area Figure 9 Location error change with different city population 6.3 Effect of city area and population Figures 8 and 9 show the relative location error change for different city areas and populations. The figures show the regression lines of the median lo cation error values. Both figures indicate better ac curacy with increasing city area and population. The reason for these accuracy changes is the source of location information that come from traffic measure ment (more populated places generate more Internet traffic). This also holds for crawling and datamining the web servers for location data. Figure 10 Absolute location error of nodes belonging to same-ISP Figure 8 Location error change with different city area Location error change with different city area The specific numbers of this evaluation are shown in Table 7. The database with the lowest median location error is again Skyhook. However, the median value for the database Skyhook is about 50 km worse, com pared to the general multi-ISP scenario. Figure 9 Location error change with different city population 6.6 Large errors Correct city estimations for nodes belonging to same-ISP A common problem of IP geolocation is that some lo cations are returned with a great error. The previous observation shows the percentages of such large-er rors in Figure 7. It shows that there is a percentage of location errors over 200 km for each database. The database GeoBytes shows the worst results for loca tion errors over 200 km. Correct city estimations for nodes belonging to same-ISP We demonstrate the problem of large errors in Fig ure 13. The green/bright marks show the locations estimated with some large errors. Additionally, one of the red/dark marks shows an estimated location with an extremely large error. The node is correctly situat ed in Paris, France but the estimated location points to Istanbul, Turkey. 6.4 Effect of ISP assignment The databases typically return the same or a small set of locations for the nodes assigned to the same ISP. We particularly study this phenomenon in Figure 10. The 341 2017/3/46 Information Technology and Control We also study relative errors for the same-ISP nodes. Figure 11 shows the correctly estimated cities. We con clude that the databases estimate approximately one quarter (on average) of the cities correctly when the same ISPs are used compared to multiple ISPs usage. We also study relative errors for the same-ISP nodes. Figure 11 shows the correctly estimated cities. We con clude that the databases estimate approximately one quarter (on average) of the cities correctly when the same ISPs are used compared to multiple ISPs usage. a relative location only when a certain level of trust worthiness is reached. We also note that these num bers do not indicate the location accuracy as some databases return the capital city or the centre of a country when a better estimation is not known. This is better seen when Figures 12 and 6 are compared. Figure 13 Figure 13 Example of very large location errors The previous results show that the databases do not resolve (do not return) a location for every node re quested. Figure 12 shows the not-returned relative lo cations (country, region, and city) and the coordinates. The results indicate that the best database in terms of the resolved locations is DB-IP (100 % locations re turned), IP2Location IP (100 % locations returned), Neustar, and GeoBytes. We note that Skyhook returns Example of very large location errors Figure 14 of small cities involved in the dataset (the median city population was 17 000). Databases differences for large location errors (last decile) The city population and area have a significant impact on location accuracy. Furthermore, node association to the ISPs also have a significant impact on location accuracy. Multiple nodes that were assigned to the same ISP but in different cities were located less ac curately than the nodes that were assigned to different ISPs. There was a 50 km worse median location error for the best performance database Skyhook. Also, the databases estimated approximately one quarter (on average) of the cities correctly when the same ISPs were used compared to multiple ISPs usage. The percentages of returned locations do not correlate with location accuracy. This was particularly apparent for the correctly estimated and unresolved cities. 7. Conclusions Finally, we noticed great differences between absolute and relative location accuracy. Some databases may return a city or region only when there is a high prob ability of a correct match so as not to confuse the users. The accuracy of client-independent IP location was high at the country level where it reached almost 100 % of correct estimations (except the database GeoBytes). In this case, IP geolocation databases can be trusted. The region-level accuracy was of a worse value, best databases gave around 50 % of correct es timations. The best city-level accuracy was around 30 %. Considering the error distances between the estimated and correct coordinates, the best median error was 10 km for the database Skyhook. We note that all these values were obtained for a high number Acknowledgments Research described in this paper was financed by the National Sustainability Program under grant LO1401. Infrastructure of the SIX (Sensor, Information and Communication Systems) Center was used. Figure 12 Figure 12 Unresolved locations Unresolved locations Differences between the databases for such large er rors may be seen better by plotting a matrix of the mu tual differences for the last decile of location errors. The heatmap in Figure 14 indicates that some of the database pairs (MaxMind and Eurek, ipinfo and DB- IP) have very small location error differences. On the other hand, GeoBytes seems to be quite unreliable as it has large differences from the other databases reaching a range of hundreds of kilometers. Therefore this database should be excluded from the technique that uses the centre of gravity of multiple location re sults as the final estimated location. 342 342 2017/3/46 of small cities involved in the dataset (the median city population was 17 000). Vehicular Networking Conference (VNC) 2011, 2011, 70-77. https://doi.org/10.1109/VNC.2011.6117126 Vehicular Networking Conference (VNC) 2011, 2011, 70-77. https://doi.org/10.1109/VNC.2011.6117126 erties for IP Geolocation. Information Technology and Control, 2016, 45(1), 77-85. https://doi.org/10.5755/j01. itc.45.1.11062 7. Graham, M., Hale, S., Gaffney, D. Where in the World Are You? Geolocation and Language Identification in Twitter. The Professional Geographer, 2014, 66(4), 568- 578. https://doi.org/10.1080/00330124.2014.907699 18. Komosny, D., Voznak, M., Kathiravelu, G., Sathu, H. Esti mation of Internet Node Location by Latency Measure ments – The Underestimation Problem. Information Technology and Control, 2015, 44(3), 279-286. https:// doi.org/10.5755/j01.itc.44.3.8353 8. Gueye, B., Uhlig, S., Fdida, S. Investigating the Impre cision of IP Block-Based Geolocation. In: Passive and Active Network Measurement, Lecture Notes in Com puter Science, Springer, 2007, 4427, 237-240. https:// doi.org/10.1007/978-3-540-71617-4_26 19. Krajsa, O., Fojtova, L. RTT Measurement and Its De pendence on the Real Geographical Distance. IEEE 34th International Conference on Telecommunica tions and Signal Processing, 2011, 231-234. https://doi. org/10.1109/TSP.2011.6043737 9. Gueye, B., Ziviani, A., Crovella, M., Fdida, S. Con straint-Based Geolocation of Internet Hosts. IEEE/ ACM Transactions on Networking, 2006, 14(6), 1219- 1232. https://doi.org/10.1109/TNET.2006.886332 20. Laki, S., Matray, P., Haga, P., Sebok, T., Csabai, I., Vat tay, G. Spotter: A Model Based Active Geolocation Service. IEEE Conference on Computer Communica tions, 2011, 3173-3181. https://doi.org/10.1109/INF COM.2011.5935165 10. Guo, Ch., Liu, Y., Shen, W., Wang, H., Yu, Q., Zhang, Y. Mining the Web and the Internet for Accurate IP Ad dress Geolocations. IEEE INFOCOM 2009, 2009, 2841- 2845. 21. Moravek, P., Komosny, D., Burget, R., Sveda, J., Handl, T., Jarosova L. Study and Performance of Localization Methods in IP Based Networks: Vivaldi Algorithm. Jour nal of Network and Computer Applications, 2011, 34(1), 351-367. https://doi.org/10.1016/j.jnca.2010.06.014 11. Hegr, T., Bohac, L., Kocur, Z., Voznak, M., Chlumsky, P. Methodology of the Direct Measurement of the Switch ing Latency. Przeglad Elektrotechniczny, 2013, 89(7), 59-63. 22. Moravek, P., Komosny, D., Simek, M., Sveda, J., Handl, T. Vivaldi and Other Localization Methods. TSP 2009: 32nd International Conference on Telecommunica tions and Signal Processing, Asszisztencia Congress Bureau, 2009, 214-218. 12. Huffaker, B., Fomenkov, M., Claffy, K. Geocompare: a Comparison of Public and Commercial Geolocation Databases. CAIDA Tech Report, University of Califor nia, 2011. 13. Katz-Bassett, E., John, P., Krishnamurthy, A., Wether all, D., Anderson, T., Chawathe, Y. Towards IP Geolo cation Using Delay and Topology Measurements. Pro ceedings of the 6th ACM SIGCOMM Conference on Internet Measurement, ACM, 2006, 71-84. https://doi. org/10.1145/1177080.1177090 23. Percacci, R., Vespignani, A. Scale-Free Behavior of the Internet Global Performance. References 1. Backstrom, L., Sun, E., Marlow, C. Find Me if You Can: Improving Geographical Prediction with Social and Spatial Proximity. Proceedings of the 19th Internation al Conference on World Wide Web, ACM, 2010, 61-70. https://doi.org/10.1145/1772690.1772698 ence on Computational Linguistics (COLING 2012), 2012, 1045-1062. 4. Burget, R., Komosny, D., Kathiravelu, G. Topology Aware Feedback Transmission for Real-Time Control Protocol. Journal of Network and Computer Applica tions, 2012, 35(2), 723-730. https://doi.org/10.1016/j. jnca.2011.11.005 2. Balakrishnan, H., Lakshminarayanan, K., Ratna samy, S., Shenker, S., Stoica, I., Walfish, M. A Lay ered Naming Architecture for the Internet. Pro ceedings of the 2004 Conference on Applications, Technologies, Architectures, and Protocols for Com puter Communications, ACM, 2004, 343-352. https:// doi.org/10.1145/1015467.1015505 5. Fioreze, T., Heijenk, G. Extending DNS to Support Geo casting Towards VANETS: A Proposal. IEEE Vehicular Networking Conference (VNC) 2010, 2010, 271-277. https://doi.org/10.1109/VNC.2010.5698258 6. Fioreze, T., Heijenk, G. Extending the Domain Name System (DNS) to Provide Geographical Addressing To wards Vehicular Ad-Hoc Networks (VANETs). IEEE 3. Bo, H., Cook, P., Baldwin, T. Geolocation Prediction in Social Media Data by Finding Location Indicative Words. Proceedings of the 24th International Confer 343 Information Technology and Control 2017/3/46 Vehicular Networking Conference (VNC) 2011, 2011, 70-77. https://doi.org/10.1109/VNC.2011.6117126 The European Physical Journal B – Condensed Matter and Complex Systems, 2003, 32(4), 411-414. https://doi.org/10.1140/epjb/ e2003-00123-6 24. Poese, I., Uhlig, S., Kaafar, M., Donnet, B., Gueye, B. IP Geolocation Databases: Unreliable? ACM SIGCOMM Computer Communication Review, 2011, 41, 53-56. https://doi.org/10.1145/1971162.1971171 14. Komosny, D., Balej, J., Sathu, H., Shukla, R., Dolezel, P., Simek, M. Cable Length Based Geolocalisation. Prze glad Elektrotechniczny, 2012, 88(7a), 26-32. 15. Komosny, D., Pang, S., Pruzinsky, J., Ilko, P., Polasek, J. PlanetLab Europe as Geographically-Distributed Test bed for Software Development and Evaluation. Advanc es in Electrical and Electronic Engineering, 2015, 13(2), 137-146. https://doi.org/10.15598/aeee.v13i2.1245 25. Shavitt, Y., Zilberman, N. A Geolocation Databases Study. IEEE Journal on Selected Areas in Communica tions, 2011, 29(10), 2044-2056. https://doi.org/10.1109/ JSAC.2011.111214 26. Sousa, A., Costa, A., Santos, A., Meneses, F., Nicolau, M. Using DNS to Establish a Localization Service. IEEE International Conference on Indoor Positioning and Indoor Navigation 2014, 2014, 385-392. https://doi. org/10.1109/IPIN.2014.7275506 16. Komosny, D., Pruzinsky, J., Ilko, P., Polasek, J., Masek, P., Kocatepe, O. On Geographic Coordinates of PlanetLab Europe. IEEE 38th International Conference on Tele communications and Signal Processing (TSP), 2015, 642-646. https://doi.org/10.1109/TSP.2015.7296342 27. Triukose, S., Ardon, S., Mahanti, A., Seth, A. Geolocat ing IP Addresses in Cellular Data Networks. Passive 17. Komosny, D., Voznak, M., Bezzateev, S., Kathiravelu, G. The Use of European Internet Communication Prop 344 344 2017/3/46 Information Technology and Control and Active Measurement, 13th International Con ference, Springer, 2012, 7192, 115-167. https://doi. org/10.1007/978-3-642-28537-0_16 29. Wong, B., Stoyanov, I., Sirer, E. Octant: A Comprehen sive Framework for the Geolocalization of Internet Hosts. Proceedings of the 4th USENIX Conference on Networked Systems Design & Implementation, USE NIX Association, 2007, 313-326. 28. Voznak, M., Rozhon, J., Ilgin, H. Network Delay Varia tion Model Consisting of Sources with Poisson‘s Proba bility Distribution. Proceedings of the 15th WSEAS In ternational Conference on Computers, WSEAS, 2011, 240-244. 30. Zander, S. How Accurate is IP Geolocation Based on IP Allocation Data? Technical Report 120524A, Swin burne University of Technology, 2012. Summary / Santrauka This paper deals with finding the geographical location of Internet nodes remotely with no need to communi cate with the nodes located (client-independently). IP geolocation is used in a number of areas, such as content personalisation, on-line fraud prevention and detection, and digital media law enforcement. One of the main concerns when studying the accuracy of client-independent geolocation is the groundtruth dataset. As we show in the related work, the used groundtruth influences the results a lot. We construct an error-free groundtruth dataset consisting of nodes with GPS-precise locations. We also record the country, region, city, and ISP for each groundtruth node. Using the created groundtruth, we study the accuracy of eight IP location databases in a number of scenarios, such as effect of city area and population, effect of ISP assignment, and number of not-returned locations. Straipsnyje aptariamas geografinės interneto mazgų padėties aptikimas nuotoliniu būdu, kai nereikia susisiekti su pačiais mazgais (nepriklausomais nuo kliento). IP adresų geografinė informacija naudojama daugelyje sričių, pavyzdžiui, turinio personalizavimas, sukčiavimo prevencija ir aptikimas internete bei skaitmeninės žiniasklai dos teisėsauga. Vienas iš svarbiausių aspektų, tyrinėjant nuo kliento nepriklausomą geografinę informaciją, yra etaloninių reikšmių duomenų rinkinys. Kaip rodo ankstesnieji tyrimai, naudojamos etaloninės reikšmės turi daug įtakos rezultatams. Autoriai pristato savo sukurtą klaidų neturintį etaloninių reikšmių duomenų rinkinį, sudarytą iš mazgų su tiksliomis, GPS pagalba nustatytomis lokacijomis. Kiekvienam etaloninės reikšmės mazgui taip pat padaromas šalies, regiono, miesto ir ISP įrašas. Naudodamiesi savo sukurtomis etaloninėmis reikšmėmis, autoriai tiria aštuonių IP lokacijų duomenų bazių tikslumą skirtinguose scenarijuose – tokiuose, kaip miesto vie tovės ir populiacijos įtaka, interneto ryšio tiekėjo paskyrimo ir negautų atsakymų, įtaka.
https://openalex.org/W1534937555	https://www.scielo.br/j/sa/a/7LGYxhmRTFx4kxxGNYfY64C/?lang=en&format=pdf	English	null	Scaling to generalize a single solution of Richards' equation for soil water redistribution	Scientia Agrícola	2,011	cc-by	7,355	582 582 Scaling to generalize a single solution of Richards' equation for soil water redistribution Morteza Sadeghi1, Bijan Ghahraman1, Kamran Davary1, Seyed Majid Hasheminia1, Klau Reichardt2 Morteza Sadeghi1, Bijan Ghahraman1, Kamran Davary1, Seyed Majid Hasheminia1, Klaus Reichardt2 1Ferdowsi University of Mashhad/College of Agriculture – Dept. of Water Engineering – 91779-4897 – Mashhad, Iran. 2USP/CENA – Lab. de Física do Solo, C.P. 96 – 13418-900 – Piracicaba, SP – Brasil. Corresponding author <m.sadeghi.um@gmail.com> Edited by: Jussara Borges Regitano ABSTRACT: Using scaling methods, a single solution of Richards' equation (RE) will suffice for numerous specific cases of water flow in unsaturated soils. In this study, a new method is developed to scale RE for the soil water redistribution process. Two similarity conditions are required: similarity in the shape of the soil water content profiles as well as of the water flux density curves. An advantage of this method is that it is not restricted to a specific soil hydraulic model – hence, all such models can be applied to RE. To evaluate the proposed method, various soil textures and initial conditions were considered. After the RE was solved numerically using the HYDRUS-1D model, the solutions were scaled. The scaled soil water content profiles were nearly invariant for medium- and fine-textured soils when the soil profile was not deeply wetted. The textural range of the soils in which the similarity conditions are held decreases as the initial conditions deal with a deeply wetted profile. Thus, the scaling performance was poor in such a condition. This limitation was more pronounced in the coarse-textured soils. Based on the scaling method, a procedure is suggested by which the solution of RE for a specific case can be used to approximate solutions for many other cases. Such a procedure reduces complicated numerical calculations and provides additional opportunities for solving the highly nonlinear RE as in the case of unsaturated water flow in soils. Keywords: Nielsen-similarity, spatial variability, invariant solutions Introduction by Warrick et al. (1985), and Vogel et al. (1991). In the second class, the scaling factors are defined by considering the imposed boundary and/or initial conditions. A clear advantage of these methods over the first class is that the scaled RE is invariant to the boundary and/or initial conditions. However, these methods are limited to specific hydrological processes and/or soil hydraulic models. For example, the method of Warrick and Hussen (1993), developed for infiltration and redistribution, applies only for the Brooks-Corey hydraulic functions. Scaling methods based on the "similar media" concept (Miller and Miller, 1956) were developed to cope with the spa- tial variability of soils (Warrick et al., 1977; Sharma et al., 1980; Ahuja and Williams, 1991; Kosugi and Hopmans, 1998; Tuli et al., 2001; Kozak and Ahuja, 2005; Roth, 2008; Sadeghi et al., 2010). Vereecken et al. (2007) comprehensively reviewed the scaling methods developed during the past years. Scaling has proven its success also as a tool for numerical analyses. Using scaling methods, a single solution of Richards' equation (RE) will suffice for numerous specific cases of un- saturated water flow. Hence, these methods considerably reduce the calculations required for heterogeneous soils (Warrick and Hussen, 1993). So far, various methods for scaling RE have been proposed (Reichardt et al., 1972; Warrick and Amoozegar- Fard, 1979; Warrick et al., 1985; Vogel et al., 1991; Kutilek et al., 1991; Warrick and Hussen, 1993; Neuweiler and Cirpka, 2005). Using specific scaling factors, these methods allow a linear transformation of RE variables to achieve invariant solutions for a set of similar soils. This similarity may be defined based on microscopic-scale geometry (Miller and Miller 1956), shape of soil hydraulic functions (Simmons et al., 1979), or a linear variability concept (Vogel et al., 1991). The objective of this study, following the second class, is to scale RE focused on the redistribution process. Scaling of RE solutions is proposed instead of solving the scaled RE. By this procedure, this new method is not restricted to a specific hydraulic model – hence, all existing and future models can be used for the redistribution solutions. Theory The following initial conditions are considered: θ(z, 0) = θfi (0 < z < zfi) (04a) θ(z, 0) = θi (z > zfi) (04b) (04a) (04b) where θfi and zfi are the initial values of the wetting front water content θf(t) and depth zf(t), respectively; θι is the initial soil water content below the wetting front (WF). Figure 1 graphi- cally describes the imposed boundary and initial conditions on the solution domain. (08) (08) A θ-based solution of Eq. (1), considering conditions (2) to (4), yields soil water content profiles (SWCP), θ(z), during redistribution. Considering the shape of the SWCPs during redistribution, two general forms can be found: the SWCP (a) with the θ gradient everywhere positive and with the θ distribution almost uniform above a sharp WF, and (b) with a further wetting below advancing as a step-like profile while the soil desaturates near the surface (Youngs, 1990). where subscripts A and B correspond to cases A and B. Eq. (8) gives: (09) (09) Theory Consider a one-dimensional Richards’ equation (RE) of the form: (01) (01) where θ [L3 L–3] is the volumetric soil water content, K [L T–1] the unsaturated hydraulic conductivity, h [L] the soil water matric po- tential head (i.e. absolute value of the soil water pressure head), z the vertical position coordinate below soil surface, and t the time. Scaling methods can be divided into two classes (Kutilek and Nielsen, 1994). In the first class, the scaling factors are derived to unify the soil hydraulic functions into a single curve and therefore, are invariant quantities for each soil. Using these methods, scaled RE will be invariant for similar soils provided that the scaled boundary and initial conditions are the same. This provision limits the applicability of such methods developed, for example, Considering a soil water redistribution process without surface evaporation, the following conditions are imposed on the upper (z = 0) and lower boundary (z = L) of the solution Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Richards' equation for soil water redistribution 583 θ (z, t = 0) = 1 (0 < z < 1) (07a) θ* (z, t = 0) = 0 (z > 1) (07b) domain dealing with zero water flux density and free drainage, respectively: domain dealing with zero water flux density and free drainage, respectively: (07a) (07b) q(0,t)=0 (02) q(L,t)=K(L,t) (03) q(0,t)=0 (02) In Eqs. (5) to (7) θ* and z* are the scaled soil water content and depth, respectively. It is assumed that t can be scaled using a constant scaling factor τ (i.e. t=τt, where t is the scaled time) so that the scaled solutions of RE, θ(z,t), be invariant for a set of cases (i.e. specific soils and initial conditions). To do so, a primary requirement is that the shape of the SWCPs be similar for all the cases. Assume two cases A and B for which this con- dition is held. Since the scaled initial conditions are invariant, the scaled solutions will be the same for these two cases only if the scaled WF advance velocity (i.e. dzf /dt, where zf is the scaled WF depth) is the same at each scaled time: q(L,t)=K(L,t) (03) where q [L T–1] is the water flux density. Scaling Method here vf [T–1] represents dzf /dt, and vﬁ is the initial value of vf. Based on Eq. (9), vﬁ will be the best choice for the time scaling factor, τ: Following relationships are proposed to scale θ and z: (05) (06) factor, τ: (05) t = vﬁ t (10) t* = vﬁ t (10) To determine vfi, Darcy’s equation is considered in the form of: (06) (06) of: (11) (11) by which the scaled values of θfi and zfi will become unity and the scaled initial conditions will be invariant as follows: Integrating Eq. (11) from the soil surface to the WF depth at t = 0, we have: Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/ Figure 1 – Graphical description of the imposed boundary and initial conditions on the soil proﬁ le during redistribution process. (12) (12) which yields: which yields: (13) (13) In Eqs. (12) and (13), hi [L] is the matric potential head corresponding to θi, hfi [L] and Kfi [LT–1] are the matric potential head and hydraulic conductivity corresponding to θfi, respectively, θfi is the initial value of downward flux density at the WF, Ks [LT–1] is the saturated hydraulic Figure 1 – Graphical description of the imposed boundary and initial conditions on the soil proﬁ le during redistribution process. Figure 1 – Graphical description of the imposed boundary and initial conditions on the soil proﬁ le during redistribution process. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sadeghi et al. 584 (19) conductivity, and G [L] is WF matric head at t = 0 defined as: (19) (14) where θr and θs are soil residual and saturated water contents, respectively, and α, n, and m are empirical parameters with the assumption that m=1-1/n. (14) Soils of twelve textural classes were considered by applying parameters of Carsel and Parrish (1988) which are the default parameters of HYDRUS for van Genuchten functions (Table 1). Two sets of initial conditions, presented in Table 2, were considered using various combinations of zfi, hfi and hi. Set A (A1 to A6) was considered to evaluate the effect of the total water added to the soil profile, W, while Set B (B1 to B8) was considered to separately evaluate the effects of zfi, hfi and hi. Scaling Method (17) implies in another condition necessary e scaling, dealing with the shape similarity of the flux den- urves over the scaled soil profile, such that it can be scaled near transformation. This kind of similarity was previously ed by Simmons et al. (1979) and, as stated by Sposito and 1985), is referred to as "Nielsen-similarity". Materials and Methods chards' Equation was solved using HYDRUS-1D, Version munek et al., 2008) for various soils and initial conditions. To ate the SWCPs during redistribution, zero water flux and free ge were set as the upper and lower boundary conditions, ctively. Van Genuchten (1980) hydraulic functions based on m's (1976) model were adopted: (18) Table 1 – Van Genuchten parameters of 12 soils (Carsel and Parrish, 1988) used for the numerical studies. Soil texture θr θs α n Ks cm–1 cm per day sand 0.045 0.43 0.145 2.68 712.8 loamy sand 0.057 0.41 0.124 2.28 350.2 sandy loam 0.065 0.41 0.075 1.89 106.1 loam 0.078 0.43 0.036 1.56 24.96 silt 0.034 0.46 0.016 1.37 6.00 silt loam 0.067 0.45 0.020 1.41 10.80 sandy clay loam 0.100 0.39 0.059 1.48 31.44 clay loam 0.095 0.41 0.019 1.31 6.24 silty clay loam 0.089 0.43 0.010 1.23 1.68 sandy clay 0.100 0.38 0.027 1.23 2.88 silty clay 0.070 0.36 0.005 1.09 0.48 clay 0.068 0.38 0.008 1.09 4.80 September/October 2011 n necessary he flux den- an be scaled s previously Sposito and 1D, Version nditions. To flux and free conditions, ns based on (18) Table 1 – Van Genuchten parameters of 12 soils (Carsel and Parrish, 1988) used for the numerical studies. Soil texture θr θs α n Ks cm–1 cm per day sand 0.045 0.43 0.145 2.68 712.8 loamy sand 0.057 0.41 0.124 2.28 350.2 sandy loam 0.065 0.41 0.075 1.89 106.1 loam 0.078 0.43 0.036 1.56 24.96 silt 0.034 0.46 0.016 1.37 6.00 silt loam 0.067 0.45 0.020 1.41 10.80 sandy clay loam 0.100 0.39 0.059 1.48 31.44 clay loam 0.095 0.41 0.019 1.31 6.24 silty clay loam 0.089 0.43 0.010 1.23 1.68 sandy clay 0.100 0.38 0.027 1.23 2.88 silty clay 0.070 0.36 0.005 1.09 0.48 clay 0.068 0.38 0.008 1.09 4.80 Table 1 – Van Genuchten parameters of 12 soils (Carsel and Parrish, 1988) used for the numerical studies. Scaling Method To prevent the divergence of the solutions, the abrupt increase of the matric potential head at the WF was avoided. To do so, the matric potential head gradually increased from hfi and hi through four space steps at the WF. It should be noted that HYDRUS works with the pressure head (negative values) instead of the absolute value of the matric head. Mass conservation law gives: (15) (15) Combining (13) and (15) results in: Combining (13) and (15) results in: (16) (16) SWCPs obtained by HYDRUS were scaled using Eqs. (5), (6), and (10). The value of vfi was determined from Eq. (16) with G being computed from Eq. (14). The integral of Eq. (14) was approximated by the trapezoidal rule. For convenience of application, it is worth to note that hfi=0 and hi Æ ∞, G approaches the effective capillary drive (HcM) defined by Morel-Seytoux and Khanji (1974): Using Eq. (16), vfi can be calculated from the initial con- ditions (i.e. θfi, θi and zfi). The soil hydraulic functions should be known to determine Kfi and hfi from θfi as well as hi from θi (hfi and hi are required to calculate G using Eq. (14)). Although there is no restriction on a specific form of the soil hydraulic models, it should be noticed that the selected hydraulic models should be the same for solving RE and cal- culating vfi using Eq. (16). Based on the mass conservation law, ∂θ / ∂t = –∂q / ∂z , Eqs. (5), (6), and (10) suggest the following relationship to scale the flux density: (20) (20) (17) (17) When van Genuchten hydraulic models with the assumption of m=1-1/n are applied, HcM can be approximated as follows (Morel-Seytoux et al., 1996): where q* is the scaled flux density. To scale the redistribution process for a set of soils and initial conditions, q(z) should be a unified curve. Eq. (17) implies in another condition necessary for the scaling, dealing with the shape similarity of the flux den- sity curves over the scaled soil profile, such that it can be scaled by a linear transformation. This kind of similarity was previously adopted by Simmons et al. (1979) and, as stated by Sposito and Jury (1985), is referred to as "Nielsen-similarity". gric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 ied curve. Eq. Materials and Methods Richards' Equation was solved using HYDRUS-1D, Version 4 (Simunek et al., 2008) for various soils and initial conditions. To simulate the SWCPs during redistribution, zero water flux and free drainage were set as the upper and lower boundary conditions, respectively. Van Genuchten (1980) hydraulic functions based on Mualem's (1976) model were adopted: (18) (18) Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Richards' equation for soil water redistribution 585 (21) where θ* mi is the arithmetic mean of all θ* values at each 0.1 in- crement of z* at which θ* i is located, and N is the total number of the evaluated points. Profile of θ* m shows a mean scaled SW- CPs, and therefore, MAES and RMSES indicate the deviations of the scaled SWCPs from the mean scaled SWCPs. When all the scaled SWCPs coalesce, these criteria will be equal to zero, suggesting an ideal performance of the scaling method. (21) with minimal errors (< 2 %) over the range of m from 0.05 to 0.7. Eq. (21) was used to find a proper value for increments of h in the approximation of G using the trapezoidal rule. With 0.1 cm increments of h, approximations of the trapezoidal rule were close to those of Eq. (21). Results and Discussion To quantitatively evaluate the performance of the proposed scaling method, for each set of the scaled SWCPs, two criteria – the mean absolute error of scaling (MAES) and the root mean squared error of scaling of scaling (RMSES) – were defined as: Figure 2 shows HYDRUS model outputs for the 12 soils of Table 1 and initial conditions of A1 which were scaled by Eqs. (5), (6), and (10). Except for the sand, loamy sand and sandy loam, the remaining nine soils were reasonably well scaled and manifest a nearly unique scaled SWCP. A reason for the undesirable deviations in the three sandy soils is that the SWCPs in these soils with the imposed initial conditions are not similar in shape to those of the other soils. However, it seems that the main reason for the deviations is that, regarding the Nielsen-similarity condition, these soils are not similar to the other soils. To clarify this issue, the similarity condition should be evaluated. (22) (23) (22) (22) (23) (23) As mentioned earlier, the so-called Nielsen-similarity deals with the shape similarity of the flux density curves during redis- tribution. It is assumed that the flux curves are described by the following power model (Jury and Horton, 2003): Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Table 2 – Two sets of initial conditions (A and B) used for the numerical studies. Code A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 B7 B8 zfi (cm) 5 10 15 20 30 50 5 50 5 50 5 50 5 50 hfi (cm) 10 8 6 4 2 0 10 10 0 0 10 10 0 0 hi (× 104 cm) 500 100 50 10 5 1 500 500 500 500 1 1 1 1 Figure 2 – Scaled soil water content proﬁ les for the 12 soils of Carsel and Parrish (1988) (see Table 1) and initial conditions of A1 (see Table 2) at (a) t* = 1, (b) t* = 5, and (c) t* = 10. Table 2 – Two sets of initial conditions (A and B) used for the numerical studies. Table 2 – Two sets of initial conditions (A and B) used for the numerical studies. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Results and Discussion Figure 2 – Scaled soil water content proﬁ les for the 12 soils of Carsel and Parrish (1988) (see Table 1) and initial conditions of A1 (see Table 2) at (a) t* = 1, (b) t* = 5, and (c) t* = 10. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sadeghi et al. 586 q=a(t+b)c (24) We conclude that the scaling is more sensitive to W for the very coarse- and very fine- textured soils. The scaled SWCPs for the loam are approximately invariant with respect to the initial conditions. For the sand, when W is relatively small (A1 and A2), the scaled SWCPs show a delay, while the scaling is adequate for larger values of W. On the other hand, the scaled SWCPs in the clay coalesced for all initial conditions except for A6 – the condition corresponding to the greatest value of W. To separately study the effects of zfi, hfi and hi on the scal- ing performance, RE was solved by applying set B of the initial conditions in which, for zfi, hfi and hi, extreme values of set A are considered. The scaled solutions are presented in Figure 7 for sand, loam and clay at t* = 5. The figure indicates that, in We conclude that the scaling is more sensitive to W for the very coarse- and very fine- textured soils. The scaled SWCPs for the loam are approximately invariant with respect to the initial conditions. For the sand, when W is relatively small (A1 and A2), the scaled SWCPs show a delay, while the scaling is adequate for larger values of W. On the other hand, the scaled SWCPs in the clay coalesced for all initial conditions except for A6 – the condition corresponding to the greatest value of W. where a, b, and c are empirical fitting parameters. When time t in Eq. (24) is longer than a day or so, the model shows a linear behavior. Therefore, the equality in the slope of the log-log plot of q(t) verifies the similarity. We studied the similarity condition in three soils of sand, loam, and clay textures under the conditions corresponding to Figure 2. For these soils, log-log transformed curves of q(t) at z=zfi (z=1) are shown in Figure 3. Results and Discussion The slope of the sand is defi- nitely greater than those of the loam and clay which are nearly more equal (Figure 3). Therefore, we consider that the curves for the loam and clay are nearly similar but different from that of the sand. To separately study the effects of zfi, hfi and hi on the scal- ing performance, RE was solved by applying set B of the initial conditions in which, for zfi, hfi and hi, extreme values of set A are considered. The scaled solutions are presented in Figure 7 for sand, loam and clay at t = 5. The figure indicates that, in Figure 4 shows the scaled SWCPs for the 12 soils and initial conditions of A6 which considers a deeply wetted initial profile. A comparison of Figures 2 and 4 indicates that by increasing W (i.e. total water added to the soil profile) the scaling performance decreases. Since dzf /dt at t=0 is used as the scaling factor of time, the scaled SWCPs increasingly diverge as the time increases. The poor performance of the scaling in Figure 4 can be similarly justified by invalidity of the two similarity conditions required for scaling. For the sand, loam, and clay of Figure 4, log-log transformed curves of q(t) at z=zfi (z=1) are shown in Figure 5. The figure shows that the slopes are significantly different indicating that the Nielsen-similarity condition is not held in Figure 4. It can be concluded that by increasing W, the flow properties become more sensitive to soil texture. In other words, the textural range of the soils in which the similarity condition is held decreases as W increases. Figure 3 – Log-log transformed curves of the water ﬂ ux density at z = zﬁ (z* = 1) for three soils of sand, loam, and clay of Carsel and Parrish (1988) (see Table 1) under the conditions corresponding to Figure 2. S represents the slope of the lines ﬁ tted to the points. Results and Discussion -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 log q (cm per day) log t (day) sand loam clay S = -1.09 S = -1.00 S = -1.31 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 log q (cm per day) log t (day) sand loam clay S = -1.09 S = -1.00 S = -1.31 Figure 3 – Log-log transformed curves of the water ﬂ ux density at z = zﬁ (z* = 1) for three soils of sand, loam, and clay of Carsel and Parrish (1988) (see Table 1) under the conditions corresponding to Figure 2. S represents the slope of the lines ﬁ tted to the points. Figure 3 – Log-log transformed curves of the water ﬂ ux density at z = zﬁ (z* = 1) for three soils of sand, loam, and clay of Carsel and Parrish (1988) (see Table 1) under the conditions corresponding to Figure 2. S represents the slope of the lines ﬁ tted to the points. The impact of W on the scaling performance, studied in detail by varying the initial conditions from A1 to A6, is shown in Figure 6 for the sand, loam, and clay at t* = 5. S i A i (Pi i b B z ) 68 5 p 582 591 S pt b /O t b 2011 Figure 4 – Scaled soil water content proﬁ les for the 12 soils of Carsel and Parrish (1988) (see Table 1) and initial conditions of A6 (see Table 2) at (a) t* = 1, (b) t* = 5, and (c) t* = 10. Figure 4 – Scaled soil water content proﬁ les for the 12 soils of Carsel and Parrish (1988) (see Table 1) and initial conditions of A6 (see Table 2) at (a) t* = 1, (b) t* = 5, and (c) t* = 10. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Richards' equation for soil water redistribution 587 very coarse- or fine-textured soils, the scaling performance is more sensitive to zfi, hfi and hi. The scaled SWCPs are approxi- mately invariant for the loam while this is not the case for the sand and clay. is small. The results confirm that the flow properties are not significantly affected by variation of hi. Results and Discussion In the sand, the impact of hi is significant only if hfi is large. This is the case for the clay if simultaneously, hfi is large and zfi is small. Impacts of zfi, hfi and hi are dependent to each other. Based on the results, hfi seems to be the most effective param- eter on the scaling performance especially for fine-textured soils. Variation of this parameter significantly changes the flow rate and therefore, the shape of the flux curves. Thus, cases with an extreme value of hfi will be far from the other cases regarding the Nielsen-similarity condition. Also, increasing zfi decreases the scaling performance (Figure 7). This is the case for the sand when hfi is large, however, for the clay when hfi f f Generally, the above discussions suggest that the proposed scaling method can be successfully applied for medium- and fine-textured soils provided that the initial profile is not deeply wetted. Figure 8 shows the scaled SWCPs at three scaled times of 1, 5, and 10 for the nine medium- and fine-textured soils of Table 1 (i.e. from loam to clay) having initial conditions A1 through A5. Even though the figure contains 45 various scenarios for the solution of RE, the scaled SWCPs are nearly invariant with limited scattering around the mean scaled SW- CPs (the white points in the figure). The 45 cases considered in Figure 8 are approximately Nielsen-similar. For these 45 cases, Figure 9a shows the log-log transformed curves of q(t) at z=zfi (z=1). It is obvious that the slopes are approximately equal with an average of -0.955 (standard deviation=0.127). Therefore, the flux curves could be well scaled using Eq. (17), which are presented in Figure 9b. Although the flux values fall in a wide range, Figure 9b indicates that the scaled fluxes coalesced into a unified curve and could be well described using a power model similar to Eq. (24), q=0.71(t+0.069)–1.03, with a determination coefficient of 0.995. Recognizing that the two assumed similarity conditions are held for the range of soils and selected initial conditions, we propose that the scaling method can be generalized to numerous other cases in this range leading to approximations of the solutions of RE. Results and Discussion Figure 5 – Log-log transformed curves of the water ﬂ ux density at z = zﬁ (z = 1) for three soils of sand, loam, and clay of Carsel and Parrish (1988) (see Table 1) under the conditions corresponding to Figure 4. S represents the slope of the lines ﬁ tted to the points. -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.0 0.5 1.0 1.5 log q (cm per day) log t (day) sand loam clay S = -1.021 S = -1.355 S = -0.714 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.0 0.5 1.0 1.5 log q (cm per day) log t (day) sand loam clay S = -1.021 S = -1.355 S = -0.714 Figure 5 – Log-log transformed curves of the water ﬂ ux density at z = zﬁ (z* = 1) for three soils of sand, loam, and clay of Carsel and Parrish (1988) (see Table 1) under the conditions corresponding to Figure 4. S represents the slope of the lines ﬁ tted to the points. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Generalizing a Single Solution The ability of the scaling method to approximate numeri- cal solutions of RE using a single solution was evaluated. To Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Figure 6 – Scaled soil water content proﬁ les for (a) sand, (b) loam, and (c) clay of Carsel and Parrish (1988) (see Table 1) and set A of initial conditions (see Table 2) at t* = 5. Figure 6 – Scaled soil water content proﬁ les for (a) sand, (b) loam, and (c) clay of Carsel and Parrish (1988) (see Table 1) and set A of initial conditions (see Table 2) at t* = 5. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 588 Sadeghi et al. Figure 7 – Scaled soil water content proﬁ les for (a) sand, (b) loam, and (c) clay of Carsel and Parrish (1988) (see Table 1) and set B of initial conditions (see Table 2) at t* = 5. 588 Figure 7 – Scaled soil water content proﬁ les for (a) sand, (b) loam, and (c) clay of Carsel and Parrish (1988) (see Table 1) and set B of initial conditions (see Table 2) at t* = 5. Figure 8 – Scaled soil water content proﬁ les for medium- and ﬁ ne-textured soils of Table 1 (from loam to clay) and set A of initial conditions except A6 (see Table 2) at (a) t* = 1, (b) t* = 5, and (c) t* = 10. White points show the mean scaled SWCPs. Figure 8 – Scaled soil water content proﬁ les for medium- and ﬁ ne-textured soils of Table 1 (from loam to clay) and set A of initial conditions except A6 (see Table 2) at (a) t* = 1, (b) t* = 5, and (c) t* = 10. White points show the mean scaled SWCPs. A5 were considered. To determine the van Genuchten param- eters of the four soils of UNSODA, van Genuchten hydraulic models, Eqs. (18) and (19), were simultaneously fitted to the measured data using the RETC software (van Genuchten et al., 1991). Table 4 presents the van Genuchten parameters of the soils as well as the initial conditions. Generalizing a Single Solution do so, six medium- and fine-textured soils were selected from the literature: a Beit Netofa Clay (van Genuchten et al., 1980), a Pima Clay Loam (Elmaloglou and Malamos, 2003), and four other soils taken from UNSODA database (Leij et al., 1999) specified by codes 1300, 1370, 3360, and 4030. Some general information of these soils, including texture, taxonomic class and geographical location, are presented in le 3. For each soil, randomly produced initial conditions in the range of A1 to It was assumed that, for these six soils, the scaled solutions of RE are invariant and identical to the mean scaled SWCPs Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Richards' equation for soil water redistribution 589 Figure 9 – (a) Unscaled and (b) scaled water ﬂ ux density curves at z = zﬁ (z* = 1) for 45 cases of Figure 8. S represents the slope of the lines ﬁ tted to the points. -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0 0.2 0.4 0.6 0.8 1 1.2 log q (cm per day) log t (day) (a) average of S = -0.955 standard deviation of S = 0.127 1.E-04 1.E-03 1.E-02 1.E-01 1.E+00 0 100 200 300 400 q* t* (b) q* = 0.71 × (t* + 0.069)^-1.03 R-squared = 0.995 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0 0.2 0.4 0.6 0.8 1 1.2 log q (cm per day) log t (day) (a) average of S = -0.955 standard deviation of S = 0.127 1.E-04 1.E-03 1.E-02 1.E-01 1.E+00 0 100 200 300 400 q* t* (b) q* = 0.71 × (t* + 0.069)^-1.03 R-squared = 0.995 t* Figure 9 – (a) Unscaled and (b) scaled water ﬂ ux density curves at z = zﬁ (z* = 1) for 45 cases of Figure 8. S represents the slope of the lines ﬁ tted to the points. Table 3 – General information of the six selected soils: Beit Netofa Clay (van Genuchten, 1980), Pima Clay Loam (Elmaloglou and Malamos, 2003), and four other soils taken from UNSODA database (Leij et al., 1999). Generalizing a Single Solution An advantage of this method is that it is not restricted to a specific soil hydraulic model. A disadvantage is that the method does not adequately scale water content redistribu- tion profiles of sands and other coarse-textured soils wetted partially, or those of fine-textured soils wetted deeply. Textural range of the soils in which the similarity conditions are held decreases as the initial conditions deals with a deeply wetted profile. In such a condition, a classification of soils and initial conditions and then separately scaling of each class may allevi- ate the problem. The method is promising to reduce compli- Table 4 –Van Genuchten parameters of the six selected soils: Beit Netofa Clay (Van Genuchten et al., 1980), Pima Clay Loam (Elmaloglou and Malamos, 2003), and four other soils taken from UNSODA database (Leij et al., 1999), as well as the randomly produced initial diti Table 4 –Van Genuchten parameters of the six selected soils: Beit Netofa Clay (Van Genuchten et al., 1980), Pima Clay Loam (Elmaloglou and Malamos, 2003), and four other soils taken from UNSODA database (Leij et al., 1999), as well as the randomly produced initial conditions. Soil name θr θs α n Ks zfi hfi hi cm–1 cm per day ------------- cm ------------- × 106 cm Beit Netofa Clay 0.000 0.446 0.0015 1.17 0.08 22 8 2.49 Pima Clay Loam 0.200 0.550 0.0321 1.28 9.91 9 6 1.64 1300 0.000 0.371 0.0225 1.26 9.59 20 7 1.31 1370 0.190 0.608 0.0089 1.32 15.89 14 2 0.68 3360 0.064 0.362 0.0062 1.39 2.08 18 6 1.31 4030 0.000 0.415 0.0432 1.41 1.16 14 3 1.51 Generalizing a Single Solution Soil name Texture Taxonomic class Geographical location Beit Netofa Clay clay Rhodustalfs Beit Netofa valley, Lower Galilee, Israel (32º 44’ N, 35º 26’ E) Pima Clay Loam clay laom Cumulic Haplustoll Marana, Arizona, USA (32º 24’ N, 111º 10’ W) 1300 sandy clay loam Thermic Typic Torrifluvents Las Cruces, New Mexico, USA (USA - 32º 19’ N, 106º 45’ W) 1370 loam Gley-Pseudogley Muenchehagen (Loccum), Germany (52º26’ N, 9º11’ E) 3360 silt loam Mesic Typic Hapludalf Goettingen-Weende, Germany (51º 33’ N, 9º55’ E) 4030 silt loam Typic Hapludalf Helecine (Leuven), Belgium(50º 44’ N, 4º 41’ E) of Figure 6. Subsequently, the mean scaled SWCPs were de- scaled (i.e. converted to the real scale) for the six soils using the Eqs. (5), (6) and (10). Hence, for each soil, SWCPs were approximated during redistribution. To evaluate the accuracy of the approximations, the SWCPs for the same soils and initial conditions were individually simulated using HYDRUS. The simulated and approximated SWCPs were compared using the mean relative error criterion, MRE (i.e. the mean of the absolute errors between the simulated and approximated values relative to the simulated values). Figure 10 shows the simulated and approximated SWCPs for the six soils of Table 3 at two times corresponding to t=1 and t=10. With the MRE values never exceeding 5%, the scaling method satisfactorily approximated the numerical solutions of RE for the selected soils and initial conditions. Table 4 –Van Genuchten parameters of the six selected soils: Beit Netofa Clay (Van Genuchten et al., 1980), Pima Clay Loam (Elmaloglou and Malamos, 2003), and four other soils taken from UNSODA database (Leij et al., 1999), as well as the randomly produced initial conditions. Soil name θr θs α n Ks zfi hfi hi cm–1 cm per day ------------- cm ------------- × 106 cm Beit Netofa Clay 0.000 0.446 0.0015 1.17 0.08 22 8 2.49 Pima Clay Loam 0.200 0.550 0.0321 1.28 9.91 9 6 1.64 1300 0.000 0.371 0.0225 1.26 9.59 20 7 1.31 1370 0.190 0.608 0.0089 1.32 15.89 14 2 0.68 3360 0.064 0.362 0.0062 1.39 2.08 18 6 1.31 4030 0.000 0.415 0.0432 1.41 1.16 14 3 1.51 Conclusion Scaled soil water content profiles were found to be nearly invariant during scaled redistribution times for medium- to fine-textured soils when the initial profile was not deeply wet- ted. Conclusion Figure 10 – Simulated (solid lines) and approximated (doted lines) soil water content proﬁ les for the six selected soils: Beit Netofa Clay (van Genuchten et al., 1980), Pima Clay Loam (Elmaloglou and Malamos, 2003), and four other soils taken from UNSODA database (Leij et al., 1999) at two times corresponding to t* = 1 and t* = 10. cated numerical calculations and opens a new window to easily obtain approximate solutions of highly nonlinear equation of Richards for water flow in unsaturated soils, within prescribed levels of error. Elmaloglou, S.T.; Malamos, N. 2003. A method to estimate soil water movement under a trickle surface line source, with water extraction by roots. Irrigation and Drainage 52: 273-284. Jury, W.A.; Horton, R. 2003. Soil Physics. 6ed. John Wiley, New York, NY, USA. Kozak, J.A.; Ahuja, L.R. 2005. Scaling of inﬁ ltration and redistribution of water across soil textural classes. Soil Science Society of America Journal 69: 816-827 Conclusion of Figure 6. Subsequently, the mean scaled SWCPs were de- scaled (i.e. converted to the real scale) for the six soils using the Eqs. (5), (6) and (10). Hence, for each soil, SWCPs were approximated during redistribution. To evaluate the accuracy of the approximations, the SWCPs for the same soils and initial conditions were individually simulated using HYDRUS. The simulated and approximated SWCPs were compared using the mean relative error criterion, MRE (i.e. the mean of the absolute errors between the simulated and approximated values relative to the simulated values). Scaled soil water content profiles were found to be nearly invariant during scaled redistribution times for medium- to fine-textured soils when the initial profile was not deeply wet- ted. An advantage of this method is that it is not restricted to a specific soil hydraulic model. A disadvantage is that the method does not adequately scale water content redistribu- tion profiles of sands and other coarse-textured soils wetted partially, or those of fine-textured soils wetted deeply. Textural range of the soils in which the similarity conditions are held decreases as the initial conditions deals with a deeply wetted profile. In such a condition, a classification of soils and initial conditions and then separately scaling of each class may allevi- ate the problem. The method is promising to reduce compli- Figure 10 shows the simulated and approximated SWCPs for the six soils of Table 3 at two times corresponding to t=1 and t=10. With the MRE values never exceeding 5%, the scaling method satisfactorily approximated the numerical solutions of RE for the selected soils and initial conditions. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 590 Sadeghi et al. Figure 10 – Simulated (solid lines) and approximated (doted lines) soil water content proﬁ les for the six selected soils: Beit Netofa Clay (van Genuchten et al., 1980), Pima Clay Loam (Elmaloglou and Malamos, 2003), and four other soils taken from UNSODA database (Leij et al., 1999) at two times corresponding to t* = 1 and t* = 10. 590 Sadeghi et al. Acknowledgement Kosugi, K.; Hopmans, J.W. 1998. Scaling water retention curves for soils with lognormal pore-size distribution. Soil Science Society of America Journal 62: 1496-1504. To Prof. D.R. Nielsen and Prof. A.W. Warrick, for their thorough review and helpful comments. The last author thanks CNPq for granting his productivity fellowship. Kutilek, M.; Nielsen, D.R. 1994. Soil Hydrology. Catena, Germany. Kutilek, M.; Zayani, K.; Haverkamp, R.; Parlange, J.Y.; Vachaud, G. 1991. Scaling of Richards' equation under invariant ﬂ ux boundary conditions. Water Resources Research 27: 2181-2185. References Water Resources Research 27: 2181-2185. Leij, F.J.; Alves, W.J.; van Genuchten, M.T.; Williams, J.R. 1999. The UNSODA unsaturated soil hydraulic database. p. 1269-1281. In: van Genuchten, M.T.; Leij, F.J.; Wu. L., eds. Characterization and measurement of the hydraulic properties of unsaturated porous media. University of California, Riverside, CA, USA. Miller, E.E.; Miller, R.D. 1956. Physical theory for capillary ﬂ ow phenomena. Journal of Applied Physics 27: 324-332. Leij, F.J.; Alves, W.J.; van Genuchten, M.T.; Williams, J.R. 1999. The UNSODA unsaturated soil hydraulic database. p. 1269-1281. In: van Genuchten, M.T.; Leij, F.J.; Wu. L., eds. Characterization and measurement of the hydraulic properties of unsaturated porous media. University of Ahuja, L.R.; Williams, R.D. 1991. Scaling water characteristic and hydraulic conductivity based on Gregson-Hector-McGowan approach. Soil Science Society of America Journal 55: 308-319. Carsel, R.F.; Parrish, R.S. 1988. Developing joint probability distributions of soil water characteristics. Water Resources Research 24: 755-769. California, Riverside, CA, USA. Miller, E.E.; Miller, R.D. 1956. Physical theory for capillary ﬂ ow phenomena. Journal of Applied Physics 27: 324-332. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 Richards' equation for soil water redistribution 591 Morel-Seytoux, H.J.; Khanji, J. 1974. Derivation of an equation of inﬁ ltration. Water Resources Research 10: 795-800. Van Genuchten, M.T. 1980 A closed-form equation for predicting the hydraulic conductivity of unsaturated soils. Soil Science Society of America Journal 44: 892-898. Morel-Seytoux, H.J.; Meyer, P.D.; Nachabe, M.; Touma, J,; van Genuchten, M.T.; Lenhard, R.J. 1996. Parameter equivalence for the Brooks-Corey and van Genuchten soil characteristics: Preserving the effective capillary drive. Water Resources Research 32: 1251-1258. Van Genuchten, M.T.; Leij, F.J.; Yates, S.R. 1991. The RETC Code for Quantifying the Hydraulic Functions of Unsaturated Soils. Robert S. Kerr Environmental Research Laboratory, Ada, OK, USA. drive. Water Resources Research 32: 1251-1258. Kerr Environmental Research Laboratory, Ada, OK, USA. Mualem, Y. 1976. A new model for predicting the hydraulic conductivity of unsaturated porous media. Water Resources Research 12: 513-22. Vereecken, H.; Kasteel, R.; Vanderborght, J.; Harter, T. 2007. Upscaling hydraulic properties and soil water ﬂ ow processes in heterogeneous soils: a review. Vadose Zone Journal 6:1-28. Neuweiler, I.; Cirpka, O.A. 2005. Homogenization of Richards equation in permeability ﬁ elds with different connectivities. Water Resources Research 41: W02009, doi:10.1029/2004WR003329. J Vogel, T.; Cislerova, M.; Hopmans, J.W. 1991. Porous media with linearly hydraulic properties. Water Resources Research 27: 2735-2741. Sci. Agric. (Piracicaba, Braz.), v.68, n.5, p.582-591, September/October 2011 References Reichardt, K.; Nielsen, D.R.; Biggar, J.W. 1972. Scaling of horizontal inﬁ ltration into homogeneous soils. Soil Science Society of America Proceedings 36: 241-245. Warrick, A.W.; Mullen, G.J.; Nielsen, D.R. 1977. Scaling of ﬁ eld measured hydraulic properties using a similar media concept. Water Resources Research 13: 355-362. Roth, K. 2008. Scaling of water ﬂ ow through porous media and soils. European Journal of Soil Science 59: 125-130. Warrick, A.W.; Amoozegar-Fard, A. 1979. Infiltration and drainage calculations using spatially scaled hydraulic properties. Water Resources Research 15: 1116-1120. Sadeghi, M.; Gohardous-Monfared, M.R.; Ghahraman, B. 2010. Scaling of soil hydraulic conductivity function using effective capillary drive. Journal of Water and Soil 24: 189-197 (in Persian). Warrick, A.W.; Lomen, D.O.; Yates, S.R. 1985. A generalized solution to inﬁ ltration. Soil Science Society of America Journal 49: 34-38. of Water and Soil 24: 189-197 (in Persian). Simmons, C.S.; Nielsen, D.R.; Biggar, J.W. 1979. Scaling of ﬁ eld-measured soil-water properties. Hilgardia 47: 77-173. Warrick, A.W.; Hussen, A.A. 1993. Scaling of Richards’ equation for inﬁ ltration and drainage. Soil Science Society of America Journal 57: 15-18. Simunek, J.; Sejna, M.; Saito, H.; Sakai, M.; van Genuchten, M.T. 2008. The HYDRUS-1D Software Package for Simulating the One-Dimensional Movement of Water, Heat, and Multiple Solutes in Variably-Saturated Media, Version 4.0. University of California, Riverside, CA, USA. Youngs, E.G. 1990. Application of scaling to soil-water movement considering hysteresis. p. 23-37. In: Hillel, D.; Elrick, D.E., eds. Scaling in soil physics: principles and applications. Soil Science Society of America, Madison, WI, USA. Sharma, M.L.; Gander, G.A.; Hunt, C.G. 1980. Spatial variability of inﬁ ltration in a watershed. Journal of Hydrology 45: 101-122. in a watershed. Journal of Hydrology 45: 101-122. Tuli, A.; Kosugi, K.; Hopmans, J.W. 2001. Simultaneous scaling of soil water retention and unsaturated hydraulic conductivity functions assuming lognormal pore-size distribution. Advance in Water Resources 24: 677-688. Received July 22, 2010 Accepted April 20, 2011
https://openalex.org/W4200036202	https://digitalcommons.usf.edu/cgi/viewcontent.cgi?article=3333&context=geo_facpub	English	null	Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams	Remote sensing	2,021	cc-by	14,958	University of South Florida University of South Florida Digital Commons @ University of Digital Commons @ University of South Florida South Florida School of Geosciences Faculty and Staff Publications School of Geosciences 2022 Using Remote Sensing and Machine Learning to Locate Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams Groundwater Discharge to Salmon-Bearing Streams Mary E. Gerlach University of South Florida Kai C. Rains University of South Florida, krains@usf.edu Edgar J. Guerrón-Orejuela University of South Florida William J. Kleindl Montana State University Joni Downs University of South Florida, downs@usf.edu See next page for additional authors Follow this and additional works at: https://digitalcommons.usf.edu/geo_facpub Part of the Earth Sciences Commons Scholar Commons Citation Scholar Commons Citation Gerlach, Mary E.; Rains, Kai C.; Guerrón-Orejuela, Edgar J.; Kleindl, William J.; Downs, Joni; Landry, Shawn M.; and Rains, Mark C., "Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams" (2022). School of Geosciences Faculty and Staff Publications. 2326. https://digitalcommons.usf.edu/geo_facpub/2326 This Article is brought to you for free and open access by the School of Geosciences at Digital Commons @ University of South Florida. It has been accepted for inclusion in School of Geosciences Faculty and Staff Publications by an authorized administrator of Digital Commons @ University of South Florida. For more University of South Florida University of South Florida Digital Commons @ University of Digital Commons @ University of South Florida South Florida School of Geosciences Faculty and Staff Publications School of Geosciences 2022 Using Remote Sensing and Machine Learning to Locate Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams Groundwater Discharge to Salmon-Bearing Streams Mary E. Gerlach University of South Florida Kai C. Rains University of South Florida, krains@usf.edu Edgar J. Guerrón-Orejuela University of South Florida William J. Kleindl Montana State University Joni Downs University of South Florida, downs@usf.edu See next page for additional authors Follow this and additional works at: https://digitalcommons.usf.edu/geo_facpub Part of the Earth Sciences Commons Scholar Commons Citation Scholar Commons Citation Gerlach, Mary E.; Rains, Kai C.; Guerrón-Orejuela, Edgar J.; Kleindl, William J.; Downs, Joni; Landry, Shawn M.; and Rains, Mark C., "Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams" (2022). School of Geosciences Faculty and Staff Publications. 2326. https://digitalcommons.usf.edu/geo_facpub/2326 This Article is brought to you for free and open access by the School of Geosciences at Digital Commons @ University of South Florida. It has been accepted for inclusion in School of Geosciences Faculty and Staff Publications by an authorized administrator of Digital Commons @ University of South Florida. For more information, please contact digitalcommons@usf.edu. Part of the Earth Sciences Commons Authors Authors Mary E. Gerlach, Kai C. Rains, Edgar J. Guerrón-Orejuela, William J. Kleindl, Joni Downs, Shawn M. Landry, and Mark C. Rains This article is available at Digital Commons @ University of South Florida: https://digitalcommons.usf.edu/ geo_facpub/2326 Scholar Commons Citation Scholar Commons Citation Gerlach, Mary E.; Rains, Kai C.; Guerrón-Orejuela, Edgar J.; Kleindl, William J.; Downs, Joni; Landry, Shawn M.; and Rains, Mark C., "Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams" (2022). School of Geosciences Faculty and Staff Publications. 2326. https://digitalcommons.usf.edu/geo_facpub/2326 Gerlach, Mary E.; Rains, Kai C.; Guerrón-Orejuela, Edgar J.; Kleindl, William J.; Downs, Joni; Landry, Shawn M.; and Rains, Mark C., "Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams" (2022). School of Geosciences Faculty and Staff Publications. 2326. https://digitalcommons.usf.edu/geo_facpub/2326 This Article is brought to you for free and open access by the School of Geosciences at Digital Commons @ University of South Florida. It has been accepted for inclusion in School of Geosciences Faculty and Staff Publications by an authorized administrator of Digital Commons @ University of South Florida. For more information, please contact digitalcommons@usf.edu. Citation: Gerlach, M.E.; Rains, K.C.; Guerrón-Orejuela, E.J.; Kleindl, W.J.; Downs, J.; Landry, S.M.; Rains, M.C. Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams. Remote Sens. 2022, 14, 63. https://doi.org/10.3390/rs14010063 Academic Editor: Mark S. Lorang Received: 9 October 2021 Accepted: 23 December 2021 Published: 24 December 2021 Citation: Gerlach, M.E.; Rains, K.C.; Guerrón-Orejuela, E.J.; Kleindl, W.J.; Downs, J.; Landry, S.M.; Rains, M.C. Using Remote Sensing and Machine Learning to Locate Groundwater Discharge to Salmon-Bearing Streams. Remote Sens. 2022, 14, 63. https://doi.org/10.3390/rs14010063 Keywords: seeps; springs; geology; topography; aquifer outcrops; topographic indices; geospatial modeling; Kenai Peninsula Lowlands; Alaska Mary E. Gerlach 1, Kai C. Rains 1, Edgar J. Guerrón-Orejuela 1 , William J. Kleindl 2, Joni Downs 1 Shawn M. Landry 1 and Mark C. Rains 1,* 1 School of Geosciences, University of South Florida, Tampa, FL 33620, USA; marygerlach@usf.edu (M.E.G.); krains@usf.edu (K.C.R.); edgarguerron@usf.edu (E.J.G.-O.); downs@usf.edu (J.D.); landry@usf.edu (S.M.L.) 2 Land Resources and Environmental Sciences, Montana State University, Bozeman, MT 59717, USA; william.kleindl@montana.edu * C d i @ f d T l 1 813 974 3310 * Correspondence: mrains@usf.edu; Tel.: +1-813-974-3310 * Correspondence: mrains@usf.edu; Tel.: +1-813-974-3310 Abstract: We hypothesized topographic features alone could be used to locate groundwater discharge, but only where diagnostic topographic signatures could ﬁrst be identiﬁed through the use of limited ﬁeld observations and geologic data. We built a geodatabase from geologic and topographic data, with the geologic data only covering ~40% of the study area and topographic data derived from airborne LiDAR covering the entire study area. We identiﬁed two types of groundwater discharge: shallow hillslope groundwater discharge, commonly manifested as diffuse seeps, and aquifer-outcrop groundwater discharge, commonly manifested as springs. We developed multistep manual proce- dures that allowed us to accurately predict the locations of both types of groundwater discharge in 93% of cases, though only where geologic data were available. However, ﬁeld veriﬁcation suggested that both types of groundwater discharge could be identiﬁed by speciﬁc combinations of topographic variables alone. We then applied maximum entropy modeling, a machine learning technique, to predict the prevalence of both types of groundwater discharge using six topographic variables: proﬁle curvature range, with a permutation importance of 43.2%, followed by distance to ﬂowlines, eleva- tion, topographic roughness index, ﬂow-weighted slope, and planform curvature, with permutation importance of 20.8%, 18.5%, 15.2%, 1.8%, and 0.5%, respectively. The AUC values for the model were 0.95 for training data and 0.91 for testing data, indicating outstanding model performance. Authors Authors remote sensing remote sensing remote sensing 1. Introduction Many ecosystems depend on groundwater discharge, including many wetlands [1,2], lakes [3,4], streams [5,6], and estuaries [7,8]. Groundwater discharge to streams is particularly prevalent and critical, being the sole source of baseflow by definition [9] and commonly a substantive subcomponent of stormflow [10]. Though regionally variable, estimates suggest that groundwater discharge provides 14–90% of all stream flow in the conterminous United States [5]. In addition to subsidizing stream flow, groundwater discharge to streams can also modulate stream temperature [11,12] and deliver nutrients and organic carbon [13,14], thereby playing important roles in structuring habitats from the benthos [15] to the fish [16]. Groundwater is also an important water supply component, with 321,000,000 m3 of ground- water withdrawals comprising 26% of all water use in the United States in 2015 [17]. Many of these withdrawals are centralized, including withdrawals for thermoelectric power gen- eration (41%), public water supply (12%), and industrial water supply (5%). Others are more dispersed, including irrigation water supply (37%) and domestic water supply (1%). Effective management and protection of groundwater resources is critical, therefore, to a diverse suite of natural and human users [18]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/remotesensing Remote Sens. 2022, 14, 63. https://doi.org/10.3390/rs14010063 Remote Sens. 2022, 14, 63 2 of 18 2 of 18 The ﬁrst step toward protecting groundwater discharge to ecosystems is to determine the types of groundwater discharge (e.g., sourced from local versus regional groundwater ﬂow systems), the locations where groundwater discharge occurs, and their support for downgradient ecosystems (e.g., ﬂuvial ecosystems). Field studies are often essential in iden- tifying types and locations of groundwater discharge, especially in geologically complex regions where there may be more than one type of groundwater discharge from more than one type of geologic unit [19,20]. Field mapping of these types of groundwater discharge is possible in some situations (e.g., [21]) but is impractical over large spatial scales and/or in difﬁcult-to-access regions. 1. Introduction In these instances, remote sensing, geospatial modeling, and/or machine learning have been used to map remote locations where groundwater discharge occurs, with some degree of success (e.g., [22,23]). These tools are receiving increased attention for general applications in hydrology as computer processing power increases and remote sensing data become more easily available. The ways remote sensing, geospatial modeling, and/or machine learning are used in hydrologic studies depends on the question being addressed; the spatial and temporal scale of the question; and the type, amount, and quality of the available data [24–26]. Nevertheless, these tools have been incorporated into strategies to forecast groundwater levels [27–30], groundwater quality [31–33], saltwater intrusion and groundwater salin- ity [34], and groundwater resource availability [35,36]. Using these approaches to better understand and predict groundwater discharge is particularly challenging (e.g., [22,23]). In many cases, groundwater discharge occurs where erosion or tectonic uplift has exposed aquifers, creating aquifer outcrops. This means that better understanding and predicting groundwater discharge requires an understanding of both topography and geology, with subsurface lithology commonly being poorly known [22,23] yet nevertheless playing a disproportionately important role [30]. The primary controls on groundwater recharge, ﬂow, and discharge are climate, geology, and topography [37]. Climate is typically constant across large study areas, and regional-scale geologic data are difﬁcult to obtain, so studies typically rely upon topography to characterize generalized hydrology [38,39] and locations where groundwater discharge is likely to occur [23,40,41]. However, geologic heterogeneity often plays a controlling role in groundwater recharge, ﬂow, and discharge [42], leading some to suggest that geologic data are more important than topographic data when characterizing hydrological processes (e.g., [30,43]). However, accurate prediction of groundwater discharge is often desired in regions where the geology is heterogeneous and anisotropic, poorly understood, and/or inadequately documented. We therefore hypothesized that topographic features alone could be used to locate groundwater discharge, but only where diagnostic topographic signatures could ﬁrst be identiﬁed through ﬁeld observations and geologic data covering a characteristic subset of the study area. We based this hypothesis on the understanding that groundwater levels and discharges play important roles in structuring local- and watershed- scale geomorphology, thereby affecting topography [44,45], and that groundwater ﬂow systems are typically attracted to the land surface at concave surfaces, such as hillslope failures and toeslopes [2,46,47]. 1. Introduction We tested this hypothesis in south-central Alaska, in a large area that is difﬁcult to access and where geologic data are incompletely available but where remotely sensed LiDAR-based topographic data are widely available. 2. Materials and Methods 2.1. Site Description The study was conducted on the Kenai Peninsula Lowlands in south-central Alaska (Figure 1). The study area is a 1655 km2 area comprising five watersheds: Anchor River, Stariski Creek, Happy Creek, Deep Creek, and the Ninilchik River, from south to north respectively. All except Happy Creek are salmon-bearing and therefore support vibrant sport and commercial fisheries that are central to the regional economy [48,49]. Groundwater discharge plays a critical role in controlling the structure and function of these streams, by augmenting stream flow, modulating stream temperatures, and delivering nutrient Remote Sens. 2022, 14, 63 3 of 18 vibrant round- subsidies [12,14]. Most of the study area is roadless or accessible only by unimproved roads. However, more than 80% of the land is privately owned and has begun seeing steadily increasing development pressure [50], particularly in the western region of the study area and primarily to support single-family homes and farm-to-fork agriculture [51]. Groundwater is the primary source of water for domestic, commercial, and industrial uses [52] and is also threatened by land-use/land-cover change [53], aggregate mining [49], and a drying trend in the climate [54–56]. streams, by augmenting stream flow, modulating stream temperatures, and delivering nutrient subsidies [12,14]. Most of the study area is roadless or accessible only by unim- proved roads. However, more than 80% of the land is privately owned and has begun seeing steadily increasing development pressure [50], particularly in the western region of the study area and primarily to support single-family homes and farm-to-fork agricul- ture [51]. Groundwater is the primary source of water for domestic, commercial, and in- dustrial uses [52] and is also threatened by land-use/land-cover change [53], aggregate mining [49], and a drying trend in the climate [54–56]. subsidies [12,14]. Most of the study area is roadless or accessible only by unimproved roads. However, more than 80% of the land is privately owned and has begun seeing steadily increasing development pressure [50], particularly in the western region of the study area and primarily to support single-family homes and farm-to-fork agriculture [51]. Groundwater is the primary source of water for domestic, commercial, and industrial uses [52] and is also threatened by land-use/land-cover change [53], aggregate mining [49], and a drying trend in the climate [54–56]. streams, by augmenting stream flow, modulating stream temperatures, and delivering nutrient subsidies [12,14]. Most of the study area is roadless or accessible only by unim- proved roads. 2. Materials and Methods 2.1. Site Description However, more than 80% of the land is privately owned and has begun seeing steadily increasing development pressure [50], particularly in the western region of the study area and primarily to support single-family homes and farm-to-fork agricul- ture [51]. Groundwater is the primary source of water for domestic, commercial, and in- dustrial uses [52] and is also threatened by land-use/land-cover change [53], aggregate mining [49], and a drying trend in the climate [54–56]. Figure 1. General location of the five watersheds that comprise the study area on the Kenai Penin sula Lowlands. Base map source [57]. Figure 1. General location of the ﬁve watersheds that comprise the study area on the Kenai Peninsula Lowlands. Base map source [57]. Figure 1. General location of the five watersheds that comprise the study area on the Kenai Penin- sula Lowlands. Base map source [57]. Figure 1. General location of the ﬁve watersheds that comprise the study area on the Kenai Peninsula Lowlands. Base map source [57]. The climate is transitional from maritime to coastal and consists of short summers and long winters (HOMER 8 NW, ALASKA [503672], 1981–2010). The mean annual minimum temperature is −0.8 ◦C, and the mean annual maximum temperature is 6.1 ◦C. Total annual precipitation is 748 mm, with approximately one-third falling as snow and approximately half falling during the wet season (i.e., August–November). The study area underwent at least five major Pleistocene glaciations and two minor post-Pleistocene glacial advances, each variously recorded in ice-scoured landforms, drift sheets, moraines, and discordant drainage relations separated by unconformities and weathering profiles [58]. Most of the study region is now covered with younger glacial outwash and valley train; glaciolacustrine; Remote Sens. 2022, 14, 63 4 of 18 and other minor terminal, recessional, lateral, medial, and ground moraine deposits [59], some reworked by the recent minor glacial advances. Groundwater is found in both surficial deposits, often in wetlands, and in deeper deposits, commonly in thin, discontinuous, and poorly lithified sandstone aquifers formed in buried channel lag and bar deposits [60]. Overall topographic relief ranges from 0 to 889 m above mean sea level (AMSL). Local topography is also commonly steep, as streams have deeply dissected the landscape during the Quaternary. 2.2. Overall Approach The study proceeded in three phases. During the first phase, we created a geographic information system (GIS) geodatabase from geologic and topographic data. Geologic data were sourced from publicly available well logs available for ~40% of the study area; topo- graphic data were sourced from airborne LiDAR available for the entire study area. During the second phase, we stayed within the subset of the study area where geological data were available, using the geodatabase and field observations to identify two types of groundwa- ter discharge and locations where they occurred. The geologic data and geodatabase were essential to the initial identification of one of those two types of groundwater discharge and locations where it occurred. However, field observations suggested that these loca- tions could also be identified by specific combinations of topographic variables alone even where geologic data were not available (e.g., numerous narrow gullies and other deeply incised headwater stream channels that abruptly start along the same topographic contour interval on a hillslope). In the final phase, we used a machine learning approach using only topographic data to predict the likelihood that either type of groundwater discharge occurs. 2.3. Geodatabase Development 2.3.1. Geologic Data 2.3. Geodatabase Development 2.3.2. Topographic Data Topographic data were derived from airborne LiDAR (2008 Kenai Watershed Forum Topographic LiDAR: Kenai Peninsula, Alaska; https://www.fisheries.noaa.gov/inport/ item/49620; accessed on 25 February 2019). The LiDAR-based digital elevation model (DEM) was acquired at 1 × 1 m pixel size but was resampled to a 3 × 3 m pixel size, which both reduced run times and smoothed microtopographic anomalies. The DEM was also modified to remove areas that were below the estimated tide level at the time of data collection (~3 m AMSL). This resampled and modified DEM was used to produce all topographic data using standard tools in ArcGIS 10.5 or ArcGIS Pro 2.7.1 (ESRI, Redlands, CA, USA). ( ) Topographic data directly extracted from the DEM included elevation, slope, proﬁle curvature, proﬁle curvature range, planform curvature, and planform curvature range. Slope records the steepness of the terrain expressed as a percentage. Steep slopes can be indicative of steep hydraulic gradients driving shallow groundwater ﬂow [42], and long steep slopes may be indicative of locations where aquifers might outcrop and therefore where deep groundwater discharge might occur. Proﬁle curvature measures convexity or concavity of the slope parallel to the direction of the slope; planform curvature measures convexity or concavity of the slope perpendicular to the direction of the slope. The range of proﬁle and planform curvatures were calculated within a 3 × 3 cell (9 × 9 m) window to measure changes in curvature over short distances, which can be an indicator of slope failures like those induced by groundwater discharge [46,47], the headward extents of channels formed by groundwater discharge [62,63], and/or locations where water tables might be close to or above the land surface [2,64]. g Topographic data derived from the DEM included flowlines, terrain ruggedness in- dex (TRI), flow-weighted slope (FWS), and topographic wetness index (TWI). Flowlines were defined by categorizing flow accumulation values higher than 2000 as streams and converting those into vector format. Flowlines may represent locations where water tables might be close to or above the land surface [6], and the headward extents of flowlines likely correlate with the headward extents of channels formed by groundwater discharge [63]. 2.3.1. Geologic Data Subsurface geologic data were obtained from well logs in the publicly available Well Log Tracking System (WELTS) maintained by the Alaska Department of Natural Resources (https://dnr.alaska.gov/welts/; accessed on 29 May 2019). Records from >800 well logs within and immediately adjacent to the study area were used to quantify the locations, depths, thicknesses, and geologic characteristics of the water-bearing formations, i.e., the aquifers. q Depths and thicknesses of the aquifers were converted to top and bottom elevations of the aquifers. The aquifer materials are unconsolidated to poorly lithified buried channel lag and bar deposits and therefore vary slightly in thickness and slope gently in the original direction of drainage. Therefore, a user-specified 5 m vertical buffer was added to the top and subtracted from the bottom elevations of the aquifers. These vertically buffered aquifers were then projected outward from the well logs in concentric circles of increasing radii using the Inverse-Distance Weighting (IDW) interpolation tool. Areas where the buffered aquifers intersected the ground surface were found by intersecting the aquifer boundaries with a digital elevation model (DEM, see below) using the Raster Calculator tool and were mapped as potential aquifer outcrops. The final step was to determine the horizontal spatial scale over which the aquifer interpolations were valid. We did so using standard geologic mapping techniques. Geologic mapping is an interpretive method in which field observations are commonly recorded as qualitative data, such as sketches and narratives [61]. We made such qualitative observations at increasing radial distances from wells, looking for aquifer outcrops of the same material and at the same approximate elevations as described in the corresponding well log. We initially tested circles of 1000 m radius and then tested circles of 2000 and 3000 m radius as we continued to find aquifer outcrops at the outer edges of the projections, though with decreasing frequency with increasing radial distance. We then tested circles of 5000 m radius, finding no aquifer outcrops of the same material at the same approximate elevations as described in the corresponding well log. We concluded that the horizontal spatial scale over which the aquifer interpolations were valid ended somewhere between 3000 and 5000 m, and we adopted the more-conservative limit of 3000 m. This Remote Sens. 2.3.1. Geologic Data 2022, 14, 63 5 of 18 5 of 18 resulted in >800 overlapping circles of 3000 m radius covering ~40% of the study area, which is sufficiently representative of the entire study area. Many of these overlapping circles intersect the ground surface and therefore indicate locations where groundwater discharge from aquifer outcrops likely occurs. 2.3.2. Topographic Data TRI measures topographic heterogeneity, calculated as the square root of the average squared differences in elevation between a pixel and its eight neighbors, and is defined per pixel as: TRI = hXij −X00 2i 1 2 , (1) (1) where Xij is the elevation of all eight pixels neighboring pixel X00 [65]. TRI was computed using Arc Hydro in ArcGIS Pro. TRI is an indicator of slope failures like those induced by groundwater discharge, the headward extents of channels formed by groundwater discharge, and narrow gullies and other deeply incised headwater stream channels [66]. FWS indicates the degree to which water is concentrated and then driven downslope by topography, and it is defined per subcatchment as: FWS = ∑(βi ∗FACi)/∑FACi, (2) (2) where βi is the slope (in percent) at a particular pixel, FACi is ﬂow accumulation for that pixel, and ∑(FACi) is the summation of ﬂow accumulation for all pixels within the subcatchment. FWS was calculated using Arc Hydro in ArcMap 10.8. Arc Hydro was ﬁrst used to calculate ﬂow direction and ﬂow accumulation and deﬁne, segment, and link streams. These were then used to delineate catchments using the Arc Hydro catchment grid delineation tool. The catchment grid was then converted into a polygon feature class using the Arc Hydro catchment polygon processing tool. The stream link layer was then Remote Sens. 2022, 14, 63 6 of 18 converted into a drainage line feature class using the Arc Hydro drainage line processing tool. Finally, the Arc Hydro adjoint catchment processing tool was used to generate the aggregated upstream catchments from the catchment feature class. FWS for each catchment was then calculated from these layers using the raster calculator. FWS has been shown to correlate both with groundwater discharge [12] and stream water chemistry which itself may be a function of groundwater discharge [67]. TWI indicates where water is likely to accumulate, and it is deﬁned per pixel as: converted into a drainage line feature class using the Arc Hydro drainage line processing tool. Finally, the Arc Hydro adjoint catchment processing tool was used to generate the aggregated upstream catchments from the catchment feature class. FWS for each catchment was then calculated from these layers using the raster calculator. FWS has been shown to correlate both with groundwater discharge [12] and stream water chemistry which itself may be a function of groundwater discharge [67]. 2.3.2. Topographic Data TWI indicates where water is likely to accumulate, and it is deﬁned per pixel as: TWI = ln A Tanβ0 , (3) (3) where A is the area that contributes ﬂow to a particular pixel and Tanβ0 is the tangent of the slope of the pixel being analyzed [68,69]. TWI was calculated using Arc Hydro and the TWI tool in TauDEM Version 5 (Terrain Analysis Using Digital Elevation Models; https://hydrology.usu.edu/taudem/taudem5/; accessed on 7 May 2019) in ArcMap 10.8. The D-inﬁnity (DINF) tool in Arc Hydro was ﬁrst used to calculate a slope-sensitive ﬂow direction. The DINF is an iterative process which guarantees that each ﬂat pixel ultimately drains to a lower elevation, eliminating the possibility of inconsistencies such as loops in the ﬂow direction angle [70]. The DINF contributing area tool in Arc Hydro was then used to calculate a grid of pixel-speciﬁc catchment areas. TWI for each pixel was then calculated using the TWI tool from the TauDEM. TWI has also been called Wetx and Compound Topographic Index (CTI); all three utilize the same formula to represent likelihood of water ﬂow over landscapes [68,69,71]. ld 2.3.4. Field Work 2.3.4. Field Work Field work was conducted during the summers of 2018 and 2019. Init was focused on identifying the types of groundwater discharge that occur a tions under which they occur. We then developed and tested procedures f identification of these types of groundwater discharge using the full geolo graphic portions of the geodatabase (i.e., both the geologic and topographi these manual procedures, we identified 67 locations in the Anchor Rive Creek watersheds, the southernmost two watersheds in the study area (F manual procedures predicted that groundwater discharge did occur at 54 tions and did not occur at 13 of these locations. We then visited each of thes obtaining geographic positioning system (GPS) coordinates at each locatio min Rino 650 handheld GPS unit (Garmin, Olathe, KS, USA) and noting if discharge actually did or did not occur. Where groundwater discharge did o ature, pH, and specific conductance were measured using a YSI MPS 556 Springs, OH, USA). Specific conductance was particularly important becau for water–rock contact time, with precipitation having no water–rock con relatively low specific conductance, shallow soil water having relatively sho contact time and relatively moderate specific conductance, and deep aquifer relatively long water–rock contact time and relatively high specific cond [72]). Therefore, it was a useful proxy for distinguishing between you 2.3.4. Field Work Field work was conducted during the summers of 2018 and 2019. Init was focused on identifying the types of groundwater discharge that occur a tions under which they occur. We then developed and tested procedures f identification of these types of groundwater discharge using the full geolo graphic portions of the geodatabase (i.e., both the geologic and topographi these manual procedures, we identified 67 locations in the Anchor Rive Creek watersheds, the southernmost two watersheds in the study area (F manual procedures predicted that groundwater discharge did occur at 54 tions and did not occur at 13 of these locations. We then visited each of thes obtaining geographic positioning system (GPS) coordinates at each locatio min Rino 650 handheld GPS unit (Garmin, Olathe, KS, USA) and noting if discharge actually did or did not occur. Where groundwater discharge did o ature, pH, and specific conductance were measured using a YSI MPS 556 Springs, OH, USA). 2.3.3. Layers Derived from the Geologic and Topographic Data 2.3.3. Layers Derived from the Geologic and Topographic Data Multiple geologic and topographic layers were derived from the geologic and topo- graphic data (Figure 2). The geologic data were obtained from >800 well logs associated with domestic, commercial, and/or industrial wells, all located proximal to roads in the more- developed western and southern parts of the study area. The topographic data were derived from a DEM which covered the entire, mostly roadless, 1655 km2 study area. Therefore, the GIS layers which represent the geologic data are situated predominantly in the western and southern portions of the study area while the layers representing the topographic data cover the full extent of the study area. REVIEW Figure 2. Cont. Figure 2. Cont. Figure 2. Cont. Remote Sens. 2022, 14, 63 7 of 18 7 of 18 Figure 2. Primary geologic and topographic layers include: (a) well log points and m outcrops; (b) DEM, represented by a shaded relief to emphasize terrain, with the 67 f for training and testing; (c) flowlines; (d) TRI; (e) FWS; and (f) TWI. Here, only th Watershed, the southernmost of the five watersheds, is shown in full. Figure 2. Primary geologic and topographic layers include: (a) well log points and m outcrops; (b) DEM, represented by a shaded relief to emphasize terrain, with the 67 f for training and testing; (c) flowlines; (d) TRI; (e) FWS; and (f) TWI. Here, only th Watershed, the southernmost of the five watersheds, is shown in full. Figure 2. Primary geologic and topographic layers include: (a) well log points and modeled aquifer outcrops; (b) DEM, represented by a shaded relief to emphasize terrain, with the 67 ﬁeld points used for training and testing; (c) ﬂowlines; (d) TRI; (e) FWS; and (f) TWI. Here, only the Anchor River Watershed, the southernmost of the ﬁve watersheds, is shown in full. Figure 2. Primary geologic and topographic layers include: (a) well log points and m outcrops; (b) DEM, represented by a shaded relief to emphasize terrain, with the 67 fi for training and testing; (c) flowlines; (d) TRI; (e) FWS; and (f) TWI. Here, only th Watershed, the southernmost of the five watersheds, is shown in full. Figure 2. 2.3.3. Layers Derived from the Geologic and Topographic Data Primary geologic and topographic layers include: (a) well log points and m outcrops; (b) DEM, represented by a shaded relief to emphasize terrain, with the 67 fi for training and testing; (c) flowlines; (d) TRI; (e) FWS; and (f) TWI. Here, only th Watershed, the southernmost of the five watersheds, is shown in full. Figure 2. Primary geologic and topographic layers include: (a) well log points and modeled aquifer outcrops; (b) DEM, represented by a shaded relief to emphasize terrain, with the 67 ﬁeld points used for training and testing; (c) ﬂowlines; (d) TRI; (e) FWS; and (f) TWI. Here, only the Anchor River Watershed, the southernmost of the ﬁve watersheds, is shown in full. 2.3.5. Modeling The study area is large and difficult to access, and geologic data are only available for ~40% of the study area. Furthermore, field observations indicated that the locations where groundwater discharge occurred could be identified by specific combinations of topographic features alone. Therefore, we applied maximum entropy modeling, a machine learning technique, to predict the likelihood groundwater discharge occurs using only the topographic portion of the geodatabase. We chose a Maxent modeling approach to map the prevalence of seeps and springs across the study area, as it is a robust method that relies on presence-only data. Maxent works by relating occurrence data, in the form of points, to layers of environmental data, which are sometimes called predictors or covariates [73,74]. The method works by using maximum likelihood functions to best distinguish presence points from the landscape. Specifically, the algorithm finds the model that minimizes the relative entropy between the probability density of the presence points and the proba- bility density of background locations, as measured in covariate space. We used Maxent version 3.4 (http://biodiversityinformatics.amnh.org/open_source/maxent; accessed on 1 September 2020) to predict locations of seeps and springs with respect to environmental variables. The 51 seeps and springs identified in the field were used as the presence points, while 10 topographic layers from the geodatabase were used as the predictors: elevation, slope, planform curvature, planform curvature range, profile curvature, profile curvature range, distance to flowlines, TRI, FWS, and TWI. g , , , , We modeled the prevalence of seeps and springs using a logistic model with the default parameters, except for specifying a prevalence value of 0.10. The value of 0.10 was selected because we expected seep and spring formation to occur uncommonly, over an estimated 10% of the area. We used a systematic approach to evaluate and reduce the number of environmental layers to obtain a final model. First, the set of candidate variables was reduced by removing highly correlated layers, as collinearity can cause bias and make relationships between individual variables difficult to discern [75,76]. Pairwise correlations were calculated between all candidate layers; a threshold of r > 0.70 was used to identify correlated variables. Then, single-variable Maxent models were run for each correlated variable, with the most predictive variable from each pair, as measured using a jackknife test, retained for further analysis. Second, a Maxent model was run on all remaining, uncorrelated variables. ld 2.3.4. Field Work Specific conductance was particularly important becau for water–rock contact time, with precipitation having no water–rock con relatively low specific conductance, shallow soil water having relatively sho contact time and relatively moderate specific conductance, and deep aquifer relatively long water–rock contact time and relatively high specific cond [72]). Therefore, it was a useful proxy for distinguishing between you Field work was conducted during the summers of 2018 and 2019. Initial ﬁeld work was focused on identifying the types of groundwater discharge that occur and the condi- tions under which they occur. We then developed and tested procedures for the manual identiﬁcation of these types of groundwater discharge using the full geologic and topo- graphic portions of the geodatabase (i.e., both the geologic and topographic data). Using these manual procedures, we identiﬁed 67 locations in the Anchor River and Stariski Creek watersheds, the southernmost two watersheds in the study area (Figure 2). Our manual procedures predicted that groundwater discharge did occur at 54 of these locations and did not occur at 13 of these locations. We then visited each of these 67 locations, obtaining geographic positioning system (GPS) coordinates at each location with a Garmin Rino 650 handheld GPS unit (Garmin, Olathe, KS, USA) and noting if groundwater discharge actually did or did not occur. Where groundwater discharge did occur, temperature, pH, and speciﬁc conductance were measured using a YSI MPS 556 (YSI, Yellow Springs, OH, USA). Speciﬁc conductance was particularly important because it is a proxy for water–rock contact time, with precipitation having no water–rock contact time and relatively low speciﬁc conductance, shallow soil water having relatively short water–rock contact time and relatively moderate speciﬁc conductance, and deep aquifer water having relatively long water–rock contact time and relatively high speciﬁc conductance (e.g., [72]). Therefore, it was a useful proxy for distinguishing between younger, shallow hillslope groundwater (e.g., recent precipitation, including snowmelt, moving downslope along the surface and in the shallow subsurface) from older, deep aquifer groundwater (e.g., precipitation, includ- ing snowmelt, that had inﬁltrated and recharged deeper aquifers, then traveled laterally to discharge from an aquifer outcrop). We simultaneously also made observations that indicated we might otherwise identify these types of groundwater discharge using only the topographic portion of the geodatabase (i.e., only the topographic data). Remote Sens. 2022, 14, 63 8 of 18 8 of 18 2.3.5. Modeling The permutation importance of each variable was examined, and any variables with no contribution to the model were removed. Third, a Maxent model consisting only of uncorrelated, contributing variables was run to predict spring prevalence. Finally, a cross-validation procedure was used to test the predictive performance of the final model. p p p Our manual procedures previously predicted groundwater discharge occurred at 54 locations. Field verification indicated that groundwater discharge actually occurred at 51 of these 54 locations. These 51 presence-only occurrences were used as training and testing data, with 70% (n = 36) used as training data and 30% (n = 15) used as testing data. The performance of the final model was assessed by computing the area under the receiver operating curve (AUC), which measures the probability that a randomly selected presence location will be ranked higher than a randomly selected background location. 3.2. Manual Identification of Groundwater D 3.2.1. Hillslope Groundwater Discharge 3.2.1. Hillslope Groundwater Discharge Hillslope groundwater discharge is likely to occur on large, concave, and steep hillslopes that accumulate, concentrate, and drive shallow groundwater downgradient toward concave midslope and/or toeslope positions. These factors are reflected in FWS, which is a function of the flow accumulation area and slope. FWS is partly a function of slope, so it tends to be highest in the steep terrain characteristic of the eastern section of the study area where high-elevation headwaters are common (Figure 2). Previous work in this study area has demonstrated that hillslopes with relatively moderate–high FWS are commonly associated with groundwater discharge to streams [12]. Flowlines are also a function of flow accumulation area. Therefore, a simple two-step workflow using FWS and flowlines was found to be sufficient for identifying locations where hillslope ground- Hillslope groundwater discharge is likely to occur on large, concave, and steep hill- slopes that accumulate, concentrate, and drive shallow groundwater downgradient toward concave midslope and/or toeslope positions. These factors are reﬂected in FWS, which is a function of the ﬂow accumulation area and slope. FWS is partly a function of slope, so it tends to be highest in the steep terrain characteristic of the eastern section of the study area where high-elevation headwaters are common (Figure 2). Previous work in this study area has demonstrated that hillslopes with relatively moderate–high FWS are commonly associated with groundwater discharge to streams [12]. Flowlines are also a function of ﬂow accumulation area. Therefore, a simple two-step workﬂow using FWS and ﬂowlines was found to be sufﬁcient for identifying locations where hillslope groundwater discharge was likely to occur, which could then be veriﬁed in the ﬁeld (Figure 4). REVIEW 10 of 20 water discharge was likely to occur, which could then be verified in the field (Figure 4). Figure 4. Example of implementing the two-step workflow to locate hillslope groundwater dis- charge. FWS is first used to identify hillslopes with relatively high FWS. Flowlines are then used to identify specific locations where channels may initiate. Diffuse seeps are commonly found in these settings, including at the field location in this example. 3 2 2 Aquifer Outcrop Groundwater Discharge Figure 4. Example of implementing the two-step workﬂow to locate hillslope groundwater discharge. FWS is ﬁrst used to identify hillslopes with relatively high FWS. Flowlines are then used to identify speciﬁc locations where channels may initiate. 3.1. Types of Groundwater Discharge Two types of groundwater discharge were identiﬁed in the study area, hillslope groundwater discharge and aquifer-outcrop groundwater discharge (Figure 3). Hillslope groundwater discharge occurs where rainfall and snowmelt inﬁltrate into the shallow subsurface, move laterally downslope through the shallow subsurface, and discharge as diffuse seeps and small springs at groundwater-induced slope failures and valley-bottom toeslopes. Aquifer-outcrop groundwater discharge occurs where rainfall and snowmelt inﬁltrate into the deep subsurface, move laterally through aquifers, and discharge as larger springs at aquifer outcrops in valleys carved by modern streams. Remote Sens. 2022, 14, 63 9 of 18 wmelt rge as Figure 3. Types of groundwater discharge include (a) hillslope groundwater discharge and (b) aq- uifer-outcrop groundwater discharge. Illustrations drawn by Conrad Field from field sketches and notes prepared by Mark Rains Figure 3. Types of groundwater discharge include (a) hillslope groundwater discharge and (b) aquifer- outcrop groundwater discharge. Illustrations drawn by Conrad Field from field sketches and notes prepared by Mark Rains. Figure 3. Types of groundwater discharge include (a) hillslope groundwater discharge and (b) aq- uifer-outcrop groundwater discharge. Illustrations drawn by Conrad Field from field sketches and notes prepared by Mark Rains Figure 3. Types of groundwater discharge include (a) hillslope groundwater discharge and (b) aquifer- outcrop groundwater discharge. Illustrations drawn by Conrad Field from field sketches and notes prepared by Mark Rains. notes prepared by Mark Rains. 3.2. Manual Identiﬁcation of Groundwater Discharge 3.2.2. Aquifer-Outcrop Groundwater Discharge 3.2.2. Aquifer-Outcrop Groundwater Discharge 3.2.2. Aquifer-Outcrop Groundwater Discharge 3.2.2. Aquifer-Outcrop Groundwater Discharge 3.2.2. Aquifer-Outcrop Groundwater Discharge 3.2.2. Aquifer-Outcrop Groundwater Discharge Aquifer-outcrop groundwater discharge is likely to occur where aquifers outcrop and topography indicates the initiation of channelized ﬂow. Aquifer outcrops are reﬂected in the aquifer outcrop layer, a created layer that covers only the western and southern, i.e., more-developed, settings where well log information was available (Figure 2). These aquifer outcrops commonly support large springs which form the headward extent of prominent channels, typically aligned roughly parallel to one another and abruptly initiating along the same contour interval. The spatially limited aquifer outcrop data product was then used to explore the topographic data that reﬂected the initiation of channelized ﬂow, including the headward extent of incised topography, the initiation of ﬂowlines, and the sudden concentration of the TWI. Therefore, a simple four-step workﬂow using the aquifer outcrops overlaid on contour lines, ﬂowlines, and TWI was found to be sufﬁcient for identifying locations where aquifer-outcrop groundwater discharge was likely to occur, which could then be veriﬁed in the ﬁeld (Figure 5). Aquifer-outcrop groundwater discharge is likely to occur where aquifers outcrop and topography indicates the initiation of channelized flow. Aquifer outcrops are re- flected in the aquifer outcrop layer, a created layer that covers only the western and south- ern, i.e., more-developed, settings where well log information was available (Figure 2). These aquifer outcrops commonly support large springs which form the headward extent of prominent channels, typically aligned roughly parallel to one another and abruptly in- itiating along the same contour interval. The spatially limited aquifer outcrop data prod- uct was then used to explore the topographic data that reflected the initiation of channel- ized flow, including the headward extent of incised topography, the initiation of flow- lines, and the sudden concentration of the TWI. Therefore, a simple four-step workflow using the aquifer outcrops overlaid on contour lines, flowlines, and TWI was found to be sufficient for identifying locations where aquifer-outcrop groundwater discharge was likely to occur, which could then be verified in the field (Figure 5). Figure 5. Example of implementing the four-step workflow to locate aquifer-outcrop groundwater discharge. Aquifer outcrops are first used to indicate regions where large volumes of groundwater Figure 5. Example of implementing the four-step workflow to locate aquifer-outcrop groundwater discharge. Aquifer outcrops are first used to indicate regions where large volumes of groundwater discharge likely occur. 3.2.2. Aquifer-Outcrop Groundwater Discharge 3.2.2. Aquifer-Outcrop Groundwater Discharge Then each of the three topographic layers, i.e., contour lines, the initiation of flowlines, and sudden increases in TWI, are used to identify locations where channelized flows initiate. Springs are commonly found in these settings. In this case, the lowermost field point was preselected and found in the field to be 13 m from a spring. The uppermost field point was then visited, and the static water level was found to be ~2 m below the ground surface in a hand-dug well. Figure 5. Example of implementing the four-step workflow to locate aquifer-outcrop groundwater discharge. Aquifer outcrops are first used to indicate regions where large volumes of groundwater Figure 5. Example of implementing the four-step workflow to locate aquifer-outcrop groundwater discharge. Aquifer outcrops are first used to indicate regions where large volumes of groundwater discharge likely occur. Then each of the three topographic layers, i.e., contour lines, the initiation of flowlines, and sudden increases in TWI, are used to identify locations where channelized flows initiate. Springs are commonly found in these settings. In this case, the lowermost field point was preselected and found in the field to be 13 m from a spring. The uppermost field point was then visited, and the static water level was found to be ~2 m below the ground surface in a hand-dug well. 3.2. Manual Identification of Groundwater D 3.2.1. Hillslope Groundwater Discharge Diffuse seeps are commonly found in these settings, including at the ﬁeld location in this example. water discharge was likely to occur, which could then be verified in the field (Figure Figure 4. Example of implementing the two-step workflow to locate hillslope groundwater dis- charge. FWS is first used to identify hillslopes with relatively high FWS. Flowlines are then used to identify specific locations where channels may initiate. Diffuse seeps are commonly found in these settings, including at the field location in this example. Figure 4. Example of implementing the two-step workﬂow to locate hillslope groundwater discharge. FWS is ﬁrst used to identify hillslopes with relatively high FWS. Flowlines are then used to identify speciﬁc locations where channels may initiate. Diffuse seeps are commonly found in these settings, including at the ﬁeld location in this example. Figure 4. Example of implementing the two-step workflow to locate hillslope groundwater dis- charge. FWS is first used to identify hillslopes with relatively high FWS. Flowlines are then used to identify specific locations where channels may initiate. Diffuse seeps are commonly found in these settings, including at the field location in this example. Figure 4. Example of implementing the two-step workﬂow to locate hillslope groundwater discharge. FWS is ﬁrst used to identify hillslopes with relatively high FWS. Flowlines are then used to identify speciﬁc locations where channels may initiate. Diffuse seeps are commonly found in these settings, including at the ﬁeld location in this example. Remote Sens. 2022, 14, 63 10 of 18 n used to in these 3.3. Modeled Identiﬁcation of Groundwater Discharge The ﬁnal Maxent model included six topographic variables. Proﬁle curvature range contributed the most information to the model with a permutation importance of 43.2%, followed by distance to ﬂowlines, elevation, TRI, FWS, and planform curvature, with permutation importance of 20.8%, 18.5%, 15.2%, 1.8%, and 0.5%, respectively (Table 2). Predicted prevalence of seeps and springs was highest where proﬁle curvature ranges were large, distances to ﬂowlines were low, elevation was low, TRI was high (i.e., terrain was rugged), FWS was high, and planform curvature values were large (Figure 6). Collectively, the model predicts groundwater discharge where topography changes abruptly over small distances in close proximity to ﬂowlines at lower elevations (Figure 7). The model predicts that seeps and springs are widespread over the study area, with high prevalence locations particularly at the headward extent of and alongside streams and along coastal bluffs. The AUC values for the model were 0.95 for training data and 0.91 for testing data, indicating outstanding performance [79]. VIEW 12 of 20 Figure 6. Predicted probability of prevalence (y-axis) for six topographic variables used in the final Maxent model to predict seeps and springs: (a) profile curvature range, (b) distance to flowlines, (c) elevation, (d) TRI, (e) FWS, and (f) planform curvature. The curves represent the dependence of predicted prevalence on both the individual topographic variables and the correlations between them. Figure 6. Predicted probability of prevalence (y-axis) for six topographic variables used in the final Maxent model to predict seeps and springs: (a) profile curvature range, (b) distance to flowlines, (c) elevation, (d) TRI, (e) FWS, and (f) planform curvature. The curves represent the dependence of predicted prevalence on both the individual topographic variables and the correlations between them. igure 6. Predicted probability of prevalence (y-axis) for six topographic variables used in the final Maxent model to predict seeps and springs: (a) profile curvature range, (b) distance to flowlines, (c) levation, (d) TRI, (e) FWS, and (f) planform curvature. The curves represent the dependence of redicted prevalence on both the individual topographic variables and the correlations between hem. Figure 6. Predicted probability of prevalence (y-axis) for six topographic variables used in the final Maxent model to predict seeps and springs: (a) profile curvature range, (b) distance to flowlines, (c) elevation, (d) TRI, (e) FWS, and (f) planform curvature. 3.2.3. Field Veriﬁcation The procedures for identifying groundwater discharge were ﬁeld veriﬁed by visiting 67 ﬁeld locations, 54 where groundwater discharge was predicted to occur and 13 where groundwater discharge was predicted not to occur. Groundwater discharge was logged as occurring if a seep or spring was observed within 30 m of the predicted location. Results are tabulated in a confusion matrix (Table 1). The sensitivity (i.e., correctly predicted posi- tives/total actual positives) is 50/51, or 98%, while the precision (i.e., correctly predicted positives/total predicted positives) is 50/54, or 93%. Accuracy, calculated as the percentage of correct predictions, is 62/67, or 93%. That is, overall, the manual procedures accurately predicted the presence or absence of groundwater discharge in 93% of cases. The kappa coefﬁcient (κ), which takes into account the possibility of the agreement occurring by chance, is 0.78, which indicates substantial strength of agreement with the ﬁeld data [77,78]. Remote Sens. 2022, 14, 63 11 of 18 11 of 18 Table 1. Confusion matrix of ground-truth points collected to verify the accuracy of the geodatabase predictions. Table 1. Confusion matrix of ground-truth points collected to verify the accuracy of the geodatabase predictions. Predicted No Predicted Yes Total Actual No 12 4 16 Actual Yes 1 50 51 Total 13 54 67 Table 1. Confusion matrix of ground-truth points collected to verify the accuracy of the geodatabase predictions. 3.3. Modeled Identiﬁcation of Groundwater Discharge The curves represent the dependence of predicted prevalence on both the individual topographic variables and the correlations between them. 12 of 18 13 of 20 12 of 18 13 of 20 Remote Sens. 2022, 14, 63 Remote Sens. 2022, 14, x FO Figure 7. Predicted prevalence of seeps and springs in the entire study area. Seeps and springs ar most likely to occur where spring prevalence values are highest. The inset highlights the small bo in the southeast of the study area, which is an example area where the probability of the occurrenc of seeps and springs is particularly high. Figure 7. Predicted prevalence of seeps and springs in the entire study area. Seeps and springs are most likely to occur where spring prevalence values are highest. The inset highlights the small box in the southeast of the study area, which is an example area where the probability of the occurrence o seeps and springs is particularly high. Figure 7. Predicted prevalence of seeps and springs in the entire study area. Seeps and springs are most likely to occur where spring prevalence values are highest. The inset highlights the small box in the southeast of the study area, which is an example area where the probability of the occurrence of seeps and springs is particularly high. Figure 7. Predicted prevalence of seeps and springs in the entire study area. Seeps and springs are most likely to occur where spring prevalence values are highest. The inset highlights the small box in the southeast of the study area, which is an example area where the probability of the occurrence of seeps and springs is particularly high. Figure 7. Predicted prevalence of seeps and springs in the entire study area. Seeps and springs are most likely to occur where spring prevalence values are highest. The inset highlights the small box in the southeast of the study area, which is an example area where the probability of the occurrence of seeps and springs is particularly high. Figure 7. Predicted prevalence of seeps and springs in the entire study area. Seeps and springs are most likely to occur where spring prevalence values are highest. The inset highlights the small box in the southeast of the study area, which is an example area where the probability of the occurrence of seeps and springs is particularly high. 4. 3.3. Modeled Identiﬁcation of Groundwater Discharge Discussion Though the primary controls on groundwater flow and discharge are climate, geol- ogy, and topography [37], we demonstrated that the locations where groundwater dis- charge occurs can be predicted based solely on topography if key diagnostic topographic signatures can be first identified using ancillary field observations and geologic data in a representative subset of the study area. Here, we modeled two types of groundwater dis- charge: hillslope groundwater discharge and aquifer-outcrop groundwater discharge (Figure 3). We constructed a robust geodatabase comprising field observations and geo- logic data from >800 well logs covering a representative subset of the study area and topo- graphic data from an airborne LiDAR-derived DEM covering the entire study area (Figure 2). We then developed and refined procedures to manually identify the two types of groundwater discharge in the representative subset of the study area where the field ob- Table 2. Permutation importance for variables used to predict seeps and springs using the Maxent model. Variable Permutation Importance (%) Proﬁle curvature range 43.2 Distance to ﬂowlines 20.8 Elevation 18.5 Terrain ruggedness index 15.2 Flow-weighted slope 1.8 Planform curvature 0.5 4. Discussion Though the primary controls on groundwater flow and discharge are climate, geol ogy and topography [37] we demonstrated that the locations where groundwater dis Table 2. Permutation importance for variables used to predict seeps and springs using the Maxent model. servations, geo While doing s 4. Discussion The modeling benefited greatly from previous field observations by Callahan et al. [12], which in turn benefited greatly from other previous field observations by Walker et al. [66] and King et al. [80]. These studies showed that topography correlates with the structure and function of streams in the Kenai Peninsula Lowlands, including stream flow and stream water temperature [12], stream water chemistry [66], and stream biota [80]. These studies were conducted at 18 shared study sites in the Anchor River, Stariski Creek, Deep Creek, and Ninilchik Creek watersheds, four of the five watersheds included in this study. Callahan et al. [12] made the key insight that motivated our study. Their field observations indicated that a topographic feature, i.e., FWS, could be used to predict the location of hillslope groundwater discharge to streams. We further refined this understanding, noting that, for example, hillslopes with high FWS also had a prevalence of small headwater streams that originated at seeps and small springs. These are evident in the topographic data in a number of ways, including sudden changes in curvature (i.e., profile curvature range), flowlines, and TWI (Figure 2). This then allowed the accurate manual and modeled identification of hillslope groundwater discharge (Figures 4 and 7). p g g g The modeling also benefited greatly from the availability of >800 publicly available well logs (Figure 2). Surficial geology data are available for the entirety of the Kenai Peninsula Lowlands, at the 1:350,000 scale [59]. Such data can be useful in predicting potential groundwater recharge zones (e.g., [81]). However, such coarse data alone cannot be used to map thin confined aquifers and their outcrops, as was necessary for this study. The well logs allowed us to do so. Then subsequent field work further allowed us to refine our understanding of the spatial scale over which the well logs were predictive of aquifer outcrops (Figure 2). This allowed us to find numerous springs, which we then used to explore the topographic data that reflected the initiation of channelized flow, including the headward extent of incised topography, the initiation of flowlines, and the sudden concentration of the TWI. servations, geo While doing s 4. Discussion While doing so, we made observations that indicated we might otherwise identify these two types of groundwater discharge using only the topographic data. We therefore devel- oped and refined procedures to model the two types of groundwater discharge through- out the entire study area from the topographic data alone (Table 2; Figure 7). Devito et al. [43] previously argued that topography was the last control to consider in explaining hy- drologic processes, after climate and geology. Rahmati et al. [30] concurred, suggesting that geologic data (e.g., lithology) was a relatively strong predictor of groundwater levels Though the primary controls on groundwater flow and discharge are climate, geology, and topography [37], we demonstrated that the locations where groundwater discharge occurs can be predicted based solely on topography if key diagnostic topographic signatures can be first identified using ancillary field observations and geologic data in a represen- tative subset of the study area. Here, we modeled two types of groundwater discharge: hillslope groundwater discharge and aquifer-outcrop groundwater discharge (Figure 3). We Remote Sens. 2022, 14, 63 13 of 18 13 of 18 constructed a robust geodatabase comprising field observations and geologic data from >800 well logs covering a representative subset of the study area and topographic data from an airborne LiDAR-derived DEM covering the entire study area (Figure 2). We then devel- oped and refined procedures to manually identify the two types of groundwater discharge in the representative subset of the study area where the field observations, geologic data, and topographic data were available (Table 1; Figures 4 and 5). While doing so, we made observations that indicated we might otherwise identify these two types of groundwater discharge using only the topographic data. We therefore developed and refined procedures to model the two types of groundwater discharge throughout the entire study area from the topographic data alone (Table 2; Figure 7). Devito et al. [43] previously argued that topography was the last control to consider in explaining hydrologic processes, after climate and geology. Rahmati et al. [30] concurred, suggesting that geologic data (e.g., lithology) was a relatively strong predictor of groundwater levels while topographic data (e.g., slope) was a relatively weak predictor of groundwater levels. Here, topography was in fact the only control we considered, but only after topography was contextualized with the field observations and geologic data in the representative subset of the study area. servations, geo While doing s 4. Discussion Once these relationships were identified, the topography could in many cases be used as a proxy for the geology, such as in cases where aquifer outcrops were instead indicated by the initiation of multiple, parallel channelized flows along the same contour intervals on the same and/or opposite hillslopes (e.g., Figure 5). This then allowed the accurate manual and modeled identification of aquifer-outcrop groundwater discharge (Table 2; Figures 5 and 7). g The novelty of our modeling approach lies in the integration between field observations, remote-sensing data, and machine learning. Workflows for the manual identification of groundwater discharge were used to locate hillslope and aquifer-outcrop groundwater discharges in the field, with an overall accuracy of 93% (Table 1; Figures 4 and 5). Though labor-intensive, this approach enabled the field identification of a large enough sample of seep and spring locations to develop an “outstanding” predictive model for the entire study area using topographic data alone, with an AUC of 0.95 and 0.91 for training and Remote Sens. 2022, 14, 63 14 of 18 14 of 18 testing data, respectively (Table 2; Figure 7). Using only topographic data was ideal in our study area because well logs and therefore crucial geologic data (i.e., aquifer-outcrop locations) were only available over ~40% of the study area. Maxent modeling in particular was advantageous because it uses presence-only data and therefore can be used to make widespread predictions over a large study area with limited data over only a subset of the study area (e.g., [82]). Another advantage of the Maxent approach is its ability to quantify the relationships between feature prevalence and the environmental predictors [72,73]. Our model confirms field observations that groundwater discharge is most likely to occur where topography changes abruptly over small distances in close proximity to flowlines, supporting the findings of other studies (e.g., [2,46,47,63]). pp g g g Both field observations and modeling results indicate that seeps and springs are com- monly located proximal to streams, both the headward extent of streams and along hillslopes adjacent to streams (e.g., Figure 7). Following the five major Pleistocene glaciations and two minor post-Pleistocene glacial advances, the Kenai Peninsula Lowlands comprised mixed ice-scoured landforms, drift sheets, and moraines separated by unconformities and weathering profiles, much covered with younger glacial outwash and valley train, glacio- lacustrine, and other minor moraine deposits [58,59]. servations, geo While doing s 4. Discussion This heterogeneity was reflected at the surface, where local topographic relief was sufficient to direct surface-water flows into the earliest watersheds, and in the subsurface, where aquifers were commonly thin and discontinuous, often formed in thin glacial outwash and valley train deposits. Subsequent downcutting by the streams shaped and steepened valley hillslopes, thereby creating and enhancing hillslope groundwater discharge, and exposed aquifer outcrops, thereby creating and enhancing aquifer-outcrop groundwater discharge (Figure 3). This enhanced stream flow and therefore stream power, creating a positive feedback which further enhanced downcutting by the streams. g y This groundwater discharge is essential for the proper functioning of streams on the Kenai Peninsula Lowlands. Groundwater discharge to these streams augments stream flow, providing approximately half of the summer stream flow and likely all of the winter stream flow [12]. Groundwater discharge to these streams also modulates stream temperatures, providing cold-water refugia in summer and warm-water refugia in winter [12]. Salmonids are cold-water species with life-history stages sensitive to high stream water temperatures, including sublethal temperatures which can affect everything from cellular function to behavior [83,84]. Therefore, cold-water refugia in summer are crucial, and increasingly so in light of climate-induced warming trends in Alaska’s salmon-bearing streams [85]. Juvenile salmonids must overwinter in these streams prior to outmigrating the following spring. Therefore, warm-water refugia in winter are also crucial, keeping some reaches unfrozen and available as overwintering habitats [86]. Lastly, much of this groundwater first passes through and interacts with nitrogen-fixing alder patches on adjacent hillslopes, delivering nitrogen-rich groundwater to riparian wetlands and these streams [14], where it enhances primary productivity in the riparian wetlands [14,87] and controls rates of in-stream nitrogen fixation and respiration [15,85]. The nutrient subsidies to these streams are then evident in the juvenile salmonids, who preferentially use abundant allochthonous sources, especially in the headwater settings [88]. Groundwater discharge is therefore thought to at least partly explain the predictable species composition along specific reaches in these streams, especially in headwater settings [66]. This new understanding of the importance of groundwater discharge to proper functioning of streams on the Kenai Peninsula Lowlands has led to groundwater being adopted as a central feature of the conceptual model underlying the management of the salmonid resources that underlie important sport and commercial fisheries [49]. Meanwhile, groundwater is the primary source of water for domestic, commercial, and industrial uses on the Kenai Peninsula Lowlands [52]. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Publicly available datasets were analyzed in this study. This data can be found here: doi:10.6084/m9.ﬁgshare.16586903. Acknowledgments: This project beneﬁtted immeasurably from in-kind support provided by the Kachemak Bay National Estuarine Research Reserve, which provided lodging, local knowledge, introductions to stakeholders, the coordination of formal stakeholder engagements, and more. Coowe Walker and Syverine Bentz were particularly instrumental. Conrad Field illustrated Figure 3 from ﬁeld sketches and notes prepared by M Rains. Annalyssa Hernandez assisted in some ﬁeld work. A special thank you to all of the many stakeholders who provided their time, local knowledge, and access to private properties. Conﬂicts of Interest: The authors declare no conﬂict of interest. 6. Winter, T.C. Relation of streams, lakes, and wetlands to groundwater ﬂow systems. Hydrogeol. J. 1999, 7, 28–45. [CrossRef] 7. Moore, W.S.; Blanton, J.O.; Joye, S. Estimates of ﬂushing times, submarine groundwater discharge, and nutrient ﬂuxes to Okatee Estuary, South Carolina. J. Geophys. Res. Space Phys. 2006, 111, 111. [CrossRef] y p y p y Moore, W.S. The Effect of Submarine Groundwater Discharge on the Ocean. Annu. Rev. Mar. Sci. 2010, 2, 5 y J p y p y [ ] 8. Moore, W.S. The Effect of Submarine Groundwater Discharge on the Ocean. Annu. Rev. Mar. Sci. 2010, 2, 59–88. [CrossRef] 5. Winter, T.C.; Harvey, J.W.; Franke, O.L.; Alley, W.M. Ground Water and Surface Water: A Single Resource; Circular 1139; US Geological Survey: Reston, VA, USA, 1998. [CrossRef] servations, geo While doing s 4. Discussion Most wells are domestic and are drilled by, maintained, and operated at the sole discretion and expense of the individual landowner. Drilling costs are calculated per unit depth, so there is little incentive to drill beyond the shallowest aquifer that can provide sufﬁcient quantities of water. Well logs indi- Remote Sens. 2022, 14, 63 15 of 18 15 of 18 cate that these aquifers are thin and discontinuous and commonly yield ~0.01–0.1 m3/min (see also [60]). These then are the same aquifers that often outcrop on nearby hillslopes, commonly at the headward extent of streams and along hillslopes adjacent to streams (e.g., Figures 3 and 7). These aquifers are therefore the nexus of a potential conﬂict over limited groundwater resources between natural and human users. These results have height- ened awareness, with recent and ongoing work focused on using this new understanding to explore sources and locations of acute groundwater vulnerability and connecting this new understanding to decision-making by building capacity to support both peer and institutional discussions [49]. Author Contributions: This paper was the result of a broad, collaborative effort by all authors. Con- ceptualization, M.C.R.; Methodology, M.E.G., K.C.R., E.J.G.-O., J.D. and M.C.R.; Validation, M.E.G., K.C.R., E.J.G.-O., W.J.K., J.D. and M.C.R.; Formal Analysis, M.E.G., E.J.G.-O. and J.D.; Investigation, M.E.G., K.C.R., E.J.G.-O., W.J.K., J.D., S.M.L. and M.C.R.; Data Curation, M.E.G., K.C.R., E.J.G.-O. and S.M.L.; Writing—Original Draft Preparation, M.E.G. and M.C.R.; Writing—Review & Editing, K.C.R., E.J.G.-O., W.J.K., J.D. and S.M.L.; Visualization, S.M.L. and J.D.; Supervision, K.C.R. and M.C.R.; Project Administration, K.C.R. and M.C.R.; Funding Acquisition, M.C.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded primarily by the National Estuarine Research Reserve System Science Collaborative under Grant No. 54584 (https://nerrssciencecollaborative.org/project/Walker17; accessed on 23 December 2021). Additional faculty support was funded by the National Science Foundation under Grant No. 1702029 (https://www.nsf.gov/awardsearch/showAward?AWD_ID=17 02029; accessed on 23 December 2021). Additional student scholarships were funded by the National Science Foundation under Grant No. 1930451 (https://nsf.gov/awardsearch/showAward?AWD_ID= 1930451; accessed on 23 December 2021). References 1. Rains, M.C.; Fogg, G.E.; Harter, T.; Dahlgren, R.A.; Williamson, R.J. The role of perched aquifers in hydrological connectivity and biogeochemical processes in vernal pool landscapes, Central Valley, California. Hydrol. Process. 2006, 20, 1157–1175. [CrossRef] 2. Neff, B.P.; Rosenberry, D.O.; Leibowitz, S.G.; Mushet, D.M.; Golden, H.E.; Rains, M.; Brooks, J.R.; Lane, C.R. A Hydrologic Landscapes Perspective on Groundwater Connectivity of Depressional Wetlands. Water 2019, 12, 50. [CrossRef] [PubMed] 3. Kornelsen, K.; Coulibaly, P. Synthesis review on groundwater discharge to surface water in the Great Lakes Basin. J. Great Lakes Res. 2014, 40, 247–256. [CrossRef] 4 S l M X L d O M Q R lli A D F M í D E W i l P C i i f h ll l k 1. Rains, M.C.; Fogg, G.E.; Harter, T.; Dahlgren, R.A.; Williamson, R.J. The role of perched aquifers in hydrological connectivity and biogeochemical processes in vernal pool landscapes, Central Valley, California. Hydrol. Process. 2006, 20, 1157–1175. [CrossRef] 1. Rains, M.C.; Fogg, G.E.; Harter, T.; Dahlgren, R.A.; Williamson, R.J. The role of perched aquifers in hydrological connectivity and biogeochemical processes in vernal pool landscapes, Central Valley, California. Hydrol. Process. 2006, 20, 1157–1175. [CrossRef] 2. Neff, B.P.; Rosenberry, D.O.; Leibowitz, S.G.; Mushet, D.M.; Golden, H.E.; Rains, M.; Brooks, J.R.; Lane, C.R. A Hydrologic Landscapes Perspective on Groundwater Connectivity of Depressional Wetlands. Water 2019, 12, 50. [CrossRef] [PubMed] 1. Rains, M.C.; Fogg, G.E.; Harter, T.; Dahlgren, R.A.; Williamson, R.J. The role of perched aquifers in hydrological connectivity and biogeochemical processes in vernal pool landscapes, Central Valley, California. Hydrol. Process. 2006, 20, 1157–1175. [CrossRef] 1. Rains, M.C.; Fogg, G.E.; Harter, T.; Dahlgren, R.A.; Williamson, R.J. The role of perched aquifers in hydrological connectivity and biogeochemical processes in vernal pool landscapes, Central Valley, California. Hydrol. Process. 2006, 20, 1157–1175. [CrossRef] 2. Neff, B.P.; Rosenberry, D.O.; Leibowitz, S.G.; Mushet, D.M.; Golden, H.E.; Rains, M.; Brooks, J.R.; Lane, C.R. A Hydrologic Landscapes Perspective on Groundwater Connectivity of Depressional Wetlands. Water 2019, 12, 50. [CrossRef] [PubMed] 3. Kornelsen, K.; Coulibaly, P. Synthesis review on groundwater discharge to surface water in the Great Lakes Basin. J. Great Lakes Res. 2014, 40, 247–256. [CrossRef] g p p p , y, y , , [ ] 2. Neff, B.P.; Rosenberry, D.O.; Leibowitz, S.G.; Mushet, D.M.; Golden, H.E.; Rains, M.; Brooks, J.R.; Lane, C.R. A Hydrologic Landscapes Perspective on Groundwater Connectivity of Depressional Wetlands. Water 2019, 12, 50. g y y g J 7. Moore, W.S.; Blanton, J.O.; Joye, S. Estimates of ﬂushing times, submarine groundwater discharge, and Estuary, South Carolina. J. Geophys. Res. Space Phys. 2006, 111, 111. [CrossRef] References 2022, 14, 63 16 of 18 9. Misra, D.; Daanen, R.P.; Thompson, A.M. Base Flow/Groundwater Flow. In Encyclopedia of Snow, Ice and Glaciers; Encyclopedia of Earth Sciences Series; Singh, V.P., Singh, P., Haritashya, U.K., Eds.; Springer: Dordrecht, The Netherlands, 2011. g g y p g 10. Guérin, A.; Devauchelle, O.; Robert, V.; Kitou, T.; Dessert, C.; Quiquerez, A.; Allemand, P.; Lajeunesse, E. Stream-Discharge Surges Generated by Groundwater Flow. Geophys. Res. Lett. 2019, 46, 7447–7455. [CrossRef] g y p y 11. Caissie, D. The thermal regime of rivers: A review. Freshw. Biol. 2006, 51, 1389–1406. [CrossRef] g 12. Callahan, M.K.; Rains, M.; Bellino, J.C.; Walker, C.M.; Baird, S.J.; Whigham, D.F.; King, R.S. Controls on Temperature in Salmonid- Bearing Headwater Streams in Two Common Hydrogeologic Settings, Kenai Peninsula, Alaska. JAWRA J. Am. Water Resour. Assoc. 2014, 51, 84–98. [CrossRef] 13. Luke, S.H.; Luckai, N.J.; Burke, J.M.; Prepas, E.E. Riparian areas in the Canadian boreal forest and linkages with water quality in streams. Environ. Rev. 2007, 15, 79–97. [CrossRef] 14. Callahan, M.K.; Whigham, D.F.; Rains, M.; Rains, K.C.; King, R.S.; Walker, C.M.; Maurer, J.R.; Baird, S.J. Nitrogen Subsidies from Hillslope Alder Stands to Streamside Wetlands and Headwater Streams, Kenai Peninsula, Alaska. JAWRA J. Am. Water Resour. Assoc. 2017, 53, 478–492. [CrossRef] 15. Hiatt, D.L.; Robbins, C.J.; Back, J.A.; Kostka, P.K.; Doyle, R.D.; Walker, C.M.; Rains, M.C.; Whigham, D.F.; King, R.S. Catchment- scale alder cover controls nitrogen ﬁxation in boreal headwater streams. Freshw. Sci. 2017, 36, 523–532. [CrossRef] 16. Power, G.; Brown, R.S.; Imhof, J.G. Groundwater and Fish—Insights from Northern North America. 401–422. [CrossRef] 17. Dieter, C.A.; Maupin, M.A.; Caldwell, R.R.; Harris, M.A.; Ivahnenko, T.I.; Lovelace, J.K.; Barber, N.L.; Linsey, K.S. Estimated Use of Water in the United States in 2015; Circular 1441; US Geological Survey: Reston, VA, USA, 2018. [CrossRef] 18. Falkenmark, M.; Rockström, J. Balancing Water for Humans and Nature: The New Approach in Ecohydrology; Earthscan: London, UK; Sterling, VA, USA, 2004; ISBN 978-1-85383-927-6. 19. Khalil, M.A.; Bobst, A.; Mosolf, J. Utilizing 2D Electrical Resistivity Tomography and Very Low Frequency Electromagnetics to Investigate the Hydrogeology of Natural Cold Springs Near Virginia City, Southwest Montana. Pure Appl. Geophys. PAGEOPH 2018, 175, 3525–3538. [CrossRef] [ ] 20. Gleason, C.L.; Frisbee, M.D.; Rademacher, L.K.; Sada, D.W.; Meyers, Z.P.; Knott, J.R.; Hedlund, B.P. Hydrogeology of desert springs in the Panamint Range, California, USA: Geologic controls on the geochemical kinetics, ﬂowpaths, and mean residence times of springs. References [CrossRef] [PubMed] g p p p y y 2. Neff, B.P.; Rosenberry, D.O.; Leibowitz, S.G.; Mushet, D.M.; Golden, H.E.; Rains, M.; Brooks, J.R.; Lane, C.R. A Hydrologic Landscapes Perspective on Groundwater Connectivity of Depressional Wetlands. Water 2019, 12, 50. [CrossRef] [PubMed] , ; y, ; , ; , ; , ; , ; , J ; , y g Landscapes Perspective on Groundwater Connectivity of Depressional Wetlands. Water 2019, 12, 50. [CrossRef] [PubMed] 3. Kornelsen, K.; Coulibaly, P. Synthesis review on groundwater discharge to surface water in the Great Lakes Basin. J. Great Lakes Res. 2014, 40, 247–256. [CrossRef] p p y p , , [ ] [ ] 3. Kornelsen, K.; Coulibaly, P. Synthesis review on groundwater discharge to surface water in the Great Lakes Basin. J. Great Lakes Res. 2014, 40, 247–256. [CrossRef] p p y p 3. Kornelsen, K.; Coulibaly, P. Synthesis review on groundwater discharge to surface water in the Great Lakes Basin. J. Great Lakes Res. 2014, 40, 247–256. [CrossRef] 4. Solana, M.X.; Londoño, O.M.Q.; Romanelli, A.; Donna, F.; Martínez, D.E.; Weinzettel, P. Connectivity of temperate shallow lakes to groundwater in the Pampean Plain, Argentina: A remote sensing and multi-tracer approach. Groundw. Sustain. Dev. 2021, 13, 100556. [CrossRef] 4. Solana, M.X.; Londoño, O.M.Q.; Romanelli, A.; Donna, F.; Martínez, D.E.; Weinzettel, P. Connectivity of temperate shallow lakes to groundwater in the Pampean Plain, Argentina: A remote sensing and multi-tracer approach. Groundw. Sustain. Dev. 2021, 13, 100556. [CrossRef] 4. Solana, M.X.; Londoño, O.M.Q.; Romanelli, A.; Donna, F.; Martínez, D.E.; Weinzettel, P. Connectivity of temperate shallow lakes to groundwater in the Pampean Plain, Argentina: A remote sensing and multi-tracer approach. Groundw. Sustain. Dev. 2021, 13, 100556. [CrossRef] 5. Winter, T.C.; Harvey, J.W.; Franke, O.L.; Alley, W.M. Ground Water and Surface Water: A Single Resource; Circular 1139; US Geological Survey: Reston, VA, USA, 1998. [CrossRef] y 6. Winter, T.C. Relation of streams, lakes, and wetlands to groundwater ﬂow systems. Hydrogeol. J. 1999, 7, 28–45. [CrossRef] 7. Moore, W.S.; Blanton, J.O.; Joye, S. Estimates of ﬂushing times, submarine groundwater discharge, and nutrient ﬂuxes to Okatee Estuary, South Carolina. J. Geophys. Res. Space Phys. 2006, 111, 111. [CrossRef] g y y g J [ ] 7. Moore, W.S.; Blanton, J.O.; Joye, S. Estimates of ﬂushing times, submarine groundwater discharge, and nutrient ﬂuxes to Okatee Estuary, South Carolina. J. Geophys. Res. Space Phys. 2006, 111, 111. [CrossRef] Remote Sens. References Adv. Water Resour. 2020, 141, 103595. [CrossRef] 29. Afan, H.A.; Osman, A.I.A.; Essam, Y.; Ahmed, A.N.; Huang, Y.F.; Kisi, O.; Sherif, M.; Sefelnasr, A.; Chau, K.-W.; El-Shaﬁe, A. Modeling the ﬂuctuations of groundwater level by employing ensemble deep learning techniques. Eng. Appl. Comput. Fluid Mech. 2021, 15, 1420–1439. [CrossRef] 30. Rahmati, O.; Pourghasemi, H.R.; Melesse, A.M. Application of GIS-based data driven random fores models for groundwater potential mapping: A case study at Mehran Region, Iran. Catena 2016, 137, 36 30. Rahmati, O.; Pourghasemi, H.R.; Melesse, A.M. Application of GIS-based data driven random forest and maximum entropy models for groundwater potential mapping: A case study at Mehran Region, Iran. Catena 2016, 137, 360–372. [CrossRef] 31. El Bilali, A.; Taleb, A.; Brouziyne, Y. Groundwater quality forecasting using machine learning algorithms for irrigation purposes. Agric. Water Manag. 2021, 245, 106625. [CrossRef] 30. Rahmati, O.; Pourghasemi, H.R.; Melesse, A.M. Application of GIS-based data driven random forest and maximum entropy models for groundwater potential mapping: A case study at Mehran Region, Iran. Catena 2016, 137, 360–372. [CrossRef] 31 El Bil li A T l b A B i Y G d li f i i hi l i l i h f i i i 31. El Bilali, A.; Taleb, A.; Brouziyne, Y. Groundwater quality forecasting using machine learning algori Agric. Water Manag. 2021, 245, 106625. [CrossRef] g g 32. Shiri, N.; Shiri, J.; Yaseen, Z.M.; Kim, S.; Chung, I.-M.; Nourani, V.; Zounemat-Kermani, M. Development of artificial intelligence models for well groundwater quality simulation: Different modeling scenarios. PLoS ONE 2021, 16, e0251510. [CrossRef] [PubMed] 33 T Z Y Q Zh Y M hi L i M d l f G d t A i S ti l Di t ib ti i B l d h I ﬂ f 32. Shiri, N.; Shiri, J.; Yaseen, Z.M.; Kim, S.; Chung, I.-M.; Nourani, V.; Zounemat-Kermani, M. Development of artificial intelligence models for well groundwater quality simulation: Different modeling scenarios. PLoS ONE 2021, 16, e0251510. [CrossRef] [PubMed] 33. Tan, Z.; Yang, Q.; Zheng, Y. Machine Learning Models of Groundwater Arsenic Spatial Distribution in Bangladesh: Inﬂuence of Holocene Sediment Depositional History. Environ. Sci. Technol. 2020, 54, 9454–9463. [CrossRef] [PubMed] models for well groundwater quality simulation: Different modeling scenarios. PLoS ONE 2021, 16, e0251510. [CrossRef] [PubMed] 33. Tan, Z.; Yang, Q.; Zheng, Y. Machine Learning Models of Groundwater Arsenic Spatial Distribution in Bangladesh: Inﬂuence of Holocene Sediment Depositional History. Environ. References Hydrol. Process. 2020, 34, 2923–2948. [CrossRef] p g y 21. Mocior, E.; Rzonca, B.; Siwek, J.; Plenzler, J.; Płaczkowska, E.; Dabek, N.; Jaskowiec, B.; Potoniec, P.; Roman, S.; Zdziebko, D. Determinants of the distribution of springs in the upper part of a ﬂysch ridge in the Bieszczady Mountains in southeastern Poland. Episodes 2015, 38, 21–30. [CrossRef] p 22. Howard, J.; Merriﬁeld, M. Mapping Groundwater Dependent Ecosystems in California. PLoS ONE 2010, 5, e11249. [CrossRef] 23. Pourtaghi, Z.S.; Pourghasemi, H.R. GIS-based groundwater spring potential assessment and mapping in the Birjand Township, southern Khorasan Province, Iran. Hydrogeol. J. 2014, 22, 643–662. [CrossRef] p 22. Howard, J.; Merriﬁeld, M. Mapping Groundwater Dependent Ecosystems in California. PLoS ONE 201 , J ; , pp g p y , , [ ] 23. Pourtaghi, Z.S.; Pourghasemi, H.R. GIS-based groundwater spring potential assessment and mapping in the Birjand Township, southern Khorasan Province, Iran. Hydrogeol. J. 2014, 22, 643–662. [CrossRef] 24. Lange, H.; Sippel, S. Machine Learning Applications in Hydrology. In Forest-Water Interactions; Levia, D.F., Carlyle-Moses, D.E., Iida, S., Michalzik, B., Nanko, K., Tischer, A., Eds.; Ecological Studies; Springer International Publishing: Cham, Switzerland, 2020; Volume 240, pp. 233–257. ISBN 978-3-030-26085-9. pp 25. Nearing, G.S.; Kratzert, F.; Sampson, A.K.; Pelissier, C.S.; Klotz, D.; Frame, J.M.; Prieto, C.; Gupta, H.V. What Role Does Hydrological Science Play in the Age of Machine Learning? Water Resour. Res. 2021, 57, e2020WR028091. [CrossRef] pp 25. Nearing, G.S.; Kratzert, F.; Sampson, A.K.; Pelissier, C.S.; Klotz, D.; Frame, J.M.; Prieto, C.; Gupta, H.V. What Role Does Hydrological Science Play in the Age of Machine Learning? Water Resour. Res. 2021, 57, e2020WR028091. [CrossRef] 26. Shen, C.; Chen, X.; Laloy, E. Editorial: Broadening the Use of Machine Learning in Hydrology. Front. Water 2021, 3, 681023. [CrossRef] Hydrological Science Play in the Age of Machine Learning? Water Resour. Res. 2021, 57, e2020WR028091. [CrossRef] 26. Shen, C.; Chen, X.; Laloy, E. Editorial: Broadening the Use of Machine Learning in Hydrology. Front. Water 2021, 3, 681023. [CrossRef] 26. Shen, C.; Chen, X.; Laloy, E. Editorial: Broadening the Use of Machine Learning in Hydrology. Front. Water 2021, 3, 681023. [CrossRef] 27. Sahoo, S.; Russo, T.A.; Elliott, J.; Foster, I. Machine learning algorithms for modeling groundwater level changes in agricultural regions of the U.S. Water Resour. Res. 2017, 53, 3878–3895. [CrossRef] g 28. Rahman, A.S.; Hosono, T.; Quilty, J.M.; Das, J.; Basak, A. Multiscale groundwater level forecasting: learning approaches with wavelet transforms. References Groundwater discharge: A common generator of diverse geologic and morphologic phenomena. Int. Assoc. Sci. Hydrol. Bull. 1971, 16, 7–24. [CrossRef] 45. Huang, X.; Niemann, J.D. Modelling the potential impacts of groundwater hydrology on long-term drainage basin evolution. Earth Surf. Process. Landforms 2006, 31, 1802–1823. [CrossRef] 46. Iverson, R.M.; Reid, M.E. Gravity-driven groundwater ﬂow and slope failure potential: 1. Elastic Effective-Stress Model. Water Resour. Res. 1992, 28, 925–938. [CrossRef] 47. Reid, M.E.; Iverson, R.M. Gravity-driven groundwater ﬂow and slope failure potential: 2. Effects of slo properties, and hydraulic heterogeneity. Water Resour. Res. 1992, 28, 939–950. [CrossRef] properties, and hydraulic heterogeneity. Water Resour. Res. 1992, 28, 939–950. [CrossRef] 48. UACED (University of Alaska Center for Economic Development). Kenai Peninsula 2021–2026 Comprehensive Economic Development Strategy; Kenai Peninsula Economic Development District: Kenai AK USA 2021; p 97 48. UACED (University of Alaska Center for Economic Development). Kenai Peninsula 2021–2026 Comprehen Strategy; Kenai Peninsula Economic Development District: Kenai, AK, USA, 2021; p. 97. 49. Walker, C.M.; Whigham, D.F.; Bentz, I.S.; Argueta, J.M.; King, R.S.; Rains, M.C.; Simenstad, C.A.; Guo, C.; Baird, S.J.; Field, C.J. Linking landscape attributes to salmon and decision-making in the southern Kenai Lowlands, Alaska, USA. Ecol. Soc. 2021, 26, 1. [CrossRef] 50. ADLWD (Alaska Department of Labor and Workforce Development). Alaska Population Projections 2019– of Labor and Workforce Development: Juneau, AK, USA, 2020. 51. HSWCD (Homer Soil and Water Conservation District). Growing Local Food: A Survey of Commercial Producers on the Southern Kenai Peninsula; Homer Soil and Water Conservation District: Homer, AK, USA, 2018. 52. Glass, R. Ground-Water Conditions and Quality in the Western Part of Kenai Peninsula, Southcentral Alaska; Open-File Rep. 96-446; US Geological Survey: Reston, VA, USA, 1996. [CrossRef] g y [ ] Baughman, C.A.; Loehman, R.A.; Magness, D.R.; Saperstein, L.B.; Sherriff, R.L. Four Decades of Land-Cover 53. Baughman, C.A.; Loehman, R.A.; Magness, D.R.; Saperstein, L.B.; Sherriff, R.L. Four Decades of Land-Cover Change on the Kenai Peninsula, Alaska: Detecting Disturbance-Inﬂuenced Vegetation Shifts Using Landsat Legacy Data. Land 2020, 9, 382. [CrossRef] 53. Baughman, C.A.; Loehman, R.A.; Magness, D.R.; Saperstein, L.B.; Sherriff, R.L. Four Decades of Land-Cover Change on the Kenai Peninsula, Alaska: Detecting Disturbance-Inﬂuenced Vegetation Shifts Using Landsat Legacy Data. Land 2020, 9, 382. References Sci. Technol. 2020, 54, 9454–9463. [CrossRef] [PubMed] 33. Tan, Z.; Yang, Q.; Zheng, Y. Machine Learning Models of Groundwater Arsenic Spatial Distribution Holocene Sediment Depositional History. Environ. Sci. Technol. 2020, 54, 9454–9463. [CrossRef] [Pu 34. Tran, D.A.; Tsujimura, M.; Ha, N.T.; Nguyen, V.T.; Van Binh, D.; Dang, T.D.; Doan, Q.-V.; Bui, D.T.; Ngoc, T.A.; Phu, L.V.; et al. Evaluating the predictive power of different machine learning algorithms for groundwater salinity prediction of multi-layer coastal aquifers in the Mekong Delta, Vietnam. Ecol. Indic. 2021, 127, 107790. [CrossRef] 17 of 18 Remote Sens. 2022, 14, 63 35. Hussein, E.A.; Thron, C.; Ghaziasgar, M.; Bagula, A.; Vaccari, M. Groundwater Prediction Using Machine-Learning Tools. Algorithms 2020, 13, 300. [CrossRef] g , , [ ] 36. Jaafarzadeh, M.S.; Tahmasebipour, N.; Haghizadeh, A.; Pourghasemi, H.R.; Rouhani, H. Groundwater recharge potential zonation using an ensemble of machine learning and bivariate statistical models. Sci. Rep. 2021, 11, 1–18. [CrossRef] g g p 37. Winter, T.C. The concept of hydrologic landscapes. JAWRA J. Am. Water Resour. Assoc. 2001, 37, 335 g p The concept of hydrologic landscapes. JAWRA J. Am. Water Resour. Assoc. 2001, 37, 335–349. [CrossRef] , p y g p J J , , [ ] 38. Wolock, D.M.; Winter, T.C.; McMahon, G. Delineation and Evaluation of Hydrologic-Landscape Regions in the United States Using Geographic Information System Tools and Multivariate Statistical Analyses. Environ. Manag. 2004, 34, S71–S88. [CrossRef] [PubMed] 39. Wigington, P.J.; Leibowitz, S.G.; Comeleo, R.L.; Ebersole, J. Oregon Hydrologic Landscapes: A Classiﬁcation Framework1. JAWRA J. Am. Water Resour. Assoc. 2012, 49, 163–182. [CrossRef] ng Groundwater Discharge Areas Using Only Digital Elevation Models as Input Data; Swedish Nuclear Fuel and Stockholm, Sweden, 2006; p. 18. 40. Brydsten, L. Modelling Groundwater Discharge Areas Using Only Digital Elevation Models as Input Data; Sw Waste Management: Stockholm, Sweden, 2006; p. 18. g p 41. Tweed, S.O.; Leblanc, M.; Webb, J.; Lubczynski, M.W. Remote sensing and GIS for mapping groundwater recharge and discharge areas in salinity prone catchments, southeastern Australia. Hydrogeol. J. 2006, 15, 75–96. [CrossRef] 42. Haitjema, H.M.; Mitchell-Bruker, S. Are Water Tables a Subdued Replica of the Topography? Ground Water 2005, 43, 781–786. [CrossRef] [PubMed] 43. Devito, K.; Creed, I.; Gan, T.; Mendoza, C.; Petrone, R.; Silins, U.; Smerdon, B. A framework for broad-scale classiﬁcation of hydrologic response units on the Boreal Plain: Is topography the last thing to consider? Hydrol. Process. 2005, 19, 1705–1714. [CrossRef] 44. Toth, J. 62. Heine, R.A.; Lant, C.L.; Sengupta, R.R. Development and Comparison of Approaches for Automated Mapping of Stream Channel Networks. Ann. Assoc. Am. Geogr. 2004, 94, 477–490. [CrossRef] References [CrossRef] 54 Kl i E B E E Di l R W l d d i d i h K i P i l L l d h l Al k C J Peninsula, Alaska: Detecting Disturbance-Inﬂuenced Vegetation Shifts Using Landsat Legacy Data. Land 2020, 9, 382. [CrossRef] 54. Klein, E.; Berg, E.E.; Dial, R. Wetland drying and succession across the Kenai Peninsula Lowlands, south-central Alaska. Can. J. For. Res. 2005, 35, 1931–1941. [CrossRef] g g g g y 54. Klein, E.; Berg, E.E.; Dial, R. Wetland drying and succession across the Kenai Peninsula Lowlands, south-central Alaska. Can. J. For. Res. 2005, 35, 1931–1941. [CrossRef] 55. Berg, E.E.; Hillman, K.M.; Dial, R.; DeRuwe, A. Recent woody invasion of wetlands on the Kenai Peninsula Lowlands, south- central Alaska: A major regime shift after 18000 years of wet Sphagnum–sedge peat recruitment. Can. J. For. Res. 2009, 39, 2033–2046. [CrossRef] 56. Magness, D.R.; Morton, J.M. Using climate envelope models to identify potential ecological trajectories on the Kenai Peninsula, Alaska. PLoS ONE 2018, 13, e0208883. [CrossRef] [PubMed] 57. USGS (U.S. Geological Survey). The National Map U.S. Geological Survey’s (USGS) National Geospatial Program.. Available online: https://www.usgs.gov/core-science-systems/national-geospatial-program/national-map (accessed on 30 August 2021). 58. Karlstrom, T.N. Quaternary Geology of the Kenai Lowland and Glacial History of the Cook Inlet Region, Alask US Geological Survey: Reston, VA, USA, 1964. [CrossRef] ternary Geology of the Kenai Lowland and Glacial History of the Cook Inlet Region, Alaska; Professional Paper 443 ey: Reston, VA, USA, 1964. [CrossRef] 59. Wilson, F.H.; Hults, C.P. Geology of the Prince William Sound and Kenai Peninsula Region, Alaska; Scientiﬁc Investigations Map 3110; US Geological Survey: Anchorage, AK, USA, 2012. [CrossRef] g y g 60. Nelson, G.; Johnson, P. Ground-Water Reconnaissance of Part of the Lower Kenai Peninsula, Alaska; Open-File Rep. 81-905; US Geological Survey: Reston, VA, USA, 1981. [CrossRef] 60. Nelson, G.; Johnson, P. Ground-Water Reconna Survey: Reston, VA, USA, 1981. [CrossRef] 61. Spencer, E.W. Geologic Maps: A Practical Guide to Preparation and Interpretation; Waveland Press: Long Grove, IL, USA, 2017; ISBN 1-4786-3488-X. 62. Heine, R.A.; Lant, C.L.; Sengupta, R.R. Development and Comparison of Approaches for Automated Mapping of Stream Channel Networks. Ann. Assoc. Am. Geogr. 2004, 94, 477–490. [CrossRef] 18 of 18 Remote Sens. 2022, 14, 63 63. Jaeger, K.L.; Montgomery, D.R.; Bolton, S.M. Channel and Perennial Flow Initiation in Headwater Streams: Management Implications of Variability in Source-Area Size. Environ. Manag. 2007, 40, 775–786. References [CrossRef] p y g 64. Detty, J.M.; McGuire, K.J. Topographic controls on shallow groundwater dynamics: Implications of hydrologic connectivity between hillslopes and riparian zones in a till mantled catchment. Hydrol. Process. 2010, 24, 2222–2236. [CrossRef] 65. Riley, S.J.; DeGloria, S.D.; Elliot, R. A Terrain Ruggedness Index That Quantiﬁes Topographic Heterogeneity. Intermt. J. Sci. 1999, 5, 23–27. 66. Korzeniowska, K.; Pfeifer, N.; Landtwing, S. Mapping gullies, dunes, lava fields, and landslides via surface roughness. Geomorphology 2018, 301, 53–67. [CrossRef] 67. Walker, C.M.; King, R.S.; Whigham, D.F.; Baird, S.J. Landscape and Wetland Inﬂuences on Headwater Stream Chemistry in the Kenai Lowlands, Alaska. Wetlands 2012, 32, 301–310. [CrossRef] 68. Beven, K.J.; Kirkby, M.J. A physically based, variable contributing area model of basin hydrology/Un m zone d’appel variable de l’hydrologie du bassin versant. Hydrol. Sci. Bull. 1979, 24, 43–69. [CrossRef] pp y g y 69. Sørensen, R.; Zinko, U.; Seibert, J. On the calculation of the topographic wetness index: Evaluation of different methods based on ﬁeld observations. Hydrol. Earth Syst. Sci. 2006, 10, 101–112. [CrossRef] y y 70. Tarboton, D. A new method for the determination of ﬂow directions and upslope areas in grid digital elevation models. Water Resour. Res. 1997, 33, 309–319. [CrossRef] 71. Horvath, E.K.; Christensen, J.R.; Mehaffey, M.H.; Neale, A.C. Building a potential wetland restoration indicator for the contiguous United States. Ecol. Indic. 2017, 83, 463–473. [CrossRef] 72. Rains, M.; Mount, J.F. Origin of shallow ground water in an alluvial aquifer as determined by isotopic and chemical procedures. Ground Water 2002, 40, 552–563. [CrossRef] [PubMed] , M. Modeling of species distributions with Maxent: New extensions and a comprehensive evaluation 61–175. [CrossRef] 73. Phillips, S.J.; Dudík, M. Modeling of species distributions with Maxent: New extensions and a Ecography 2008, 31, 161–175. [CrossRef] g p y 74. Elith, J.; Phillips, S.J.; Hastie, T.; Dudík, M.; Chee, Y.E.; Yates, C.J. A statistical explanation of MaxEnt for ecologists. Divers. Distrib. 2011, 17, 43–57. [CrossRef] [ ] 75. Merow, C.; Smith, M.J.; Silander, J.A. A practical guide to MaxEnt for modeling species’ distributions: What it does, and why inputs and settings matter. Ecography 2013, 36, 1058–1069. [CrossRef] 76. Feng, X.; Park, D.S.; Liang, Y.; Pandey, R.; Pape¸s, M. Collinearity in ecological niche modeling: Confusions and challenges. Ecol. Evol. 2019, 9, 10365–10376. [CrossRef] 77. Cohen, J. A Coefﬁcient of Agreement for Nominal Scales. Educ. Psychol. Meas. 1960, 20, 37–46. [CrossR 78. Landis, J.R.; Koch, G.G. 88. Dekar, M.P.; King, R.S.; Back, J.A.; Whigham, D.; Walker, C.M. Allochthonous inputs from grass-dominated wetlands support juvenile salmonids in headwater streams: Evidence from stable isotopes of carbon, hydrogen, and nitrogen. Freshw. Sci. 2012, 31, 121–132. [CrossRef] References An Application of Hierarchical Kappa-type Statistics in the Assessment of Majority Agreement among Multiple Observers. Biometrics 1977, 33, 363. [CrossRef] 79. Mandrekar, J.N. Receiver Operating Characteristic Curve in Diagnostic Test Assessment. J. Thorac. Oncol. 2010, 5, 1315–1316. [CrossRef] 80. King, R.S.; Walker, C.M.; Whigham, D.F.; Baird, S.J.; Back, J.A. Catchment topography and wetland geomorphology drive macroinvertebrate community structure and juvenile salmonid distributions in south-central Alaska headwater streams. Freshw. Sci. 2012, 31, 341–364. [CrossRef] 81. Souissi, D.; Msaddek, M.H.; Zouhri, L.; Chenini, I.; El May, M.; Dlala, M. Mapping groundwater recharge potential zones in arid region using GIS and Landsat approaches, southeast Tunisia. Hydrol. Sci. J. 2018, 63, 251–268. [CrossRef] 82. Rhoden, C.M.; Peterman, W.E.; Taylor, C.A. Maxent-directed ﬁeld surveys identify new populations of narrowly endemic habitat specialists. PeerJ 2017, 5, e3632. [CrossRef] [PubMed] 83. Taylor, S.G. Climate warming causes phenological shift in Pink Salmon, Oncorhynchus gorbuscha, behavior at Auke Creek, Alaska. Glob. Chang. Biol. 2008, 14, 229–235. [CrossRef] 84. Bowen, L.; von Biela, V.R.; McCormick, S.D.; Regish, A.M.; Waters, S.C.; Durbin-Johnson, B.; Britton, M.; Settles, M.L.; Donnelly, D.S.; Laske, S.M.; et al. Transcriptomic response to elevated water temperatures in adult migrating Yukon River Chinook salmon (Oncorhynchus tshawytscha). Conserv. Physiol. 2020, 8, coaa084. [CrossRef] y y y 85. Shaftel, R.S.; King, R.S.; Back, J.A. Breakdown rates, nutrient concentrations, and macroinvertebrate colonization of bluejoint grass litter in headwater streams of the Kenai Peninsula, Alaska. J. N. Am. Benthol. Soc. 2011, 30, 386–398. [CrossRef] 86. Gutsch, M.K. Dentiﬁcation and Characterization of Juvenile Coho Salmon Overwintering Habitats and Early Spring Outmigration in the Anchor River Watershed, Alaska; University of Alaska, Fairbanks: Fairbanks, AK, USA, 2011. 87. Whigham, D.; Walker, C.; Maurer, J.; King, R.; Hauser, W.; Baird, S.; Keuskamp, J.; Neale, P. Watershed inﬂuences on the structure and function of riparian wetlands associated with headwater streams—Kenai Peninsula, Alaska. Sci. Total Environ. 2017, 599, 124–134. [CrossRef] [PubMed] 88. Dekar, M.P.; King, R.S.; Back, J.A.; Whigham, D.; Walker, C.M. Allochthonous inputs from grass-dominated wetlands support juvenile salmonids in headwater streams: Evidence from stable isotopes of carbon, hydrogen, and nitrogen. Freshw. Sci. 2012, 31, 121–132. [CrossRef]
https://openalex.org/W2810402821	https://ebuah.uah.es/dspace/bitstream/10017/33097/1/estudiante_villares_RIECS_2018_v.%203%2c_n.%201.pdf	es		El estudiante de Medicina: Un ficus en formación	Revista de investigación y educación en ciencias de la salud	1,970	cc-by	6,452	Facultad de Medicina y Ciencias de la Salud UAH Comunicación Delegación Estudiantes de Medicina UAH en III Congreso Nacional de Bioética para estudiantes. El estudiante de Medicina: Un ficus en formación Alejandro Villares López 1,, Paula Rubio García 2 1 Estudiante de Medicina (promoción 2013-2019); Universidad de Alcalá Estudiante de Medicina (promoción 2013-2019); Universidad de Alcalá Autor correspondencia: alexvillares.lopez@gmail.com delegados1319@gmail.com 2 Recibido: 01/04/2018; Aceptado: 23/04/2018; Publicado: 01/05/2018 Resumen: Actualmente, la formación universitaria en el Grado en Medicina consta de la realización de seis años durante los cuales se adquieren una serie de conocimientos teóricos y prácticos mediante la asistencia a las prácticas clínicas en los diferentes centros asociados a las instituciones universitarias en cuestión. Concretamente la formación ofertada por la Universidad de Alcalá consta de un plan estructurado en el que la formación práctica se reduce a un sexto del total de horas invertidas para lograr alcanzar el título de graduado. Ante ello, se nos plantean una serie de preguntas ¿Son estas suficientes horas de prácticas y son aprovechadas? ¿Cómo es la realidad de las prácticas clínicas? ¿Es posible que el estudiante pueda realizar una formación adicional al currículum y disposiciones de su Grado? La realidad es que, en ocasiones, las prácticas clínicas no son aprovechadas, ya que los estudiantes se convierten en elementos decorativos del espacio sanitario como consecuencia de errores en la capacidad de integración del mismo en el equipo asistencial siendo prioritaria la asistencia al paciente sobre la formación del estudiante; así mismo, asumimos la falta de motivación, cooperación e interés por parte del estudiantado, tanto en el ámbito de las prácticas clínicas como en el de la formación humanista, investigadora y profesional. Por tanto, el estudiante de medicina ¿es un ficus en formación? Hemos pretendido abordar en el presente trabajo dicha resolución realizando una revisión de los diferentes planes de estudios de las universidades nacionales e internacionales, resaltando aquellos más llamativos. Palabras Clave: Estudiantes; Grado en Medicina; Ficus; Aprendizaje activo; Competencia clínica; Docencia; Innovación. Abstract: Nowadays, it takes six years to obtain a medical degree. During their time at university, the students acquire theoretical and practical knowledge through clinical rotations in partner institutions. Regarding the University of Alcalá, these rotations were reduced by one sixth in its study plan. The student is the said to have acquired the skills necessary to graduate from university. In the light of these considerations, some questions arise: Are these training hours enough? Are students being adequately trained during their clinical rotations? What is current situation of the clinical rotations? Is it feasible for the student to receive additional training outside the established study plan? The truth is, this practical training is not always helpful for the students, as the feel useless in an environment where their skills are not strengthened and used. This is due to their lack of integration in the health-care team, as patient care is of greater importance than student training. Moreover, it is also noteworthy the lack of motivation, cooperation and interest among students during their practical, humanistic, research and professional training. In order to analyze what has been said heretofore, this study is going to examine the different study plans of national and international universities, emphasizing those with the most conspicuous features. Key words: Student; Medicine degree; Ficus; Active learning; Clinic competence; Teaching; Innovation. RIECS 2018, 3, 1; ISSN: 2530-2787 www.riecs.es RIECS 2018, 3, 1 94 1. Introducción La figura del estudiante de Medicina como “ficus en formación” surgió en el seno de la representación estudiantil con el objetivo de hacer ver mediante una imagen, una planta que se emplea para la decoración de interiores, la situación que pueden vivir los estudiantes durante el desarrollo de sus prácticas clínicas. Frente a ello, varias universidades españolas e internacionales, así como asociaciones de representación estudiantil presentan diferentes métodos, campañas de concienciación y diversas innovaciones en docencia que posteriormente quedan recogidas en la presente publicación. La evolución de estas campañas ha pasado del lema “No soy un ficus” al de “Riégame en prácticas” para fomentar el aprendizaje activo en la docencia clínica del hospital; siendo una de las campañas más recientes la conocida como “Nos quemamos” una de las que más repercusión mediática tuvo en relación al “burnout” que sufre el propio estudiante de Medicina por la necesidad de mejora en materia de educación médica. La actualidad e importancia del tema que tratamos queda reflejada en la voluntad expresada por parte del máximo organismo de representación estudiantil en el ámbito nacional, el Consejo Estatal de Estudiantes de Medicina (CEEM), durante la celebración de las últimas Jornadas Estatales de Estudiantes de Medicina (JEEM) en Murcia (marzo 2018) donde se aprobó la elaboración de un “Estatuto del Estudiante en prácticas” así como la constitución de una comisión de trabajo para lograr tal fin. Dicha comisión de trabajo está formada por los propios estudiantes de Medicina, procedentes de todas las facultades de Medicina de España. Será en las próximas JEEM que se celebrarán en la Universidad Jaume I cuando se apruebe este primer borrador como documento oficial. 2. Material y Métodos Realización de una revisión bibliográfica en relación a modelos pedagógicos universitarios, centrando la búsqueda en modelos basados en el aprendizaje activo. Realización de revisión bibliográfica a propósito de planes docentes relacionados con las prácticas clínicas de la Medicina en las universidades españolas: revisión de los planes y guías docentes de tales asignaturas de las facultades de Medicina de dichas instituciones (búsqueda on-line de guías docentes disponibles en los diferentes portales universitarios). Revisión de campañas y posicionamientos aprobados y publicados por el CEEM relacionadas la comisión de Educación Médica y docencia [1]. Realización de entrevistas personales a personal médico de asistencia de primer y segundo nivel, así como a Médicos Internos Residentes. 3. Discusión 3.1 Recuerdo histórico: de las madrassas a los hospitales universitarios. El que la formación médica se encuentre alojada entre las paredes de la Universidad como institución se debe, en su génesis, a la reglamentación propulsada en el s. XIII por el emperador Federico II para el Reino de Sicilia y que, poco a poco, fue extendiéndose a lo largo de toda la Baja Edad Media por todas las universidades europeas. Es relevante señalar en este punto la marcada distancia que se erige entre la formación médica (clínica, en un lenguaje cercano) y la formación quirúrgica y que no ha de resolverse hasta el siglo XIX. Concretamente en España, la enseñanza de la Medicina se inicia en el siglo XIII en la Universidad de Salamanca, en el seno de la Corona de Castilla, y en la Universidad de Montpellier, en la Corona de Aragón. Debemos alcanzar a entender la sociedad del momento como un crisol de culturas en la que el estudio de la Medicina se realizaba a través de los ojos de las tres religiones, tal y como sucedía en la villa de Toledo. Asimismo, se debe recordar la posterior conversión de dicha sociedad enriquecida en la cultura en otra, donde el oscurantismo cultural promovido por las instituciones eclesiásticas favoreció la pérdida de dicha enriquecida tradición docente. La formalización de la facultad de Medicina en la Universidad de Alcalá se realizó en 1508. RIECS 2018, 3, 1 95 Respecto del inicio del conocimiento práctico de la Medicina, es decir, la disección de cadáveres se introdujo en la Corona de Aragón siguiendo las corrientes universitarias italianas realizando de una a tres autopsias de un reo a muerte en los meses de invierno, siendo en el siglo XV en la Escuela de Cirugía del Reino de Valencia donde se comenzó la práctica de la disección de cadáveres humanos con fines docentes e incorporándose en la complutense en 1534 tras la solicitud por parte de los propios estudiantes. “No se puede tener noticia verdadera mediante la lectura ni el entendimiento en la medida que lo hacen los ojos corporales”. El nacimiento de las cátedras de cirugía en el resto de las universidades españolas se produjo a finales del siglo XVI y principios del XVII ya que, hasta este momento, dicho conocimiento teórico-práctico estaba desterrado en su totalidad de la institución docente, exceptuando la Escuela de Valencia. No es hasta mediados del siglo XVIII, bajo el influjo pensamiento ilustrado, cuando se decide comenzar a incorporar al estudio puramente teórico de los grandes textos la asistencia diaria a la visitación médica por parte de los estudiantes en los hospitales. De esta forma, se inició la promoción por parte de Gregorio Mayans de una reforma en los estudios de Medicina basada en la realización de cuatro años de estudios teóricos y dos prácticos asistiendo de esta forma a la aparición de la primera cátedra de clínica en el año 1776 en la Universidad de Granada. Así mismo, se comenzó a realizar una docencia práctica de la Medicina y Cirugía en instituciones extrauniversitarias, como en el Hospital General de Madrid (actual Museo Nacional Centro de Arte Reina Sofía), de esta forma se asistió al ascenso social e institucional de la Cirugía respecto de la Medicina que a su vez estuvo respaldado por la necesidad de adquirir una puntera cirugía militar. Fue en 1799 cuando se asistió a la unificación de los Colegios de Medicina y Cirugía en Barcelona, Cádiz y Madrid, mientras que continuaba la separación en las instituciones universitarias. Se inició de esta forma un proceso reformista que iría transformando el panorama de la docencia en Medicina y Cirugía a lo largo del siglo XIX y que tuvo en la Facultad de Madrid un importante exponente, asociada esta al Hospital San Carlos. Es en dicha facultad donde se promueve la implantación de un abordaje del conocimiento médico desde la cabecera en los hospitales, es decir, enseñanza práctica y clínica más allá de lo puramente teórico, que se inició en Francia en el siglo anterior, quedándose fuera la promulgación de la investigación médica desde dichas instituciones. El siglo XIX constituye, en resumen, un periodo de gran agitación en el panorama de la docencia en Medicina donde los aspectos políticos influyeron en el proceso reformista siendo este arduo y lento. Es, en gran medida, gracias a la figura de Santiago Ramón y Cajal cuando se consigue establecer una Facultad de Medicina en Madrid con cátedras, repartidas en siete años para obtener el Doctorado, refiéranse como teóricas, otras clínicas y quirúrgicas que se aproximan a la construcción de los planes de estudios actuales. Otro de los grandes hitos respecto de la formación médica y que afecta concretamente a la enseñanza universitaria es la aparición de la especialización médica en los hospitales españoles, hecho que ocurre a mediados del siglo XX, siendo punteros en dichos programas de formación el Hospital de la Santa Creu i Sant Jordi de Barcelona y el Hospital de Basurto, en Vizcaya. Este sistema de especialización de los licenciados en Medicina y Cirugía comienza a reglarse en los años sesenta comenzando en 1966 el actual sistema MIR (médico interno residente) y finalizando dicha legislación en el año 2008. El hecho de la aparición del médico residente en los hospitales universitarios supone la asunción de un nuevo eslabón en la jerarquía propia de los hospitales quedando la figura como estudiante relegada a un plano más secundario. Esto hace que, en la práctica diaria, la docencia práctica de la Medicina sea de manera simultánea para la especialización y para la formación universitaria, lo que en muchas ocasiones plantea serios problemas éticos respecto de la integridad del paciente que está siendo asistido, a consecuencia de la sobresaturación del sistema de enseñanza en los hospitales universitarios. RIECS 2018, 3, 1 96 3.2 La docencia práctica de la Medicina: una necesidad. Pedagógicamente, no se puede entender la Medicina sin experimentarla. Un buen modelo para entender las competencias clínicas es el propuesto por Miller (Ver figura 1) que estructura la competencia clínica en cuatro escalones: 1. La base de la Pirámide corresponde al «Saber», por tanto, son los conocimientos que debemos adquirir mediante la asistencia a las clases en la Facultad o en los Hospitales, así como con el estudio diario y la investigación personal. 2. El segundo escalón se corresponde con el «saber cómo», es decir, saber aplicar los conocimientos a problemas concretos relacionados con el manejo de los pacientes. Estos dos primeros escalones se circunscriben al dominio de los conocimientos siendo el último un inicio de aplicación práctica. 3. El tercer escalón corresponde al «mostrar cómo». El profesional ha de demostrar el dominio de la praxis, generalmente en un primer momento y especialmente en la etapa del grado en un medio simulado. En este apartado quedan encuadradas, entre otras, las habilidades técnicas y procedimentales. 4. La cúspide de la pirámide corresponde al «hacer» en la práctica real e incluye todos los aspectos anteriores más aquellos referidos a las actitudes, ética, toma de decisiones, y desarrollo profesional. Los dos últimos escalones se circunscriben al dominio del comportamiento. En ellos se pone de manifiesto la necesidad de hacer del estudio de la Medicina un aprendizaje activo en el que el estudiante vaya más allá de las clases magistrales y aplique de manera directa las competencias adquiridas y aún por adquirir. Un aprendizaje desarrollado en un nivel cognitivo más elevado que supone la interacción con el paciente y con cualquier miembro del equipo asistencial. Cada una de estas interacciones nutre al estudiante (y genera un feedback entre los miembros implicados) en un ámbito de aplicación diferente. Figura 1 Pirámide de Miller de la competencia clínica. Modificado de: [2] Miller, G.E. «The assessment of clinical skills/competence/performance». Academic Medicine (Suplemento) 1990; 65: S63-S67 En conclusión, teniendo en cuenta todas las anteriores consideraciones podemos acabar definiendo la competencia clínica como el grado con el que un médico utiliza los conocimientos, aptitudes, actitudes y buen juicio (recursos internos) asociados a la profesión médica, así como los del entorno: colegas, otros profesionales, documentación (recursos externos) para poder RIECS 2018, 3, 1 97 desempeñarse de manera eficaz en todas las situaciones clínicas que corresponden al campo de su práctica profesional. Ello implica la integración del saber, saber hacer y saber ser. Este análisis nos lleva directamente a la realidad de nuestras prácticas clínicas diarias. Y lo que se concluye es una falta de funcionalidad del propio estudiante: No cumple una función efectiva, no es práctico “tener” un estudiante en la consulta, en el despacho… En definitiva, en el ejercicio diario de la profesión médica. 3.3 La docencia práctica de la medicina: entorno del estudiante y del docente En el presente epígrafe procedemos a realizar un análisis comparativo de la situación en la que se encuentran los dos principales integrantes del tema que tratamos: los estudiantes y los docentes. 3.3.1 Entorno del estudiante universitario Actualmente, no están regladas las funciones, ni el “rol” del estudiante dentro del hospital. La situación ideal sería la redacción y aprobación de una Norma donde se refleje el compromiso ético que supone, la responsabilidad de la confidencialidad y las labores a desempeñar por el estudiante. Si existiese una modificación en el sistema que regulase ese aprendizaje activo, se concluirían unos “derechos y deberes” del estudiante para con sus prácticas hospitalarias. Y no solo el estudiante, sino también sus tutores y miembros que, con su actividad profesional diaria, participan de manera indirecta en dichas rotaciones clínicas. Es fundamental promover este modelo pedagógico a todos los niveles formativos. Gracias al aprendizaje activo, el estudiante de Medicina:  Pasa del concepto a la dimensión vital: la enfermedad no es un capítulo presente en los múltiples manuales de Medicina Interna, sino un estado y percepción de una persona que vive y sufre.  Se nutre del modus operandi del equipo asistencial, de la forma de expresarse de manera científica dentro del mismo y de aclarar y enfocar la información al paciente.  Se reafirma en su vocación como médico y se motiva ante la comprensión del conocimiento teórico.  Genera un debate con otros compañeros e incluso con docentes y tutores sobre aquellos aspectos que considera relevantes. Figura 2 Cono del aprendizaje de Edgar Dale: Modificado de: [3] Rey, Corsino. (2017). Proyecto docente e investigador de pediatría. 10.13140/RG.2.2.18976.38400 Actualmente, se ha demostrado que el estudiante es capaz de recordar un 10% de lo que lee y el 20% de lo que escucha, el 30% de lo que ve y el 50% de lo que ve y escucha; el 70% de lo que escribe y RIECS 2018, 3, 1 98 el 90% de lo que hace y enseña a otros (Ver figura 2). Con la aplicación del Plan Bolonia y, por tanto, la entrada en el Espacio Europeo de Educación Superior se ha dado un paso adelante en el cambio de la docencia universitaria pasando de un modelo tradicional centrado en el docente a uno en el que el estudiante hace del aprendizaje una experiencia individualizada. Nosotros, como representantes, estudiantes y autores del presente documento incentivamos a seguir construyendo esta visión en el Grado de Asistencia al Paciente y a plantearnos la resistencia del estudiante a formar parte de este aprendizaje activo abandonando su papel de Estudiante pasivo. 3.3.2 Entorno del docente. De la misma manera, es destacable el hecho de que muchos docentes realizan cursos de formación pedagógica, así como la labor realizada por parte de los Médicos Residentes Internos (MIR) del hospital, en relación a la impartición de una docencia no reconocida, ni compensada. Así mismo, realizamos a propósito de la presente, diversas entrevistas a varios facultativos en activo en los diferentes niveles de la asistencia sanitaria, esto es, varios especialistas en Medicina de Familia y Comunitaria, un Facultativo Adjunto Especialista en el Servicio de Urgencias, así como Médicos Internos Residentes en diferentes años de formación de las que resultaron las siguientes conclusiones:  La docencia es un ejercicio básico para cualquier médico, desde el plano de la Ética, como el de la formación científica y pedagógica.  Este ejercicio es entendido en la mayoría de los casos como una actividad voluntaria a la que se accede de manera altruista.  El estudiante es visto desde muchos matices: participación activa, aunque también pasiva.  En la mayoría de los casos, se recalca un feedback entre estudiantes y docentes.  No existe una regulación en relación a la docencia universitaria de los Médicos Internos Residentes, fuera de profesores asociados a la universidad.  Falta de tiempo para realizar una formación individualizada durante la asistencia. 3.4 La docencia práctica de la Medicina: planes de mejora e innovación Finalmente, después de haber realizado este recorrido desde el origen histórico de la docencia práctica de la Medicina, habiendo comprendido la necesidad del aprendizaje activo como modelo de docencia universitario y realizando un análisis de la situación que viven estudiantes y docentes, resumimos en este último apartado un conjunto de planes, ideas, proyectos de mejora y concienciación, en definitiva, que se llevan a cabo o se implantarán en el ámbito nacional. 3.4.1 Inmersión clínica precoz (ICP) [4] Este método pedagógico, creado por Fernando Caballero, es aplicado en universidades madrileñas como la Universidad Francisco de Vitoria y la Universidad Autónoma de Madrid, y consiste en el desarrollo de unas prácticas de rotación clínica en los primeros cursos de la carrera (esto es primero y segundo de grado), únicamente, en Atención Primaria, y en los servicios hospitalarios de Urgencias, Cuidados paliativos y Psiquiatría. La labor del estudiante es analizar, dentro del ámbito introspectivo de la rotación, la importancia del médico, cómo se han sentido ellos mismos, los aspectos éticos y emociones que resultan de la práctica clínica. De esta manera, se fomenta la atención al paciente desde el modelo biopsicosocial e integral de la persona. Además, se ha demostrado que con este tipo de prácticas clínicas se influye positivamente en la mejora de la sensibilidad ética y empatía del estudiante de manera progresiva y significativa conforme avanzan de curso. RIECS 2018, 3, 1 99 3.4.2 Programa Docente de Historia Clínica Electrónica. Este tipo de método formativo existe actualmente en Estados Unidos. Se basa en un programa informático similar al existente en España para la actualización de las historias clínicas de los pacientes (Historia Clínica Electrónica) donde cada estudiante puede acceder con una cuenta personal a las historias de su hospital anonimizando todo dato personal del paciente. De esta manera, los estudiantes realizan sus prácticas clínicas tal y como si fueran un miembro más dentro del servicio: pronunciando un diagnóstico, pautando un tratamiento, escribiendo un evolutivo, etc. Asimismo, el estudiante siente la responsabilidad de tomar decisiones y su médico-tutor es el encargado de revisarlas, corregirlas y evaluar el aprendizaje del mismo. Como ventajas, además, permite dar continuidad entre diferentes estudiantes de distintos cursos o diferentes periodos de rotación que realicen sus prácticas dentro de un mismo servicio. Todo esto queda exclusivamente registrado en el programa informático destinado a la docencia y no en el oficial ya existente para el personal médico del centro hospitalario. 3.4.3 Método de evaluación 3 60 Este tipo de evaluación trata de hacer más objetiva la calificación que reciben los estudiantes; es la misma que se emplea para la evaluación del programa de formación de los Médicos Internos Residentes. Consiste en la cumplimentación por parte de todo el personal que ha estado en contacto con el estudiante: equipo médico, enfermería, celadores, pacientes, familiares, etc. de manera que se concluya una valoración multidisciplinar y de los diferentes ámbitos de las competencias clínicas del estudiante. 3.4.4 Infectar de escepticemia [5-6] “La escepticemia (término acuñado en 1989 por Petr Skrabanek y James McCormick) es una enfermedad de baja contagiosidad contra la que se intenta vacunar a los estudiantes en las facultades de medicina, tal “enfermedad” se debe al pensamiento crítico, y genera oposición a las afirmaciones que carecen de fundamento empírico verificable y contrastado. Su vacuna conlleva una triple pérdida de capacidad: de crítica, de ética y de propuestas de mejora. Como fruto final de un proyecto iniciado a raíz del Seminario de Innovación en Atención Primaria (SIAP) celebrado en su fase presencial en Bilbao (País Vasco, España) en febrero de 2016, sobre “Pacientes que lloran y otras consultas sagradas”, nace la “Guía para infectar de escepticemia a estudiantes de Medicina”. Propuso esta iniciativa Paula Rodríguez Molino (estudiante de Medicina en la Universidad de Santiago de Compostela) y con la ayuda clave y constante de Borja de Apellániz Aparicio (estudiante de Medicina en la Universidad de Zaragoza) y la participación de múltiples clínicos, residentes y estudiantes participantes en el SIAP-Bilbao, en una primera fase de tormenta de ideas, se pudo llegar a la actual redacción, con apoyo de Juan Gérvas (coordinador de los SIAP). Se aceptan sugerencias, críticas y recomendaciones de mejora. Para contagiar de escepticemia a estudiantes de medicina se recogieron las siguientes propuestas: a) Propuestas para estudiantes de medicina:  Valorar críticamente lo que enseñan nuestros docentes pues nadie es infalible. Su interpretación de los hechos no es una verdad absoluta. Es importante identificar los sesgos.  Preguntar, participar, debatir con estudiantes y docentes. La ciencia y el aprendizaje avanzan con dudas y debates.  Profundizar, leer y ampliar conocimientos. Estudiar para uno mismo y los pacientes, no para un examen. “El peor de los libros suele ser mejor que el mejor de los apuntes” e incomparable con un documento PowerPoint. RIECS 2018, 3, 1  100 Buscar activamente formación. Averiguar qué iniciativas existen ya en el entorno del estudiante (dentro y fuera de la facultad). Organizar actividades para compartir este conocimiento con más estudiantes.  Abrazar la formación personal: las revistas, las instituciones, los blogs y las redes sociales tienen recursos generalmente más actualizados que lo dicho en las aulas.  “El médico que sólo Medicina sabe, ni Medicina sabe”. Aprender de otras culturas y valores con otras ciencias, profesiones, pacientes y activistas.  Organizarse, quejarse y proponer mejoras. Conocer los derechos y deberes dentro de la facultad. Participar activamente en los órganos de decisión, pues en la facultad y los docentes agradecen la crítica constructiva.  El médico que serás mañana lo define el estudiante que eres hoy. No escudarse en la falta de estímulos externos, ejerce la autocrítica. No caer en el cinismo, siempre se puede trabajar y mejorar “lo que hay”. Promover el cambio predicando con el ejemplo.  Ser proactivo, preguntar y mantenerse motivado. Tener los ojos abiertos ya que desde espacios como la consulta hasta la cafetería o la sala de espera constituyen núcleos de aprendizaje.  Aprovechar los recursos y personas disponibles. Valorar la experiencia los conocimientos del tutor, pues de toda oportunidad es posible extraer una lección. Los profesionales y estudiantes de otras ramas, así como pacientes y familiares, pueden aportar mucho si se pregunta con respeto y sin miedo. Apoyarse en los médicos residentes.  Comportarse con los pacientes y sus familiares como el profesional que se quiere ser, no caer en la rutina deshumanizada, aunque nos rodee. Presentarse siempre, dar la mano y mantener un lenguaje no verbal acogedor. Mostrar empatía, mirar a los ojos, pedir permiso antes de explorar. Ser respetuoso. Explorar los miedos e inseguridades de los pacientes, sin frivolizarlos. Tranquilizar y abrazar cuando sea necesario, acompañar la vulnerabilidad.  “No hay enfermedades, sino enfermos”. Conocer y atender a cada paciente en su situación personal.  b) Ser humilde, mantener la mente abierta a críticas y sugerencias. Propuestas para docentes de Medicina:  Presentarse siempre y explicar conflictos de interés y sesgos a estudiantes y pacientes.  Ayudar a formar estudiantes críticos y éticos a través del ejemplo. No temer compartir dudas propias, el ejercicio de la ética de la ignorancia es un aspecto clave en la relación médico-paciente y docente-estudiante.  Debatir en clase y en la clínica. Lo básico se puede aprender en casa, usar el tiempo presencial para destacar los aspectos más polémicos. Dedicar una parte de tu tiempo docente a presentar temas controvertidos, de actualidad o que el alumnado vea valioso discutir. Si el tiempo presencial se queda corto, proponer espacios virtuales.  Ofrecer lecturas complementarias de forma que quepan enfoques diferentes. Los estudiantes deben aprender a defender posturas razonadas y fundadas en la mejor ciencia.  Mover a los estudiantes a participar activamente. Al explicar, centrarse en los motivos y valores de porqué hacerlo. RIECS 2018, 3, 1  101 Investigar otros grupos e iniciativas externos a la facultad que puedan interesar a los estudiantes, participar en ellos y difundirlos.  Ayudar a garantizar que los estudiantes tengan voz independiente dentro de la facultad. Asegurar tiempo y medios para hablar de problemas sobre el método docente y el plan de estudios. Ser humilde y pedir opinión acerca de la docencia personal.  La evaluación es parte de la docencia. Admitir anulaciones e impugnaciones basadas en libros, documentos y artículos, en la presentación usada en clase. No exacerbar la competitividad, será contraproducente. El objetivo no es aprobar sino aprender. Crear evaluaciones que discriminen de manera adecuada y asegurar medidas para evitar trampas.  Enseñar a los estudiantes para que resistan la frustración ante los pacientes que no se curan, que son la mayoría, y para que acepten que el objetivo de la medicina es a veces curar, en muchos casos evitar y siempre aliviar y acompañar.  Contribuir a que los estudiantes se especialicen en Humanidad antes de graduarse en Medicina. Que aprendan a explorar y a respetar lo físico, lo psíquico y lo social; la cultura, las expectativas y los valores de los pacientes-familias-comunidades. Que sepan identificar y enfrentarse a los errores clínicos. Que sean científicos, humanos, empáticos y asertivos. Que aprendan las habilidades sociales necesarias para atender de manera digna a todo paciente”. Para concluir, reincidimos en la siguiente idea: “El estudiante que eres hoy definirá el médico que serás en el futuro”. 3.4.5 El alumno interno [7]. Se entiende por “Alumno Interno” aquel estudiante que decide participar de su formación de una forma más comprometida mediante la inclusión en el cuerpo de trabajo de un Departamento de la Facultad en la que se encuentra cursando sus estudios de Medicina. Esto supone la puesta en contacto con incontables oportunidades de formación en el ámbito de la Investigación, Docencia, así como la Asistencia Sanitaria resultando una formación profesional más avanzada en relación a la del estudiante universitario estándar. El Consejo Estatal de Estudiantes de Medicina (CEEM) realizó en el año 2011 un estudio para evaluar diferentes ítems en relación a la figura del Alumno Interno. Las conclusiones que se obtienen de dicho estudio ponen de manifiesto que tal programa formativo no se encuentra disponible en todas las universidades españolas (de una muestra de 17 universidades tan solo 9 pudieron participar definitivamente en el estudio puesto que el resto carecían de tal plan); así mismo se concluyó que en aquellas facultades donde sí existe tal vacante no se cubre de manera óptima la difusión de lo que supone este modelo de formación universitaria. La duda que se plantea ante este modelo formativo es la posibilidad de compatibilización entre la formación universitaria estándar en Medicina con dicho proceso. Los datos señalan que un 49% de los encuestados afirma ser un proceso totalmente compatible, un 13% escasamente compatible y en un 13% restante la compatibilidad quedaba relegada a acuerdos entre los departamentos y el propio estudiante (en el 25% restante no se obtuvieron datos). Por tanto, se puede concluir una valoración positiva en rentabilidad, en cuanto a recurso “tiempo” por parte del estudiantado. Respecto al modo de acceso a tal programa, se registra una heterogeneidad en los mismos: convocatoria de concurso-oposición por cada Departamento (evaluación del solicitante mediante examen de desarrollo de temas propuestos, examen oral o escrita con lectura ante Tribunal), por orden de solicitud de la plaza desde inicio convocatoria de las mismas, mediante acuerdo entre el Departamento y el estudiante. En ocasiones es necesario haber superado un porcentaje de créditos relativos a las asignaturas de formación básica. En relación al compromiso que adquiere el Alumno Interno para con su Departamento, hemos encontrado varios estatutos (en general no se dispone de una regulación establecida de tales RIECS 2018, 3, 1 102 programas) de universidades españolas (Universidad de Granada y Universidad de Navarra) donde quedan registrados los derechos y deberes del estudiante como tal miembro departamental. En tales documentos se afirma la obligada compatibilización entre las actividades formativas obligatorias y éstas “voluntarias”, la posibilidad de certificación de las horas y proyectos realizados por el estudiante dentro del Departamento, valorarle en la coautoría de las investigaciones que se llevan a cabo dentro del equipo, así como las competencias básicas a adquirir por el mismo durante su formación. La existencia de un proyecto formativo como este supone nuevamente la mejora en la adquisición de los conceptos teórico-prácticos de la Medicina, el cultivo del espíritu y labor de investigación, así como la del profesional docente. Todo ello enmarcado en un contexto de formación individualizada y personal, donde las relaciones entre profesores y estudiantes se concluyen como satisfactorias. 3.4.6 Proyecto Wikicencia Se trata de un proyecto de innovación docente basado en el último escalón de la pirámide del aprendizaje de Edgar Dale: recordamos el 90% de lo que enseñamos a otros. De esta forma, estudiantes de cursos superiores realizan talleres prácticos en relación con las habilidades clínicas que serán evaluadas en el examen ECOE y de otras que no se adquieren en el Grado en cuestión, siendo aplicable a todos los Grados de Ciencias de la Salud. Un médico general puede conocer la realización de un ejercicio rehabilitador, la toma de constantes vitales… de la misma forma que un estudiante de enfermería puede reconocer soplos cardíacos e interpretar una analítica. Se trata de realizar una docencia en Wiki, entre todos, donde formamos parte del Grado en Atención al paciente. Dicho proyecto fue presentado por su autor (Alejandro Villares López) el año pasado en la Universidad de Alcalá y se consiguió la realización de talleres de Historia clínica, Auscultación Cardio-Respiratoria y Diccionario Médico, teniendo una gran acogida entre los estudiantes. 3.4.7 Residente 0: proyecto de aprendizaje clínico colaborativo y centrado en el estudiante en las prácticas clínicas del grado en Medicina Este proyecto de innovación docente fue presentado en 2017 en la Universidad de Alcalá por su autora, Dolores Ruiz Berdún, estando en el momento actual en aplicación al grado. Los objetivos de dicho proyecto son el de integrar al estudiante de tercer curso en el concepto de las prácticas clínicas, ahondar en el modelo docente de residente 0 para los estudiantes del sexto curso, en relación al ámbito docente, con perspectivas de aplicación a su próxima residencia y el de “optimizar el tiempo y los aprendizajes en las prácticas clínicas y mejorar la satisfacción del alumnado con estas”. Así mismo, se incorporan en este proyecto de mejora, una serie de guías de acogida, elaboradas por la propia Delegación de Estudiantes de Medicina UAH, a las prácticas clínicas, cuyo contenido ofrece información acerca de los servicios, horarios, aspectos éticos de las prácticas, etc. 4. Conclusiones Las principales ideas que se pueden extraer del presente texto vienen a resaltar la dualidad en la situación del estudiante de Medicina como ficus por decisión propia o como resultado de los múltiples factores externos a él mismo que hemos procedido a analizar y describir. Así como la necesidad de realizar un documento oficial que sirva de estatuto en relación a los derechos y deberes de los estudiantes de Medicina y la realización de las prácticas clínicas. La importancia de definir las competencias para que, una vez definidas, desde las Facultades de Medicina se trabaje en un proceso de reforma curricular que conduzca a la elaboración del plan de estudios basado en las competencias definidas. Se debe elaborar una estrategia que contemple el proceso participativo mediante el cual los agentes implicados, desarrollen e implementen un modelo curricular y unas metodologías de aprendizaje que permitan alcanzar dichas competencias. El modelo curricular debe permitir el contacto precoz del estudiante con la realidad médica y favorecer el trabajo en equipo interdisciplinar [8-14]. Asimismo, la búsqueda de nuevos planes de mejora e innovación tomando RIECS 2018, 3, 1 103 como base el aprendizaje activo y realizando una reinterpretación y actualización del mismo; infectar de escepticemia al colectivo universitario para lograr una mayor implicación y calidad de las prácticas clínicas. Nos gustaría terminar citando a Confucio: “Dime algo, y lo olvidaré. Enséñame algo, y lo recordaré. Hazme partícipe de algo, y lo aprenderé” y reinterpretando sus palabras en relación al presente documento: Vive la Medicina y la enseñaré. Agradecimientos: En primer lugar, agradecemos al Decanato de la Facultad de Medicina y Ciencias de la Salud la posibilidad de hacer posible la comunicación de este tema de actualidad en el ámbito universitario durante el pasado III Congreso Nacional de Bioética para Estudiantes de Medicina, así como al Comité Organizador del mismo. En segundo lugar, agradecemos al Equipo Editorial de la presente revista electrónica por haberse puesto en contacto con nosotros y dar continuidad a nuestro trabajo de investigación. Así mismo, queremos agradecer a la doctora María Dolores Ruiz Berdún, cuya ayuda fue indispensable para poder abordar el origen histórico de las prácticas clínicas de la Medicina. Finalmente, no menos importantes, al equipo humano componente de la Delegación de Estudiantes de Medicina UAH, cuya colaboración fue fundamental en el apoyo institucional durante el citado congreso, y para la presentación y defensa de continuos proyectos de mejora e innovación en la docencia en Medicina; A: Celia Estrada Costas, Paula Carrasco Pintor, Rosa López Martínez, Alba Aguilar Monge, Fernando Rodado Aranguren, María García García, Samuel Díaz Planellas, Amalio Fernández Leal, Mª Teresa Alcolea Sáez, Paula Velasco Alcalde, David Cabañas Moreno, Alejandro de Jesús y Alejandro Iñarra Navarro por colaborar en la grabación de los vídeos que proyectamos en la ponencia. Finalmente, agradecemos a Isabel Otero Barderas por su colaboración en la realización del abstract. Contribución de los autores: Paula Rubio García y Alejandro Villares López han concebido y diseñado el estudio y presentación de la comunicación; Paula Rubio García y Alejandro Villares López realizaron las entrevistas citadas; Paula Rubio García y Alejandro Villares López escribieron el artículo. Conflictos de Intereses: Los autores no declaran conflicto de intereses. Los patrocinadores fundadores no tenían ningún papel en el diseño del estudio; en la colección, análisis o interpretación de los datos; en la escritura del manuscrito y en la decisión de publicar los resultados. Abreviaturas Las siguientes abreviaturas son usadas en este manuscrito: MIR: Médico Interno Residente. UAH: Universidad de Alcalá de Henares. CEEM: Consejo Estatal de Estudiantes de Medicina. JEEM: Jornadas Estatales de Estudiantes de Medicina. ICP: Inmersión Clínica Precoz. SIAP: Seminario de Innovación en Atención Primaria. Referencias Bibliográficas 1. 2. 3. Posicionamiento en Docencia. Libro de Posicionamientos. CEEM (Consejo Estudiantes de Medicina), abril 2013. Miller, G.E. «The assessment of clinical skills/competence/performance». Academic Medicine (Suplemento) 1990; 65: S63-S67. Rey, Corsino. (2017). PROYECTO DOCENTE E INVESTIGADOR DE PEDIATRÍA. 10.13140/RG.2.2.18976.38400. Disponible en: https://www.researchgate.net/publication/311807763_PROYECTO_DOCENTE_E_INVESTIGADOR_DE_ PEDIATRIA RIECS 2018, 3, 1 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 104 D. Monge Martín, R. Abengózar Muela, C. García de Leonardo, F. Caballero Martínez. “Evaluación de un programa de inmersión clínica precoz en un grado de medicina mediante el análisis del impacto en la sensibilidad ética del alumno. Páginas 337, nº 81 Cuaderno de Bioética 2013. https://secuelasdeescepticemia.wordpress.com Guía para infectar de escepticemia a estudiantes de medicina, SIAP- Bilbao, 2016 Alumno interno, Comisión Educación Médica, CEEM (Consejo Estudiantes de medicina), enero 2011 Guía para la evaluación de la práctica clínica en las Facultades de Medicina. Promovido bajo las direcciones de la Cátedra de Educación Médica Fundación Lilly-UCM, por la Sociedad Española de Educación Médica (SEDEM) y la Conferencia Nacional de Decanos de Facultades de Medicina (CNDFM) Recomendaciones para un nuevo proceso de reforma curricular en las facultades de Medicina Española. Documento realizado Sociedad Española de Educación Médica (SEDEM), la Associació Catalana d’Educació Mèdica (ACEM), la Sociedad de Educación Médica de Euskadi (SEMDE) y la Sociedad Aragonesa de Educación Médica (SADEM) y aprobado en la reunión de la Junta Directiva de la SEDEM, celebrada en Madrid el día 22 de noviembre de 2004. The assessment of global minimum essential requirements in medical education. David T. Stern a; Andrzej Wojtczak b; M. Roy Schwarz ca Departments of Internal Medicine and Medical Education, University of Michigan, Ann Arbor, MI, USA b Institute for International Medical Education, White Plains, NY, USA c China Medical Board of New York, New York, USA European Core Curriculum. EMSA-IFMSA. 5th International Follow-up conference on the Bologna Process in Medical Education. July, 2006, Bristol (UK) La Educación Médica y el Espacio Europeo Superior. Comisión de Educación Médica, CEEM, Enero 2011. Libro Blanco. Título de Grado en Medicina. Por la Agencia Nacional de Evaluación de la Calidad y Acreditación (ANECA) La enseñanza de la medicina en la Universidad Española. José Danón. Fundación Uriach 1838, Colección Ilustre de Ciencias de la Salud. https://www.youtube.com/watch?v=B87_pdTkkF4&t=3s © 2018 por los autores; Esta obra está sujeta a la licencia de Reconocimiento 4.0 Internacional de Creative Commons. Para ver una copia de esta licencia, visite http://creativecommons.org/licenses/by-nc-nd/4.0/.
https://openalex.org/W2269873530	https://europepmc.org/articles/pmc2968895?pdf=render	English	null	Poly[[[diaquacobalt(II)]-bis[<i>μ<sub>2</sub></i>-1,1′-(butane-1,4-diyl)diimidazole-<i>κ<sup>2</sup></i><i>N</i><sup>3</sup>:<i>N</i><sup>3′</sup>]] dichloride tetrahydrate]	Acta crystallographica. Section E	2,009	cc-by	3,324	metal-organic compounds Experimental Crystal data [Co(C10H14N4)2(H2O)2]Cl24H2O Mr = 618.43 Triclinic, P1 a = 7.969 (6) A˚ b = 9.979 (6) A˚ c = 10.259 (7) A˚ = 114.97 (2) = 90.83 (3) = 93.70 (3) V = 737.3 (8) A˚ 3 Z = 1 Mo K radiation = 0.81 mm1 T = 291 K 0.44 0.37 0.22 mm Data collection Rigaku R-AXIS RAPID diffractometer Absorption correction: multi-scan (ABSCOR; Higashi, 1995) Tmin = 0.718, Tmax = 0.842 7288 measured reﬂections 3348 independent reﬂections 3018 reﬂections with I > 2(I) Rint = 0.017 Reﬁnement R[F 2 > 2(F 2)] = 0.028 wR(F 2) = 0.084 S = 1.14 3348 reﬂections 169 parameters H-atom parameters constrained max = 0.33 e A˚ 3 min = 0.23 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Table 2 Hydrogen-bond geometry (A, ). D—H A D—H H A D A D—H A O1—H15 O3ii 0.85 1.94 2.781 (2) 169 O1—H16 Cl1 0.85 2.35 3.1728 (19) 165 O2—H17 Cl1ii 0.85 2.32 3.172 (2) 176 O2—H18 Cl1iii 0.85 2.44 3.292 (3) 175 O3—H19 O2 0.85 1.99 2.829 (3) 171 O3—H20 Cl1 0.85 2.41 3.261 (3) 174 Symmetry codes: (ii) x þ 1; y þ 1; z þ 1; (iii) x 1; y; z. metal-organic compounds metal-organic compounds Table 1 In the title compound, {[Co(C10H14N4)2(H2O)2]Cl24H2O}n, the CoII atom and the mid-point of the 1,10-butane-1,4- diyldiimidazole ligands lie on inversion centers. The CoII atom is six-coordinated in a slightly distorted octahedral environ- ment by four N atoms from four different ligands and by two O atoms from the water molecules. The CoII atoms are bridged by the ligands into a (4,4) net. Adjacent nets are linked to the chloride anions and uncoordinated water molecules via O— H Cl and O—H O hydrogen bonds, generating a three- dimensional supramolecular structure. Table 2 Hydrogen-bond geometry (A˚ , ). Yu Su, Yan-Jun Hou, Zhi-Zhong Sun, Guang-Feng Hou and Jin-Sheng Gao* College of Chemistry and Materials Science, Heilongjiang University, Harbin 150080, People’s Republic of China Correspondence e-mail: hgf1000@163.com Received 26 February 2009; accepted 1 March 2009 Received 26 February 2009; accepted 1 March 2009 Key indicators: single-crystal X-ray study; T = 291 K; mean (C–C) = 0.003 A˚; R factor = 0.028; wR factor = 0.084; data-to-parameter ratio = 19.8. Table 1 Selected geometric parameters (A˚ , ). Co1—N1 2.1265 (18) Co1—N3 2.1355 (18) Co1—O1 2.1819 (17) Symmetry code: (i) x þ 1; y; z þ 1. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: NG2551). Related literature For the synthesis of 1,10-butane-1,4-diyldiimidazole, see: Ma et al.(2003); Yu et al. (2008). For a related Co complex, see: Dong & Zhang (2006). Data collection: RAPID-AUTO (Rigaku, 1998); cell reﬁnement: RAPID-AUTO; data reduction: CrystalStructure (Rigaku/MSC, 2002); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97. The authors acknowledge ﬁnancial support from the National Natural Science Foundation of China (grant Nos. 20872030), the Research Foundation of Heilongjiang Provin- cial Education Department (grant Nos. 11513073), the Project of the Special Fund of the Science and Technology Innovation People of Harbin (grant Nos. RC2006QN018001) and Heilongjiang University. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: NG2551). m370 Su et al. Acta Cryst. (2009). E65, m370–m371 doi:10.1107/S1600536809007478 Rigaku/MSC (2002). CrystalStructure. Rigaku/MSC Inc., The Woodlands, Texas, USA. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Yu, Y.-H., Shi, A.-E., Su, Y., Hou, G.-F. & Gao, J.-S. (2008). Acta Cryst. E64, m628. Acta Cryst. (2009). E65, m370–m371 Su et al. [Co(C10H14N4)2(H2O)2]Cl24H2O m371 References Dong, G.-C. & Zhang, R.-C. (2006). Acta Cryst. E62, m1847–m1849. Higashi, T. (1995). ABSCOR. Rigaku Corporation, Tokyo, Japan. Ma, J.-F., Yang, J., Zheng, G.-L. & Liu, J.-F. (2003). Inorg. Chem. 42, 7531–7534. Rigaku (1998). RAPID-AUTO. Rigaku Corporation, Tokyo, Japan. References Rigaku/MSC (2002). CrystalStructure. Rigaku/MSC Inc., The Woodlands, Texas, USA. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Yu, Y.-H., Shi, A.-E., Su, Y., Hou, G.-F. & Gao, J.-S. (2008). Acta Cryst. E64, m628. Su et al. [Co(C10H14N4)2(H2O)2]Cl24H2O m371 Acta Cryst. (2009). E65, m370–m371 Experimental L was prepared from imidazole and 1,4-dibromobutane in DMSO (Ma et al., 2003). L (0.76 g, 4 mmol) and cobalt dichloride (0.51 g, 4 mmol) were dissolved in hot aqua solution (10 ml) to give a clear solution. The resulting solution was allowed to stand in a desiccator at room temperature for a week, red crystals of (I) were obtained. Comment The L molecules as a flexible ligand exhibit a variety of supramolecular aggregation patterns (Ma et al., 2003; Dong et al., 2006; Yu et al., 2008). In this paper, we report the new title compound, (I), synthssized by the reaction of L molecules and cobalt dichloride in aqua solution. In (I), each CoII atom is located on a inversion centre and is six-coordinated in an octahedral environment by four N atoms from four different L molecules and two O atoms form the two water molecules (Fig. 1). The Co—N and Co—O distances are normal (Table 1). The CoII atoms are bridged by ligands, generating a two-dimensional (4,4)-network (Fig. 2). The hydrogen bonding cluster, containing the O—H···Cl and O—H···O hydrogen bonding interaction between the chlor- ide anions, uncoordinated water molecules and the coordinated water molecules (Fig. 3), which linke the adjacent fishnet planes to a three-dimensional supramolecular structure (Fig. 4, Table 2). Poly[[[diaquacobalt(II)]-bis[ 2-1,1'-(butane-1,4-diyl)diimidazole- 2N3:N3']] dichloride tetrahy- drate] Y. Su, Y.-J. Hou, Z.-Z. Sun, G.-F. Hou and J.-S. Gao Y. Su, Y.-J. Hou, Z.-Z. Sun, G.-F. Hou and J.-S. Gao supplementary materials supplementary materials Acta Cryst. (2009). E65, m370-m371 [ doi:10.1107/S1600536809007478 ] Acta Cryst. (2009). E65, m370-m371 [ doi:10.1107/S1600536809007478 ] Refinement H atoms bound to C atoms were placed in calculated positions and treated as riding on their parent atoms, with C—H = 0.93 Å (aromatic), C—H = 0.97 Å (methylene), and with Uiso(H) = 1.2Ueq(C). Water H atoms were initially located in a difference Fourier map, but they were treated as riding on their parent atoms with O—H = 0.85 Å and with with Uiso(H) = 1.5Ueq(O). Figures Fig. 1. The molecular structure of (I), showing displacement ellipsoids at the 30% probability level for non-H atoms. Dashed lines indicate the hydrogen-bonding interactions [Symmetry code; (I) -x + 1, -y + 1, -z + 1; (II) -x + 2, -y, -z + 2: (III) -x, -y + 1, -z + 2] Figures Poly[[[diaquacobalt(II)]-bis[µ2-1,1'-(butane-1,4-diyl)diimidazole- κ2N3:N3']] dichloride tetrahydrate] Crystal data [Co(C10H14N4)2(H2O)2]Cl2·4H2O Z = 1 Mr = 618.43 F000 = 325 Triclinic, P1 Dx = 1.393 Mg m−3 Hall symbol: -P 1 Mo Kα radiation λ = 0.71073 Å a = 7.969 (6) Å Cell parameters from 6505 reflections b = 9.979 (6) Å θ = 3.3–27.5º c = 10.259 (7) Å µ = 0.81 mm−1 α = 114.97 (2)º T = 291 K β = 90.83 (3)º Block, red γ = 93.70 (3)º 0.44 × 0.37 × 0.22 mm V 737 3 (8) Å3 Crystal data [Co(C10H14N4)2(H2O)2]Cl2·4H2O Z = 1 Mr = 618.43 F000 = 325 Triclinic, P1 Dx = 1.393 Mg m−3 Hall symbol: -P 1 Mo Kα radiation λ = 0.71073 Å a = 7.969 (6) Å Cell parameters from 6505 reflections b = 9.979 (6) Å θ = 3.3–27.5º c = 10.259 (7) Å µ = 0.81 mm−1 α = 114.97 (2)º T = 291 K β = 90.83 (3)º Block, red γ = 93.70 (3)º 0.44 × 0.37 × 0.22 mm V = 737.3 (8) Å3 Data collection Rigaku R-AXIS RAPID diffractometer 3348 independent reflections Radiation source: fine-focus sealed tube 3018 reflections with I > 2σ(I) Monochromator: graphite Rint = 0.017 T = 291 K θmax = 27.5º ω scan θmin = 3.3º Absorption correction: Multi-scan (ABSCOR; Higashi, 1995) h = −10→10 Tmin = 0.718, Tmax = 0.842 k = −12→12 7288 measured reflections l = −13→13 Radiation source: fine-focus sealed tube 7288 measured reflections sup-2 supplementary materials Refinement Refinement on F2 Secondary atom site location: difference Fourier map Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites R[F2 > 2σ(F2)] = 0.028 H-atom parameters constrained wR(F2) = 0.084 w = 1/[σ2(Fo 2) + (0.0444P)2 + 0.1566P] where P = (Fo 2 + 2Fc 2)/3 S = 1.14 (Δ/σ)max < 0.001 3348 reflections Δρmax = 0.33 e Å−3 169 parameters Δρmin = −0.22 e Å−3 Primary atom site location: structure-invariant direct methods Extinction correction: none Refinement Refinement on F2 Secondary atom site location: difference Four Least-squares matrix: full Hydrogen site location: inferred from neighbo sites R[F2 > 2σ(F2)] = 0.028 H-atom parameters constrained wR(F2) = 0.084 w = 1/[σ2(Fo 2) + (0.0444P)2 + 0.1566P] where P = (Fo 2 + 2Fc 2)/3 S = 1.14 (Δ/σ)max < 0.001 3348 reflections Δρmax = 0.33 e Å−3 169 parameters Δρmin = −0.22 e Å−3 Primary atom site location: structure-invariant direct methods Extinction correction: none Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.028 wR(F2) = 0.084 S = 1.14 3348 reflections 169 parameters Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained H-atom parameters constrained w = 1/[σ2(Fo 2) + (0.0444P)2 + 0.1566P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.33 e Å−3 Δρmin = −0.22 e Å−3 w = 1/[σ2(Fo 2) + (0.0444P)2 + 0.1566P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.33 e Å−3 Δρmin = −0.22 e Å−3 w = 1/[σ2(Fo 2) + (0.0444P)2 + 0.1566P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.33 e Å−3 Δρmin = −0.22 e Å−3 Primary atom site location: structure-invariant direct methods Extinction correction: none Figures Fig. 1. The molecular structure of (I), showing displacement ellipsoids at the 30% probability level for non-H atoms. Dashed lines indicate the hydrogen-bonding interactions [Symmetry code; (I) -x + 1, -y + 1, -z + 1; (II) -x + 2, -y, -z + 2: (III) -x, -y + 1, -z + 2] sup-1 supplementary materials Fig. 2. A partial packing view, showing the two-dimensional (4,4)-network. C-bond H atoms have beeb omitted. Fig. 3. A showing of the hydrogen bonding cluster in I. Fig. 4. A Partial packing view, shoving the three-dimensional supramolecular structure. Dashed lines indicate the hydrogen-bonding interactions and no involving H atoms have beeb omitted. Fig. 2. A partial packing view, showing the two-dimensional (4,4)-network. C-bond H atoms have beeb omitted. Fig. 4. A Partial packing view, shoving the three-dimensional supramolecular structure. Dashed lines indicate the hydrogen-bonding interactions and no involving H atoms have beeb omitted. supplementary materials supp e e ta y ate a s sup-4 C9 0.0058 (2) 0.3775 (2) 0.7958 (2) 0.0406 (4) H11 −0.1151 0.3629 0.7760 0.049* H12 0.0495 0.4529 0.7663 0.049* C10 0.0423 (3) 0.43001 (19) 0.9552 (2) 0.0438 (4) H13 0.1630 0.4487 0.9751 0.053* H14 0.0038 0.3522 0.9832 0.053* Cl1 0.74821 (7) 0.35621 (5) 0.32791 (5) 0.04818 (14) Co1 0.5000 0.0000 0.5000 0.02144 (9) N1 0.58238 (16) 0.03688 (14) 0.71136 (13) 0.0274 (3) N2 0.68668 (16) −0.00004 (15) 0.89242 (14) 0.0299 (3) N3 0.26947 (15) 0.08534 (14) 0.59164 (13) 0.0265 (3) N4 0.08195 (17) 0.23842 (15) 0.71246 (15) 0.0312 (3) O1 0.59361 (16) 0.22377 (12) 0.53595 (13) 0.0381 (3) H15 0.5827 0.3049 0.6089 0.057* H16 0.6273 0.2429 0.4670 0.057* O2 0.1615 (2) 0.38041 (17) 0.36469 (18) 0.0668 (5) H17 0.1812 0.4490 0.4489 0.100* H18 0.0554 0.3700 0.3490 0.100* O3 0.4231 (2) 0.49280 (17) 0.24618 (19) 0.0670 (5) H19 0.3378 0.4575 0.2734 0.100* H20 0.5121 0.4639 0.2693 0.100* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 C1 0.0329 (8) 0.0426 (9) 0.0241 (7) 0.0052 (7) 0.0005 (6) 0.0074 (7) C2 0.0328 (8) 0.0345 (8) 0.0280 (7) 0.0091 (6) 0.0016 (6) 0.0086 (7) C3 0.0268 (7) 0.0319 (8) 0.0256 (7) 0.0010 (6) −0.0018 (6) 0.0120 (6) C4 0.0357 (9) 0.0503 (10) 0.0395 (9) −0.0060 (7) −0.0089 (7) 0.0314 (8) C5 0.0354 (9) 0.0321 (8) 0.0369 (8) 0.0016 (6) −0.0083 (7) 0.0188 (7) C6 0.0291 (8) 0.0340 (8) 0.0418 (9) 0.0078 (6) 0.0106 (7) 0.0205 (7) C7 0.0335 (8) 0.0283 (8) 0.0337 (8) 0.0033 (6) 0.0067 (6) 0.0110 (7) C8 0.0306 (8) 0.0358 (8) 0.0378 (8) 0.0041 (6) 0.0110 (7) 0.0161 (7) C9 0.0412 (10) 0.0369 (9) 0.0508 (10) 0.0190 (7) 0.0179 (8) 0.0228 (8) C10 0.0513 (11) 0.0324 (9) 0.0497 (11) 0.0197 (8) 0.0149 (9) 0.0166 (8) Cl1 0.0565 (3) 0.0506 (3) 0.0353 (2) −0.0058 (2) 0.0008 (2) 0.0175 (2) Co1 0.02117 (15) 0.02402 (15) 0.01902 (14) 0.00459 (10) 0.00162 (10) 0.00862 (11) N1 0.0255 (6) 0.0329 (7) 0.0226 (6) 0.0051 (5) 0.0004 (5) 0.0104 (5) N2 0.0247 (6) 0.0420 (7) 0.0260 (6) 0.0001 (5) −0.0020 (5) 0.0179 (6) N3 0.0235 (6) 0.0316 (6) 0.0256 (6) 0.0061 (5) 0.0038 (5) 0.0127 (5) N4 0.0290 (7) 0.0328 (7) 0.0353 (7) 0.0105 (5) 0.0103 (5) 0.0166 (6) O1 0.0505 (8) 0.0261 (6) 0.0355 (6) 0.0007 (5) 0.0115 (5) 0.0110 (5) O2 0.0597 (10) 0.0521 (9) 0.0657 (10) 0.0088 (7) −0.0033 (8) 0.0025 (8) O3 0.0763 (12) 0.0472 (9) 0.0683 (10) 0.0111 (8) 0.0051 (9) 0.0147 (8) Geometric parameters (Å, °) C1—C2 1.354 (3) C8—N4 1.363 (2) C1—N2 1.367 (2) C8—H10 0.9300 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq C1 0.6521 (2) 0.1455 (2) 0.94674 (17) 0.0354 (4) H1 0.6695 0.2157 1.0416 0.043 C2 0.5873 (2) 0.16729 (19) 0.83506 (17) 0.0330 (3) H2 0.5516 0.2565 0.8409 0.040* C3 0.64306 (19) −0.06103 (17) 0.75067 (16) 0.0283 (3) H3 0.6543 −0.1596 0.6885 0.034* C4 0.7646 (2) −0.0765 (2) 0.9698 (2) 0.0382 (4) H4 0.7167 −0.1783 0.9312 0.046* H5 0.7386 −0.0288 1.0705 0.046* C5 0.9543 (2) −0.07561 (17) 0.95821 (18) 0.0335 (3) H6 0.9958 −0.1443 0.9931 0.040* H7 0.9803 −0.1107 0.8576 0.040* C6 0.2259 (2) 0.22255 (18) 0.64249 (18) 0.0331 (3) H8 0.2873 0.2993 0.6314 0.040* C7 0.1451 (2) 0.00888 (18) 0.63070 (18) 0.0325 (3) H9 0.1409 −0.0916 0.6093 0.039* C8 0.0303 (2) 0.10224 (18) 0.70471 (18) 0.0344 (4) H10 −0.0658 0.0784 0.7432 0.041* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 supplementary materials Atomic displacement parameters (Å2) sup-4 supplementary materials C1—H1 C2—N1 C2—H2 C3—N1 C3—N2 C3—H3 C4—N2 C4—C5 C4—H4 C4—H5 C5—C5i C5—H6 C5—H7 C6—N3 C6—N4 C6—H8 C7—C8 C7—N3 C7—H9 C2—C1—N2 C2—C1—H1 N2—C1—H1 C1—C2—N1 C1—C2—H2 N1—C2—H2 N1—C3—N2 N1—C3—H3 N2—C3—H3 N2—C4—C5 N2—C4—H4 C5—C4—H4 N2—C4—H5 C5—C4—H5 H4—C4—H5 C5i—C5—C4 C5i—C5—H6 C4—C5—H6 C5i—C5—H7 C4—C5—H7 H6—C5—H7 N3—C6—N4 N3—C6—H8 N4—C6—H8 C8—C7—N3 C8—C7—H9 0.9300 C9—N4 1.380 (2) C9—C10 0.9300 C9—H11 1.319 (2) C9—H12 1.348 (2) C10—C10ii 0.9300 C10—H13 1.468 (2) C10—H14 1.518 (3) Co1—N1iii 0.9700 Co1—N1 0.9700 Co1—N3 1.513 (3) Co1—N3iii 0.9700 Co1—O1 0.9700 Co1—O1iii 1.316 (2) O1—H15 1.345 (2) O1—H16 0.9300 O2—H17 1.347 (2) O2—H18 1.378 (2) O3—H19 0.9300 O3—H20 106.40 (14) C9—C10—H13 126.8 C10ii—C10—H13 126.8 C9—C10—H14 109.55 (16) C10ii—C10—H14 125.2 H13—C10—H14 125.2 N1iii—Co1—N1 111.34 (14) N1iii—Co1—N3 124.3 N1—Co1—N3 124.3 N1iii—Co1—N3iii 112.55 (14) N1—Co1—N3iii 109.1 N3—Co1—N3iii 109.1 N1iii—Co1—O1 109.1 N1—Co1—O1 109.1 N3—Co1—O1 107.8 N3iii—Co1—O1 113.60 (19) N1iii—Co1—O1iii 108.8 N1—Co1—O1iii 108.8 N3—Co1—O1iii 108.8 N3iii—Co1—O1iii 108.8 O1—Co1—O1iii 107.7 C3—N1—C2 111.80 (15) C3—N1—Co1 124.1 C2—N1—Co1 124.1 C3—N2—C1 109.45 (15) C3—N2—C4 125.3 C1—N2—C4 0.9300 C9—N4 1 1.380 (2) C9—C10 1 0.9300 C9—H11 0 1.319 (2) C9—H12 0 1.348 (2) C10—C10ii 1 0.9300 C10—H13 0 1.468 (2) C10—H14 0 1.518 (3) Co1—N1iii 2 0.9700 Co1—N1 2 0.9700 Co1—N3 2 1.513 (3) Co1—N3iii 2 0.9700 Co1—O1 2 0.9700 Co1—O1iii 2 1.316 (2) O1—H15 0 1.345 (2) O1—H16 0 0.9300 O2—H17 0 1.347 (2) O2—H18 0 1.378 (2) O3—H19 0 0.9300 O3—H20 0 106.40 (14) C9—C10—H13 1 126.8 C10ii—C10—H13 1 126.8 C9—C10—H14 1 109.55 (16) C10ii—C10—H14 1 125.2 H13—C10—H14 1 125.2 N1iii—Co1—N1 1 111.34 (14) N1iii—Co1—N3 9 124.3 N1—Co1—N3 8 124.3 N1iii—Co1—N3iii 8 112.55 (14) N1—Co1—N3iii 9 109.1 N3—Co1—N3iii 1 109.1 N1iii—Co1—O1 8 109.1 N1—Co1—O1 9 109.1 N3—Co1—O1 8 107.8 N3iii—Co1—O1 9 113.60 (19) N1iii—Co1—O1iii 9 108.8 N1—Co1—O1iii 8 108.8 N3—Co1—O1iii 9 108.8 N3iii—Co1—O1iii 8 108.8 O1—Co1—O1iii 1 107.7 C3—N1—C2 1 111.80 (15) C3—N1—Co1 1 124.1 C2—N1—Co1 1 124.1 C3—N2—C1 1 109.45 (15) C3—N2—C4 1 125.3 C1—N2—C4 1 0.9300 C9—N4 1.466 (2) 1.380 (2) C9—C10 1.508 (3) 0.9300 C9—H11 0.9700 1.319 (2) C9—H12 0.9700 1.348 (2) C10—C10ii 1.518 (3) 0.9300 C10—H13 0.9700 1.468 (2) C10—H14 0.9700 1.518 (3) Co1—N1iii 2.1265 (18) 0.9700 Co1—N1 2.1265 (18) 0.9700 Co1—N3 2.1355 (18) 1.513 (3) Co1—N3iii 2.1355 (18) 0.9700 Co1—O1 2.1819 (17) 0.9700 Co1—O1iii 2.1819 (17) 1.316 (2) O1—H15 0.8500 1.345 (2) O1—H16 0.8501 0.9300 O2—H17 0.8501 1.347 (2) O2—H18 0.8499 1.378 (2) O3—H19 0.8500 0.9300 O3—H20 0.8501 106.40 (14) C9—C10—H13 109.1 126.8 C10ii—C10—H13 109.1 126.8 C9—C10—H14 109.1 109.55 (16) C10ii—C10—H14 109.1 125.2 H13—C10—H14 107.8 125.2 N1iii—Co1—N1 180.0 111.34 (14) N1iii—Co1—N3 93.49 (6) 124.3 N1—Co1—N3 86.51 (6) 124.3 N1iii—Co1—N3iii 86.51 (6) 112.55 (14) N1—Co1—N3iii 93.49 (6) 109.1 N3—Co1—N3iii 180.0 109.1 N1iii—Co1—O1 88.40 (6) 109.1 N1—Co1—O1 91.60 (6) 109.1 N3—Co1—O1 88.99 (6) 107.8 N3iii—Co1—O1 91.01 (6) 113.60 (19) N1iii—Co1—O1iii 91.60 (6) 108.8 N1—Co1—O1iii 88.40 (6) 108.8 N3—Co1—O1iii 91.01 (6) 108.8 N3iii—Co1—O1iii 88.99 (6) 108.8 O1—Co1—O1iii 180.0 107.7 C3—N1—C2 105.50 (14) 111.80 (15) C3—N1—Co1 126.67 (11) 124.1 C2—N1—Co1 127.81 (12) 124.1 C3—N2—C1 107.20 (14) 109.45 (15) C3—N2—C4 125.39 (15) 125.3 C1—N2—C4 127.34 (14) sup-5 supplementary materials supplementary materials N3—C7—H9 125.3 C6—N3—C7 105.19 (14) C7—C8—N4 106.96 (15) C6—N3—Co1 128.87 (11) C7—C8—H10 126.5 C7—N3—Co1 125.19 (11) N4—C8—H10 126.5 C6—N4—C8 106.59 (14) N4—C9—C10 111.41 (14) C6—N4—C9 126.86 (15) N4—C9—H11 109.3 C8—N4—C9 126.17 (14) C10—C9—H11 109.3 Co1—O1—H15 128.5 N4—C9—H12 109.3 Co1—O1—H16 121.2 C10—C9—H12 109.3 H15—O1—H16 108.9 H11—C9—H12 108.0 H17—O2—H18 106.8 C9—C10—C10ii 112.6 (2) H19—O3—H20 109.6 Symmetry codes: (i) −x+2, −y, −z+2; (ii) −x, −y+1, −z+2; (iii) −x+1, −y, −z+1. Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A O1—H15···O3iv 0.85 1.94 2.781 (2) 169 O1—H16···Cl1 0.85 2.35 3.1728 (19) 165 O2—H17···Cl1iv 0.85 2.32 3.172 (2) 176 O2—H18···Cl1v 0.85 2.44 3.292 (3) 175 O3—H19···O2 0.85 1.99 2.829 (3) 171 O3—H20···Cl1 0.85 2.41 3.261 (3) 174 Symmetry codes: (iv) −x+1, −y+1, −z+1; (v) x−1, y, z. N3—C7—H9 125.3 C6—N3—C7 105.19 (14) C7—C8—N4 106.96 (15) C6—N3—Co1 128.87 (11) C7—C8—H10 126.5 C7—N3—Co1 125.19 (11) N4—C8—H10 126.5 C6—N4—C8 106.59 (14) N4—C9—C10 111.41 (14) C6—N4—C9 126.86 (15) N4—C9—H11 109.3 C8—N4—C9 126.17 (14) C10—C9—H11 109.3 Co1—O1—H15 128.5 N4—C9—H12 109.3 Co1—O1—H16 121.2 C10—C9—H12 109.3 H15—O1—H16 108.9 H11—C9—H12 108.0 H17—O2—H18 106.8 C9—C10—C10ii 112.6 (2) H19—O3—H20 109.6 Symmetry codes: (i) −x+2, −y, −z+2; (ii) −x, −y+1, −z+2; (iii) −x+1, −y, −z+1. Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A O1—H15···O3iv 0.85 1.94 2.781 (2) 169 O1—H16···Cl1 0.85 2.35 3.1728 (19) 165 O2—H17···Cl1iv 0.85 2.32 3.172 (2) 176 O2—H18···Cl1v 0.85 2.44 3.292 (3) 175 O3—H19···O2 0.85 1.99 2.829 (3) 171 O3—H20···Cl1 0.85 2.41 3.261 (3) 174 Symmetry codes: (iv) −x+1, −y+1, −z+1; (v) x−1, y, z. sup-6 supplementary materials 1 Fig. 1 sup-7 supplementary materials Fig. 2 Fig. 2 Fig. 2 sup-8 supplementary materials Fig. 3 supplementary materials Fig. 3 sup-9 supplementary materials Fig. 4 Fig. 4 Fig. 4 sup-10
https://openalex.org/W2090629246	https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1003553&type=printable	English	null	Metabolic Features of Protochlamydia amoebophila Elementary Bodies – A Link between Activity and Infectivity in Chlamydiae	PLOS pathogens	2,013	cc-by	21,395	Received January 8, 2013; Accepted June 28, 2013; Published August 8, 2013 Copyright: 2013 Sixt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was funded by the Austrian Science Fund FWF (I291-B09) and the German Federal Ministry of Education and Research in the framework of the ERA-NET project ‘‘Pathogen-host metabolomics and interactomics (Pathomics).’’ BSS is a recipient of a DOC-fFORTE fellowship of the Austrian Academy of Sciences at the Department of Microbial Ecology, University of Vienna, Austria. MH acknowledges support from the European Research Council (ERC StG ‘‘EvoChlamy’’). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: horn@microbial-ecology.net * E-mail: horn@microbial-ecology.net . These authors contributed equally to this work. Chlamydiae, infect host species as diverse as vertebrates, inverte- brates, and even protozoa [4,5]. Their impact on human health is not yet well understood, though there is evidence that some species might represent emerging pathogens [4,6]. Among the most well- studied representatives of the environmental chlamydiae are members of the family Parachlamydiaceae, in particular Protochlamydia amoebophila [7] and Parachlamydia acanthamoebae [8]. Although primarily considered to be symbionts of amoebae [4,9], there are indications that they may be associated with human diseases, and in particular respiratory tract infections [10,11]. Metabolic Features of Protochlamydia amoebophila Elementary Bodies – A Link between Activity and Infectivity in Chlamydiae Barbara S. Sixt1., Alexander Siegl1., Constanze Mu¨ ller2., Margarete Watzka3, Anna Wultsch1, Dimitrios Tziotis2, Jacqueline Montanaro1, Andreas Richter3, Philippe Schmitt-Kopplin2, Matthias Horn1* Barbara S. Sixt1., Alexander Siegl1., Constanze Mu¨ ller2., Margarete Watzka3, Anna Wultsch1, Dimitrios Tziotis2, Jacqueline Montanaro1, Andreas Richter3, Philippe Schmitt-Kopplin2, Matthias Horn1* 1 Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria, 2 Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum Mu¨nchen, Neuherberg, Germany, 3 Division of Terrestrial Ecosystem Research, Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria 1 Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria, 2 Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum Mu¨nchen, Neuherberg, Germany, 3 Division of Terrestrial Ecosystem Research, Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria PLOS Pathogens \| www.plospathogens.org Abstract The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from 13C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity. Citation: Sixt BS, Siegl A, Mu¨ller C, Watzka M, Wultsch A, et al. (2013) Metabolic Features of Protochlamydia amoebophila Elementary Bodies – A Link between Activity and Infectivity in Chlamydiae. PLoS Pathog 9(8): e1003553. doi:10.1371/journal.ppat.1003553 Editor: Ming Tan, University of California Irvine, United States of America Received January 8, 2013; Accepted June 28, 2013; Published August 8, 2013 Received January 8, 2013; Accepted June 28, 2013; Published August 8, 2013 August 2013 \| Volume 9 \| Issue 8 \| e1003553 Author Summary The Chlamydiae are a group of bacteria that strictly rely on eukaryotic host cells as a niche for intracellular growth. This group includes major pathogens of humans and animals as well as symbionts of protists. Unlike most other bacteria, chlamydiae alternate between two distinct developmental stages. Here we provide novel insights into the infective stage, the elementary body (EB), which has been described almost a century ago and is commonly referred to as an inert spore-like particle. Our analyses of EBs of the amoeba symbiont Protochlamydia amoebophila provide a detailed overview of their metabolism outside of, and independent from, their natural host cells. We demonstrated that these EBs are capable of respiration and are active in the major routes of central carbon metabolism, including glucose import, biosynthetic reac- tions, and catabolism for energy generation. Glucose starvation resulted in a rapid decline of metabolic activity in P. amoebophila EBs and a concomitant decrease in their potential to infect new host cells. The human pathogen Chlamydia trachomatis was also dependent on nutrient availability for extracellular survival. The extent of meta- bolic activity in chlamydial EBs and its consequences for infectivity challenge long-standing textbook knowledge and demonstrate that the infective stage is far more dependent on its environment than previously recognized. Due to these structural and biochemical features, EBs have been thought to be completely metabolically inert particles. However, this concept has been challenged by recent studies. EB proteomes of diverse chlamydial species, including members of the Chlamyd- iaceae, as well as P. amoebophila, have been shown to comprise remarkably complete sets of proteins involved in transcription, translation, and energy metabolism [36–40]. Moreover, we recently observed that the chemically defined medium DGM- 21A, which had originally been developed for cultivation of Acanthamoeba spp. [41,42], sustains host-free activity of the infectious stage of chlamydiae [43]. More specifically, EBs of P. amoebophila and C. trachomatis incubated in DGM-21A maintained their ability to take up the amino acid L-phenylalanine in a process that could be reversibly inhibited by an ionophore, which demonstrated that EBs are dependent on a membrane potential and are able to reenergize their membrane [43]. Most recently, sustained metabolic activity of chlamydial EBs was shown in a study that demonstrated transcription and protein biosynthesis in host-free C. trachomatis EBs [44]. In the current study we focused on an in-depth investigation of the metabolic potential of P. Introduction The Chlamydiaceae are a group of obligate intracellular bacteria that have been well-known for more than a century and include some of the most successful bacterial pathogens. Two species in particular are considered to represent a major threat to human health, Chlamydia trachomatis, a well-established agent of trachoma and sexually transmitted disease [1,2], and Chlamydia pneumoniae, which causes pneumonia and has also been associated with numerous chronic diseases [3]. More recently, the discovery of the so-called ‘‘environmental chlamydiae’’ has radically changed our perception of chlamydial diversity and distribution in nature. These newly discovered species, which are related to the Chlamydiaceae yet represent separate families within the phylum Although we are just beginning to understand aspects of host range, host-symbiont interactions, and potential pathogenicity of these newly discovered species, it is well-established that all characterized environmental chlamydiae share distinctive key August 2013 \| Volume 9 \| Issue 8 \| e1003553 1 PLOS Pathogens \| www.plospathogens.org Metabolic Activity of Protochlamydia EBs nutrients [25,27,28]. EBs, moreover, have a distinct ultrastructure that is characterized by highly condensed chromatin [24]. DNA compaction, achieved by the action of chlamydial histone-like proteins [29–32], has been proposed to cause a complete shutdown of transcriptional activity in EBs [33,34], which is also consistent with their reduced RNA to DNA ratio compared to the replicative stage [35]. Author Summary Accordingly, this developmental form was reported to be more resistant to harsh conditions and mechanical stress than the fragile replicative form due to its rigid, highly cross-linked outer membrane [25,26] that has been suggested to represent a permeability barrier inhibiting uptake of Investigations of the genomic repertoire of several members of the Chlamydiae have revealed that they harbor highly reduced metabolic capacities, presumably as a consequence of their adaptation to intracellular life [12–18]. Although environmental chlamydiae, including P. amoebophila, have a significantly larger genome size than the Chlamydiaceae, their metabolic potential is only slightly increased, and they are thus also strictly dependent on eukaryotic host cells [14,16–19]. Despite being auxotrophic for most amino acids, cofactors, and nucleotides and being able to take up host-derived ATP, chlamydiae have at least partially maintained metabolic pathways devoted to carbon metabolism and energy generation [19–21]. However, little is known about the role of these metabolic features and the specific nutrient requirements throughout chlamydial development. As a conse- quence, axenic growth of chlamydiae has not yet been achieved. PLOS Pathogens \| www.plospathogens.org Author Summary amoebophila EBs in order to decipher the nature and biological significance of their activities. By applying a comprehensive combination of fluorescence microsco- py- and mass spectrometry-based techniques, we could demon- strate respiratory activity and D-glucose utilization in EBs and, furthermore, could obtain first insights into the host-free central carbon metabolism of P. amoebophila. Importantly, our investiga- tions revealed that the availability of a metabolizable substrate during host-free incubation significantly extends maintenance of infectivity in both, P. amoebophila and C. trachomatis, indicating a biological role for metabolic activity in the infectious stage. aspects of chlamydial biology with the Chlamydiaceae [4]. These include a strict dependence on a eukaryotic host cell as a replicative niche as well as a biphasic developmental cycle, both of which have major implications on the metabolic traits of this group of bacteria. Investigations of the genomic repertoire of several members of the Chlamydiae have revealed that they harbor highly reduced metabolic capacities, presumably as a consequence of their adaptation to intracellular life [12–18]. Although environmental chlamydiae, including P. amoebophila, have a significantly larger genome size than the Chlamydiaceae, their metabolic potential is only slightly increased, and they are thus also strictly dependent on eukaryotic host cells [14,16–19]. Despite being auxotrophic for most amino acids, cofactors, and nucleotides and being able to take up host-derived ATP, chlamydiae have at least partially maintained metabolic pathways devoted to carbon metabolism and energy generation [19–21]. However, little is known about the role of these metabolic features and the specific nutrient requirements throughout chlamydial development. As a conse- quence, axenic growth of chlamydiae has not yet been achieved. The chlamydial developmental cycle comprises two major stages: the intracellular replicative reticulate bodies (RBs) and the extracellular, non-dividing, infectious elementary bodies (EBs). Transition stages are referred to as intermediate bodies (IBs) [22]. After their uptake into a host cell EBs differentiate into RBs, which then replicate within a membrane-enclosed compartment. At the end of the infection cycle the bacteria differentiate back into EBs, which are subsequently released into the environment to infect neighboring cells [23]. Chlamydial EBs, which thus serve as dispersal stage, have been shown to differ from RBs in several structural and biochemical features, which is thought to reflect adaptation to their main biological functions of extracellular survival and reinfection [24]. Results S2E), demonstrating that residual host-derived activity is not stable under these conditions. Figure 1. Respiratory activity of P. amoebophila developmental stages and effect of D-glucose deprivation. Fractions of P. amoebophila developmental forms were subjected to host-free incubation in DGM-D (or DGM-L, if indicated) containing 5 mM CTC, either immediately after purification (‘‘0 h pre-incub’’) or after a 40 h pre-incubation in the respective medium (‘‘40 h pre-incub’’), followed by the detection of bacteria with the DNA dye DAPI. Heat-inactivated EBs were included as control. The percentage of CTC-positive DAPI- stained bacteria was determined and subsequently corrected based on observed differences in the detectability of bacteria among fractions. Displayed data represent means and standard deviations of three independent experiments. For each experiment and condition, in total 1500 bacteria were considered for the quantification of CTC-positive bacteria, and 450 bacteria for the subsequent correction. Statistically significant differences compared to the EB fraction (ANOVA) are indicated by stars located directly above the bars, significant differences between immediate activity and activity after pre-incubation (t-test) are indicated by the stars at the upper edge of the diagram (*, p#0.001; , p#0.01; , p#0.05). In order to directly compare the respiratory activity between developmental forms in freshly purified suspensions, the propor- tion of DAPI-stained chlamydiae able to reduce CTC was determined. Examination using differential interference contrast (DIC) microscopy additionally revealed that most bacteria in EB and intermediate fractions were detectable by DAPI (90.0% (64.1) and 79.1% (64.0), respectively), whereas a significant proportion of bacteria in the RB fraction (49.1% (62.7)) were not. This suggests that, consistent with the frequently reported fragility of this developmental stage [22,35,44], many RBs were damaged during the purification procedure or rapidly lysed during the short host-free incubation with CTC. Proportions of active bacteria were therefore corrected to account for the different detectability with DAPI. This analysis revealed that only 24.4% (62.2) of the bacterial particles in the RB fraction were active, whereas 52.3% (62.9) of the bacteria in the EB fraction contained intracellular CTC crystals (Fig. 1). p p doi:10.1371/journal.ppat.1003553.g001 Collectively, these findings demonstrate that respiratory activity in EB fractions cannot be solely attributed to a contamination with other developmental stages or host-derived components, but truly occurs in P. amoebophila EBs. They furthermore indicate that EB activity is stable in a host-free environment, whereas activity declines more rapidly in RB fractions. Results amoebophila developmental forms were subjected to host-free incubation in DGM-D (or DGM-L, if indicated) containing 5 mM CTC, either immediately after purification (‘‘0 h pre-incub’’) or after a 40 h pre-incubation in the respective medium (‘‘40 h pre-incub’’), followed by the detection of bacteria with the DNA dye DAPI. Heat-inactivated EBs were included as control. The percentage of CTC-positive DAPI- stained bacteria was determined and subsequently corrected based on observed differences in the detectability of bacteria among fractions. Displayed data represent means and standard deviations of three independent experiments. For each experiment and condition, in total 1500 bacteria were considered for the quantification of CTC-positive bacteria, and 450 bacteria for the subsequent correction. Statistically significant differences compared to the EB fraction (ANOVA) are indicated by stars located directly above the bars, significant differences between immediate activity and activity after pre-incubation (t-test) are indicated by the stars at the upper edge of the diagram (, p#0.001; , p#0.01; , p#0.05). analysis, bacteria were additionally stained with the DNA dye 49,6- diamidino-2-phenylindole (DAPI). Cells containing red fluorescent crystals were observed in all fractions, irrespective of whether the assessment of activity was started directly after the purification or after a 40 h pre-incubation in DGM-D (Fig. S2A–C). Heat- inactivated bacteria were inactive, indicating that CTC reduction was not an artifact caused by constituents of the incubation medium (Fig. S2D). In order to exclude that residual host cell components that may be present in suspensions of purified bacteria contributed to observed activities, the CTC reducing capacity of lysates of uninfected Acanthamoeba was assessed as control. When freshly prepared lysates were analyzed, formation of CTC crystals could also be observed in the absence of bacteria (Fig. S2E). However, these signals, which were most likely derived from host mitochon- dria, could be clearly distinguished from active bacteria, due to their larger size and irregular shape. In addition, they did not co- localize with bright DAPI signals that can typically be observed for living bacteria, but not for mitochondria or other components in host cell lysates, which are only weakly stained with this dye. CTC crystals of similar appearance, potentially formed by remaining host components, were occasionally observed in fractions of purified chlamydiae, but were not considered during quantitative assessments of bacterial activity. Most importantly, CTC reduction was not detected in host lysates that were pre-incubated for 40 h before analysis (Fig. Results Respiratory Activity in Host-Free P. amoebophila EBs P. amoebophila developmental stages were purified from amoeba host cells and physically separated from each other by density gradient centrifugation. This approach was originally described almost 50 years ago [35,45] and is today widely applied for the analysis of Chlamydiaceae developmental forms (e.g. [39,44]). We optimized this purification method for P. amoebophila in a previous study and have quantitatively evaluated the purity of obtained EB- and RB-enriched fractions using transmission electron microscopy (TEM) [43] (Fig. S1). The chlamydial developmental cycle comprises two major stages: the intracellular replicative reticulate bodies (RBs) and the extracellular, non-dividing, infectious elementary bodies (EBs). Transition stages are referred to as intermediate bodies (IBs) [22]. After their uptake into a host cell EBs differentiate into RBs, which then replicate within a membrane-enclosed compartment. At the end of the infection cycle the bacteria differentiate back into EBs, which are subsequently released into the environment to infect neighboring cells [23]. Chlamydial EBs, which thus serve as dispersal stage, have been shown to differ from RBs in several structural and biochemical features, which is thought to reflect adaptation to their main biological functions of extracellular survival and reinfection [24]. Accordingly, this developmental form was reported to be more resistant to harsh conditions and mechanical stress than the fragile replicative form due to its rigid, highly cross-linked outer membrane [25,26] that has been suggested to represent a permeability barrier inhibiting uptake of Host-free activity of P. amoebophila was initially analyzed by using the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which is a non-fluorescent soluble molecule that is reduced by metabolically active cells, leading to intracellular deposition of bright red fluorescent crystals [46,47]. Due to its good correlation with other measures of cellular respiration and studies indicating an involvement of electron transport chain activity, CTC reduction is considered to be an indicator for respiratory activity [47–50]. Purified EBs and RBs, as well as an intermediate fraction representing a mixture of all developmental stages of P. amoebophila, were incubated for 2 h in host-free modified DGM-21A medium (in this study referred to as DGM-D to indicate the presence of D- glucose) containing 5 mM CTC. Prior to fluorescence microscopic August 2013 \| Volume 9 \| Issue 8 \| e1003553 2 Metabolic Activity of Protochlamydia EBs Figure 1. Respiratory activity of P. amoebophila developmental stages and effect of D-glucose deprivation. Fractions of P. Results This observation, together with the observed instability of host-derived activity, was exploited in the following experiments, in which a pre-incubation of purified bacteria was intentionally applied not only to ensure that truly host-free metabolic activity is assessed, but also to exclude significant contributions of co-purified RBs or host components. When bacterial activity was assessed after a host-free incubation for 40 h in DGM-D, a highly significant decrease in the proportion of active bacteria was observed in the RB fraction, in which only 11.5% (61.4) of the DIC-detectable particles had maintained their ability to reduce CTC (Fig. 1) (t-test, p#0.001). This observation can most likely be explained by significant lysis of the more fragile RBs during the extracellular incubation. However, the degree of bacterial disintegration might be greatly underestimated by our assessment due to the fact that completely lysed bacteria might not be detectable in DIC. Based on this consideration and the fact that, according to a recent ultrastruc- tural analysis, suspensions of purified RBs initially still contain about 5% EBs [43], it appears most likely that this residual activity originates from the more stable EBs in this fraction. In fact, the proportion of active bacteria in the EB fraction, which initially contained about 76% mature EBs and only 8% RBs [43], was still 51.3% (64.6) after 40 h incubation and thus almost unchanged compared to the active proportion detected in freshly purified suspensions (Fig. 1). PLOS Pathogens \| www.plospathogens.org Effect of D-Glucose Deprivation on Host-Free Respiratory Activity in EBs The observation of sustained respiratory activity in P. amoebophila EBs during host-free incubation suggested that the DGM-D medium contains substrates that the bacteria can utilize to fuel metabolic activities. The defined medium includes a variety of potential carbon compounds, including all proteinogenic amino acids, some of which may also be fed into energy generating metabolic pathways according to the predictions from genome annotation [14]. DGM-D, however, also contains D-glucose, and we recently observed that host-free activity of chlamydiae could not be sustained in the incubation medium reported by Hatch August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 3 Metabolic Activity of Protochlamydia EBs Figure 2. Visualization of D-glucose uptake by host-free P. amoebophila EBs. Living or heat-inactivated P. amoebophila EBs were subjected to host-free incubation in DGM-D/2 containing 100 mM of the green-fluorescent D-glucose analog 2-NBDG. The incubation was conducted either immediately after purification or after a 40 h pre- incubation in DGM-D. Representative fluorescence and DIC images are shown. The bar indicates 10 mm. doi:10.1371/journal.ppat.1003553.g002 [43], which as a major difference to DGM-D does not contain any carbohydrates [51]. This consideration and the fact that the P. amoebophila genome encodes a complete pathway for D-glucose catabolism [14] prompted us to test whether D-glucose depriva- tion would affect host-free activity of P. amoebophila EBs. Complete withdrawal of glucose from DGM-D would change the physico- chemical characteristics of the medium, such as the osmolarity, and it is unknown whether this could have an effect on bacterial activity. Respiratory activity of EBs was thus analyzed in a modified medium termed DGM-L, in which D-glucose was not simply omitted but replaced by its stereoisomer L-glucose, which cannot be metabolized by most organisms [52–54]. This replacement of substrates led to a significant decrease in the proportion of active bacteria, as inferred from their capability to reduce CTC (Fig. 1 and S2F) (Analysis of variance (ANOVA), p#0.01). Moreover, though directly after purification from host cells 27.0% (61.9) of the EBs detectable in DIC appeared to be metabolically active in DGM-L, a large reduction in active cells (to 6.0% (63.1)) was observed when purified EBs were analyzed after 40 h pre-incubation in this D-glucose-free medium (Fig. 1). These findings demonstrate that the nutrient composition of the host-free incubation medium affects P. amoebophila EB activity as well as its maintenance. Effect of D-Glucose Deprivation on Host-Free Respiratory Activity in EBs Moreover, they suggest that D-glucose might serve as an energy source for the bacteria during host-free incubation. Figure 2. Visualization of D-glucose uptake by host-free P. amoebophila EBs. Living or heat-inactivated P. amoebophila EBs were subjected to host-free incubation in DGM-D/2 containing 100 mM of the green-fluorescent D-glucose analog 2-NBDG. The incubation was conducted either immediately after purification or after a 40 h pre- incubation in DGM-D. Representative fluorescence and DIC images are shown. The bar indicates 10 mm. doi:10.1371/journal.ppat.1003553.g002 PLOS Pathogens \| www.plospathogens.org D-Glucose Uptake by Host-Free P. amoebophila EBs In order to further explore the potential for D-glucose utilization by P. amoebophila EBs, we next investigated whether the bacteria were able to import this substrate under host-free conditions by applying 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)a- mino]-2 deoxy-D-glucose (2-NBDG), a fluorescent analog that has previously been used as an indicator for D-glucose uptake in living cells [55]. In addition, isotope ratio mass spectrometry (IRMS), a method suitable for the analysis of the isotopic composition of biological samples [56], was applied as a supplementary technique to directly prove the import of non-derivatized sugar molecules. bacteria able to import the fluorescent analog 2-NBDG once more shows that this activity is not restricted to a small proportion of possibly co-purified RBs or transition stages, but occurs in the EB stage. The consistency between the percentage of respiratorily active bacteria and bacteria that imported D-glucose further supports the concept that P. amoebophila EBs may not only import, but also metabolize D-glucose under host-free conditions. For the fluorescence-based assay, purified living and heat- inactivated EBs were incubated for 10 h with 100 mM 2-NBDG in DGM-D/2, a medium containing a reduced concentration of non- fluorescent D-glucose, followed by microscopic examination. 2- NBDG uptake could be detected in living but not in heat- inactivated bacteria, irrespective of whether they were assessed directly after purification or after pre-incubation in DGM-D. The proportion of chlamydiae able to take up 2-NBDG remained stable over time and was similar to the proportion of respiratorily active cells, comprising 47.8% (61.7) or 53.4% (63.4) of all bacterial particles before or after pre-incubation, respectively (Fig. 2). August 2013 \| Volume 9 \| Issue 8 \| e1003553 Catabolism of D-Glucose by Host-Free P. amoebophila EBs Catabolism of D-Glucose by Host-Free P. amoebophila EBs Host-free incubations with D-[U-13C6]-glucose were carried out in gas-tight vials. Thus, not only incorporation of the label into the biomass, but also respiratory activity, inferred from 13CO2 release, could be analyzed. For this purpose, gas samples that were collected from the headspace of the incubations were subjected to IRMS analysis to measure the amount of CO2 and the atom percent 13C (At%13C) in the CO2. When expressed as enrichment relative to values obtained for blank incubations of bacteria-free media, these serve as measure for CO2 production and for the enrichment of 13C, respectively. For IRMS analysis, a pre-incubated EB-enriched fraction was further incubated for 48 h in DGM-D-13C, a medium in which D-glucose was replaced by its fully 13C-labeled isotopolog (D-[U- 13C6]-glucose). Bacterial biomass was then subjected to IRMS. Based on the measured carbon content of the biomass and the ratio of 13C to 12C in the sample, the amount of 13C that was incorporated by purified EBs during the whole period of host-free incubation could be calculated. Although a slight enrichment in 13C (11.0 (61.9) nmol 13C/mg dry weight (DW)) was also detected for heat-inactivated bacteria (Fig. S3), presumably as a result of substrate adsorption to the bacterial surface, a significantly higher incorporation (48.1 (67.4) nmol 13C/mg DW) was observed for living bacteria (t-test, p#0.001). In general, CO2 amounts in the headspace of incubations were increased compared to the composition of normal lab air even in the absence of bacteria, suggesting that a part of the newly formed CO2 may be derived from outgassing carbonate. However, the presence of living bacteria led to a marked increase of 146.5 (633.9) ppm CO2/ml (Fig. 3A). Moreover, a significant enrich- ment of 13C in CO2 (atom percent 13C enrichment (APE13C) 30.7 (64.5)) could be detected in the presence of D-[U-13C6]-glucose (ANOVA, p#0.001). In contrast, biological CO2 production was not observed when heat-inactivated bacteria were used. Taken together these findings clearly demonstrate active uptake of D-glucose by host-free P. amoebophila. The high proportion of Taken together, these findings provide further evidence that EBs of P. amoebophila are metabolically active under host-free August 2013 \| Volume 9 \| Issue 8 \| e1003553 4 Metabolic Activity of Protochlamydia EBs Figure 3. D-glucose catabolism by host-free P. amoebophila EBs revealed by IRMS. Purified P. Catabolism of D-Glucose by Host-Free P. amoebophila EBs Statistically significant differences are indicated (for CO2 release and APE13C at the upper or lower edge of the diagram, respectively) in respect to living bacteria incubated in DGM-D (ANOVA) (, p#0.001; , p#0.01; , p#0.05). Note that bacterial numbers that were applied per incubation were similar between replicate experiments (between 3.96109 and 5.96109 bacteria (A); between 6.36108 and 1.06109 bacteria (B)), but were significantly different between (A) and (B), explaining observed differences in the extents of CO2 production. doi:10.1371/journal.ppat.1003553.g003 conditions and demonstrate that the observed CO2 formation can, at least partly, be attributed to D-glucose catabolism. DGM-D-6-13C. These media contained unlabeled D-glucose, fully 13C-labeled D-glucose (D-[U-13C6]-glucose), D-glucose labeled exclusively at carbon 1 (D-[1-13C]-glucose), or D-glucose labeled at carbon 6 (D-[6-13C]-glucose), respectively. Moreover, incubation of EBs in DGM-L was included to analyze the effect of D-glucose deprivation on CO2 production. Host cell lysates incubated in DGM-D-13C were handled in parallel to test for potential contributions of residual host-derived activity. Catabolism of D-Glucose by Host-Free P. amoebophila EBs amoebophila EBs (EB-enriched fraction (A); highly pure EB fraction (B)) were pre-incubated for 40 h in DGM-D, followed by 48 h incubation in different media (DGM-D or DGM-D-13C (A); DGM-D, DGM-D-1-13C, DGM-D-6-13C, DGM-D-13C, or DGM-L (B)) and subsequent CO2 measurement in the headspace of incubations by IRMS. As control, heat-inactivated bacteria (‘‘EB-hi’’) (A) or host cell lysates (B) incubated in DGM-D-13C were handled in parallel. CO2 production (in ppm CO2/ml; white circles) and the APE13C in the CO2 (black diamonds) are displayed. Diamonds and circles indicate results from individual replicates, bars display mean values. The black solid line highlights the base line for CO2 release and APE13C, which corresponds to values observed in the blanks, i.e. bacteria-free incubations of the respective media. Results from three independent experiments each consisting of two replicate incubations per condition are shown. An exception was the incubation in DGM-L, for which only two experiments were conducted. Statistically significant differences are indicated (for CO2 release and APE13C at the upper or lower edge of the diagram, respectively) in respect to living bacteria incubated in DGM-D (ANOVA) (, p#0.001; , p#0.01; , p#0.05). Note that bacterial numbers that were applied per incubation were similar between replicate experiments (between 3.96109 and 5.96109 bacteria (A); between 6.36108 and 1.06109 bacteria (B)), but were significantly different between (A) and (B), explaining observed differences in the extents of CO2 production. doi:10.1371/journal.ppat.1003553.g003 y y Figure 3. D-glucose catabolism by host-free P. amoebophila EBs revealed by IRMS. Purified P. amoebophila EBs (EB-enriched fraction (A); highly pure EB fraction (B)) were pre-incubated for 40 h in DGM-D, followed by 48 h incubation in different media (DGM-D or DGM-D-13C (A); DGM-D, DGM-D-1-13C, DGM-D-6-13C, DGM-D-13C, or DGM-L (B)) and subsequent CO2 measurement in the headspace of incubations by IRMS. As control, heat-inactivated bacteria (‘‘EB-hi’’) (A) or host cell lysates (B) incubated in DGM-D-13C were handled in parallel. CO2 production (in ppm CO2/ml; white circles) and the APE13C in the CO2 (black diamonds) are displayed. Diamonds and circles indicate results from individual replicates, bars display mean values. The black solid line highlights the base line for CO2 release and APE13C, which corresponds to values observed in the blanks, i.e. bacteria-free incubations of the respective media. Results from three independent experiments each consisting of two replicate incubations per condition are shown. An exception was the incubation in DGM-L, for which only two experiments were conducted. Metabolic Activity of Protochlamydia EBs Both ordinations, which included dead bacteria incubated in DGM-D-13C15N and living bacteria incubated either in DGM-D or in DGM-D-13C15N, revealed a separation between living and inactivated bacteria that reflects the presence of different molecular patterns (Fig. S5C–D). Metabolites that are discriminative for living EBs, i.e. compounds that can be detected in living, but not or only in low amounts, in inactivated bacteria, might represent valuable indicators for an active metabolism. As a next step we applied a partial least square discriminative analysis (PLS-DA) model including data from DGM-D-incubated living and DGM-D-13C15N-incubated inac- tivated EBs (R2Y(cum) = 0.926,Q2 = 0.834) (Fig. S6). MassTRIX annotation of the most relevant m/z features revealed that metabolites from the amino acid-, nucleotide-, and central carbon metabolism were discriminative for living EBs, whereas the pattern of metabolites assigned to lipid metabolism appeared to be less affected (Fig. 4). y Liberation of carbon 6 from D-glucose as CO2 is usually only observed as a consequence of tricarboxylic acid (TCA) cycle activity and is therefore evidence for a functional sugar catabolism in extracellular EBs of P. amoebophila. However, our data do not support a scenario in which all assimilated D-glucose would be fully catabolized to CO2 via glycolysis and/or pentose phosphate pathway (PPP) combined with TCA cycle activity, as this would result in equal amounts of released carbon 1 and carbon 6. The preferred release of carbon 1 indicates that D-glucose also passes through the oxidative part of the PPP without being coupled to a subsequent complete breakdown of its products. Moreover, the observed extent of 13CO2 production from fully labeled D-glucose additionally indicates that also carbon atoms other than carbon 1 are released from D-glucose as CO2 in metabolic reactions that do not lead to complete catabolism. Thus, a proportion of the metabolized D-glucose may be devoted to anabolic reactions, including for example pathways such as fatty acid and isoprenoid synthesis, which begin with a decarboxylation of pyruvate. We focused on the three metabolic scenarios outlined above to model their contributions to CO2 production using the calculation described in detail in Fig. S4. Our data would be consistent with a theoretical ratio of 6.2 (putative anabolic reactions):3.8 (oxidative part of PPP):1.0 (complete catabolism). This finding suggests that the experimentally obtained data are plausible and can be explained by a reasonable simplified metabolic model. Metabolic Activity of Protochlamydia EBs D-glucose by L-glucose, reduced CO2 formation to background levels (Fig. 3B). Respiratory activity was also not observed in amoebal lysates. An enrichment of 13C in CO2 was detected for all incubations of living bacteria in media containing 13C-labeled D- glucose. However, the degree of labeling was highly variable depending on the variant of labeled substrate included. As expected the greatest formation of 13CO2 was observed in the presence of the fully labeled D-glucose (APE13C 15.2 (63.4)), which was about 4.4 times higher than that observed for D-[1- 13C]-glucose and about 21.2 times higher than that observed for D-[6-13C]-glucose. The calculated ratio for the APE13C was 21.2 (DGM-D-13C):4.8 (DGM-D-1-13C):1.0 (DGM-D-6-13C). Pro- duction of 13CO2 could not be detected for host cell lysates. Prior to the analysis, metabolites were extracted from an EB- enriched fraction that had been pre-incubated and subsequently incubated for 48 h in DGM-D-13C15N, a modified medium containing 13C-labeled D-glucose and 13C15N-labeled L-phenyl- alanine, or in DGM-D, containing the corresponding unlabeled substrates. Heat-inactivated bacteria incubated in DGM-D- 13C15N were also analyzed. Labeled L-phenylalanine was included in this experiment as a control, as it is a substrate that is taken up by host-free EBs [43] and which exhibits only limited potential of being further metabolized by P. amoebophila according to predictions from the genome [14]. ICR/FT-MS analysis enabled the assignment of 1674 and 1767 m/z features to DGM-D-13C15N- or DGM-D-incubated bacteria, respectively. In order to gain a first impression on the sample composition, masses that were detected in extracts from DGM-D-incubated EBs were submitted to MassTRIX for annotation [61]. This analysis revealed that metabolites from several different biochemical pathways were detected; including compounds involved in amino acid-, nucleotide-, sugar-, and lipid metabolism (Fig. 4). Spectra of living bacteria were highly similar to each other irrespective of the applied incubation medium, indicating that the presence of the stable isotope-labeled substrates did not affect the metabolite pattern (Fig. S5A). To verify this visual impression, we investigated the data using principal component analysis (PCA). Extracts from living bacteria that were incubated either in DGM-D-13C15N or in DGM-D clustered together in the PCA ordination, which confirms that the samples were comparable in their general composition (Fig. S5B). We additionally produced PCA ordinations including data from the heat-inactivated EBs. Insights into D-Glucose Metabolism in Host-Free P. amoebophila EBs by IRMS Though CO2 production evidences metabolic activity it does not necessarily indicate complete catabolism of a given substrate, as several biochemical pathways involve only partial substrate breakdown. Defining the exact position of carbon atoms in the substrate molecule that are liberated as CO2 thus aids in a better understanding of the activity of certain metabolic pathways. In the present study, the contribution of different carbon atoms in D- glucose to CO2 produced by host-free EB activity was investigated by IRMS. For this purpose, a pre-incubated EB fraction was further incubated for 48 h in DGM-D, DGM-D-13C, DGM-D-1-13C, or CO2 production was observed for all incubations of living bacteria in media containing D-glucose, as inferred from an average increase in detectable CO2 of 29.0 (611.1) ppm/ml relative to amounts detected in blank incubations of bacteria-free media (Fig. 3B). Taking into account that about six times fewer bacterial cells were applied per incubation, the extent of CO2 release appeared to be very similar to that observed in the previous experiment (Fig. 3A). Substrate deprivation, i.e. the replacement of August 2013 \| Volume 9 \| Issue 8 \| e1003553 August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 5 Metabolic Activity of Protochlamydia EBs Metabolic Activity of Protochlamydia EBs The calculation, furthermore, suggests that a major proportion of D- glucose is not completely degraded, but may enter anabolic pathways. Central Carbon Metabolism of Host-Free P. amoebophila EBs Revealed by Mass Spectrometric Metabolite Analysis Central Carbon Metabolism of Host-Free P. amoebophila EBs Revealed by Mass Spectrometric Metabolite Analysis M/z features detected by ICR/FT-MS in samples of DGM-D-incubated EBs were annotated by MassTRIX [61], followed by data analysis using PLS-DA (Fig. S6) to extract the most discriminative compounds characterizing living compared to inactivated EBs. The PLS-DA model included data from DGM-D-incubated living and DGM-D-13C15N-incubated inactivated EBs. Bars indicate the total number of annotated metabolites that were assigned to specific KEGG pathways. The number of metabolites that were found to be discriminative (black) or non-discriminative (white) for living EBs are additionally indicated. Note that metabolites from carbohydrate, nucleotide, cofactor, vitamin, and amino acid metabolism were more abundant in living EBs, whereas the pattern of lipid species was more similar between living and inactivated bacteria. doi:10.1371/journal.ppat.1003553.g004 Figure 4. Overview of ICR/FT-MS-detected annotated compounds and of metabolites discriminative for living compared to inactivated EBs. An EB-enriched fraction of P. amoebophila was pre-incubated for 40 h in DGM-D, followed by 48 h incubation in DGM-D or DGM- D-13C15N and subsequent mass spectrometric analysis of metabolite extracts. Heat-inactivated EBs incubated in DGM-D-13C15N were included as control. M/z features detected by ICR/FT-MS in samples of DGM-D-incubated EBs were annotated by MassTRIX [61], followed by data analysis using PLS-DA (Fig. S6) to extract the most discriminative compounds characterizing living compared to inactivated EBs. The PLS-DA model included data from DGM-D-incubated living and DGM-D-13C15N-incubated inactivated EBs. Bars indicate the total number of annotated metabolites that were assigned to specific KEGG pathways. The number of metabolites that were found to be discriminative (black) or non-discriminative (white) for living EBs are additionally indicated. Note that metabolites from carbohydrate, nucleotide, cofactor, vitamin, and amino acid metabolism were more abundant in living EBs, whereas the pattern of lipid species was more similar between living and inactivated bacteria. doi:10.1371/journal.ppat.1003553.g004 metabolites were also detected as unlabeled molecules in extracts from DGM-D-13C15N-incubated EBs, indicating co-utilization of D-[U-13C6]-glucose with additional unlabeled carbon com- pounds. The observed abundance ratios of 12C and 13C atoms in the detected labeled metabolites suggest a rather minor glycolytic catabolic activity in host-free EBs and a predominant catabolism of D-glucose by the PPP. Central Carbon Metabolism of Host-Free P. amoebophila EBs Revealed by Mass Spectrometric Metabolite Analysis EBs Revealed by Mass Spectrometric Metabolite Analysis In order to obtain deeper insights into the metabolism of host- free P. amoebophila EBs and to explore the intracellular fate of 13C- labeled D-glucose we next conducted a mass spectrometric metabolite analysis combining ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS) and ultra-perfor- mance liquid chromatography mass spectrometry (UPLC-MS). ICR/FT-MS offers ultra-high resolution, enabling the distinction of several thousands of ions. This technique provides extremely high mass accuracy, which allows direct calculation of the elemental composition of detected compounds and thereby facilitates metabolite annotation and clear discrimination between isotopologs [57,58]. However, as chromatographic separation of analytes prior to mass spectrometric analysis carries several advantages over direct injection experiments such as decreased matrix effects, separation of isobaric compounds, and delivery of additional information about physicochemical properties of analytes by their retention time [59,60], we additionally conducted UPLC-MS as second analytical technique, in particular to verify the detection of labeled metabolic intermediates. To obtain deeper insights into the central carbon metabolism of host-free EBs, further data analysis focused on 13C-labeled metabolites that were derived from 13C-labeled D-glucose. Labeled metabolites were identified based on their accurate mass and presence of their unlabeled metabolite analogs in extracts of bacteria incubated in DGM-D by application of mass difference- based networks. Peaks corresponding to fully labeled glucose (C6H12O6) and phenylalanine (C9H11NO2) could be readily detected in extracts from bacteria incubated in DGM-D- 13C15N (Table 1). Both metabolites were, however, also detected in ICR/FT-MS and UPLC-MS spectra from heat-inactivated bacteria. This suggests adsorption of these substrates to bacterial surfaces, a phenomenon that was already noted in the IRMS- based analysis (Fig. S3). Beside glucose and phenylalanine, 34 additional fully or partially 13C-labeled metabolites were detected in ICR/FT-MS spectra of living, but not heat-inactivated, bacteria incubated in DGM-D-13C15N (Table 1 and S1). According to their predicted chemical composition, some of these metabolites August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 6 Metabolic Activity of Protochlamydia EBs Figure 4. Overview of ICR/FT-MS-detected annotated compounds and of metabolites discriminative for living compared to inactivated EBs. An EB-enriched fraction of P. amoebophila was pre-incubated for 40 h in DGM-D, followed by 48 h incubation in DGM-D or DGM- D-13C15N and subsequent mass spectrometric analysis of metabolite extracts. Heat-inactivated EBs incubated in DGM-D-13C15N were included as control. PLOS Pathogens \| www.plospathogens.org Metabolic Activity of Protochlamydia EBs In order to exclude that D-glucose plays a role in infection beyond fueling metabolic reactions, we also assessed whether addition of D-glucose to bacteria that were incubated in DGM-L and thus starved could restore their infection capacity. However, this treatment did not affect the rapid decline of infectivity in DGM-L (Fig. S7), indicating that the supportive effect of D-glucose cannot be explained by a potential promotion of bacterial attachment or entry into host cells that might be mediated by sugar molecules adsorbed to the surface of EBs. Collectively, these findings demonstrate that the availability of D-glucose in the host-free environment is critical for P. amoebophila EBs as it significantly extends maintenance of their major biological role, which is the capacity to successfully infect new host cells and to initiate a new round of intracellular replication. Figure 5. ESI(-)ICR/FT-MS spectra indicating host-free synthesis Figure 5. ESI(-)ICR/FT-MS spectra indicating host-free synthesis of hexose-P by P. amoebophila EBs. An enlarged view on the mass range 265.00–265.12 in ESI(-)ICR/FT-MS spectra of DGM-D- and DGM-D- 13C15N-incubated living and DGM-D-13C15N-incubated inactivated bacteria is shown. Note that the peak indicating fully 13C-labeled hexose-P (13C6H13O9P) can only be seen in spectra from DGM-D- 13C15N-incubated living EBs, but not in the controls, demonstrating host-free synthesis of hexose-P by P. amoebophila EBs. doi:10.1371/journal.ppat.1003553.g005 Figure 5. ESI(-)ICR/FT-MS spectra indicating host-free synthesis of hexose-P by P. amoebophila EBs. An enlarged view on the mass range 265.00–265.12 in ESI(-)ICR/FT-MS spectra of DGM-D- and DGM-D- 13C15N-incubated living and DGM-D-13C15N-incubated inactivated bacteria is shown. Note that the peak indicating fully 13C-labeled hexose-P (13C6H13O9P) can only be seen in spectra from DGM-D- 13C15N-incubated living EBs, but not in the controls, demonstrating host-free synthesis of hexose-P by P. amoebophila EBs. doi:10.1371/journal.ppat.1003553.g005 Influence of Nutrient Availability on Maintenance of C. trachomatis Infectivity In order to explore whether a dependency of host-free EBs on nutrient availability can be observed for the pathogenic Chlamydiaceae, we compared maintenance of C. trachomatis (serovar L2) infectivity in different media. C. trachomatis, in contrast to P. amoebophila, lacks a gene encoding a glucokinase [12,14]. This chlamydial species is thus unable to utilize D- glucose directly and is expected to rely on the phosphorylated derivative instead. The media used for host-free incubation of C. trachomatis consequently included nutrient-free Dulbecco’s phos- phate-buffered saline (DPBS), DGM-D, and DGM-L, as well as DGM containing D-glucose-6-phosphate instead of D-glucose (DGM-D6P) or containing both D-glucose and D-glucose-6- phosphate (DGM-DD6P). Assessment of infectivity for HeLa 229 cells conducted 30 minutes after suspension of purified bacteria in the respective incubation media indicated that already after this short exposure infectivity was significantly decreased in all media devoid of D-glucose-6-phosphate compared to DGM-D6P or DGM-DD6P (p#0.01; ANOVA) (Fig. 7C). In addition, while infectivity remained relatively stable in DGM-D6P (90.8%64.1) and DGM-DD6P (87.3%67.5) during the first 2 h of host-free incubation, a larger reduction was observed in media lacking D-glucose-6-phosphate (DGM-D (59.1%610.0), DGM-L (65.4%63.5), and DPBS (53.0%66.6)) (Fig. 7C–D). Surprisingly, infectivity was almost completely lost after 24 h host-free incubation in all tested media, which contrasts strongly to the prolonged extracellular survival of P. amoebophila. further support the occurrence of anabolic activities in host-free P. amoebophila EBs. Taken together, the ICR/FT-MS and UPLC-MS analyses were fully consistent with the results obtained by IRMS measurements; they provide first detailed information on the central carbon metabolism in host-free P. amoebophila EBs and show the occurrence of both catabolic as well as anabolic reactions. Metabolic Activity of Protochlamydia EBs Metabolic Activity of Protochlamydia EBs Figure 5. ESI(-)ICR/FT-MS spectra indicating host-free synthesis of hexose-P by P. amoebophila EBs. An enlarged view on the mass range 265.00–265.12 in ESI(-)ICR/FT-MS spectra of DGM-D- and DGM-D- 13C15N-incubated living and DGM-D-13C15N-incubated inactivated bacteria is shown. Note that the peak indicating fully 13C-labeled hexose-P (13C6H13O9P) can only be seen in spectra from DGM-D- 13C15N-incubated living EBs, but not in the controls, demonstrating host-free synthesis of hexose-P by P. amoebophila EBs. doi:10.1371/journal.ppat.1003553.g005 be explained by a potential toxicity of L-glucose, as incubation in DGM-DL, a medium containing both sugar stereoisomers, revealed a similar infectivity curve than observed for DGM-D (Fig. 7A). A rapid decline in the number of infectious particles was also observed in PBS and 0.6% NaCl solution. We also noted that in these nutrient-free media the initial infectivity after 2 h incubation was already significantly reduced compared to that of bacteria incubated in DGM-D (p#0.001; ANOVA). This finding suggests that despite having a similar pH and osmolarity these media may also lack other essential components or differ in physicochemical properties required for chlamydial survival or stability. In order to exclude that D-glucose plays a role in infection beyond fueling metabolic reactions, we also assessed whether addition of D-glucose to bacteria that were incubated in DGM-L and thus starved could restore their infection capacity. However, this treatment did not affect the rapid decline of infectivity in DGM-L (Fig. S7), indicating that the supportive effect of D-glucose cannot be explained by a potential promotion of bacterial attachment or entry into host cells that might be mediated by sugar molecules adsorbed to the surface of EBs. be explained by a potential toxicity of L-glucose, as incubation in DGM-DL, a medium containing both sugar stereoisomers, revealed a similar infectivity curve than observed for DGM-D (Fig. 7A). A rapid decline in the number of infectious particles was also observed in PBS and 0.6% NaCl solution. We also noted that in these nutrient-free media the initial infectivity after 2 h incubation was already significantly reduced compared to that of bacteria incubated in DGM-D (p#0.001; ANOVA). This finding suggests that despite having a similar pH and osmolarity these media may also lack other essential components or differ in physicochemical properties required for chlamydial survival or stability. Central Carbon Metabolism of Host-Free P. amoebophila EBs Revealed by Mass Spectrometric Metabolite Analysis In fact, whereas labeled metabolites in the PPP were only detected as fully labeled molecules, clearly demonstrating their origin from D-[U-13C6]- glucose, the mass signal corresponding to biphosphorylated hexoses only indicated the presence of a partially labeled metabolite, which is inconsistent with its generation by glycolytic breakdown of fully labeled D-glucose, but may result from joining of one unlabeled C3 and one fully labeled C3 body by a reverse aldolase reaction during gluconeogenesis. This interpretation is consistent with the detection of traces of partially labeled phosphorylated hexose. In addition, the observed synthesis of a partially labeled disaccharide and traces of a labeled trisaccharide could be annotated as phosphorylated hexose (C6H13O9P, hexose- P) (Fig. 5), biphosphorylated hexose (C6H14O12P2, hexose-PP), a short chain hydroxy acid (C6H12O7, e.g. gluconate), a phosphor- ylated heptose (C7H15O10P, heptose-P), a short chain tricarboxylic acid (C6H8O7; e.g. citrate), a disaccharide (C12H22O11), and a trisaccharide (C18H32O16). UPLC-MS analysis confirmed the presence of these metabolites. Only the detection of bipho- sphorylated hexose could not be verified, due to the absence of the corresponding peak of the labeled and unlabeled compound in UPLC-MS spectra. Moreover, UPLC-MS detected several addi- tional labeled metabolites, such as phosphorylated pentose (C5H11O8P, pentose-P), phosphorylated tetrose (C4H9O7P, tet- rose-P), and a short chain acyl phosphate (C3H5O6P; e.g. phosphoenolpyruvate) (Table 1). could be annotated as phosphorylated hexose (C6H13O9P, hexose- P) (Fig. 5), biphosphorylated hexose (C6H14O12P2, hexose-PP), a short chain hydroxy acid (C6H12O7, e.g. gluconate), a phosphor- ylated heptose (C7H15O10P, heptose-P), a short chain tricarboxylic acid (C6H8O7; e.g. citrate), a disaccharide (C12H22O11), and a trisaccharide (C18H32O16). UPLC-MS analysis confirmed the presence of these metabolites. Only the detection of bipho- sphorylated hexose could not be verified, due to the absence of the corresponding peak of the labeled and unlabeled compound in UPLC-MS spectra. Moreover, UPLC-MS detected several addi- tional labeled metabolites, such as phosphorylated pentose (C5H11O8P, pentose-P), phosphorylated tetrose (C4H9O7P, tet- rose-P), and a short chain acyl phosphate (C3H5O6P; e.g. phosphoenolpyruvate) (Table 1). Altogether, detection of these labeled metabolites indicates synthesis of key intermediates of glycolysis/gluconeogenesis, PPP, and the TCA cycle in host-free P. amoebophila EBs (Fig. 6). Beside the detection of the 13C-labeled isotopologs, most of those August 2013 \| Volume 9 \| Issue 8 \| e1003553 7 Influence of Nutrient Availability on Maintenance of P. amoebophila Infectivity To further explore the biological relevance of the host-free metabolic activity in EBs, which represent the infectious non- replicative developmental stage of chlamydiae [24], we analyzed the effect of nutrient availability on P. amoebophila infectivity. For this purpose host-free incubations were conducted in different media, including DGM-D, the modified medium in which D- glucose was replaced by L-glucose (DGM-L), a medium containing both D- and L-glucose (DGM-DL), as well as a nutrient-free buffer (phosphate-buffered saline (PBS)) or salt solution (0.6% NaCl solution) of similar pH and osmolarity. The capability of incubated bacteria to infect amoebae was then assessed at selected time points during a period of one week. Overall, numbers of infectious particles appeared to decline over time in all tested media (Fig. 7A). In fact, under the applied incubation and infection conditions virtually no infection capability was left after a host-free period of 7 days. Initial infectivity, assessed after 2 h incubation, was similar in DGM-D and DGM-L. However, while it remained stable for the first two days of host-free incubation in DGM-D, a rapid reduction to 8.6% (68.3) infectivity (relative to the initial value in DGM-D) was observed for bacteria incubated for this period of time in DGM-L (Fig. 7A–B) (p#0.001; ANOVA). This finding cannot These findings demonstrate that a dependency of infectivity maintenance on the availability of a metabolizable substrate is not restricted to the amoeba symbiont P. amoebophila, but can also be observed for the human pathogen C. trachomatis. This suggests that sustained metabolic activity in EBs may represent a more general and important feature of chlamydial biology. August 2013 \| Volume 9 \| Issue 8 \| e1003553 August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 8 Metabolic Activity of Protochlamydia EBs Table 1. Annotated 13C-labeled metabolites detected in DGM-D-13C15N-incubated EBs by a combination of ICR/FT-MS and UPLC- MS Table 1. Annotated 13C-labeled metabolites detected in DGM-D-13C15N-incubated EBs by a combination of ICR/FT-MS and UPLC- MS. Annotated 13C-labeled metabolites detected in DGM-D-13C15N-incubated EBs by a combination of ICR/FT Table 1. Annotated 13C-labeled metabolites detected in DGM-D-13C15N-incubated EBs by a combination of ICR/FT-MS and UPLC- MS. Discussion insights into the biology of Chlamydiaceae RBs and EBs [22,39,44,64]. By taking into account the purity of P. amoebophila EB and RB fractions defined by TEM (Fig. S1) [43] in combination with the application of single-cell based assays (Fig. 1, 2, and S2), our data clearly allow us to attribute metabolic activity to the EB stage for the reasons outlined below. The chlamydial EB has long been regarded as a completely metabolically inert particle that can only be reactivated after entry into a suitable eukaryotic host cell. This notion has recently been challenged [43,44] and the data presented in the present study confirm and greatly extend the concept of metabolic activity in the infective stage of chlamydiae. Our findings demonstrate that host- free EBs of P. amoebophila at least temporarily interact with their environment and maintain both catabolic as well as anabolic activities. Furthermore, these activities are of major biological relevance as they contribute to prolonged survival of infectious EBs. Several lines of evidence demonstrate that the observations reported in this study cannot be explained by residual host-derived activities: (i) respiratory activity, inferred from CTC reduction, was detectable in individual bacteria and could be clearly distinguished from the short-lived activity in lysates of uninfected host cells that was lost completely after pre-incubation (Fig. 1 and S2); (ii) import of 2-NBDG, as indicator for D-glucose uptake, could be detected at the single-cell level (Fig. 2); and (iii) D-glucose catabolism, as inferred from the release of 13CO2 from 13C-labeled D-glucose, could be observed in pre-incubated purified EBs, whereas no CO2 production was detected in equally treated highly concentrated host cell lysates (Fig. 3B). The purification of chlamydial developmental forms and hence also a direct investigation of their biological properties, is a challenging task. A perfect enrichment and a complete removal of transition forms, some of which may be close to RBs or EBs but not yet fully differentiated, cannot be achieved [35,43,62,63] nor can a contamination with host proteins be avoided completely [36–40]. Nevertheless, density gradient centrifugation and TEM, as used in the present study, currently represent the most powerful approach for the separation of and discrimination between chlamydial developmental stages and has enabled important Our data also clearly show that metabolic activity cannot be solely explained by co-purified RBs or transition stages, as CTC reduction and 2-NBDG uptake were detectable in about 50% of all bacterial cells (Fig. Influence of Nutrient Availability on Maintenance of P. amoebophila Infectivity b)Labeled metabolites were considered to be present in extracts of DGM-D-13C15N-incubated EBs when peaks corresponding to the exact mass of the unlabeled metabolites were detected in the DGM-D-incubated controls and observed mass shifts were consistent with the exchange of 12C by 13C atoms. Metabolites containing single 13C atoms were also observed in extracts from DGM-D-incubated EBs, due to the natural isotopic distribution of carbon, and were thus excluded. c)The detection method (A: ICR/FT-MS; B: UPLC-MS) is indicated, as well as the ion species detected (H: [M-H+]2; Cl: [M+Cl2]2). d)These metabolites were detected by ICR/FT-MS analysis in purchased D-[U-13C6]-glucose and were thus not considered as host-free synthesized metabolites. doi:10.1371/journal.ppat.1003553.t001 PLOS Pathogens \| www.plospathogens.org August 2013 \| Volume 9 \| Issue 8 \| e1003553 Influence of Nutrient Availability on Maintenance of P. amoebophila Infectivity Detected m/za) Elemental composition Annotation (example) [M-H+]2 [M+Cl2]2 Number of 13C atomsb) Detection methodc) C6H12O6 Glucose 185.07624 221.05292 6d) A(H,Cl); B(H,Cl) 184.07288 220.04957 5d) A(H,Cl); B(H,Cl) C6H13O9P Hexose-P 265.04257 6 A(H); B(H) 264.03923 5 A(H); B(H) 262.03246 3 A(H); B(H) – traces C6H14O12P2 Hexose-PP 377.97560 3 A(Cl) C3H5O6P Phosphoenol-pyruvate 205.961 3 B(Cl) C6H8O7 Citrate 197.040 6 B(H) 196.037 5 B(H) 195.03315 4 A(H); B(H) 194.02979 3 A(H); B(H) 193.026 2 B(H) C5H11O8P Pentose-P 234.029 5 B(H) C7H15O10P Heptose-P 296.05649 7 A(H); B(H) 295.05314 6 A(H); B(H) C4H9O7P Tetrose-P 203.015 4 B(H) 202.011 3 B(H) C6H12O7 Gluconate 201.07116 6 A(H) C12H22O11 Disaccharide 353.14920 389.12587 12d) A(H,Cl); B(H,Cl) 352.146 388.12252 11d) A(Cl); B(H) 383.10575 6 A(Cl) C18H32O16 Trisaccharide 557.19883 18 A(Cl) – traces a)The detected m/z is given for each ion species with the instrument given accuracy. b)Labeled metabolites were considered to be present in extracts of DGM-D-13C15N-incubated EBs when peaks corresponding to the exact mass of the unlabeled metabolites were detected in the DGM-D-incubated controls and observed mass shifts were consistent with the exchange of 12C by 13C atoms. Metabolites containing single 13C atoms were also observed in extracts from DGM-D-incubated EBs, due to the natural isotopic distribution of carbon, and were thus excluded. c)The detection method (A: ICR/FT-MS; B: UPLC-MS) is indicated, as well as the ion species detected (H: [M-H+]2; Cl: [M+Cl2]2). d)These metabolites were detected by ICR/FT-MS analysis in purchased D-[U-13C6]-glucose and were thus not considered as host-free synthesized metabolites. doi:10.1371/journal.ppat.1003553.t001 a)The detected m/z is given for each ion species with the instrument given accuracy. a)The detected m/z is given for each ion species with the instrument given accuracy. b)Labeled metabolites were considered to be present in extracts of DGM-D-13C15N-incubated EBs when peaks corresponding to the exact mass of the unlabeled metabolites were detected in the DGM-D-incubated controls and observed mass shifts were consistent with the exchange of 12C by 13C atoms. Metabolites containing single 13C atoms were also observed in extracts from DGM-D-incubated EBs, due to the natural isotopic distribution of carbon, and were thus excluded. c)The detection method (A: ICR/FT-MS; B: UPLC-MS) is indicated, as well as the ion species detected (H: [M-H+]2; Cl: [M+Cl2]2). d)These metabolites were detected by ICR/FT-MS analysis in purchased D-[U-13C6]-glucose and were thus not considered as host-free synthesized metabolites. doi:10.1371/journal.ppat.1003553.t001 a)The detected m/z is given for each ion species with the instrument given accuracy. Discussion Percentage values next to the bars denote the relative abundance of the isotopologs calculated from the mass signal intensity or peak area of the respective peak (for ICR/FT-MS or UPLC-MS data, respectively) compared to peaks of the unlabeled metabolite detected in DGM-D-incubated bacteria. For citrate, for which all possible isotopologs were detected by UPLC-MS, instead of bars the complete isotopolog profile observed in DGM- D-13C15N-incubated living EBs is shown. In (B), mass signal intensities and peak areas (for ICR/FT-MS or UPLC-MS data, respectively) of selected fully 13C-labeled (‘‘13C’’) and corresponding unlabeled (‘‘12C’’) metabolites are displayed for DGM-D-13C15N-incubated living bacteria and the controls. Note the absence of fully 13C-labeled isotopologs in extracts from inactivated bacteria and the appearance of labeled intermediates and the concomitant reduction in the amount of the corresponding unlabeled metabolite in samples of DGM-D-1315N-incubated living bacteria compared to DGM-D-incubated bacteria. Exceptions were the detection of labeled glucose and phenylalanine in extracts of inactivated bacteria, presumably due to substrate adsorption to the surface of the bacterial cells. doi:10.1371/journal.ppat.1003553.g006 metabolism in P. amoebophila EBs deduced from mass spectrometry-based metabolite analysis. A schemati 13 proportion of active bacteria in the RB fraction dropped to a level similar to the expected proportion of co-purified EBs (Fig. 1). We therefore concluded that RB activity was essentially lost during this 40 h incubation. Based on this observation we exploited the instability of RBs and host-derived activity by including an initial 40 h host-free incubation step as pre-treatment of EB fractions prior to the detailed characterization of their central carbon metabolism. Alternative pre-treatment procedures, which have been applied by investigators in earlier studies on host-free activities of Chlamydiaceae – such as protease treatment to remove host cell-derived enzymes [65–67], sonication [28], or the addition of detergents to cause lysis of the more fragile RBs [68,69] – were avoided in the present study due to potential detrimental effects on bacterial surface proteins or EB viability. that consists of about 76% mature EBs (defined as bacteria containing only electron-dense and electron-lucent material). The high proportion of inactive bacteria may be explained by the presence of dead bacteria. This would be consistent with previous observations that revealed that directly after harvesting of bacteria from infected amoeba cultures a significant proportion of the bacteria could be stained with the membrane-impermeable DNA dye propidium iodide, indicating that they had lost their membrane integrity [43]. Discussion 1 and 2) in a highly enriched EB fraction August 2013 \| Volume 9 \| Issue 8 \| e1003553 August 2013 \| Volume 9 \| Issue 8 \| e1003553 9 Metabolic Activity of Protochlamydia EBs Figure 6. Central carbon metabolism in P. amoebophila EBs deduced from mass spectrometry-based metabolite analysis. A schematic representation of the central carbon metabolism in P. amoebophila is shown in (A). 13C-labeled metabolites detected by ICR/FT-MS or UPLC-MS in extracts of DGM-D-13C15N-incubated living bacteria are indicated. The isotopologs that were observed are additionally specified by bars, consisting of a number of units equal to the number of C atoms in the molecules, whereby each unit of the bar indicates either a 12C (white) or a 13C (gray) atom. Percentage values next to the bars denote the relative abundance of the isotopologs calculated from the mass signal intensity or peak area of the respective peak (for ICR/FT-MS or UPLC-MS data, respectively) compared to peaks of the unlabeled metabolite detected in DGM-D-incubated bacteria. For citrate, for which all possible isotopologs were detected by UPLC-MS, instead of bars the complete isotopolog profile observed in DGM- D-13C15N-incubated living EBs is shown. In (B), mass signal intensities and peak areas (for ICR/FT-MS or UPLC-MS data, respectively) of selected fully 13C-labeled (‘‘13C’’) and corresponding unlabeled (‘‘12C’’) metabolites are displayed for DGM-D-13C15N-incubated living bacteria and the controls. Note the absence of fully 13C-labeled isotopologs in extracts from inactivated bacteria and the appearance of labeled intermediates and the concomitant reduction in the amount of the corresponding unlabeled metabolite in samples of DGM-D-1315N-incubated living bacteria compared to DGM-D-incubated bacteria. Exceptions were the detection of labeled glucose and phenylalanine in extracts of inactivated bacteria, presumably due to substrate adsorption to the surface of the bacterial cells. doi:10.1371/journal.ppat.1003553.g006 Figure 6. Central carbon metabolism in P. amoebophila EBs deduced from mass spectrometry-based metabolite analysis. A schematic representation of the central carbon metabolism in P. amoebophila is shown in (A). 13C-labeled metabolites detected by ICR/FT-MS or UPLC-MS in extracts of DGM-D-13C15N-incubated living bacteria are indicated. The isotopologs that were observed are additionally specified by bars, consisting of a number of units equal to the number of C atoms in the molecules, whereby each unit of the bar indicates either a 12C (white) or a 13C (gray) atom. Discussion Consistent with reports on Chlamydiaceae [22,35,44], in particular the RBs of P. amoebophila appeared to be very fragile, and hence clear differences in the host-free maintenance of respiratory activity were observed between developmental stages of P. amoebophila. Whereas EBs maintained their ability to reduce CTC during a 40 h incubation period, the August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 10 Metabolic Activity of Protochlamydia EBs Figure 7. Effect of substrate availability on maintenance of infectivity. P. amoebophila and C. trachomatis cells were harvested from infected amoeba and HeLa 229 cell cultures, respectively, and incubated for indicated periods of time in different host-free media. Subsequently, incubated bacteria were used to infect amoebae (P. amoebophila) or HeLa 229 cells (C. trachomatis), which were then fixed at 48 h or 24 h p.i., respectively. Bacteria were detected by FISH (P. amoebophila) or immunostaining (C. trachomatis). The observed infectivity, relative to that observed for 2 h incubation in DGM-D (P. amoebophila) or 30 min incubation in DGM-D6P (C. trachomatis) is depicted in (A) and (C), respectively. Data represent means and standard deviations from at least three replicate host-free incubations. For each sample a minimum of 600 amoebae (A) or 300 HeLa 229 cells (C) was counted. Statistically significant differences compared to the values obtained for DGM-D (A) or DGM-D6P (C) are indicated (ANOVA; , p#0.001; , p#0.01; , p#0.05). In (B) representative fluorescence and DIC images of amoebae infected with P. amoebophila after 48 h host-free incubation in the indicated media are shown (FISH, red). The bar indicates 10 mm. In (D) representative confocal fluorescence images of HeLa 229 cells infected with C. trachomatis after 2 h host-free incubation in the indicated media are shown. Bacteria were detected by immunostaining (red), host cells and DNA were stained using HCS cytoplasmic stain (grey) and DAPI (blue), respectively. The bar indicates 25 mm. doi:10.1371/journal.ppat.1003553.g007 Metabolic Activity of Protochlamydia EBs Figure 7. Effect of substrate availability on maintenance of infectivity. P. amoebophila and C. trachomatis cells were harvested from infected amoeba and HeLa 229 cell cultures, respectively, and incubated for indicated periods of time in different host-free media. Subsequently, incubated bacteria were used to infect amoebae (P. amoebophila) or HeLa 229 cells (C. trachomatis), which were then fixed at 48 h or 24 h p.i., respectively. Bacteria were detected by FISH (P. amoebophila) or immunostaining (C. trachomatis). PLOS Pathogens \| www.plospathogens.org Metabolic Activity of Protochlamydia EBs Metabolic Activity of Protochlamydia EBs and EBs, but so far mainly attributed to the replicative stage, remained stable for at least two days [66,73]. This finding appears to be inconsistent with the known fragility of RBs and thus may provide further evidence for metabolic activity in the infective stage. A biological role for metabolic activities in the EB stage was furthermore indicated by a direct comparison of the protein complements of C. trachomatis EBs and RBs, which demonstrated that proteins required for the central metabolism and glucose catabolism were, in fact, even predominant in the infective stage [39]. synthesis were detected in host-free Chlamydiaceae [76–79]. In contrast, while our findings from IRMS and the mass spectrom- etry-based metabolite analysis also indicate an involvement of the PPP in sugar metabolism and suggest occurrence of host-free anabolic reactions in P. amoebophila EBs, they additionally demonstrate that these bacteria are able to use non-phosphory- lated D-glucose and that this sugar is at least to some extent completely catabolized via the TCA cycle (Fig. 3B, 5, and 6, Table 1). and EBs, but so far mainly attributed to the replicative stage, remained stable for at least two days [66,73]. This finding appears to be inconsistent with the known fragility of RBs and thus may provide further evidence for metabolic activity in the infective stage. A biological role for metabolic activities in the EB stage was furthermore indicated by a direct comparison of the protein complements of C. trachomatis EBs and RBs, which demonstrated that proteins required for the central metabolism and glucose catabolism were, in fact, even predominant in the infective stage [39]. Some of the host-free metabolic activities detected in P. amoebophila EBs are indeed not expected to occur in exactly the same manner in EBs of other chlamydial species due to known differences in their genomic repertoire. P. amoebophila, in contrast to Chlamydiaceae, encodes a glucokinase (glk, pc0935, UniProtKB Q6MCP0), which is required to activate D-glucose for metabolic reactions, and a complete enzyme set required for host-indepen- dent operation of the TCA cycle [14,21]. Glucokinase, as well as most other enzymes involved in glycolysis/gluconeogenesis, PPP, TCA cycle, and in the electron transport chain, were recently also detected in the P. amoebophila EB proteome [40]. The mechanism by which D-glucose is imported in P. amoebophila remains to be elucidated, as the genome encodes a putative glucose-6-phosphate transporter (uhpC, pc0387, UniProtKB Q6ME88), the C. Discussion The observed infectivity, relative to that observed for 2 h incubation in DGM-D (P. amoebophila) or 30 min incubation in DGM-D6P (C. trachomatis) is depicted in (A) and (C), respectively. Data represent means and standard deviations from at least three replicate host-free incubations. For each sample a minimum of 600 amoebae (A) or 300 HeLa 229 cells (C) was counted. Statistically significant differences compared to the values obtained for DGM-D (A) or DGM-D6P (C) are indicated (ANOVA; , p#0.001; , p#0.01; , p#0.05). In (B) representative fluorescence and DIC images of amoebae infected with P. amoebophila after 48 h host-free incubation in the indicated media are shown (FISH, red). The bar indicates 10 mm. In (D) representative confocal fluorescence images of HeLa 229 cells infected with C. trachomatis after 2 h host-free incubation in the indicated media are shown. Bacteria were detected by immunostaining (red), host cells and DNA were stained using HCS cytoplasmic stain (grey) and DAPI (blue), respectively. The bar indicates 25 mm. doi:10.1371/journal.ppat.1003553.g007 Altogether, the findings presented in this study clearly demonstrate metabolic activity in P. amoebophila EBs and thus strongly support recent appeals to revise the dogma of the metabolic inertness of the infective stage of chlamydiae [43,44]. New methodological approaches, differences in media composi- tion and purification and pre-treatment of EBs before assessment of activity might explain conflicting observations in previous studies [51,68–70]. Indeed, the possibility of sustained activities in EBs, although not generally recognized by the scientific community, has already been indicated in a few earlier studies. For example, it has been shown that nucleoid decondensation during the redifferentiation of C. trachomatis EBs to RBs shortly after their uptake into host cells depends on bacterial de novo transcription and translation, implying that the capability of a certain level of activity must be maintained [71]. Consistently, Sarov and Becker could show earlier that purified EBs of C. trachomatis were able to synthesize RNA [72], and our previous findings suggested de novo protein synthesis in host-free C. trachomatis and P. amoebophila EBs [43]. Both findings could very recently be confirmed for C. trachomatis [44]. In the context of carbon metabolism, early investigations of host-free Chlamydiaceae also indicated that activities detected in mixed suspensions of RBs August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 11 Metabolic Activity of Protochlamydia EBs Metabolic Activity of Protochlamydia EBs [80], but does not provide evidence for a known importer for the non-phosphorylated sugar [14]. Early studies on host-free activities of Chlamydiaceae indicated that phosphorylation of D-glucose in suspensions of bacteria may also occur through the activity of a co- purified host-derived hexokinase [65]. However, this activity was shown to be strictly dependent on the availability of extensive amounts of ATP in the incubation medium [73]. Due to the pre- incubation step and the absence of externally added ATP we thus exclude significant contributions of host-derived kinase activity in the experiments presented in the present study. indispensable for maintaining the intracellular redox homeostasis, which in turn sustains protein function and counteracts oxidative stress [81]. An additional physiological function of D-glucose metabolism in EBs might be indicated by the detected synthesis of a disaccharide (Table 1) that could play a role in osmoprotection, though other functions, such as carbon storage, may be equally plausible. The biological relevance of host-free activity of chlamydiae and its implications for their lifestyle have, to our knowledge, not been assessed before. A recent study that focused on C. trachomatis and several environmental chlamydiae reported differences in infec- tivity maintenance in nutrient-rich growth medium compared to sterile tap water [82]. The effects of the fundamentally different physicochemical properties of these incubation media, however, prohibited a correlation of the observed differences with nutrient availability. This is well illustrated by our observation of that P. amoebophila infectivity is markedly decreased after only a 2 h incubation in PBS or NaCl solution (Fig. 7A). In the present study, however, we demonstrate that the absence of D-glucose alone during host-free incubation of P. amoebophila, achieved by an exchange with its non-metabolizable stereoisomer, which does not affect the medium osmolarity, pH, or overall composition, is sufficient to cause a rapid decrease in the number of infectious particles (Fig. 7A–B). Likewise, exchange of D-glucose-6-phos- phate with D-glucose resulted in a more rapid decline of C. trachomatis infectivity, consistent with the inability of this chlamyd- ial species to utilize the non-phosphorylated compound (Fig. 7C– D). Addition of D-glucose to starved P. amoebophila failed to restore infectivity (Fig. S7), which indicates that a continuous supply of metabolizable substrates is required for the host-free survival of infectious EBs and thus demonstrates that metabolic activity in EBs is linked to their biological role as a dispersal stage. Metabolic Activity of Protochlamydia EBs The more rapid decline of infectivity observed for C. trachomatis compared to P. amoebophila during host-free incubation may reflect differences in their metabolic potential, their requirement for host-free survival, or their adaptation to a host-free environment. Several lines of evidence suggest that host-free P. amoebophila EBs co-utilize medium-derived D-glucose with other internal or external carbon compounds that may even partially substitute for the sugar in its absence. Accordingly, during conditions of D- glucose deprivation, i.e. in DGM-L, CTC reducing activity was still detectable in a small proportion of the bacteria (Fig. 1 and S2F), suggesting that EBs may contain storage compounds, such as glycogen that may compensate D-glucose shortage for a period of time. The abundance patterns of differently labeled metabolic intermediates and the co-occurrence of unlabeled metabolites detected during the mass spectrometry-based analysis indicate an additional contribution of alternative substrates even when D- glucose is present in the extracellular medium. Thus, the fact that partially labeled citrate that contained predominately either three or four 13C atoms could be detected after incubation with fully labeled D-glucose (Fig. 6, Table 1) can only be explained by a mixed entry of labeled and unlabeled molecules into the TCA cycle. However, the high relative abundance of labeled metabolites in the PPP (59–94%, Fig. 6), as well as the fact that the absolute abundance of unlabeled molecules increased with proximity to the TCA cycle, contradicts extensive utilization of D-glucose from storage compounds during incubation in the nutrient-rich medium, but rather suggests a predominant co-utilization of substrates that can enter the central carbon metabolism at a level further downstream. These may include products from protein or lipid degradation or, even more likely considering their availability in the medium, imported amino acids. In fact, a potential utilization of amino acids is supported by a recent IRMS-based analysis conducted in our lab, which indicated that besides L- phenylalanine also L-glutamate, L-aspartate, and L-threonine could be imported by host-free P. amoebophila (data not shown). In addition, L-glutamate has also been proposed to represent a major carbon source for C. trachomatis based on their predicted metabolic repertoire [12]. However, the fact that CO2 production could not be detected for P. amoebophila incubated in DGM-L (Fig. 3B) and the rapid loss of its metabolic activity in this medium (Fig. Metabolic Activity of Protochlamydia EBs 1 and S2), demonstrate that these additional carbon compounds are less effective than D-glucose, which thus appears to be an essential nutrient for maintenance of metabolic activity in host-free P. amoebophila EBs under the applied conditions. A similar substrate dependence may occur in C. trachomatis EBs, for which it was very recently shown that host-free metabolic activity can be greatly enhanced by the presence of D-glucose-6-phosphate [44]. In conclusion, we provide evidence for D-glucose utilization and metabolic activity in host-free P. amoebophila EBs, which disagrees with the current perception of the infectious stage of chlamydiae as being metabolically inert and further establishes the chlamydial EB as being a developmental stage with a defined metabolic activity. The observed link between the availability of a metabolizable substrate and survival of infectious particles observed for P. amoebophila and C. trachomatis implies that the detection of metabolic activity in EBs is relevant for the main biological function of this infective stage, which thus appears to be more sensitive to its environment than has been thus far appreciated. PLOS Pathogens \| www.plospathogens.org Metabolic Activity of Protochlamydia EBs pneumo- niae homolog of which has recently been functionally characterized In addition to these conceptual considerations, the present study represents a hitherto unmatched detailed metabolomic analysis of a member of the Chlamydiae. It provides invaluable insights into the central carbon metabolism of P. amoebophila (Fig. 8) and indicates both similarities as well as major differences to observations reported for the Chlamydiaceae. While it was initially proposed that the latter can metabolize D-glucose under host-free conditions, as inferred from the production of 14CO2 from 14C-labeled D- glucose [74,75], it was subsequently shown that the starting point for sugar catabolism in these bacteria is D-glucose-6-phosphate [65]. Moreover, experiments with substrates labeled at various carbon atoms revealed a combined action of glycolysis and PPP, yet complete TCA cycle activity could not be demonstrated [66,74,75]. In addition, anabolic reactions such as lipid and folate Figure 8. Schematic representation of host-free activity of P. amoebophila EBs. A metabolic model based on current knowledge is shown. The representation integrates observations from our previous investigations [43] and new findings obtained in the current study. Metabolic pathways and enzymes are illustrated in red, detected and postulated metabolites in blue. The techniques that provided experimental support for the indicated activities are shown in green (‘‘ICR & UPLC’’ denotes activities confirmed by both, ICR/FT-MS and UPLC-MS). Dotted lines indicate metabolic reactions, whose occurrence in EBs was suggested, but not demonstrated. The electron transport chain, amino acid transporters, as well as a putative D-glucose importer, are depicted in the bacterial membrane as gray boxes. doi:10.1371/journal.ppat.1003553.g008 Figure 8. Schematic representation of host-free activity of P. amoebophila EBs. A metabolic model based on current knowledge is shown. The representation integrates observations from our previous investigations [43] and new findings obtained in the current study. Metabolic pathways and enzymes are illustrated in red, detected and postulated metabolites in blue. The techniques that provided experimental support for the indicated activities are shown in green (‘‘ICR & UPLC’’ denotes activities confirmed by both, ICR/FT-MS and UPLC-MS). Dotted lines indicate metabolic reactions, whose occurrence in EBs was suggested, but not demonstrated. The electron transport chain, amino acid transporters, as well as a putative D-glucose importer, are depicted in the bacterial membrane as gray boxes. doi:10.1371/journal.ppat.1003553.g008 August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 12 Media for Extracellular Incubation Extracellular incubations of P. amoebophila and C. trachomatis were conducted in media that were based on the chemically defined Acanthamoeba medium DGM-21A [42], but that, as a modification, contained L-phenylalanine (5.4 mM) instead of the racemic mixture DL-phenylalanine and were additionally supplemented with 0.25 g/l NaHCO3. Specific media used in this study included DGM-D, which like the original DGM-21A contained 83.2 mM D-glucose, DGM-D/2 with a reduced D-glucose concentration of 41.6 mM, DGM-L containing 83.2 mM L-glucose instead of D- glucose, DGM-DL containing both 83.2 mM D-glucose and 83.2 mM L-glucose, DGM-D6P containing 83.2 mM D-glucose- 6-phosphate instead of D-glucose, and DGM-DD6P containing both 83.2 mM D-glucose and 83.2 mM D-glucose-6-phosphate. In addition, DGM-D-based media in which certain endogenous substrates were completely replaced by stable isotope-labeled variants were also used. These media included DGM-D-13C (containing fully 13C-labeled D-glucose (D-[U-13C6]-glucose, 99%)), DGM-D-1-13C (containing D-glucose labeled at carbon 1 (D-[1-13C]-glucose, 98–99%)), DGM-D-6-13C (containing D- glucose labeled at carbon 6 (D-[6-13C]-glucose, 99%)), and DGM- D-13C15N (containing D-[U-13C6]-glucose and fully 13C15N- labeled L-phenylalanine (L-[U-13C9,15N]-phenylalanine, 97– 99%)). The composition of all incubation media is summarized in Table S2. Stable isotope-labeled substrates were purchased from Euriso-top. Cell Culture Acanthamoeba sp. UWC1 containing P. amoebophila UWE25 [7] and symbiont-free isogenic amoebae were maintained at 20uC in TSY medium (30 g/l trypticase soy broth, 10 g/l yeast extract). HeLa 229 cells (ATCC, CCL-2.1) were cultivated at 37uC, 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitro- gen) supplemented with 10% fetal bovine serum (PAA). C. trachomatis L2 was propagated in HeLa 229 cells by transfer of supernatant from infected to uninfected cultures every 2–3 days. Cultures were regularly screened for contamination by fluores- cence microscopy using the DNA dye DAPI and fluorescence in situ hybridization (FISH). Mammalian cells were additionally shown to be free of contamination with Mycoplasma spp. by using The exclusive detection of only partially labeled six-carbon compounds in the glycolytic/gluconeogenic pathway, most likely derived from joining of 12C and 13C precursors, together with the observation that for all detected intermediates in the PPP a high proportion of the metabolite pool appeared to be fully labeled (Fig. 6), suggests that the latter represents the preferred route of D- glucose catabolism in host-free P. amoebophila. In addition to being an alternative pathway for the breakdown of sugars into products that can be further metabolized for ATP generation, the PPP represents the main route for regeneration of NADPH, a reducing agent that is not only required for lipid synthesis, but is also August 2013 \| Volume 9 \| Issue 8 \| e1003553 13 Metabolic Activity of Protochlamydia EBs the Venor GeM PCR kit (Minerva Biolab).The identity of the chlamydiae was verified by 16S rRNA gene sequence analysis, as described recently [83]. Acanthamoeba sp. UWC1 were harvested at 32006 g, washed with PAS, and resuspended in 6.5 ml sucrose-phosphate-glutamate buffer (75 g/l sucrose, 0.52 g/l KH2PO4, 1.53 g/l Na2H- PO462H2O, 0.75 g/l glutamic acid) per 1 g wet weight. After cell disruption on ice by using a dounce homogenizer (Wheaton), the suspension was filtrated (1.2 mm). Cell debris was collected at 12 8606 g (10 min, 4uC), resuspended in DGM-D, and analyzed immediately or after a 40 h pre-incubation (27uC, 200 rpm). Sample Preparation for IRMS Analysis 13 For the detection of 13C-D-glucose uptake and catabolism by IRMS, a pre-incubated EB-enriched fraction or a highly pure EB fraction were applied for the first and second experimental approach, respectively. After pre-incubation, the suspension of bacteria was mixed by vortexing and transferred to 1.5 ml tubes, so that each tube contained the same number of cells. Bacteria were then collected by centrifugation (10 6206 g, 10 min), Purification of P. amoebophila EBs and RBs Recently, we developed a protocol for an efficient purification of P. amoebophila EBs and RBs and investigated the purity of obtained fractions by an ultrastructural analysis (Fig. S1) [43]. In the current study a slightly modified protocol, including thicker layers in the density gradients for improved phase separation, was applied. Briefly, after host cell disruption and filtration, released bacteria were directly subjected to density gradient centrifugation using a gradient consisting of 3.5 ml 30% (v/v) gastrografin (Bayer Schering Pharma) in Page’s amoebic saline (PAS; 0.12 g/l NaCl, 0.004 g/l MgSO467H2O, 0.004 g/l CaCl262H2O, 0.142 g/l Na2HPO4, 0.136 g/l KH2PO4) and 3.5 ml 50% (w/v) sucrose. The gradient applied during the second centrifugation consisted either of 3.5 ml 34% (v/v) and 3.5 ml 40% (v/v) gastrografin or of 3.5 ml 40% (v/v) and 3.5 ml 46% (v/v) gastrografin. RB-enriched and intermediate fractions were collected at the 34/40% or 40/ 46% interface, respectively. A highly enriched EB fraction was collected at the bottom of the 40/46% gradient. For applications requiring large amounts of biomass, such as the analysis of biomass by IRMS or the preparation of metabolite extracts for ICR/FT- MS and UPLC-MS analysis, the EB-enriched pellet of the 34/ 40% gastrografin gradient (in this study explicitly referred to as ‘‘EB-enriched fraction’’) was used instead. Purified bacteria were washed once in 10 ml PAS (12 8606 g, 10 min), resuspended in DGM-D, and analyzed immediately or after a 40 h pre-incubation (27uC, 200 rpm). Bacteria that were heat-inactivated (30 min, 80uC, 700 rpm) directly before assessment of activity were included as control when indicated. Fluorescence Microscopic Detection of D-Glucose Uptake Purified living and heat-inactivated EBs were analyzed for D- glucose uptake either immediately or after pre-incubation. For that purpose, bacteria were collected by centrifugation (10 6206 g, 10 min, 4uC), resuspended in 220 ml DGM-D/2 containing 100 mM 2-NBDG (Invitrogen), and incubated for 10 h (27uC, 200 rpm). After the incubation, bacteria were washed once with 1 ml PBS and transferred to microscope slides. Images were obtained as described above. The percentage of stained cells was determined for three separate incubations (representing 2 inde- pendent experiments), for each of which at least 500 bacterial cells were evaluated. Fluorescence Microscopic Detection of Respiratory Activity y Respiratory activity in fractions of living or heat-inactivated bacteria and in host lysates was analyzed immediately or after pre- incubation. For that purpose, bacteria or cell debris were collected by centrifugation (10 6206g, 10 min, 4uC), resuspended in 220 ml DGM-D containing 5 mM CTC (Sigma-Aldrich), and incubated for 2 h (27uC, 200 rpm). For the assessment of the effect of D- glucose deprivation, purified bacteria were washed twice in 1 ml DGM-L directly after the purification and both the pre-incubation and the incubation with CTC were subsequently conducted in DGM-L. After the incubation with CTC, bacteria and amoebal cell debris were collected at 10 6206 g (10 min), fixed in 400 ml 4% formaldehyde (15 min, room temperature), and washed once with 1 ml PBS (10 mM NaxPO4, 0.76% NaCl (if not stated otherwise), pH 7.3). Pellets were then resuspended in a small volume PBS and transferred to microscope slides. Bacteria and cell debris were dried (46uC), stained with DAPI (0.5 mg/ml in PBS, 10 min), washed once with PBS, and embedded in mowiol [84]. Images were taken with a CCD camera (AxioCam HRc; Carl Zeiss) connected to an epifluorescence microscope (Axioplan 2 imaging; Carl Zeiss). The percentage of DAPI-stained bacteria containing red fluorescent CTC crystals was determined. At least 1500 bacteria per sample, 500 for each of three replicate wells on the microscope slide, were counted. In addition, the efficiency of DAPI staining, i.e. the percentage of bacteria observable in DIC that could be detected by DAPI staining, was also assessed. For that purpose, at least 450 bacteria per sample, including 150 per replicate well, were considered. Data were collected for three independent experiments (throughout this study, the term ‘‘independent experiments’’ refers to experiments conducted with separate purifications of bacteria). Purification of P. amoebophila EBs and RBs PLOS Pathogens \| www.plospathogens.org August 2013 \| Volume 9 \| Issue 8 \| e1003553 Preparation of Host Cell Lysates CO2 production ppm=ml ½ ~ CO2 ½ Sample{ CO2 ½ Blank1z CO2 ½ Blank2 2 ð1Þ APE13C~At%13CSample{ At%13CBlank1zAt%13CBlank2 2 ð2Þ CO2 production ppm=ml ½ ~ CO2 ½ Sample{ CO2 ½ Blank1z CO2 ½ Blank2 2 ð1Þ ð1Þ APE13C~At%13CSample{ At%13CBlank1zAt%13CBlank2 2 ð2Þ ð2Þ In these equations, [CO2] and At%13C represent the measured values for CO2 (in ppm/ml) and At%13C for the sample and for the two corresponding blank incubations (i.e. incubations of bacteria-free media) of the respective experiment. Based on the APE13C observed for incubations in media with differently labeled D-glucose isotopologs the contributions of different metabolic pathways to 13CO2 release were estimated using a script written in Python 2.7.2. The working procedure of the calculation is depicted in Fig. S4. Bacterial 13C incorporation (per mg DW) was calculated for each sample by applying equation (3). 13C incorporation nmol13C mg DW ~ Amt%C=100 ð Þ APE13C 100 1=M13C ð Þ 106 ð3Þ ð3Þ In this equation, Amt%C indicates the measured proportional amount of carbon in the sample and M13C the atomic mass of 13C (13.00335). The APE13C is calculated from the measured At%13C of the sample by subtracting the mean of the At%13C of the two blank incubations of the respective experiment (i.e. bacteria incubated in DGM-D) (as described in equation (2)). Sample Preparation for Metabolite Analysis For the analysis of metabolite extracts, a pre-incubated EB- enriched fraction was mixed by vortexing and transferred to 1.5 ml tubes, so that each tube contained the same number of cells. After centrifugation (10 6206 g, 10 min), bacteria were resuspended in 1 ml of the respective incubation medium (DGM- D or DGM-D-13C15N) and incubated for 48 h (27uC, 200 rpm). Bacteria that were heat-inactivated prior to incubation in DGM- D-13C15N were included as control. For preparation of metab- olite extracts, bacteria were centrifuged (20 8206 g, 5 min, 4uC) and washed once with 1.5 ml cold PBS. Bacterial pellets were then resuspended in 400 ml cold (220uC) methanol ($99.9%, CHRO- MASOLV, Fluka), frozen in liquid nitrogen (1 min), and thawed on ice, followed by two cycles consisting of vortexing (30 sec) and 5 min incubation on ice. After centrifugation (20 8206 g, 5 min, 4uC) extracts were transferred to pre-cooled 1.5 ml tubes and pellets were extracted a second time with 400 ml of a cold (220uC) 1:1 mixture of methanol ($99.9%, CHROMASOLV) and water (LC-MS Ultra CHROMASOLV, Fluka) by the same procedure as described above. Both extracts were pooled and stored at 280uC until analysis. As extraction blank, empty 1.5 ml tubes were subjected to the same extraction procedure. Metabolite extracts were obtained for three independent experiments each consisting of two replicate incubations per incubation condition. Bacterial numbers, which were assessed by quantification of bacterial particles in an aliquot that was withdrawn from the bacterial suspension during the pre-incubation, were highly similar between replicate experiments, ranging from 2.66109 to 2.86109 bacteria per incubation. Preparation of Host Cell Lysates Host cell lysates, that were included as negative control when indicated, were prepared analogously to the first steps applied during the purification of bacteria [43]. Briefly, uninfected August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 14 Metabolic Activity of Protochlamydia EBs resuspended in 1 ml of the respective incubation medium (DGM- D, DGM-L, DGM-D-13C, DGM-D-1-13C, or DGM-D-6-13C), and transferred to 15 ml glass vials that were subsequently closed with gas impermeable butyl rubber stoppers (GMT) and incubated for 48 h (27uC, 200 rpm). Prior to the incubation in DGM-L, bacteria were washed twice in 1 ml of this medium. When indicated, incubations of heat-inactivated bacteria or host cell lysates in DGM-D-13C were included as control. After the 48 h period, gas and biomass samples were collected and pre-processed for IRMS analysis. For the CO2 measurements, defined volumes (between 6.5 and 7.5 ml per sample) of the head-space of the incubations, as well as of parallel incubations of bacteria-free media (that served as blanks for the gas measurements), were collected with a syringe, transferred to evacuated glass tubes (exetainers, 12 ml, Labco), and brought to a volume of 15 ml with N2 gas prior to IRMS analysis. For the biomass measurements, incubated bacteria were collected by centrifugation (20 8206 g, 5 min, 4uC) and washed twice with 1 ml PBS. Prior to the wash steps, bacteria incubated in DGM-D, which served as blank for biomass measurements, were shortly washed in DGM-D-13C to allow substrate adsorption to the surface of the bacteria. Bacterial pellets were finally heat-inactivated (80uC, 10 min), dried for 6 h in a speedvac (Concentrator 5301, Eppendorf) and subsequently overnight at 60uC. Defined amounts of biomass (between 0.3 and 0.5 mg per sample) were transferred to tin capsules and subjected to IRMS analysis. Prior to the processing of biomass for IRMS, aliquots were withdrawn from bacterial suspensions for the quantification of bacterial particles using a previously described procedure [83]. Applied bacterial numbers were similar between replicate experiments (between 3.96108 and 5.96109 or between 6.36108 and 1.06109 bacteria per incubation, for the first and second experimental approach, respectively). The applied amount of host cell lysate exceeded that of the bacterial biomass, based on the size of the pellets. Samples for IRMS measurements were obtained from three independent experiments each consisting of two replicate incubations per condition. Preparation of Host Cell Lysates An exception was the incubation in DGM-L, for which only two independent experi- ments were conducted. resuspended in 1 ml of the respective incubation medium (DGM- D, DGM-L, DGM-D-13C, DGM-D-1-13C, or DGM-D-6-13C), and transferred to 15 ml glass vials that were subsequently closed with gas impermeable butyl rubber stoppers (GMT) and incubated for 48 h (27uC, 200 rpm). Prior to the incubation in DGM-L, bacteria were washed twice in 1 ml of this medium. When indicated, incubations of heat-inactivated bacteria or host cell lysates in DGM-D-13C were included as control. After the 48 h period, gas and biomass samples were collected and pre-processed for IRMS analysis. For the CO2 measurements, defined volumes (between 6.5 and 7.5 ml per sample) of the head-space of the incubations, as well as of parallel incubations of bacteria-free media (that served as blanks for the gas measurements), were collected with a syringe, transferred to evacuated glass tubes (exetainers, 12 ml, Labco), and brought to a volume of 15 ml with N2 gas prior to IRMS analysis. For the biomass measurements, incubated bacteria were collected by centrifugation (20 8206 g, 5 min, 4uC) and washed twice with 1 ml PBS. Prior to the wash steps, bacteria incubated in DGM-D, which served as blank for biomass measurements, were shortly washed in DGM-D-13C to allow substrate adsorption to the surface of the bacteria. Bacterial pellets were finally heat-inactivated (80uC, 10 min), dried for 6 h in a speedvac (Concentrator 5301, Eppendorf) and subsequently overnight at 60uC. Defined amounts of biomass (between 0.3 and 0.5 mg per sample) were transferred to tin capsules and subjected to IRMS analysis. Prior to the processing of biomass for IRMS, aliquots were withdrawn from bacterial suspensions for the quantification of bacterial particles using a previously described procedure [83]. Applied bacterial numbers were similar between replicate experiments (between 3.96108 and 5.96109 or between 6.36108 and 1.06109 bacteria per incubation, for the first and second experimental approach, respectively). The applied amount of host cell lysate exceeded that of the bacterial biomass, based on the size of the pellets. Samples for IRMS measurements were obtained from three independent experiments each consisting of two replicate incubations per condition. An exception was the incubation in DGM-L, for which only two independent experi- ments were conducted. Infectivity Assay (P. amoebophila) For the analysis of the effect of nutrient availability on infectivity, P. amoebophila were purified from host cells using a protocol without density gradient centrifugation in order to mimic more closely the natural situation of bacterial dispersal in the environment. Briefly, Acanthamoeba sp. UWC1 infected with P. amoebophila, as well as released bacteria in supernatants of respective cultures, were harvested and washed, followed by disruption of host cells, as described for the purification of EBs by density gradient centrifugation [43]. After filtration (1.2 mm), bacteria were collected by centrifugation (12 8006 g, 10 min), washed once with 20 ml PBS, and then resuspended in a small volume of PBS. A small aliquot was withdrawn for the quantification of bacterial particles (see above). The bacterial suspension was then transferred into 1.5 ml tubes for parallel host- free incubations in different media, including a 0.6% NaCl solution, PBS (containing 0.6% NaCl), DGM-D, DGM-L, and DGM-DL. After centrifugation, bacteria were resuspended in the respective media and incubated at 27uC (200 rpm) for 2, 48, 94, or 168 h before being added to amoebae that had been seeded into a 24-well plate (Nunc; 16105 cells per well) at a multiplicity of infection (MOI) of 5. Amoebae and bacteria in each well were mixed by pipetting, followed by 15 min incubation at 27uC, centrifugation (1306g, 15 min, 23uC), and incubation at 27uC for 48 h. Amoebae were then transferred to microscope slides, fixed with 4% formaldehyde (10 min, room temperature), and washed with PAS. For the detection of bacteria by FISH, cells were dehydrated by incubation in increasing concentrations of ethanol (50%, 80% and 96%, 3 min incubation with each) and hybridized with a combination of two Cy3-labeled probes, targeting different positions at the 16S rRNA, to increase signal strength. Applied probes (purchased from Thermo Fisher Scientific) included the Chlamydiae-specific probe Chls-0523 [89] and the probe E25-454 (59-GGA TGT TAG CCA GCT CAT-39) that had been designed to target P. amoebophila. Hybridization occurred at 46uC for 1.5 h at a formamide concentration of 20%, using hybridization and wash buffers described elsewhere [90]. Cells were embedded in mowiol and images were taken as described above. The percentage of infected cells (containing at least six intracellular bacteria) was determined taking into account a minimum of 600 cells per sample. Infectivity was expressed relative to the infectivity observed for bacteria incubated for 2 h in DGM-D. Infectivity Assay (P. amoebophila) Data were collected for at least three independent experiments, each consisting of three parallel host-free incubations per incubation medium and duration. UPLC-MS Measurements and Data Analysis UPLC-MS analysis of metabolite extracts was conducted on an UHR QqToF instrument (maXis, Bruker Daltonics) hyphenated to an ACQUITY UPLC (Waters). Prior to injection, extracts were dried (SpeedVac Concentrator, Savant SPD 121P, Thermo Fisher Scientific) and re-solved in solvent A (see below), using half of the original sample volume. Separation was performed on a ACQUITY UPLC BEH Amide column (15062.1 mm, 1.5 mm, Waters) using a 3 min gradient from 10% to 90% solvent B, followed by 2 min plateau on 90% B (solvent A: 80% acetonitrile, 20% water, 0.1% (v/v) ammonium hydroxide; solvent B: 70% water, 30% acetonitrile, 0.1% (v/v) ammonium hydroxide). A column equilibration time of 5 min was applied after each analysis. The flow rate was optimized to 0.1 ml/min with a column temperature of 45uC. ToF mass spectra were acquired in negative ESI mode. Parameters were tuned for best resolution and sensitivity in the mass range of about 100 to 400 Da. A quality control consisting of an aliquot of all samples and a mixture of different standard compounds (including raffinose, ribose, arabi- nose, galactose, fructose, fucose, gentiobiose, erythrol, glucose, pyruvate, and citrate) was used to monitor drifts in retention time and mass accuracy. The acquired spectra were calibrated internally against naturally abundant fatty acids (DataAnalysis ICR/FT-MS Measurements and Data Analysis ICR/FT-MS Measurements and Data Analysis ICR/FT-MS Measurements and Data Analysis Ultrahigh resolution mass spectra were acquired on a solariX ICR/FT mass spectrometer (Bruker Daltonics) equipped with an Apollo II electrospray source (Bruker Daltonics) and a 12 Tesla super conducting magnet (Magnex Scientific). The mass spec- trometer was tuned in order to obtain highest sensitivity for metabolites in the m/z range of about 150 to 500 Da in broad band detection mode with a time domain transient of 2 Megaword. The instrument was calibrated with a 1 ppm arginine solution. A mass error below 100 ppb and a resolving power of ,300 000 at m/z 300 was achieved. Negative electrospray ionization (ESI) mode was chosen due to favored ionization of carbohydrates and their metabolic derivatives by proton loss or chloride attachment [85]. Diluted extracts (1:100 in methanol ($99.9%, CHROMASOLV) were cooled (8uC) and injected (2 ml/min flow rate) through a Gilson autosampler (sample changer 223, Gilson). In total 600 scans were acquired for one spectrum of each sample. The obtained spectra were internally calibrated against naturally abundant fatty acids and analyzed in DataAnalysis 4.0 SP2 (Bruker Daltonics). Mass lists were generated with a signal-to-noise ratio (S/N) of four, exported, and combined to one data matrix by applying a 1 ppm window [86]. Subsequently, mass lists were filtered very conservatively. Masses that were also detected in the extraction blanks were excluded if their detected intensity in the samples did not exceed ten times the detected intensity in the blank. Furthermore, for each incubation condition, masses found in less than two out of the three independent biological experiments were also excluded. Detected metabolites in extracts from DGM-D-incubated EBs were annotated with MassTRIX (,1 ppm) [61]. For the detection of 13C-labeled metabolites an application of mass difference-based networks [87] was developed and applied. Hereby, mass differences of peaks detected in samples from DGM-D- and DGM-D-13C15N-incubated bacteria were compared by a poly- nomial-time algorithm. Labeled metabolites were considered to be present in extracts of DGM-D-13C15N-incubated EBs when peaks corresponding to the exact mass of the unlabeled metabolites were detected in the DGM-D-incubated controls and observed mass shifts were consistent with the exchange of 12C by 13C atoms. Metabolic Activity of Protochlamydia EBs Metabolic Activity of Protochlamydia EBs ICR/FT-MS Measurements and Data Analysis 4.0 SP2, Bruker Daltonics) and exported to MZmine 2.7 [88] for data evaluation. Annotation of the m/z features in spectra from DGM-D-incubated bacteria was carried out with MassTRIX applying a maximal mass error of 0.005 Da [61]. Corresponding masses of fully or partially labeled metabolites were calculated and their presence in spectra of DGM-D-13C15N-incubated samples was checked manually. Masses of putatively labeled metabolites were taken in consideration if the retention time and peak shape matched with the corresponding parameters of unlabeled metab- olites detected in DGM-D-incubated EBs and the mass difference between putatively labeled and unlabeled metabolites was consistent with a shift corresponding to the exchange of 12C by 13C atoms. IRMS Measurements and Data Analysis Analysis of bacterial biomass for total carbon and C isotopes (13C, 12C) was conducted using an elemental analyzer (EA 1110, CE Instruments) interfaced via a ConFlo III device (Thermo Finnigan) to a continuous flow stable isotope ratio mass spectrometer (DeltaPLUS, Thermo Finnigan). A mixture of proline and sucrose was used as standard, which was regularly calibrated against international standards (IAEA) for 13C and against atropine for total C content. Stable isotopes (13C, 12C) in CO2 of gas samples were analyzed against CO2 reference gas by an isotope ratio mass spectrometer (Delta Advantage V, Thermo Fisher Scientific) coupled to a headspace gas sampler (GasBench II, Thermo Fisher Scientific) with a GC-PAL autosampler (CTC Analytics). CO2 reference gas was calibrated using ISO-TOP gas standards with certified 13C concentrations (Air Liquide) and counterchecked by elemental analysis coupled with IRMS of international standards (IAEA). The averages of peak area and At%13C of three consecutive injections of each sample were used to calculate concentrations of CO2 and 13C, respectively. For calibration of CO2 concentration, gas standards containing 495 and 1020 ppm were analyzed together with the samples. For gas samples, measures for bacterial CO2 production and 13C enrichment in CO2 were calculated based on equations (1) and (2), respectively. August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 15 Statistical Analysis Throughout the study values are given as means with standard deviations. Unpaired two-sided student’s t-test and one-way ANOVA were carried out using the software PASW statistics 17.0 (SPSS Inc.). Dunnett’s T3 test was chosen as post-hoc test for ANOVA due to the fact that experimental data did frequently not comply with the criteria of variance homogeneity. The following notation of significance levels was used throughout the study: , p#0.001; , p#0.01; , p#0.05. The multivariate modeling was done in SIMCA-P 9 (Umetrics). PCA models were used for data visualization and discovery of naturally occurring differences in the metabolite pattern of living and heat-inactivated EBs and of EBs incubated in different media. Mean centering in combination with unit variance scaling was applied. Data were further analyzed with PLS-DA to extract the most discriminative compounds charac- terizing living EBs compared to heat-inactivated bacteria. A seven- fold cross-validation, as well as a permutation test using 200 iterations, was conducted for model validation. Figure S4 Working scheme for the estimation of the contributions of different metabolic pathways to 13CO2 release. In respect to the release of certain carbon atoms from D- glucose as CO2, three major metabolic scenarios (A–C) were distinguished, which are indicated in the gray box and will here be termed ‘‘pathways’’ for simplicity. The pathway ratio was calculated based on the APE13C in CO2 observed for incubations in media with different labeled D-glucose isotopologs (blue box). Initially, these experimental values were used to calculate an experimental APE13C ratio and the pathway ratio was set to 0:0:1 (A:B:C), assuming that all D-glucose is completely catabolized (red box). Based on this pathway ratio a corresponding expected ratio of APE13C for the differently labeled substrates was calculated, assuming that pathways A, B, and C lead to a release of 1.83, 1, and 6, respectively, carbons from D-[U-13C6]-glucose, 0, 1, and 1, respectively, carbons from D-[1-13C]-glucose, and 0, 0, and 1, respectively, carbons from D-[6-13C]-glucose. The assumed release of 1.83 carbon atoms per molecule D-[U-13C6]-glucose by pathway A was based on the assumption that pyruvate, as starting molecule for scenario A, is produced with equal Infectivity Assay (C. trachomatis) Thus, bacteria containing reticulated material and a relaxed nucleoid were considered as mature RBs (white arrows), bacteria containing only electron- dense and electron-lucent material were considered as mature EBs (black arrows), and intermediate morphologies were considered as IBs (gray arrowheads). The bar indicates 1 mm. (TIF) Figure S2 Visualization of respiratory activity in P. amoebophila developmental stages and the effect of D- glucose deprivation. Activity of P. amoebophila developmental forms was assessed by application of CTC as indicator for respiration. RB (A), intermediate (B), and EB (C) fractions of P. amoebophila were subjected to host-free incubation in DGM-D containing 5 mM CTC either immediately after purification or after a 40 h pre-incubation in DGM-D. A heat-inactivated EB fraction (D) and a lysate of uninfected amoebae (E) were included as controls. The effect of D-glucose deprivation on EB activity was tested by replacement of DGM-D with DGM-L during pre- incubation and incubation with CTC (F). Incubation with CTC was followed by formaldehyde fixation and DNA staining with DAPI. Fluorescence and corresponding DIC images are shown (reduced CTC, red; DAPI, blue). The bar indicates 10 mm. (TIF) Figure S3 D-glucose uptake in host-free P. amoebophila EBs revealed by IRMS. An EB-enriched fraction of P. amoebophila was pre-incubated for 40 h in DGM-D, followed by 48 h incubation in DGM-D or DGM-D-13C and subsequent analysis of bacterial biomass by IRMS. Heat-inactivated bacteria incubated in DGM-D-13C were included as control. The amount of incorporated 13C (in nmol 13C/mg DW) in DGM-D-13C- incubated bacteria was calculated by considering the 13C content of DGM-D-incubated bacteria as blank. Diamonds indicate results from individual replicates, bars display mean values. Results from three independent experiments each consisting of two replicate incubations per condition are shown. Bacterial numbers applied per incubation were similar between replicate experiments (between 3.96109 and 5.96109 bacteria). The observed difference in 13C incorporation between living and heat-inactivated bacteria was statistically significant (t-test; , p#0.001). (TIF) Infectivity Assay (C. trachomatis) HeLa 229 cells infected with C. trachomatis were harvested at 40 h p.i (when significant host cell lysis was first observed), washed with DPBS (Invitrogen), resuspended in SPG buffer [83], and August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 16 Metabolic Activity of Protochlamydia EBs disrupted by two rounds of freezing (dry ice/ethanol bath) and thawing (37uC). After removal of host cell debris by centrifugation (2506 g, 5 min, 4uC), the supernatant was filtered (1.2 mm). Bacteria were collected by centrifugation (15 5576 g, 10 min, 4uC) and resuspended in SPG buffer. The bacterial suspension was then transferred into 1.5 ml tubes for parallel host-free incubations in different media, including DPBS, DGM-D, DGM-L, DGM- D6P, and DGM-DD6P. After centrifugation (12 8516g, 10 min, 4uC), bacteria were resuspended in the respective media and incubated at 37uC (200 rpm) for 30 min, 2 h, 6 h, or 24 h before being added to HeLa 229 cells that had been seeded into a 24-well plate (Nunc; 76104 cells per well). Multi-well plates were incubated at 37uC, 5% CO2 for 24 h. Cells were fixed with 4% formaldehyde (1 h, room temperature) and washed with DPBS. For the detection of bacteria by immunostaining, cells were first permeabilized in 0.2% Triton-X-100 in PBS for 15 min, followed by incubation in blocking solution (2% BSA in PBS, 20 min), and subsequent incubations with primary antibodies (raised against recombinant P. amoebophila heat shock protein DnaK [91]) and secondary antibodies (Cy3-labled, Dianova) diluted in blocking solution (for 1 h each). HeLa 229 cells were counter-stained with HCS CellMask Deep Red cytoplasmic stain (Life technologies; 10 mg/ml, 20 min) and DNA was stained with DAPI (0.5 mg/ml in PBS, 10 min). Cells were embedded in Mowiol and images were taken at a confocal laser scanning microscope (TCS SP8 X, Leica). The percentage of infected cells was determined taking into account at least 300 cells per sample. Infectivity was expressed relative to the infectivity observed for bacteria incubated for 30 min in DGM-D6P. Data were collected for three parallel host- free incubations per incubation medium and duration. IB, 8% RB) and intermediate fractions represented a more uniform mixture of all stages (38% EB, 35% IB, 27% RB). Bacteria were classified as RBs, EBs, and IBs based on their characteristic morphological features. Supporting Information Figure S1 Transmission electron micrographs of puri- fied P. amoebophila developmental stages. P. amoebophila developmental forms were purified from amoebal host cells and separated from each other by density gradient centrifugation, using a previously established protocol [43]. TEM was carried out as described in the respective study [43]. Micrographs of a highly pure EB fraction (collected below 46% gastrografin), as well as of an RB-enriched and an intermediate fraction (collected above 40% gastrografin or at the 40/46% interface, respectively), are shown. A quantitative evaluation indicated a high enrichment of the replicative stage in RB fractions (5% EB, 34% IB, 61% RB), whereas EB fractions were highly enriched in EBs (76% EB, 16% August 2013 \| Volume 9 \| Issue 8 \| e1003553 17 PLOS Pathogens \| www.plospathogens.org Metabolic Activity of Protochlamydia EBs probability from glycolysis and PPP activity, the latter of which already releases carbon 1 as CO2 and thus yields a lower amount of pyruvate per molecule glucose. The calculated expected APE13C ratio was then compared to the experimental APE13C ratio. This was followed by stepwise adjustments of B and A until the expected APE13C ratio coincided with the experimentally observed ratio (yellow box). The finally obtained pathway ratio represents an estimation of the contributions of the three considered metabolic scenarios to 13CO2 release by host-free P. amoebophila EBs (green box). (TIF) Figure S7 Lack of infectivity restoration by addition of D-glucose to starved P. amoebophila. P. amoebophila were harvested from amoebal host cell cultures followed by host-free incubation for the indicated periods of time in DGM-D or DGM- L. Subsequently, bacteria incubated in DGM-L were supplement- ed with D-glucose (83.2 mM final concentration). After an additional incubation of the bacteria for 15 min (27uC, 200 rpm), amoebae were infected at a MOI of 4.3 and the percentage of infected cells was assessed at 48 h p.i. after detection of intracellular bacteria by FISH. The observed infectivity, relative to that observed for 2 h incubation in DGM-D, is shown. Data represent means and standard deviations of three independent experiments, each consisting of three replicate host-free incuba- tions. For each sample at least 600 cells were counted. Statistically significant differences in infectivity observed between the incuba- tion media at selected time points after start of host-free incubation are indicated (t-test; , p#0.001; *, p#0.01). Acknowledgments The authors would like to thank David Berry, Martina Grill, Alexander Galushko, Lena Ko¨nig, Michael Wagner, and Wolfgang Wanek from the Department of Microbiology and Ecosystem Science (University of Vienna), as well as Volker Egelhofer from the Department of Ecogenomics and Systems Biology (University of Vienna), for excellent advice and valuable discussions at various stages during this study. Waltraud Klepal and the team of the Core Facility Cell Imaging and Ultrastructure Research (University of Vienna) are gratefully acknowledged for advice and assistance with electron microscopy. The authors thank Katharina Herbst and Georg Ha¨cker from the Institute for Medical Microbiology and Hygiene (University Hospital Freiburg) for kindly donating HeLa 229 cells and C. trachomatis L2. Figure S6 PLS-DA analysis for the extraction of metab- olites discriminative for living compared to heat-inacti- vated EBs. ICR/FT-MS spectra were analyzed by PLS-DA to extract the most discriminative compounds characterizing living compared to inactivated EBs. The PLS-DA model included data from DGM-D-incubated living and DGM-D-13C15N-incubated inactivated EBs. The assignment of discriminative and non- discriminative metabolites to KEGG pathways is displayed in Fig. 4. Table S2 Media for extracellular incubation of P. amoebophila. (DOCX) Table S2 Media for extracellular incubation of P. amoebophila. (DOCX) Supporting Information (TIF) Figure S5 Comparison of ICR/FT-MS metabolite pro- files from DGM-D- and DGM-D-13C15N-incubated living and heat-inactivated EBs. In (A) representative ESI(- )ICR/FT-MS spectra of DGM-D- and DGM-D-13C15N-incu- bated living bacteria, as well as of DGM-D-13C15N-incubated inactivated bacteria, are shown. Spectra illustrate the m/z range of 150–800. In addition, an enlarged view on the m/z range of 175– 190 (indicated in gray in the overview spectra), is displayed. A comparison of spectra from living bacteria incubated in the two different media revealed a shift of the D-glucose peak from m/z ,179 (12C-D-glucose) to m/z ,185 (13C-D-glucose), while the remaining pattern was unaltered. The exchange of D-glucose and L-phenylalanine in the incubation medium against their stable isotope-labeled analogs thus did not profoundly change the metabolite profile of P. amoebophila EBs. This visual impression, as well as the effect of heat-inactivation, was further tested by pairwise PCA comparisons of spectra from DGM-D- and DGM- D-13C15N-incubated living bacteria (B), and of spectra from DGM-D-1315N-incubated inactivated bacteria with those from DGM-D-incubated living EBs (C) or DGM-D-13C15N-incubated living EBs (D). Note that this analysis revealed a separation of spectra from living and dead bacteria, whereas no separation could be observed for samples from living bacteria incubated in the two different media. (TIF) Author Contributions Conceived and designed the experiments: BSS AS CM PSK MH. Performed the experiments: BSS AS CM MW AW JM. Analyzed the data: BSS AS CM DT PSK MH. Contributed reagents/materials/analysis tools: AR. Wrote the paper: BSS AS CM PSK MH. References 1. Wright HR, Turner A, Taylor HR (2008) Trachoma. Lancet 371: 1945–1954. 2 B b C d B b B (2009) G i l Chl di h i i f i Cli 8. Amann R, Springer N, Schonhuber W, Ludwig W, Schmid EN, et al. (1997) Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp. Appl Environ Microbiol 63: 115–121. 2. Be´be´ar C, de Barbeyrac B (2009) Genital Chlamydia trachomatis infections. Clin Microbiol Infect 15: 4–10. 2. Bebear C, de Barbeyrac B Microbiol Infect 15: 4–10. 9. Corsaro D, Venditti D (2006) Diversity of the parachlamydiae in the environment. Crit Rev Microbiol 32: 185–199. 3. Blasi F, Tarsia P, Aliberti S (2009) Chlamydophila pneumoniae. Clin Microbiol Infect 15: 29–35. 10. Haider S, Collingro A, Walochnik J, Wagner M, Horn M (2008) Chlamydia-like bacteria in respiratory samples of community-acquired pneumonia patients. FEMS Microbiol Lett 281: 198–202. 4. Horn M (2008) Chlamydiae as symbionts in eukaryotes. Annu Rev Microbiol 62: 113–131. 5. Kuo CC, Horn M, Stephens RS (2011) The order Chlamydiales. In: Krieg NR, Staley JT, Brown DR, Hedlund BP, Paster BJ, et al., editors. Bergey’s Manual of Systematic Bacteriology - The Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes. New York: Springer. pp. 844–845. 11. Greub G (2009) Parachlamydia acanthamoebae, an emerging agent of pneumonia. Clin Microbiol Infect 15: 18–28. 12. Stephens RS, Kalman S, Lammel C, Fan J, Marathe R, et al. (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282: 754–759. 6. Corsaro D, Greub G (2006) Pathogenic potential of novel chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria. Clin Microbiol Rev 19: 283–297. 13. Kalman S, Mitchell W, Marathe R, Lammel C, Fan J, et al. (1999) Comparative genomes of Chlamydia pneumoniae and C. trachomatis. Nat Genet 21: 385–389. 7. Collingro A, Toenshoff ER, Taylor MW, Fritsche TR, Wagner M, et al. (2005) ‘Candidatus Protochlamydia amoebophila’, an endosymbiont of Acanthamoeba spp. Int J Syst Evol Microbiol 55: 1863–1866. 7. Collingro A, Toenshoff ER, Taylor MW, Fritsche TR, Wagner M, et al. (2005) ‘Candidatus Protochlamydia amoebophila’, an endosymbiont of Acanthamoeba spp. Int J Syst Evol Microbiol 55: 1863–1866. 14. Horn M, Collingro A, Schmitz-Esser S, Beier CL, Purkhold U, et al. (2004) Illuminating the evolutionary history of chlamydiae. Science 304: 728–730. Table S1 Non-annotated 13C-labeled metabolites de- tected by ICR/FT-MS in DGM-D-13C15N-incubated EBs. Table S1 Non-annotated 13C-labeled metabolites de- tected by ICR/FT-MS in DGM-D-13C15N-incubated EBs. (DOCX) 3. Blasi F, Tarsia P, Aliberti S (2009) Chlamydophila pneumoniae. Clin Microbiol Infect 15: 29–35. Metabolic Activity of Protochlamydia EBs Matsumoto A (1988) Structural characteristics of chlamydial bodies. In: Barron AL, editor. Microbiology of Chlamydia. Boca Raton, FL: CRC Press. pp. 21–45. 53. Sun HJ, Saccomanno V, Hedlund B, McKay CP (2009) Stereo-specific glucose consumption may be used to distinguish between chemical and biological reactivity on Mars: a preliminary test on Earth. Astrobiology 9: 443–446. 23. Abdelrahman YM, Belland RJ (2005) The chlamydial developmental cycle. FEMS Microbiol Rev 29: 949–959. 24. Hatch TP (1999) Developmental Biology. In: Stephens RS, editor. Chlamydia. Washington DC: American Society for Microbiology. pp. 29–67. y p y gy 54. Warmflash D, Chu H, Siefert J, Fox GE (2009) Life detection using glucose and tetrasaccharide enantiomer pairs. Astrobiology 9: 297–303. 54. Warmflash D, Chu H, Siefert J, Fox GE (2009) Life detection tetrasaccharide enantiomer pairs. Astrobiology 9: 297–303. 25. Hackstadt T, Todd WJ, Caldwell HD (1985) Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae? J Bacteriol 161: 25–31. 55. Yoshioka K, Takahashi H, Homma T, Saito M, Oh KB, et al. (1996) A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim Biophys Acta 1289: 5–9. 26. Hatch T (1996) Disulfide cross-linked envelope proteins: the functional equivalent of peptidoglycan in chlamydiae? J Bacteriol 178: 1–5. y p y 56. Muccio Z, Jackson GP (2009) Isotope ratio mass spectrometry. Analyst 134: 213–222. 27. Bavoil P, Ohlin A, Schachter J (1984) Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infect Immun 44: 479–485. 57. Moseley HN (2010) Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry. BMC Bioinformatics 11: 139. 28. Peeling RW, Peeling J, Brunham RC (1989) High-resolution 31P nuclear magnetic resonance study of Chlamydia trachomatis: induction of ATPase activity in elementary bodies. Infect Immun 57: 3338–3344. 58. Ohta D, Kanaya S, Suzuki H (2010) Application of Fourier-transform ion cyclotron resonance mass spectrometry to metabolic profiling and metabolite identification. Curr Opin Biotechnol 21: 35–44. 29. Hackstadt T, Baehr W, Ying Y (1991) Chlamydia trachomatis developmentally regulated protein is homologous to eukaryotic histone H1. Proc Natl Acad Sci U S A 88: 3937–3941. 59. Dunn WB (2008) Current trends and future requirements for the mass spectrometric investigation of microbial, mammalian and plant metabolomes. Phys Biol 5: 011001. 30. Metabolic Activity of Protochlamydia EBs Barry CE, Hayes SF, Hackstadt T (1992) Nucleoid condensation in Escherichia coli that express a chlamydial histone homolog. Science 256: 377–379. 60. Lei Z, Huhman DV, Sumner LW (2011) Mass spectrometry strategies in metabolomics. J Biol Chem 286: 25435–25442. 31. Perara E, Ganem D, Engel JN (1992) A developmentally regulated chlamydial gene with apparent homology to eukaryotic histone H1. Proc Natl Acad Sci U S A 89: 2125–2129. 61. Suhre K, Schmitt-Kopplin P (2008) MassTRIX: mass translator into pathways. Nucleic Acids Res 36: W481–484. 62. Tamura A, Higashi N (1963) Purification and chemical composition of meningopneumonitis virus. Virology 20: 596–604. 32. Christiansen G, Pedersen LB, Koehler JE, Lundemose AG, Birkelund S (1993) Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA. J Bacteriol 175: 1785–1795. 63. Kahane S, Kimmel N, Friedman MG (2002) The growth cycle of Simkania negevensis. Microbiology 148: 735–742. 33. Barry CE, 3rd, Brickman TJ, Hackstadt T (1993) Hc1-mediated effects on DNA structure: a potential regulator of chlamydial development. Mol Microbiol 9: 273–283. 64. Nelson DE (2012) The chlamydial cell envelope. In: Ming T, Bavoil PM, editors. Intracellular pathogens I: Chlamydiales. Washington, DC: ASM Press. pp. 74– 96. 34. Pedersen LB, Birkelund S, Christiansen G (1996) Purification of recombinant Chlamydia trachomatis histone H1-like protein Hc2, and comparative functional analysis of Hc2 and Hc1. Mol Microbiol 20: 295–311. 65. Vender J, Moulder JW (1967) Initial step in catabolism of glucose by the meningopneumonitis agent. J Bacteriol 94: 867–869. 66. Weiss E (1967) Transaminase activity and other enzymatic reactions involving pyruvate and glutamate in Chlamydia (psittacosis-trachoma group). J Bacteriol 93: 177–184. 35. Tamura A, Matsumoto A, Higashi N (1967) Purification and chemical composition of reticulate bodies of the meningopneumonitis organisms. J Bacteriol 93: 2003–2008. 67. Weiss E, Neptune EM, Jr., Gaugler RW (1968) Influence of gas environment on catabolic activities and on reoxidation of reduced nicotinamide adenine dinucleotide phosphate in Chlamydia. J Bacteriol 96: 1567–1573. 36. Vandahl BB, Birkelund S, Demol H, Hoorelbeke B, Christiansen G, et al. (2001) Proteome analysis of the Chlamydia pneumoniae elementary body. Electrophoresis 22: 1204–1223. 68. Hatch TP, Al-Hossainy E, Silverman JA (1982) Adenine nucleotide and lysine transport in Chlamydia psittaci. J Bacteriol 150: 662–670. 37. Shaw AC, Gevaert K, Demol H, Hoorelbeke B, Vandekerckhove J, et al. (2002) Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2. Proteomics 2: 164–186. p y 69. Hatch TP (1988) Metabolism of Chlamydia. Metabolic Activity of Protochlamydia EBs Metabolic Activity of Protochlamydia EBs 15. Dean D, Myers GS, Read TD (2006) Lessons and challenges arising from the ‘first wave’ of Chlamydia genome sequencing. In: Bavoil PM, Wyrick PB, editors. Chlamydia - genomics and pathogenesis. Norfolk: Horizon Bioscience. pp. 1–24. 46. Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992) Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol 58: 1801–1808. 47. Creach V, Baudoux AC, Bertru G, Rouzic BL (2003) Direct estimate of active bacteria: CTC use and limitations. J Microbiol Methods 52: 19–28. 16. Greub G, Kebbi-Beghdadi C, Bertelli C, Collyn F, Riederer BM, et al. (2009) High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach. PLoS One 4: e8423. 48. Cook KL, Garland JL (1997) The relationship between electron transport activity as measured by CTC reduction and CO2 production in mixed microbial communities. Microb Ecol 34: 237–247. of a new pathogen: the dirty genome approach. PLoS One 4: e8 17. Bertelli C, Collyn F, Croxatto A, Ruckert C, Polkinghorne A, et al. (2010) The Waddlia genome: a window into chlamydial biology. PLoS One 5: e10890. 49. Smith JJ, McFeters GA (1997) Mechanisms of INT (2-(4-iodophenyl)-3-(4- nitrophenyl)-5-phenyl tetrazolium chloride), and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli K-12. J Microbiol Methods 29: 161–175. 18. Collingro A, Tischler P, Weinmaier T, Penz T, Heinz E, et al. (2011) Unity in variety - the pan-genome of the Chlamydiae. Mol Biol Evol 28: 3253–3270. 19. Horn M, Collingro A, Schmitz-Esser S, Wagner M (2006) Environmental chlamydia genomics. In: Bavoil PM, Wyrick PB, editors. Chlamydia: genomics and pathogenesis. Norfolk, UK: Horizon Bioscience. pp. 25–44. 50. Smith EM (1998) Coherence of microbial respiration rate and cell-specific bacterial activity in a coastal planktonic community. Aquat Microb Ecol 16: 27– 35. p g pp 20. Iliffe-Lee ER, McClarty G (1999) Glucose metabolism in Chlamydia trachomatis: the ‘energy parasite’ hypothesis revisited. Mol Microbiol 33: 177–187. 51. Hatch TP, Miceli M, Silverman JA (1985) Synthesis of protein in host-free reticulate bodies of Chlamydia psittaci and Chlamydia trachomatis. J Bacteriol 162: 938–942. 21. McClarty G (1999) Chlamydial metabolism as inferred from the complete genome sequence. In: Stephens RS, editor. Chlamydia. Washington DC: American Society for Microbiology. pp. 69–100. 52. Rudney H (1940) The utilization of L-glucose by mammalian tissues and bacteria. Science 92: 112–113. y gy pp 22. References August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 18 Metabolic Activity of Protochlamydia EBs In: Barron AL, editor. Microbiology of Chlamydia. Boca Raton, Florida: CRC Press. pp. 97–109. 38. Skipp P, Robinson J, O’Connor CD, Clarke IN (2005) Shotgun proteomic analysis of Chlamydia trachomatis. Proteomics 5: 1558–1573. 70. Plaunt MR, Hatch TP (1988) Protein synthesis early in the developmental cycle of Chlamydia psittaci. Infect Immun 56: 3021–3025. 39. Saka HA, Thompson JW, Chen YS, Kumar Y, Dubois LG, et al. (2011) Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms. Mol Microbiol 82: 1185–1203. 71. Grieshaber NA, Fischer ER, Mead DJ, Dooley CA, Hackstadt T (2004) Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis. Proc Natl Acad Sci U S A 101: 7451–7456. 40. Sixt BS, Heinz C, Pichler P, Heinz E, Montanaro J, et al. (2011) Proteomic analysis reveals a virtually complete set of proteins for translation and energy generation in elementary bodies of the amoeba symbiont Protochlamydia amoebophila. Proteomics 11: 1868–1892. 72. Sarov I, Becker Y (1971) Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in purified trachoma elementary bodies - effect of sodium chloride on ribonucleic acid transcription. J Bacteriol 107: 593–598. 41. Byers TJ, Akins RA, Maynard BJ, Lefken RA, Martin SM (1980) Rapid growth of Acanthamoeba in defined media; induction of encystment by glucose-acetate starvation. J Protozool 27: 216–219. 73. Weiss E (1965) Adenosine triphosphate and other requirements for the utilization of glucose by agents of the psittacosis-trachoma group. J Bacteriol 90: 243–253. 42. Schuster FL (2002) Cultivation of pathogenic and opportunistic free-living amebas. Clin Microbiol Rev 15: 342–354. 74. Ormsbee RA, Weiss E (1963) Trachoma agent: glucose utilization by purified suspensions. Science 142: 1077–1078. 43. Haider S, Wagner M, Schmid MC, Sixt BS, Christian JG, et al. (2010) Raman microspectroscopy reveals long-term extracellular activity of chlamydiae. Mol Microbiol 77: 687–700. p 75. Weiss E, Myers WF, Dressler HR, Chun-Hoon H (1964) Glucose metabolism by agents of the psittacosis-trachoma group. Virology 22: 551–562. 76. Colo´n JI (1960) Enzymes for formation of citrovorum factor in members of the psittacosis group of microorganisms. J Bacteriol 79: 741–746. 76. Colo´n JI (1960) Enzymes for formation of citrovorum factor in 44. Omsland A, Sager J, Nair V, Sturdevant DE, Hackstadt T (2012) Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium. Proc Natl Acad Sci U S A 109: 19781–19785. 45. Metabolic Activity of Protochlamydia EBs 79. Weiss E, Wilson NN (1969) Role of exogenous adenosine triphosphate in catabolic and synthetic activities of Chlamydia psittaci. J Bacteriol 97: 719–724. 86. Marianna L, Fekete A, Frommberger M, Schmitt-Kopplin P (2011) Metabo- lomics: High-resolution tools offer to follow bacterial growth on a molecular level. In: de Bruijn FJ, editor. Handbook of Molecular Microbial Ecology I: Metagenomics and Complementary Approaches. Hoboken, NJ, , USA: John Wiley & Sons, Inc. 80. Schwo¨ppe C, Winkler HH, Neuhaus HE (2002) Properties of the glucose-6- phosphate transporter from Chlamydia pneumoniae (HPTcp) and the glucose-6- phosphate sensor from Escherichia coli (UhpC). J Bacteriol 184: 2108–2115. 87. Tziotis D, Hertkorn N, Schmitt-Kopplin P (2011) Kendrick-analogous network visualisation of ion cyclotron resonance Fourier transform mass spectra: improved options for the assignment of elemental compositions and the classification of organic molecular complexity. Eur J Mass Spectrom (Chichester, Eng) 17: 415–421. 81. Li J, Ward KM, Zhang D, Dayanandam E, Denittis AS, et al. (2012) A bioactive probe of the oxidative pentose phosphate cycle: Novel strategy to reverse radioresistance in glucose deprived human colon cancer cells. Toxicol In Vitro 27: 367–377. 82. Coulon C, Eterpi M, Greub G, Collignon A, McDonnell G, et al. (2012) Amoebal host range, host-free survival and disinfection susceptibility of environmental chlamydiae as compared to Chlamydia trachomatis. FEMS Immunol Med Microbiol 64: 364–373. 88. Pluskal T, Castillo S, Villar-Briones A, Oresic M (2010) MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics 11: 395. 89. Poppert S, Essig A, Marre R, Wagner M, Horn M (2002) Detection and differentiation of Chlamydiae by fluorescence in situ hybridization. Appl Environ Microbiol 68: 4081–4089. 83. Sixt BS, Hiess B, Ko¨nig L, Horn M (2012) Lack of effective anti-apoptotic activities restricts growth of Parachlamydiaceae in insect cells. PloS one 7: e29565. 84. Heinz E, Rockey DD, Montanaro J, Aistleitner K, Wagner M, et al. (2010) Inclusion membrane proteins of Protochlamydia amoebophila UWE25 reveal a conserved mechanism for host cell interaction among the Chlamydiae. J Bacteriol 192: 5093–5102. 90. Daims H, Stoecker K, Wagner M (2005) Fluorescence in situ hybridization for the detection of prokaryotes. In: Osborn AM, Smith CJ, editors. Advanced Methods in Molecular Microbial Ecology. Abingdon, UK: Bios-Garland. pp. 213–239. 85. Boutegrabet L, Kanawati B, Gebefugi I, Peyron D, Cayot P, et al. Metabolic Activity of Protochlamydia EBs Friis RR (1972) Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J Bacteriol 110: 706–721. 44. Omsland A, Sager J, Nair V, Sturdevant DE, Hackstadt T (2012) Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium. Proc Natl Acad Sci U S A 109: 19781–19785. psittacosis group of microorganisms. J Bacteriol 79: 741–746. 77. Colo´n JI (1962) The role of folic acid in the metabolism of members of the psittacosis group of microorganisms. Ann N Y Acad Sci 98: 234–249. 45. Friis RR (1972) Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J Bacteriol 110: 706–721. 78. Gaugler RW, Neptune EM, Adams GM, Sallee TL, Weiss E, et al. (1969) Lipid synthesis by isolated Chlamydia psittaci. J Bacteriol 100: 823–826. August 2013 \| Volume 9 \| Issue 8 \| e1003553 August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 19 Metabolic Activity of Protochlamydia EBs Metabolic Activity of Protochlamydia EBs (2012) Attachment of chloride anion to sugars: mechanistic investigation and discovery of a new dopant for efficient sugar ionization/detection in mass spectrometers. Chemistry 18: 13059–13067. 91. Aistleitner K, Heinz C, Ho¨rmann A, Heinz E, Montanaro J, et al. (2013) Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens. PLoS One 8: e55010. August 2013 \| Volume 9 \| Issue 8 \| e1003553 PLOS Pathogens \| www.plospathogens.org 20 PLOS Pathogens \| www.plospathogens.org
https://openalex.org/W2953935440	https://periodicos.ufv.br/elo/article/download/8232/3412	Portuguese	null	Construindo saberes sobre desenvolvimento rural sustentável e agroecologia: uma experiência com extensão universitária	Elo	2,019	cc-by	6,288	Building knowledge on sustainable rural development and agroecology: an experience with university extension Abstract: This article aims to share an experience with university extension on the construction of knowledge to promote sustainable rural development from agroecology in the municipality of the São Thomé das Letras - MG. Ten meetings and twelve community activities were held in twenty months. Initially a participatory diagnosis was carried out and all the actions were constructed collectively considering the demands of the community. During the course of the project, there were some limitations mainly related to lack of funding and participation in meetings. As potentialities, the various possibilities of the social actors involved in learning, discussing and teaching about sustainable rural development and agroecology are highlighted. This experience was marked by dialogue and exchange of knowledge, seeking to overcome the idea of transmitting knowledge from university to society. Keywords: University Extension. Sustainable rural development. Agroecology. Construindo saberes sobre desenvolvimento rural sustentável e agroecologia: uma experiência com extensão universitária André Wagner Barata Silva1, Viviane Santos Pereira2, Luiz Antônio Augusto Gomes3,4 André Wagner Barata Silva1, Viviane Santos Pereira2, Luiz Antônio Augusto Gomes3, Resumo: Este artigo tem como objetivo compartilhar uma experiência com extensão universitária sobre a construção de saberes, para a promoção do desenvolvimento rural sustentável, a partir da agroecologia no município São Thomé das Letras - MG. Foram realizadas dez reuniões e doze atividades com a comunidade em vinte meses. Inicialmente foi realizado um diagnóstico participativo, e todas as ações foram construídas de forma coletiva, considerando-se as demandas da comunidade. Observaram-se, no decorrer do projeto, algumas limitações, principalmente, relacionadas à falta de financiamento e à participação às reuniões. Como potencialidades, ressaltam-se as diversas possibilidades dos atores sociais envolvidos em aprender, dialogar e ensinar sobre o desenvolvimento rural sustentável e agroecologia. Essa experiência foi marcada pelo diálogo e troca de saberes, ao buscar superar a ideia de transmissão de conhecimentos da universidade para a sociedade. Palavras-chave: Extensão universitária. Desenvolvimento rural sustentável. Agroecologia. Área Temática: Agroecologia. Meio Ambiente. Construindo saberes sobre desenvolvimento rural sustentável e agroecologia: uma experiência com extensão universitária André Wagner Barata Silva1, Viviane Santos Pereira2, Luiz Antônio Augusto Gomes3,4 Construindo saberes sobre desenvolvimento rural sustentável e agroecologia: uma experiência com extensão universitária André Wagner Barata Silva1, Viviane Santos Pereira2, Luiz Antônio Augusto Gomes3,4 Professora da Universidade Federal de Lavras. Coordenadora do projeto de extensão. End.: Departamento de Adm de Lavras. Caixa postal 37. Lavras/MG. CEP 37200-000. Tel.(35)3829-1459. E-mail: vivianepereira@ufla.br 1 Doutorando no Programa Pós-Graduação em Agronomia/Fitotecnia da Universidade Federal de Lavras. g ç g / 4 Professor na Universidade Federal de Uberlândia - Campus Patos de Minas/MG. de Lavras. Caixa postal 37. Lavras/MG. CEP 37200-000. Tel.(35)3829-1459. E-mail: vivianepereira@ufla.br 3 Professor no Programa Pós-Graduação em Agronomia/Fitotecnia da Universidade Federal de Lavras. 4 P f U i id d F d l d Ub l di C P d Mi /MG 1 Doutorando no Programa Pós-Graduação em Agronomia/Fitotecnia da Universidade Federal de Lavras. 2 Professora da Universidade Federal de Lavras. Coordenadora do projeto de extensão. End.: Departamento de A g ç g / 2 Professora da Universidade Federal de Lavras. Coordenadora do projeto de extensão. End.: Departamento de Administração e Economia da Universidade Federal de Lavras. Caixa postal 37. Lavras/MG. CEP 37200-000. Tel.(35)3829-1459. E-mail: vivianepereira@ufla.br 3 Professor no Programa Pós-Graduação em Agronomia/Fitotecnia da Universidade Federal de Lavras. 4 P f U i id d F d l d Ub lâ di C P t d Mi /MG 1 Doutorando no Programa Pós-Graduação em Agronomia/Fitotecnia da Universidade Federal de Lavras. 2 Professora da Universidade Federal de Lavras. Coordenadora do projeto de extensão. End.: Departamento de Administração e Economia da Universidade Federal de Lavras. Caixa postal 37. Lavras/MG. CEP 37200-000. Tel.(35)3829-1459. E-mail: vivianepereira@ufla.br 3 Professor no Programa Pós-Graduação em Agronomia/Fitotecnia da Universidade Federal de Lavras. 4 Professor na Universidade Federal de Uberlândia - Campus Patos de Minas/MG. Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 1 Doutorando no Programa Pós-Graduação em Agronomia/Fitotecnia da Universidade Federal de Lavras. 3 Professor no Programa Pós-Graduação em Agronomia/Fitotecnia da Universidade 4 Professor na Universidade Federal de Uberlândia - Campus Patos de Minas/MG. Introdução Em decorrência do atual modelo econômico, causador de desequilíbrios ambientais, a sociedade vem passando por mudanças substanciais, pois o atual modelo de crescimento econômico tem gerado enormes desequilíbrios ao meio ambiente. Os avanços obtidos pela sociedade atual são tão visíveis quanto os prejuízos que os modelos convencionais de desenvolvimento rural e urbano provocaram. A noção de desenvolvimento, historicamente relacionada ao crescimento econômico, vai além do domínio da economia, à medida que incorpora aspectos sociais, ambientais, culturais e políticos, sustentando-se em novas visões pela busca de soluções para enfrentar os problemas socioambientais. Isso representa uma mudança, no paradigma dominante sobre os caminhos para o desenvolvimento, reconhecendo a incompletude e a insuficiência dos modelos econômicos e sociais que têm servido de apoio e orientação aos processos de desenvolvimento. Nota-se que as estratégias convencionais de desenvolvimento são insuficientes para minimizar as crescentes condições de desigualdade, exclusão social e degradação do meio ambiente. Estudos e contribuições, decorrentes das novas visões sobre o processo de desenvolvimento, passaram a incorporar, de forma cada vez mais evidente, a problemática da sustentabilidade (CAPORAL E COSTABEBER, 2004). A procura por uma sustentabilidade, no desenvolvimento rural, implica reconhecer a existência de saberes construídos, mediante uma lógica indutiva, que vai sendo estabelecida na história dos grupos sociais. Para Moreira e Carmo (2004), a sustentabilidade e a estratégia de desenvolvimento rural devem ser definidas a partir da participação e da identidade etnoecossistêmica de cada localidade a ser considerada. Nesse sentido, a integração de diferentes conhecimentos científicos, em diálogo com outras formas de conhecimento, tem ganhado cada vez mais espaço, para a construção de um processo de desenvolvimento rural sustentável, embasando-se na agroecologia que tem oferecido princípios, bases técnicas e metodológicas que visam apoiar essa transição. Tendo o entendimento da agroecologia, enquanto campo de estudos de caráter multidisciplinar, tomaram-se, como base conceitual, os trabalhos de Caporal e Costabeber (2004), Altieri (2002), Casado, Molina e Guzmán (2000) e Gliesman (2000). Casado, Molina e Guzmán (2000) afirmam que a agroecologia é um enfoque científico que reúne vários campos de conhecimento, incorporando reflexões teóricas e avanços científicos de distintas disciplinas. Conforme Gliessman (2000), a agroecologia é entendida como aplicação de princípios e conceitos no manejo e desenho de agroecossistemas sustentáveis. Já Altieri (2002) declara que a agroecologia é uma ciência que fornece os princípios ecológicos básicos, para estudar, desenhar e manejar agroecossistemas produtivos, que conservem recursos naturais, que sejam culturalmente apropriados, socialmente justos e economicamente viáveis. Construyendo conocimientos sobre desarrollo rural sostenible y agroecología: una experiencia con extensión universitaria Resumen: Este artículo tiene como objetivo compartir una experiencia con extensión universitaria sobre construcción de saberes para promover el desarrollo rural sostenible a partir de la agroecología en el municipio São Thomé das Letras - MG. Se realizaron diez reuniones y doce actividades con la 15 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Silva, A.W.B. et al. comunidad en veinte meses. Inicialmente se realizó un diagnóstico participativo y todas las acciones fueron construidas de forma colectiva considerando las demandas de la comunidad. En el transcurso del proyecto se observaron algunas limitaciones principalmente relacionadas con la falta de financiamiento y la participación en las reuniones. Como potencialidades, se resaltan las diversas posibilidades de los actores sociales involucrados de aprender, dialogar y enseñar sobre desarrollo rural sostenible y agroecología. Esta experiencia fue marcada por el diálogo y el intercambio de saberes, buscando superar la idea de transmisión de conocimientos de la universidad a la sociedad. Palabras clave: Extensión universitaria. Desarrollo rural sostenible. Agroecología. 16 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Objetivos O projeto de extensão, aqui apresentado, teve como objetivo estimular a construção de saberes, para a promoção do desenvolvimento rural sustentável, a partir da agroecologia no município de São Thomé das Letras/MG, por meio da promoção de espaços de diálogo e aprendizagem. [...] a agroecologia se nutre de outros campos de conhecimento e de outras disciplinas científicas, assim como de saberes, conhecimentos e experiências dos próprios agricultores, o que permite o estabelecimento de marcos conceituais, metodológicos e estratégicos [...] (CAPORAL e COSTABEBER, 2004 p. 108). [...] a agroecologia se nutre de outros campos de conhecimento e de outras disciplinas científicas, assim como de saberes, conhecimentos e experiências dos próprios agricultores, o que permite o estabelecimento de marcos conceituais, metodológicos e estratégicos [...] (CAPORAL e COSTABEBER, 2004 p. 108). [...] a agroecologia se nutre de outros campos de conhecimento e de outras disciplinas científicas, assim como de saberes, conhecimentos e experiências dos próprios agricultores, o que permite o estabelecimento de marcos conceituais, metodológicos e estratégicos [...] (CAPORAL e COSTABEBER, 2004 p. 108). Sob esta ótica, a integração de diferentes conhecimentos científicos, em diálogo com o conhecimento tradicional e saber local, tem ganhado cada vez mais espaço, para a construção de um processo de desenvolvimento rural sustentável, sendo a interação entre a universidade e a sociedade, por meio da extensão universitária, um importante caminho para alcançar esse objetivo. De acordo com o FORPROEX (2012), ao longo do tempo, a Extensão Universitária tornou-se instrumento de inter-relação da Universidade com a sociedade, de oxigenação da própria Universidade, de democratização do conhecimento acadêmico, assim como de (re) produção desse conhecimento por meio da troca de saberes com as comunidades. A Extensão Universitária denota uma postura da Universidade, na sociedade em que se insere, entendida como um processo interdisciplinar, educativo, cultural, científico e político, por meio do qual se promove uma interação transformadora. Para que essa interação seja dialógica, faz-se necessária a aplicação de metodologias que estimulem a participação e a democratização do conhecimento, destacando a contribuição de atores não universitários em sua produção e multiplicação. Por isso, a extensão deve ser um processo educativo que considera a comunicação, a cultura, a visão de mundo e a realidade local, a fim de problematizar questões, ao estabelecer diálogos entre o conhecimento científico e outras formas de conhecimento em qualquer temática. Ressalta-se, nesse cenário, um dos objetivos da Política Nacional de Extensão Universitária que é o de estimular a educação ambiental e o desenvolvimento sustentável como componentes da atividade extensionista (FORPROEX, 2012). Nesse contexto, torna-se relevante o desenvolvimento de ações de extensão universitária, alinhado a uma perspectiva de extensão, que busca contribuir para a promoção do desenvolvimento rural sustentável, adotando uma abordagem sistêmica e multidisciplinar, mediante a utilização de métodos participativos. Diante do exposto, este artigo apresenta um projeto de extensão universitária sobre desenvolvimento rural sustentável. Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Introdução Segundo Caporal e Costabeber (2004), a intenção da agroecologia vai além de aspectos agronômicos de produção, incorporando dimensões mais abrangentes e complexas da produção agrícola. Desta forma, a agroecologia orienta, também, processos de desenvolvimento rural sustentável. Nessa perspectiva, para ser sustentável, o processo de desenvolvimento rural precisa considerar as diferentes dimensões da sustentabilidade (social, econômica, ambiental, cultural, política e ética) em uma perspectiva que não abandona a solidariedade intra e intergeracional (COSTABEBER E CAPORAL, 2003). Assim, de acordo com Caporal et. al (2009), a proposta de desenvolvimento rural sustentável necessita de esforços à identificação e à construção de saberes ecológicos, agronômicos, econômicos e sociais que permitam, de forma participativa, desenvolver processos toleráveis de exploração da natureza e compatíveis com as exigências de reprodução social das famílias. 16 Construindo saberes sobre desenvolvimento rural sustentável e agroecologia: uma experiência com extensão universitária Metodologia Neste projeto, optou-se por realizar um processo de “intervenção na concepção participativa”, conforme proposto por France (2005 p. 113), visando a uma construção compartilhada de conhecimentos entre pessoas com formações distintas, pontuando os princípios gnosiológicos da concepção construtivista de aprendizagem - ato de conhecer como um processo em construção. Assim, não ocorre apenas a assimilação de novos conhecimentos, mas a transformação dos conhecimentos já incorporados pela prática. Nesse processo, há participação do grupo atingido “desde os atos de problematização até os de decisão” (FRANCE, 2005). O Diagnóstico Rural Participativo (DRP), também, foi utilizado como ferramenta metodológica. De acordo com Verdejo (2006), o DRP é um conjunto de técnicas e ferramentas que permite que as comunidades façam o seu próprio diagnóstico e, a partir daí, comecem a autogerenciar o seu planejamento e desenvolvimento. Dessa maneira, os participantes podem compartilhar experiências e analisar os seus conhecimentos, a fim de melhorar as suas habilidades de planejamento e ação. Em sua realização, o projeto de extensão intitulado “Construção de saberes para a promoção do desenvolvimento rural sustentável pela agroecologia em São Thomé das Letras” estava vinculado ao Departamento de Administração e Economia (DAE) da Universidade Federal de Lavras (UFLA) e foi desenvolvido no município de São Thomé das Letras - MG. A Sociedade Brasileira de Eubiose da referida cidade (SBE-STL), ainda, foi parceira na realização do projeto. 17 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Silva, A.W.B. et al. A equipe do projeto foi composta, incialmente, por uma professora da UFLA, também coordenadora do projeto e por um doutorando do Programa de Pós-Graduação em Fitotecnia da universidade. Posteriormente, a equipe teve a participação de dois estudantes de graduação (Agronomia e Administração). As ações se deram entre abril de 2017 e dezembro 2018 e ocorreram, sobretudo, por meio de reuniões e atividades. Nas reuniões, as pautas eram construídas coletivamente. Eram definidas e organizadas as atividades, que seriam realizadas, de acordo com o tema e formato, tais como: oficinas, visitas, palestras, cursos, entre outros. Ao longo desses vinte meses, foram realizadas dez reuniões e doze atividades teóricas e práticas com a comunidade. Os participantes do projeto totalizaram vinte e cinco moradores da zona rural e urbana do município. As reuniões tiveram duração de quatro horas e houve, em média, a participação de nove pessoas. Resultados e discussão A idealização deste projeto surgiu, a partir do convívio de um dos membros da equipe (doutorando) com a realidade do município, ao longo dos últimos dez anos. Após sua participação em movimentos sociais, como membro do Conselho Municipal de Defesa e Conservação do Meio Ambiente (CODEMA) e representante do município no Comitê da Bacia Hidrográfica do Rio Verde, percebeu-se, no que diz respeito a inúmeros conflitos socioambientais no município, o interesse e demanda da comunidade, em aprofundar seus conhecimentos sobre a temática do desenvolvimento rural sustentável, aproximando-se de sua realidade. A partir de abril de 2017, foram realizadas reuniões entre a equipe do projeto e a organização parceira (SBE/STL), a fim de iniciar o contato e o convite à comunidade do município. A organização parceira disponibilizou seu espaço físico, para a realização das reuniões e atividades. Após elaborar uma carta-convite – (explicando o propósito do projeto de extensão) – foi divulgada aos interessados, por meio das redes sociais da comunidade, para as organizações locais, além de abordagens pessoais com moradores da zona rural. A primeira reunião foi realizada, no início de agosto e dela participaram onze moradores tanto da zona rural como da zona urbana do município. Inicialmente houve a apresentação dos presentes, seguida de exposição e discussão do projeto e intervalo para café com prosa. Logo depois do intervalo, foi realizado um diagnóstico participativo, sendo problematizada a percepção da realidade do município pelos participantes e levantamento de demandas com temas e atividades de interesse. Dentre as principais demandas, ressaltam-se, por ordem de importância, o interesse por espaços teóricos e práticos sobre a conservação de recursos hídricos; alternativas para emprego e renda, via fortalecimento da economia solidária; capacitações em agroecologia, alimentação e aproveitamento dos alimentos; produção orgânica e identificação dos produtores rurais do município. Todos os temas apresentados, nas demandas dos participantes, têm relação com a agroecologia e podem fortalecer processos para a promoção do desenvolvimento rural sustentável. Como encaminhamentos dessa primeira reunião, definiu-se que a equipe do projeto ficaria responsável por contatar docentes e profissionais que pudessem contribuir, compartilhando saberes, em espaços dialógicos, relacionados às temáticas de interesse do grupo. Além disso, foi estipulado que os próprios participantes do projeto também iriam compartilhar seus saberes. Assim, as pautas das reuniões foram construídas, coletivamente, por meio das demandas e encaminhamentos de reuniões anteriores. Metodologia A comunicação entre os participantes e a equipe do projeto ocorria, principalmente, por meio das redes sociais em que eram socializadas informações sobre as temáticas de interesse, sínteses das reuniões, definição de pautas, avisos e articulação entre os participantes. Cabe ressaltar que esse projeto não obteve financiamento, sendo as despesas com viagem, materiais de escritório e alimentação custeadas pelos membros da equipe. 18 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Resultados e discussão Apesar de as dificuldades encontradas, uma parcela do grupo se manteve assídua, ao longo do tempo, participando das reuniões e desenvolvendo uma série de atividades que foram avaliadas pelos participantes como muito enriquecedoras. Destacam-se o empenho e a participação do grupo, o qual se manteve frequente às ações do projeto, seja na definição das temáticas, data e local, formato das atividades, assim como na divulgação e articulação com a comunidade. Segue uma breve descrição de cada uma das doze atividades desenvolvidas ao longo dos vinte meses do projeto. A primeira atividade organizada foi com a participação de quatro pessoas do projeto, na etapa do Circuito Sul Mineiro de Agroecologia de 2018, que ocorreu na cidade de Maria da Fé/MG. Esse foi um evento organizado pela “Orgânicos Sul de Minas” com duração de um dia. Nesse evento, os membros da Associação de Produtores de Agricultura Natural de Maria da Fé/APANFÉ receberam outros produtores e demais participantes, a fim de problematizar, compartilhar, realizar capacitações sobre produção orgânica e, também, trocar sementes crioulas em uma das propriedades dos membros da APANFÉ. Foram abordadas as técnicas de produção de adubo orgânico bokashi e de microorganismos eficientes (E.M). Os quatro participantes do projeto compartilharam essa experiência com os demais, na reunião seguinte, de forma oral, por meio de resumo escrito e fotos, salientando as técnicas e as aprendizagens, bem como a satisfação em participar de espaços de trocas de experiências. A segunda atividade foi uma palestra sobre desenvolvimento rural sustentável e Agroecologia conduzida pelo doutorando membro da equipe do projeto. A palestra, que foi expositiva e dialogada, teve duração de duas horas e problematizou os modelos convencionais de desenvolvimento e como essa crise socioambiental cria possibilidades, para maiores questionamentos sobre os princípios, valores e técnicas, que orientam o desenvolvimento rural. Foram apresentados dados sobre a realidade rural brasileira, com o objetivo de estimular a reflexão e o debate sobre como a concepção de desenvolvimento pode contribuir, para aumentar ou diminuir as desigualdades sociais, sobrevivência e manutenção da população no campo, assim como a agroecologia, para a promoção de um desenvolvimento rural sustentável. A terceira atividade organizada foi uma palestra expositiva dialogada sobre preservação de recursos hídricos, com duração de duas horas, à qual compareceram 15 pessoas. Resultados e discussão As reuniões, geralmente, ocorriam no primeiro ou terceiro sábado do mês, em função das atividades desenvolvidas pela organização parceira Sociedade Brasileira da Eubiose. Ao início das reuniões, costumava-se fazer um breve resgate da pauta anterior e, logo depois, dos itens seguintes. A coordenadora do projeto conduzia a reunião, fazendo papel de mediadora, e o doutorando o papel de cofacilitador que auxiliava a mediadora, buscando agilizar e dinamizar o trabalho, favorecendo um ambiente de aprendizagem. Ambos se responsabilizaram por organizar o tempo de fala de cada participante, a fim de se evitar centralizações; controlar o tempo das discussões sobre cada ponto de pauta, para que se 18 Construindo saberes sobre desenvolvimento rural sustentável e agroecologia: uma experiência com extensão universitária alcançassem os objetivos propostos em cada reunião; fazer os relatórios de cada encontro; estimular o debate e a reflexão; fazer sínteses sobre as tomadas de decisão e encaminhamentos, buscando alinhar, dar maior clareza e resgatar os aspectos centrais ao longo das reuniões. Em quatro reuniões, os estudantes de graduação puderam participar e, também, colaboraram por meio de mediação e realização de relatórios. Durante as reuniões, eram definidas as temáticas, organizadas as atividades que seriam realizadas (data e formato, por exemplo), assim como as responsabilidades de cada participante. Após cada reunião, era disponibilizada uma síntese contendo data, participantes presentes, os pontos de pauta com respectivas discussões e decisões, informes e encaminhamentos. Geralmente às reuniões compareciam nove pessoas, em média, cinco mais assíduas e as demais menos frequentes. Algumas tiveram dificuldades, para frequentar todas as reuniões, no entanto acompanhavam o grupo e participavam dele, mais ativamente, pelas redes sociais. Essas dificuldades tornaram-se assuntos de duas das reuniões e estavam relacionadas a diversos fatores tais como: impossibilidade de conciliar o dia da reunião com o trabalho, pois as principais fontes de renda do município advêm do turismo e da mineração; tempo de duração das reuniões, pois tomavam a tarde toda; e logística, visto que a maioria residia na zona rural e não conseguia carona, ou mesmo pelas condições das estradas e do tempo. Durante o desenvolvimento do projeto, foram realizadas duas avaliações, (em dezembro de 2017 e dezembro de 2018), que tiveram como objetivo analisar as reuniões e as atividades desenvolvidas, potencializando os aspectos positivos, compreender as dificuldades vivenciadas e pensar estratégias e sugestões para aperfeiçoar os processos. Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Resultados e discussão Ela foi conduzida por uma economista e mestranda do Programa de Pós-Graduação em Desenvolvimento Sustentável e Extensão da UFLA (PPGDE). A oficina teve início com uma exposição dialogada pelos sete participantes sobre o conceito e os princípios da economia solidária. Foram apresentadas e discutidas algumas iniciativas em Minas Gerais e em todo Brasil. Posteriormente ocorreu uma discussão sobre alternativas de geração de renda, para o município, em que se destacou o fortalecimento da produção orgânica e o acesso a políticas públicas como o Programa Nacional de Alimentação Escolar (PNAE). Percebeu-se que a proposta de uma economia solidária estava alinhada ao pensamento dos participantes, à medida que o espaço de discussão estimulava trocas, socializações e propostas alternativas tanto no que se refere à permuta de materiais e produtos, quanto a informações e conhecimentos. Essa oficina instigou os participantes do projeto a utilizarem mais os espaços de encontro e as redes sociais, para compartilhar informações, livros, mudas, sementes, dentre outras. A sexta atividade ocorreu, por meio de uma palestra sobre gestão de bacias hidrográficas e conservação de estradas rurais, conduzida por outro professor do Departamento de Engenharia da UFLA, Gilberto Coelho. A palestra teve duração de três horas, na qual se discutiram a importância das bacias hidrográficas e o papel dos comitês de bacia, assim como algumas demandas relacionadas ao impacto ambiental sobre o Ribeirão Canta Galo - decorrentes da retirada de água para o abastecimento da zona urbana do município. Quanto à conservação das estradas, foram apresentadas algumas fotos de estradas rurais do município e, além disso, foi articulada junto ao professor a possibilidade de ministrar um curso teórico e prático sobre a conservação de estradas rurais para os profissionais da prefeitura. Os participantes deram ideias de locais, para sua realização e comunicaram à prefeitura essa probalidade. A sétima atividade foi uma oficina sobre (re)aproveitamento integral de alimentos e ocorreu em casa de uma das participantes do projeto com a presença de 15 pessoas. Esse evento teve duração de 5 horas e foi conduzido por dois participantes do projeto que prepararam um espaço teórico e prático sobre esse tema, seguido de uma confraternização. Na atividade prática, foram preparadas quatro receitas (caskalouca de banana, arroz com talos, suco verde e salada de frutas) que foram oferecidas como refeição ao grupo durante a confraternização. Resultados e discussão A roda foi conduzida pelo professor de hidrologia, Marcelo Ribeiro Viola, do Departamento de Engenharia da UFLA, que abordou a importância da conservação dos recursos hídricos, a legislação sobre o tema e conversou sobre o projeto de pesquisa que estava sendo desenvolvido sobre o monitoramento hidrológico do Ribeirão Canta Galo, principal afluente da Área de Proteção Ambiental (APA) - São Thomé, no qual estão inseridas as cachoeiras que são os principais pontos turísticos do município. Além disso, os interessados foram convidados a participar da atividade prática de coleta de dados para o monitoramento do ribeirão. 19 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Silva, A.W.B. et al. A quarta atividade foi sobre métodos de monitoramento hidrológico, também, conduzida pelo professor de hidrologia do Departamento de Engenharia e por um técnico da UFLA. Essa atividade teve duração de dois dias e a participação de três membros do projeto de extensão que acompanharam as atividades de pesquisa do professor. Tais atividades consistiram na coleta de dados referentes à velocidade de infiltração da água no solo e à instalação de sensores e régua de nível no ribeirão, sendo assim, foram instalados pluviômetros e estações meteorológicas na microbacia do Ribeirão Canta Galo. Ressalta-se a importância da temática da água para os participantes. Ao longo do tempo, eles perceberam que o curso do ribeirão, apesar de essencial para o abastecimento do município e para a promoção de um desenvolvimento rural sustentável, tem se tornado cada vez mais escasso e poluído. O município tem o turismo como importante segmento, para a geração de ocupação e renda, sendo baseado em recursos hídricos como as cachoeiras e as belezas paisagísticas naturais. Nesse contexto, surgem conflitos socioambientais relacionados à sua degradação, em decorrência do turismo em massa e do crescimento desordenado das populações rural e urbana que trazem muitos desafios em torno do tema da água no município. Existe, inclusive, um grupo informal no município, denominado Movimento Todos pela Água, que tem atuado, nos últimos anos, em defesa das águas do município e alguns participantes do projeto de extensão são integrantes desse movimento. A quinta atividade organizada foi uma oficina sobre economia solidária e alternativa para a geração de renda. 20 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Resultados e discussão A décima primeira e a décima segunda atividades fizeram parte da semana do meio ambiente do município de São Thomé, uma vez que os participantes do projeto foram convidados a colaborar com o evento. Realizaram-se duas atividades com estudantes da escola estadual do município, quais sejam, uma oficina sobre aproveitamento integral de alimentos e oito palestras sobre o controle biológico de insetos-praga em sistemas orgânicos. A oficina sobre aproveitamento integral dos alimentos foi conduzida por três participantes do projeto (uma nutricionista, um proprietário de restaurante e um analista de mídias sociais) e teve como objetivo difundir os conhecimentos teórico-práticos a respeito desse tema com as merendeiras da escola que também ajudaram a preparar a receita da caskalouca de banana, para ser servida com pão, no lanche (merenda), ao turno da tarde, para cem estudantes do ensino fundamental. Essa oficina foi compartilhada, na reunião seguinte, como sendo algo muito gratificante. Segundo relatos, o fato de a caskalouca se parecer muito com uma carne causou muita surpresa e interesse por parte dos estudantes e dos colaboradores da escola. Nessa mesma semana e escola, ocorreram palestras sobre controle biológico de insetos-praga em sistemas orgânicos, sendo repetidas oito vezes, ao longo do turno da manhã e tarde, para estudantes do ensino fundamental e médio da escola (200 estudantes). A oficina sobre controle biológico foi conduzida pelo professor Luís Cláudio Paterno Silveira do Departamento de Entomologia da UFLA; um estudante de doutorado em Entomologia; um doutorando em Fitotecnia, também membro da equipe e, ainda, contou com apoio de três participantes do projeto de extensão. O objetivo da oficina foi despertar nas crianças e adolescentes o entendimento sobre a importância dos insetos ao meio ambiente. Para isso, foi-lhes apresentada uma palestra rápida, bastante ilustrativa e, em seguida, os alunos puderam ver a diversidade das ordens de insetos, por meio de coleções entomológicas, assim como puderam manipular insetos vivos criados no Departamento de Entomologia. Destacaram-se a curiosidade e o interesse dos estudantes no assunto, como também a satisfação daqueles que conduziram ou colaboraram para que essa atividade acontecesse. Após a realização de dez reuniões e doze atividades com a comunidade, observaram-se dificuldades e potencialidades. Resultados e discussão A receita de caskalouca, que utiliza a casca da banana e o uso dos talos no arroz, chamou muito a atenção quanto às possibilidades de aproveitamento integral de alimentos, além de outras sugestões culinárias, como conservação de alimentos, compartilhadas entre todos. A oitava e nona atividade surgiu, a partir da organização dos participantes, para viabilizar dois cursos gratuitos ofertados pelo Senar/MG, ambos com duração 20 horas (curso de recuperação de nascentes e de recuperação de áreas degradadas) que ocorreram nas propriedades rurais dos participantes do projeto. Uma vez que a temática da água era de grande interesse dos partícipes, tema central ou transversal de outras atividades e, diante da disponibilidade da realização dos cursos, o grupo se mobilizou estimulando moradores de seus bairros e demais interessados. Verificou-se que ter disponibilidade de tempo, para se dedicar a um curso de 20 horas, é um desafio, pois é necessário priorizar essa escolha em detrimento de outras atividades que fazem parte da rotina, compromissos e necessidades dos moradores. 20 Construindo saberes sobre desenvolvimento rural sustentável e agroecologia: uma experiência com extensão universitária O curso de recuperação de nascentes reuniu 15 pessoas, às quais foi aplicada a técnica de recuperação de nascentes, na propriedade de um membro do projeto. Como resultado, providenciou- se o aumento da vazão da nascente e a articulação de um mutirão solidário, para a recuperação de nascentes em outras propriedades, visando contribuir com a melhoria do meio ambiente do município e, também, praticar, consolidar e compartilhar a técnica aprendida. O curso de recuperação de áreas degradadas ocorreu em propriedade de outro participante do projeto e estiveram presentes 16 pessoas, sendo recuperada uma área degradada, apresentando início de voçoroca, provocada por processos erosivos causados pela água. A décima atividade se referiu a uma oficina sobre Plano de Manejo de APA da qual participaram 20 pessoas e teve duração de quatro horas. Foi conduzida pelo professor substituto do Instituto Federal do Sudoeste de Minas, Vinícius do Couto Carvalho, que abordou a temática, capacitando o conceito, papel, aspectos legais e importância de um plano de manejo, para uma Unidade de Conservação, como, por exemplo, uma APA, assunto de grande interesse dos participantes do projeto de extensão. Destacou-se a importância da APA - São Thomé, para o município e sua relação quanto à conservação dos recursos hídricos, paisagísticos e turísticos, demostrando a necessidade do envolvimento da comunidade nesse processo. Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Resultados e discussão Quanto às dificuldades, ressaltam-se as limitações pela falta de financiamento, para cobrir despesas com materiais de escritório, viagens e alimentação, mas, por outro lado, sempre houve empenho em encontrar soluções para minimizá-las, tais como organização (por parte da equipe e dos participantes do projeto) de lanches, hospedagem e alimentação solidária ofertada aos profissionais convidados, entre outros. Um aspecto limitante se referiu à dificuldade de estímulo à participação de pessoas, nas reuniões de elaboração, estruturação e execução das atividades. Entretanto houve grande comprometimento e dedicação por parte daqueles se que se prontificaram a participar, com frequência, tanto das reuniões como das atividades. Além dessa potencialidade, notou-se a satisfação dos participantes diante da possibilidade de se capacitar, aprender, dialogar e ensinar sobre temáticas que são relevantes à sua realidade, sendo demandadas e entendidas como formidáveis a eles e que estão alinhadas com a proposta de promoção do desenvolvimento rural sustentável. Essa nova perspectiva de desenvolvimento, segundo Costabeber e Caporal (2003), em sua formulação mais ampla, significa a realização de potencialidades sociais, culturais e econômicas de 21 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Silva, A.W.B. et al. uma sociedade, em sintonia com o seu entorno ambiental e com seus valores políticos e éticos. Esses autores defendem o desenvolvimento rural sustentável como um processo gradual de mudança que encerra, em sua construção e trajetória, a consolidação de processos educativos e participativos que envolvem as populações, conformando uma estratégia impulsionadora de dinâmicas socioeconômicas mais ajustadas ao imperativo ambiental, aos objetivos de equidade e aos pressupostos de solidariedade intra e intergeracional, tendo a agroecologia como base desse novo paradigma de desenvolvimento. uma sociedade, em sintonia com o seu entorno ambiental e com seus valores políticos e éticos. Esses autores defendem o desenvolvimento rural sustentável como um processo gradual de mudança que encerra, em sua construção e trajetória, a consolidação de processos educativos e participativos que envolvem as populações, conformando uma estratégia impulsionadora de dinâmicas socioeconômicas mais ajustadas ao imperativo ambiental, aos objetivos de equidade e aos pressupostos de solidariedade intra e intergeracional, tendo a agroecologia como base desse novo paradigma de desenvolvimento. As temáticas das atividades desenvolvidas, durante o projeto, possuem interface com a agroecologia à medida que estão alinhadas a suas dimensões ecológica, social, econômica, cultural, política e ética. Considerações finais Esta experiência foi marcada pelo diálogo e troca de saberes, pois construir e realizar dez reuniões e doze atividades com a comunidade constituiu-se resultado de um esforço coletivo entre a universidade e a sociedade. Essa interação promoveu, além disso, o enriquecimento da experiência docente e discente em termos teóricos, metodológicos e pessoais. O uso de metodologias sob uma concepção participativa propiciou a criação de espaços dialogicamente interativos e discursivamente mediados, conforme assinalado por Coelho (2005), propiciando estabelecer uma relação de reciprocidade e de compromisso entre as partes. Foram valorizadas as demandas e o conhecimento local, a fim de superar a ideia de que a Universidade possuía um saber acabado que seria oferecido à sociedade. O projeto estimulou reflexões e práticas sobre o desenvolvimento rural sustentável e agroecologia que pudessem fortalecer a construção de uma sociedade mais democrática e sustentável. Muitos ainda são os desafios, tanto no que se refere à interação dialógica e à participação, quanto à mudança de concepção de desenvolvimento com ações práticas. Essa mudança, a transição para um desenvolvimento de uma sociedade mais sustentável, requer que as pessoas e as instituições reflitam sobre seus valores, suas escolhas, possíveis consequências e o tipo de relações que pretendem estabelecer. O desenvolvimento rural sustentável foi pensado neste projeto, a partir da comunicação entre as pessoas, pessoas e natureza e da análise das prioridades e disponibilidade dos participantes. Nesse sentido, é essencial que haja iniciativas de interação da universidade com a sociedade que visem consolidar a agroecologia e o desenvolvimento rural sustentável, por meio da criação de espaços de diálogo, valorizando os saberes e as demandas da comunidade. Resultados e discussão Ressalta-se que a agroecologia pode ser entendida como um caminho direcionado ao desenvolvimento rural sustentável e consiste na busca de local para, a partir daí, recriar a heterogeneidade do mundo rural, por meio de formas de ação social coletiva, pautadas pelo protagonismo dos atores locais no processo de desenvolvimento (GUSMÁN, 2005). Nesse sentido, este projeto procurou explorar alternativas que propiciassem uma participação ativa, bem como permitiu a sua aproximação com os agentes nele envolvidos, visando estabelecer um processo educativo de comunicação. Todos os espaços estimularam maior afinidade entre os participantes (amizades, trocas, parcerias, mutirões, entre outras), participantes e a sociedade civil organizada (como o movimento “Todos pela água”) e, também, potencializaram maior proximidade dos participantes junto ao poder público (levando demandas à prefeitura e participando em espaços públicos como reuniões da câmara dos vereadores do município). Agradecimentos Agradecemos à Universidade Federal de Lavras, à Sociedade Brasileira de Eubiose (SBE), Departamento de São Thomé das Letras - MG (SBE-STL) e a todos os participantes deste projeto de extensão, sem os quais este trabalho não teria sentido. 22 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Data de submissão: 30/3/2019. Data de aceite: 25/4/2019. Referências ALTIERI, M. A. Agroecologia: bases científicas para uma agricultura sustentável. Guaíba: Agropecuária; AS-PTA, 2002. 592 p. CAPORAL, F. R.; COSTABEBER, J. A. (Org.). Agroecologia e Extensão Rural Sustentável: Contribui- ções para a promoção do desenvolvimento rural sustentável. Brasília: MDA/SAF/DATER/IICA, v.1, 2004. 166 p. 22 Revista ELO - Diálogos em Extensão Volume 08, número 01 - junho de 2019 Construindo saberes sobre desenvolvimento rural sustentável e agroecologia: uma experiência com extensão universitária CAPORAL, F. R.; RAMOS, L.F.; CAPORAL, D. S., COSTABEBER, J. A.; PAULUS, G. (Org.). Exten- são Rural e Agroecologia: temas sobre um novo desenvolvimento rural sustentável. 1.ed. Brasília: MDA/SAF, v.1, 2009. 408 p. CASADO, G. G.; MOLINA, M. G. de; GUZMÁN, E. S. (coord.). Introducción a la Agroecología como desarrollo rural sostenible. Madrid: Ediciones Mundi-Prensa, 2000. COELHO, F. M. G. A arte das orientações técnicas no campo: concepções e métodos. Viçosa: UFV, 2005. 139 p. COSTABEBER, J. A.; CAPORAL, F. R. Possibilidades e alternativas do desenvolvimento rural sus- tentável. In: VELA, H. (Org.). Agricultura familiar e desenvolvimento rural sustentável no Mercosul. Santa Maria: Editora da UFSM/Pallotti, 2003. p.157-194. FORPROEX - FÓRUM DE PRÓ-REITORES DE EXTENSÃO DAS UNIVERSIDADES PÚBLICAS BRASILEIRAS. Política Nacional de Extensão Universitária. Manaus: FORPROEX, 2012. GLIESSMAN, S. R. Agroecologia: processos ecológicos em agricultura sustentável. Porto Alegre: Editora da UFRGS, 2000. 654 p. GUZMÁN, E. S. Agroecologia e Desenvolvimento Rural Sustentável. In: AQUINO, A. M.; ASSIS, R. L. (Ed.). Agroecologia: Princípios e Técnicas para uma Agricultura Orgânica Sustentável. Brasília: Embrapa Informação Tecnológica, 2005. p. 101-132. MOREIRA, R. M. CARMO, M. S. do. Agroecologia na construção do desenvolvimento rural susten- tável. Agriculturas em São Paulo. São Paulo, v. 51, n. 2, p. 37-56, jul./dez. 2004. VERDEJO, M. E. Diagnóstico rural participativo: guia prático DRP. Brasília, DF: MDA/Secretaria da Agricultura Familiar, 2006. 62 p. Data de submissão: 30/3/2019. Data de aceite: 25/4/2019. 23
https://openalex.org/W4205910454	https://bjgp.org/content/bjgp/72/718/e342.full.pdf	English	null	Accessing primary care and the importance of ‘human fit’: a qualitative participatory case study	British journal of general practice	2,021	cc-by	7,415	Research Research Jennifer Voorhees, Simon Bailey, Heather Waterman and Kath Checkland Jennifer Voorhees, Simon Bailey, Heather Waterman and Kath Checkland Access theory: capturing complexity International scholars studying healthcare access have long recognised a disparity between the simplistic approaches to access evident in policy and the complexities of how individuals actually access care,2 with policies often focused on timeliness or numbers of appointments, and tending to see continuity of care as something to be traded off against better access. Access theories, by contrast, aim to capture the complexity of individuals’ needs alongside characteristics of health systems and populations, issues which are often missing from policy. Donabedian identified ‘accessibility’ as ‘characteristics of the service that facilitate or obstruct use by potential clients.’ 3 Penchansky and Thomas elaborated on this, defining access ‘as a concept representing the degree of fit between clients and the system.’ 4 Conclusion Email: jennifer.voorhees@manchester.ac.uk An understanding of access as ‘human fit’ has the potential to address longstanding problems of access within general practice, focusing attention on the need for staff training and support, and emphasising the importance of continuity of care. Submitted: 21 June 2021; Editor’s response: 23 August 2021; final acceptance: 17 November 2021. Submitted: 21 June 2021; Editor’s response: 23 August 2021; final acceptance: 17 November 2021. Accessing primary care and the importance of ‘human fit’: a qualitative participatory case study ©The Authors This is the full-length article (published online 8 Feb 2022) of an abridged version published in print. Cite this version as: Br J Gen Pract 2022; DOI: https://doi.org/10.3399/BJGP.2021.0375 Background Background Good access to primary care is an important determinant of population health. While the academic literature on access to care emphasises its complexity, policies aimed at improving access to general practice in the UK have tended to focus on measurable aspects, such as timeliness or number of appointments. Address for correspondence Address for correspondence Centre for Primary Care and Health Services Research, University of Manchester, Oxford Road, Manchester M13 9PL, UK. Method Levesque et al’s conceptual framework of patient- centred access was applied and the study used multiple qualitative methods (interviews, focus groups, and observation). Analysis was ongoing, iterative, inductive, and abductive with the theory. Aim To fill the gap between the complex understanding of primary care access in the literature and the narrow definition of access assumed in UK policies. Qualitative research about patient experiences has defined additional concepts such as candidacy, which describes ‘the ways in which people’s eligibility for medical attention and intervention is jointly negotiated between individuals and health services.’ 9 Related work exploring access for people with mental health problems in the UK applied candidacy, alongside concordance (‘the match between users’ and practitioners’ narratives and resources for successful access’ ), and recursivity (‘the interdependency of users’ experiences of health services and future actions in regards to health and help-seeking’ ) to capture the nuances of people’s lived experiences.10 Importantly, in these patient- centred approaches, continuity of care is seen as a potential contributor to better access. Design and setting Qualitative, community-based participatory case study within the geographic footprint of a clinical commissioning group in the north west of England. Data collection took place from October 2015 to October 2016. Purposive sampling and snowball approaches were used to achieve maximum variation among service users and providers across general practice settings. INTRODUCTION Aday and Andersen’s multiple models of access derived from patterns of utilisation, incorporating patients’ ‘predisposing, enabling, and need components’ as well as differentiating ‘potential’ from ‘realised’ access.2,5–7 More recent conceptual work has embraced these theories, defining access as ‘… a multidimensional concept based on the interaction (or degree of fit) between health care systems and individuals, households, and communities.’ 8 Abstract The ability of citizens to access the health care they need is one of the most important attributes of any health system, with access to primary care particularly important for addressing population health inequalities.1 This study applies an existing conceptual framework for understanding access to primary care and develops it further, drawing conclusions relevant to contemporary policy approaches to this important issue. The ability of citizens to access the health care they need is one of the most important attributes of any health system, with access to primary care particularly important for addressing population health inequalities.1 This study applies an existing conceptual framework for understanding access to primary care and develops it further, drawing conclusions relevant to contemporary policy approaches to this important issue. Results The comprehensiveness of Levesque et al’s access theory resonated with diverse participant experiences. However, while its strength was to highlight the importance of people’s abilities to access care, this study’s data suggest equal importance of healthcare workforce abilities to make care accessible. Thus, the authors present a definition of access as the ‘human fit’ between the needs and abilities of people in the population and the abilities and capacity of people in the healthcare workforce, and provide a modified conceptual framework reflecting these insights. In 2013, Levesque et al, synthesized a conceptual framework for ‘patient- centred access to health care’ (Figure 1),11 Address for correspondence Centre for Primary Care and Health Services Research, University of Manchester, Oxford Road, Manchester M13 9PL, UK. Email: jennifer.voorhees@manchester.ac.uk Submitted: 21 June 2021; Editor’s response: 23 August 2021; final acceptance: 17 November 2021. ©The Authors This is the full-length article (published online 8 Feb 2022) of an abridged version published in print. Cite this version as: Br J Gen Pract 2022; DOI: https://doi.org/10.3399/BJGP.2021.0375 Voorhees J (ORCID 0000-0003-3327-0026), MD, PhD, MHS, National Institute for Health Research clinical lecturer; Checkland K (ORCID 0000-0002- 9961-5317), MBBS, BMedSci, MA(Econ), PhD, MRCGP, professor of health policy and primary care, Centre for Primary Care and Health Services Research, University of Manchester, Manchester. Bailey S (ORCID 0000-0001-9142-2791), BSc, MA, PhD, research fellow, Centre for Health Services Studies, University of Kent, Kent. Waterman H (ORCID 0000-0001-7052-2734), BNurs, PhD, DipN, OND, RN, former head of school of health sciences, Cardiff University, Cardiff. METHOD This was a qualitative participatory case study23 in the north west of England, using the Levesque et al theory11 as a tool within data collection and analysis. The study was based in the geographical area covered by the Tameside and Glossop (T&G) Clinical Commissioning Group (CCG). CCGs have delegated responsibility for overseeing primary care provision, commissioning care, and managing contracts, including implementing national access policies. Situating the study in a single CCG area allowed for in-depth exploration of the contextual factors affecting access to care in a mixed area covering both urban and rural communities. Keywords access; general practice; population health; i i ti i liti ti it f access; general practice; population health; service organisation; inequalities; continuity of care. service organisation; inequalities; continuity of care. e342 British Journal of General Practice, May 2022 that timeliness was not more important to patients or doctors than continuity.16–18 How this fits in In England in the mid-2010s, policies to provide 7-day extended access to general practice services dominated. These were costly, not based on any recognised theory of access, and rolled out without any evidence that they were needed, further undermining continuity.19–21 Current access policy is, in part, driven by the 2019 Conservative election manifesto promise of 50 million more appointments a year in general practice.22 While superficially attractive as a simple and easily understood policy, this approach again prioritises volume of appointments over appropriateness or need. Access to general practice is an important topic that receives much attention. While research literature recognises access issues as complex, policy on access to general practice in the UK tends to define it narrowly, with unintended consequences. This study found that an existing comprehensive model of access resonated with frontline experiences. The study offers further modifications to the theory to make it practically useful for both policymakers and clinicians to address longstanding issues of access to general practice. This research therefore aimed to fill the gap between the complex understanding of access in the literature and the narrow definition of access assumed in many policies. The study explored how access theory, as depicted by Levesque et al ’s conceptual framework of access to care,11 resonated with a broad range of stakeholders, and considered how the theory could be developed to inform policy. which juxtaposes five dimensions of accessibility of the healthcare system (approachability, acceptability, availability and accommodation, affordability, and appropriateness) with the abilities of patients to identify health needs, seek, reach, and utilise care. This conceptualisation builds on previous access literature and has multiple strengths, including conceptualising continuity as a component of access (within the dimension of appropriateness). While Levesque et al ’s definition of access does not include the concept of ‘fit’, their framework clearly depicts this dynamic, resonating with previous theories of access and concepts such as candidacy described above. For these reasons, this conceptualisation of access was applied within this research. UK access policy: narrow conceptualisation with unintended consequences Despite the complexity evident in empirical studies of service access and manifest in associated theories, policies addressing access to general practice in the UK often assume a narrow definition of access, linked to measurable targets such as timeliness or number of appointments.12,13 This narrow approach fails to embrace the real-life complexity captured in theoretical literature, and has potentially worsened health inequalities and inequities of access to care.14 Importantly, rather than embracing continuity of care as a component of access, policy casts continuity as something separate, to be traded off against better access. Important policy examples include the focus on providing access within 48 hours in the 2000s, and the widespread adoption of ‘advanced access’ systems to meet those targets.15 British Journal of General Practice, May 2022 e343 Community-based participatory research Thi h d t d h d i d Community-based participatory research This research adopted an approach derived from community-based participatory research.24,25 Building on initial contacts made with the CCG and relevant groups, such as Healthwatch, a community-based participatory research team was formed consisting of 12 members of the T&G community, including patients, carers, GPs, practice staff, commissioners, and the voluntary sector. Levesque et al ’s conceptual framework was used11 to introduce the community research team to the complexities of access to care. The community research team contributed to project design and to data collection and analysis, meeting 35 times over 4.5 years. The community Such policy solutions were adopted even though available evidence demonstrated British Journal of General Practice, May 2022 e343 Professional values, norms, culture, and gender Transparency Outreach Information Screening Approachability Ability to perceive Ability to seek Ability to reach Ability to pay Ability to engage Acceptability Availability and accommodation Affordability Appropriateness Geographic location Accommodation Hours of opening Appointments mechanisms Direct costs Indirect costs Opportunity costs Technical and interpersonal quality Adequacy Coordination and continuity Healthcare consequences Healthcare reaching Healthcare seeking Healthcare needs Health literacy Health beliefs Trust and expectations Personal and social values, culture, gender, autonomy Living environments Transport Mobility Social support Income Assets Social capital Health insurance Empowerment Information Adherence Caregiver support Perception of needs and desire for care Healthcare utilisation Economic Satisfaction Health Primary access Secondary access team members contributed to purposive sampling decisions. Interviews and focus groups explored participants’ experiences of accessing or providing care. Community research team members assisted with focus group facilitation. Non-participant observation in communal general practice spaces focused on receptionist activities and interactions with service users. Non- participant observation in relevant public meetings focused on how access was discussed by service users, providers, and commissioners. Levesque et al ’s conceptual framework of access to care11 was utilised as a visual prompt26 to support discussion during data collection in interviews and focus groups, and occasionally during observation sessions. The 54 interview and focus group participants included service users (patients, carers, and voluntary sector roles) and service providers (GPs, practice managers, receptionists, commissioners, and other relevant NHS roles). Community-based participatory research Thi h d t d h d i d The 39 service user participants included: people possessing each of the nine protected characteristics under the UK Equality Act 201032 (age, disability, gender reassignment, marriage and civil partnership, race, religion or belief, sex, and sexual orientation), as well as patients with various health needs (both physical and mental); carers of people with chronic diseases, including dementia, mental health problems, and learning disabilities; people facing economic deprivation; people for whom English was not their first language; and members of relevant voluntary organisations. Patient and carer participants ranged in age from 26–79 years, with carers discussing patients ranging from age zero to 101 years. The 15 service provider participants had between four and 26 years of experience. The surgery sites included small and large surgeries in all five neighbourhoods of T&G, and the 7-day access hub sites. The meetings and events observed included CCG Governing Body and Primary Care Joint Committee meetings, and CCG and Healthwatch public events. In all, the study collected data that spanned experiences across approximately 36 of the 45 general practice/hub sites in T&G. research team represented a wide range of backgrounds and professional roles, and this diversity provided an important resource for the project. Figure 1. A conceptual framework for patient-centred access to health care (reproduced from Levesque et al, 2013) 11 Data collection Data collection took place from October 2015 to October 2016, and included 19 semi- structured interviews,26 7 focus groups,27 and 71 hours of observation,28,29 (summarised in Table 1). Purposive sampling27 and snowball approaches30,31 were used to achieve maximum variation among service users and providers across general practice settings. Community research Table 1. Summary of interview, focus group, and observation data generated in this study Method Events, n Participants, n Approximate hours Interviews: service users 9 9 12 Interviews: service providers 10 10 (3 also patients in area) 12 Focus groups: service users 6 30 10 Focus groups: service 1 5 1.5 providers Observation: surgeries 13 8 sites (including interactions 45 with approximately 40 receptionists, GPs, practice managers, practice nurses, administrative staff, and patients) Observation: meetings and 12 Approximately 70 individuals 26 events across all meetings/events Total 51 54 (+ approximately 110) 106.5 e344 British Journal of General Practice, May 2022 Table 1. Summary of interview, focus group, and observation data generated in this study Method Events, n Participants, n Approximate hours Interviews: service users 9 9 12 Interviews: service providers 10 10 (3 also patients in area) 12 Focus groups: service users 6 30 10 Focus groups: service 1 5 1.5 providers Observation: surgeries 13 8 sites (including interactions 45 with approximately 40 receptionists, GPs, practice managers, practice nurses, administrative staff, and patients) Observation: meetings and 12 Approximately 70 individuals 26 events across all meetings/events Total 51 54 (+ approximately 110) 106.5 e344 British Journal of General Practice, May 2022 Table 1. Summary of interview, focus group, and observation data generated in this study Table 1. Summary of interview, focus group, and observation data generated in this study Data analysis An ongoing, iterative analysis process was influenced by grounded theory,33 the five- step framework approach,34,35 and abductive analysis,36 balancing inductive insights from the data with insights from theory. The community research team and academic research team members contributed to each of the five stages of analysis: familiarisation, building the coding tree, coding, charting, and mapping and interpretation. ‘Familiarisation’ included immersion in the raw data. Interviews and focus groups were recorded and independently transcribed, checked for accuracy, and anonymised by the research team before sharing with community team members. Handwritten fieldnotes were made during observation sessions and then expanded in typed form shortly after the sessions. ‘Building the coding tree’ included discussions with team members about memos and notes made during familiarisation, notes made during interviews on printouts of the Levesque et al model, and important emerging concepts. These discussions directed further sampling until it was agreed saturation37,38 was met when it was felt a diverse range of experiences across of a variety of participants and practice sites was understood. ‘Coding’ of the data using NVivo (version 11) was informed by the rich discussions with team members during data collection and analysis. Coded transcripts were discussed further with team members to ensure consistency with early discussions and their insights. ‘Charting’ consisted of arranging data by code and further analysing content within each code39 through continued discussion with team members. ‘Mapping and interpretation’ involved integration of insights, including advancing the access theory, building on the previous analysis stages. capabilities and with varying capacity to engage. This research therefore presents a definition of access as the ‘human fit’ of the needs and abilities of the population with the capacity and abilities of the healthcare workforce, in the context of particular societal conditions and organisational structures and processes. The modified theory is depicted in Figure 2. This new formulation retains many of Levesque et al ’s categories, but does two additional things: first, it emphasises that human abilities are important on both sides of the access equation, with those of the healthcare workforce as important as those of the population; second, it shows that both societal factors (affecting the population) and system factors (constraining staff) are mediated by these human abilities, and that both operate within a wider context characterised by the state of population health and the availability of healthcare workers. Data analysis Furthermore, it illustrates how continuity matters throughout the human interactions at all stages of accessing care, not just within the dimension of appropriateness. Below, study data are presented, which support these modifications to the conceptualisation of access. Each responder has a unique code in which IR denotes ‘interview responder’ and FG denotes 'focus group' with individual responders. Stakeholder perceptions of access. Study participants (both service users and service providers) were enthusiastic about the complex depiction of access in the applied theory.11 This conceptualisation of access chimed with their experiences in a way that contemporary policy discourses did not. For example, following a full discussion of experiences of accessing care, a patient participant viewing the visual prompt commented: Stakeholder perceptions of access. Study participants (both service users and service providers) were enthusiastic about the complex depiction of access in the applied theory.11 This conceptualisation of access chimed with their experiences in a way that contemporary policy discourses did not. For example, following a full discussion of experiences of accessing care, a patient participant viewing the visual prompt commented: RESULTS The study found that Levesque et al ’s11 depiction of access as a complex interaction between individuals seeking care and the structural and cultural characteristics of the healthcare-providing organisation was intuitively understood by both patients and care providers, and in observations the study was able to recognise elements of the framework as they occurred. However, it was found that the framework, as depicted by Levesque et al, failed to explain all of the features of the situations that were observed or heard described. This led to modification of the framework, and offering an incremental advancement of Levesque et al ’s original theory. The new formulation builds on the definition implicit in Levesque et al ’s model (access as a ‘fit’ between human needs and capabilities and the structures and organisational processes in healthcare organisations), but puts additional emphasis on the human interactions facilitating (or hindering) access, highlighting the fact that organisational structures and processes are mediated by individuals with different ‘It reflects, I think, the discussions we’ve just had, and it … fits with the way I’ve been trying to describe things, because I can recognise all of those, and I’ve alluded to them.’ (IR01, patient) A GP interview participant responded similarly: A GP interview participant responded similarly: A GP interview participant responded similarly: Jennifer Voorhees (JV), interviewer: ‘How do you feel this idea of access resonates with you?’ IR04, GP: ‘Really strongly. I think it’s very, very useful and tells you — it has just so much more in it than how people are usually seeing access.’ Jennifer Voorhees (JV), interviewer: ‘How do you feel this idea of access resonates with you?’ Jennifer Voorhees (JV), interviewer: ‘How do you feel this idea of access resonates with you?’ IR04, GP: ‘Really strongly. I think it’s very, very useful and tells you — it has just so much more in it than how people are usually seeing access.’ British Journal of General Practice, May 2022 e345 ‘I think it’s relevant ... Especially things like this: “the ability to reach, the ability to engage” … “the abilities of people” … I think that’s really, really, really important … because I don’t think they think about that enough … Also — and I don’t know whether it should be on here … there’s the abilities of GPs to be able to engage ... A GP interview participant responded similarly: I think that is massively important … ’ (IR10, NHS staff member) ACCESS = the human fit of the needs and abilities of the population with the capacity and abilities of the healthcare workforce with the capacity and abilities of the healthcare workforce Population Healthcare workforce Individuals, households, and communities Individuals, teams, organisations, and systems Human interaction Population health Human factors: Attitudes Beliefs Empowerment Expectations Experiences Knowledge Resources Roles Workforce capacity Societal factors Population abilities Workforce abilities System factors Healthcare needs Health literacy Health beliefs Trust Expectations Ability to perceive Ability to approach or be approachable Transparency Outreach Information Screening Perception of needs and desire for care Personal and social values: culture, gender, and autonomy Ability to seek Ability to accept or be acceptable Professional values Norms Culture Gender Seeking health care Living environments Transport Mobility Social support Ability to reach Ability to be available and to accomodate Location Hours of opening Appointment mechanisms Reaching health care Income assets Social capital Health Insurance Ability to pay Ability to be afforded Direct costs Indirect costs Opportunity costs Healthcare utilisation Empowerment Information Adherence Caregiver support Ability to engage Ability to be appropriate Technical and interpersonal quality Adequacy Coordination Continuity Healthcare consequences Better fit between abilities of population and healthcare workforce: population needs met/health improved, workforce optimised Healthcare workforce Individuals, teams, organisations, and systems Human interaction This participant insight was one of several about the importance of the abilities of people within the workforce, and is explored further in the following section. Population health Human fit: people and their abilities in context and the importance of continuity. Patients told us that they wanted to be treated as individuals, that they appreciated when receptionists knew them, and that this aspect of care had deteriorated compared with their experiences in the past. One commissioner explained how during engagement activities with members of the community around aspects of general practice, the top patient priority was to be treated as an individual, and that this was contrary to the current policy emphasis on timeliness of appointments: ‘When I read back what people said and summarised it … people put at the top that they really liked being treated as an individual first. e346 British Journal of General Practice, May 2022 Improving access: relevance of the concept of ‘human fit’ In addition to illuminating current access issues as experienced by patients and staff, applying the new conception of access as the human fit between the abilities of both patients and staff can help to define ‘better’ access, providing specific targets for change. For example, the data suggest that improving access could mean improving continuity, providing more opportunity to achieve that human fit between staff and patients. Participants felt that the access theory was useful for this purpose as well: Participants recognised that continuity was important across the clinical team: ‘Yeah … going back to this team and longevity in the practice, having a stable team of somebody saying, “I always go to [name], the healthcare assistant. He’s great. I’ve been going for five years.”’ (IR07, commissioner) It was noticeable that, while patients requested clinicians who were known to them, many practice appointment systems were unable to accommodate this, leading to conflicts. An exchange from a focus group of practice managers demonstrates workload and doctor turnover limiting capacity to offer continuity: ‘What you can almost come out with is a toolkit to help you in your area look at access seriously. So this could be [pointing to diagram], these could be the ways you could start to look at access. Not “the answer”, it’s, “Before you look at that, think about this in your patch.”’ (IR07, commissioner) The complexity of human factors affecting access and the need for a proactive outreach to patients with unmet needs were relevant issues that participants felt were not appreciated by politicians. This GP explained: ‘We had an example just this morning, a patient came in, and admittedly she’d come down because she said she couldn’t get through on the phone … She’s known to us, quite bad asthmatic, she was having a flare-up of her asthma, but only wanted to see one of two particular GPs. One was actually off on long-term sick, don’t know when he’s coming back, the other one was fully booked, but we had an appointment with a locum and another GP. We could have given her an appointment within 40 minutes of her turning up at the window, and it wasn’t good enough for her … She went away saying that she was going to complain. A GP interview participant responded similarly: For example, an NHS staff member with public engagement experience appreciated the depiction of ‘abilities of people’ on the population side, but offered an insight about the importance of abilities of the healthcare workforce: ‘… some GPs can be challenging because of their own stress levels or their workload … In one surgery I’ll have a GP who is … the kind of person that would talk like you and I talk, and then say, “Is there anything else that I can help you with?” and, “How are you?” and will look you in the eye and will e346 British Journal of General Practice, May 2022 me, if you’re really ill … you’ll see anybody who’s qualified to see you, and you don’t refuse an appointment within 40 minutes. I actually think that that’s quite good to be offered something so quick, but it wasn’t good enough, and I’m fully expecting, when I’m back in … that she’ll have written in and complained, and this is the kind of thing that you’re up against all the time.’ (FG1R2, practice manager) go away from their computer screen, and that’s how you want them to be. And with others, they will sit down, and they’ll say … “What do you want?” … and that’s not helpful … If you were somebody who wasn’t feeling very comfortable or wasn’t feeling very well or wasn’t feeling very in the mood to have a debate … you’d feel, “Well actually I’ll just take the prescription, thanks.”’ (IR15 patient/ voluntary sector worker) To treat someone as an individual requires some knowledge of them or at least openness to that knowledge. This was observed to occur more readily in smaller practices. For example, in small practices receptionists often provided significant continuity for the patients, knowing them and their circumstances well. Some GPs recognised the role receptionists play in knowing patients within the practice population: Thus, the data demonstrates how continuity can be an important component of good access, but emphasises that it is also a complex construct. In particular, the human skills of all members of the practice team are important in determining whether or not patients feel ‘known’ and therefore understood. Moreover, the current context of general practice with high workload and shortages of staff requires patients to also be adaptable and to show understanding of the pressures being experienced by staff. A GP interview participant responded similarly: That was their ultimate priority … Appointments was important, but people didn’t sit there and all say “I can’t get an appointment.” They thought the topic needed looking at, but they had mixed experiences.’ (IR07, commissioner) ‘When I read back what people said and summarised it … people put at the top that they really liked being treated as an individual first. That was their ultimate priority … Appointments was important, but people didn’t sit there and all say “I can’t get an appointment.” They thought the topic needed looking at, but they had mixed experiences.’ (IR07, commissioner) Healthcare consequences As depicted in Figure 2, the modified theory reflects that feeling as if you are being treated as an individual relates to the receptionists’ and other care team members’ abilities to be approached, to accept, to accommodate, and to be appropriate, as well as their knowledge of particular individuals. Furthermore, it is not just the receptionist or clinician’s innate or learned abilities, but their capacity to use those abilities within the context of available resources, system factors, and workload pressures. A voluntary sector worker and patient reflected on how clinicians’ innate abilities were constrained by their workload: Better fit between abilities of population and healthcare workforce: population needs met/health improved, workforce optimised Better fit between abilities of population and healthcare workforce: population needs met/health improved, workforce optimised Figure 2. Access as human fit (adapted from Levesque et al’s conceptual framework for access to health care) 11 JV: ‘You mentioned that this isn’t necessarily the way access is usually talked about.’ IR04, GP: ‘No, it’s all about whether you can get an appointment within a certain length of time.’ Participants offered additional insights and critiques based on their experiences. A GP interview participant responded similarly: ‘And they also build up a knowledge of the individual characters who are ringing us up and how to handle them. So yeah, they’re on the same journey as us, in that respect.’ (IR13, GP) British Journal of General Practice, May 2022 e347 Implications for research and practice Implications for research and practice The concept of access as human fit can be applied to inform the design of interventions to improve access and evaluations of access policies and interventions. Further research is also needed with practices and primary care networks to determine how to best apply this understanding of access in practical ways to improve fit for those in the population with unmet needs. Such research should continue to take a participatory approach in order to ensure patient and public perspectives shape the research efforts. Politicians and policymakers can apply this understanding of access to convey a different goal for improving access to care: better human fit. Policies that take account of this conceptualisation would see continuity as an important contributor to better access, with attendant improvements in a wide range Improving access: relevance of the concept of ‘human fit’ Now to ‘I don’t think the politicians appreciate any of that … They just see it as: if you’ve … got the flu, how quickly can you get to see your GP? Not that you need to see your GP with the flu, but that’s their own direct experience of doctors, and they don’t give any thought to the opportunistic stuff, the preventative stuff, the vulnerable people who don’t actually ask for appointments, but need probably more British Journal of General Practice, May 2022 e347 care than most voting adults. And so yeah, there is just an impoverished debate around it. There’s a lack of imagination about the true nature of the problem.’ (IR13, GP) provided a detailed understanding from multiple perspectives,41 and the participatory approach ensured that the research was grounded in and steered by the lived experiences of the local community. Some GPs recognised that understanding access in this way and rethinking some of the rules within general practice around appointments were key to addressing health inequalities: A potential limitation is that the study occurred in one area. However, the ability to forge partnerships in the community and to explore the area in depth supported successful recruitment, including from groups sometimes termed ‘hard to reach’. A further limitation is the potential for social desirability bias, in that participants could respond positively to the access theory in order to please the researcher. However, participants were encouraged to tell their own story before mentioning the theory, and efforts were made to encourage critique. ‘There should never be a one-size-fits- all rule, should there? I think flexibility is definitely the route to helping with health inequalities.’ (IR04, GP) This research suggests that applying the ‘access as human fit’ conceptualisation has the potential to stimulate the imagination, highlight the breadth of issues to be addressed, and broaden targets for change. For example, by moving beyond timeliness to consider the human abilities and factors affecting interactions between the population and healthcare staff, as depicted in Figure 2. Comparison with existing literature Comparison with existing literature This work resonates with existing theoretical literature on access to health care, as well as with previous calls for access theory to be applied within service improvement and research.14,42 It resonates with other theories such as candidacy that embrace a recursive and dynamic view of access interactions.9 While Levesque et al ’s original theory includes aspects of the human capabilities of service providers under the heading of ‘appropriateness’, the study suggests that these capabilities are important across all of the dimensions of access. Thus, for example, ‘ability to be approachable’ highlights that the extent to which a service is experienced as approachable will be determined by both the design of the system and the human abilities of those greeting service users. This study offers an incremental adjustment of the original theory, which is a novel contribution. DISCUSSION Summary y This study demonstrates the relevance of a broad and complex theory of access, and advances a modified concept of access as the ‘human fit’ between the needs and abilities of the population with the capacity and abilities of the healthcare workforce. This modified concept of access, as depicted in Figure 2, builds on Levesque et al ’s patient-centred access concept11 to highlight the importance of both human and contextual factors within society and the healthcare system. Through qualitative and participatory research, this study demonstrates diverse service user and workforce stakeholder perceptions of accessing primary care in the UK. The relevance of continuity as a component of access, rather than something separate, has been emphasised. The multiple human and contextual factors of access, as depicted by the model in Figure 2, create opportunities to address components of access that have been overshadowed by historical policy emphasis on timeliness or number of appointments. The data presented demonstrate that attention to these other factors would be welcomed by both service users and the healthcare workforce, who have long recognised their value and contribution to longstanding access problems. e348 British Journal of General Practice, May 2022 Competing interests The authors have declared no competing interests. Ethical approval Ethical approval was obtained from the Greater Manchester West Research Ethics Committee (reference: 15/NW/0740) alongside NHS Research Governance Approval (reference: 177650). Funding This research was funded by the National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care (NIHR CLAHRC) Greater Manchester, now recommissioned as NIHR Applied Research Collaboration Greater Manchester. The views expressed in this publication are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. Acknowledgements The authors sincerely thank the members of the community-based participatory research team in Tameside and Glossop — Lesley Surman, Tina Greenhough, Jean Hurlston, Joanna Bircher, Jamie Douglas, Manoj Mistry, Catherine Poulton, Peter Denton, Tracey Turley, Julie Beech, Laura Browse, and Elaine Parker-Boyd — for their significant contributions to this research. The authors would also like to thank the participants who shared their experiences, and all those who facilitated this research. Thank you to Katherine Cairoli for technical assistance with Adobe InDesign to adapt the access model. Strengths and limitations The study’s main strength lies in the application and advancement of a theoretical framework.40 Triangulation of methods e348 British Journal of General Practice, May 2022 Funding Although this research took place before the COVID-19 pandemic, the understanding of access as human fit continues to be relevant. There were major changes to general practice appointments due to the pandemic,47 that is, remote consultation, which occurred out of necessity and without reference to patient preferences, and are likely to be associated with worse experiences of access for some groups, while potentially improving access for others. Further research can utilise the concept of human fit to understand the impacts of changes made, including on continuity of care. It could be used to support practices to think through which aspects of changes should be retained and which require modification to address potentially worsening inequalities. of outcomes (including mortality,43 accident and emergency use,44 hospital admissions,45 and patient and provider satisfaction,17,18) rather than as something that must be traded away for improved speed. Wider application of this conceptualisation may support commissioners and providers to work in partnership with patients to improve access as human fit. Interventions are often aimed at the health service side, including creating more services,46 but this framework supports focusing on the abilities of people in the population, including issues of health literacy, as well as supporting training for practice staff to enable them to work more effectively within current constraints. This might, for example, support staff in understanding how they can, without increasing their workload, find ways of helping patients to ‘feel known’. British Journal of General Practice, May 2022 e349 Open access This article is Open Access: CC BY 4.0 licence (http://creativecommons.org/licences/ by/4.0/). REFERENCES 23. Stake RE. Case studies. In: Denzin NK, ed. Strategies of qualitative inquiry. Los Angeles, CA: Sage Publications, 2003, 134–164. 24. Minkler M, Wallerstein N. Community-based participatory research for health: from process to outcomes. 2nd edn. San Francisco, CA: Jossey-Bass, 2008. 1. Starfield B, Shi, L, Mackinko, J. Contribution of primary care to health systems and health. Milbank Q 2005; 83(3): 457–502. 25. Israel BA, Eng E, Schulz AJ, Parker EA. Methods for community-based participatory research for health. 2nd edn. San Francisco, CA: Jossey-Bass, 2012. 2. Aday LA, Andersen R. A framework for the study of access to medical care. Health Serv Res 1974; 9(3): 208–220. 3. Donabedian A. Aspects of medical care administration: specifying requirements for health care. Cambridge: Harvard University Press,1973. 26. McCracken GD. The long interview. Newbury Park, CA: Sage Publications, 1988. for health care. Cambridge: Harvard University Press,1973. 4. Penchansky R, Thomas JW. The concept of access: definition and relationship to consumer satisfaction. Med Care 1981; 19(2): 127–140. 27. Green J, Thorogood N. Qualitative methods for health research. 2nd edn. Los Angeles, CA: Sage Publications, 2009. 5. Ricketts TC, Goldsmith LJ. Access in health services research: the battle of the frameworks. Nurs Outlook 2005; 53(6): 274–280. 28. Emerson RM, Fretz RI, Shaw LL. Writing ethnographic fieldnotes. 2nd edn. Chicago, IL: The University of Chicago Press, 2011. 6. Aday LA, Andersen RM. Equity of access to medical care: a conceptual and empirical overview. Med Care 1981; 19(12): 4–27. 29. Madison DS. Critical ethnography: method, ethics, and performance. 2nd edn. Thousand Oaks, CA: Sage Publications, 2012. 7. Andersen RM, McCutcheon A, Aday LA, et al. Exploring dimensions of access to medical care. Health Serv Res 1983; 18(1): 49–74. 30. Biernacki P, Waldorf D. Snowball sampling — problems and techniques of chain referral sampling. Sociol Methods Res 1981; 10(2): 141–163. 8. McIntyre D, Thiede M, Birch S. Access as a policy-relevant concept in low- and middle-income countries. Health Econ Policy Law 2009; 4(Pt 2): 179–193. 31. Goodman LA. Comment: On respondent-driven sampling and snowball sampling in hard-to-reach populations and snowball sampling not in hard-to- reach populations. Sociol Methodol 2011; 41(1): 347–353. 9. Dixon-Woods M, Cavers D, Agarwal S, et al. Conducting a critical interpretive synthesis of the literature on access to healthcare by vulnerable groups. BMC Med Res Methodol 2006; 6: 35. 32. UK Government. Equality Act 2010. https://www.legislation.gov.uk/ ukpga/2010/15/contents (accessed 1 Feb 2022). 10. REFERENCES Kovandzic M, Chew-Graham C, Reeve J, et al. Access to primary mental health care for hard-to-reach groups: from ‘silent suffering’ to ‘making it work’. Soc Sci Med 2011; 72(5): 763–772. 33. Corbin J, Strauss A. Basics of qualitative research: techniques and procedures for developing grounded theory. 3rd edn. Los Angeles, CA: Sage Publications, 2008. 11. Levesque JF, Harris MF, Russell G. Patient-centred access to health care: conceptualising access at the interface of health systems and populations. Int J Equity Health 2013; 12: 18. 34. Pope C, Ziebland S, Mays N. Qualitative research in health care. Analysing qualitative data. BMJ 2000; 320(7227): 114–116. 12. Simpson JM, Checkland K, Snow S, et al. Access to general practice in England: time for a policy rethink. Br J Gen Pract 2015; DOI: https://doi. org/10.3399/bjgp15X687601. 35. Ritchie J, Spencer L. Qualitative data analysis for applied policy research. In: Bryman A, Burgess RG, eds. Analyzing qualitative data. Oxon: Routledge, 1994. 36. Tavory I, Timmermans S. Abductive analysis: theorizing qualitative research. Chicago, IL: The University of Chicago Press, 2014. 13. Boyle S, Appleby J, Harrison A. A rapid view of access to care. An inquiry into the quality of general practice in England. 2010. https://www.kingsfund.org. uk/sites/default/files/field/field_document/rapid-view-access-care-gpinquiry- research-paper-mar11.pdf (accessed 1 Feb 2022). 37. Malterud K, Siersma VD, Guassora AD. Sample size in qualitative interview studies: guided by information power. Qual Health Res 2016; 26(13): 1753–1760. 38. Hennink MM, Kaiser BN, Marconi VC. Code saturation versus meaning saturation: how many interviews are enough? Qual Heal Res 2017; 27(4): 591–608. 14. Chapman JL, Zechel A, Carter YH, Abbott S. Systematic review of recent innovations in service provision to improve access to primary care. Br J Gen Pract 2004; 54(502): 374–381. 15. Salisbury C, Banks J, Goodall S, et al. An evaluation of Advanced Access in general practice. Final report. Report for the National Co-ordinating Centre for NHS Service Delivery and Organisation R&D (NCCSDO). 2007. https:// fundingawards.nihr.ac.uk/award/08/1310/070 (accessed 1 Feb 2022). 39. Bazeley P, Jackson K. Qualitative data analysis with NVivo. 2nd edn. Los Angeles, CA: Sage Publications, 2013. 40. Kislov R. Engaging with theory: from theoretically informed to theoretically informative improvement research. BMJ Qual Saf 2019; 28(3): 177–179. 16. Bower P, Roland M, Campbell J, Mead N. Setting standards based on patients’ views on access and continuity: secondary analysis of data from the general practice assessment survey. BMJ 2003; 326(7383): 258. 41. Mays N, Pope C. Discuss this article Discuss this article Contribute and read comments about this article: bjgp.org/letters Discuss this article Contribute and read comments about this article: bjgp.org/letters British Journal of General Practice, May 2022 e349 REFERENCES REFERENCES Qualitative research in health care. Assessing quality in qualitative research. BMJ 2000; 320(7226): 50–52. 42. Campbell JL, Salisbury C. Research into practice: accessing primary care. Br J Gen Pract 2015; DOI: https://doi.org/10.3399/bjgp15X688057. 17. Gerard K, Salisbury C, Street D, et al. Is fast access to general practice all that should matter? A discrete choice experiment of patients’ preferences. J Health Serv Res Policy 2008; 13(2): 3–10. 43. Pereira Gray DJ, Sidaway-Lee K, White E, et al. Continuity of care with doctors — a matter of life and death? A systematic review of continuity of care and mortality. BMJ Open 2018; 8(6): e021161. 18. Guthrie B, Wyke S. Personal continuity and access in UK general practice: a qualitative study of general practitioners’ and patients’ perceptions of when and how they matter. BMC Fam Pract 2006; 7(1): 11. 44. Huntley A, Lasserson D, Wye L, et al. Which features of primary care affect unscheduled secondary care use? A systematic review. BMJ Open 2014; 4(5): e004746. 19. Morse A. Improving patient access to general practice. 2017. https://www. nao.org.uk/wp-content/uploads/2017/01/Improving-patient-access-general- practice.pdf (accessed 1 Feb 2022). 45. Barker I, Steventon A, Deeny SR. Association between continuity of care in general practice and hospital admissions for ambulatory care sensitive conditions: cross sectional study of routinely collected, person level data. BMJ 2017; 356: j84. 20. Ford JA, Jones AP, Wong G, Steel N. Weekend opening in primary care: analysis of the General Practice Patient Survey. Br J Gen Pract 2015; DOI: https://doi. org/10.3399/bjgp15X687673. 21. MacDonald M. Prime Minister’s Challenge Fund: improving access to general practice. First evaluation report: October 2015. 2015. https://www.england.nhs. uk/wp-content/uploads/2015/10/pmcf-wv-one-eval-report.pdf (accessed 1 Feb 2022). 46. Richard L, Furler J, Densley K, et al. Equity of access to primary healthcare for vulnerable populations: the IMPACT international online survey of innovations. Int J Equity Health 2016;15: 64. 47. Murphy M, Scott LJ, Salisbury C, et al. Implementation of remote consulting in UK primary care following the COVID-19 pandemic: a mixed-methods longitudinal study. Br J Gen Pract 2021; DOI: https://doi.org/10.3399/ BJGP.2020.0948. 22. Conservative and Unionist Party. Get Brexit done. Unleash Britain’s potential. The Conservative and Unionist Party manifesto 2019. 2019. https://www. conservatives.com/our-plan/conservative-party-manifesto-2019 (accessed 1 Feb 2022). e350 British Journal of General Practice, May 2022
https://openalex.org/W4243280077	https://bmcpregnancychildbirth.biomedcentral.com/track/pdf/10.1186/s12884-021-03637-4	English	null	Intervention fidelity and its determinants of focused antenatal care package implementation, in South Wollo, Northeast Ethiopia.	Research Square (Research Square)	2,020	cc-by	6,628	Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 https://doi.org/10.1186/s12884-021-03637-4 Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 https://doi.org/10.1186/s12884-021-03637-4 (2021) 21:150 Tessema et al. BMC Pregnancy and Childbirth https://doi.org/10.1186/s12884-021-03637-4 Open Access Intervention fidelity and its determinants of focused antenatal care package implementation, in south Wollo zone, Northeast Ethiopia Tessema1* , Abebaw Gebeyehu1, Solomon Mekonnen2, Kassahun Alemu3 and Zemene Tigabu Asressie Molla Tessema1* , Abebaw Gebeyehu1, Solomon Mekonnen2, Kassahun Alemu3 and Abstract Background: Focused antenatal care is directed at sustaining maternal health and improving fetal wellbeing to ensure birth of a healthy neonate. Failure to implement focused antenatal care can result in inability to reduce maternal and perinatal morbidity and mortality in low income countries. Due to evidence-practice gaps, however, thousands of maternal, fetal and neonatal lives are still lost every day, mostly from preventable causes. This study aimed to assess focused antenatal care package’s intervention fidelity and its determinant factors in South Wollo Zone, Northeast Ethiopia. Methods: A cross-sectional study design was employed and a total of 898 women who gave birth in the last 6 months prior to data collection were included. Also 16 health extension workers, working in ten selected health posts, were included. Interviews and self-administered questionnaires were used to collect data from mothers and health extension workers. Ten [10] health posts were audited to assess availability and functionality of drugs and supplies to provide focused antenatal care. Mothers were asked whether or not the required level of care was provided. Health extension workers were provided with self-administered questionnaires to assess socio-demographic characteristics, reception of training, facilitation strategies for the implementation of focused antenatal care and ability to classify danger signs. Multilevel linear regression analysis was performed to identify individual and organizational level’s factors influencing focused antenatal care package intervention fidelity. Results: Overall weighted average focused antenatal care package intervention fidelity (implemented as intended/ planned) was 49.8% (95% CI: 47.7–51.8), which means the average number of focused antenatal care package interventions women received is 49.8%. Health extension workers implemented 55.1% and skilled providers (nurses, midwives, health officers or medical doctors) 44.9% of focused antenatal care package interventions. Overall antenatal care coverage, irrespective of frequency (at least one visit), was 752/898 women (83.7%; 95% CI: 81.3–86.1); 263/752 women (35.0%; 95% CI: 31.6–38.4) received at least four antenatal visits and only 46/752 women (6.1%; 95% CI: 4.4–7.8) received all recommended components of focused antenatal care. Previous pregnancy-related problems, paternal education and implementation of facilitation strategies were found to be significant factors enhancing focused antenatal care package intervention fidelity. Abstract (C ti d t ) * Correspondence: asressie@gmail.com 1Institute of Public Health, University of Gondar, Gondar, Ethiopia Full list of author information is available at the end of the article * Correspondence: asressie@gmail.com 1Institute of Public Health, University of Gondar, Gondar, Ethiopia Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: asressie@gmail.com 1Institute of Public Health, University of Gondar, Gondar, Ethiopia Full list of author information is available at the end of the article Background Focused antenatal care (FANC) is evidence based, women oriented, goal directed and individualized care for preg- nant women to improve maternal, perinatal and neonatal outcomes. FANC includes clinical assessment of pregnant women and their fetus during pregnancy in order to achieve favorable outcomes for both women and fetus. FANC’s interventions include identification and manage- ment of obstetric complications and infections, promoting using skilled attendants and healthy behavior [1]. FANC activities are directed at sustaining maternal health and improving fetal wellbeing to ensure birth of healthy neo- nates. Failure to implement FANC can result in inability to reduce maternal, perinatal and neonatal morbidity and mortality in low income countries [2, 3]. Evidence shows that public health interventions during the antenatal period are effective to reduce maternal, peri- natal and neonatal mortality [4]. In all studies reviewed here, interventions during pregnancy significantly reduced neonatal mortality in addition to improving fetal and ma- ternal health. Studies conducted in Indonesia, Bangladesh, sub-Saharan Africa and India indicated that increasing the number of antenatal visits has shown to decrease mater- nal, perinatal and neonatal mortality [5–8]. Several studies demonstrated that prenatal iron and folic acid supplemen- tation [9–15], tetanus toxoid vaccination [9, 16–19], use of insecticide-impregnated bed nets during pregnancy [4] and syphilis screening and treatment [15] have shown to reduce maternal, perinatal and neonatal mortality. Import- antly, randomized trials and large observational studies showed significant reductions in neonatal mortality and improvement of maternal and childcare uptake after implementing these interventions as a package in commu- nity settings [4, 20–27]. The bare number of antenatal visits does not have a significant reducing effect on those mortalities [22]. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 Page 2 of 8 (Continued from previous page) Conclusion: Focused antenatal care package intervention fidelity in the study area was low; this may imply that the current level of maternal, perinatal and neonatal mortality might be partly due to the low level of focused antenatal care intervention fidelity. Improving implementation of facilitation strategies is highly required to contribute to the reduction of those mortalities. Keywords: Focused antenatal care packages, Intervention fidelity, Neonatal mortality, Implementation of facilitation strategies Keywords: Focused antenatal care packages, Intervention fidelity, Neonatal mortality, Implementation of facilitation strategies collaborative strategy of Ethiopian government composed of 25–30 unpaid women volunteers in neighboring house- holds [29–32]. It aims at early identification of pregnant women and provision of FANC by linking community levels of care with health extension workers (HEWs) in community health posts to primary health care units. Even after the introduction of FANC, however, maternal and child health indicators in Ethiopia are still among the highest in the world. The main question here is, why those maternal, neonatal and child health indicators remained high while these effective intervention packages are imple- mented? We hypothesize that these interventions may not be properly implemented as per standard, commonly known as evidence-practice gaps. To our knowledge, no study thus far assessed whether FANC is implemented with fidelity or not, and facilitators enhancing and barriers inhibiting FANC intervention fidelity influenced its imple- mentation. Intervention fidelity refers to the degree to which interventions are implemented as planned in the original implementation document [33]. Therefore, FANC package intervention fidelity is defined as the degree to which the FANC package is implemented as described by community-based newborn care (CBNC) plan, which was developed by the Ministry of Health of Ethiopia. This study aimed to assess FANC package intervention fidelity and its determinants in South Wollo Zone, Northeast Ethiopia. Methods Design Cross-sectional study design was used for evaluating intervention fidelity of FANC package in south Wollo Zone, North east Ethiopia. Statistical analysis FANC requires a continuum of care provided at house- hold, health post, health center and hospital levels. Main goal of the intervention package is to transform evidence into action for reducing maternal, perinatal and neonatal morbidity and mortality by increasing the reach to all pregnant women and newborns in the community. FANC includes provision of four antenatal visits, coun- seling on nutrition, impregnated bed net use, danger signs and mother to child HIV-transmission. It also in- cludes birth preparedness and complication readiness planning, treatment of diagnosed sexually transmitted infections (STI), blood pressure, height and weight measurement in addition to identification of maternal danger signs and referral if necessary, provision of two doses of tetanus toxoid vaccination, promotion of facility birth, iron and folate supplementation and detection and management of complications. Facilitation strategies in- clude weekly supervision and support of HEWs by health center staff, monthly supervision by Woreda health office, community and HDA support [28]. y Antenatal care coverage, frequency and content were computed by considering the recommended amount of FANC as a reference. Antenatal care coverage was deter- mined as the proportion of women who have been con- tacted at least once by health care providers during pregnancy. Since the recommended number of FANC visits was at least four, getting antenatal care frequency less than four was weighed (as 1 4 ; 2 4 ; 3 4 ; >4 4 ) by considering > four as one (reference). For antenatal care contents, taking the total 17 antenatal care issues as maximum (Table 1), mothers who received less than the recommended con- tents during pregnancy were weighted accordingly (as 1 17 ; 2 17 ; 3 17 ; 4 17 ; … 17 17 ). As there was no previous study that assessed intervention fidelity, equal weights were given for coverage, components and frequency to compute fidelity [37, 38]. FANC package intervention fidelity was calcu- lated by taking the mean of the weighted product of ante- natal care coverage, frequency, and contents. Health posts were audited for the presence and functionality of supplies and equipment necessary for FANC. Multilevel statistical model was considered because mothers were nested from the health post and the sampling method was cluster sam- pling (by kebeles). Before jumping to multilevel model, intra-cluster correlation coefficients (ICC) were computed and > 5% was used as a cutoff point. Context FANC package is a combined effective and efficient pub- lic health intervention provided at household, health post, health center and hospital levels. Main implemen- ters are HDAs, HEWs, and skilled health providers in health centers. HEWs, who are young females with 10th grade education completed, have been trained and certi- fied to provide family health care at community level, in- cluding FANC, diseases prevention and control, hygiene and environmental sanitation, health education and Ethiopia is implementing FANC package in community settings since 2013 to achieve a reduction in maternal, perinatal and neonatal mortality [28]. One-to-five net- works are a household-based government strategy, con- sisting of one leader with five member households for reaching women and their children. Five to six of such networks can make one-to-thirty health development army (HDA) teams. HDA is an innovative, inclusive and Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 Page 3 of 8 communication [28, 34, 35]. HEWs work in health posts under supervision and support of health centers. communication [28, 34, 35]. HEWs work in health posts under supervision and support of health centers. visits and components provided for the mothers were considered as frequency and content. Sample size determination The study was conducted in South Wollo Zone of the Amhara region, which is 400 kms north of Addis Ababa, capital of Ethiopia. There were 900 rural and 150 urban HEWs, 499 health posts, 126 health centers and 9 hospi- tals (one zonal) in the Zone. All mothers who gave birth in the last 6 months of data collection, HEWs and health posts in the selected kebeles’ were included in the study. In Ethiopia, Kebele is the smallest administrative unit. Considering 52% of pregnant mothers who received 4+ antenatal care visits and all contents of antenatal care, 95% confidence level, 5% margin of error with 10% non- response rate, 422 participants were required [36]. How- ever, due to cluster sampling of kebeles, we collected data from 898 mothers. In addition, sixteen HEWs were included, and ten health posts where those 16 HEWs worked, were audited. Subgroup (sampling) Kebeles from South Wollo Zone were selected randomly using computer-generated random numbers. All mothers who gave birth in the last 6 months (for individual-level variables and fidelity assessment), all HEWs and all health posts in the selected kebeles (for cluster-level variables) were included. Mothers were interviewed in their homes and HEWs completed the questionnaires by themselves while their facility was audited. Facility audit is a review of a facility’s assets, important for provision of FANC. Statistical analysis Exploratory data ana- lysis was performed using SPSS version 20 and statistical modeling was conducted by R statistical software. Both Akaike’s Information Criteria (AIC) and Bayesian Infor- mation Criteria (BIC) were used for checking model fitness. This study used standards for reporting implemen- tation research guidelines (StaRI) [33]. Results Primary outcome of this study, FANC package interven- tion fidelity was computed by the weighted average of program reach (contact coverage), adherence to FANC contents and frequency. Program reach was measured by the proportion of mothers who visited any health fa- cility at least once and provided by any health care pro- vider during recent pregnancy. The number of antenatal Socio-demographic characteristics Mean age of women at the time of interview was 30.96 + 7.22 years. Of 898 women, 449 (50%) were between 25 and 36 years of age. Six hundred thirty eight (71.4%) did not attend any formal education, 768 (85.5%) were mar- ried and 662 (74%) of them were housewives. Tessema et al. Socio-demographic characteristics Out of those 752 women, ANC was provided by HEWs for 397 (52.8%; 95% CI: 52.7–52.9) and for 355 (47.2%; 95% CI: 47.1–47.3) by skilled providers. Mean time of first antenatal care visit was 4.14 + 2 months. Interestingly, 344/752 (45.7, 95% CI: 44.1–47.7) attended ANC in the first trimester of gestation (less than 12 weeks). HEWs’ mean age was 26 + 3.67 years and 13 (81.3%) of them were married and they walked an average of around 3 h (95% CI: 2:05–3:05) to reach to the most far away mother’s home. Socio-demographic characteristics BMC Pregnancy and Childbirth (2021) 21:150 Page 4 of 8 Table 1 Components of focused antenatal care package provided by HEWs and skilled providers’ qualification in South Wollo Zone, Ethiopia Contents Number of mothers (%) By HEWs (%) By skilled provider (%) Weight measured Yes 629 (83.6) 296 (47.1) 333 (52.9) No 123 (16.4) 101 (82.1) 22 (17.9) Height measured Yes 488 (64.9) 252 (51.6) 236 (48.4) No 264 (35.1) 145 (54.9) 119 (45.1) Blood pressure measured Yes 558 (74.2) 305 (54.7) 253 (45.3) No 194 (25.8) 92 (47.4) 102 (52.6) Advised for institutional birth Yes 686 (91.2) 372 (54.2) 314 (45.8) No 66 (8.8) 25 (37.9) 41 (62.1) Advised for BPCRa Yes 645 (85.8) 350 (54.3) 295 (45.7) No 107 (14.2) 47 (43.9) 60 (56.1) Advised on danger signs during pregnancy and birth Yes 606 (80.6) 327 (54.0) 279 (46.0) No 146 (19.4) 70 (47.9) 76 (52.1) Advised on personal hygiene Yes 695 (92.4) 372 (53.5) 323 (46.5) No 57 (7.6) 25 (43.9) 32 (56.1) Advised for PMTCTa Yes 633 (84.2) 320 (50.6) 313 (49.4) No 119 (15.8) 77 (64.7) 42 (35.3) Advised and screened for STIa Yes 622 (82.7) 308 (49.5) 314 (50.5) No 130 (17.3) 89 (68.5) 41 (31.5) Advised for bed net use Yes 527 (70.1) 299 (56.7) 228 (43.3) No 225 (29.9) 98 (43.6) 127 (56.4) Mothers tested for HIV Yes 678 (90.2) 345 (509) 333 (49.1) No 74 (9.8) 52 (70.3) 22 (29.7) Advised for nutrition during pregnancy Yes 634 (84.3) 350 (55.2) 284 (44.8) No 118 (15.7) 47 (39.8) 71 (60.2) Told to seek care for pregnancy danger signs Yes 669 (89.1) 363 (54.3) 306 (45.7) No 82 (10.9) 34 (41.5) 48 (58.5) Number of TTa vaccine received No 58 (7.7) 23 (39.7) 35 (60.3) TT1 287 (38.2) 185 (64.5) 102 (35.5) TT2+ 407 (54.1) 189 (46.4) 218 (53.6) Iron folic acid received Yes 425 (56.5) 203 (47.8) 222 (52.2) No 327 (43.5) 194 (59.3) 133 (40.7) Referred for institutional birth Yes 475 (63.2) 284 (59.8) 191 (40.2) No 277 (36.8) 113 (40.8) 164 (59.2) Expected date of birth told Yes 462 (61.4) 228 (49.4) 234 (50.6) No 290 (38.6) 169 (58.3) 121 (41.7) aBPCR Birth preparedness & complication readiness, PMTCT Prevention of mother to child transmission of HIV, STI Sexually transmitted infection, TT Tetanus toxoid Table 1 Components of focused antenatal care package provided by HEWs and skilled providers’ qualification in South Wollo Zone, Ethiopia aBPCR Birth preparedness & complication readiness, PMTCT Prevention of mother to child transmission of HIV, STI Sexually transmitted infection, TT Tetanus toxoi aBPCR Birth preparedness & complication readiness, PMTCT Prevention of mother to child pregnancy, making an overall antenatal care coverage of 83.7% (95% CI: 81.28–86.12). Coverage of FANC Seven hundred fifty two of 898 women were contacted by health care providers at least once during their recent Page 5 of 8 Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 Frequency and contents of FANC Support strategies set by Ministry of Health were assessed from health center, district health offices, com- munity and development armies’ perspective. Eleven (68.8%) HEWs reported that implementation of support either from the community, health development armies or district health office was lower than the planned standard. Mean number of antenatal visits were 3 + 1.6, and 263/ 752 women (35.0%; 95% CI: 31.6–38.4) attended at least four ANC visits. Higher numbers of antenatal visits were related with increased FANC package contents provided to mothers (Fig. 1). Only 46/752 women (6.1%; 95% CI 4.4–7.8) received all contents of FANC. Thirty three of those (4.3%) re- ceived care from HEWs and 13 (1.7%) from skilled pro- viders (Table 1). Women-related factors Only 180 women (20%) were living within 15-min’ walk- ing distance from the health post, while 333 (37.1%) of them had to travel > 45 min on foot. Of those who re- ceived antenatal care, 685 (91.1%) were self-referrals. One hundred eighty seven (20.8%) encountered pregnancy- related problems like bleeding, convulsions or high temperature in their previous pregnancies. Overall weighted average FANC package intervention fidelity (implementation as planned or average number of FANC package interventions women received) was 0.498 (49.8%; 95% CI 47.7–51.8); HEWs provided 0.62 (62.0%; 95% CI: 59.7–64.3) while skilled providers pro- vided 0.566 (56.6%; 95% CI 53.9–59.2). Only 20 women (2.2%) received the whole recommended FANC package with full fidelity. Discussion Facilitators’ and barriers’ of FANC intervention fidelity y The ICC observed in the model was 17.7%, which in- dicates that 17.7% of the variation in FANC package intervention fidelity is explained by health post (clus- ter) level factors. This shows FANC package interven- tion fidelity varies between health posts and there are health post level factors which affect implementation of the package. Antenatal care coverage was 83.7%; 6.1% women re- ceived all recommended components and 35% received at least four ANC visits. Moreover, over 90% were self- referrals to antenatal care. The overall weighted average FANC package intervention fidelity was 49.8% and of these, 62.0% were by HEWs and 56.6% by skilled pro- viders. Having pregnancy-related medical problems, formally educated partners and implementation of facili- tation strategies were significant facilitators for FANC package intervention fidelity. In the first level model, maternal age, distance from the health post, maternal education, pregnancy-related problems in previous pregnancies, partner’s education and total number of abortions were considered. Facili- tation strategies, distance from the farthest household and availability of supplies in the health post were considered in the second level model. Finally, pregnancy-related problems in previous pregnancies, partner’s education and facilitation strategies were found to be statistically significant facilitators for FANC package intervention fidelity (Table 2). In this study, women with pregnancy-related problems in previous pregnancies had a 9% increase of FANC package intervention fidelity as compared to those without. Women who had formally educated partners had an 8% increase in the levels of FANC package intervention fidelity in recent pregnancies as com- pared to their counterparts. An average increased im- plementation of health post level facilitation strategies resulted in a 4% increase in the level of FANC pack- age intervention fidelity. In this study, the weighted average FANC package intervention fidelity was too low compared to the stand- ard from the implementation plan. Durlak et.al. sug- gested that the level of an intervention implementation should be around 60% to produce positive results [39]. This might imply that the FANC package intervention fidelity, according to our findings, is too low to result in an anticipated reduction of maternal, perinatal and neonatal mortality and morbidity. We have also shown that the level of FANC frequency was 35.0% and content of 6.1%. Non-conformity with prescribed frequency (≥4 visits) and recommended components of FANC was ex- tremely evident. Discussion In the study, the observed increase in antenatal care visits was accompanied by implementa- tion of more FANC components, which is in line with findings from 41 countries’ demographic and health sur- veys [40]. Mere increase in the number of ANC visits does not lead to increased provision of expected compo- nents and intervention fidelity of the FANC package. In the final model, ICC was reduced to 4.7% and both Akaike’s Information Criteria (AIC) and Bayesian Information Criteria (BIC) decreased to 187.3 and 168.6 from the initial model (AIC 334.6 and BIC 349.0). It is indicated in this study that history of pregnancy related problems increases the use of FANC intervention package which is contrary to findings of other studies in Ethiopia that assessed the factors affecting contact Table 2 The following table shows the initial (maternal level) and final (combined) variables with the corresponding beta coefficient and confidence intervals of mixed effect model Variables Estimate 95% Confidence interval Level 1 variables Age of mothers (in years) 0.004 0.0003–0.008 Maternal problems in previous pregnancy No 1 Yes 0.06 0.01–0.11 Total number of abortions 0.01 0.005–0.022 Husband education No formal education 1 Attend formal education (1–8) 0.10 0.05–0.15 Combined model Pregnancy related-medical problems in previous pregnancy No 1 Yes 0.09 0.02–0.15 Husband education No formal education 1 Attend formal education (1–8) 0.08 0.02–0.13 Implementation of supportive/ facilitation strategy 0.04 0.02–0.05 Total number of abortions Husband education Provider-related factors No health post had all required functional equipment and medical supplies for FANC. Birth preparedness and complication readiness forms, supervision check- lists, blood pressure cuffs, pregnant women registra- tion books, stethoscopes and tape measures were the most frequently mentioned unavailable items in health posts. Twelve (75%) HEWs were ever trained on FANC pack- age while only 2 (12.5%) of them received refreshment training in the last 3 months. Only two health posts were supervised weekly from the catchment health cen- ter and 9 (56.3%) HEWs received onsite assistance for difficult cases. Nine of them responded that they were able to provide FANC. Fig. 1 Percentage of women who received ANC contents by number of visit and providers in South Wollo Administrative Zone, Ethiopia tage of women who received ANC contents by number of visit and providers in South Wollo Administrative Zone, Ethiopia Fig. 1 Percentage of women who received ANC contents by number of visit and providers in South Wollo Administrativ Page 6 of 8 Page 6 of 8 Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 Funding Th d This study was funded by the University of Gondar, Ethiopia. AMT received a financial support from the Federal Ministry for Economic Cooperation and Development (BMZ) through German Academic Exchange program (DAAD), PhD scholarship under the In-Country/In-Region Programme Ethiopia. When facilitation strategies, put in place to optimize implementation of FANC package intervention, in- creased, intervention fidelity of FANC may be optimized to its expected level. When HEWs get support from community, HDA, health center and district health of- fice staff as planned, provision of FANC package inter- vention will be enhanced. Measuring facilitating effects of supportive strategies is essential for optimizing and harmonizing the FANC package intervention implemen- tation [43]. Weakness in the facilitation strategy could be one of possible reasons for the observed low level of FANC package intervention fidelity, thereby contributing to the high level of maternal, perinatal, neonatal mortal- ity and morbidity in Ethiopia. Author details 1 1Institute of Public Health, University of Gondar, Gondar, Ethiopia. 2Department of Human Nutrition, Institute of Public Health, University of Gondar, Gondar, Ethiopia. 3Department of Epidemiology and Biostatistics, Institute of Public Health, University of Gondar, Gondar, Ethiopia. 4 Consent for publication Not applicable. Consent for publication Not applicable. Combined model Husband education Implementation of supportive/ facilitation strategy Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 Page 7 of 8 Page 7 of 8 coverage [41]. The difference might be due to the differ- ence in outcome of interest as our study is the combin- ation of coverage, frequency and content. This may indicate that mothers’ pregnancy-related risk perceptions play an important role for their adherence to the recom- mended FANC package interventions. CBNC: Community-Based Newborn Care; HDA: Health Development Army; WDA: Women Development Army; KMs: Kilo-Meters; HIV: Human Immunodeficiency Virus; STI: Sexually Transmitted Infection; ICC: Intra-cluster Correlation Coefficient; SPSS: Statistical Package for Social Sciences; AIC: Akaike’s Information Criteria; BIC: Bayesian Information Criteria; StaRI: Standards for reporting implementation research; ANC: Antenatal Care; MCH: Maternal and Child Health CBNC: Community-Based Newborn Care; HDA: Health Development Army; WDA: Women Development Army; KMs: Kilo-Meters; HIV: Human Immunodeficiency Virus; STI: Sexually Transmitted Infection; ICC: Intra-cluster Correlation Coefficient; SPSS: Statistical Package for Social Sciences; AIC: Akaike’s Information Criteria; BIC: Bayesian Information Criteria; StaRI: Standards for reporting implementation research; ANC: Antenatal Care; MCH: Maternal and Child Health Partners’ attendance of formal education facilitates FANC package intervention fidelity, contrary to women with the same level of education. Paternal education, even at the lowest level, thus contributes to improved uptake and adherence to the recommended package of care which is in line with another systematic review and primary study [41, 42]. Maternal and child (MCH) health care uptake decision-making may depend on partners, particularly for mothers with low levels of edu- cation. Therefore, MCH policy development and imple- mentation needs to involve partners, particularly for mothers with low levels of education in rural areas. Conclusion This study showed FANC package intervention fidelity to be low. High levels of maternal, perinatal and neo- natal mortality indicators may be partly due to low levels of FANC package intervention fidelity, pointing to im- plementation problems. Maternal previous pregnancy- related problems, partner’s education and implementa- tion of facilitation strategies were significant facilitators of FANC package intervention fidelity. Paternal educa- tion and implementation of facilitation strategies play significant roles for improved FANC package interven- tion fidelity. Further studies are needed that focus on why facilitation strategies were relatively underused dur- ing implementation. 4Department of Pediatrics and Child Health, University of Gondar, Gondar, Ethiopia. 4Department of Pediatrics and Child Health, University of Gondar, Gondar, Ethiopia. Received: 6 April 2020 Accepted: 10 February 2021 Received: 6 April 2020 Accepted: 10 February 2021 Acknowledgments h h First the authors would like to express their deepest gratitude for Estifanos Yalew Baye (PhD) for his generous and critical review of the manuscript. We would like also acknowledge all study participants, data collectors, and South Wollo Zone’s district health officials. Availability of data and materials The dataset used and analyzed for this study is available from the corresponding author. This data can be made available upon reasonable request. Authors’ contributions AMT d h d AMT conceived the idea, develop and implemented the proposal, analyzed and interpreted data and wrote the manuscript under the supervision of AG, SM, KA, and ZT. AG, SM, KA, and ZT critically reviewed the manuscript. All authors read and approved the final manuscript. References References 1. Warren C, Daly P, Toure L, Mongi P. Opportunities for Africa ’ s Newborns; 2006. p. 80. 2. Villar J, Bergsjo P. WHO Antenatal Care Randomized Trial: Manual for the implementation of the New Model. WHO/RHR/01.30; 2002. p. 1–42. 3. Ebunoluwa Oshinyemi T, Ojo Aluko J, Abimbola Oluwatosin O. Focused antenatal care: re-appraisal of current practices. Int J Nurs Midwifery. 2018; 10(8):90–8. 4. Hodgins S, Tielsch J, Rankin K, Robinson A, Kearns A, Caglia J. A new look at Care in Pregnancy: simple, effective interventions for neglected populations. PLoS One. 2016;11(8):1–73. 5. Ibrahim J, Yorifuji T, Tsuda T, et al. Frequency of antenatal care visits and neonatal mortality in Indonesia. J Trop Pediatr. 2012;58(3):184–8. 6. Roy S, Haque MA. Effect of antenatal care and social well-being on early neonatal mortality in Bangladesh. BMC Pregnancy Childbirth. 2018;18(1):4–9. 1. Warren C, Daly P, Toure L, Mongi P. Opportunities for Africa ’ s Newborns; 2006. p. 80. 2. Villar J, Bergsjo P. WHO Antenatal Care Randomized Trial: Manual for the implementation of the New Model. WHO/RHR/01.30; 2002. p. 1–42. 3. Ebunoluwa Oshinyemi T, Ojo Aluko J, Abimbola Oluwatosin O. Focused antenatal care: re-appraisal of current practices. Int J Nurs Midwifery. 2018; 10(8):90–8. 4. Hodgins S, Tielsch J, Rankin K, Robinson A, Kearns A, Caglia J. A new look at Care in Pregnancy: simple, effective interventions for neglected populations. PLoS One. 2016;11(8):1–73. 5. Ibrahim J, Yorifuji T, Tsuda T, et al. Frequency of antenatal care visits and neonatal mortality in Indonesia. J Trop Pediatr. 2012;58(3):184–8. 5. Ibrahim J, Yorifuji T, Tsuda T, et al. Frequency of antenatal care visits and neonatal mortality in Indonesia. J Trop Pediatr. 2012;58(3):184–8. 6. Roy S, Haque MA. Effect of antenatal care and social well-being on early neonatal mortality in Bangladesh. BMC Pregnancy Childbirth. 2018;18(1):4–9. sSA: Sub-Saharan Africa; SDG: Sustainable Development Goals; FANC: Focused Antenatal Care; HEWs: Health Extension Workers; Competing interests Nonetheless, information was collected from the mothers by non-health professional data collectors dur- ing the last 6 months that the issue of social desirability bias needs to be considered in interpreting our findings. The authors declare that there is no competing interest. Ethics approval and consent to participate Ethical approval was obtained from the ethical board of the University of Gondar (Ref. No O/V/P/RCS/05/810/2018), and written permission letters were granted from Amhara regional state health bureau, South Wollo Zone and district health offices. Written informed consent was taken from all participants. 4. Hodgins S, Tielsch J, Rankin K, Robinson A, Kearns A, Caglia J. A new look at Care in Pregnancy: simple, effective interventions for neglected populations. PLoS One. 2016;11(8):1–73. Abbreviations b h 6. Roy S, Haque MA. Effect of antenatal care and social well-being on early neonatal mortality in Bangladesh. BMC Pregnancy Childbirth. 2018;18(1):4–9. Page 8 of 8 Page 8 of 8 Page 8 of 8 Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 7. V Doctor H. Assessing antenatal care and newborn survival in sub-Saharan Africa within the context of renewed commitments to save newborn lives. AIMS Public Heal. 2016;3(3):432–47. reproductive , maternal , newborn and child healthcare practices : a cross- sectional study in four regions of Ethiopia. BMC Pregnancy Childbirth. 2018; 18:373. reproductive , maternal , newborn and child healthcare practices : a cross- sectional study in four regions of Ethiopia. BMC Pregnancy Childbirth. 2018; 18:373. 30. Wang H, Tesfaye R, Ramana GNV, Chekagn CT. Ethiopia Health Extension Program; 2016. 8. Singh A, Pallikadavath S, Ram F, et al. Do antenatal care interventions improve neonatal survival in India? Health Policy Plan. 2014;29(7):842–8. 9. Abir T, Ogbo FA, Stevens GJ, et al. The impact of antenatal care, iron–folic acid supplementation and tetanus toxoid vaccination during pregnancy on child mortality in Bangladesh. PLoS One. 2017;12(11):e0187090. 31. Yibeltal A, Yalemzewod A, Hill PS, et al. Community health extension program of Ethiopia , 2003–2018 : successes and challenges toward universal coverage for primary healthcare services. Glob Health. 2019;15(21):1–11. for primary healthcare services. Glob Health. 2019;15(21):1–11. 32. Health Sector Development Programme IV. Federal Democratic Republic of Ethiopia Monistry of Health. 2010. 10. Nisar YB, Dibley MJ. Iron/folic acid supplementation during pregnancy prevents neonatal and under-five mortality in Pakistan: propensity score matched sample from two Pakistan demographic and health surveys. Glob Health Action. 2016;9(1):29621. 33. Pinnock H, Barwick M, Carpenter CR, et al. Standards for report 33. Pinnock H, Barwick M, Carpenter CR, et al. Standards for reporting implementation studies (StaRI): explanation and elaboration document. BMJ Open. 2017;7(4):e013318. implementation studies (StaRI): explanation and elaboration document. BMJ Open. 2017;7(4):e013318. 11. Titaley CR, Dibley MJ, Roberts CL, et al. Iron and folic acid supplements and reduced early neonatal deaths in Indonesia. Bull World Health Organ. 2010; 88(7):500–8. 4. Langer J, Cagliaannie K. Health extension Workers in Ethiopia; 201 35. Ethiopian Federal ministry of health. Community based newborn care traning for health extension workers: Facilitators guide. 2013. 12. Mosha D, Liu E, Hertzmark E, et al. Abbreviations b h Int J Environ Res Public Health. 2019;16(748). 17. Giles ML, Krishnaswamy S, Wallace EM. Maternal immunisation: What have been the gains? Where are the gaps? What does the future hold? F1000Research. 2018;7(F1000 Faculty Rev):1733. 43. Carroll C, Patterson M, Wood S, Booth A, Rick J, Balain S. A conceptual framework for implementation fidelity. Implement Sci. 2007;2(1):40. 43. Carroll C, Patterson M, Wood S, Booth A, Rick J, Balain S. A conceptual framework for implementation fidelity. Implement Sci. 2007;2(1):40. 18. Singh A, Pallikadavath S, Ogollah R, et al. Maternal tetanus toxoid vaccination and neonatal mortality in rural North India. PLoS One. 2012; 7(11):e48891. Publisher’s Note 19. Boone P, Eble A, Elbourne D, et al. Community health promotion and medical provision for neonatal health—CHAMPION cluster randomised trial in Nagarkurnool district, Telangana (formerly Andhra Pradesh). India PLOS Med. 2017;14(7):e1002324. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 20. Baqui AH, Williams E, El-Arifeen S, et al. Effect of community-based newborn care on cause-specific neonatal mortality in Sylhet district, Bangladesh: findings of a cluster-randomized controlled trial. J Perinatol. 2016;36(1):71–6. 20. Baqui AH, Williams E, El-Arifeen S, et al. Effect of community-based newborn care on cause-specific neonatal mortality in Sylhet district, Bangladesh: findings of a cluster-randomized controlled trial. J Perinatol. 2016;36(1):71–6. 21. Lassi ZS, Mansoor T, Bhutta ZA, et al. Essential pre-pregnancy and pregnancy interventions for improved maternal, newborn and child health. Reprod Health. 2014;11:S2. 21. Lassi ZS, Mansoor T, Bhutta ZA, et al. Essential pre-pregnancy and pregnancy interventions for improved maternal, newborn and child health. Reprod Health. 2014;11:S2. 22. Lassi ZS, Das JK, Bhutta ZA, et al. Evidence from community level inputs to improve quality of care for maternal and newborn health: interventions and findings. Reprod Health. 2014;11:S2. 23. Trivedi D. Cochrane review summary : community-based intervention packages for reducing maternal and neonatal morbidity and mortality and improving neonatal outcomes. Prim Health Care Res Dev. 2019;17:317–8. 23. Trivedi D. Cochrane review summary : community-based intervention packages for reducing maternal and neonatal morbidity and mortality and improving neonatal outcomes. Prim Health Care Res Dev. 2019;17:317–8. 24. Lassi ZS, Bhutta ZA. Community-based intervention packages for reducing maternal and neonatal morbidity and mortality and improving neonatal outcomes. Cochrane Database Syst Rev. 2015;3. 24. Lassi ZS, Bhutta ZA. Community-based intervention packages for reducing maternal and neonatal morbidity and mortality and improving neonatal outcomes. Cochrane Database Syst Rev. 2015;3. 25. Baqui AH, El-Arifeen S, Darmstadt GL, et al. Effect of community-based newborn-care intervention package implemented through two service- delivery strategies in Sylhet district, Bangladesh: a cluster-randomised controlled trial. Lancet. 2008;371:1936–44. 26. Darmstadt GL, Bhutta ZA, Cousens S, et al. Neonatal Survival 2 Evidence- based , cost-effective interventions : how many newborn babies can we save ? Lancet. 2005;365:977–88. 26. Darmstadt GL, Bhutta ZA, Cousens S, et al. Neonatal Survival 2 Evidence- based , cost-effective interventions : how many newborn babies can we save ? Lancet. 2005;365:977–88. 27. Neupane S, Doku DT. Abbreviations b h Dietary iron and calcium intakes during pregnancy are associated with lower risk of prematurity, stillbirth and neonatal mortality among women in Tanzania. Public Health Nutr. 2017; 20(4):678–86. 36. Benova L, Tunçalp Ö, Moran AC, et al. Not just a number: examining coverage and content of antenatal care in low-income and middle-income countries. BMJ Glob Heal. 2018;3(2):e000779. 7. Hamilton M, MEASUREMENT IN, MEDICINE. Lancet. 1958;271(7028 13. Titaley CR, Dibley MJ, Roberts CL, et al. Combined iron / folic acid supplements and malaria prophylaxis reduce neonatal mortality in 19 sub- Saharan African countries 1–3. Am J Clin Nutr. 2010;92(1):235–43. 38. Greco S, Ishizaka A, Tasiou M, et al. On the Methodological Framework of Composite Indices : A Review of the Issues of Weighting , Aggregation , and Robustness. Soc Indic Res. 2018;141:61–94. 14. Titaley CR, Dibley MJ. Antenatal iron/folic acid supplements, but not postnatal care, prevents neonatal deaths in Indonesia: analysis of Indonesia demographic and health surveys 2002/2003–2007 (a retrospective cohort study). BMJ Open. 2012;2(6):e001399. 39. Durlak JA, Dupre ÆEP. Implementation matters : a review of research on the influence of implementation on program outcomes and the factors affecting implementation. Am J Community Psychol. 2008;41:327–50. 39. Durlak JA, Dupre ÆEP. Implementation matters : a review of research on the influence of implementation on program outcomes and the factors affecting implementation. Am J Community Psychol. 2008;41:327–50. 40. Hodgins S, D’Agostino A. The quality–coverage gap in antenatal care: toward better measurement of effective coverage. Glob Heal Sci Pract. 2014; 2(2):173–81. 40. Hodgins S, D’Agostino A. The quality–coverage gap in antenatal care: toward better measurement of effective coverage. Glob Heal Sci Pract. 2014; 2(2):173–81. 15. Bhutta ZA, Das JK, Bahl R, et al. Can available interventions end preventable deaths in mothers, newborn babies, and stillbirths, and at what cost? Lancet. 2014;384(9940):347–70. 41. Okedo-Alex IN, Akamike IC, Ezeanosike OB, Uneke CJ. Determinants of antenatal care utilisation in sub-Saharan Africa: a systematic review. BMJ Open. 2019;9(10):e031890. 16. Blencowe H, Lawn J, Vandelaer J, et al. Tetanus toxoid immunization to reduce mortality from neonatal tetanus. Int J Epidemiol. 2010; 39(Supplement 1):i102–9. 42. Mekonnen T, Dune T, Perz J, Ogbo FA. Trends and Determinants of Antenatal Care Service Use in Ethiopia between 2000 and 2016. Int J Environ Res Public Health. 2019;16(748). 42. Mekonnen T, Dune T, Perz J, Ogbo FA. Trends and Determinants of Antenatal Care Service Use in Ethiopia between 2000 and 2016. Tessema et al. BMC Pregnancy and Childbirth (2021) 21:150 Publisher’s Note Association of the quality of antenatal care with neonatal mortality: meta-analysis of individual participant data from 60 low- and middle-income countries. Int Health. 2019;11(6):596–604. 27. Neupane S, Doku DT. Association of the quality of antenatal care with neonatal mortality: meta-analysis of individual participant data from 60 low- and middle-income countries. Int Health. 2019;11(6):596–604. 28. Ethiopian Federal ministry of health. Community-Based Newborn Care Implementation Plan. 2013. 28. Ethiopian Federal ministry of health. Community-Based Newborn Care Implementation Plan. 2013. 29. Betemariam W, Zigene ZD, Fesseha N. Correlates of the Women ’ s Development Army strategy implementation strength with household 29. Betemariam W, Zigene ZD, Fesseha N. Correlates of the Women ’ s Development Army strategy implementation strength with household
https://openalex.org/W4361847418	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_Legend_from_FANCJ_Localization_by_Mismatch_Repair_Is_Vital_to_Maintain_Genomic_Integrity_after_UV_Irradiation/22401489/1/files/39847281.pdf	English	null	Supplementary Figure Legend from FANCJ Localization by Mismatch Repair Is Vital to Maintain Genomic Integrity after UV Irradiation	null	2,023	cc-by	1,139	Supplemental Figure Legends: Supplemental Figure 1. FANCJ is recruited to sites of UV-induced damage in an NER dependent manner (A) XP-F cells expressing vector, XPFWT, or XPFD676A were globally UV irradiated (5 J/m2). At 3h after UV irradiation, cells were processed and co-immunostained with the indicated Abs. (B) Quantification of cells with FANCJ-positive foci. (C) A549 cells containing unique shRNA vectors targeting FANCJ or NSC were UV irradiated through micropore filters, co-immunostained with the indicated Abs at several time points and quantified for cells with ERCC1 positive LUDS and (D) XPC positive LUDs. Where shown, error bars represent the standard deviation of the mean of three independent experiments Supplemental Figure 2. MMR localizes FANCJ to UV induced LUDs (A) U2OS cells containing a second shRNA vector targeting MSH2 or NSC were analyzed by immunoblot and (B) UV irradiated through micropore filters and quantified for cells with FANCJ- or 6-4 PP-positive LUDs. (C) U2OS cells containing two distinct shRNA vectors targeting MSH6 or NSC were analyzed by immunoblot and (D) treated as in B and quantified for cells with FANCJ- or 6-4 PP-positive LUDs. (E) U2OS cells containing shRNA vectors targeting MSH2 or NSC were UV irradiated through micropore filters and co-stained with XPC and ERCC1 abs. Representative images are shown 2 h after UV irradiation. (F) Quantification of cells with XPC- or ERCC1-positive LUDs. (G) XP-F cells complemented with empty vector or XPFWT were UV irradiated through micropore filters and co-stained with MSH2 and ERCC1 abs. Representative images are shown 2 h after UV irradiation. (H) Quantification of XP-F cells with MSH2- or ERCC1-positive LUDs. (I) XP-F cells containing shRNA vectors targeting MSH2 or NSC were UV irradiated through micropore filters, pre-extracted with 0.5% Triton-X in PBS prior to fixation, and co-immunostained with the indicated Abs, a representative image is shown 2 hrs post UV irradiation. Where shown, error bars represent the standard deviation of the mean of three independent experiments Supplemental Figure 3. FANCJ localizes to UV induced LUDs predominantly in S-Phase and contributes to the UV induced checkpoint (A) 48BR cells were UV irradiated through micropore filters prior to incubation with 10uM EdU for 3 hrs. Cells were then processed for EdU incorporation and co-immunostained with the indicated Abs. Supplemental Figure Legends: Supplemental Figure Legends: Supplemental Figure Legends: Supplemental Figure Legends: (B) Quantification of co-localization of XPF, FANCJ, MLH1, or MSH2 with LUDs in non-S phase cells representing sites of gap filling and (C) quantification of co-localization of XPF, FANCJ, MLH1, or MSH2 with LUDs in S-phase cells. (D) Whole cell extracts of MCF7 cells containing shRNA vectors targeting FANCJ or NSC were analyzed by immunoblot with the indicated Abs at the indicated time points after UV irradiation. The ratio of phospho-protein/total protein by densitometry using Image J software is quantified. Supplemental Figure 4. FANCJ and MMR function in a common pathway for RPA phosphorylation and 6-4 Photoproduct elimination, but not for gap repair. (A) A549 cells containing shRNA vectors targeting FANCJ or NSC were stably depleted of MSH2 versus a second NSC and analyzed for immunoblot or (B) UV irradiated through 5 um filters to generate LUDs and co- immunostained with the indicated Abs at several time points and quantified for cells with phospho- serine4/8 RPA32-positive LUDs and (C) 6-4 PP-positive LUDs. (D) 48BR cells containing shRNA vectors targeting XPF, FANCJ-1, FANCJ-2, MLH1, MSH2 or NSC were UV irradiated through micropore filters prior to incubation with 10uM EdU for 3 hrs and quantified for gap-filling in non-S cells. (E) MSH2 isogenic mouse embryonic fibroblasts were analyzed by immunoblot, treated as in A, and quantified for gap filling in non- S phase cells. Where shown, error bars represent the standard deviation of the mean of three independent experiments. and quantified for gap filling in non- S phase cells. Where shown, error bars represent the standard deviation of the mean of three independent experiments. Supplemental Figure 5. FANCJ precipitates with UV modified PCNA and CPD from chromatin extracts. (A) A549 cells were left untreated or globally UV irradiated and collected in 150 nM NETN buffer. Cells were then spun down and the insoluble pellet was re-suspended in RIPA buffer and sonicated. The RIPA fraction was spun down and the chromatin lysate was used for input or for CPD immuno-precipitation (IP). IPs were then analyzed by immunoblot with the indicated Abs. Supplemental Figure 6. FANCJ deficient cells are sensitive to DNA interstrand crosslinking agents and FANCJ suppresses UV-induced mutations (A) A549 cells expressing shRNA vectors targeting FANCJ or NSC were left untreated or treated with Cisplatin (CPPD) and analyzed for colony survival. (B) Quantification of surviving colonies. Supplemental Figure Legends: (C) FA-J cells expressing empty vector control or FANCJWT were UV irradiated and analyzed for survival in 6-TG relative to untreated cells (D) Empty vector control- or FANCJWT-complemented FA-J cells were analyzed for relative survival after mitomycin C treatment. Cells were stained with crystal violet, solubilized, and absorbance was measured at 590nm. The relative absorbance of treated/untreated was quantified as relative survival. (E) FA-J cells were treated as in D, except analyzed for relative survival after UV irradiation. Where shown, error bars represent the standard deviation of the mean of three independent experiments. Supplemental Figure 7. Mutations in FANCJ and MMR loci occur in melanoma genomes (A) FANCJ protein coding mutations were identified from sequenced melanoma genomes in cBioPortal and the Catalogue of Somatic Mutations in Cancer (CoSMiC) databases. Mutations are located throughout the FANCJ sequence as indicated. Two specific amino acids were previously identified, in hereditary breast cancer (blue) and in Fanconi Anemia (green). Mutations found in the iron-sulfur domain (red). Sites where FANCJ interacts with MLH1 (K141/K142) and BRCA1 (S990) are indicated. (B) FANCJ protein coding mutations (C) protein coding mutations identified in MSH2 as in A, (D) MSH6, (E) MLH1, and (F) PMS2. Supplemental Figure 8. Ectopic expression of catalytic inactive FANCJK52R disrupts clearance of UV induced lesions in U2OS cells (A) U2OS cells were transfected with pCDNA3 constructs containing vector, FANCJWT, or FANCJK52R and analyzed by immunoblot wit the indicated Abs. (B) Cells were UV irradiated through 5 um filters to generate LUDs and co-immunostained with the indicated Abs 16h post treatment and (C) quantified for cells with phospho-serine4/8 RPA32-positive LUDs and 6-4 PP-positive LUDs. Supplemental Figure 9. FANCJ expression promotes UV induced GFP-polh foci formation in U2OS cells (A) U2OS cells were transfected with GFP-polh and left untreated or globally UV irradiated and analyzed on a fluorescent microscope. (B) Cells were co-transfected with GFP-polh and siRNA reagents targeting luciferase control, FANCJ, or RAD18 and analyzed by immunoblot with the indicated Abs. (C) Cells were left untreated or globally UV irradiated quantified for cells with >5 GFP-polh foci. Supplemental Table 1. FANCJ suppresses UV-induced mutations at the HPRT locus. Classification of clones with HPRT-inactivating mutations from A549 cells expressing shRNAs to FANCJ (combined) vs. NSC.
https://openalex.org/W2614335936	http://eap-iea.org/index.php/eap/article/download/853/827	French	null	In regard to Levi-Strauss’s motive of oblivion	Etnoantropološki problemi	2,009	cc-by-sa	14,804	* Ce texte est le résultat d’une participation au projet Anthropologie au XX siècle: portée théorique et méthodologique qui est en totalité financé par le Ministère de la Science et de la Technologie de la République de Serbie ( n°. 147037). Tekst je nastao kao rezultat rada na projektu br. 147037 Antropologija u XX veku: teorijski i metodološki dometi koji u celosti finansira MNTR RS. Dragana Antonijevi Département d'ethnologie et d'anthropologie Faculté de Philosophie, Université de Belgrade daa@eunet.rs Département d'ethnologie et d'anthropologie Faculté de Philosophie, Université de Belgrade daa@eunet.rs ARTICLE ARTICLE UDC: 316.77:165.75 ARTICLE * Ce texte est le résultat d’une participation au projet Anthropologie au XX siècle: portée théorique et méthodologique qui est en totalité financé par le Ministère de la Science et de la Technologie de la République de Serbie ( n°. 147037). Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) g p q ( ) Tekst je nastao kao rezultat rada na projektu br. 147037 Antropologija u XX veku: teorijski i metodološki dometi koji u celosti finansira MNTR RS. 1 Traduction en croate:"Asdiwalova junaka djela" dans: Strukturalna antropolo- gija 2, Zagreb: Školska knjiga, 1988, pp. 132-183 (trad. D. Buan et V. Mimica). En serbe"Le Mythe d’Asdiwal" a été publié dans la revue Savremenik 7-8, 1986: 7-24 (I partie) et Savremenik 9-10, 1986: 230-249 (II partie) mais sans le post scriptum (trad. D. Antonijevi). 2 j ) 2 F. Boas a noté les versions de 1895, 1912 et 1916 du fleuve Skeene, et en 1902, il a noté le mythe sur le fleuve Nass. 3 À propos du concept de l’oubli de Lévi-Strauss Structure de communication perturbée et styles de pensée dans la Serbie en transition * Résumé: Le point de départ du présent travail est le concept sémantique de Lévi-Strauss que constituent l’oubli, le malentendu, l’indiscrétion et la nostal- gie. Après l’introduction du nouveau syntagme de communication perturbée et du terme d’obscurité, l’auteure offre une correction du concept de Lévi- Strauss et analyse les implications sémantiques et cognitives des notions in- cluses dans le système de "communication perturbée". Par la convergence de deux modèles – celui de la communication perturbée et de la structure de la modalité véridictoire de Greimas, sont proposés et analysés des exemples illustratifs de communication perturbée et des styles de pensée dans la Serbie en transition afin de définir leur position structurale, cognitive et communica- tionnelle dans la création d’une carte cognitive de l’oubli et de la mémoire des citoyens. Mots-clés: le concept de l’oubli de Lévi-Strauss, communication pertur- bée, styles et communautés de pensée, transition, fonction socio-normative de la sémantique de l’oubli. Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 168 DRAGANA ANTONIJEVI Claude Lévi-Strauss et la sémantique de l’oubli 3 Lévi-Strauss n’utilise qu’une fois le terme"pathologie dans la communication" (Lévi-Strauss 1983: 254), d’où je conclus qu’il ne le considérait pas comme trop im- portant pour le phénomène qu’il étudiait. Pour ma part, cette expression ne m’a pas plu, car je considère que le mot"pathologique" surcharge ces catégories cognitives qui apparaissent souvent dans la communication de tous les jours. .. . 4. .2 (2009) 1 Traduction en croate:"Asdiwalova junaka djela" dans: Strukturalna antropolo- gija 2, Zagreb: Školska knjiga, 1988, pp. 132-183 (trad. D. Buan et V. Mimica). En serbe"Le Mythe d’Asdiwal" a été publié dans la revue Savremenik 7-8, 1986: 7-24 (I partie) et Savremenik 9-10, 1986: 230-249 (II partie) mais sans le post scriptum (trad. D. Antonijevi). 2 F. Boas a noté les versions de 1895, 1912 et 1916 du fleuve Skeene, et en 1902, il a noté le mythe sur le fleuve Nass. 3 Lévi-Strauss n’utilise qu’une fois le terme"pathologie dans la communication" (Lévi-Strauss 1983: 254), d’où je conclus qu’il ne le considérait pas comme trop im- portant pour le phénomène qu’il étudiait. Pour ma part, cette expression ne m’a pas plu, car je considère que le mot"pathologique" surcharge ces catégories cognitives qui apparaissent souvent dans la communication de tous les jours. 2 F. Boas a noté les versions de 1895, 1912 et 1916 du fleuve Skeene, et en 1902, il a noté le mythe sur le fleuve Nass. 3 Lévi-Strauss n’utilise qu’une fois le terme"pathologie dans la communication" (Lévi-Strauss 1983: 254), d’où je conclus qu’il ne le considérait pas comme trop im- portant pour le phénomène qu’il étudiait Pour ma part cette expression ne m’a pas Claude Lévi-Strauss et la sémantique de l’oubli C’est dans le post-scriptum de l’analyse du mythe d’Asdiwal (La geste d’Asdiwal) dans son Anthropologie structurale II (Lévi-Strauss 1973: 229- 231)1 que Claude Lévi-Strauss s’est penché pour la première fois sur le "mo- tif de l’oubli". C’est alors qu’il constate que le motif de l’oubli ne représente pas un simple élément narratif servant à faire débuter l’intrigue, mais une catégorie mythique importante avec une signification précise – celle d’omissions dans la communication. En comparant quatre versions du mythe d’Asdiwal Tshimshian de 1895, 1902, 1912 et de 1916,2 il est amené à la con- clusion que la mort du héros – Asdiwal, Asiwe ou Waux (selon les différentes versions) est expliquée par une des possibilités qui apparaissent comme des transformations à l’intérieur du champ sémantique unique de "l’oubli". Dans l’impossibilité de concilier les différentes formes de vie qu’il vit succes- sivement, le héros du mythe meurt victime de la nostalgie qu’il ressent et qui lui fait commettre la faute de l’oubli ou de l’indiscrétion. Lévi-Strauss fait alors un schéma des transformations des sémentèmes communicationnels: l’oubli, l’indiscrétion et le malentendu qui se créent sur des axes d’opposition constitués de couples "soi : autrui" et "excès : défaut de communication". Bien que les mythes lui parlent clairement de la nostalgie que ressent Asdiwal et qui lui inspire un comportement qui va résulter par la tragédie, Lévi-Strauss n’introduit pas ce quatrième élément dans le schéma. Dans une note, cependant, il reconnaît son omission (Lévi-Strauss 1973: 230). Pour pouvoir nommer plus facilement et avec plus de précision le champ sémantique que forment les termes qui participent à l’excès/le défaut de com- munication, j’ai introduit l’expression communication perturbée, convaincue qu’elle est suffisamment valable et opératoire pour le concept examiné. Il importe de noter que ce syntagme, que j’utilise dans mon travail, est à moi et que Lévi-Strauss ne s’en sert pas. 3 3 Lévi-Strauss n’utilise qu’une fois le terme"pathologie dans la communication" (Lévi-Strauss 1983: 254), d’où je conclus qu’il ne le considérait pas comme trop im- portant pour le phénomène qu’il étudiait. Pour ma part, cette expression ne m’a pas plu, car je considère que le mot"pathologique" surcharge ces catégories cognitives qui apparaissent souvent dans la communication de tous les jours. .. . 4. Claude Lévi-Strauss et la sémantique de l’oubli .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 169 Lévi-Strauss a proposé le schéma de transformations du champ sémantique de l’oubli (schéma 1), que je vais ici compléter par la nostalgie qui manque dans son schéma original: Schéma 1: Schéma 1: INDISCRETION MALENTENDU OUBLI NOSTALGIE Excès/ Défaut + - - + Autrui / Soi + + - - C’est ainsi qu’Asdiwal est sans cesse rongé par la nostalgie au mauvais moment et au mauvais endroit – lorsqu’il vit sur mer, il s’ennuie des monta- gnes, lorsqu’il chasse dans les montagnes, il regrette la mer. Ces déplacements entre les montagnes et la mer, et particulièrement entre les deux fleuves, sont en rapport avec un fait important de sa vie – les périodes de grande famine que les Tshimshian vivent à la fin de l’hiver, lorsque ils attendent avec impa- tience le début de la saison (c’est cette faim qui pousse le héros dans des ex- péditions de chasse et de pêche). Les règles matrimoniales apparaissent comme la deuxième opposition, imposant le mariage entre des clans rivaux, et le besoin d’être simultanément le chef du clan de la mère et de la femme, ce qui est source d’instabilité sociale dans la société matrilinéaire des Tshims- hian (le héros est en conflit permanent avec les frères de sa femme). Par con- séquent, Lévi-Strauss souligne que ce mythe transmet fidèlement les crises et les difficultés de la vie sociale qui représentent un problème insoluble pour les Tshimshian, et dont ils trouvent l’issue dans l’état d’inertie. Cependant, en étudiant la version du mythe racontée par une partie du peuple Kwakiutl, Lévi-Strauss conclut que le héros de sa version d’Asdiwal parvient à dépasser les contradictions en les conciliant et en les unissant, en quoi il évite la mort comme punition pour la communication perturbée. Cette version reflète le succès des Kwakiutl à trouver d’une part une solu- tion sociale (le héros du mythe parvient à être successivement chef du clan de sa mère et de sa femme, en se débarrassant des beaux-frères fâcheux, et à dépasser de cette manière l’antinomie entre la filiation et l’alliance), et d’autre part de choisir comme leur résidence permanente la vallée du fleuve, à mi-chemin entre les montagnes et la mer, en chassant sur les deux rives quand il le faut (le nom même du héros du mythe renvoie au succès dans le dépassement de cette contradiction – "Le-plus-joli-des-chasseurs" et "Chef- de-la-pleine-mer"). La tribu Koeksotenok se voit comme un peuple fluvial qui n’a aucun problème mental avec l’antinomie "montagne : mer", étant donné que leur mode de vie, sans déplacements saisonniers, unit efficace- ment les deux. De cette manière, les valeurs négatives de la communication perturbée, dans les versions Tshimshian, se transforment en valeurs posi- tives chez les Kwakiutl. INDISCRETION MALENTENDU OUBLI NOSTALGIE Excès/ Défaut + - - + Autrui / Soi + + - - INDISCRETION MALENTENDU OUBLI NOSTALGIE Excès/ Défaut + - - + Autrui / Soi + + - - À partir du schéma on peut voir que l’indiscrétion consiste à dire à autrui plus qu’il ne fallait ou qu’il n’était souhaitable de le faire (excès dans la com- munication avec autrui); que le malentendu naît comme conséquence de la déduction que l’on a dit quelque chose qui en vérité n’a pas été dit, ou que l’on n’a pas voulu dire (défaut de communication avec autrui); que l’oubli représente un défaut de communication avec soi-même – nous avons oublié quelque chose/quelqu’un; et que la nostalgie signifie que nous avons exagéré dans la communication avec nous-mêmes, étant donné que nous nous disons plus qu’il n’est nécessaire ou souhaitable de se dire. Lévi-Strauss souligne l’exceptionnelle importance sémantique de "l’oubli" comme d’une catégorie de la pensée mythique; mais dans L’Anthropologie structurale II il n’élabore pas davantage son observation intéressante et origi- nale. Il reviendra à cette question une dizaine d’années plus tard dans son livre Le regard éloigné (Lévi-Strauss 1983) où il a à nouveau discuté sur la signifi- cation du motif de l’oubli dans ses deux travaux. Dans son texte De la possibilité mythique à l'existence sociale (Lévi- Strauss 1983: 215-240) Lévi-Strauss compare les versions Tshimshian du mythe d’Asdiwal avec la version correspondante de la tribu Kwakiutl qui vit sur une berge du fleuve Nass. Les Kwakiutl, plus précisément les Koeksote- nok, une de leurs tribus, ont emprunté le mythe aux voisins Tshimshian (ver- sion de la tribu Nisqua de 1902), ont inversé la structure de la version tshims- hian en l’adaptant à leur mode de vie et ont ainsi essentiellement changé le message du mythe. En quoi consistait la différence? Les oppositions mythiques originelles consistent dans l’impossibilité des Tshimshian de trouver une bonne réponse aux prémisses basiques de leur organisation sociale et économique. D’un côté il y a leurs déplacements sai- sonniers réguliers et les formes d’activité utilitaire qui s’y rattachent – entre la chasse à l’ours et à la chèvre sauvage dans les montagnes, et la pêche au pois- son-chandelle et au saumon en été dans les vallées des fleuves (Skeena et Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 170 DRAGANA ANTONIJEVI Nass), ou aux phoques et à d’autres espèces de poissons sur la côté du Pacifi- que. .. . 4. .2 (2009) INDISCRETION MALENTENDU OUBLI NOSTALGIE Excès/ Défaut + - - + Autrui / Soi + + - - Ce qui nous importe, c’est la conclusion qu’en tire Lévi-Strauss: c’est la société qui façonne les sémantèmes mentionnés et leurs transformations comme des facteurs positifs ou négatifs. La pensée mythique est indifférente envers eux, elle nous montre les combinaisons des notions, "elle ne semble jamais satisfaite d’apporter une seule réponse à un problème: sitôt formulée, cette réponse s’insère dans un jeu de transformation où toutes les autres ré- ponses possibles s’engendrent ensemble ou successivement" (Lévi-Strauss 1983: 232). Cependant, la réponse dépend de l’interprétation des communau- .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 171 tés humaines concrètes et de leur destin social, économique, historique et politique concret. Dans le second texte Mythe et oubli (Lévi-Strauss 1983: 253-260), Lévi- Strauss se demandera finalement quelle est la fonction de la communication perturbée. Dans ce texte il a comparé les versions Tshimshian et Kwakiutl d’Asdiwal, puis les mythes de la tribu Hidatsa sur leur origine, ainsi que trois mythes grecs sur lesquels Jean-Pierre Vernant a attiré son attention. Dans ces mythes, Plutarque explique comment les joueurs de flûte ont été frappés de l’interdiction d’entrer au temple de Ténès à l’île de Ténédos, ainsi que de prononcer dans ce temple le nom d’Achille, alors que Pindare interprète l’origine du rite de sacrifice sans feu au Rodos, et l’origine des droits rituels du monarque sur le territoire sacré, en les rattachant au mythe des Argonautes et de Médée. Dans tous les mythes évoqués, le champ de la "communication perturbée" joue un rôle important, dans sa fonction normative: celle d’introduire certaines règles matrimoniales dans la société (Tshimshian et Kwakiutl); d’expliquer l’origine et l’identité de la tribu, puis les raisons de sa séparation en deux peuples, Crow-Hidatsa et Awaxawi, alors que dans les mythes grecs cette fonction est d’établir les rituels et le calendrier des coutu- mes et des formes de comportement rituel en rapport avec eux, autant d’interdictions que de prescriptions. Autrement dit, d’assurer la mémoire et la continuité des règles face à l’oubli qui a provoqué une perturbation dans la communication, et par là dans la société en tant qu’unité. 4"Car c’est bien cette continuité que vient de briser l’oubli dans l’ordre mental : nous le reconnaissons nous-mêmes en parlant de ’trous de mémoire’" (Lévi-Strauss 1983: 259). 4"Car c’est bien cette continuité que vient de briser l’oubli dans l’ordre mental : nous le reconnaissons nous-mêmes en parlant de ’trous de mémoire’" (Lévi-Strauss 1983: 259). 5 Dans le recueil de Vuk St. Karadi, Srpske narodne pripovetke, Beograd: Državna štamparija kraljevine Jugoslavije, 1937, il existe deux versions de ce conte. ) 5 Dans le recueil de Vuk St. Karadi, Srpske narodne pripovetke, Beograd: Državna štamparija kraljevine Jugoslavije, 1937, il existe deux versions de ce conte. INDISCRETION MALENTENDU OUBLI NOSTALGIE Excès/ Défaut + - - + Autrui / Soi + + - - Comme troisième conclusion importante de Lévi-Strauss, on peut relever le constat de la fonction normative de la sémantique de l’oubli: le motif sert, selon Lévi-Strauss, à instaurer les pratiques rituelles qui vont assurer la conti- nuité qui, de son côté, va briser l’oubli dans l’ordre mental, que nous appelons généralement "trou de mémoire".4 A part dans les mythes, le motif de l’oubli est un lieu privilégié dans les contes merveilleux, particulièrement dans ceux qui traitent des époux. Il s’agit du type de contes généralement connu sous le nom de "La belle et la bête", dans les mythes grecs son prototype est "Amour et Psyché", alors que dans le catalogue d’ Aarne–Thompson ils portent le nom de Recherche du mari perdu (AT 425), ou de Mari transformé en bête (AT 425 C). Dans les contes serbes que j’ai analysés (Antonijevi 1991), ce type est présent dans sa version la plus connue Le serpent jeune marié.5 Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 172 DRAGANA ANTONIJEVI Le motif de l’oubli qui apparaît dans ces contes, se rencontre dans le cadre du système de communication perturbée. En effet, il s’agit du lien existant entre l’indiscrétion de la femme et l’oubli du mari. Le principal sujet du type 425C peut être réduit à l'histoire suivante: la jeune fille se marie avec un ser- pent, mais la nuit, dans le lit, l’animal se transforme en un bel homme. C’est cependant un secret que la femme ne doit dévoiler à personne avant l’écoulement d’un certain laps de temps, tant que dure l’envoûtement de son mari. Dès que l’interdiction est posée, d’après les règles du conte, elle doit être transgressée. Dans la version serbe, la femme dévoile le secret à sa belle- mère trop curieuse (indiscrétion), avide de savoir comment la femme vit avec le serpent, ou plus précisément ce qui se passe la nuit dans leur alcôve. De concert avec sa belle-mère, la femme dérobe au mari endormi sa peau de ser- pent pour la brûler et ainsi le munir d'une figure humaine durable. A ce mo- ment-là, le mari se réveille, comprend que la femme l’a trahi en transgressant l’interdiction de dévoiler le secret, et il la quitte en l’avertissant qu’elle le cherchera "tant qu’elle n’aura pas cassé le bâton de fer et déchiré les opanci* de fer" ce qui est, pour ainsi dire, une tâche impossible à accomplir. INDISCRETION MALENTENDU OUBLI NOSTALGIE Excès/ Défaut + - - + Autrui / Soi + + - - Une fois qu’il l’a quittée, le mari l’oublie immédiatement. Pour le retrouver et lui rap- peler son existence et leur vie conjugale, la femme entreprend une longue et pénible quête du mari perdu. Elle le retrouve dans un empire éloigné, en ré- alité – dans l’au-delà, ce par quoi le conte nous suggère que le héros, en raison du péché d’indiscrétion de sa femme et de sa mère, est en vérité – mort. Son oubli de la vie antérieure est égal à la mort réelle. Dans les mythes et les contes, donc, les héros meurent ou sont d’une autre manière punis pour des omissions dans la communication, ce qui nous avertit ainsi de la gravité de ce problème. On peut comprendre pourquoi la punition est aussi sévère: si la culture est basée sur un système complexe de communi- cation à différents niveaux, la perturbation représente alors un problème social réel et une transgression. Oubli et mémoire: Concepts du temps et de la durée culturelle L'homologie capitale établie entre la mort et l’oubli représente le fonde- ment de presque tous les systèmes religieux et mythiques – ces deux notions représentent d’importantes lignes de démarcation entre ce monde et l’au-delà, le passé et le présent. L’oubli se rattache essentiellement au passage du temps .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 173 et à la mortalité humaines.6 Dans les mythes grecs, le Lèthè était un fleuve au royaume d’Hadès où les morts venaient boire pour oublier leur vie terrestre. L’âme qui savait éviter de boire au fleuve de l’Oubli, au carrefour de la vie et de la mort, se tournait vers Mnèmosunè, pour s’abreuver au lac de la Mé- moire, à la fontaine de l’Immortalité (Vernant 1982, 1: 88, 92). C’est ainsi que nous parvenons au couple indissociable dans son opposi- tion et sa complémentarité, à l’oubli et à la mémoire. Dans les mythes, la mé- moire sert à combler le fossé entre ce monde et l’au-delà, à briser l’oubli par le rappel du passé. Dans le système plus ancien des croyances grecques, ce rappel ne comprenait pas seulement le temps qui précède le temps courant, mais plus encore – c’était un savoir sur les tous débuts des choses et le temps primordial, mais également le savoir sur les événements futurs. Le passé ainsi compris apparaît comme une dimension de l’au-delà (ibid, 87). Dans des in- terprétations philosophiques et eschatologiques postérieures, la mémoire et l’oubli vont se réduire à la dimension de la vie humaine individuelle et histo- rique et sa temporalité mesurable. Pour se définir et se donner un sens dans l’histoire, les sociétés et les indi- vidus créent et utilisent le concept du temps de différentes manières, luttant contre l’oubli et la peur de la disparition biologique et symbolique. Les con- cepts du temps socialement construit peuvent être cycliques, linéaires et limi- naux (oniriques, concepts d’époques); bien que ces concepts soient parallèles et reliés entre eux, dans chaque société, l’un d’entre eux va prévaloir en fonc- tion de l’orientation sociale vers le passé, le présent ou le futur (Rot 2000: 163). Il est clair, alors, pourquoi la mémoire a une fonction normative: elle donne des repères à la société et des points d’appui à l’identité à l’intérieur de la temporalité cyclique, linéaire ou onirique. 6 Dans les interprétations mystiques et les doctrines de la réincarnation, chaque phase de l’âme humaine est reliée avec l’oubli de l’existence précédente. Dans les systèmes antérieurs de croyances, la tristesse de l’oubli des morts se rapportait à la perte des joies terrestres et de la vie ; plus tard on croit que cette tristesse se rapporte à l’oubli des splendeurs de la vie d’au-delà de l’âme qui, par la réincarnation, est con- damnée aux souffrances de l’existence terrestre, et par conséquent à l’oubli des vérités éternelles qu’elle avait pu contempler avant de se réincarner. Opanci – Sorte de chaussures rustiques, généralement en cuir tanné. N. de la T. 7 A. J. Greimas considérait, avec de bonnes raisons, que ses modèles sémiotiques peuvent être transmis du niveau de l’étude de la langue et du texte sur un champ dis- cursif plus large, dans le domaine de la sociologie et du comportement social (Grei- mas 1989a: 543). 8 Nous le formulons par des expressions comme"reviens à toi", "rappelle-toi", "remets-toi","remémore-toi","figure-toi". ) 8 Nous le formulons par des expressions comme"reviens à toi", "rappelle-toi", "remets-toi","remémore-toi","figure-toi". 7 A. J. Greimas considérait, avec de bonnes raisons, que ses modèles sémiotiques peuvent être transmis du niveau de l’étude de la langue et du texte sur un champ dis- cursif plus large, dans le domaine de la sociologie et du comportement social (Grei- mas 1989a: 543). Oubli et mémoire: Concepts du temps et de la durée culturelle En ce sens, l’oubli et la mémoire représentent réellement un couple psychologique et cognitif important. Nous allons voir maintenant comment ils sont intégrés dans le processus important pour la survie de la société et de la culture. q p p Opanci – Sorte de chaussures rustiques, généralement en cuir tanné. N. de la T. Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 174 DRAGANA ANTONIJEVI * .. . 4. .2 (2009) * Les sémioticiens français A. J. Greimas et Joseph Courtés ont proposé le champ sémantique du savoir (Courtés 1976: 81). Bien que leur intention pre- mière ne fût pas de traiter le motif de l’oubli mais la modalité sémiologique du "savoir", ils ont placé l’opposition "oubli : mémoire" à l’intérieur d’une structure cognitive plus générale qui règle leur rapport, créeant un nouveau couple antithétique prédominent: "savoir : non-savoir". (schéma 2). Schéma 2: Modalité du savoir conscience SAVOIR oubli ignorance NON - SAVOIR mémoire Schéma 2: Modalité du savoir SAVOIR conscience NON - SAVOIR J’interpréterai de la manière suivante ce modèle sémiotique7, étudié sous l’angle de la culture et du comportement social. La conscience et la mémoire forment la catégorie du savoir qui par sa ré- pétitivité et sa créativité assure la continuité des représentations mentales et des pratiques correspondantes en contribuant à la création, au maintien et à la durée de la culture humaine. La conscience fait revenir la mémoire et brise l’oubli,8 permet au savoir de s’accumuler dans le temps et se conserve dans la .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 175 mémoire qui est, comme le dit Michel de Certeau," ... cachée, (elle n’a pas de lieu repérable), jusqu’à l’instant où elle se révèle, au ’moment opportun’ [...] moyennant ce déplacement de l’espace au temps" (de Certeau 1988: 82,83), donc, un espace où pourront s’exprimer et agir l’expérience acquise, le savoir et le savoir-faire. A l’autre extrémité de l’opposition, à la position du non- savoir, se trouvent l’oubli et l’ignorance, les épouvantails mentaux qui déchi- rent le fin et complexe réseau de communication. Mais l’oubli et l’ignorance sont eux-mêmes en opposition. L’ignorance est un vide, un rien cognitif (nous ne pouvons nous rappeler quelque chose que nous ne savons pas) qu’il est possible de remplir par un certain contenu (le savoir); alors que l’oubli vide le savoir actuel, le diminue quantitativement, le détruit, crée un "trou de mé- moire", le transformant en savoir virtuel qui, lui, par le rappel, peut à nouveau être actualisé. *** En ce sens, l’oubli peut être traité comme un processus actif et destructif, alors que l’ignorance est un état passif, une possibilité (une imma- nence existant dans les limites de la conscience) que l’on est à même de chan- ger et de mettre en mouvement par le processus de l’étude et du savoir. Si la culture humaine repose sur la transmission du savoir acquis, la répé- tition de l’appris et l’accroissement du savoir existant au moyen de nouvelles découvertes et connaissances, alors la mémoire et le souvenir influent de ma- nière décisive sur ce processus. C’est pourquoi l’oubli est un péché dans le système mythique, c’est-à-dire une faute, un ennui et un problème dans la communication quotidienne. L’importance de ce fait est démontrée par l’histoire des pertes des différentes connaissances et de savoir-faire que les hommes, au cours de leur existence, concevaient et découvraient pour les oublier, pour toujours ou temporairement. Autant cela vaut-il pour les cultures qui connaissent l’écriture et les différentes technologies d’archiver les savoirs, qu’il est possible alors de perdre et de détruire, encore plus cela vaut-il pour les cultures orales, qui, jusqu'à il y a peu, représentaient la grande majorité des communautés humaines et dont l'archivage mental, maintenu à l’aide de diffé- rentes mnémotechniques et transmis par la tradition orale, et qu’il est si facile de détruire parce qu'il s’appuie sur la mémoire fragile des hommes. 9 Je remercie Ivan Kovaevi, professeur d université, dont les suggestions m ont aidée à clarifier les incertitudes sémantiques et logiques liées à l’emploi que fait Lévi- Strauss du terme de"malentendu". Je remercie également la traductrice madame Ta- mara Vali qui m a aidée à comprendre les nuances dans la signification des termes français qu’utilise Lévi-Strauss. 10 Il aurait tout de même pu facilement s’en rendre compte lui-même. Lévi-Strauss dit clairement que dans la version du mythe d’Asdiwal de 1916, la mort de son fils Waux est causée par la distraction, mais que la mort de la femme de Waux est le ré- sultat du malentendu de sa part – elle n’a pas correctement interprété les mots que Waux lui a adressés, en d’autres termes,"elle n’a pas bien compris son ordre", elle s’est gorgée de graisse, a éclaté est s’est changée en silex veiné! (Lévi-Strauss 1988: 178) [Lévi-Strauss 1973: 228]. Il est réellement étonnant que Lévi-Strauss n’ait pas distingué la différence, bien qu’il ait écrit lui-même qu’il s’agissait d’une inteprétation inexacte de la part de la femme de Waux (donc, destinataire du message). .. . 4. .2 (2009) Le système de communication perturbée : une structure un peu mieux équilibrée La sémantique de l’oubli participe ainsi des deux structures: du système communicationnel et du système cognitif. On peut se poser la question com- ment mettre en relation la structure cognitive étudiée avec le système de communication perturbée. Pour ce faire, il y a de bonnes raisons pour d’abord Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 176 DRAGANA ANTONIJEVI changer, compléter et clarifier certains éléments importants du schéma de Lévi-Strauss. Le problème fondamental apparaît dans l’emploi que fait Lévi-Strauss du terme malentendu. Ce défaut de communication avec autrui est-il quantitatif – une insuffisance, un manque de mots ; ou est-il qualitatif – compréhension erronée des mots? À Lévi-Strauss utilise les deux notions sans faire de différence. À un moment donné il dit "défaut de communication avec autrui", puis immédiatement après il explique que "le malentendu consiste à comprendre, dans ce que quelqu’un a dit, autre chose que ce qu’il a voulu dire" (Lévi-Strauss 1973: 230). Mon hé- sitation a, en outre, été provoquée par la traduction croate d’Asdiwal, dans laquelle l’antinomie est traduite par "excès : faute" (Lévi-Strauss 1988: 180,181). Cependant, il ne s’agit pas seulement d’une confusion linguistique. Vu d’un point de vue rationnel, le contraire de l’"excès" ou du "surcroît de quelque chose" est le"manque" ou "la carence de quelque chose". En outre, le contraire d’"erroné" ou d’"inexact" est "exact", "correct", et non pas "l’excès".9 Toute l’affaire est cependant facile à démêler si nous revenons à la série communicationnelle initiale. Il s’agit du destinateur et du destinataire du mes- sage. Lévi-Strauss a dans son explication non seulement confondu le défaut quantitatif et qualitatif dans la communication, mais il a également confondu ces deux groupes d’individus!10 Il a fait un schéma élégant et simple, mais tout de même en partie inexact. Il a d’abord parlé de destinateur qui peut dire au destinataire du message moins que ce qui est nécessaire/souhaitable. Dans ce cas, étant donné qu’il s’agit d’un manque quantitatif de mots, de privation ou d’insuffisance d’information, je vais désigner cette catégorie du nom d’obscurité. À l’autre extrémité de la chaîne de communication se trouve le destinataire du message 9 Je remercie Ivan Kovaevi, professeur d université, dont les suggestions m ont aidée à clarifier les incertitudes sémantiques et logiques liées à l’emploi que fait Lévi- Strauss du terme de"malentendu". Le système de communication perturbée : une structure un peu mieux équilibrée Je remercie également la traductrice madame Ta- mara Vali qui m a aidée à comprendre les nuances dans la signification des termes français qu’utilise Lévi-Strauss. 9 Je remercie Ivan Kovaevi, professeur d université, dont les suggestions m ont aidée à clarifier les incertitudes sémantiques et logiques liées à l’emploi que fait Lévi- Strauss du terme de"malentendu". Je remercie également la traductrice madame Ta- mara Vali qui m a aidée à comprendre les nuances dans la signification des termes français qu’utilise Lévi-Strauss. 10 Il aurait tout de même pu facilement s’en rendre compte lui-même. Lévi-Strauss dit clairement que dans la version du mythe d’Asdiwal de 1916, la mort de son fils Waux est causée par la distraction, mais que la mort de la femme de Waux est le ré- sultat du malentendu de sa part – elle n’a pas correctement interprété les mots que Waux lui a adressés, en d’autres termes,"elle n’a pas bien compris son ordre", elle s’est gorgée de graisse, a éclaté est s’est changée en silex veiné! (Lévi-Strauss 1988: 178) [Lévi-Strauss 1973: 228]. Il est réellement étonnant que Lévi-Strauss n’ait pas distingué la différence, bien qu’il ait écrit lui-même qu’il s’agissait d’une inteprétation inexacte de la part de la femme de Waux (donc, destinataire du message). .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 177 qui est chargé d’une tâche interprétative – celle d’interpréter de manière exacte ou erronée les mots du destinateur, et dans ce cas une erreur qualitative peut apparaître, à savoir la mauvaise compréhension de ce qui a été dit, ce que je continuerai à désigner par la notion de malentendu, étant donné que celle-ci relève du champ de l’interprétation. Il existe, certes, le cas d’erreur qualitative de la part du destinateur du mes- sage, lorsqu’il donne délibérément une fausse information au destinataire, lui mentant par exemple, ou l’abusant. Le mensonge est un phénomène cognitif et moral important qui provoque aussi de graves malentendus et conflits. Nous pouvons traiter le non-savoir ou l’ignorance comme une erreur qualitative dans la communication avec soi-même, ce qui peut avoir pour conséquence de se leurrer soi-même (la conviction que nous savons quelque chose que nous ne savons pas), soit envers autrui dans le sens d’une désinformation involon- taire par ignorance. Cependant, Lévi-Strauss n’a pas examiné ces cas d’erreurs qualitatives de la part du destinateur du message. .. . 4. .2 (2009) Le système de communication perturbée : une structure un peu mieux équilibrée Je pense qu’il est maintenant clair que toutes les quatre catégories quanti- tatives de communication perturbée – l’oubli, l’indiscrétion, l’obscurité et la nostalgie peuvent dans le processus d’interprétation provoquer un malentendu chez le destinataire du message, que le destinataire soit ou non la même per- sonne que le destinateur du message. Je traiterai la communication tournée vers soi-même comme une action "réflexive", et celle tournée vers autrui comme "transitive". J’introduirai également le segment du défaut/de l’erreur qualitatifs dans la communication de la part du destinateur, pour indiquer les combinaisons et les variantes des différentes possibilités de communication perturbée. C’est pour cette raison qu’il est possible d’établir le schéma cogni- tif et communicationnel suivant (schéma 3) qui à mon avis, déterminerait avec plus de précision les rapports sémantiques entre les notions données. Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) Schéma 3: Schéma cognitivo-communicationnel de communication perturbée DESTINATEUR S1 DESTINATAIRE S1 RÉFLEXIF INTERPRÉTATIF Défaut quantitatif Défaut qualitatif Interprétation consécutive SOI SOI SOI DÉFAUT EXCÈS FAUX FAUX VRAI Oubli Nostalgie Non-savoir Malentendu avec soi Oubli de soi-même Leurre de soi- même Souvenir Prise de conscience Connaissance Schéma 3: Schéma cognitivo-communicationnel de communication perturbée Schéma 3: Schéma cognitivo-communicationnel de communication perturbée DESTINATEUR S1 DESTINATAIRE S1 RÉFLEXIF INTERPRÉTATIF Défaut quantitatif Défaut qualitatif Interprétation consécutive SOI SOI SOI DÉFAUT EXCÈS FAUX FAUX VRAI Oubli Nostalgie Non-savoir Malentendu avec soi Oubli de soi-même Leurre de soi- même Souvenir Prise de conscience Connaissance Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 178 DRAGANA ANTONIJEVI DESTINATEUR S1 DESTINATAIRE S2 TRANSITIF INTERPRETATIF Défaut quantitatif Défaut qualitatif Interprétation consécutive AUTRUI AUTRUI AUTRUI DEFAUT EXCÈS FAUX FAUX VRAI Obscurité /Mystère/ Indiscrétion /Divulgation du secret / Mensonge Leurre Désinformation Malentendu avec autrui Incompréhension Non-savoir Cafardage Accord Com- préhension Savoir Révélation DESTINATEUR S1 DESTINATAIRE S2 TRANSITIF INTERPRETATIF Défaut quantitatif Défaut qualitatif Interprétation consécutive AUTRUI AUTRUI AUTRUI DEFAUT EXCÈS FAUX FAUX VRAI Obscurité /Mystère/ Indiscrétion /Divulgation du secret / Mensonge Leurre Désinformation Malentendu avec autrui Incompréhension Non-savoir Cafardage Accord Com- préhension Savoir Révélation La question de la crédibilité du discours et des différents points de vue La question de la crédibilité du discours et des différents points de vue La question de la crédibilité du discours et des différents points de vue La question des différents points de vue du destinateur et du destinataire du message entre dans le cadre du problème qui concerne le malentendu, c’est-à- dire des significations connotatives possibles, mais aussi du problème de la véracité et de la crédibilité du discours. Il ne faudrait pas se laisser abuser par le fait que les participants de la chaîne de communication semblent "enfermés" dans l’échange monovocal de savoir /information, bien que l’on suppose leur accord, ne serait-ce qu’implicite sur ce qui est vrai ou, du moins, ce qui a l’apparence du vrai. Greimas a désigné cet accord tacite par l’expression "contrat de véracité" (veridiction contract) qui est à la base des discours individuels et sociaux. Dans les communautés homo- gènes, archaïques, on parvient à ce "contrat" grâce à la vérité et aux normes établies par la tradition, alors que dans les sociétés contemporaines complexes et hétérogènes on y parvient à travers le processus de négociation sur les postu- lats axiologiques et cognitifs qui vont produire ce que Greimas a nommé "l’effet de signification du vrai" (étant donné qu’il n’y a plus, comme il l’a fait remar- quer, de "vérités immuables" dans les sociétés post-industrielles) (Greimas 1989b: 657). Toutefois, alors que le destinateur est responsable du succès ou de l’insuccès de son discours qu’il peut manipuler, "la question ultime de la con- firmation de la véracité repose sur le destinataire du message et sa confiance dans ce qui lui est dit", et ce qui devrait correspondre à ses attentes (ibid, 657, 658). Il est clair que de nombreux malentendus sont possibles sur ce chemin de la manipulation des discours et leur crédibilité, découlant soit de l’erreur quanti- tative soit de l’erreur qualitative. Prenons l’exemple de l’erreur qualitative – le mensonge. Le destinateur peut consciemment mentir au destinataire qui, lui, peut considérer cette in- .. . 4. .2 (2009) 179 A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS formation comme vraie (malentendu) tant qu’il n’aura pas démasqué le desti- nateur. S’il est en mesure de réagir, le destinataire du message peut accuser le destinateur de l’abuser délibérément et de diffuser de fausses informations. Une telle communication va provoquer différents ennuis, puis entraîner cer- taines sanctions. Ou bien, prenons le cas de l’indiscrétion. La question de la crédibilité du discours et des différents points de vue Dire plus qu’il n’est permis/souhaitable/nécessaire de dire est généralement compris comme la divulgation d’un secret / le cafardage / le bavardage. Bien que les catégories évoquées appartiennent à la communication perturbée, elles ne sont pas équi- valentes par leur signification et leurs conséquences. En outre, il peut y avoir une troisième personne dans la chaîne de communication qui est, par exemple, concernée par le secret et qui a donné l’interdiction à sa divulgation (le cas du conte "Le serpent jeune marié" que nous avons décrit ci-dessus, mais aussi les versions du mythe d’Asdiwal de 1895). Enfin, retournons au cas de l’oubli. Que se passe-t-il lorsque l’oubli passe de la catégorie réflexive à la catégorie persuasive11 et transitive, autrement dit, lorsque l’oubli de quelque chose et/ou de quelqu’un nous est imposé institutionnellement? Il est évident que dans ces cas-là le destinateur et le destinataire ne sont pas égaux. Leur communication se déroule alors selon un rapport hiérarchique de domination et de subordina- tion, ce qui peut avoir pour conséquence soit l’adoption de l’opinion imposée soit la confrontation de points de vue, la méfiance, voire un conflit. Il en découle clairement que les problèmes de communication perturbée peuvent s’aggraver du fait des points de vue divergents des participants, de l’hiérarchie de leurs positions et rapports, de leurs motifs personnels et des implications morales incluses dans l’échange d’informations, enfin de la ques- tion de confiance dans la crédibilité de l’énoncé. En postulant la modalisation véridictoire (schèma 4) où chaque discours peut être désigné comme vrai, faux, mensonger et secret, Greimas a lié "le contrat de véracité" à la question de lecture et d’interprétation de la signi- fication dans la dimension cognitive, autrement dit, à la participation du desti- nateur et du destinataire dans la communication dans laquelle l’axe de crédibi- lité est fait de la reconnaissance, c’est-à-dire, du passage du faux savoir au savoir vrai, ce qui permet de dissiper le malentendu existant dans la commu- nication perturbée (Greimas et Courtés 1976:440-441). Si elle vient en temps utile, la "reconnaissance" va permettre d’éviter le conflit et la fin tragiques, et elle représente notamment la pointe finale du discours comique, mais ce n’est pas toujours le cas. 12 Greimas considèrait que dans les sociétés contemporaines post-industrielles le fossé entre le vrai et le certain, le savoir et la croyance est manifeste, et que le destina- teur du message ne ressent plus l’obligation de dire la vérité mais ce qui a l’apparence de vérité. D’où il conclut que la cohésion sociale dans les sociétés modernes repose sur une communication qui ressemble à des contes de folklore appelés contes de fri- pons, où les interlocuteurs se trompent à tour de rôle, se laissant tromper après avoir eux-mêmes trompé /menti (à) l’interlocuteur (Greimas 1989b: 657, 659). .. . 4. .2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie Toute époque est marquée par son propre style de pensée façonné selon les intérêts de la classe dirigeante. Mary Douglas (Daglas 2001: 99) Toute époque est marquée par son propre style de pensée façonné selon les intérêts de la classe dirigeante. Mary Douglas (Daglas 2001: 99) J’ai emprunté les expressions styles de pensée et communautés de pensée à Mary Douglas (Daglas 2001) pour les besoins de ce travail, parce qu’elles me semblent utiles pour l’analyse des manières dont certaines communautés, composées de groupes sociaux différents, peuvent dans leur discours public défendre des opinions semblables ou identiques. Douglas a à son tour em- prunté la notion au philosophe Ludwik Fleck qui considèrait que le style de pensée d’une communauté "pose la condition préalable à toute cognition et détermine ce qui va être considéré comme question raisonnable et réponse vraie ou fausse. Il assure le contexte et pose les limites à tout jugement sur la réalité objective" (Daglas 2001:23). Étant donné que dans ce travail les pro- blèmes cognitifs de l’oubli et du souvenir, puis du vrai et du faux, ont jusque là été analysés, j’ai considéré la notion de "styles" comme appropriée préci- sément parce que les catégories plus vastes de pensée de certaines commu- nautés, sont plus larges que la notion de "groupe social" (dont j’ai voulu éviter la définition précise). La fluidité et la multidiscursivité des différents styles de pensée me semblent particulièrement importantes pour les pays en transition, dont fait partie la Serbie, étant donné qu’ils sont confrontés au changement du système socio-économique et de l’idéologie, luttant pour instaurer de nou- veaux modèles culturels, axiologiques et cognitifs. En réalité, ces pays ont la difficile tâche de créer un nouveau style de pensée conforme à l’ordre social qui est en train de se développer à travers le processus de transformation post- socialiste. Comme ce processus n’est pas simple dans ces pays, comme d’ailleurs dans toute société en effervescence, le conflit éclate entre les diffé- rentes visions du monde. Les sociétés ex-socialistes en transition ont un problème particulier à défi- nir leur attitude envers le passé, c’est-à-dire envers ce qui sera jugé digne de la mémoire sociale et représentera une base pour la création d’une vision du monde collective nouvelle. La question de la crédibilité du discours et des différents points de vue Dans de nombreux récits, la reconnaissance arrive trop 11 On sous-entend par communication persuasive"la communication émotionnelle des règles sociales", c’est-à-dire, le type de communication culturelle qui a pour l’objectif de motiver l’action en provoquant les émotions, et sert habituellement à la transmission des valeurs et règles sociales sous forme de normes et d’exemples d’application de ces normes (Ferrara 1974: 245, 246). Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 180 DRAGANA ANTONIJEVI tard, ce qui les transforme en tragédies; dans le récit d’Asdiwal il n’y a point de reconnaissance, ce qui a amené Lévi-Strauss à la conclusion sur le profond pessimisme de ce mythe. Schéma 4: La modalisation véridictoire Schéma 4: La modalisation véridictoire être VRAI SECRET MENSONGE non - paraître non - être FAUX paraître VRAI FAUX Le problème s’accroit, cependant, dans des sociétés contemporaines, mul- tivocales dont Greimas avait une vision pessimiste, en tant que de nouvelles "tours de Babylone", parce qu’en elles il y a rivalité des voix, où chacun des nombreux discours se mêle et lutte avec les autres pour son droit de parole, pour "sa vérité", devenant ainsi un moyen pour les "connotations terrorisantes dans la nouvelle ère d’incroyance" (Greimas 1989b: 656).12 Enfin, il a conclu, avec une certaine dose d’ironie, qu’il fallait inverser la conception existante sur le rapport du discours et du contexte culturel, dans le sens où "ce sont les contextes culturels qui sont définis par l’interprétation connotative du dis- cours", et non l’inverse (ibid, 655). Cela nous mène à l’analyse de l’application du champ sémantique de la communication perturbée et du contrat de véracité dans les différents styles .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 181 de pensée dans la Serbie en transition, qui, elle, offre une image typique de la société multidiscursive luttant à travers la transition pour l’établissement de nouveaux/anciens postulats cognitifs et axiologiques. Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie Etant donné que "la mémoire sociale légitime l’ordre social" (Konerton 2002: 11), il est clair que les sociétés en transition sont à la recherche des contenus qui donneront une signification aux rapports Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 182 DRAGANA ANTONIJEVI politiques et sociaux nouvellement instaurés, choisissant soigneusement dans le passé les éléments qui vont soutenir le nouveau système,".. car il appartient à la mémoire, à savoir à l’État qui est le propriétaire de la mémoire, de créer l’illusion d’un passé unique et de trouver dans ce passé des appuis communs: d’unir les générations, les ancêtres et les contemporains, d’anticiper leur va- leur dans les temps nouveaux et de styliser le passé, voire une de ses versions, pour les besoins de ce nouveau temps" (eri 2009: 67, italique de D. A.). Etant donné, donc, que "notre sentiment du présent repose dans une large mesure sur notre savoir sur le passé", comme le dit Paul Connerton, voici où apparaît le problème: "le présent peut être vécu de différentes manières, en fonction de différents passés auxquels nous pouvons le rattacher" (Konerton 2002: 10). D’où les difficultés des sociétés en transition: à quel passé se ratta- cher? Fuyant le passé communiste et la mémoire historique dirigée, encore très présents dans la mémoire et l’expérience de ses citoyens, elles se tour- naient vers différents segments du passé dans leur marche à travers l’histoire, à partir de la Deuxième Guerre mondiale comme premier jalon temporel sûr, puis revenant en arrière aux racines séculaires et millénaires. Le point d’appui était recherché dans différentes identités – nationale, régionale, européenne, religieuse, dans la culture traditionnelle, autochtone ou étrangère, rurale et/ou urbaine etc. La Serbie, à l’instar des autres pays en transition, est à la recherche d’une identité nouvelle/ancienne, de cette "version stylisée du passé" qui va donner un sens aux événements qui ont marqué sa situation spécifique de transforma- tion post-socialiste. Le problème de la Serbie s’aggrave par le fait que "son" projet du 20e siècle, celui de créer un nouvel état appelé Yougoslavie, a échoué au début des années 90 dans les flammes de la guerre civile; la Serbie a en outre été accusée pour la "fomentation" de cette guerre, ce qui a initié de nombreuses remises en question de son rôle social et national, de son impor- tance et de son efficacité dans l’histoire des temps modernes. .. . 4. .2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie Toute personne qui a pu, dans la dernière vingtaine d’années, suivre en Serbie les débats pu- blics et les polémiques sur le thème du passé national, de son rôle dans la création et le démantèlement de la Yougoslavie, puis de ce qui est digne de mémoire sociale, sait que des "luttes violentes" ont périodiquement été me- nées dont le résultat est, toujours, une image encore embrouillée de la position et de l’identité actuelle et future. Ces points de vue plurivoques et multiples, parfois complètement opposés entre eux, m’ont incitée à concevoir l’idée de l’existence de la communication perturbée. Cependant, je ne considère pas ce processus avec pessimisme. Je pars de l’hypothèse, donc, que la transition est une période où s’accentuent les processus de remise en question et de réévaluation, où se rencontrent différentes opinions qui dans les temps instables peuvent provo- quer des malentendus et des conflits dans la société, mais qui, en dernière .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 183 analyse, mènent au façonnement des positions et à la formation des idées et des représentations collectives plus stables. Pour présenter l’image de la communication perturbée en Serbie, je n’évoquerai, pour illustrer, que quelques exemples de styles de pensée en conflit et de références de ces styles aux différents segments du passé, sans vouloir les analyser en détail ni étudier tous les débats possibles menés entre les différents styles de pensée dans les vingt dernières années. Je voudrais montrer le mode et le lieu auquel se positionnent dans la structure de la "communication perturbée" et la "modalité véridictoire", les points de vue qui se forment par rapport à elles et calculer les chances qu’ils ont de devenir partie intégrante du "style de pensée" prescrit et officiel. Je tiens compte de la fluidité des concepts et des idées, du flottement mental, puis de la variabilité du contexte et des intérêts, notamment politiques, que certaines communautés de pensée peuvent avoir ; c’est pourquoi, par l’analyse proposée ici, je ne plaide pour aucune structure cognitive fermée, mais simplement désire rendre compte de la capacité et de l’aptitude de certains styles de pensée à s’intégrer avec (ou sans) succès au processus de construction de la mémoire socio-natio- nale et de l’identité dans la transformation post-socialiste. Styles de pensée dans le système de communication perturbée: cas de la Serbie Dans l’analyse je vais croiser les schémas de la "communication pertur- bée" et de la "modalité véridictoire" dans un rapport de congruence des posi- tions des sémantèmes proposés. Je vais d’abord analyser les termes sur des axes horizontaux, antithétiques, stables structurellement et opposés entre eux, qui représentent le maximum (l’axe supérieur) et le minimum (l’axe inférieur) des traits souhaitables d’identification. Les axes verticaux ou les déixis repré- sentent, eux, des concepts instables aspirant en principe à se poser, grâce au processus de "reconnaissance", sur l’un des axes cognitifs antithétiques, deve- nant ainsi des énoncés vrais ou faux. Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) Communication perturbée soi OUBLI NOSTALGIE OBSCU- RITÉ excès autrui INDISCRÉTION défaut Communication perturbée soi OUBLI NOSTALGIE OBSCU- RITÉ excès autrui INDISCRÉTION défaut Communication perturbée INDISCRÉTION Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 184 DRAGANA ANTONIJEVI Modalité véridictoire être VRAI SECRET MENSONGE non-paraître non-être FAUX paraître VRAI Position "de l’oubli" et "du vrai". Ici l’on peut ranger toutes ces repré- sentations et discours sur les événements historiques et politiques que la so- ciété à travers l’oubli d’elle-même s’efforce de refouler de la mémoire ou de reconsidérer leur importance, ou bien, celles qui s’imposent à la société à travers l’oubli institutionnel. Ces deux processus – réflexif et transitif – peu- vent et peuvent ne pas être identiques par leur contenu. Dans le cas de l’oubli de soi-même, le processus commence spontanément au niveau de la commu- nauté, mais le mot de la fin est aux institutions sociales qui prescrivent le con- tenu de l’histoire mémorisée/oubliée en formant une carte cognitive de la mémoire de ses citoyens. Qu’est-ce que les sociétés oublient elles-même ?13 La réponse à cette ques- tion intéressante peut être trouvée dans le parallèle avec l’oubli individuel et le refoulement des expériences désagréables, traumatisantes ou de celles qui nous présentent sous un mauvais jour. La nostalgie contribue aussi à l’oubli (de soi-même), ce que Lévi-Strauss a explicitement montré sur l’exemple d’Asdiwal. Les sociétés se comportent semblablement, ce que confirment les matériaux folkloriques, notamment les récits historico-culturels et les chan- sons épiques, où est manifeste un manque de contenu concernant les défaites et les traumas collectifs et nationaux. 13 Mary Douglas a remarqué que"les anthropologues sont moins enclins à se de- mander pourquoi les hommes oublient. D’après eux, la particularité qu’il faut expli- quer est la mémoire" (Daglas 2001: 78). .. . 4. .2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie En réalité, il existe toute une série de sujets du passé national qui résistent à la folklorisation car ils sont incompati- bles avec le besoin de la société de glorifier ses héros, ses ancêtres, son his- toire et son pays natal, et à travers cela – la nation elle-même. Si la société ne .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 185 parvient pas, par une révision ultérieure de la mémoire et sa narrativisation, à transformer sa défaite en une victoire, ou en un martyre et un sacrifice qu’elle va exalter et ainsi se munir d’une justification pour sa communauté et des raisons pour une mémoire collective (par l’identification du coupable dans le groupe étranger et/ou l’identification du coupable dans son propre groupe, et de l’autre côté dans la glorification du héros-martyr, comme dans le cas du mythe épique serbe sur la bataille de Kosovo), elle tentera de refouler ces souvenirs, de les préformuler en une philosophie nationale de la souffrance et de la constance (forme d’auto-justification), ou de les exprimer dans des gen- res qui stylisent les matériaux d’une manière plus objective et rendent possible la reconstruction historique (bien qu’elle soit elle-même sujette aux mystifi- cations). De tels cas de refoulement de la mémoire dans le matériaux folklori- ques, mais également dans le discours publique, sont par exemple la grande migration des Serbes de 1690 (v. Miloevi-orevi 2000), l échec catastro- phique de la Première insurrection serbe de 1813 (v. Antonijevi 2007a, 2007b), le grand pillage du peuple à travers la nationalisation, la confiscation et la collectivisation de la part du pouvoir communiste (v. Antonijevi 2009), ou la révision du rôle et le refoulement de la culpabilité par rapport à la guerre civile en Yougoslavie de 1991-1995. Ce dernier exemple déclenche en Serbie encore aujourd’hui de nombreuses polémiques entre, d’une part, ceux qui s’efforcent de refouler ces événements de la mémoire ou de minimiser leur importance, et ceux, d’autre part, qui tentent de ne pas oublier et de ne pas permettre au peuple serbe de remettre en question sa propre culpabilité et sa part dans ces événements, considérant qu’ils possèdent le savoir exact qu’il ne faut pas remettre en question. Ces deux styles s’expriment parfois dans des formes extrêmes et irritantes qui ne contribuent pas à l’interprétation objective des événements historiques traumatisants. 14 Les destins de certaines personnalités du Parti communiste de Yougoslavie et qui ont vécu en Serbie, représentent des exemples paradigmatiques d’oubli, à travers un gommage quasi littéral de toute mention publique: Milovan ilas-ido, dont la tâche était de s’occuper dans le cadre d’Agitprop du purisme et de l’orthodoxie poli- tiques, a été exclu du Parti communiste en 1954 et est devenu plus tard le dissedent et le critique yougoslave du communisme le plus connu, arrêté et emprisonné à maintes reprises ; Aleksandar-Leka Rankovi, chef de tous les services policiers et secrets, expulsé du Parti en 1966. sous l’accusation qu’il avait mis sur écoute le président Tito, sombrant plus tard dans le silence et l’anonymat; enfin Jovanka Broz, l’épouse du président Tito, subitement éloignée de la vie publique à la fin des années 70 sans raison apparente, tenue pendant presque 30 ans sous résidence surveillée, privée de droits civiques qui ne lui ont été rendus que tout récemment. Styles de pensée dans le système de communication perturbée: cas de la Serbie Cet exemple illustre bien la multi- discursivité et les différents points de vue qui existent dans la société, et bien qu’il concerne un passé tout récent, rend compte des difficultés de formuler "l’effet du vrai" au niveau du discours officiel. L’oubli institutionnel représente le second cas de figure. La devise du cha- pitre, empruntée à Mary Douglas, est univoque dans la constatation que les classes dirigeantes sont celles qui possèdent le pouvoir politique nécessaire pour imposer à la société l’oubli ou bien le souvenir, comme processus paral- lèle, des événements historiques et des individus qui leur importent ou les dérangent au sein d’une idéologie donnée, pour former de cette manière leur propre style de pensée. La pression de l’oubli institutionnel sur la société est d’autant plus ferme et énergique que le régime est autoritaire et inquiet du succès de son idéologie. Toutes les sociétés en transition, qui depuis la fin de la Deuxième Guerre mondiale jusqu’au début des années 90 du 20e siècle ont vécu dans le régime autoritaire du communisme, connaissent bien cette forme d’oubli institution- Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 186 DRAGANA ANTONIJEVI nellement imposé de tout le passé qui a précédé le régime socialiste, et parti- culièrement l’oubli de toute la tradition de la culture bourgeoise, mais égale- ment celui de nombreux éléments et symboles de la culture et de l’histoire nationales non conformes au style de pensée communiste. En outre, ces régi- mes ont infailliblement imposé le pouvoir de l’oubli institutionnel même quand il s’agissait de leurs propres hommes – "rénégats et pécheurs".14 Le refoulement des souvenirs du passé pré-communiste était, dans l’ex-Yougo- slavie, suivi d’un énergique effort d’imprimer les souvenirs de la lutte des partisans et des mérites des communistes et de leurs chefs et héros pendant la Deuxième Guerre mondiale et la reconstruction socialiste ultérieure. Avec la chute du communisme, le boomerang historique s’est retourné – les contenus refoulés de la mémoire nationale ont à nouveau ressurgi à la surface, certains avec la force et la violence caractéristiques des souvenirs éveillés et des émo- tions jusque là durement réprimées et étouffées."L’Europe tout entière a été témoin du fléau de rebaptisation des rues et des places dans les anciens pays socialistes et de la démolition des monuments odieux des leaders encore plus odieux" (Rihtman-Auguštin 2000: 37). .. . 4. .2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie En réalité, ce processus est semblable à celui qui avait eu lieu il y a cinquante ans, lorsque les communistes avaient pris le pouvoir, seulement cette fois dans le sens inverse. "Le fléau de rebaptisation" a commencé en Serbie dans les années 90, à l’époque où Slobodan Miloševi était au pouvoir, et s est poursuivi après l’année 2000 et les changements démocratiques survenus dans le pays. Ce processus se déployait principalement à travers les changements dans l’utilisation des symboles, très importante, dans la toponymie urbaine et les noms de villes, dans la rédaction de nouveaux manuels d’histoire et de géo- graphie, à travers l’introduction des fêtes et des symboles nationaux nouveaux ou le retour des anciens, et particulièrement, à travers la réhabilitation dans ses droits civiques des descendants de la famille royale des Karaorevi au- trefois bannie, le retour d’une partie des biens, ainsi que leur droit au retour au .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 187 pays et l’autorisation de prendre part à la vie protocolaire.15 À l’instar des autres pays en transition, la Serbie s’est tournée vers son passé national jus- qu’en 1945. Cependant, à la différence du régime communiste, le pouvoir démocratique actuel fait preuve de tolérance envers certains contenus de la période communiste et de la Yougoslavie de Tito, mais il les pousse sur les marges de la mémoire et de la mention publique. Une telle politique ne provoque généralement pas de malentendus chez les citoyens étant donné qu’après l’échec de l’idéologie communiste, il existe un consensus social sur le retour en vie des éléments et des symboles du passé pré-communiste. Quelquefois, des malentendus surviennent avec ceux des groupes de citoyens qui continuent à se sentir proches de l’idéologie commu- niste et le souvenir du système socialiste, et qui sont fâchés et vexés lorsque, par exemple, l’on rebaptise les rues appelées selon les héros de la Lutte pour la libération nationale ou, lorsque par un acte, on abolit le souvenir des évé- nements importants de la lutte partisane anti-fasciste et de la Yougoslavie de Tito.16 15 Les noms des rues à Belgrade ont été changés à plusieurs reprises dans les 15 dernières années. Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie En principe, la politique de changement des noms de rues consiste à revenir aux noms d’avant 1945, comme me l’a affirmé dans un entretien le professeur Ivan Kovaevi, président du Comité pour la dénomination des rues dans la période de 1997-2000, et comme l’a confirmé dans une interview le président actuel du Co- mité, Branko Beli. www.b92.net/info/vesti/index.php?yyyy=2006&mm=11&dd=30&nav_category=12& nav_id=221921 Sur l’analyse des contenus des manuels d’histoire en Serbie, Croatie et Bosnie et Herzégovine au cours et après la guerre civile en Yougoslavie, et l’analyse compara- tive des manuels historiques dans la période post-socialiste dans les pays de l’Europe du Sud-est, l’analyse effectuée par un groupe spécial composé d’historiens de plu- sieurs pays, voir le texte de l historienne dr Dubravka Stojanovi"Konstrukcija pro- šlosti : sluaj srpskih udbenika iz istorije", et l’interview de la même auteure "Širom otvorene oi", Vreme 779, decembar 2005, accessible sur : 16 De grandes polémiques ont été menées par exemple, au sujet de la suppression du nom Boulevard de l’AVNOJ à Nouvelle Belgrade et sa rebaptisation en Boulevard de Zorana inia (le premier ministre démocratique de Serbie, assassiné en 2003). En revanche, le pouvoir en Serbie a mis du temps, après la disparition de la Yougo- slavie, avant d’abolir la fête du 29. novembre – le jour où a été"créée" la Yougoslavie socialiste lors de la convention de l’AVNOJ (Conseil anti-fasciste de libération natio- nale de la Yougoslavie) à Jajce en 1943. La suppression des noms de rues et de places nommées d’après le Maréchal Tito a généralement été menée sans grandes protesta- tions, mais il n’en demeure pas moins qu’il y a quelques milliers de rues qui portent toujours ce nom. Il est intéressant de noter qu’un étrange compromis a été fait à Bel- Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 188 DRAGANA ANTONIJEVI Il est clair qu’à la fin de ce processus de réévaluation l’emportera le style de pensée de ceux qui représentent déjà la classe dirigeante ou le feront dans le futur, comme l’a si justement fait remarquer Mary Douglas, et que grâce à cette position ils vont prescrire le contenu de la mémoire et de l’oubli sociaux, c’est-à-dire qu’ils vont permettre "la reconnaissance" de la vérité dominante, ou mieux, "l’effet du vrai" – comme l’aurait dit Greimas. L’objectif du rema- niement de l’histoire n’est pas d’arriver à "une optique parfaitement plane. grade en 1991 après l’abolition du nom Rue du maréchal Tito, l’une des artères cen- trales de la ville. Elle devait son nom antérieur au roi Milan (Obrenovi) et pour cer- taines raisons le pouvoir d’alors n’a pas été enchanté de rebaptiser la rue par le nom de ce roi; alors, un nouveau nom, idéologiquement neutre, a été donné à la rue, celui de la – Rue des princes serbes (Srpskih vladara). Cela a soulevé des polémiques et des protestations de ceux qui plaidaient pour un retour conséquent des noms d’avant 1945. Ce n’est qu’en 1997 que le pouvoir municipal démocratique a rendu l’ancien nom à cette rue – Rue du roi Milan. Styles de pensée dans le système de communication perturbée: cas de la Serbie Le miroir, et c’est l’histoire, déforme autant qu’il le faisait avant. L’objectif du remaniement est que les déformations s’adaptent à l’esprit du temps présent" (Daglas 2001: 77). Dans la mesure où cela conviendra au temps présent – celui de la transition, et le futur – celui de l’après-transition, la vérité se for- mera à travers la reconnaissance de ceux des segments du passé collectif qui s’intégreront dans l’image souhaitée de la société. Position de l’"indiscrétion" et du "faux". Il est indubitable que les diffé- rentes formes d’indiscrétion – la divulgation du secret, la dénonciation, le cafardage, la curiosité excessive ou le bavardage – sont considérées comme une forme incorrecte et inappropriée de comportement social qui, en règle générale, provoque des malentendus et entraîne différentes réactions négatives – réprobation, colère, accusations, punitions, engouffrement dans l’oubli so- cial etc. Cette position est occupée par les communautés de pensée formelles et in- formelles dont l’activité publique est vécue comme une des formes d’excès de communication avec autrui évoquées ci-dessus, et cela au détriment, réel ou imaginaire, de sa société. Dans le cas de la transition, si ces "autres" sont des étrangers dont l’opinion et le jugement influent sur l’évaluation de la voie des réformes em- pruntée par la Serbie et sur le financement et d’autres formes d’aide et de soutien qui s’y rattachent, la rédaction des rapports empreints d’esprit critique "là où il faut" (Washington, Bruxelles) de la part de certaines organisations non-gouvernementales en Serbie, est alors vécue par une partie du public comme "cafardage", "mouchardage", "diffamation" de son propre peuple et le "travail de délation" pour des organisations étrangères "hostiles". Pour le .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 189 moins, leurs interventions dans l’espace public sont-elles vécues comme une instigation agressive à imposer leur propre opinion à l’encontre de l’opinion et du climat majoritaires. Si nous nous posons la question sur le rapport envers le passé de ces communautés de pensée, il est évident qu’il s’agit d’un passé tout récent – à partir de la guerre civile dans l’ex-Yougoslavie, puis l’époque de Slobodan Miloevi et la floraison des idéologies nationalistes et radicales, jusqu’au temps présent où sont apostrophés certaines instances du pouvoir, les partis politiques, les institutions et les personnes qui "ne se comportent pas comme il faut" d’après le jugement des membres de ces communautés de pensée "indiscrètes". 17 Sur les motifs des critiques et des attaques sur la présidente du Comité de Hel- sinki pour les droits de l’homme en Serbie en raison du rapport pour l’année 2007, ainsi que sur les arguments en faveur du Comité de Helsinki et de son activité, voir : http://www.helsinki.org.yu/serbian/hajka.html; http://www.danas.rs/vesti/politika/or ganizovana_hajka_stizu_i_pretnje.56.html?news_id=140477; http://www.nin.co.rs/ pages/article.php?id=40353; http://www.glas-javnosti.rs/clanak/drustvo/glas-javnosti- 21-09-2008/ciscenje-univerziteta; http://www.novosti.rs/code/navigate.php?Id=4& status=jedna&vest=129259&datum=2008-09-26 j 18 La liste des intellectuels"indésirables", selon la présidente du Comité de Helsin- ki, voir dans le texte de S. Antoni"ienje Univerziteta". Accessible sur: Styles de pensée dans le système de communication perturbée: cas de la Serbie Bien qu’elles affirment qu’elles le font dans l’intention de "redresser les injustices", de sauver de l’oubli les crimes commis au nom de la Serbie au cours de la guerre civile dans les années 90, d’attirer l’attention sur les per- sonnes dont l’engagement et l’écriture, à leurs yeux, sont nationalistes et par conséquent indésirables, ou bien sur les institutions qui n’ont pas encore commencé les réformes souhaitées, la manière et le contenu de leurs interven- tions, souvent radicales, auprès du public serbe et étranger, irrite bien des gens, sans pour autant bénéficier du soutien et de la sympathie importants auprès des institutions officielles. Les conséquences de ces actions pour les membres des communautés de pensée "indiscrètes" peuvent être plus que désagréables – une partie du public leur répond par des paroles agressives et offensantes, des menaces, et même par des agressions physiques. La dernière polémique violente qui a été menée autour de ces rapports en Serbie, a eu lieu en automne 2008 après la publication du rapport du Comité de Helsinki pour les droits de l’homme pour l’année 2007 sous le titre"Auto- isolation: réalité et objectif", publié en mai 2008.17 Les principales critiques et accusations sur le compte de la présidente du Comité de Helsinki S. Biserko concernent le fait que dans le rapport ont été publiés les noms d’une soixan- taine de professeurs d’université, d’écrivains et de journalistes qui, selon ce rapport, sont "de droite et créent un esprit anti-occidental, anti-européen et attisent des idées nationalistes parmi les jeunes en Serbie" (Glas javnosti 21. 09. 2008),18 ce que les apostrophés ont compris comme une dénonciation de Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 190 DRAGANA ANTONIJEVI leur travail et le "lynchage" de leur droit de penser comme ils pensent, signa- lant au public qu’une telle activité de la présidente du Comité de Helsinki leur rappelle irrésistiblement la pratique communiste de "sanction pour délit ver- bal". "Tous nos rapports dans les huit, neuf dernières années ressemblent à celui-ci et sont concentrés de la même manière sur les personnalités et les institutions dans l’idée de présenter le contexte politique qui rend impossible le progrès de la Serbie... .. . 4. .2 (2009) http://www.glas-javnosti.rs/clanak/drustvo/glas-javnosti-21-09-2008/ciscenje- univerziteta 19 Source: http://bs.wikipedia.org/wiki/Jugonostalgija .. . 4. .2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie .2 (2009 191 A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS l’idéologie communiste et au socialisme, ce pourquoi dans la perspective de la transition, la vie dans l’ex-Yougoslavie leur apparaît comme "l’époque la plus heureuse" et le système social "le plus juste", ce qui leur fait regretter l’échec de son idéologie. Yougonostalgiques sont aussi, de temps en temps, certains citoyens d’âge mûr qui regrettent simplement leur jeunesse, toujours belle vue d’un point de vue ultérieur dans la vie, et comme ils l’ont passée dans l’ex- Yougoslavie, que ce soit à tort ou à raison, ils l’analysent sous un jour positif. Enfin, il y a également ceux qui sont attachés à certaines formes de culture et de loisirs populaires yougoslaves (musique, films, séries télévisées, concerts et festivals, production littéraire, vacances d’été, voyages etc.) gravées dans leur mémoire et regrettent la perte de certaines valeurs socio-culturelles et expériences vécues dans la patrie commune. Certains des membres de cette communauté de pensée se relient à des ni- veaux officieux différents, à travers la coopération culturelle et artistique – par exemple Le Lexique de la mythologie yougoslave (Leksikon Yu mitologije)20, à travers la célébration informelle des dates importantes de la Yougoslavie de Tito, des visites à la maison de Tito à Kumrovec (Croatie) et à la Maison des fleurs à Belgrade où se trouve la tombe de Tito, à travers la participation dans la "Yougoslavie virtuelle" sur des sites Internet21 etc. Il ne s’agit manifestement pas de contenus oubliés, mais ils perdent de leur importance et de leur pouvoir dans la formation des représentations collecti- ves dans la Serbie actuelle. Ils n’ont pas d’influence considérable dans la vie publique, bien que l’on envisage leur discours avec une certaine dose de tolé- rance. Les malentendus qu’un tel style de pensée peut provoquer concernent les groupes ou les individus de la vie publique qui avec des émotions négati- ves prononcées jugent la vie dans l’ex-Yougoslavie et qui pour cette raison n’éprouvent pas de sympathie envers les "yougonostalgiques", ou ne veulent plus se souvenir de la Yougoslavie et toute évocation superflue de ce pays disparu les dérange. Dans la "modalité véridictoire" c’est le discours de la nostalgie qui corres- pond à la position du secret. Styles de pensée dans le système de communication perturbée: cas de la Serbie Nous avons, dans notre rapport, détecté cette ten- dance anti-européenne" – explique la présidente du Comité de Helsinki, men- tionnant qu’elle est pour cette raison exposée à des insultes et des menaces de la part des particuliers et des groupes de droite, et qu’une véritable chasse aux sorcières médiatique est organisée à son encontre (Danas 25.09.2008). La position structurale des styles de pensée indiscrets, au côté opposé du vrai, démontre que leur effort d’imposer leur opinion et de fuir l’oubli (en tant que forme de sanction) est relativement important, et leur influence éven- tuelle sur le public des plus faibles, étant donné que l’axe antithétique est con- sidéré au sens structural comme "vidé’ – comme une double négation des sèmes positifs supérieurs. Il s’agit donc de styles de pensée indiscrets qui ont le minimum de traits souhaitables d’identification, ce pourquoi ils sont consi- dérés comme faux. Bien qu’il s’agisse de groupes d’influence relativement marginale sur les événements sociaux généraux, le malentendu et l’aversion qu’ils inspirent dans l’opinion publique ne sont pas négligeables. Position de la "nostalgie" et du "secret". À cette position, j’évoquerais ceux des récits et des discours qui sont désignés par le nom commun de you- gonostalgie qui, elle, "sert à qualifier un phénomène social dans les pays cons- titués suite à la dislocation de l’ex-Yougoslavie, ce qui en fait une notion so- ciologique assez récente. Elle désigne principalement l’attitude qui idéalise la complète situation économique, culturelle et sécuritaire dans la Yougoslavie socialiste dans la période de 1945 jusqu’en 1991. La yougonostalgie est géné- ralement limitée à des particuliers ou des groupes relativement restreints qui ont passé la plus grande partie de leur vie dans l’ex-Yougoslavie, et consi- dèrent cette période comme positive et heureuse par rapport à l’époque de la guerre et de l’insécurité économique, survenue après 1991."19 Ce style de pensée existe chez des groupes assez hétéroclites dans des me- sures, intensités, significations différentes. D’une part, s’y trouvent tous ceux qui se considèrent comme des perdants existentiels en raison de la dislocation de l’état commun ou regrettent d’une autre manière "les histoires de vie gom- mées". Puis, il y a ceux qui sont encore rattachés au souvenir de Tito, à .. . 4. 20 Voir le site : www.leksikon-yu-mitologije.net 21 Voir par ex. les sites: www.miniyu.org.yu/;www.slobodnajugoslavija.com/ 20 Voir le site : www.leksikon-yu-mitologije.net 21 Voir par ex. les sites: www.miniyu.org.yu/;www.slobodnajugoslavija.com/ Styles de pensée dans le système de communication perturbée: cas de la Serbie Il s’agit d’une position instable car elle est placée structurellement sur l’axe de déixis, ce qui signifie qu’elle oscille entre les concepts stables du "vrai" et du "faux" (c’est-à-dire, les positions du maxi- mum et du minimum des traits d’identification souhaitables). C’est le sort des idéologies "détrônées", des concepts dépassés et des symboles supprimés qui continuent à vivre comme des idées cachées, marginalisées et refoulées. Le sort réservé à ce style de pensée est très probablement de sombrer dans l’oubli avec la disparition biologique des yougonostalgiques, subsistant encore dans les souvenirs et les récits de leurs descendants comme une sorte de mu- Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 192 DRAGANA ANTONIJEVI sée mental, ou à travers les objets matériels dans les musées historiques et ethnographiques, les films et les cinémathéques ou sur les DVD, les chansons sur des disques ou des CD, etc. Leur influence subversive est cependant pos- sible sur des représentations collectives déjà fixées, se frayant le chemin pour relancer dans l’avenir certains de ses contenus comme des valeurs que nous n’allons pas désavouer ou en avoir honte seulement parce qu’elles portent le préfixe "yougo". "Notre nostalgie est une manière informelle de commenter et de donner un sens à l’histoire, qui révèle nos désirs de changements sociaux" (Shircliffe 2001: 62). Position de l’"obscurité" et du "mensonger". Bien des exemples pour- raient être cités pour la forme du confus et de l’obscur dans la communication publique, ce qui laisse de la place pour différentes "réécritures", interpréta- tions erronées, d’où naissent des malentendus dans la société. Il n’est pas rare que l’énoncé obscur soit considéré comme mensonger, c’est-à-dire celui par lequel pour des raisons quelconques l’on dissimule volontairement la vérité. Les acteurs politiques, enclins aux manipulations, utilisent souvent l’obscurité comme stratégie afin d’éviter des réponses franches et honnêtes ("je ne sais pas", "je n’ai pas été informé", "je préfère ne pas en parler" etc). Les discours publiques obscurs peuvent avoir des conséquences négatives pour la société, faisant naître chez les citoyens le doute, la confusion, le sentiment d’être trompés et bernés, mais également une base pour le foisonnement de mythes et de rumeurs politiques. C’est un cas de figure assez fréquent lorsque les pouvoirs et les systèmes se succèdent, lorsqu’on veut décréter secret d’état ou d’une autre manière dissimuler la vérité, et nous pouvons en trouver des exemples dans presque toutes les sociétés. 22 Sur la recherche de la tombe de Draa Mihailovi et les différentes réactions voir par exemple sur: http://www.politika.rs/rubrike/Drustvo/Streljanje-Draze-vishe-nije- drzavna-tajna.lt.html; http://www.politika.rs/rubrike/Drustvo/Tajna-groba-Draze-Mihai- lovica.lt.html; http://www.politika.rs/rubrike/Drustvo/Tajne-sluzbe-ne-znaju-gde-je- .. . 4. .2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie J’ai choisi, pour l’illustrer, un exemple de dissimulation délibérée de la vérité dans le règlement de comptes des idéologies, ce qui a résulté par un grand mensonge social subsistant avec tenacité jusqu’à nos jours et provoquant des malentendus et des conflits dans une partie du public en Serbie. En effet, l’actuel gouvernement de Serbie a formé une Commission d’État à laquelle il a confié la tâche d’établir la vérité sur les peines capitales exécutées sans procès dans la période de 1944 à 1946. La plus importante parmi ces vérités à découvrir reste l’élucidation des circonstances dans les- quelles la peine capitale a été exécutée sur Dragoljub Draa Mihailovi, leader des Tchétniks et commandant en chef de l’Armée yougoslave dans le pays, ainsi que la découverte de sa sépulture et de sa dépouille.22 Le pouvoir com- .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 193 muniste l’a fusillé de nuit, en cachette, le 17 juillet 1946, en tant qu’"ennemi du peuple"; soixante-trois ans plus tard le public n’a toujours pas été informé de l’endroit de son exécution ni de l’emplacement de sa sépulture. Actuellement, 95% des documents qui dévoilent la terrible vérité sur ces exécutions ont été retrouvés dans les archives, mais ce n’est pas le cas de ceux qui concernent Draa Mihailovi, ce qui fait naître la conviction chez les enquê- teurs de la Commission du Gouvernement que c’est précisément l’emplacement de sa sépulture qu’il s’agissait pour les communistes de garder dans le secret le plus absolu. Aujourd’hui, les exécuteurs ne sont plus au nombre des vivants, et ils "sont restés redevables au public serbe l’explication pourquoi cet emplace- ment reste le secret d’état le mieux gardé... Comme cela arrive souvent, dans l’absence de versions officielles, sur cette mort et le lieu d’enterrement se tissent de nombreuses histoires, presque fantastiques, et des informations ’confiden- tielles’ se transmettent d’homme à homme" (Politika on-line, 24.03.2009). ( ) Le pouvoir démocratique de Serbie a, en formant cette Commission, satisfait à de nombreuses demandes des membres et des sympathisants du mouvement tchétnik et de leurs descendants en Serbie et dans la diaspora, ainsi qu’aux fa- milles de ceux qui ont péri dans des purges communistes dans les premières années d’après guerre, pour que leurs parents péris aient le droit à la réhabilita- tion et eux mêmes, enfin – le droit à la vérité. Drazin-grob.sr.html; http://www.politika.rs/rubrike/Drustvo/SUBNOR-protiv-rehabilita- cije-Draze-Mihailovica.lt.html; http://www.politika.rs/rubrike/Drustvo/Za-i-protiv-isti- ne-o-Drazi.lt.html; http://www.politika.rs/rubrike/Drustvo/Homen-Tuzilashtvo-da-trazi- Drazin-grob.lt.html; http://www.b92.net/info/vesti/index.php?yyyy=2009&mm=07 &dd=18&nav_ category=12&nav_id=371907 Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) Styles de pensée dans le système de communication perturbée: cas de la Serbie D’un autre côté, cette enquête a déclenché un violent tollé de certains groupes de citoyens qui considèrent un tel acte injustifié et offensant pour tous ceux qui ont combattu, comme les parti- sans de Tito, contre les tchétniks ou ont été leurs victimes dans la Deuxième Guerre mondiale. Ceux qui considèrent que de tels ou de semblables sujets re- présentent une charge inutile pour "le présent et le passé du peuple et de l’État" se sont aussi adressés au public. Ces groupements de citoyens, qui représentent à eux seuls une véritable communauté de pensée, ne sont manifestement pas dérangés par le mensonge et l’injustice infligée aux victimes et à leurs familles, probablement de peur qu’ainsi ne soient découvertes de bien pires vérités et ne soit déclenchée une nouvelle avalanche d’accusations qui pourrait déstabiliser la société. Cependant, la revendication des descendants des familles de ceux qui ont péri dans des purges communistes est aussi légitime que toute autre reven- dication de toutes autres vérités lorsqu’il est question de crimes massifs commis au nom d’une idéologie quelconque. g q q Le passé flou et embrouillé représente, donc, quelque chose que les uns aimeraient oublier à tout prix, et d’autres clarifier et aboutir à la vérité. Peut- Drazin-grob.sr.html; http://www.politika.rs/rubrike/Drustvo/SUBNOR-protiv-rehabilita- cije-Draze-Mihailovica.lt.html; http://www.politika.rs/rubrike/Drustvo/Za-i-protiv-isti- ne-o-Drazi.lt.html; http://www.politika.rs/rubrike/Drustvo/Homen-Tuzilashtvo-da-trazi- Drazin-grob.lt.html; http://www.b92.net/info/vesti/index.php?yyyy=2009&mm=07 &dd=18&nav_ category=12&nav_id=371907 Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 194 DRAGANA ANTONIJEVI être faudrait-il, pour finir, citer le commentaire d’un des membres de la Commission comme une illustration du sens que prend la tentative d’abolir le mensonge et le secret: "Notre objectif est de clarifier les imprécisions, sans aucune intention d’accuser ou disqualifier quiconque, ou de laisser l’emporter un quelconque fanatisme sur la vérité historique, pour qu’une telle démarche puisse nous acheminer vers un meilleur avenir de notre descendance, sans plus avoir de comptes à régler" (Politika on-line, 24.03.2009).23 La position structurale de l’obscur et du mensonger se trouve sur la déixis, ce qui rend cette position instable, et en outre sémantiquement très négative- ment marquée, tendant à ce que l’énoncé ou l’événement obscurs soient clari- fiés, le secret dévoilé, le mensonge démasqué, les doutes et les imprécisions levés pour que la situation puisse devenir stable du point de vue cognitif et axiologique – vraie ou fausse. 23 Accessible sur: http://www.politika.rs/rubrike/Drustvo/Za-i-protiv-istine-o- Drazi.lt.html 4 2 (2009) 23 Accessible sur: http://www.politika.rs/rubrike/Drustvo/Za-i-protiv-istine-o- Drazi.lt.html .. . 4. .2 (2009) Références: Antonijevi, Dragana. 1991. Znaenje srpskih bajki. Beograd: Etnografski institut SANU, Posebna izdanja knj. 33. j j Antonijevi, Dragana. 2007a. Karaore i Milo: Izmeu istorije i predan- ja, Srpski genealoški centar, Etnološka biblioteka, knj. 32, Beograd. Antonijevi, Dragana. 2007b. Karaore i Milo: Mit i politika. Srpski genealoški centar, Etnološka biblioteka, knj. 33, Beograd. Antonijevi Dragana. 2009. Okviri prouavanja linih i porodinih pria o materijalnom gubitku i porazu. Etnoantropološki problemi. n.s. 4 (1) : 13-35. de Certeau, Michel. 1984. The Practice of Everyday Life. University of California Press. Courtés, Joseph. 1976. Introduction à la sémiotique narrative et discur- sive. Paris: Hachette. Daglas, Meri. 2001. Kako institucije misle. Beograd: Samizdat B92. eri, Gordana. 2009. Drutveno pamenje i primenjena kritika: O pretva- ranju poezije u ideološku batinu. Etnoantropološki problemi, n.s. 4 (1) : 17-85. Ferrara, Fernando. 1974. Theory and Model for the Structural Analysis of Fiction. New Literary History, Changing Views of Character, 5 (2): 245-268. Ferrara, Fernando. 1974. Theory and Model for the Structural Analysis of Fiction. New Literary History, Changing Views of Character, 5 (2): 245-268. Greimas, A. J. 1989a. On Meaning. New Literary History, Greimassian Semiotics, 20 (3): 539-550. y y g g ( ) Greimas, A. J. 1989a. On Meaning. New Literary History, Greimassian Semiotics, 20 (3): 539-550. Greimas, A.J. 1989b. The Veridiction Contract. New Literary History, Greimassian Semiotics 20 (3): 651-660. Greimas. A. J. and J. Courtés. 1976. The Cognitive Dimension of Narra- Greimas. A. J. and J. Courtés. 1976. The Cognitive Dimension of Narra- tive Discourse. New Literary History, Thinking in the Arts, Sciences, and Literature 7 (3) : 433-447 tive Discourse. New Literary History, Thinking in the Arts, Sciences, and Literature 7 (3) : 433 447 Literature, 7 (3) : 433-447. Konerton, Pol. 2002. Kako društva pamte. Beograd: Samizdat B 92. Lévi-Strauss, Claude. 1973. Anthropologie structurale deux. Paris: Plon. Lévi-Strauss, Claude. 1983. Le regard éloigné. Paris: Plon. Š Konerton, Pol. 2002. Kako društva pamte. Beograd: Samizdat B 92. Konerton, Pol. 2002. Kako društva pamte. Beograd: Samizdat B 92. Lévi-Strauss, Claude. 1973. Anthropologie structurale deux. Paris: Plo Lévi-Strauss, Claude. 1983. Le regard éloigné. Paris: Plon. Š Lévi-Strauss, Claude. 1973. Anthropologie structurale deux. Paris: Plon. Lévi-Strauss, Claude. 1983. Le regard éloigné. Paris: Plon. Lévi-Strauss, Claude. 1988. Strukturalna antropologija 2. Zagreb: Školska knjiga. Miloevi-orevi, Nada. 2000. Seoba i srpska kulturnoistorijska predan- ja. Od bajke do izreke. Beograd: Društvo za srpski jezik i književnost, biblio- teka "Književnost i jezik" knj. Aperçu critique Mon point de départ a été le champ sémantique de l’oubli de Lévi-Strauss, ce qui m’a ensuite permis de concevoir mon propre champ de communication perturbée. En le croisant avec la modalité véridictoire de Greimas, je me suis efforcée de montrer, sur des exemples choisis de styles de pensée, comment se comportent les phénomènes de communication perturbée dans la société de transition, comment ils influent sur la formation de l’identité sociale, de la mémoire et de l’oubli, puis quelles peuvent être les conséquences pour la so- ciété compte tenu des malentendus nés en raison d’une communication per- turbée. La société se protège des malentendus de plusieurs manières: elle neu- tralise les styles de pensée en conflit en donnant à l’un d’entre eux l’avantage et en le promouvant en discours officiel, ou bien elle les pousse dans l’oubli ou sur les marges de la vie sociale en tant que formes de pensée et de compor- tement erronés et dépassées, ou encore, elle nie et ignore leur existence "en mettant la tête dans le sable". La pire des variantes est, sans aucun doute, que la société "soit paralysée" au niveau des malentendus chroniques qu’elle ne parvient pas à résoudre, tout comme Asdiwal s’est littéralement pétrifié dans une des versions Tshimshian. .. . 4. .2 (2009) A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 195 Références: 7, 140-150. j j j Rihtman-Auguštin, Dunja. 2000. Ulice moga grada. Beograd: XX vek. Rot, Klaus. 2000. Slike u glavama. Beograd: XX vek. j j j Rihtman-Auguštin, Dunja. 2000. Ulice moga grada. Beograd: XX vek. Rot, Klaus. 2000. Slike u glavama. Beograd: XX vek. Shircliffe, Barbara. 2001. "We Got the best of that World". A Case for thr Study of Nostalgia in the Oral History of School Segregation. Oral History Review, 28 (2): 59-84. Vernant, Jean-Pierre. 1982. Mythe et pensée chez les Grecs, I. Paris: PCM. Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009) 196 DRAGANA ANTONIJEVI Dragana Antonijevi Povodom Levi-Strosovog koncepta Zaborava. Struktura poremeene komunikacije i stilovi miljenja u tranzicijskoj Srbiji .. . 4. .2 (2009) Povodom Levi-Strosovog koncepta Zaborava. Struktura poremeene komunikacije i stilovi miljenja u tranzicijskoj Srbiji Povodom Levi-Strosovog koncepta Zaborava. Struktura poremeene komunikacije i stilovi miljenja u tranzicijskoj Srbiji U izlaganju se polazi od Levi-Strosovog koncepta semantike zaborava koji ine zaborav, nesporazum, indiskrecija i nostalgija. Analizirajui indijanske i grke mitove, Levi-Stros dolazi do zakljuka da "semantiko polje zaborava" ima vano znaenje koje se, pre svega, tie normativne funkcije i uspostavl- janja društveno-kulturnih pravila i rituala. Kroz uvoenje nove sintagme poremeena komunikacija i termina nedoreenost, autorka nudi korekciju Levi-Strosovog koncepta i razmatra semantike i kognitivne implikacije poj- mova ukljuenih u sistem poremeene komunikacije, take gledita koje se stvaraju spram razliitih diskursa i pitanje njihove verodostojnosti. U radu se, dalje, razmatra period tranzicije koji predstavlja nestabilno vreme u kome drutvo pregovara o znaenjima i u kome se nadmeu razliiti stilovi mišljenja u nameri da utiu na kolektivne procese zaborava i seanja. Polje njihove komunikacije moe se posmatrati kao polje "poremeene komu- nikacije" koje, u krajnjoj konsekvenci, ima funkciju da utemelji i normira stavove i predstave koji se tiu kolektivnog identiteta i nacionalne prolosti i budunosti. Konvergencijom dvaju modela – poremeene komunikacije i Gremasove strukture modaliteta istinitosti, razmatraju se ilustrativni primeri poremeene komunikacije u tranzicijskoj Srbiji s ciljem da se odredi njihova strukturalna, kognitivna i komunikacijska pozicija u kreiranju mape seanja graana, kao i njihov potencijal da nametnu svoja gledita u javnosti. Kljune rei: Levi-Strosovov koncept zaborava, poremeena komunikaci- ja, stilovi i zajednice mišljenja, tranzicija, društevno-normativna funkcija se- mantike zaborava A PROPOS DU CONCEPT DE MOTIF DE L’OUBLI DE LÉVI-STRAUSS 197 Dragana Antonijevi Dragana Antonijevi In regard to Levi-Strauss’s "motive of oblivion". Structure of disturbed communication and styles of thoughts in transitory Serbia In regard to Levi-Strauss’s "motive of oblivion". Structure of disturbed communication and styles of thoughts in transitory Serbia This paper starts with Levi-Strauss’s semantic concept that consists of ob- livion, misunderstanding, indiscretion and nostalgia. Through his analysis of North American and Greek myths, Levi-Strauss concluded that "semantic filed of oblivion" has an important meaning especially in the construction of particular rules and rituals; or, in other words, it has an important part in the introduction of culture to the nature of cognitive and social processes. After the introduction of concept Structure of disturbed communication and some corrections to the Levi-Strauss’s concept, I start with the proposi- tion that the period of transition represents unstable and ‘slippery’ time in which society negotiates different meanings. That is the time when different styles of thoughts, represented by different and powerful groups that have an impact on current social, political and ideological processes, compete with each other fighting for supremacy. Their field of communication can be seen as a field of "disturbed communication", which in the final instance has a normative function: to reinforce and regulate certain attitudes, ideas and knowledge. This is achieved through the narratives which symbolise a com- munity of newly established order in the moment of its supposed socio- historical stabilisation. Finally, combining two different theoretical models – Levi-Strauss’s one described above with Greimas’s ideas about structures of modes of veridic- tion, this paper predicts chances of particular paradigmatic forms of thought in transitory Serbia to became dominant modes of thought, despite of their cur- rent low visibility in the public sphere. In mythical terms, it seems though that their domineering efforts are predetermined to success or fail, since they posi- tion themselves according to the laws immanent to these structures them- selves, which on their part a priori position these structures as powerful or powerless, influential or non-influential communities of thought. Key words: Levi-Strauss’s "motive of oblivion", structure of disturbed communication, styles and communities of thoughts, modes of veridiction, transition, socio-normative function of the semantics of the oblivion Problèmes d’ethnologie et d’anthropologie n.s. vol. 4. is. 2 (2009)
https://openalex.org/W2997391801	https://www.researchsquare.com/article/rs-1496/v2.pdf?c=1585619079000	English	null	De novo sequencing of the transcriptome reveals regulators of the floral transition in Fargesia macclureana (Poaceae)	BMC genomics	2,019	cc-by	11,070	De novo sequencing of the transcriptome of Fargesia macclureana (Poaceae) reveals regulators of the floral transition and ecological adaptations to high altitude De novo sequencing of the transcriptome of Fargesia macclureana (Poaceae) reveals regulators of the floral transition and ecological adaptations to high altitude Ying Li International Center for Bamboo and Rattan h Chunxia Zhang Nanjing Forestry University Kebin Yang International Center for Bamboo and Rattan Jingjing Shi International Center for Bamboo and Rattan Yulong Ding Nanjing Forestry University Zhimin Gao (  gaozhimin@icbr.ac.cn ) https://orcid.org/0000-0003-4464-7159 Research article Keywords: Transcriptome, Floral transition, Bamboo, Qinghai–Tibet Plateau Posted Date: October 3rd, 2019 DOI: https://doi.org/10.21203/rs.2.10521/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on December 30th, 2019. See the published version at https://doi.org/10.1186/s12864-019-6418-2. Page 1/25 Abstract Background Fargesia macclureana (Poaceae) is a woody bamboo species found on the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3800 m above sea level. It rarely blossoms in the QTP, but it flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. To date, little is known regarding the molecular mechanism of bamboo flowering, and no studies of flowering have been conducted on wild bamboo plants growing in extreme environments. Here, we report the first de novo transcriptome sequence for F. macclureana to investigated the putative mechanisms underlying the flowering time control used by F. macclureana to adapt to its environment. Results Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, assembled into 99,056 unigenes. A comprehensive analysis of the broadly, specifically and differentially expressed unigenes (BEUs, SEUs, and DEUs) and a weighted gene co- expression network analysis (WGCNA) revealed that changes in expressions of unigenes related to the circadian cycle may account for the differences in the floral transition of F. macclureana after being transplanted from the QTP to a laboratory outside. In addition, there were differences in active carbohydrate metabolism and signal transduction between the flowering and non-flowering plants. Moreover, we detected the expression of unigenes related to DNA repair and plant-pathogen interactions, which may be of adaptive importance. Finally, we detected 9,296 simple sequence repeats (SSRs) that may be useful for further molecular marker-assisted breeding. Conclusions F. macclureana may have evolved specific reproductive strategies for flowering-related pathways in response to photoperiodic cues to ensure long vegetation growing period. Our findings will provide new insights to future investigations into the mechanisms of flowering time control and adaptive evolution in plants growing at high altitudes. Background However, samples collected in these analyses were limited to mature spikelets or to different spikelets at different development stages. Thus, it is likely that dynamic changes in genes occurring at different development stages may be missing. In addition, the specific response of particular tissues to internal and external cues and how plants integrate these signals to regulate different phases of reproductive development (including the floral transition, florigen transport, and floral organ specification) has not yet been elucidated in bamboo. Furthermore, no studies of flowering have been conducted on wild bamboo plants growing in extreme environments. Here, we took advantage of an unexpected flowering event in highland arrow bamboo, Fargesia macclureana [25], and performed the first de novo transcriptome analysis. This transcriptome includes data from six different tissues collected at different development stages, including inflorescences in the initial and peak flower stage (I- and P- spikelets), branchlets, and leaves from both flowering and non-flowering bamboo plants (F/NF-branchlets and F/NF -leaves). F. macclureana is a woody bamboo species found in areas 2,000 ~ 3,800 m above sea level on the Qinghai–Tibet Plateau (QTP) (Fig. 1), which is the highest and largest plateau in the world. The growth environment of the QTP is characterized by low temperature and low oxygen availability, reduced pathogen incidence, and intense radiation [26]. F. macclureana rarely blossoms in the QTP, but it flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. Our goal is to use the transcriptomic data to gain a deeper understanding of the mechanisms underlying the control of flowering time and the adaptation of F. macclureana to the complex extreme conditions of the QTP. On one hand, we expect to detect regulatory hubs involved in the flowering mechanisms. On the other hand, we aim to discover signs of the adaptive evolutionary changes in F. macclureana in response to the harsh environmental conditions in the QTP, which may, in turn, provide a broader insight into the adaptive mechanisms for plants that grow at high altitudes. Background The flowering time is of crucial importance to ensure the reproductive success of flowering plants. Previous results have indicated that the floral transition is orchestrated by several parallel and interactive genetic pathways that are regulated by a variety of environmental and endogenous signals [1]. Many key genes and regulatory networks have been identified in herbaceous annual plants such as Arabidopsis [2, 3], rice [4], gourds [5], potato [6] and sorghum [7]. However, much less is known about such regulation in perennial plants. Despite the increasing attention on perennial dicotyledonous woody plants such as poplar [8, 9], eucalyptus [10] and citrus [11] species, to date, the molecular mechanism underlying floral regulation in monocotyledonous woody plants remains elusive. Furthermore, previous studies investigated flowering mainly by artificially altering the external signals (e.g. photoperiod and light intensity) and did not assess the impact of the original environment on the adaptive evolution of species-specific reproductive strategies. Bamboo plants are an important group in the Bambusoideae subfamily of the monocotyledonous Poaceae. They exhibit a wide degree of variation in the timing (1-120 years) and nature (sporadic vs. gregarious) of flowering among species [12]. Sporadic flowering involves flowering in only a few isolated clumps, which set little or no seed and usually remain alive afterward [13]. In contrast, gregarious flowering involves all individuals of a species regardless of age and/or location within and among the populations at the same time, which is usually followed by death and seed setting [14]. And the simultaneous death of many individuals triggers serious ecological consequences, including changes in the population dynamics of neighboring plants, differences in soil properties, various effects on endangered animals that depend on bamboo [15], and the Page 2/25 Page 2/25 Page 2/25 knock-on effects on human economies in many parts of the world [16]. Therefore, dissecting the regulators that control the unique life history of bamboo may be of use for plant ecology and human society. However, to date, little is known regarding the molecular mechanisms of bamboo flowering, in part because of the sporadic occurrence of these flowering episodes and the long intervals between events. Many genes have been identified as regulators of reproductive development in different bamboo species, including the MADS-box transcription factors [17-19], CONSTANS (CO) [20] and FLOWERING LOCUS T (FT) [21], among others. In addition, studies of sequenced transcriptomes have identified microRNAs related to floral development [22-24]. De novo transcriptome assembly yielded 99,056 unigenes Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, including 471,537,304 clean reads in 18 unique samples (Additional file 1: Table S1). The average Q20 (sequencing error rate less than 1%) and Q30 (sequencing error rate less than 0.1%) percentages were 100.00% and 89.95% respectively. The GC content of all samples ranged from 53.78% to 55.86%, with an average of 54.81%. Sample data were assembled into 289,122 transcript scaffolds, with an N50 and average length of 1,765 bp and 1,183 bp, respectively. The final de novo assembly included 99,056 unigenes, with an N50 and average length of Page 3/25 Page 3/25 1,587 bp and 926 bp, respectively. Among these unigenes, 71.02% (70,354) were shorter than 1,000 bp and 12.06% (11,950) were longer than 2,000 bp (Table 1). Most unigenes were functionally annotated and classified A total of 47,306 unigenes were annotated (Additional file 2: Table S2). Of these, 45,516 (96.22%) unigenes were found to encode products that showed significant similarity to characterized proteins in the non-redundant protein sequence database (Nr) at an E-value threshold of 10-5 (Table 2). We also found that 7,027 (15.45 %) unigenes showed similarity to genes found in rice, 11.33% were similar to those found in Brachypodium distachyon, and we also found a significant proportion of the unigenes that were similar to those found in Setaria italica, Oryza brachyantha, and Zea mays (Fig. 2a). We identified 24,847 (52.52%), 28,317 (59.86%) and 43,909 (92.82%) unigenes that showed significant matches to entries in the Swiss-Prot, Pfam, and eggnog databases, respectively (Table 2). Many unigenes expressed in the F. macclureana transcriptome were functionally annotated as regulators of plant responses to evolutionarily important phenotypes, including membrane stabilization, heat stress response and pathogen defense (Additional file 2: Table S2). Functional annotation indicated that many unigenes were involved in metabolism and genetic information We were able to annotate 13,128 unigenes (27.75% of the total) in 25 different categories of the COG (clusters of orthologous groups) classification database (Fig. 2b). Of these, the cluster for “General function prediction only” (3,277, representing 24.96% of the 13,128 unigenes annotated by this database) was the largest group, followed by “Replication, recombination and repair” (2,202, 16.77%), “Transcription” (1,571, 11.97%), and “Translation, ribosomal structure and biogenesis” (1,429, 10.88%). The “Signal transduction mechanisms”, “posttranslational modification, protein turnover, chaperones”, “carbohydrate and amino acid transport and metabolism” and “transport and metabolism” categories also contained a significant proportion of the annotated unigenes. GO enrichment analysis indicated that these predicted unigenes were categorized into three main categories— i.e. biological process (BP), cellular component (CC), and molecular function (MF). As shown in Fig. 2c, for unigenes that were enriched in the BP category, they were mainly involved in biological processes related to reproduction, posttranslational modification and signal transduction; as for those in the CC category, they were mainly involved in cellular components related to membrane, ubiquitin ligase complex, mitochondrion, chloroplast and etc.; while for those in the MF category, they were mainly involved in molecular functions related to signaling transduction (e.g. “ATP binding”, “zinc ion binding”, “protein kinase activity”, and etc.) (Additional file 3: Table S3). chloroplast and etc.; while for those in the MF category, they were mainly involved in molecular functions related to signaling transduction (e.g. “ATP binding”, “zinc ion binding”, “protein kinase activity”, and etc.) (Additional file 3: Table S3). We also mapped 14,307 unigenes (representing 30.24% of the total) to six different KEGG subsystems, including metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. As shown in Fig. 3, the majority of these unigenes (7,922, representing 66.17% of the 14,307 unigenes classified using KEGG annotations) were assigned to metabolic pathways, including carbohydrate metabolism, energy metabolism, and others. In addition, 4,024 unigenes (28.13%) were assigned to genetic information processing, including transcription, translation, and folding, and 474 unigenes (3.31%) were found to be related to membrane transport and signal transduction. We also found 707 genes Page 4/25 Page 4/25 (4.94%) that were related to transport and catabolism and 377 genes (2.64%) related to environmental adaptation. Most BEUs were involved in genetic information processing, environmental adaptation and signal transduction As shown in the Venn diagram (Fig. 4a), we found nearly equal numbers of unigenes that were broadly and specifically expressed in I-spikelets, P-spikelets, F-branchlets, and F-leaves. COG analysis indicated that most BEUs were clustered in signal transduction mechanisms (T), replication, recombination and repair (L), and transcription (K), besides general function prediction only (R). GO enrichment analysis for these BEUs indicated that they were also mainly involved in reproduction, environmental adaptation and signal transduction, which was largely similar with that for all predicted unigenes (Additional file 4: Table S4-a). KEGG enrichment analysis also indicated that these BEUs were mainly enriched in pathways related to environmental adaptation (including circadian rhythm, endocytosis, and plant-pathogen interactions), signal transduction (including plant hormone signal transduction, phosphatidylinositol signaling system, and inositol phosphate metabolism) and genetic information processing (including spliceosome, mRNA surveillance, and RNA transport and degradation; Additional file 4: Table S4-b). The SEUs were mostly involved in carbohydrate metabolism, energy metabolism, and environmental adaptation As shown in Fig. 4a, we identified 10,653 unigenes that were specifically expressed in spikelets, including 5,528 and 5,025 unigenes in I- and P-spikelets, respectively. We also found 9,067 and 7,437 unigenes that were specifically expressed in F-branchlets and F-leaves, respectively. COG annotation indicated that the distribution patterns of SEUs among the 26 terms were similar, with the number of SEUs within each term varying among the three tissues (Fig. 4b). The GO enrichment analysis indicated that these SEUs not only shared some common GO terms, but also had some particular ones. As shown in Fig. 4c and Additional file 4: Table S4-c, for those SEUs that were enriched in the BP category, they were broadly involved in several important biological processes, including “protein phosphorylation”, “regulation of flower development”, “protein ubiquitination”, “regulation of transcription, DNA- templated”, “reciprocal meiotic recombination” and “meiotic chromosome segregation”. In addition, SEUs in I- and P- spikelets were also involved in some processes related to reproduction; and those in F-branchlets were mainly involved in processes related to posttranslational modification; while those in F-leaves were mainly involved in processes related to plant-pathogen interaction. As for those in the CC category, they were broadly involved in several important cellular components, including “mitochondrion”, “plasma membrane” and “plastid”. In addition, SEUs in I- and P-spikelets were also involved in ribosome and mitochondria; and those in F-branchlets were mainly involved in endoplasmic reticulum and proteasome; and those in F-leaves were mostly involved in chloroplast. As for those in the MF category, they were broadly involved in several molecular functions, including “ATP binding”, “ubiquitin-protein transferase activity” and “protein tyrosine kinase activity”. In addition, SEUs in I- and P-spikelets were also involved in DNA and microtubule binding; those in F-branchlets were also enriched in oxidoreductases activities; and those in F-leaves were also enriched in enzymes involved in carbohydrate metabolism. Page 5/25 As shown in Additional file 5: Fig. S1, KEGG pathway analysis indicated that SEUs in I- and F-spikelets mainly mapped to the ribosome pathway, with those in F-branchlets mainly mapped to the ribosome, amino acid As shown in Additional file 5: Fig. DEUs were mostly involved in carbohydrate and energy metabolism, signal transition and environmental adaptation As shown in Table 3, many unigenes showed differential expressions across all 15 groups sampled. The number of DEUs in each sample pair ranged from 970 between I- vs P-spikelets to 13,577 in NF-leaves vs I- spikelets. For most pairwise comparisons, the number of up- and down-regulated DEUs was approximately the same, except for four groups, including I- vs P-spikelets, F-branchlets vs both I- and P- spikelets, and F-leaves vs P-spikelets. The Venn diagram of DEU sets shows that 5,494 unigenes were differentially expressed in F-branchlets/F- leaves vs I- and P-spikelets. For those DEUs that were up-regulated in spikelets, they are mainly mapped to KEGG pathways related to carbohydrate metabolism, plant-pathogen interactions and DNA repair (Fig. 5a). Notably, among the 970 DEUs identified between I- and P-spikelets, 916 up-regulated DEUs were mapped to KEGG pathways related to metabolic activity (Additional file 6: Table S5). A total of 5,494 unigenes were differentially expressed in the DEU sets of spikelets/F-leaves vs F- branchlets. Upregulated DEUs in F-branchlets were mapped to KEGG pathways including phenylalanine metabolism, phenylpropanoid biosynthesis, ABC transporters, and flavone and flavonol biosynthesis (Fig. 5b). Those that were upregulated in F- and NF-leaves vs F- branchlets were mainly mapped to plant hormone signal transduction, homologous recombination, base excision repair, and mismatch repair (Additional file 6: Table S5). Notably, 3,275 (50.20% of the total) DEUs found between NF- and F-branchlets were upregulated; these were mainly mapped to KEGG pathways related to replication and recombination (Additional file 6: Table S5). Those that were downregulated were mainly mapped to carbon fixation and photosynthesis (Additional file 6: Table S5). We also found that 6,966 (43.69% of the total) DEUs found in spikelets/F-branchlets vs F-leaves were up- regulated, and were mainly mapped to KEGG pathways related to carbohydrate metabolism (Fig. 5c). 2,492 (49.52%) DEUs in NF-vs F-leaves were up-regulated, and these were mainly mapped to starch and sucrose metabolism (Additional file 6: Table S5). In contrast, downregulated DEUs were mainly mapped to KEGG pathways related to photosynthesis (Additional file 6: Table S5). Among the 5,032 DEUs identified between NF- and F-leaves, 70 were mapped to the circadian rhythm–plant KEGG pathway (Additional file 7: Fig. S2) and 10 of them showed differential expressions (Additional file 8: Table S6). Interestingly, c109220.graph_c0, a bamboo ortholog of Heading date 3a/RICE FLOWERING LOCUS T1 (RFT1) (Swissprot: PE=1 SV=1) (Additional file 9: Fig. The SEUs were mostly involved in carbohydrate metabolism, energy metabolism, and environmental adaptation S1, KEGG pathway analysis indicated that SEUs in I- and F-spikelets mainly mapped to the ribosome pathway, with those in F-branchlets mainly mapped to the ribosome, amino acid Page 5/25 biosynthesis, and carbon metabolism pathways, and those in F-leaves mainly mapped to KEGG pathways related to energy metabolism (including oxidative phosphorylation, fatty acid metabolism, and photosynthesis), environmental adaptation (e.g. proteasomes), genetic information processing, and various unrelated metabolic pathways (e.g. tryptophan metabolism, beta-alanine metabolism, and N-glycan biosynthesis). DEUs were mostly involved in carbohydrate and energy metabolism, signal transition and environmental adaptation S3), named FmHd3a, was found to be significantly upregulated in F-leaves (FDR = 4.23, log2FC = 5.55); while the other ortholog of rice FT (Additional file 9: Fig. S3), unigene c110963.graph_c4, named FmFT, was significantly downregulated in F-leaves (FDR = 4.25E-07, Page 6/25 log2FC = -4.81). RT-qPCR analysis also showed that FmHd3a was significantly more highly expressed in I- /P- spikelets and F-leaves than in NF-leaves or NF- branchlets (Additional file 10: Fig. S4). WGCNA results identified gene modules related to specific tissues WGCNA results showed that unigenes expressed in the six different tissues of flowering and nonflowering plants tested here clustered into 18 branches representing 18 different genetic modules (Additional file 11: Fig. S5a). Unigenes within each module were highly co-expressed, while those in different modules were co- expressed to a lower degree (Additional file 11: Fig. S5b). In six of the samples collected, we identified nine significant gene modules including 1,344 unigenes. Here, correlation coefficient of a module with a related trait > 0.7 was used as a threshold of significance (Additional file 11: Fig. S5c). Notably, these six tissues were more strongly divided into clades according to whether they were flowered or not rather than by the differences among tissues (Additional file 11: Fig. S5d). In addition, the unigenes in gene modules relating to I - and P- spikelets were most strongly enriched in KEGG pathways related to carbohydrate metabolism, genetic information processing, and environmental information processing. In contrast, those related to F- and NF- branchlets were mostly enriched in KEGG pathways related to metabolism, plant hormone signal transduction, and genetic information processing. The gene modules related to F-leaves were enriched in pathways related to plant hormone signal transduction and protein processing, while the gene modules related to NF-leaves were enriched in KEGG pathways related to oxidative phosphorylation (Additional file 12: Table S7). Identification of SSRs We detected a total of 9,296 SSRs in 7,668 unigenes longer than 1,000 bp (Additional file 13: Table S8). 1,628 (21.23%) unigenes contained more than one SSR. Mono-nucleotide repeats were the most common (46.28% of all SSRs) at a density of 71 SSRs per Mb, followed by tri- (26.32%) and di- (22.06%) nucleotide repeats, with densities of 40 and 32 SSRs per Mb, respectively (Fig. 6). Activated Hd3a expression probably accelerates flowering in F. macclureana FT is a key floral regulator that controls the timing of flowering and seasonal growth cessation in response to light and the circadian clock in many plant species [8, 10, 27]. In this study, FmHd3a, a bamboo ortholog of rice FT, was significantly expressed only in tissue samples collected from flowering plants. In rice, Hd3a functions as a major photoperiodic flowering regulator and participates in the OsGI–Hd1–Hd3a module, which is similar to the GI-CO-FT module in Arabidopsis [28]. Hd1 activates and suppresses Hd3a expression by promoting heading under the short day (SD) and long day (LD) conditions, respectively [29-30]. As F. macclureana rarely blossoms on the QTP, which experiences a long photoperiod with a low ratio of red to far-red light, it may have evolved specific reproductive strategies involving flowering-related pathways in response to photoperiodic cues to ensure long vegetation growing period. It is probably that the weak light intensity with a low proportion of blue light might activate Hd3a expression even in the LD conditions, thereby accelerating flowering. Notably, reproduction pathways play an important role in the mechanisms of plant adaptation to extreme environments. Page 7/25 Page 7/25 Previous studies showed that the phytochrome and flowering time regulatory protein 1 (PFT1) from Crucihimalaya himalaica, a close relative of Arabidopsis and Capsella, grows on the QTP, showed signs of positive selection for adaptive divergence [31]. We presume that the function of Hd3a in promoting flowering is likely to be conserved between bamboo and rice, because both of them belong to the Poaceae. In F-leaves, the expression of FmFT and the photoreceptor gene FmCRY (c105898.graph_c2) were both significantly downregulated, while the expression of another FT ortholog, FmHd3a, was significantly upregulated. Photoreceptors mediate light input pathways to synchronize the circadian clock [1, 5]. In A. thaliana, CRY activates FT transcription in response to blue light [3, 32]. UV-B radiation causes a multitude of low- and high-fluence responses similar to the phytochrome responses [33-34]. Thus, down-regulated FmFT expression is likely due to down-regulated FmCRY, which is, in turn, a response to a lower ratio of blue light or reduced light intensity (both in the laboratory or in the QTP). The upregulated expression may indicate that FmHd3a can function in a CRY-independent manner or be negatively regulated by FmFT. We suspect that the floral transition of F. Activated Hd3a expression probably accelerates flowering in F. macclureana macclureana is regulated by a complex regulatory network in which at least two unique FT orthologs interact with the circadian clock pathways. However, how these circadian clock pathways mediate the activation of FmHd3a and FmFT in response to light signalling remains to be elucidated by future research. Notably, we detected FmFT expressions in all four tissues collected from the flowering plants, but not in NF- leaves or NF-branchlets. Given that all plants were grown in the same conditions, we suspect the physiological states of the plant itself may be the possible cause. Additionally, the DEUs upregulated in F-leaves relative to NF-leaves were mainly enriched in KEGG pathways related to starch, sucrose, and galactose metabolism, and corresponding down-regulated DEUs were mapped to the light and carbon fixation, plant circadian rhythm, and photosynthesis pathways. Therefore, we speculate that bamboo FT orthologs might be regulated by regulators involved in other pathways. However, the nature of the mechanism responsible for this cross-regulation needs further experimental verification. Carbohydrate metabolism and signal transport may be major factors in floral transition, organogenesis, and death after flowering Bamboo exhibits excellent flexibility and fracture toughness, and so far, the presence of fibers within the bamboo culm was thought responsible for these remarkable mechanical properties [35]. And the development of plant fibers is accompanied by the of carbohydrate metabolism [36]. Perhaps this is the reason why many DEUs were involved in carbohydrate metabolism pathway. Interestingly, our results indicated that starch and sucrose metabolism was a major enriched KEGG pathway for the DEUs from several combination pairs, including NF- vs F-leaves, branchlets/leaves vs I- & P- spikelets. Transcripts and metabolic signatures of maize leaves have shown that the balance between transitory starch and sucrose is associated with the autonomous floral transition [37]. And in Lilium, carbohydrates have been found to be transported from the vascular bundles to floral organs during reproduction [38]. Yang et al., (2017) also reported that the deficiency in the resources in male flowers reduced pollen viability in Tapiscia sinensis due to biased carbohydrate transport toward the female flowers [39]. Therefore, we suspect there may be a correlation between DEUs related to starch and sucrose metabolism and arrow bamboo floral organ development. In rice, excessive uridine 5´-diphosphoglucose-glucose (UDPG) can result in programmed cell death, accumulation of reactive oxygen species and an increase in the caspase-like activity [40] and inactivate starch Page 8/25 synthase disrupted normal male reproduction by delaying programmed cell death in cotton [41], suggestive of a correlation between starch and sucrose metabolism and death. Bamboo flowering, especially in masting species, often causes plants to wilt and die after setting seed. It is possible that increased starch and sucrose metabolism might trigger the excessive accumulation of reactive oxygen species and result in the altered activity of key enzymes in important biological pathways. synthase disrupted normal male reproduction by delaying programmed cell death in cotton [41], suggestive of a correlation between starch and sucrose metabolism and death. Bamboo flowering, especially in masting species, often causes plants to wilt and die after setting seed. It is possible that increased starch and sucrose metabolism might trigger the excessive accumulation of reactive oxygen species and result in the altered activity of key enzymes in important biological pathways. In the present study, unigenes related to the signal transduction pathways were significantly upregulated in the tissues of flowering arrow bamboo plants. The transcriptomic profiles of Posidonia oceanica also showed a strong metabolic activation of hormones in the heat stress-induced flowering plants [42]. Carbohydrate metabolism and signal transport may be major factors in floral transition, organogenesis, and death after flowering Signal transduction- related genes were also found to have undergone both significant positive selection and expansion events on the adaptive evolution of Crucihimalaya himalaica [31] and cyanobacterium Trichormus sp. NMC-1 [43] on the QTP. In the present study, unigenes related to the signal transduction pathways were significantly upregulated in the tissues of flowering arrow bamboo plants. We suspect that this may be due to the long distance transport of the FT proteins, which ensures floral promotion at the shoot apex [44], or the phytohormone signaling and calcium signaling, which play diverse roles in the specification of flower organs during arrow bamboo reproductive development [45-46]. Tissues collection The studied plant species is highland arrow bamboo (Fargesia macclureana), and it grows mainly as a underbrush of coniferous forest or coniferous and broad-leaved mixed forest, and sometimes forms a pure population in the QTP at an altitude of approximately 2,000 ~ 3800 m above sea level (Fig. 9). F. macclureana was formally identified by Stapleton in 1993 [25] and detailed explanations are provided in the volume 22 of Flora of China (http://foc.iplant.cn/) [56]. A voucher specimen of this material has been deposited in the Bamboo Research Institute of Nanjing Forestry University. Six seedlings of F. macclureana were obtained from the wild with the permission of the local forestry department and collected from the Bayi District, Linzhi City, Tibet, China (29˚46′ 0.95″ N, 94̊ 44′46.36″ E, altitude: ~2,200 m). Then they were all transferred to individual pots at the State Forestry Administration Key Open Laboratory at the International Center for Bamboo and Rattan in Beijing (N: 39˚59′ 17.52″, E: 116̊ 28′46.06″, altitude ~34 m). During growth, the plants were maintained at 28 ± 1 °C and 50-55% relative humidity under a 16/8 h (light/dark) photoperiod regimen with a light intensity of 200 μmol · m-2 · s-1. All the seedlings were watered with a 1/3 B5 macronutrient nutrient solution three times a week. After twenty days, we found that four out of six seedlings flowered, while the other two didn't blossom until the time of sampling. One month later, we collected samples of six tissues for further de novo sequencing, including inflorescences in the initial flower stage (I-spikelets), inflorescences at the peak flower stage (P- spikelets), branchlets of the flowering plants (F-branchlets), leaves of the flowering plants (F-leaves), branchlets of the non-flowering plants (NF-branchlets) and leaves of the non-flowering plants (NF-leaves). We collected three independent replicates of each tissue type. Conclusions In the present study, we constructed a novel de novo transcriptome analysis for F. macclureana. Based on a comprehensive analysis of specifically and differentially expressed unigenes, combined with a WGCNA, we propose that changes in a variety of environmental signals, including the temperature, light intensity and photoperiod between in the lab and QTP, may have influenced the expressions of unigenes related to photoperiodic flowering, carbohydrate metabolism and signal transduction, thereby promoting floral transition in F. macclureana. Significant expressions of unigenes enriched in DNA repair and plant-pathogen interaction pathways may reflect the adaptation of F. macclureana to its high radiation and pathogen-specific environmen on the QTP. We identified both similarities and differences in adaptive mechanisms (e.g., disease-resistance and DNA repair pathways) and stress-induced flowering mechanisms (e.g., carbohydrate metabolism and signal transduction pathways) among plants that grow at high altitudes. Although further experimental verification is needed, our results provide insight into the regulation of flowering time in highland bamboo as well as how this species adapts to harsh and extreme environments. F. macclureana has presumably evolved an integrated mechanism to adapt to the harsh environment of the QTP We detected the broad expressions of unigenes encoding putative Hsp proteins, such as heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). Both Hsp70 and Hsp90 are important for maintaining cellular protein homeostasis under stress conditions and they function by activating other targets [47-49]. It is likely that the sudden exposure to the higher temperature of the lab (i.e. outside the QTP) triggered the expressions of these unigenes. Warmer temperatures can greatly reduce flowering synchrony among individuals from 72 woody and herbaceous species [50]. It was also reported that P. oceanica, a highly clonal and long-lived species, massively bloomed after a simulated heatwave [42]. Given the cold temperatures present at the high- altitude regions of the QTP [26], it is reasonable to presume that F. macclureana has developed into a heat- sensitive but not heat-tolerant bamboo species and flowering is probably a stress-induced response to the higher temperature in the lab. DNA repair and disease-resistance pathways have been found to play a crucial role in the highland adaptation of Tibetan highland plants [43, 51]. In the present study, we detected significant expressions of many unigenes related to pathogen response that contained either a nucleotide binding (NB)-ARC domain or a leucine-rich repeat (LRR) domain, which were present in most resistance (R) proteins [52-54]. We also identified many differentially expressed unigenes that were significantly enriched in the DNA repair pathways. Since relatively fewer species of pathogenic microorganisms and intense UV radiation exist on the QTP [55], it is reasonable to presume that F. macclureana has evolved a relatively narrow range of pathogen specificity and specific DNA- repair mechanisms. Sudden exposure to the lab environment, which contains a heavier load of pathogens and weak light intensity, may have induced an innate defensive response of F. macclureana, and those that were enriched in the plant-pathogen interaction and DNA repair response pathways may be important for F. macclureana to cope with the new environment present in the lab. Although further studies are needed to investigate the molecular mechanisms responsible for the putative adaptive evolutionary changes, this study provides insights into how plants adapt to harsh and extreme environments. Page 9/25 RNA extraction, quantification, and qualification Total RNA was extracted from each of the six unique tissues mentioned above using a RNeasy plant RNA extraction kit (Qiagen, Dusseldorf, Germany), and the extraction procedure was performed according to the manufacturer’s instructions. RNA degradation and contamination were monitored using 1% agarose gels. RNA purity was checked using a NanoPhotometer® spectrophotometer (Implen GmbH, Munich, Germany). RNA Page 10/25 Page 10/25 concentration was measured using a Qubit® RNA Assay Kit and a Qubit®2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using an RNA Nano 6000 Assay Kit run on an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). De novo transcriptome assembly Raw data in fastq format were first processed using in-house perl scripts. Clean data were obtained by removing low-quality reads and reads that contain the adapters or poly-N sequences. Meanwhile, we checked the quality of our unassembled read dataset by examining various measures including Q20, Q30, GC-content, and sequence duplication. All the downstream analyses were performed using high-quality clean data. The transcriptome was assembled using clean reads from all libraries and samples. The assembly was produced using Trinity [57] with min_kmer_cov set to 2 and all other parameters set to their respective default values. Library preparation for transcriptome sequencing Library construction and RNA-Seq were performed by the Biomarker Biotechnology Corporation (Beijing, China). A total of 3 μg RNA per sample was used for RNA preparation. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, followed by fragmentation carried out using divalent cations at elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via the exonuclease and polymerase activities. Next, the 3´ ends of the DNA fragments were adenylated and ligated to the NEBNext adaptors with hairpin loop structures to prepare samples for hybridization, this was to select cDNA fragments that are 150-200 bp in length. Library fragments were then purified using an Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA), and 3 μl USER enzyme (New England Biolabs, Ipswich, MA, USA) was added to the size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by five min at 95°C before PCR. PCR was performed using Phusion High-Fidelity DNA polymerase (Thermo Fisher, Waltham, MA, USA), universal PCR primers, and the Index (X) Primer. Finally, the PCR products were purified using the AMPure XP system and library quality was assessed on the Agilent Bioanalyzer 2100. Clustering and sequencing The clustering of index-coded samples was performed using a cBot Cluster Generation System and a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), and all experimental procedures were performed according to the manufacturer’s instructions. After that, library preparations were sequenced on an Illumina Hiseq 2000 platform and paired-end reads were generated. Weighted gene co-expression network analysis (WGCNA) WGCNA was performed on all unigenes identified using the WCGNA R package. We calculated the adjacency matrices and performed the topological overlap measures (TOMs), which show the degree of overlap in shared neighbors between pairs of genes in the network to define gene clusters in our transcriptome dataset. 1 − TOM was used as a dissimilarity measure for hierarchical clustering and module detection. Modules of the clustered genes were then selected using the Dynamic Tree Cut algorithm as implemented by WGCNA. To identify modules that are significantly related to particular tissues, expression profiles of each module were summarized by a module eigengene (ME) and the correlations between the modules and corresponding tissues were calculated. Expression analysis of broadly and specifically expressed unigenes For all unigenes, those that were expressed in all six tissues were defined as broadly expressed unigenes (BEUs). Similarly, unigenes that were specifically expressed in only one tissue were defined as specifically expressed unigenes (SEUs). The identification of BEUs and SEUs was conducted by using tools on the BMKCloud platform (http://www.biocloud.net). Functional annotation of the transcriptome Gene function was annotated using the following databases: NR (NCBI non-redundant protein sequences), Pfam (Protein family), KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins), Swiss-Prot (a database of manually annotated and reviewed protein sequences), KEGG (the Kyoto Encyclopedia of Genes and Genomes), and the GO (Gene Ontology) database. Page 11/25 Page 11/25 Quantification of gene expression levels Gene expression levels were estimated using RSEM [58] for each sample: clean data were mapped back onto the assembled transcriptome, followed by a read count for each gene. The expression levels of unigenes were calculated and normalized using FPKM (fragments per kb per million fragments) [59]. Expression analysis of differently expressed unigenes (DEUs) Before analysis, we conducted a principal component analysis (PCA) and removed one replicate that showed an inconsistent expression pattern in the NF-branchlets and NF-leaves to ensure consistency in the expression patterns of unigenes between replicates (Additional file 14: Figure S6). Expression analysis of the DEUs between pairs of tissues/groups was performed using the DESeq package (1.10.1) in R. DESeq provides statistical routines for identifying differential expression in the digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini-Hochberg method for controlling the false discovery rate [60]. Here, uni-transcripts with an absolute value of log2 ratio ≥ 2, an FDR significance score < 0.01, and an adjusted P-value < 0.05 were deemed to be differentially expressed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis To understand the higher-level functions of the observed unigenes, we performed GO term annotation and KEGG pathway enrichment analysis using BMKCloud (http://www.biocloud.net/; [61]). We used KOBAS 2.0 [62] to test the statistical enrichment of differentially expressed genes in KEGG pathways. Pathways with P values < 0.05 were considered significantly enriched. Protein-protein interactions (PPIs) Page 12/25 The DEU and SEU sequences were queried using BLASTX against the related species to predict PPIs that the DEUs and SEUs may be involved in. This search procedure was capable of identifying PPIs that may be similar to any others found in the STRING database (http://string-db.org/). These PPIs were then visualized using Cytoscape [63]. The DEU and SEU sequences were queried using BLASTX against the related species to predict PPIs that the DEUs and SEUs may be involved in. This search procedure was capable of identifying PPIs that may be similar to any others found in the STRING database (http://string-db.org/). These PPIs were then visualized using Cytoscape [63]. Validation of FmHd3a transcript levels by qRT-PCR To verify the expression pattern of the FmHd3a, we used RT-qPCR to assess the expressions of FmHd3a in six distinct tissues. First-strand cDNA was synthesized from total RNA extracted by using a reverse transcription system (Promega, Madison, WI, USA) following the manufacturer’s instructions. Each RT-qPCR amplification was performed at least three times, and NTB and TIP41 were used as internal controls [64]. Primers for these genes are listed in Additional file 15: Table S9. The relative expression levels of FmHd3a in different tissues were calculated using the 2-ΔΔCT method [65]. The statistical significance of differences in the mean levels of expression was tested using a one-way ANOVA. Significant differences in transcript abundance between different tissues were then compared using Duncan’s multiple range tests as implemented by SPSS version 17.0 (IBM SPSS, Chicago, USA). We considered mean differences at P < 0.05 and P < 0.01 to be statistically significant and highly statistically significant, respectively. Detection of SSRs Picard-tools version 1.41 and samtools version 0.1.18 were used to sort data, remove duplicated reads, and merge the bam alignment results of each sample. SSRs were identified using MISA (https://webblast.ipk- gatersleben.de/misa/). Acknowledgements Not applicable. Abbreviations BEUs: Broadly expressed unigenes; BP: Biological process; CC: Cellular component; COG: Clusters of orthologous groups; DEUs: Differentially expressed unigenes; F-branchlets: Branchlets of the flowering plants; F- leaves: Leaves of the flowering plants; FPKM: Fragments per kb per million fragments; GO: Gene ontology; I- spikelets: Inflorescences in the initial flower stage; KEGG: Kyoto Encyclopedia of Genes and Genomes; ME: Module eigengene; MF: Molecular function; NF-branchlets: Branchlets of the non-flowering plants; NF-leaves: Leaves of the non-flowering plants; NR: NCBI non-redundant protein sequences; PCA: Principal component analysis; Pfam: Protein family; PPIs: Protein-protein interactions. P-spikelets: Inflorescences at the peak flower stage; QTP: Qinghai–Tibet Plateau; SEUs: Specifically expressed unigenes; SSRs: Simple sequence repeats; TOMs: topological overlap measures. WGCNA: Weighted gene co-expression network analysis. Availability of data and materials The RNA sequencing dataset generated during the current study have been submitted to NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra) with the accession number PRJNA544133. Funding Page 13/25 This work was supported by the Special Funds for Fundamental Scientific Research on Professional Work Supported by International Center for Bamboo and Rattan (No. 1632019008) and the Sub-Project of National Science and Technology Support Plan of the Twelfth Five-Year in China (No. 2015BAD04B01). The funder is the corresponding author of this manuscript and he played an important role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Authors’ contributions Conceived and designed the experiments: ZM G. Performed the experiments: KB Y and JJ S. Analyzed the data: CX Z, YL D and Y L. Interpreted the results and wrote the paper: Y L. All authors have read and approved the final manuscript. References Bhattacharya S, Das M, Bar R, et al. Morphological and molecular characterization of Bambusa tulda with a note on flowering. Annals of Botany, 2006;98:529-535. 12. Bhattacharya S, Das M, Bar R, et al. Morphological and molecular characterization of Bambusa tulda with a note on flowering. Annals of Botany, 2006;98:529-535. 13. Yuan JL, Yue JJ, Gu XP, et al. Flowering of woody bamboo in tissue culture systems. Front Plant Sci. 2017;8:1589. 13. Yuan JL, Yue JJ, Gu XP, et al. Flowering of woody bamboo in tissue culture systems. Front Plant Sci. 2017;8:1589. 14. Tian Z, Liu X, Fan Z, et al. The next widespread bamboo flowering poses a massive risk to the giant panda. Biol Conserv. 2019;234:180-187. 15. Giordano CV, Sánchez RA, Austin AT. Gregarious bamboo flowering opens a window of opportunity for regeneration in a temperate forest of Patagonia. New Phytol. 2009;181:880-889. 16. Shih MC, Chou ML, Yue JJ, et al. BeMADS1 is a key to delivery MADSs into nucleus in reproductive tissues- De novo characterization of Bambusa edulis transcriptome and study of MADS genes in bamboo floral development. BMC Plant Biol. 2014;14:179. 17. Wang X, Zhang X, Zhao L, et al. Morphology and quantitative monitoring of gene expression patterns during floral induction and early flower development in Dendrocalamus latiflorus. Int J Mol Sci. 2014;15:12074-12093. 18. Zhang Y, Tang D, Lin X, et al. Genome-wide identification of MADS-box family genes in moso bamboo (Phyllostachys edulis) and a functional analysis of PeMADS5 in flowering. BMC Plant Biol. 2018;18:176. 18. Zhang Y, Tang D, Lin X, et al. Genome-wide identification of MADS-box family genes in moso bamboo (Phyllostachys edulis) and a functional analysis of PeMADS5 in flowering. BMC Plant Biol. 2018;18:176. 19. Chiou T. The role of microRNAs in sensing nutrient stress. Plant Cell Environ. 2007;30:323-332. 19. Chiou T. The role of microRNAs in sensing nutrient stress. Plant Cell Environ. 2007;30:323-332. 20. Hisamoto Y, Kashiwagi H, Kobayashi M. Use of flowering gene FLOWERING LOCUS T (FT) homologs in the phylogenetic analysis of bambusoid and early diverging grasses. J Plant Res. 2008;121:451-461. 20. Hisamoto Y, Kashiwagi H, Kobayashi M. Use of flowering gene FLOWERING LOCUS T (FT) homologs in the phylogenetic analysis of bambusoid and early diverging grasses. J Plant Res. 2008;121:451-461. 21. Gao J, Ge W, Zhang Y, et al. Identification and characterization of microRNAs at different flowering developmental stages in moso bamboo (Phyllostachys edulis) by high-throughput sequencing. Mol Genet Genom. 2015;290:2335-2353. References 1. Putterill J, Varkonyi-Gasic E. FT and florigen long-distance flowering control in plants. Curr Opin Plant Biol. 2016;33:77-82. 2. Wigge PA, Kim MC, Jaeger KE, et al. Integration of spatial and temporal information during floral induction in Arabidopsis. Science. 2005;309:1056-1059. 2. Wigge PA, Kim MC, Jaeger KE, et al. Integration of spatial and temporal information during floral induction in Arabidopsis. Science. 2005;309:1056-1059. 3. Liu H, Wang Q, Liu Y, et al. Arabidopsis CRY2 and ZTL mediate blue-light regulation of the transcription factor CIB1 by distinct mechanisms. Proceedings of the National Academy of Sciences of the United Page 14/25 Page 14/25 Page 14/25 States of America. 2013;110:17582-17587. States of America. 2013;110:17582-17587. 4. Du A, Tian W, Wei M, et al. The DTH8-Hd1 module mediates day-length-dependent regulation of rice flowering. Mol Plant. 2017;10:948-961. 5. Lin MK, Belanger H, Lee YJ, et al. FLOWERING LOCUS T protein may act as the long-distance florigenic signal in the cucurbits. Plant Cell. 2007;19:1488-1506. 6. Wolabu TW, Zhang F, Niu L, et al. Three FLOWERING LOCUS T-like genes function as potential florigens and mediate photoperiod response in sorghum. New Phytol. 2016;210:946-959. 6. Wolabu TW, Zhang F, Niu L, et al. Three FLOWERING LOCUS T-like genes function as potential florigens and mediate photoperiod response in sorghum. New Phytol. 2016;210:946-959. 7. Bohlenius H, Huang T, Charbonnel-Campaa L, et al. CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science, 2006;312:1040-1043. 7. Bohlenius H, Huang T, Charbonnel-Campaa L, et al. CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science, 2006;312:1040-1043. 8. Hsu CY, Liu Y, Luthe DS, et al. Poplar FT2 shortens the juvenile phase and promotes seasonal flowering. Plant Cell. 2006;18:1846-1861. 8. Hsu CY, Liu Y, Luthe DS, et al. Poplar FT2 shortens the juvenile phase and promotes seasonal flowering. Plant Cell. 2006;18:1846-1861. 9. Klocko AL, Ma C, Robertson S, et al. FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus. Plant Biotechnol J. 2016;14:808-819. 9. Klocko AL, Ma C, Robertson S, et al. FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus. Plant Biotechnol J. 2016;14:808-819. 10. Munoz-Fambuena N, Nicolas-Almansa M, Martinez-Fuentes A, et al. Genetic inhibition of flowering differs between juvenile and adult Citrus trees. Ann Bot. 2019;123:483-490. 11. Zhou X, Ruan J, Wang G, et al. Characterization and identification of microRNA core promoters in four model species. PLoS Comput Biol. 2007;3:e37. 12. References Genome of Crucihimalaya himalaica, a close relative of Arabidopsis, shows ecological adaptation to high altitude. Proc Natl Acad Sci U S A. 2019;116: 7137-7146. 31. Liu Y, Li X, Ma D, et al. CIB1 and CO interact to mediate CRY2-dependent regulation of flowering. EMBO Rep. 2018;19:DOI: 10.15252/embr.201845762 31. Liu Y, Li X, Ma D, et al. CIB1 and CO interact to mediate CRY2-dependent regulation of flowering. EMBO Rep. 2018;19:DOI: 10.15252/embr.201845762 32. Kim BC, Tennessen DJ, Last RL. UV-B-induced photomorphogenesis in Arabidopsis. Plant J. 1998;15:667- 674. 32. Kim BC, Tennessen DJ, Last RL. UV-B-induced photomorphogenesis in Arabidopsis. Plant J. 1998;15:667- 674. 33. Li J, Yang L, Jin D, et al. UV-B-induced photomorphogenesis in Arabidopsis. Protein Cell, 2013;4:485-492. 33. Li J, Yang L, Jin D, et al. UV-B-induced photomorphogenesis in Arabidopsis. Protein Cell, 2013;4:485-492. 34. Habibi MK, Lu Y. Crack propagation in bamboo's hierarchical cellular structure. Sci Rep. 2014;4:5598. 33. Li J, Yang L, Jin D, et al. UV-B-induced photomorphogenesis in Arabidopsis. P 33. Li J, Yang L, Jin D, et al. UV-B-induced photomorphogenesis in Arabidopsis. Protein Cell, 2013;4:485-492. 34. Habibi MK, Lu Y. Crack propagation in bamboo's hierarchical cellular structure. Sci Rep. 2014;4:5598. 34. Habibi MK, Lu Y. Crack propagation in bamboo's hierarchical cellular structur 35. Sun W, Gao Z, Wang J, et al. Cotton fiber elongation requires the transcription factor GhMYB212 to regulate sucrose transportation into expanding fibers. New Phytol. 2019;222:864-881. 35. Sun W, Gao Z, Wang J, et al. Cotton fiber elongation requires the transcription factor GhMYB212 to regulate sucrose transportation into expanding fibers. New Phytol. 2019;222:864-881. 36. Coneva V, Guevara D, Rothstein SJ, et al. Transcript and metabolite signature of maize source leaves suggests a link between transitory starch to sucrose balance and the autonomous floral transition. J Exp Bot. 2012;63:5079-5092. 36. Coneva V, Guevara D, Rothstein SJ, et al. Transcript and metabolite signature of maize source leaves suggests a link between transitory starch to sucrose balance and the autonomous floral transition. J Exp Bot. 2012;63:5079-5092. 37. Castro AJ, Clement C. Sucrose and starch catabolism in the anther of Lilium during its development: a comparative study among the anther wall, locular fluid and microspore/pollen fractions. Planta, 2007;225:1573-1582. 37. Castro AJ, Clement C. Sucrose and starch catabolism in the anther of Lilium during its development: a comparative study among the anther wall, locular fluid and microspore/pollen fractions. Planta, 2007;225:1573-1582. 38. Yang K, Zhou X, Wang Y, et al. References 21. Gao J, Ge W, Zhang Y, et al. Identification and characterization of microRNAs at different flowering developmental stages in moso bamboo (Phyllostachys edulis) by high-throughput sequencing. Mol Genet Genom. 2015;290:2335-2353. Page 15/25 Page 15/25 22. Wysocki WP, Ruiz-Sanchez E, Yin Y, et al. The floral transcriptomes of four bamboo species (Bambusoideae; Poaceae): support for common ancestry among woody bamboos. BMC Genomics, 2016;17:384. 23. Ge W, Zhang Y, Cheng Z, et al. Main regulatory pathways, key genes and microRNAs involved in flower formation and development of moso bamboo (Phyllostachys edulis). Plant Biotechnol J. 2017;15:82-96. 24. Stapleton CMA. Fargesia macclureana, a Tibetan bamboo in Europe. Bamboo Soc. (GB) Newsl. 1993;17:17. 25. Cao M, Jin Y, Liu N, et al. Effects of the Qinghai-Tibetan Plateau uplift and environmental changes on phylogeographic structure of the Daurian Partridge (Perdix dauuricae) in China. Mol Phylogenet Evol. 2012;65:823-830. 26. Abe M, Kobayashi Y, Yamamoto S, et al. FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science. 2005; 309:1052-1056. 26. Abe M, Kobayashi Y, Yamamoto S, et al. FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science. 2005; 309:1052-1056. 27. Song YH, Shim JS, Kinmonth-Schultz HA, et al. Photoperiodic flowering: time measurement mechanisms in leaves. Annu Rev Plant Biol. 2015;66:441-464. 27. Song YH, Shim JS, Kinmonth-Schultz HA, et al. Photoperiodic flowering: time measurement mechanisms in leaves. Annu Rev Plant Biol. 2015;66:441-464. 28. Yano M, Katayose Y, Ashikari M, et al. Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell. 2000;12:2473-2484. 28. Yano M, Katayose Y, Ashikari M, et al. Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell. 2000;12:2473-2484. 29. Hayama R, Yokoi S, Tamaki S, et al. Adaptation of photoperiodic control pathways produces short-day flowering in rice. Nature. 2003;422:719-722. 29. Hayama R, Yokoi S, Tamaki S, et al. Adaptation of photoperiodic control pathways produces short-day flowering in rice. Nature. 2003;422:719-722. 30. Zhang T, Qiao Q, Novikova PY, et al. Genome of Crucihimalaya himalaica, a close relative of Arabidopsis, shows ecological adaptation to high altitude. Proc Natl Acad Sci U S A. 2019;116: 7137-7146. 30. Zhang T, Qiao Q, Novikova PY, et al. References Carbohydrate metabolism and gene regulation during anther development in an androdioecious tree, Tapiscia sinensis. Ann Bot. 2017;120:967-977. 38. Yang K, Zhou X, Wang Y, et al. Carbohydrate metabolism and gene regulation during anther development in an androdioecious tree, Tapiscia sinensis. Ann Bot. 2017;120:967-977. 39. Xiao G, Zhou J, Lu X, et al. Excessive UDPG resulting from the mutation of UAP1 causes programmed cell death by triggering reactive oxygen species accumulation and caspase-like activity in rice. New Phytol. 2018;217:332-343. 39. Xiao G, Zhou J, Lu X, et al. Excessive UDPG resulting from the mutation of UAP1 causes programmed cell death by triggering reactive oxygen species accumulation and caspase-like activity in rice. New Phytol. 2018;217:332-343. Page 16/25 Page 16/25 40. Min L, Zhu L, Tu L, et al. Cotton GhCKI disrupts normal male reproduction by delaying tapetum programmed cell death via inactivating starch synthase. Plant J. 2013;75:823-835. 41. Marín-Guirao L, Entrambasaguas L, Ruiz JM, et al. Heat-stress induced flowering can be a potential adaptive response to ocean warming for the iconic seagrass Posidonia oceanica. Mol Ecol, 2019;28: 2486- 2501. 42. Qiao Q, Huang Y, Qi J, et al. The genome and transcriptome of Trichormus sp. NMC-1: insights into adaptation to extreme environments on the Qinghai-Tibet Plateau. Sci Rep. 2016;6: 29404. 42. Qiao Q, Huang Y, Qi J, et al. The genome and transcriptome of Trichormus sp. NMC-1: insights into adaptation to extreme environments on the Qinghai-Tibet Plateau. Sci Rep. 2016;6: 29404. 43. Turck F, Fornara F, Coupland G. Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Ann Rev Plant Biol. 2008;59:573-594. 43. Turck F, Fornara F, Coupland G. Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Ann Rev Plant Biol. 2008;59:573-594. 44. Galli M, Liu Q, Moss BL, et al. Auxin signaling modules regulate maize inflorescence architecture. Proc Nat Acad Sci USA. 2015;112:13372-13377. 44. Galli M, Liu Q, Moss BL, et al. Auxin signaling modules regulate maize inflorescence architecture. Proc Nat Acad Sci USA. 2015;112:13372-13377. 45. Yuan Z, Zhang D. Roles of jasmonate signalling in plant inflorescence and flower development. Curr Opin Plant Biol. 2015;27:44-51. 45. Yuan Z, Zhang D. Roles of jasmonate signalling in plant inflorescence and flower development. Curr Opin Plant Biol. 2015;27:44-51. 46. Mayer MP. Hsp70 chaperone dynamics and molecular mechanism. Trends Biochem Sci. 2013;38:507-514 46. Mayer MP. Hsp70 chaperone dynamics and molecular mechanism. Trends Biochem Sci. 2013;38:507-514. 46. Mayer MP. References KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucl Acids Res. 2011;39:W316-322. 61. Xie C, Mao X, Huang J, et al. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucl Acids Res. 2011;39:W316-322. 62. Shannon P, Markiel A, Ozier O, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498-2504. 62. Shannon P, Markiel A, Ozier O, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498-2504. 63. Fan C, Ma J, Guo Q, et al. Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis). PLoS One. 2013;8:e56573. 63. Fan C, Ma J, Guo Q, et al. Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis). PLoS One. 2013;8:e56573. 64. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt Methods. 2001;25:402-408. References Hsp70 chaperone dynamics and molecular mechanism. Trends Biochem Sci. 2013;38:507-514. 47. Schopf FH, Biebl MM, Buchner J. The HSP90 chaperone machinery. Nat Rev Mol Cell Biol. 2017;18:345- 360. 47. Schopf FH, Biebl MM, Buchner J. The HSP90 chaperone machinery. Nat Rev Mol Cell Biol. 2017;18:345- 360. 47. Schopf FH, Biebl MM, Buchner J. The HSP90 chaperone machinery. Nat Rev Mol Cell Biol. 2017;18:345- 360. 48. Biebl MM, Buchner J. Structure, function, and regulation of the Hsp90 machinery. CSH Perspect Biol. 2019;DOI: 10.1101/cshperspect.a034017. 48. Biebl MM, Buchner J. Structure, function, and regulation of the Hsp90 machinery. CSH Perspect Biol. 2019;DOI: 10.1101/cshperspect.a034017. 49. Zohner CM, Mo L, Renner SS, et al. Global warming reduces leaf-out and flowering synchrony among individuals. Elife. 2018; DOI: 10.7554/eLife.40214. 50. Fang O, Zhang QB. Tree resilience to drought increases in the Tibetan Plateau. Global Change Biol. 2019;25:245-253. 51. Li J, Huang H, Zhu M, et al. A plant immune receptor adopts a two-step recognition mechanism to enhance viral effector perception. Mol Plant, 2019;12:248-262. 52. Shao ZQ, Xue JY, Wang Q, et al. Revisiting the Origin of Plant NBS-LRR Genes. Trends Plant Sci. 2019;24:9- 12. 53. Takken FL, Albrecht M, Tameling WI. Resistance proteins: molecular switches of plant defence. Curr Opin Plant Biol. 2006;9:383-390. 54. Zhang XJ, Yao TD, Ma XJ, et al. Microorganisms in a high altitude glacier ice in Tibet. Folia Microbiol. 2002;47:241-245. 55. Li D, Stapleton C. Bambuseae. In: Flora of China. Edited by Wu ZY, Raven PH, Hong DY, Vol. 22. Beijing: Science Press and St. Louis USA: Missouri Botanic Garden Press. 2006:1-180. 56. Grabherr MG, Haas BJ, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644-652. 57. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. 58. Mortazavi A, Williams BA, McCue K, et al. Mapping and quantifying mammalian transcriptomes by RNA- Seq. Nat Methods. 2008;5:621-628. Page 17/25 Page 17/25 59. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple hypothesis testing. J R Stat Soc B. 1995;57:289-300. 60. Kanehisa M. KEGG Bioinformatics resource for plant genomics and metabolomics. Methods Mol Biol. 2016;1374:55-70. 60. Kanehisa M. KEGG Bioinformatics resource for plant genomics and metabolomics. Methods Mol Biol. 2016;1374:55-70. 61. Xie C, Mao X, Huang J, et al. Table 1 Length range of transcripts and unigenes identified in the transcriptome of F. macclureana. Table 1 Length range of transcripts and unigenes identified in the transcriptome of F. macclureana. Length Range Transcripts Unigenes 200-300 36,390 (12.59%) 25,291 (25.53%) 300-500 47,515 (16.43%) 21,257 (21.46%) 500-1,000 78,453 (27.13%) 23,806 (24.03%) 1,000-2,000 77,456 (26.79%) 16,752 (16.91%) 2,000+ 49,308 (17.05%) 11,950 (12.06%) Total number 289,122 99,056 Total length 341,956,623 91,685,618 N50 length 1,765 1,587 Mean length 1,182.74 925.59 Total number 289,122 99,056 Total length 341,956,623 91,685,618 N50 length 1,765 1,587 Mean length 1,182.74 925.59 Table 2 Statistics of annotation analysis of unigenes Page 18/25 Table 2 Statistics of annotation analysis of unigenes Anno_Database Annotated_Number percentage 300<=length<1,000 length>=1,000 COG_Annotation 13,128 27.75 3,261 7,515 GO_Annotation 34,055 71.99 10,659 17,855 KEGG_Annotation 14,307 30.24 4,550 7,397 KOG_Annotation 23,492 49.66 6,863 12,779 Pfam_Annotation 28,317 59.86 7,823 16,896 Swissprot_Annotation 24,847 52.52 7,450 14,500 eggNOG_Annotation 43,909 92.82 14,040 21,568 Nr_Annotation 45,516 96.22 15,031 22,271 All_Annotated 47,306 100.00 15,602 22,437 Table 3. Differentially expressed unigenes (DEUs; Fold change > 2; FDR < 0.01) among tissues of F. macclureana. DEUs_total: the total number of DEUs; DEUs_up (%): the number (and percentage) of up-regulated DEUs; DEUs_down (%): the number (and percentage) of down- regulated DEUs). Number Group DEUs_total DEUs_up (%) DEUs_down (%) 1 I-spikelets vs P-spikelets 970 916 (94.43) 54 (5.57) 2 F-branchlets vs I-spikelets 4,970 3,046 (61.29) 1,924 (38.71) 3 F-branchlets vs P-spikelets 5,124 3,338 (65.14) 1,786 (34.86) 4 F-branchlets vs F-leaves 8,467 3,967 (46.85) 4,500 (53.15) 5 F-leaves vs I-spikelets 12,829 6,791 (52.93) 6,038 (47.07) 6 F-leaves vs P-spikelets 10,791 6,625 (61.39) 4,166 (38.61) 7 NF-branchlets vs I-spikelets 11,628 6,135 (52.76) 5,493 (47.24) 8 NF-branchlets vs P-spikelets 10,809 5,893 (54.52) 4,916 (45.48) 9 NF-branchlets vs F-branchlets 6,524 3,275 (50.20) 3,249 (49.80) 10 NF-branchlets vs F-leaves 11,670 5,902 (50.57) 5,768 (49.43) 11 NF-branchlets vs NF-leaves 3,853 1,946 (50.51) 1,907 (49.49) 12 NF-leaves vs I-spikelets 13,577 6,921 (50.98) 6,656 (49.02) 13 NF-leaves vs P-spikelets 11,718 6,130 (52.31) 5,588 (47.69) 14 NF-leaves vs F-branchlets 11,659 5,606 (48.08) 6,053 (51.92) 15 NF-leaves vs F-leaves 5,032 2,492 (49.52) 2,540 (50.48) Additional Files Additional file 1: Table S1. Statistic of sequencing and assembly data. Additional file 2: Table S2. 47,306 unigenes were annotated and their predicted functions. Additional file 3: Table S3. The top10 enriched GO terms in three main categories for predicted unigenes. Additional file 4: Table S4. The most enriched GO terms and KEGG pathways for the specially expressed Additional file 3: Table S3. The top10 enriched GO terms in three main categories for predicted unigenes. Additional file 4: Table S4. The most enriched GO terms and KEGG pathways for the specially expressed unigenes (SEUs) and broadly expressed unigenes (BEUs) in all tissues collected from flowering plants. Additional file 4: Table S4. The most enriched GO terms and KEGG pathways for the specially expressed unigenes (SEUs) and broadly expressed unigenes (BEUs) in all tissues collected from flowering plants. Additional file 5: Figure S1. KEGG annotation of unigenes that were specifically expressed in P-spikelets (a), F- branchlets (b) and F-leaves (c) of arrow bamboo flowering plants. The size of dots is proportional to the Page 19/25 Page 19/25 Page 19/25 number of unigenes. number of unigenes. Additional file 6: Table S5. KEGG enrichment of differentially expressed unigenes (DEUs) between different tissues. Additional file 7: Figure S2. Hub unigenes in regulatory networks of flowering identified based on analysis of DEUs among tissues. Unigenes c109220.graph_c0 and c110963.graph_c4, showing differential expressions between NF-leaves and F-leaves, are both bamboo orthologs of FLOWERING LOCUS T (FT), which was marked with a red square; while unigenes down-regulated were marked with green squares. Additional file 8: Table S6. Expressions of 10 unigenes that were differentially expressed between F-leaves vs NF-leaves and mapping into the circadian phythm-plant KEGG pathway. Additional file 9: Figure S3. Phylogenetic relationship between FmFT, FmHd3a and their orthologs from Brachypodium distachyon and rice. The orthologs include BdFT (XP 010238588.1) and BdHd3a (XP 003565602.1) from B. distachyon, as well as OsFT (XP 015619436.1) and OsHd3a (XP 015611892.1) from rice. AP2 from Arabidopsis thaliana (AT4G36920.1) was used as the outgroup. Additional file 10: Figure S4. Expression analysis of FmHd3a in all six tissues collected by qRT-PCR. Relative expression levels were calculated using the 2−ΔΔCT method to reflect expressions relatively more veritably. The statistical significance was tested by one-way ANOVA, considering P < 0.05 and P < 0.01 as statistically significant and extremely significant, respectively. Additional Files Significant differences in transcript abundance between different tissues was then compared by Duncan’s multiple range test using SPSS 17.0 (SPSS Inc., Chicago, USA). Additional file 11: Figure S5. Weighted gene co-expression network analysis (WGCNA) of all unigenes identified in the transcriptome of F. macclureana. (a) The phylogenetic tree diagram and the heat map related to the traits. This diagram is divided into three parts: the cluster tree of gene system, the module color of corresponding genes, and the correlation between genes related to each trait in tested samples and its module. The redder the color, the more positive the correlation; conversely, blue is negatively correlated. (b) Gene co- expression network heatmaps drawn by randomly selected 1,500 genes, in which the left and the upper sides are the symmetrical system clustering tree of gene network/module, and the lower right area indicates the dissimilarity between genes, and the smaller the value is, the darker the color is. (c) Module and trait correlation heat map showing the relationship between a module and a given trait. The closer the correlation between a shape and a module is to the absolute value of 1, it is likely that this trait is related to the module gene work. (d) Systematic clustering tree of samples based on unigenes expressions. Additional file 12: Table S7. KEGG enrichment of unigenes in nine significant gene module relating to P- spikelets. Additional file 13: Table S8. 9,296 SSRs identified from 7,668 unigenes. Additional file 14: Figure S6. Principal component analysis (PCA) of unigenes expressions for 18 samples collected from inflorescences in the initial and peak flower stage (I- and P- spikelets), branchlets and leaves of flowering and non-flowering bamboo plants (F/NF-branchlets and F/NF-leaves). Page 20/25 Page 20/25 Additional file 15: Table S9. Primer pairs used for RT-qPCR. Figures Figure 1 Seedlings of Fargesia macclureana flowered shortly after being transferred from the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3,800 m above sea level to a low altitude lab. (a-b) Floret and spikelet of a flowering seedling maintained at the low altitude lab outside the QTP. (c-d) The seedling and shoot of plants growing on the QTP. (e) The original growing environment of F. macclureana. Additional file 15: Table S9. Primer pairs used for RT-qPCR. Additional file 15: Table S9. Primer pairs used for RT-qPCR. Figures Figures Figures Figure 1 Figure 1 Figure 1 Figure 1 Seedlings of Fargesia macclureana flowered shortly after being transferred from the Qinghai–Tibet Plateau (QTP) approximately 2,000 ~ 3,800 m above sea level to a low altitude lab. (a-b) Floret and spikelet of a flowering seedling maintained at the low altitude lab outside the QTP. (c-d) The seedling and shoot of plants growing on the QTP. (e) The original growing environment of F. macclureana. Page 21/25 Page 21/25 Figure 2 Figure 2 Figure 2 Figure 2 Function annotation and classification of unigenes identified from the transcriptome of F. macclureana. (a) Nr Annotation. (b) Clusters of orthologous groups (COG) annotation. Out of 45,516 Nr hits, 13,128 unigenes had a COG classification. A: RNA processing and modification B: Chromatin structure and dynamics C: Energy production and conversion D: Cell cycle control, cell division, chromosome partitioning E: Amino acid transport and metabolism F: Nucleotide transport and metabolism G: Carbohydrate transport and metabolism H: Coenzyme transport and metabolism I: Lipid transport and metabolism J: Translation, ribosomal structure and biogenesis K: Transcription L: Replication, recombination and repair M: Cell wall/membrane/envelope biogenesis N: Cell mobility O: Posttranslational modification, protein turnover, chaperones P: Inorganic ion transport and metabolism Q: Secondary metabolites biosynthesis, transport and metabolism R: General function prediction only S: Function unknown T: Signal transduction mechanism U: Intracellular trafficking, secretion, and vesicular transport V: Defense mechanisms W: Extracellular structures Y: Nuclear structure Z: Cytoskeleton. (c) GO annotation. Results were summarized in three main categories: biological process, cellular component and molecular function. The right and left y-axes indicated the number and percentage of unigenes in a certain category, respectively. Page 22/25 Figure 3 KEGG annotation of unigenes in the transcriptome of F. macclureana. The x-axis indicated the number of unigenes in a certain category. The right y-axis showed the main clusters of KEGG pathways. Figure 6 Densities of different SSR types. c and p1-6 represent mono-, di-, tri-, tetrad-, penta- and hexa-nucleotide repeats, respectively. Figure 3 KEGG annotation of unigenes in the transcriptome of F. macclureana. The x-axis indicated the number of unigenes in a certain category. The right y-axis showed the main clusters of KEGG pathways. KEGG annotation of unigenes in the transcriptome of F. macclureana. The x-axis indicated the number of unigenes in a certain category. The right y-axis showed the main clusters of KEGG pathways. Figure 4 Page 23/25 Page 23/25 Unigenes that were specifically expressed in different tissues collected from flowering plants of F. macclureana. (a) Venn diagram of unigenes expressed in spikelets in the initial flower stage (I-spikelets, A) and the peak flower stage (P-spikelets, B), branchlets (F-branchlets, C) and leaves (F-leaves, D) of flowering plants. (b) COG annotation of unigenes that were specifically expressed in I-spikelets, P-spikelets, F-branchlets and F-leaves. (c) GO enrichment of unigenes that were specifically expressed in I- & P- spikelets, F-branchlets and F-leaves. BP: biological process; CC: cellular component; MF: molecular function. Figure 5 KEGG annotation of unigenes that were specifically expressed in P- spikelets (a), F-branchlets (b) and F-leaves (c) of arrow bamboo flowering plants. The size of dots is proportional to the number of unigenes. Figure 5 KEGG annotation of unigenes that were specifically expressed in P- spikelets (a), F-branchlets (b) and F-leaves (c) of arrow bamboo flowering plants. The size of dots is proportional to the number of unigenes. Figure 6 Densities of different SSR types. c and p1-6 represent mono-, di-, tri-, tetrad-, penta- and hexa-nucleotide repeats, respectively. This is a list of supplementary files associated with this preprint. Click to download. his is a list of supplementary files associated with this preprint. Click to downloa Page 24/25 TableS9.xlsx FigureS6.docx TableS8.xlsx TableS5.xlsx FigureS3.docx TableS6.xlsx TableS3.xlsx FigureS4.docx TableS1.docx FigureS1.docx TableS7.xlsx FigureS2.docx TableS2.xlsx FigureS5.docx TableS4.xlsx FigureS2.docx TableS2.xlsx FigureS5.docx TableS4.xlsx Page 25/25
https://openalex.org/W2592265703	https://link.springer.com/content/pdf/10.1186/s40687-015-0021-1.pdf	English	null	Self-similar singularity of a 1D model for the 3D axisymmetric Euler equations	Research in the mathematical sciences/Research in the Mathematical Sciences	2,015	cc-by	14,949	Introduction and main results Whether the 3D Euler equations develop finite-time singularity is regarded as one of the most important open problems in mathematical fluid mechanics, and interested readers may consult the surveys [2,9,13] and references therein for more historical background about this outstanding problem. In this paper, we investigate the self-similar singularity of a 1D model for the 3D axisymmetric Euler equations, which approximates the dynamics of the axisymmetric Euler equations on the solid boundary of a cylindrical domain. It is hoped that this work may help to analyze the singularity of the 3D Euler equations. The investigated model is motivated by the numerical computation of Luo and Hou [21]. In that computation, the 3D axisymmetric Euler equations [22] are solved in a cylinder, u1,t + uru1,r + uzu1,z = 2u1φ1,z, (1.1a) w1,t + urw1,r + uzw1,z = u2 1 z , (1.1b) − ∂2 r + (3/r)∂r + ∂2 z φ1 = w1, (1.1c) where ur = −rφ1,z and uz = 2φ1 + rφ1,r are radial and axial velocity, and u1 = uθ/r, w1 = wθ/r, and φ1 = φθ/r are transformed angular velocity, vorticity, and stream u1,t + uru1,r + uzu1,z = 2u1φ1,z, (1.1a) w1,t + urw1,r + uzw1,z = u2 1 z , (1.1b) − ∂2 r + (3/r)∂r + ∂2 z φ1 = w1, (1.1c) where ur = −rφ1,z and uz = 2φ1 + rφ1,r are radial and axial velocity, and u1 = uθ/r, w1 = wθ/r, and φ1 = φθ/r are transformed angular velocity, vorticity, and stream function, respectively. 1,t 1,r 1,z 1 z , ( ) − ∂2 r + (3/r)∂r + ∂2 z φ1 = w1, (1.1c) where ur = −rφ1,z and uz = 2φ1 + rφ1,r are radial and axial velocity, and u1 = uθ/r, w1 = wθ/r, and φ1 = φθ/r are transformed angular velocity, vorticity, and stream function, respectively. (1.1c) where ur = −rφ1,z and uz = 2φ1 + rφ1,r are radial and axial velocity, and u1 = uθ/r, w1 = wθ/r, and φ1 = φθ/r are transformed angular velocity, vorticity, and stream function, respectively. According to the numerical results reported in [21], the solutions to (1.1) develop self-similar singularity in the meridian plane for certain initial conditions with no flow boundary condition at r = 1. Thomas Y Hou† and Pengfei Liu† Thomas Y Hou† and Pengfei Liu† Correspondence: plliu@caltech.edu †Equal contribution Computing+Mathematical Sciences, California Institute of Technology, Pasadena, USA © 2015 Hou and Liu; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Abstract We investigate the self-similar singularity of a 1D model for the 3D axisymmetric Euler equations, which approximates the dynamics of the Euler equations on the solid boundary of a cylindrical domain. We prove the existence of a discrete family of self-similar profiles for this model and analyze their far-field properties. The self-similar profiles we find are consistent with direct simulation of the model and enjoy some stability property. Hou and Liu Research in the Mathematical Sciences (2015) 2:5 DOI 10.1186/s40687-015-0021-1 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 DOI 10.1186/s40687-015-0021-1 RESEARCH ARTICLE Self-similar singularity of a 1D model for the 3D axisymmetric Euler equations Correspondence: plliu@caltech.edu †Equal contribution Computing+Mathematical Sciences, California Institute of Technology, Pasadena, USA Self-similar singularity of a 1D model for the 3D axisymmetric Euler equations Thomas Y Hou† and Pengfei Liu*† Introduction and main results The solid boundary and special symmetry of uθ, ωθ, and ψθ in the axial direction seem to make the flow in the meridian plane remain hyperbolic near the singularity point and be responsible for the observed finite-time singularity. A 1D model which approximates the dynamics of the 3D axisymmetric Euler equations along Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 2 of 26 the solid boundary of the cylindrical domain r = 1 has been proposed and investigated by Hou and Luo in [15]. The finite-time singularity of this model is proved very recently by Choi, Hou, Kiselev, Luo, Sverak, and Yao in [6]. Motivated by the new singularity for- mation scenario in [21], Kiselev and Sverak [17] constructed an example of 2D Euler solutions in a setting similar to [21] and proved that the gradient of vorticity exhibits dou- ble exponential growth in time, which is known to be the fastest possible rate of growth for the 2D Euler equations. This example provides further evidence that the new singu- larity formation scenario reported in [21] is an interesting candidate to investigate the 3D Euler singularity. Inspired by the work of [15] and [17], Choi, Kiselev, and Yao proposed the following 1D model (we call it the CKY model for short) [7] on [ 0, 1]: ∂tw + u∂xw = ∂xθ, (1.2a) ∂tθ + u∂xθ = 0, (1.2b) u(x, t) = −x 1 x w(y, t) y dy, (1.2c) w(0, t) = 0, θ(0, t) = 0, ∂xθ(0, t) = 0. (1.2d) (1.2d) This 1D model can be viewed as a simplified approximation to the 1D model proposed by Hou and Luo in [15], and its finite-time singularity from smooth initial data has been proved in [7]. Like the 1D model of Hou and Luo, the CKY model approximates the 3D axisymmetric Euler equations (1.1) on the boundary of the cylinder r = 1 with θ ∼u2 1, w ∼w1, u ∼uz. (1.3) θ ∼u2 1, w ∼w1, u ∼uz. (1.3) The positivity of θx(x, t) near the origin creates a compressive flow which is respon- sible for the finite-time singularity of this model (1.2), and we will use this fact in our construction in the next section. Numerical simulation suggests that this model develops finite-time singularity in a way similar to that of the 3D axisymmetric Euler equations on the boundary of the cylinder [21]. Introduction and main results Moreover, the singular solutions to this model also appear to develop self-similar structure. The main purpose of this paper is to prove the existence of self-similar singular solutions to this CKY model from smooth initial data. We make the following self-similar ansatz to the local singular solutions, θ(x, t) = (T −t)cθ x (T −t)cl , (1.4a) u(x, t) = (T −t)cuU x (T −t)cl , (1.4b) w(x, t) = (T −t)cwW x (T −t)cl . (1.4c) (1.4c) Plugging these self-similar ansatz into (1.2) and matching the exponents of (T −t) for each equation, we get cw = −1, cu = cl −1, cθ = cl −2. (1.5) (1.5) cw = −1, cu = cl −1, cθ = cl −2. Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 3 of 26 Page 3 of 26 And the self-similar profiles U(ξ), W(ξ), and (ξ) satisfy the following equations defined on R+, (2 −cl)(ξ) + clξ′(ξ) + U(ξ)′(ξ) = 0, (1.6a) W(ξ) + clξW ′(ξ) + U(ξ)W ′(ξ) −′(ξ) = 0, (1.6b) U(ξ) = −ξ ∞ ξ W(η) η dη. (1.6c) (1.6b) U(ξ) = −ξ ∞ ξ W(η) η dη. (1.6c) U(ξ) = −ξ ∞ ξ W(η) η dη. (1.6c) According to (1.2d), we require the following boundary condition for the self-similar profiles at ξ = 0, According to (1.2d), we require the following boundary condition for the self-similar profiles at ξ = 0, W(0) = 0, (0) = 0, ′(0) = 0. (1.7a) (1.7a) If we assume that the finite-time singularity of this CKY model is an isolated point sin- gularity, as we have observed in our numerical simulation, then the ansatz (1.4) requires the following matching condition for the self-similar profiles at infinity, (ξ) ∼O ξ1−2/cl , W(ξ) ∼O ξ−1/cl , U(ξ) ∼O ξ1−1/cl , ξ →+∞. (1.7b) (1.7b) We refer (1.6) as the self-similar equations, which can be easily verified to enjoy the following scaling-invariant property: U(ξ) →1 λU(λξ), W(ξ) →W(λξ), (ξ) →1 λ(λξ), λ > 0. (1.8) (1.8) In this paper, we investigate the solutions to the self-similar equations (1.6). Introduction and main results Moreover, W(ξ), U(ξ)ξ−1, and (ξ)ξ−1 are analytic with respect to ζ = ξ−1/cl at ζ = 0. Remark 1.1. We only consider analytic self-similar profiles in our construction, thus our results do not rule out possible existence of self-similar profiles that are non-analytic. Remark 1.1. We only consider analytic self-similar profiles in our construction, thus our results do not rule out possible existence of self-similar profiles that are non-analytic. An interesting fact for this model is that self-similar profiles (1.6) exist for a discrete set of scaling exponent cl, corresponding to different leading orders of (ξ). We also find that these self-similar profiles are consistent with direct simulation of the 1D model and enjoy some stability property in the sense that for fixed leading order of θ(x, 0) at x = 0, the singular solutions using different initial conditions converge to the same set of self-similar profiles. The self-similar profiles we construct are non-conventional in the sense that the velocity does not decay to zero at infinity but grows with certain fractional power. As a result, the velocity field at the singularity time is Hölder continuous. Such behavior was also observed in the numerical simulation of the 3D Euler equations in [15], which is very different from the Leray type of self-similar solutions of the 3D Euler equations, whose existence has been ruled out under certain decay assumptions on the self-similar profiles [3-5]. Our method of analysis is of interest by itself. The existence result relies on the use of a power series method to deal with the singularity of the self-similar equations at the origin, and some very subtle and relatively sharp estimates of the self-similar profiles. However, the method of analysis presented in this paper does not generalize directly to study the singularity formation of the full 3D Euler equations. Due to the global nature of the Biot- Savart law for the 3D Euler equations, we need a new set of techniques to control the nonlinear interaction terms. Another novelty in our analysis is the use of numerical computation with rigorous error control, which is an important step in establishing the existence of self-similar solutions. Our strategy to rigorously control the numerical error, including the truncation error of the integration scheme for an ODE system and the roundoff error introduced due to floating point operation, is quite general and can be used for other purposes. Introduction and main results A key fact for the CKY model is that the velocity and the vorticity field satisfy a local relation (1.9c), and the self-similar equation is equivalent to the following ODE system (2 −cl)(ξ) + clξ′(ξ) + U(ξ)′(ξ) = 0, (1.9a) W(ξ) + clξW ′(ξ) + U(ξ)W ′(ξ) −′(ξ) = 0, (1.9b) U(ξ) ξ ′ = W(ξ) ξ , (1.9c) (2 −cl)(ξ) + clξ′(ξ) + U(ξ)′(ξ) = 0, (1.9a) W(ξ) + clξW ′(ξ) + U(ξ)W ′(ξ) −′(ξ) = 0, (1.9b) (1.9a) (1.9b) U(ξ) ξ ′ = W(ξ) ξ , (1.9c) with a decay condition lim ξ→+∞ U(ξ) ξ = 0. lim ξ→+∞ U(ξ) ξ = 0. (1.10) (1.10) We first ignore the decay condition (1.10) and consider the ODE system (1.9) which has a singularity at the origin since the coefficients of the first-order derivatives vanish at ξ = 0. We confine ourselves to analytic solutions of (1.9) and use the power series method to construct the manifold of local solutions. We prove that for fixed cl and leading order of (ξ) at the origin, there exist unique (up to rescaling) analytic solutions to the singular ODE system, and these local solutions can be extended to the whole R+ through the ODE system (1.9). Then, we show that the decay condition (1.10) determines the scaling exponent cl, and there exist a discrete family of cl, corresponding to different leading orders of (ξ), to make the constructed self-similar profiles satisfy the decay condition (1.10). We achieve this with the assistance of numerical computation and rigorous error control. Given the decay condition (1.10), we further analyze the far-field properties of the constructed self-similar profiles and show that they satisfy the desired matching condition (1.7b) at infinity. Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 4 of 26 Our main result is the following theorem: Our main result is the following theorem: Theorem 1.1. There exist a discrete family of scaling exponents cl (determined by the decay condition (1.10)), such that the self-similar equations (1.6) have analytic solu- tions U(ξ), W(ξ), and (ξ) with boundary and far-field conditions (1.7). This family of solutions correspond to different leading orders of (ξ) at the origin, s ≥2, where s = min k ∈N+\| dk dξk (0) ̸= 0 . (1.11) (1.11) Moreover, W(ξ), U(ξ)ξ−1, and (ξ)ξ−1 are analytic with respect to ζ = ξ−1/cl at ζ = 0. Introduction and main results The rest of this pager is organized as follows. We first construct the local self-similar profiles using a power series method and extend them to the whole R+. Then we show that the decay condition in the Biot-Savart law determines the scaling exponents in the self-similar solutions. After that we prove the existence of self-similar profiles for different leading orders of (ξ) at the origin. We also analyze the far-field behavior of the self- similar profiles. Finally, we present our numerical results. Page 5 of 26 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Construction of the near-field solutions In this section, we ignore the decay condition (1.10) and use a power series method to construct the manifold of local analytic solutions to (1.9). We also show that these local solutions can be extended to the whole R+. The use of power series to analyze analytic differential equations is classical and can be traced back to the Cauchy-Kowalevski Theorem [11,18]. At a regular point of an ODE system, the manifold of local solutions can be parametrized by the initial values of the solution [8]. For the non-linear system (1.9), we consider its analytic solutions near a singular point (the origin) and show that the manifold of local analytic solutions can be parameterized by the values of the leading order of (ξ). Theorem 2.1. For fixed cl > 2, and leading order of (ξ), s ≥2, there exist a unique (up to rescaling) local analytic solution to (1.9) with boundary condition (1.7a). Proof. According to the boundary condition of the self-similar profiles (1.7a), we assume (ξ) = ∞ k=2 kξk, U(ξ) = ∞ k=1 Ukξk, W(ξ) = ∞ k=1 Wkξk. (2.1a) (2.1a) Based on the local relation in the Biot-Savart law (1.9c), we have Wk = kUk+1. (2.1b) (2.1b) Wk = kUk+1. Plugging (2.1) into (1.9) and matching the kth (k ≥1) order term ξk, we get (2 −cl) k + kclk + k−1 m=1 (k −m + 1)k−m+1Um = 0, (2.2a) (2.2a) (k −1)Uk + cl(k −1)2Uk + k−1 m=1 Um(k −m)2Uk−m+1 −kk = 0. (2.2b) (2.2b) Note that if initially the leading order of θ(x, 0) at the origin is s, then according to (1.2b), s will remain as the leading order of the solution θ(x, t) as long as the velocity field is smooth. Correspondingly, we assume that the leading order of (ξ) at the origin is s (1.11). As we have discussed in the introduction section, ∂xθ(x, t) should be positive near x = 0 to produce finite-time singularity, so in the corresponding self-similar profile (2.1a), we require that i = 0 for i < s, s > 0, s ≥2. (2.3) i = 0 for i < s, s > 0, s ≥2. (2.3) To make (2.2a) hold for 1 ≤k ≤s, we require To make (2.2a) hold for 1 ≤k ≤s, we require (2 −cl + scl + sU1) s = 0. (2.4) (2 −cl + scl + sU1) s = 0. Construction of the near-field solutions (2.4) Since s ̸= 0, we require Since s ̸= 0, we require U1 = (1 −s)cl −2 s . (2.5) q U1 = (1 −s)cl −2 s . U1 = (1 −s)cl −2 s . (2.5) (2.5) To make (2.2b) hold for 2 ≤k < s, we require To make (2.2b) hold for 2 ≤k < s, we require To make (2.2b) hold for 2 ≤k < s, we require To make (2.2b) hold for 2 ≤k < s, we require (k −1) + cl(k −1)2 + U1(k −1)2 Uk = 0. (2.6) Since cl > 2, and (k −1) + cl(k −1)2 + U1(k −1)2 > 0, we require (2.6) Uk = 0, 1 < k < s. (2.7) Uk = 0, 1 < k < s. (2.7) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 ch in the Mathematical Sciences (2015) 2:5 Pag Page 6 of 26 And to make (2.2b) hold for k = s, we require And to make (2.2b) hold for k = s, we require Us = s2s (scl −cl −s + 2) (s −1) > 0. (2.8) (2.8) Us = (scl −cl −s + 2) (s −1) > 0 For k > s, to make (2.2) hold, the coefficients k and Uk should satisfy k = −k−1 m=s Um(k −m + 1)k−m+1 (k/s −1)(cl −2) , (2.9a) Uk = kk −k−1 m=s Um(k −m)2Uk−m+1 (k −1) + (cl/s −2/s)(k −1)2 , (2.9b) (2.9a) Uk = kk −k−1 m=s Um(k −m)2Uk−m+1 (k −1) + (cl/s −2/s)(k −1)2 , (2.9b) (2.9b) which means the power series (2.1) can be determined inductively. To complete the proof, we need to verify that the constructed power series (2.1) con- verge for ξ small enough. We choose u0, θ0, r > 0 such that the following condition holds \|Us\| ≤1 s2 u0rs, \|s\| ≤1 s θ0rs, (s + 1)u0r cl/s −2/s ≤1, 9 4 θ0/u0 + u0r cl/s −2/s < 1. (2.10) (2.10) We can achieve this by choosing u0r and θ0/u0 small enough to make the last two hold and then choosing r large enough to make the first two hold. For example, let We can achieve this by choosing u0r and θ0/u0 small enough to make the last two hold and then choosing r large enough to make the first two hold. Construction of the near-field solutions If cl < 2, then cθ < 0 according to (1.5), which means θ(x, t) blows up in finite time according to (1.4). This is impossible since θ(x, t) is transported by the fluid (1.2b). The power series (2.1) that we construct only converge in a short interval near ξ = 0. However, these local self-similar profiles can be extended to +∞. Theorem 2.2. For cl > 2, the analytic solutions (2.1) that we construct in Theorem 2.1 can be extended to the whole R+, resulting in global solutions to the ODE system (1.9). Moreover, we have that for ξ > 0, Theorem 2.2. For cl > 2, the analytic solutions (2.1) that we construct in Theorem 2.1 can be extended to the whole R+, resulting in global solutions to the ODE system (1.9). Moreover, we have that for ξ > 0, Theorem 2.2. For cl > 2, the analytic solutions (2.1) that we construct in Theorem 2.1 can be extended to the whole R+, resulting in global solutions to the ODE system (1.9). Moreover, we have that for ξ > 0, W(ξ) > 0, (ξ) > 0. (2.18) W(ξ) > 0, (ξ) > 0. W(ξ) > 0, (ξ) > 0. (2.18) Proof. Since cl +U1 = (cl −2)/s > 0, s > 0, Ws = (s−1)Us > 0, based on the leading orders of the power series (2.1), we can choose ϵ < 1 r small enough such that clϵ + U(ϵ) > 0, W(ϵ) > 0, (ϵ) > 0. (2.19) (2.19) Then, we consider extending the self-similar profiles from ξ = ϵ to +∞by solving the ODE system with initial conditions given by the power series (2.1). Let ˜U(ξ) = clξ +U(ξ), then according to (1.9), ˜U(ξ), (ξ), and W(ξ) satisfy the following ODE system, ′(ξ) = (cl −2)(ξ) ˜U(ξ) , (2.20a) W ′(ξ) = (cl −2)(ξ) ˜U(ξ)2 −W(ξ) ˜U(ξ) , (2.20b) ˜U(ξ) ξ ′ = W(ξ) ξ . (2.20c) ′(ξ) = (cl −2)(ξ) ˜U(ξ) , (2.20a) W ′(ξ) = (cl −2)(ξ) ˜U(ξ)2 −W(ξ) ˜U(ξ) , (2.20b) (2.20a) ˜U(ξ) ξ ′ = W(ξ) ξ . (2.20c) ˜U(ξ) ξ ′ = W(ξ) ξ . (2.20c) The right hand side of (2.20) is locally Lipschitz continuous for ˜U(ξ) ̸= 0, ξ ̸= 0, so we can solve the ODE system from ϵ and get its solutions on interval [ ϵ, T). Construction of the near-field solutions For example, let A = min cl −2 s(s + 1), 2(cl −2) 9s , B = 2(cl −2) 9s , C = max ss AB , s4s A (scl −cl −s + 2) . (2.11) Then, the choice of Then, the choice of u0 = A C1/(s−1) , θ0 = u0B, r = C1/(s−1), (2.12) (2.12) will make (2.10) hold. And we will use induction to prove that for all k ≥s, \|Uk\| ≤1 k2 u0rk, \|k\| ≤1 k θ0rk. (2.13) (2.13) For k = s, (2.13) holds by (2.10). Assume now that for s ≤k < n, (2.13) holds, then for k = n ≥s + 1, based on (2.9a), we have \|n\| ≤ n−1 m=s \|Um\|\|(n −m + 1)\|\|n−m+1\| (n −s)(cl/s −2/s) . (2.14) (2.14) Using the induction assumption and the fact that ∞ m=2 1 m2 ≤1, we have Using the induction assumption and the fact that ∞ m=2 1 m2 ≤1, we have \|n\| ≤ θ0u0rn+1 (n −s) (cl/s −2/s) ≤θ0rn n × (s + 1)u0r cl/s −2/s ≤θ0rn n , (2.15) (2.15) where we have used the fact n ≥s + 1 in the second inequality and (2.10) in the third inequality. Thus, (2.13) holds for n. Based on (2.9b), we have \|Un\| ≤\|nn\| + n−1 m=s \|Um(n −m)2\|\|Un−m+1\| (cl/s −2/s) (n −1)2 (2.16) (2.16) Using the induction assumption and the fact that ∞ m=2 1 m2 ≤1, we get \|Un\| ≤ θ0rn + (u0)2rn+1 (cl/s −2/s) (n −1)2 ≤u0rn n2 × θ0/u0 + u0r cl/s −2/s × n2 (n −1)2 ≤u0rn n2 , (2.17) (2.17) where we have used (2.10) and the fact that n ≥3, n2/(n−1)2 ≤9/4 in the last inequality. So we get that (2.13) holds by induction, which implies that the power series (2.1) con- verge in some interval [ 0, 1/r). Note that we have one degree of freedom s (2.4) in Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 7 of 26 constructing the power series solutions, which can be easily verified to play the same role as the rescaling parameter (1.8). With this, we complete the proof of Theorem 2.1. Remark 2.1. We require cl > 2 in Theorem 2.1. If cl = 2, there exist only trivial solutions to (1.6). Construction of the near-field solutions We first prove that W(ξ) is positive on [ ϵ, T). Otherwise, denote ξ = t as the first time W(ξ) reaches 0, i.e. t = inf {s ∈[ ϵ, T) : W(s) ≤0} . (2.21) t = inf {s ∈[ ϵ, T) : W(s) ≤0} . (2.21) Then, we have W(ξ) is positive on [ ϵ, t), and Then, we have W(ξ) is positive on [ ϵ, t), and W ′(t) ≤0. (2.22) (2.22) W ′(t) ≤0. Based on (2.20c), ˜U(ξ) ξ is increasing on [ ϵ, t), thus ˜U(ξ) > ˜U(ϵ) > 0 for ξ ∈[ ϵ, t]. Then, based on (2.20a), (ξ) is increasing on [ ϵ, t], and (t) > 0. Evaluating (2.20b) at ξ = t, we get W ′(t) = (cl −2)(t) ˜U(t)2 > 0, (2.23) W ′(t) = (cl −2)(t) ˜U(t)2 > 0, (2.23) which contradicts with (2.22). So, W(ξ) > 0 and consequently (ξ) > 0 for ξ ∈[ ϵ, T). Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 8 of 26 Using the fact that W(ξ) > 0 in (2.20c), we have that for ξ > ϵ, Using the fact that W(ξ) > 0 in (2.20c), we have that for ξ > ϵ, ˜U(ξ) ≥C0ξ. (2.24) ˜U(ξ) ≥C0ξ. ˜U(ξ) ≥C0ξ. Using this lower bound in (2.20a), we get ′(ξ) ≤C1(ξ) ξ . ′(ξ) ≤C1(ξ) ξ . (2.25) This implies that for ξ > ϵ This implies that for ξ > ϵ (ξ) ≤C2ξC1. (2.26) (ξ) ≤C2ξC1. (ξ) ≤C2ξC1. (2.26) Using (2.26), (2.24) and the fact that W(ξ) is positive in (2.20b), we have Using (2.26), (2.24) and the fact that W(ξ) is positive in (2.20b), we have Using (2.26), (2.24) and the fact that W(ξ) is positive in (2.20b), we have W ′(ξ) ≤C3ξC1−2. Thus for ξ > ϵ, Thus for ξ > ϵ, Thus for ξ > ϵ, W(ξ) ≤C4ξC1. (2.28) W(ξ) ≤C4ξC1. Finally using (2.28) in (2.20c), we get that for ξ > ϵ, ˜U(ξ) ≤C5ξC1+1. (2.29) ˜U(ξ) ≤C5ξC1+1. (2.29) The C0, C1,...C5 in the above estimates are positive constants. These a priori esti- mates, (2.24), (2.29), (2.26) and (2.28), together imply that we can get solutions to (2.20) on [ ϵ, +∞), i.e., the local self-similar profiles constructed using power series can be extended to +∞. Determination of the scaling exponents (3.6c) ˆW ′(η) = −ˆW(η) η + 1/cl ˆU(η)η + (1 −2/cl) ˆ(η) (1 + 1/cl ˆU(η))2η3 , (3.6b) ˆW ′(η) = −ˆW(η) η + 1/cl ˆU(η)η + (1 −2/cl) ˆ(η) (1 + 1/cl ˆU(η))2η3 , (3.6b) ˆU′(η) = cl ˆW(η) η . (3.6c) (3.6b) ˆU′(η) = cl ˆW(η) η . (3.6c) (3.6c) According to (2.5) and (2.18) and the fact that ˆU(η) is monotone increasing, we have According to (2.5) and (2.18) and the fact that ˆU(η) is monotone increasing, we have ˆU(η) > ˆU(0) = (1 −s)cl −2 s , ˆW(η) > 0, ˆ(η) > 0, for η > 0. (3.7) ˆU(η) > ˆU(0) = (1 −s)cl −2 s , ˆW(η) > 0, ˆ(η) > 0, for η > 0. (3.7) (3.7) Before proving Theorem 3.1, we will first prove the following two lemmas. Before proving Theorem 3.1, we will first prove the following two lemmas. Lemma 3.1. For all cl > 2, G(cl) > −2. Proof. Assume that for some cl > 2, G(cl) ≤−2. Then, according to (3.7) and the fact that ˆU(η) is increasing, we have that for all η > 0, (1 −s)cl −2 s < ˆU(η) < −2. (3.8) Then, we get (1 −s)cl −2 s < ˆU(η) < −2. (3.8) (1 −s)cl −2 s < ˆU(η) < −2. (3.8) Then, we get Then, we get (2/cl −1) ˆU(η) 1 + 1/cl ˆU(η) ≥2. (3.9) ( ) 2/cl −1) ˆU(η) 1 + 1/cl ˆU(η) ≥2. (3.9) llows from (3.6a) that (2/cl −1) ˆU(η) 1 + 1/cl ˆU(η) ≥2. (3.9) It follows from (3.6a) that ˆ′(η) ≥2 ˆ(η) η . (3.10) ˆ′(η) ≥2 ˆ(η) η . (3.10) By direct integration and (3.7), we have that for η large enough, By direct integration and (3.7), we have that for η large enough, ˆ(η) ≥C1η2. (3.11) ˆ(η) ≥C1η2. (3.11) ˆ(η) ≥C1η2. Using this estimate and (3.7) in (3.6b), we get Using this estimate and (3.7) in (3.6b), we get Using this estimate and (3.7) in (3.6b), we get ˆW ′(η) ≥−C2 ˆW(η) η + C3 η . ˆW ′(η) ≥−C2 ˆW(η) η + C3 η . (3.12) (3.12) This implies This implies ηC2 ˆW(η) ′ ≥C3ηC2−1. ηC2 ˆW(η) ′ ≥C3ηC2−1. (3.13) Then, we have that for η large enough, Then, we have that for η large enough, ηC2 ˆW(η) ≥C3 C2 ηC2 −C4. (3.14) ηC2 ˆW(η) ≥C3 C2 ηC2 −C4. Determination of the scaling exponents In constructing self-similar profiles in the previous section, we did not consider the decay condition (1.10). In this section, we show that the decay condition determines the scaling exponent cl, i.e., only for certain cl do the constructed self-similar profiles satisfy the decay condition. Recall that for fixed leading order of (ξ), s, and s = 1, the constructed profiles U(ξ), (ξ), and W(ξ) depend on cl only. So we can define a function G(cl) as G(cl) = lim ξ→+∞ U(ξ) ξ . G(cl) = lim ξ→+∞ U(ξ) ξ . (3.1) (3.1) We will prove that G(cl) < +∞and it is a continuous function of cl. Then, the exis- tence of cl to make the decay condition (1.10) hold will follow from the intermediate value theorem if we can show that there exist cl l and cr l such that G cl l < 0, G cr l > 0. (3.2) G cl l < 0, G cr l > 0. (3.2) Theorem 3.1. For fixed cl > 2 and leading order of (ξ), s ≥2, construct the power series (2.1) with s = 1 and extend the profiles to R+ by solving (2.20). Then, G(cl) = lim ξ→∞ U(ξ) ξ < +∞, (3.3) G(cl) = lim ξ→∞ U(ξ) ξ < +∞, (3.3) and G(cl) is a continuous function of cl. and G(cl) is a continuous function of cl. We first make the following change of variables, η = ξ1/cl, ˆW(η) = W(ξ), ˆU(η) = U(ξ)ξ−1, ˆ(η) = (ξ)ξ−1+2/cl. (3.4) (3.4) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 9 of 26 Then, we have G(cl) = lim η→+∞ ˆU(η), (3.5) (3.5) G(cl) = lim η→+∞ ˆU(η), and the ODE system satisfied by ˆU(η), ˆ(η), and ˆW(η) is and the ODE system satisfied by ˆU(η), ˆ(η), and ˆW(η) is ˆ′(η) = (2/cl −1) ˆ(η) ˆU(η) η + 1/cl ˆU(η)η , (3.6a) ˆ′(η) = (2/cl −1) ˆ(η) ˆU(η) η + 1/cl ˆU(η)η , (3.6a) ˆW ′(η) = −ˆW(η) η + 1/cl ˆU(η)η + (1 −2/cl) ˆ(η) (1 + 1/cl ˆU(η))2η3 , (3.6b) ˆU′(η) = cl ˆW(η) η . Determination of the scaling exponents Using this lower bound in (3.6c), we get ˆU′(η) ≥C5 η − C6 ηC2+1 . ˆU′(η) ≥C5 η − C6 ηC2+1 . (3.15) (3.15) (3.15) (3.15) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 10 of 26 The constants C in the above estimates are positive and independent of η. The inequal- ity (3.15) implies that ˆU(η) →+∞as η →+∞, which contradicts with G(cl) ≤−2. This completes the proof of Lemma 3.1. We add a subscript cl to indicate the dependence of the profiles on cl for the rest part of this section: ˆUcl(η) = ˆU(η), ˆWcl(η) = ˆW(η), ˆcl(η) = ˆ(η). (3.16) (3.16) Lemma 3.2. Choose s = 1 in constructing the power series (2.1), and extend the local profiles to R+. Then for fixed η, ˆUcl(η), ˆWcl(η), and ˆcl(η) are continuous functions of cl. Proof. We need to prove that for any c0 l > 2, ˆUcl(η), ˆcl(η), and ˆWcl(η) as functions of cl are continuous at cl = c0 l . In our construction of the power series using (2.9), we can easily see that the coefficients Uk and k depend continuously on cl. And based on the condition (2.10), there exist uniform upper bounds of these coefficients \|Uk\| ≤u0rk k2 , \|k\| ≤θ0rk k , (3.17) (3.17) for cl in a neighborhood of c0 l . This means there exists a fixed ϵ small enough, such that ˆWcl(ϵ), ˆcl(ϵ), and ˆUcl(ϵ) are continuous at c0 l . Then, we use the continuous dependence of ODE solutions on initial conditions and parameter to complete the proof of this lemma. Now we begin to prove Theorem 3.1. We use an iterative method which enables us to get shaper estimates of the profiles after each iteration. We finally attain that ˆUcl(η) converges uniformly to G(cl), with which we can complete the proof of this theorem. Proof. Consider c0 l > 2, we will prove that G(c0 l ) < +∞, and G(cl) is continuous at cl = c0 l . According to Lemma 3.1 and Lemma 3.2, there exist η0 large enough and a neigh- borhood of c0 l , I0 = (c1, c2) with c1 > 2, c2 < +∞, such that for cl ∈I0 and η > η0, ˆUcl(η) > ˆUcl(η0) > −2 + ϵ1. (3.18) ˆUcl(η) > ˆUcl(η0) > −2 + ϵ1. Determination of the scaling exponents Putting this upper bound of ˆU(η) back in (3.6b), we have that for cl ∈I0, η > η0, ˆW ′ cl(η) < −C6 ˆWcl(η) η ln η + C3η−1−ϵ2, (3.25) (3.25) which by direct integration gives that for cl ∈I0, η > η0, which by direct integration gives that for cl ∈I0, η > η0, ˆWcl(η) exp η η0 C6 ζ ln ζ dζ < C7. (3.26) Thus, we have that for cl ∈I0 and η > η0, Thus, we have that for cl ∈I0 and η > η0, ˆWcl(η) < C8/ ln η. (3.27) ˆWcl(η) < C8/ ln η. (3.27) ˆWcl(η) < C8/ ln η. (3.27) Using this sharper upper bound of ˆW(η) in (3.6c), we get that for cl ∈I0, η > η0, Using this sharper upper bound of ˆW(η) in (3.6c), we get that for cl ∈I0, η > η0, ˆUcl(η) < C9 ln ln η. (3.28) ˆUcl(η) < C9 ln ln η. ˆUcl(η) < C9 ln ln η. Again putting this sharper upper bound in (3.6b), we have that for cl ∈I0, η > η0, Again putting this sharper upper bound in (3.6b), we have that for cl ∈I0, η > η0, ˆW ′ cl(η) < −C10 ˆWcl(η) η ln ln η + C3η−1−ϵ2. (3.29) ˆW ′ cl(η) < −C10Wcl(η) η ln ln η + C3η−1−ϵ2. (3.29) By direct integration, we get By direct integration, we get By direct integration, we get ˆWcl(η) exp η η0 C11 ζ ln ln ζ dζ < C12. (3.30) (3.30) Since η η0 C11 ζ ln ln ζ dζ > C13(ln η)α −C14 for some α ∈(0, 1), we have that for cl ∈I0, η > η0, η > η0, ˆWcl(η) < C15 exp −C13(ln η)α . (3.31) (3.31) C1, C2, . . . C15 in the above estimates are all positive constants independent of η. Using the upper bound of ˆWcl(η) (3.31) in (3.6c), we conclude that ˆUcl(η) converges uniformly as η →+∞for cl ∈I0 and complete the proof of this theorem. To complete the proof of our main result Theorem 1.1, we still need to verify condi- tion (3.2) for different s. And we leave this part to next section. Determination of the scaling exponents (3.18) Then for cl ∈I0 and η > η0, there exists ϵ2 > 0, such that Then for cl ∈I0 and η > η0, there exists ϵ2 > 0, such that (2/cl −1) ˆUcl(η) 1 + 1/cl ˆUcl(η) < 2 −ϵ2. (3.19) (2/cl −1) ˆUcl(η) 1 + 1/cl ˆUcl(η) < 2 −ϵ2. (3.19) Using this in (3.6a), we have that for cl ∈I0 and η > η0, Using this in (3.6a), we have that for cl ∈I0 and η > η0, ˆ′ cl(η) ≤(2 −ϵ2) ˆcl(η) η . (3.20) ˆ′ cl(η) ≤(2 −ϵ2) ˆcl(η) η . (3.20) Using direct integration and Lemma 3.2, we have that for cl ∈I0, η > η0, Using direct integration and Lemma 3.2, we have that for cl ∈I0, η > η0, ˆcl(η) ≤C1η2−ϵ2. (3.21) ˆcl(η) ≤C1η2−ϵ2. (3.21) ˆcl(η) ≤C1η2−ϵ2. Mathematical Sciences (2015) 2:5 Page 11 of 26 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 11 of 26 Using this upper bound of ˆ(η) in (3.6b), we have that for cl ∈I0, η > η0, Using this upper bound of ˆ(η) in (3.6b), we have that for cl ∈I0, η > η0, ˆW ′ cl(η) ≤ −1 1 + 1/cl ˆUcl(η) ˆWcl(η) η + C3η−1−ϵ2. (3.22) (3.22) The first term in (3.22) is negative according to (3.7) and the second term is integrable for η > η0. Then using Lemma 3.2, we have that for cl ∈I0, η > η0, The first term in (3.22) is negative according to (3.7) and the second term is integrable for η > η0. Then using Lemma 3.2, we have that for cl ∈I0, η > η0, ˆWcl(η) < C4. (3.23) ˆWcl(η) < C4. (3.23) ˆWcl(η) < C4. (3.23) Putting this upper bound in (3.6c) and using Lemma 3.2, we get that for cl ∈I0, η > η0, Putting this upper bound in (3.6c) and using Lemma 3.2, we get that for cl ∈I0, η > η0, Putting this upper bound in (3.6c) and using Lemma 3.2, we get that for cl ∈I0, η > η0, ˆUcl(η) < C5 ln η. (3.24 ˆUcl(η) < C5 ln η. (3.24) ˆUcl(η) < C5 ln η. Existence of self-similar profiles In this section, we verify (3.2) for s = 2, i.e., there exist cl l, cr l > 2, such that G(cl l) < 0, G(cr l) > 0, with which we can complete the first half of Theorem 1.1. Lemma 4.1. Consider solving equations (3.6) with initial conditions given by power series (2.1). For some η0 > 0, let u0 = ˆU(η0), θ0 = ˆ(η0), and w0 = ˆW(η0). Page 12 of 26 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 u0 > 0, then G(cl) > 0. (4.1b) If (4.1b) G(cl) > 0. If u0 > −2, u0 + clw0 + (cl −2) θ0 (u0 + 2) (1 + u0/cl) η2 0 < 0, (4.1c) (4.1c) then G(cl) < 0. (4.1d) (4.1d) G(cl) < 0. G(cl) < 0. Proof. G(cl) = limη→+∞ˆU(η), and ˆU(η) is increasing according to (3.6c) and (2.18). So if u0 > 0, then G(cl) > u0 > 0; and we finish the first part of the Lemma (4.1b). We prove the second part (4.1d) by contradiction. If G(cl) ≥0, then there exists η1 ∈ (η0, +∞] such that ˆU(η1) = 0, and for η ∈(η0, η1), ˆU(η) > u0. According to (3.6a), we have ˆ′(η) ≤(2/cl −1) u0 1 + u0/cl ˆ(η) η . (4.2a) ˆ′(η) ≤(2/cl −1) u0 1 + u0/cl ˆ(η) η . (4.2a) By direct integration, we get that for η ∈(η0, η1), By direct integration, we get that for η ∈(η0, η1), By direct integration, we get that for η ∈(η0, η1), ˆ(η) ≤θ0η (1−2/cl)u0 1+u0/cl 0 η (2/cl−1)u0 1+u0/cl . (4.2b) (4.2b) Using this upper bound of ˆ(η) and the fact that ˆU(η) < 0 for η ∈(η0, η1) in (3.6b), we get Using this upper bound of ˆ(η) and the fact that ˆU(η) < 0 for η ∈(η0, η1) in (3.6b), we get get ˆW(η)η ′ ≤ 1 −2/cl (1 + u0/cl)2 θ0η (1−2/cl)u0 1+u0/cl 0 η −u0−2 1+u0/cl . (4.3a) (4.3a) Since u0 > −2, integrating (4.3a) from η0 to η, we have that for η ∈(η0, η1), Since u0 > −2, integrating (4.3a) from η0 to η, we have that for η ∈(η0, η1), ˆW(η)η ≤w0η0 + 2/cl −1 (1 + u0/cl) (u0/cl −u0 −1)θ0 ⎛ ⎝η−1 0 −η (1−2/cl)u0 1+u0/cl 0 η −u0−1+u0/cl 1+u0/cl ⎞ ⎠. Existence of self-similar profiles (4.3b) (4.3b) Putting this upper bound of ˆW(η) in (3.6c) and integrating it from η0 to η1, we get 0 −u0 = ˆU (η1) −ˆU (η0) ≤clw0 + (cl −2) θ0 (u0 + 2) (1 + u0/cl) η2 0 , (4.4) (4.4) which contradicts (4.1c). Then, we complete the proof of this lemma. We use numerical computation with rigorous error control to verify (4.1a) or (4.1c). Computer programs have been used to prove mathematical theorems including, to name a few, the four color theorem [1], Kepler conjecture [14], and some others [10,16,19]. One method of computer-assisted proof is to use the interval arithmetic and inclusion princi- ple to ensure that the output of a numerical program encloses the solution of the original problem. One first reduces the computation to a sequence of the four elementary opera- tions and then proceeds by replacing numbers with intervals and performing elementary Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 13 of 26 operations between such intervals of representable numbers under appropriate rounding rules. To be precise, assume that x ∈[ xmin, xmax] and y ∈[ ymin, ymax], where xmin, xmin, ymin, and ymax are floating point numbers that can be represented exactly on a computer. Then, for one of the four elementary operations, ⊙∈{+, −, ∗, /}, we have x ⊙y ∈[zmin, zmax] , (4.5a) where (4.5a) x ⊙y ∈[zmin, zmax] , x ⊙y ∈[zmin, zmax] , x ⊙y ∈[zmin, zmax] , where zmin = min xmin⊙ymin, xmin⊙ymax, xmax⊙ymin, xmax⊙ymax , (4.5b) zmax = max xmin⊙ymin, xmin⊙ymax, xmax⊙ymin, xmax⊙ymax , (4.5c) (4.5c) and ⊙and ⊙refer to standard floating point operations with rounding modes set to ‘DOWNWARD’ and ‘UPWARD,’ respectively [23]. Namely, x⊙y is the largest floating number less than x ⊙y, and x⊙y is the smallest floating number larger than x ⊙y. For the case that ⊙is division, we require that 0 /∈[ ymin, ymax]. The RHS of (4.5) involve only floating point operations, so (4.5) allows us to track the numerical errors using computer programs. Using the above interval arithmetic strategy, we first numerically construct the power series (2.1) locally with s = 1 and then extend them to some η0 by solving the ODE system (3.6) to verify condition (4.1a) or (4.1c). Existence of self-similar profiles We only illustrate this computer-assisted proof procedure for the case s = 2 with cl l = 3 and cr l = 8. But the same process can be applied to other s > 2 to verify the existence of self-similar profiles. The computer pro- grams used for this part of proof can be found at https://sites.google.com/site/pengfeiliuc/ home/codes. The case s = 2 and cl = 3 We use the forward Euler scheme [20] to numerically integrate the ODE system (3.6). For a general ODE system with given initial conditions, y = (y1(x), y2(x), . . . yN(x))T, y′(x) = f (x, y), x ∈[ a, b] , y(a) = y0, (4.11) the forward Euler scheme discretizes the domain to finite points, a = x0 < x1 · · · < xm = b with step size xi −xi−1 = h, and the numerical solutions yn ≈y(xn) are obtained by y = (y1(x), y2(x), . . . yN(x))T, y′(x) = f (x, y), x ∈[ a, b] , y(a) = y0, (4.11) the forward Euler scheme discretizes the domain to finite points, a = x0 < x1 · · · < xm = b with step size xi −xi−1 = h, and the numerical solutions yn ≈y(xn) are obtained by y = (y1(x), y2(x), . . . yN(x))T, y′(x) = f (x, y), x ∈[ a, b] , y(a) = y0, (4.11) the forward Euler scheme discretizes the domain to finite points, a = x0 < x1 · · · < xm = b with step size xi −xi−1 = h, and the numerical solutions yn ≈y(xn) are obtained by (4.11) yn+1 = yn + hf (xn, yn). (4.12) (4.12) (4.12) yn+1 = yn + hf (xn, yn). For the solution of the ODE system (4.11), using Taylor expansion, we have For the solution of the ODE system (4.11), using Taylor expansion, we have y (xn+1) = y (xn) + hf (xn, y(xn)) + 1/2 y′′ 1 x∗ 1 , y′′ 2 x∗ 2 , . . . y′′ N x∗ N T h2, (4.13) where x∗ i ∈[xn, xn+1], for i = 1, 2, . . . N. Then, we have y (xn+1) = yn+1 + I1 + I2, (4.14) (4.14) y (xn+1) = yn+1 + I1 + I2, where I1 = ∇yf xn, y∗ (y(xn) −yn) h, (4.15) I2 = 1/2 y′′ 1 x∗ 1 , y′′ 2 x∗ 2 , . . . y′′ N x∗ N T h2, (4.16) (4.15) (4.16) and y∗lies between yn and y(xn). Note that I1 is the propagation of error from the previous steps and I2 is the local truncation error of the integration scheme. The case s = 2 and cl = 3 We verify that for s = 2, G(3) < 0. Step 1 We need to control the numerical error in the local power series solutions. To numerically compute (2.1), we first truncate the power series to finite terms. For the case s = 2 and cl = 3, the following choice of θ0, u0, and r makes (2.10) hold: u0 = 1 9 × 162, θ0 = 1 9 × 9 × 162, r = 162. (4.6) (4.6) d on (3.4), at ξ = 10−3, corresponding to ηs = 10−1, we have ˆU(ηs) = ∞ k=1 Ukη3k−3 s , ˆ(ηs) = ∞ k=2 kη3k−1 s , ˆW(ηs) = ∞ k=1 Wkη3k s . (4.7) (4.7) Using estimates (2.13), if we truncate the power series (4.7) to m = 20 terms, the truncation errors of the three series can be bounded respectively by Using estimates (2.13), if we truncate the power series (4.7) to m = 20 terms, the truncation errors of the three series can be bounded respectively by u0rm+1η3m s (m + 1)2 1 −rη3s , θ0 rη3 s m+1 (m + 1) 1 −rη3s ηs , u0rm+2η3m+2 s (m + 2) 1 −rη3s . (4.8) (4.8) Then, we need to estimate the truncated power series ˆU(ηs) ≈ 20 k=1 Ukη3k−3 s , ˆ(ηs) ≈ 20 k=2 kη3k−1 s , ˆW(ηs) ≈ 20 k=1 Wkη3k s . (4.9) (4.9) Using the interval arithmetic (4.5) strategy in each elementary operation of (2.9), we can inductively get computer-representable intervals enclosing the values of Uk and k Using the interval arithmetic (4.5) strategy in each elementary operation of (2.9), we can inductively get computer-representable intervals enclosing the values of Uk and k ch in the Mathematical Sciences (2015) 2:5 Page Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 14 of 26 for all k ≤21. Then, we use these intervals in computing (4.9) to get intervals enclos- ing the values of the truncated power series (4.9). Finally, we add back the the intervals (4.8) enclosing the truncation errors using interval arithmetic and get intervals strictly enclosing ˆU(ηs), ˆW(ηs), and ˆ(ηs). We denote them as I0 ˆU, I0 ˆW, I0 ˆ, (4.10) I0 ˆU, I0 ˆW, I0 ˆ, (4.10) use them as initial conditions to solve (3.6). and use them as initial conditions to solve (3.6). and use them as initial conditions to solve (3.6). The case s = 2 and cl = 3 We solve (3.6) from ηs = 10−1 to η0 = 3 with step size h = 2.9 × 10−6 and denote the node point and solutions at the nth step as ηn = 0.1 + nh, ˆUn, ˆW n, ˆnT , n = 0, . . . , 106. (4.17) (4.17) We already have I0 ˆU, I0 ˆW, and I0 ˆ (4.10) that enclose ˆU0, ˆW 0, and ˆ0. And we will update In ˆU, In ˆW, In ˆ (4.18) In ˆU, In ˆW, In ˆ (4.18) step by step and make sure that they enclose ˆUn, ˆW n, and ˆn. step by step and make sure that they enclose Un, W n, and n. Step 2 We need to control the roundoff error in computing yn+1 (4.12). In the nth step, we have intervals In ˆU, In ˆW, and In ˆ that enclose the values of the profiles at ηn. To update these intervals, we first choose the middle points of these intervals and use them as the numerical solution yn. Then, we use interval arithmetic to update (4.12) to get intervals enclosing the numerical solutions yn+1 at the n + 1-th step. Step 2 We need to control the roundoff error in computing yn+1 (4.12). In the nth step, we have intervals In ˆU, In ˆW, and In ˆ that enclose the values of the profiles at ηn. To update these intervals, we first choose the middle points of these intervals and use them as the numerical solution yn. Then, we use interval arithmetic to update (4.12) to get intervals enclosing the numerical solutions yn+1 at the n + 1-th step. Step 3 We need to control the propagation of error from previous steps, I1. Note that the values of the profiles at ηn are enclosed in intervals In ˆU, In ˆW, and In ˆ, and we have used their middle points as the numerical solution yn. So we use interval arith- metic to deduct the middle points from these intervals and get intervals enclosing Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 15 of 26 e Mathematical Sciences (2015) 2:5 Page 15 of 26 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 15 of 26 y(xn) −yn in (4.15). The case s = 2 and cl = 3 Then, we need estimates of the Jacobian matrix of RHS of (3.6), which is ∂ ˆW ′(η), ˆU′(η), ˆ′(η) ∂ ˆW, ˆU, ˆ = ⎛ ⎜⎜⎜⎜⎜⎜⎝ −cl clη+ ˆUη cl 4 ˆ−2cl ˆ+ cl+ ˆU η2 ˆW ˆU+cl 3 η3 cl(cl−2) (cl+ ˆU)2η3 cl η 0 0 0 cl(2−cl) ˆ cl+ ˆU 2 η (2−cl) ˆU clη+ ˆUη ⎞ ⎟⎟⎟⎟⎟⎟⎠ (4.19) (4.19) Using intervals In ˆU, In ˆW, and In ˆ and interval arithmetic in computing (4.19), we can get intervals enclosing each entry of ∇yf (x, y∗) in (4.15). Then using interval arithmetic in the matrix-vector multiplication ∇yf (x, y∗) (y(xn) −yn) gives us intervals enclosing I1. Step 4 We need to control the local truncation errors I2 of the scheme, which are 4 We need to control the local truncation errors I2 of the scheme, which are 1 2 ˆU′′ (η1) h2, 1 2 ˆW ′′(η2)h2, 1 2 ˆ′′ (η3) h2, (4.20) with η1, η2, η3 ∈ ηn, ηn+1 . According to (3.6), for cl = 3, we have (4.20) with η1, η2, η3 ∈ ηn, ηn+1 . According to (3.6), for cl = 3, we have ˆW ′′(η) = 3η2 3 + ˆU(η) ˆW(η) 6 + ˆU(η) + 3 ˆW(η) −6 ˆ(η) 6 + 2 ˆU(η) + 3 ˆW(η) η4(3 + ˆU(η))3 , (4.21a) ˆU′′(η) = 9 ˆ(η) −3η2 3 + ˆU(η) 6 + ˆU(η) ˆW(η) η4 3 + ˆU(η) 2 , (4.21b) ˆU′′(η) = 9 ˆ(η) −3η2 3 + ˆU(η) 6 + ˆU(η) ˆW(η) η4 3 + ˆU(η) 2 , (4.21b) ˆ′′(η) = ˆ(η) ˆU(η) 3 + 2 ˆU(η) −9 ˆ(η) ˆW(η) η2 3 + ˆU(η) 2 . (4.21c) ˆ′′(η) = ˆ(η) ˆU(η) 3 + 2 ˆU(η) −9 ˆ(η) ˆW(η) η2 3 + ˆU(η) 2 . (4.21c) (4.21c) To control the local truncation error (4.20), we need the following a priori estimates. Lemma 4.2. Consider the ODE system (3.6) with cl > 2 and initial conditions given by power series (2.1). Assuming that at ηn > 0, the solutions are ˆUn, ˆW n, and ˆn; then for η ∈[ ηn, ηn+1], we have the following a priori estimates, ˆ(η) ∈[θmin, θmax] , ˆU(η) ∈[umin, umax] , ˆW(η) ∈[wmin, wmax] . The case s = 2 and cl = 3 (4.22a) re ˆ(η) ∈[θmin, θmax] , ˆU(η) ∈[umin, umax] , ˆW(η) ∈[wmin, wmax] . (4.22a) ˆ(η) ∈[θmin, θmax] , ˆU(η) ∈[umin, umax] , ˆW(η) ∈[wmin, wmax] . (4.22a) where (4.22a) where where θmax = ˆn ηn+1/ηn2−cl+scl , θmin = ˆn ηn+1/ηn2−cl , (4.22b) umin = ˆUn, wmax = ˆW n + s2clθmaxh (cl −2)(ηn)3 , (4.22c) umax = ˆUn + wmaxh/ηn, wmin = ˆW n −h clwmax η0 (cl + umin). (4.22d) θmin = ˆn ηn+1/ηn2−cl , (4.22b) wmax = ˆW n + s2clθmaxh (cl −2)(ηn)3 , (4.22c) wmin = ˆW n −h clwmax η0 (cl + umin). (4.22d) θmax = ˆn ηn+1/ηn2−cl+scl , umin = ˆUn, umax = ˆUn + wmaxh/ηn, wmin = ˆW n −h clwmax η0 (cl + umin). (4.22d) Proof. According to (3.6a) and the lower bound of ˆU(η) (3.7), we have Proof. According to (3.6a) and the lower bound of ˆU(η) (3.7), we have ˆ′(η) ≤ ˆ(η) η (scl −cl + 2), ˆ′(η) ≥ ˆ(η) η (2 −cl). (4.23) (4.23) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 16 of 26 By direct integration, we can get θmax and θmin. ˆU(η) is increasing according to (3.6c), so we get the lower bound umin. Then using the upper bound θmax and (3.7) in (3.6b), we get ˆW ′(η) ≤ s2clθmax (cl −2)(ηn)3 . (4.24) ˆW ′(η) ≤ s2clθmax (cl −2)(ηn)3 . (4.24) By direct integration, we get the upper bound wmax. Putting the upper bound of ˆW(η) in (3.6c), we get the upper bound of ˆU(η), umax. Using the upper bound wmax and the lower bound umin in (3.6b), we have ˆW ′(η) ≥− clwmax ηn (cl + umin). (4.25) ˆW ′(η) ≥− clwmax ηn (cl + umin). (4.25) By direct integration, we can get the lower bound of ˆW(η), wmin. By direct integration, we can get the lower bound of ˆW(η), wmin. Remark 4.1. The a priori estimates (4.22) that we get are relatively sharp for small h since they deviate from the values of the profiles only by O(h). Remark 4.1. The a priori estimates (4.22) that we get are relatively sharp for small h since they deviate from the values of the profiles only by O(h). We first use intervals In ˆU, In ˆW, and In ˆ and the interval arithmetic in (4.22) to get intervals enclosing the values of the profiles in [ ηn, ηn+1]. ˆ(3) ∈[ 0.934100399788941, 9.34100399819680] , ˆ(3) ∈[ 0.934100399788941, 9.34100399819680] , ˆ(3) ∈[ 0.934100399788941, 9.34100399819680] , from which (4.1c) follows immediately, and we complete the proof that G(3) < 0. from which (4.1c) follows immediately, and we complete the proof that G(3) < 0. Remark 4.2. Since ˆW n, ˆUn, and ˆn are enclosed in the intervals In ˆW, In ˆU, and In ˆ, we can directly use interval arithmetic in (4.12) to get intervals enclosing y(xn)+hf (xn, y(xn)). This strategy avoids estimating the Jacobian matrix ∇yf (x, y) but will amplify the propagation of errors from previous steps and lead to meaningless numerical results for this problem. Remark 4.2. Since ˆW n, ˆUn, and ˆn are enclosed in the intervals In ˆW, In ˆU, and In ˆ, we can directly use interval arithmetic in (4.12) to get intervals enclosing y(xn)+hf (xn, y(xn)). This strategy avoids estimating the Jacobian matrix ∇yf (x, y) but will amplify the propagation of errors from previous steps and lead to meaningless numerical results for this problem. The case s = 2 and cl = 8 The case s = 2 and cl = 3 Then, we can use these intervals and interval arithmetic in (4.21a) to get intervals enclosing the local truncation error (4.20), I2. Step 5 Finally, adding up the intervals enclosing the numerical solutions yn+1 (step 2), the intervals enclosing the propagation of errors from previous steps I1 (step 3), and the intervals enclosing the local truncation error I2 (step 4), we get intervals enclosing the values of the profiles at ηn+1, In+1 ˆW , In+1 ˆU , and In+1 . We keep updating these intervals and finally get intervals enclosing the values of the self-similar profiles at η = 3. They are ˆU(3) ∈[ −1.61167791024607, −1.61167791022341] , ˆU(3) ∈[ −1.61167791024607, −1.61167791022341] , ˆW(3) ∈[ 0.110808868817194, 1.10808868851010] , ˆW(3) ∈[ 0.110808868817194, 1.10808868851010] , Remark 4.3. We only verify the existence of self-similar profiles for s = 2. But the same procedure can be applied to the cases s > 2 without difficulty. Remark 4.3. We only verify the existence of self-similar profiles for s = 2. But the same procedure can be applied to the cases s > 2 without difficulty. The case s = 2 and cl = 8 We verify that for s = 2, G(8) > 0. The verification of G(8) > 0 can be done in the same way. In the construction of the local solutions (2.1), we can easily verify that the choice of u0 = 1 6, 0 = 1 18, r = 6, (4.26) (4.26) makes the constraint (2.10) hold. Then, we truncate the power series (2.1) to the first 20 terms and evaluate them at ηs = 0.7. Using the same technique as the case cl = 3, we can get intervals enclosing the self-similar profiles at ηs = 0.7 and denote them as makes the constraint (2.10) hold. Then, we truncate the power series (2.1) to the first 20 terms and evaluate them at ηs = 0.7. Using the same technique as the case cl = 3, we can get intervals enclosing the self-similar profiles at ηs = 0.7 and denote them as I0 ˆW, I0 ˆU, I0 ˆ. (4.27) I0 ˆW, I0 ˆU, I0 ˆ. (4.27) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 17 of 26 Then, we begin to numerically solve (3.6) using (4.27). We use the same techniques as the previous case to control the numerical errors introduced in each step of the numerical integration and finally get intervals enclosing the profiles at η = 3. They are ˆU(3) ∈[5.66176313743309, 5.66176313745025] , ˆU(3) ∈[5.66176313743309, 5.66176313745025] , ˆW(3) ∈[1.13763978495371, 1.13763978496956] , ˆW(3) ∈[1.13763978495371, 1.13763978496956] , from which (4.1a) follows and we complete the proof that for s = 2, G(8) > 0. from which (4.1a) follows and we complete the proof that for s = 2, G(8) > 0. With G(3) < 0, G(8) > 0, we conclude that there exists a cl such that the self-similar equations (1.6) have analytic solutions with the leading order of (ξ) at ξ = 0 being s = 2. With G(3) < 0, G(8) > 0, we conclude that there exists a cl such that the self-similar equations (1.6) have analytic solutions with the leading order of (ξ) at ξ = 0 being s = 2. Behavior of the self-similar profiles at infinity In this section, we prove that the constructed self-similar profiles satisfy the matching condition (1.7b), and that the profiles are analytic with respect to a transformed variable ζ = ξ−1/cl at ζ = 0. With this, we can complete the proof of Theorem 1.1. This far-field property of the self-similar profiles explains the Hölder continuity of the velocity field at the singularity time that is observed in numerical simulation of this model. Theorem 5.1. For some cl > 2 and s ≥2, if the self-similar profiles constructed using power series (2.1) and extended to the whole R+ satisfy the decay condition (1.10), then the profiles satisfy the matching condition (1.7b). After the following change of variables, (5.1) ˆ(3) ∈[2.54776073991655, 2.54776074039048] , ˆ(3) ∈[2.54776073991655, 2.54776074039048] , ˜U(ζ), ˜W(ζ), and ˜(ζ) are analytic functions at ζ = 0. Our strategy is the following: we first prove that ˜U(ζ), ˜W(ζ), and ˜(ζ) are smooth at [ 0, +∞). Then, we show that there exist analytic solutions to the ODE system of ˜U(ζ), ˜W(ζ), and ˜(ζ) with the same initial conditions at ζ = 0. Finally, we show that smooth solutions to the ODE of ˜U, ˜W, ˜ with the given initial conditions are unique to complete the proof. Proof. If the decay condition (1.10) holds, then ˆU(η) tends to 0 in (3.6), so there exists η0 > 0 such that for η > η0, (2/cl −1) ˆU(η) 1 + 1/cl ˆU(η) ∈(0, 1/2). (5.2) (2/cl −1) ˆU(η) 1 + 1/cl ˆU(η) ∈(0, 1/2). (5.2) Then based on (3.6a), we have that for η > η0, Then based on (3.6a), we have that for η > η0, ˆ′(η) ≤1/2 ˆ(η) η , ˆ′(η) ≤1/2 ˆ(η) η , (5.3) (5.3) which implies that for η > η0, which implies that for η > η0, ˆ(η) ≤C1η1/2. (5.4) ˆ(η) ≤C1η1/2. (5.4) ˆ(η) ≤C1η1/2. (5.4) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 18 of 26 Using this estimate in (3.6b), we have that for η > η0, Using this estimate in (3.6b), we have that for η > η0, (5.5) ˆW(η)η ′ ≤C2η−3/2, which gives ˆW(η)η < C3. (5.6) ˆW(η)η < C3. (5.6) ˆW(η)η < C3. (5.6) Using the above estimate in (3.6c), we get that for η > η0, ˆU′(η) ≤C4η−2, (5.7) ˆU′(η) ≤C4η−2, (5.7) which together with ˆU(+∞) = 0 implies that for η > η0, ˆU(η) ≥−C5η−1. (5.8) ˆU(η) ≥−C5η−1. (5.8) Based on (3.6b) and (3.6c), we have ˆ′(η) = (2/cl −2) ˆ(η) ˆU(η) η + 1/cl ˆU(η)η , ˆW(η)η ′ = 1/cl ˆU(η) ˆW(η) 1 + 1/cl ˆU(η) + (1 −2/cl) ˆ(η) 1 + 1/cl ˆU(η) 2 η2 . (5.9) (5.9) Using (5.8), (5.6) and (5.4) in (5.9), we can see that \| ˆ′(η)\| and \|( ˆW(η)η)′\| are both integrable from η0 to +∞, thus ˆ(η) and ˆW(η)η converge as η →+∞, lim η→∞ˆW(η)η = ˆW∞∈[ 0, +∞), lim η→∞ ˆ(η) = ˆ∞∈(0, +∞). ˜U(ζ), ˜W(ζ), and ˜(ζ) are analytic functions at ζ = 0. (5.10) (5.10) Based on (3.6c) and the fact that ˆU(+∞) = 0, we have Based on (3.6c) and the fact that ˆU(+∞) = 0, we have Based on (3.6c) and the fact that ˆU(+∞) = 0, we have Based on (3.6c) and the fact that ˆU(+∞) = 0, we have lim η→+∞ ˆU(η)η = −cl ˆW∞. (5.11) lim η→+∞ ˆU(η)η = −cl ˆW∞. (5.11) The above limits imply that after changing variables, ˜U(ζ), ˜(ζ), and ˜W(ζ) are continuous for ζ ∈[ 0, +∞). The ODE system they satisfy for ζ ∈(0, +∞) is ˜′(ζ) = (2/cl −1) ˜(ζ) ˜U(ζ) −1 −˜U(ζ)ζ , (5.12a) ˜′(ζ) = (2/cl −1) ˜(ζ) ˜U(ζ) −1 −˜U(ζ)ζ , (5.12a) ˜W ′(ζ) = 1/cl ˜U(ζ) ˜W(ζ) + (1 −2/cl) ˜(ζ) −1/cl ˜′(ζ)ζ −1 −˜U(ζ)ζ , (5.12b) ˜U′(ζ) = − ˜U(ζ) ζ −cl ˜W(ζ) ζ , (5.12c) (5.12a) ˜W ′(ζ) = 1/cl ˜U(ζ) ˜W(ζ) + (1 −2/cl) ˜(ζ) −1/cl ˜′(ζ)ζ −1 −˜U(ζ)ζ , (5.12b) ˜U′(ζ) = − ˜U(ζ) ζ −cl ˜W(ζ) ζ , (5.12c) ˜W ′(ζ) = 1/cl ˜U(ζ) ˜W(ζ) + (1 −2/cl) ˜(ζ) −1/cl ˜′(ζ)ζ −1 −˜U(ζ)ζ , (5.12b) ˜U′(ζ) = − ˜U(ζ) ζ −cl ˜W(ζ) ζ , (5.12c) (ζ) = − ˜U(ζ) ζ −cl ˜W(ζ) ζ , with initial conditions given by (5.10) and (5.11), with initial conditions given by (5.10) and (5.11), ˜W(0) = ˆW∞, ˜(0) = ˆ∞, ˜U(0) = −cl ˆW∞. (5.12d) (5.12c) can be written as ˜U(ζ) = −cl ζ ζ 0 ˜W(η)dη. ˜U(ζ) = −cl ζ ζ 0 ˜W(η)dη. (5.13) (5.13) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 19 of 26 Using a simple bootstrap argument, we can get ˜W(ζ), ˜(ζ), and ˜U(ζ) are in C∞ [ 0, +∞) . On the other hand, given the initial conditions (5.12d), we can construct the following power series solutions to (5.12): ˜U(ζ) = −cl ˆW∞+ ∞ k=1 ˜Ukζ k, ˜W(ζ) = ˆW∞+ ∞ k=1 ˜Wkζ k, ˜(ζ) = ˆ∞+ ∞ k=1 ˜kζ k. (5.14) (5.14) Plugging these power series ansatz in (5.12) and matching the coefficients of ζ k, we can uniquely determine the coefficients ˜Uk, ˜Wk, and ˜k and prove that the power series (5.14) converge in a small neighborhood of ζ = 0. We omit the details here, because the argument is the same as that in our construction of the near field solutions. ˜U(ζ), ˜W(ζ), and ˜(ζ) are analytic functions at ζ = 0. Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 20 of 26 The above theorem implies that the self-similar profiles that we construct are non- conventional in the sense that the velocity does not decay to 0 at +∞but grows with certain fractional power. Coming back to the self-similar ansatz (1.4), we have u(x, t) = (T −t)cl−1U x (T −t)cl . (5.22) (5.22) For t close to T, based on Theorem 5.1, we have For t close to T, based on Theorem 5.1, we have For t close to T, based on Theorem 5.1, we have u(x, t) ≈C(T −t)cl−1 x (T −t)cl x (T −t)cl −1 cl = Cx1−1 cl . (5.23) (5.23) This explains the Hölder continuity of the velocity at the singularity time observed in numerical simulation of the 1D model, which was also observed for the 3D Euler equations [15]. We will numerically verify this in the next section. ˜U(ζ), ˜W(ζ), and ˜(ζ) are analytic functions at ζ = 0. Then to prove the analyticity of ˜U(ζ), ˜W(ζ), and ˜(ζ) at ζ = 0, we only need the uniqueness of smooth solutions to (5.12) with initial condition (5.12d). Assume that ˜Ui(ζ), ˜W i(ζ), ˜i(ζ), i = 1, 2 are two different solutions to (5.12) with initial condition (5.12d). And let δU(ζ), δW(ζ), and δ(ζ) be the difference of the two solutions, δ ˜U(ζ) = ˜U1(ζ) −˜U2(ζ), δ ˜W(ζ) = ˜W 1(ζ) −˜W 2(ζ), δ ˜(ζ) = ˜1(ζ) −˜2(ζ). (5.15) δ ˜U(ζ) = ˜U1(ζ) −˜U2(ζ), δ ˜W(ζ) = ˜W 1(ζ) −˜W 2(ζ), δ ˜(ζ) = ˜1(ζ) −˜2(ζ). (5.15) Then based on (5.12c), Then based on (5.12c), δ ˜U(ζ) = −cl ζ ζ 0 δ ˜W(τ)dτ. (5.16) (5.16) Using Hardy inequality [12], there exists C1 independent of ϵ such that ∥δ ˜U∥L2([0,ϵ]) ≤C1∥δ ˜W∥L2([0,ϵ]). (5.17) ∥δ ˜U∥L2([0,ϵ]) ≤C1∥δ ˜W∥L2([0,ϵ]). (5.17) Since the RHS of (5.12a) and (5.12b) are Lipschitz continuous, we have d dζ δ ˜W(ζ) + d dζ δ ˜(ζ) ≤C2 δ ˜W(ζ) \|+\| δ ˜U(ζ) \|+\| δ ˜(ζ) (5.18) (5.18) Integrating the square of both sides on the interval [ 0, ϵ] and using (5.17), we get we get δ ˜W(ζ) ′ L2([0,ϵ]) + δ ˜(ζ) ′ L2([0,ϵ]) ≤C3 δ ˜W(ζ) L2([0,ϵ])+ δ ˜(ζ) L2([0,ϵ]) . (5.19) Since δ ˜W(ζ) and δ ˜(ζ) vanish at ζ = 0, by Poincaré-Friedrichs inequality, we have δ ˜W(ζ) L2([0,ϵ]) + δ ˜(ζ) L2([0,ϵ]) ≤C4ϵ (δ ˜W(ζ))′ L2([0,ϵ]) + (δ ˜(ζ))′ L2([0,ϵ]) . (5.20) Since δ ˜W(ζ) and δ ˜(ζ) vanish at ζ = 0, by Poincaré-Friedrichs inequality, we have δ ˜W(ζ) L2([0,ϵ]) + δ ˜(ζ) L2([0,ϵ]) ≤C4ϵ (δ ˜W(ζ))′ L2([0,ϵ]) + (δ ˜(ζ))′ L2([0,ϵ]) . (5.20) The Ci in the above estimates are all positive constants independent of ϵ. Choosing ϵ small enough, we get a contradiction between (5.19) and (5.20), thus ˜W 1 = ˜W 2, ˜U1 = ˜U2, ˜1 = ˜2, (5.21) ˜W 1 = ˜W 2, ˜U1 = ˜U2, ˜1 = ˜2, (5.21) ˜W 1 = ˜W 2, ˜U1 = ˜U2, ˜1 = ˜2, (5.21) which means the solution is unique. And we complete the proof of this theorem. which means the solution is unique. And we complete the proof of this theorem. Numerical results In this section, we numerically locate the root of G(cl) for several s and construct the corresponding self-similar profiles. The obtained cl and self-similar profiles are consistent with numerical simulation of the CKY model. We also find that for fixed leading order of θ(x, 0), the singular solutions using different initial conditions converge to the same self-similar profiles, which implies that the self-similar profiles have some stability. Numerical methods for simulating the model We use a particle method to simulate the model and consider N+1 particles with position, density, and vorticity given by ⎧ ⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎩ q = (q0(t), q1(t), . . . qN(t))T , θ = (θ0(t), θ1(t), . . . θN(t))T , w = (w0(t), w1(t), . . . wN(t))T . (6.3) (6.3) In computing the velocity field, we use the trapezoidal rule to approximate (1.6c), In computing the velocity field, we use the trapezoidal rule to approximate (1.6c ui = −qi ⎛ ⎝ N−1 j=i wj + wj+1 2 qj+1 −qj ⎞ ⎠. (6.4) (6.4) In computing θx, we use the three-point rule: n computing θx, we use the three-point rule: (θx)i = ⎧ ⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ 0, i = 0, θi −θi+1 qi −qi+1 + θi −θi−1 qi −qi−1 + θi+1 −θi−1 qi+1 −qi−1 , 0 < i < N, θi −θi−2 qi −qi−2 + θi −θi−1 qi −qi−1 + θi−2 −θi−1 qi−2 −qi−1 , i = N. (6.5) i = 0, (θx)i = ⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ θi −θi+1 qi −qi+1 + θi −θi−1 qi −qi−1 + θi+1 −θi−1 qi+1 −qi−1 , 0 < i < N, θi −θi−2 qi −qi−2 + θi −θi−1 qi −qi−1 + θi−2 −θi−1 qi−2 −qi−1 , i = N. (6.5) (6.5) Initially, 105 + 1 particles are equally placed in the short interval [ 0, 10−3], which are sufficient to resolve the solutions in the self-similar regime. Outside this short interval, 105 −102 particles are equally placed. So the total number of particles is N + 1 = 2 × 105 −102. Then, we need to solve the following ODE system d dt q = u, d dt w = θx, d dt θ = 0. (6.6) (6.6) The initial condition of θ is θ(x, 0) = (1 −cos(πx))s/2, (6.7) θ(x, 0) = (1 −cos(πx))s/2, (6.7) whose leading order at x = 0 is s. whose leading order at x = 0 is s. whose leading order at x = 0 is s. We solve the ODE system (6.6) using the fourth order explicit Runge-Kutta method, and the time step dt is chosen adaptively to avoid the particles crossing each other: dti = 1 max ui−ui+1 qi+1−qi , 0 , dt = min dti 10 , 10−3 . (6.8) (6.8) At each time step, we record the maximal vorticity wmax(ti) and the position where it is attained qmax(ti). Numerical methods for solving the self-similar equations For fixed cl > 2, we first numerically compute the coefficients k and Uk in (2.1) up to k = 50 and determine the convergence radius of the power series using the following linear regression for s ≤k ≤50, log k = k log r1 + c1, log Uk = k log r2 + c2. (6.1) (6.1) We choose r = 1/2 min{1/r1, 1/r2} and construct the truncated power series (2.1) on [ 0, r/2]. Then, we continue to solve (1.9) from ξ = r/2 to ξ = 1 using the fourth order explicit Runge-Kutta method with step-size h = 1−r/2 104 . After ξ = 1, we make the change of variables (3.4) and solve (3.6) from η = 1 to η = 105 using fourth order Runge-Kutta method with step-size h = 105−1 106 . We use ˆUcl(105) as an approximation to G(cl). We use the bisection method to find the root of G(cl). After getting cl, we construct the local self-similar profiles using power series (2.1) and extend them from ξ = r/2 to ξ = 10 using the explicit fourth order Runge-Kutta method with step-size h = 9 104 . Then, we locate the maxima of W, which is Wmax = W(ξ0). For the cases that we consider, s = 2, 3, 4, 5, the located ξ0 are all less than 10. Finally, we rescale the maxima of W(ξ) to (1, 1) and get Ws(ξ) = 1 Wmax W(ξξ0), ξ ∈[ 0, 1] . (6.2) (6.2) We only compare the self-similar profiles Ws with direct simulation of the CKY model in this paper, but the numerical results for the profiles and U are similar. Mathematical Sciences (2015) 2:5 Page 21 of 26 Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 21 of 26 Numerical methods for simulating the model Numerical methods for simulating the model Numerical methods for simulating the model According to the self-similar ansatz (1.4), we have wmax(t) = C1(T −t)cw, qmax(t) = C2(T −t)cl. (6.9) wmax(t) = C1(T −t)cw, qmax(t) = C2(T −t)cl. (6.9) (6.9) Thus we can compute cl, cw, and the singularity time T through linear regressions, d dt log wmax(t) −1 ≈−1 cw t + T cw , (6.10a) d dt log qmax(t) −1 ≈−1 cl t + T cl . (6.10b) (6.10a) (6.10b) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 22 of 26 Table 1 cw obtained from linear regression (6.10a) s = 2 s = 3 s = 4 s = 5 cw −0.9747 −1.0001 −1.0006 −1.0007 We compute the time derivatives of log wmax(t) and log qmax(t) using the center differ- ence method, and the linear regressions (6.10) are done in some time interval close to the singularity time while the numerical solutions still have good accuracy. At certain time steps close to the singularity time, ti, i = 1, 2, 3, let wi be the maximal vorticity at time ti and qi be the position the maximal vorticity is attained. We rescale the numerical solution and get the self-similar profiles of w, W i s(ξ) = 1 wmax w ξqi, ti , ξ ∈[ 0, 1] . (6.11) (6.11) We will compare the self-similar profiles W i s(ξ) (6.11) obtained from direct simulation of the model, with Ws(ξ) (6.2) obtained from solving the self-similar equations (1.6). At the singularity time, the velocity field is Hölder continuous near the origin, u(x, T) ≈Cxα. (6.12) (6.12) u(x, T) ≈Cxα. Then, we can determine the Hölder exponent α through linear regression Then, we can determine the Hölder exponent α through linear regression ln u(x, T) ≈ln C + α ln x. (6.13) (6.13) ln u(x, T) ≈ln C + α ln x. We will compare the exponents α (6.13) obtained from the singular solutions, with 1 − 1/cl (5.23) obtained from analyzing the self-similar equations (1.6). Comparison results Comparison results In simulating the CKY model, we first choose w(x, 0) as In simulating the CKY model, we first choose w(x, 0) as w(x, 0) = 1 −cos(4πx). (6.14) (6.14) w(x, 0) = 1 −cos(4πx). We compute the scaling exponents cw and cl for different leading orders of θ, s = 2, 3, 4, 5 using (6.10a) and (6.10b), and the results are listed in Tables 1 and 2. The Hölder exponents of the velocity field at the singularity time (6.13) and 1 −1/cl are listed in Table 3, where the cl are obtained from solving the self-similar equations. For s = 2, the linear regressions (6.10a) and (6.10b) are done in the time interval 6.4371 × 10−1, 6.4391 × 10−1 , (6.15) 6.4371 × 10−1, 6.4391 × 10−1 , (6.15) the predicted singularity time T for (6.10a) and (6.10b) are both 6.4402×10−1. The linear regression (6.13) is done at t = 6.4391 × 10−1 and on the interval [ 10−10, 10−9]. F 3 th li i (6 10 ) d (6 10b) d i th ti i t l regression (6.13) is done at t 6.4391 × 10 and on the interval [ 10 , 10 ]. For s = 3, the linear regressions (6.10a) and (6.10b) are done in the time interval For s = 3, the linear regressions (6.10a) and (6.10b) are done in the time interval 6.804297 × 10−1, 6.804300 × 10−1 , (6.16) (6.16) 6.804297 × 10−1, 6.804300 × 10−1 , 6.804297 × 10−1, 6.804300 × 10−1 , the predicted singularity time T for (6.10a) and (6.10b) are both 6.804302 × 10−1. The linear regression (6.13) is done at t = 6.804302 × 10−1 and on the interval [ 10−10, 10−9]. Comparison results Table 2 cl obtained from linear regression (6.10b) and self-similar equations (1.6) s = 2 s = 3 s = 4 s = 5 Linear Regression 3.7942 3.3143 3.1718 3.0773 Self-Similar Equations 3.7967 3.3157 3.1597 3.0841 Table 2 cl obtained from linear regression (6.10b) and self-similar equations (1.6) s = 2 s = 3 s = 4 s = 5 Linear Regression 3.7942 3.3143 3.1718 3.0773 Self-Similar Equations 3.7967 3.3157 3.1597 3.0841 Table 2 cl obtained from linear regression (6.10b) and self-similar equations (1.6) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 23 of 26 Table 3 Hölder exponent of the velocity field at x = 0, and 1 −1/cl s = 2 s = 3 s = 4 s = 5 Hölder exponent 7.3381 × 10−1 6.9823 × 10−1 6.9131 × 10−1 6.7610 × 10−1 1 −1/cl 7.3661 × 10−1 6.9841 × 10−1 6.8351 × 10−1 6.7576 × 10−1 For s = 4, the linear regressions (6.10a) and (6.10b) are done in the time interval 6.571218 × 10−1, 6.571221 × 10−1 , (6.17) the predicted singularity time T for (6.10a) and (6.10b) are both 6.571223 × 10−1. The linear regression (6.13) is done at t = 6.571223 × 10−1 and on the interval [ 10−10, 10−9]. For s = 5, the linear regressions (6.10a) and (6.10b) are done in the time interval 5.9698511 × 10−1, 5.9698515 × 10−1 , (6.18) the predicted singularity time T for (6.10a) and (6.10b) are both 5.9698517 × 10−1. The linear regression (6.13) is done at t = 5.9698517×10−1 and on the interval [ 10−10, 10−9]. From the Tables 1, 2, and 3, we can see that the exponents cw we obtain from the sin- gular numerical solutions are close to −1 (1.5). And the cl we obtain from the singular solution (6.10b) are close to those obtained from solving the self-similar equations. At the singularity time, the Hölder exponents of the velocity field are close to 1 −1/cl. For the case s = 2, the dependence of G(cl) on cl is plotted in Figure 1. We can see that G(cl) seems to be a monotone increasing function, which implies that for fixed s, the scaling exponent cl to make the decay condition (1.10) hold is unique. Comparison results The self-similar profiles that are obtained from solving the self-similar equation (6.2) and from direct simulation of the model (6.11) are plotted in Figure 2. The lines labeled ‘exact’ are obtained from solving the self-similar equation (6.2). Others are obtained from rescaling the solution at different time steps corresponding to different maximal vorticity (6.11). Table 3 Hölder exponent of the velocity field at x = 0, and 1 −1/cl s = 2 s = 3 s = 4 s = 5 Hölder exponent 7.3381 × 10−1 6.9823 × 10−1 6.9131 × 10−1 6.7610 × 10−1 1 −1/cl 7.3661 × 10−1 6.9841 × 10−1 6.8351 × 10−1 6.7576 × 10−1 For s = 4, the linear regressions (6.10a) and (6.10b) are done in the time interval 6.571218 × 10−1, 6.571221 × 10−1 , ( For s = 4, the linear regressions (6.10a) and (6.10b) are done in the time interval (6.17) 5.9698511 × 10−1, 5.9698515 × 10−1 , (6.18) the predicted singularity time T for (6.10a) and (6.10b) are both 5.9698517 × 10−1. The linear regression (6.13) is done at t = 5.9698517×10−1 and on the interval [ 10−10, 10−9]. From the Tables 1, 2, and 3, we can see that the exponents cw we obtain from the sin- gular numerical solutions are close to −1 (1.5). And the cl we obtain from the singular solution (6.10b) are close to those obtained from solving the self-similar equations. At the singularity time, the Hölder exponents of the velocity field are close to 1 −1/cl. For the case s = 2, the dependence of G(cl) on cl is plotted in Figure 1. We can see that G(cl) seems to be a monotone increasing function, which implies that for fixed s, the scaling exponent cl to make the decay condition (1.10) hold is unique. The self-similar profiles that are obtained from solving the self-similar equation (6.2) and from direct simulation of the model (6.11) are plotted in Figure 2. The lines labeled ‘exact’ are obtained from solving the self-similar equation (6.2). Others are obtained from rescaling the solution at different time steps corresponding to different maximal vorticity (6.11). Figure 1 Dependence of G(cl) on cl for s = 2. Figure 1 Dependence of G(cl) on cl for s = 2. Comparison results Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 24 of 26 (A) (B) (C) (D) Figure 2 Self-similar profiles of w using initial condition w(x,0) = 1 −cos(4πx). (A) The re-scaled solutions and self-similar profiles we construct s = 2. (B) The re-scaled solutions and self-similar profiles we construct s = 3. (C) The re-scaled solutions and self-similar profiles we construct s = 4. (D) The re-scaled solutions and self-similar profiles we construct s = 5. (A) (C) (B) (C) (D) (C) (D) Figure 2 Self-similar profiles of w using initial condition w(x,0) = 1 −cos(4πx). (A) The re-scaled solutions and self-similar profiles we construct s = 2. (B) The re-scaled solutions and self-similar profiles we construct s = 3. (C) The re-scaled solutions and self-similar profiles we construct s = 4. (D) The re-scaled solutions and self-similar profiles we construct s = 5. To demonstrate the stability the self-similar profiles, we consider another initial condition, To demonstrate the stability the self-similar profiles, we consider another initial condition, w(x, 0) = x −x2. (6.19) w(x, 0) = x −x2. (6.19) The profiles obtained from rescaling the singular solutions (6.11) are plotted in Figure 3. From Figures 2 and 3, we can see that after rescaling, the singular solutions at differ- ent time steps before the singularity time are very close, which implies that the solutions develop self-similar singularity. Besides, the self-similar profiles obtained from direct sim- ulation of the model (6.11) agree very well with the self-similar profiles (6.2) we construct by solving the self-similar equations (1.6). Moreover, for fixed leading order of θ(x, 0) at the origin, the singular solutions with different initial conditions converge to the same set of self-similar profiles, which implies that the profiles have some stability property. Remark 6.1. If the initial leading order of θ(x, 0) is s ≥3, and a small perturbation of θ, which we denote by ϵ ˜θ(x, t), has leading order 2 ≤˜s < s, then the profiles of the perturbed singular solutions will be determined by ˜s, not s. From this point of view, only the self-similar profiles for s = 2 are stable in the sense of perturbation. Concluding remarks The existence of a discrete family of analytic self-similar profiles corresponding to differ- ent leading orders of the solutions at the origin for the CKY model has been established. The profiles are constructed using a power series method near the origin and then Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 25 of 26 (A) (B) Figure 3 Self-similar profiles of w using initial condition w(x,0) = x −x2. (A) s = 2. (B) s = 3. (A) (B) Figure 3 Self-similar profiles of w using initial condition w(x,0) = x −x2. (A) s = 2. (B) s = 3. (B) Figure 3 Self-similar profiles of w using initial condition w(x,0) = x −x2. (A) s = 2. (B) s = 3. extended to infinity by solving an ODE system. The decay condition in the Biot-Savart law determines the scaling exponents in the self-similar solutions. Numerical compu- tation together with rigorous error estimation is used to prove the existence of these self-similar profiles. Far-field properties of the self-similar profiles are analyzed. The constructed self-similar profiles are consistent with direct simulation of the model and enjoy some stability property. The current method of analysis does not generalize directly to study the 3D Euler singularity, and a new set of techniques are required to deal with the non-local Biot-Savart law. The existence of self-similar singularity for the 3D Euler equations is under investigation. Acknowledgements h h ld l k g The authors would like to thank Professors Russel Caflisch and Guo Luo for a number of stimulating discussions. We would also like to thank Professors Alexander Kiselev and Yao Yao for their interest in our work and for their valuable comments. The research was in part supported by NSF FRG Grant DMS-1159138. Received: 27 July 2014 Accepted: 13 February 2015 Received: 27 July 2014 Accepted: 13 February 2015 References l Hou, TY, Luo, G: On the f arXiv:1311.2613 (2013) 17. Kiselev, A, Sverak, V: Small scale c preprint arXiv:1310.4799 (2013) 18. Kovalevskaja, SV: Zur theorie der partiellen differentialgleichungen (1874) 18. Kovalevskaja, SV: Zur theorie der partiellen differentialgleichungen (1874) j g g 19. Lanford, OE III: A computer-assisted proof of the feigenbaum conjectures. Bull. Am. Math. Soc. 6(3), 427–434 (1982) 20 LeVeque RJ Finite difference methods for ordinary and partial differential equations steady state and 19. Lanford, OE III: A computer-assisted proof of the feigenbaum conjectures. Bull. Am. Math. Soc. 6(3), 427–434 19. Lanford, OE III: A computer-assisted proof of the feigenbaum conjectures. Bull. Am. Math. Soc. 6(3), 427–434 19. Lanford, OE III: A computer-assisted proof of the feigenbaum , p p g j ( ), ( ) 20. LeVeque, RJ: Finite difference methods for ordinary and partial differential equations: steady-state and 20. LeVeque, RJ: Finite difference methods for ordinary and partial differential equations: steady-state and time-dependent problems, volume 98. Siam (2007) 20. LeVeque, RJ: Finite difference methods for ordinary and partial differential equations: steady-state and d d bl l S ( ) 20. LeVeque, RJ: Finite difference methods for ordinary a time-dependent problems, volume 98. Siam (2007) 21. Luo, G, Hou, TY. Potentially singular solutions of the 3d incompressible euler equations. arXiv preprint arXiv:1310.0497 (2013) j , , , y p , g y ( ) 23. Zuras, D, Cowlishaw, M, Aiken, A, Applegate, M, Bailey, D, Bass, S, Bhandarkar, D, Bhat, M, Bindel, D, Boldo, S, Canon, S, Carlough, SR, Cornea, M, Cowlishaw, M, Crawford, JH, Darcy, JD, Das Sarma, D, Daumas, M, Davis, B, Davis, M, Delp, D, Demmel, J, Erle, MA, Fahmy, HAH, Fasano, JP, Fateman, R, Feng, E, Ferguson, WE, Fit-Florea, A, Fournier, L, et al: IEEE standard for floating-point arithmetic. IEEE Std 754-2008, 1–70 (2008) 23. Zuras, D, Cowlishaw, M, Aiken, A, Applegate, M, Bailey, D, Bass, S, Bhandarkar, D, Bhat, M, Bindel, D, Boldo, S, Canon, S, Carlough, SR, Cornea, M, Cowlishaw, M, Crawford, JH, Darcy, JD, Das Sarma, D, Daumas, M, Davis, B, Davis, M, Delp, D, Demmel, J, Erle, MA, Fahmy, HAH, Fasano, JP, Fateman, R, Feng, E, Ferguson, WE, Fit-Florea, A, Fournier, L, et al: IEEE standard for floating-point arithmetic. References l References 1. Appel, K, Haken, W: Proof of 4-color theorem. Discrete Mathematics. 16(2), 179–180 (1976) Hou and Liu Research in the Mathematical Sciences (2015) 2:5 Page 26 of 26 2. Bardos, C, Titi, E: Euler equations for incompressible ideal fluids. Russ. Math. Surveys. 62(3), 409 (2007) 3. Chae, D: Nonexistence of asymptotically self-similar singularities in the euler and the navier–stokes equations. Mathematische Annalen. 338(2), 435–449 (2007) 4. Chae, D: Nonexistence of self-similar singularities for the 3d incompressible euler equations. Commun. Math. Phys. 273(1), 203–215 (2007) 5. Chae, D: On the self-similar solutions of the 3d euler and the related equations. Commun. Math. Phys. 305(2), 333–349 (2011) 6. Choi, K, Hou, TY, Kiselev, A, Luo, G, Sverak, V, Yao, Y: On the fiinite-time blowup of a 1d model for the 3d axisymmetric euler equations. arXiv preprint arXiv:1407.4776 (2014) 6. Choi, K, Hou, TY, Kiselev, A, Luo, G, Sverak, V, Yao, Y: On the euler equations. arXiv preprint arXiv:1407.4776 (2014) 7. Choi, K, Kiselev, A, Yao, Y: Finite time blow up for a 1d model of 2d boussinesq system. arXiv preprint arXiv:1312.4913 (2013) 8. Coddington, EA: Norman Levinson: Theory of ordinary differential equations (1955) 9. Constantin, P: On the euler equations of incompressible fluids. Bull. Am. Math. Soc. 44(4), 603–621 (2007) 10. Fefferman, CL, Seco, LA: Interval arithmetic in quantum mechanics. In: Applications of interval computations, pp. 145–167. Springer, (1996) 11. Folland, GB: Introduction to partial differential equations. Princeton University Press (1995) 11. Folland, GB: Introduction to partial differential equations. Princeton University Press (1995) 12. Garling, DJH: Inequalities: a journey into linear analysis, volume 19. Cambridge University Press, Cambridge (2007) 13. Gibbon, JD: The three-dimensional euler equations: Where do we stand? Physica D: Nonlinear Phenom. 237(14), 1894–1904 (2008) g, q j y y , g y , g ( ) 13. Gibbon, JD: The three-dimensional euler equations: Where do we stand? Physica D: Nonlinear Phenom. 237(14), 1894–1904 (2008) 13. Gibbon, JD: The three-dimensional euler equations: Where do we stand? Physica D: Nonlinear Phenom. 237(14), 1894–1904 (2008) Hales, TC: A proof of the kepler conjecture. Ann. Mathematics. 162 15. Hou, TY, Luo, G: On the finite-time blowup of a 1d model for the 3d incompressible euler equations. arXiv preprint arXiv:1311.2613 (2013) 15. Hou, TY, Luo, G: On the finite-time blowup of a 1d model for the 3d incompressible euler equations. arXiv preprint arXiv:1311.2613 (2013) 15. References l IEEE Std 754-2008, 1–70 (2008) Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com
https://openalex.org/W2830833519	https://zenodo.org/record/6622879/files/4218antj01.pdf	Latin	null	On Optimization of Manufacturing of Field-Effect Heterotransistors Frame-work a Single-Stage Multi-path Operational Amplifier, To Increase their Density	Advanced nanoscience and technology : an international journal	2,018	cc-by	16,641	Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 ABSTRACT We consider an approach for increasing density of field-effect heterotransistors in a single-stage multi-path operational amplifier. At the same time one can obtain decreasing of dimensions of the above transistors. Dimensions of the elements could be decreased by manufacturing of these elements in a heterostructure with specific structure. The manufacturing is doing by doping of required areas of the heterostructure by diffusion or ion implantation with future optimization of annealing of dopant and/or radiation defects. KEYWORDS field-effect heterotransistors; single-stage multi-path operational amplifier; optimization of manufacturing. ON OPTIMIZATION OF MANUFACTURING OF FIELD EFFECT HETEROTRANSISTORS FRAMEWORK A SINGLE STAGE MULTIPATH OPERATIONAL AMPLIFIER, TO INCREASE THEIR DENSITY E.L. Pankratov Nizhny Novgorod State University, 23 Gagarin avenue, Nizhny Novgorod, 603950, Russia 2. METHOD OF SOLUTION 2. METHOD OF SOLUTION We calculate distribution of concentration of dopant in space and time by solving the following equation ( ) = t t z y x C ∂ ∂ , , , ( ) ( ) ( )     +     +     = z t z y x C D z y t z y x C D y x t z y x C D x C C C ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , (1) ) = t y ∂ , , , t ∂ ( ) ( ) ( )     +     +     = z t z y x C D z y t z y x C D y x t z y x C D x C C C ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , (1) ( ) ( ) ( )     +     +   z t z y x C D z y t z y x C D y x t z y x C C C ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , (1) Boundary and initial conditions for the equation could be written as Boundary and initial conditions for the equation could be written as ( ) 0 , , , 0 = ∂ ∂ = x x t z y x C , ( ) 0 , , , = ∂ ∂ = x L x x t z y x C , ( ) 0 , , , 0 = ∂ ∂ = y y t z y x C , (2) ( ) 0 , , , = ∂ ∂ = y L x y t z y x C , ( ) 0 , , , 0 = ∂ ∂ = z z t z y x C , ( ) 0 , , , = ∂ ∂ = z L x z t z y x C , C (x,y,z,0)=f (x,y,z). C (x,y,z,0)=f (x,y,z). C (x,y,z,0)=f (x,y,z). 1. INTRODUCTION In the present time it is attracted an interest increasing of performance of elements integrated circuits and increasing of their density. At the same time dimensions of these elements decreases with decreasing of their density. Dimensions of elements of integrated circuit could be decreases by manufacturing of them in thin-film heterostructures [1-4]. As an alternative approach for the decreasing one can use laser and microwave types annealing [5-7]. Using these types of annealing leads to generation inhomogeneous distribution of temperature. Temperature dependence of dopant diffusion coefficient and other parameters of process leads to their inhomogeneity due to inhomogeneity of temperature. The inhomogeneity could leads to decreasing of dimensions of elements of integrated circuits. Properties of electronic materials could be also changes by using radiation processing of these materials [8,9]. In this paper we consider a single-stage multi-path operational amplifier described in Ref. [10] (see Fig.1). We assume, that element of the considered circuit has been manufactured in heterostructure from Fig. 1. The heterostructure includes into itself a substrate and an epitaxial layer. The epitaxial layer consist of a main material and several sections manufactured by using another materials in the main material. The sections should be doped for generation into them DOI: 10.5121/antj.2018.4201 1 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 required type of conductivity (n or p). Most simple types of doping are diffusion or ion implantation in the case. Main aim of the paper we analyzing of redistribution of dopant and radiation defects during the doping to formulate conditions to decrease of dimensions of the considered circuit. Fig. 1a. Circuit of a pass transistor single-stage multi-path operational amplifier [10] Fig. 1b. Heterostructure with substrate and epitaxial layer. The epitaxial layer in-cludes into itself several sections Fig. 1a. Circuit of a pass transistor single-stage multi-path operational amplifier [10] Fig. 1b. Heterostructure with substrate and epitaxial layer. The epitaxial layer in-cludes into itself several sections Fig. 1a. Circuit of a pass transistor single-stage multi-path operational amplifier [10] Fig. 1b. Heterostructure with substrate and epitaxial layer. The epitaxial layer in-cludes into itself several sections 2 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 2. METHOD OF SOLUTION 4, No.2, June 2018 ( ) ( ) ( ) ( ) ( ) +     ∂ ∂ ∂ ∂ +     ∂ ∂ ∂ ∂ = ∂ ∂ y t z y x I T z y x D y x t z y x I T z y x D x t t z y x I I I , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( )− −     ∂ ∂ ∂ ∂ + t z y x V t z y x I T z y x k z t z y x I T z y x D z V I I , , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) +     ∂ ∂ ∂ ∂ +     ∂ ∂ ∂ ∂ = y t z y x I T z y x D y x t z y x I T z y x D x I I , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( ) +   ∂ ∂ +   ∂ ∂ = ∂ y T z y x D y x T z y x D x t I I , , , , , , ( ) ( ) ( ) ( ) ( )− −     ∂ ∂ ∂ ∂ + t z y x V t z y x I T z y x k z t z y x I T z y x D z V I I , , , , , , , , , , , , , , , , ( ) ( )t z y x I T z y x k I I , , , , , , 2 , − (4) ( ) ( ) ( ) ( ) ( ) +     ∂ ∂ ∂ ∂ +     ∂ ∂ ∂ ∂ = ∂ ∂ y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( )− −     ∂ ∂ ∂ ∂ + t z y x V t z y x I T z y x k z t z y x V T z y x D z V I V , , , , , , , , , , , , , , , , ( ) ( )t z y x V T z y x k 2 ( ) ( ) ( ) ( ) ( )− −     ∂ ∂ ∂ ∂ + t z y x V t z y x I T z y x k z t z y x I T z y x D z V I I , , , , , , , , , , , , , , , , ( ) ( )t z y x I T z y x k I I , , , , , , 2 , − (4) ( ) ( ) ( ) ( ) ( ) +     ∂ ∂ ∂ ∂ +     ∂ ∂ ∂ ∂ = ∂ ∂ y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( )− −     ∂ ∂ ∂ ∂ + t z y x V t z y x I T z y x k z t z y x I T z y x D z V I I , , , , , , , , , , , , , , , , ( ) ( )t z y x I T z y x k I I , , , , , , 2 , − (4) ( ) ( ) ( ) ( ) ( ) +     ∂ ∂ ∂ ∂ +     ∂ ∂ ∂ ∂ = ∂ ∂ y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( )− −     ∂ ∂ ∂ ∂ + t z y x V t z y x I T z y x k z t z y x V T z y x D z V I V , , , , , , , , , , , , , , , , ( ) ( )t z y x I T z y x k I I , , , , , , 2 , − (4) ( ) ( )t z y x I T z y x k I I , , , , , , 2 , − (4) ( ) ( ) ( ) ( ) ( ) +     ∂ ∂ ∂ ∂ +     ∂ ∂ ∂ ∂ = ∂ ∂ y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V , , , , , , , , , , , , , , , (4) ( ) ( ) ( ) ( ) ( ) +     ∂ ∂ ∂ ∂ +     ∂ ∂ ∂ ∂ = ∂ ∂ y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( )− −     ∂ ∂ ∂ ∂ + t z y x V t z y x I T z y x k z t z y x V T z y x D z V I V , , , , , , , , , , , , , , , , ( ) ( )t z y x V T z y x k V V , , , , , , 2 , − ( ) ( )t z y x V T z y x k V V , , , , , , 2 , − Boundary and initial conditions for these equations could be written as Boundary and initial conditions for these equations could be written as Boundary and initial conditions for these equations could be written as Boundary and initial conditions for these equations could be written as ( ) 0 , , , 0 = ∂ ∂ = x x t z y x ρ , ( ) 0 , , , = ∂ ∂ = x L x x t z y x ρ , ( ) 0 , , , 0 = ∂ ∂ = y y t z y x ρ , ( ) 0 , , , = ∂ ∂ = y L y y t z y x ρ , ( ) 0 , , , 0 = ∂ ∂ = x x t z y x ρ , ( ) 0 , , , = ∂ ∂ = x L x x t z y x ρ , ( ) 0 , , , 0 = ∂ ∂ = y y t z y x ρ , ( ) 0 , , , = ∂ ∂ = y L y y t z y x ρ , ( ) 0 , , , 0 = ∂ ∂ = z z t z y x ρ , ( ) 0 , , , = ∂ ∂ = z L z z t z y x ρ , ρ (x,y,z,0)=fρ (x,y,z). 2. METHOD OF SOLUTION Function C(x,y,z,t) describes distribution of concentration of dopant in space and time; T is the temperature of annealing; DС is the dopant diffusion coefficient. Dopant diffusion coefficient could be varying with changing of materials, speed of heating and cooling of heterostructure. Approximation of dopant diffusion coefficient could be written as [9,11,12] ( ) ( ) ( ) ( ) ( ) ( )       + +    + = 2 * 2 2 * 1 , , , , , , 1 , , , , , , 1 , , , V t z y x V V t z y x V T z y x P t z y x C T z y x D D L C ς ς ξ γ γ . (3) Here DL (x,y,z,T) is the dependence of dopant diffusion coefficient on coordinate and temperature; P (x,y,z,T) is the dependence of limit of solubility of dopant diffusion coefficient on coordinate and temperature; parameter γ should be integer in the interval γ ∈[1,3] [9]; V (x,y,z,t) is the dependence of distribution of concentration of radiation vacancies on coordinate and time with the equilibrium distribution V*. Dependence of dopant diffusion coefficient on dopant concentration has been discussed in details in [9]. It should be noted, that using infusion of dopant did not leads to generation radiation defects, i.e. ζ1= ζ2= 0. We determine distributions of concentrations of point defects on space and time by solving the following system of equations [11,12] 3 3 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 2. METHOD OF SOLUTION 4, No.2, June 2018 ( ) ( ) ( ) ( ) ( ) +     Φ +     Φ = Φ Φ Φ y t z y x T z y x D y x t z y x T z y x D x t t z y x I I I I I ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( ) ( )t z y x I T z y x k t z y x I T z y x k z t z y x T z y x D z I I I I I , , , , , , , , , , , , , , , , , , 2 , − +     Φ + Φ ∂ ∂ ∂ ∂ (6) ( ) ( ) ( ) ( ) ( ) +     Φ +     Φ = Φ Φ Φ y t z y x T z y x D y x t z y x T z y x D x t t z y x V V V V V ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( ) ( )t z y x V T z y x k t z y x V T z y x k z t z y x T z y x D z V V V V V , , , , , , , , , , , , , , , , , , 2 , − +     Φ + Φ ∂ ∂ ∂ ∂ ( ) ( ) ( ) ( ) ( ) +     Φ +     Φ = Φ Φ Φ y t z y x T z y x D y x t z y x T z y x D x t t z y x I I I I I ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( ) ( )t z y x I T z y x k t z y x I T z y x k z t z y x T z y x D z I I I I I , , , , , , , , , , , , , , , , , , 2 , − +     Φ + Φ ∂ ∂ ∂ ∂ (6) (6) ( ) ( ) ( ) ( ) ( ) +     Φ +     Φ = Φ Φ Φ y t z y x T z y x D y x t z y x T z y x D x t t z y x V V V V V ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , ( ) ( ) ( ) ( ) ( ) ( )t z y x V T z y x k t z y x V T z y x k z t z y x T z y x D z V V V V V , , , , , , , , , , , , , , , , , , 2 , − +     Φ + Φ ∂ ∂ ∂ ∂ Boundary and initial conditions for these equations could be written as Boundary and initial conditions for these equations could be written as ( ) 0 , , , 0 = ∂ Φ ∂ = x x t z y x ρ , ( ) 0 , , , = ∂ Φ ∂ = x L x x t z y x ρ , ( ) 0 , , , 0 = ∂ Φ ∂ = y y t z y x ρ , ( ) 0 , , , = ∂ Φ ∂ = y L y y t z y x ρ , ( ) 0 , , , 0 = ∂ Φ ∂ = z z t z y x ρ , ( ) 0 , , , = ∂ Φ ∂ = z L z z t z y x ρ , (7) ΦI (x,y,z,0)=fΦI (x,y,z), ΦV (x,y,z,0)=fΦV (x,y,z). 2. METHOD OF SOLUTION ( ) 0 , , , 0 = ∂ ∂ = z z t z y x ρ , ( ) 0 , , , = ∂ ∂ = z L z z t z y x ρ , ρ (x,y,z,0)=fρ (x,y,z). (5) ( ) 0 , , , 0 = ∂ ∂ = z z t z y x ρ , ( ) 0 , , , = ∂ ∂ = z L z z t z y x ρ , ρ (x,y,z,0)=fρ (x,y,z). (5) (5) Here ρ =I,V; function I (x,y,z,t) describe distribution of concentration of radiation interstitials in space and time; function Dρ(x,y,z,T) describe distribution of diffusion coefficients of radiation interstitials and vacancies on coordinate and temperature; squared terms on concentration of defects (i.e. V2(x,y,z,t) and I2(x,y,z,t)) describe dependences of parameter of generation of divacancies and diinterstitials on coordinate and temperature, respectively; function kI,V(x,y,z,T) describe distribution of the parameter of recombination of point radiation defects on coordinate and temperature; function kρ,ρ(x,y,z,T) describe distribution of the parameters of generation of simplest complexes of point radiation defects on coordinate and temperature. We calculate dependences of concentrations of divacancies ΦV (x,y,z,t) and diinterstitials ΦI (x,y,z,t) on coordinate and temperature by solving the following system of equations [11,12] 4 4 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 2. METHOD OF SOLUTION ( ) 0 , , , 0 = ∂ Φ ∂ = x x t z y x ρ , ( ) 0 , , , = ∂ Φ ∂ = x L x x t z y x ρ , ( ) 0 , , , 0 = ∂ Φ ∂ = y y t z y x ρ , ( ) 0 , , , = ∂ Φ ∂ = y L y y t z y x ρ , ( ) 0 , , , 0 = ∂ Φ ∂ = z z t z y x ρ , ( ) 0 , , , = ∂ Φ ∂ = z L z z t z y x ρ , (7) ΦI (x,y,z,0)=fΦI (x,y,z), ΦV (x,y,z,0)=fΦV (x,y,z). (7) ΦI (x,y,z,0)=fΦI (x,y,z), ΦV (x,y,z,0)=fΦV (x,y,z). Functions DΦρ(x,y,z,T) describe dependences diffusion coefficients of complexes of radiation defects on coordinate and temperature; functions kρ(x,y,z,T) describe dependences of parameters of decay of complexes of radiation defects on coordinate and temperature. Functions DΦρ(x,y,z,T) describe dependences diffusion coefficients of complexes of radiation defects on coordinate and temperature; functions kρ(x,y,z,T) describe dependences of parameters of decay of complexes of radiation defects on coordinate and temperature. We calculate distributions of concentrations of dopant and radiation defects in space and time by using method of averaging of function corrections [13] with decreased quantity of iteration steps [14]. First of all we used solutions of linear Eqs. (1), (4) and (6) and with averaged values of diffusion coefficients D0L, D0I, D0V, D0ΦI, D0ΦV as initial-order approximations of the considered concentrations. 2. METHOD OF SOLUTION ( ) ( ) ( ) ( ) ( ) ∑ + = Φ ∞ = Φ Φ Φ 1 0 1 2 , , , n n n n n n z y x z y x I t e z c y c x c F L L L L L L F t z y x I I I , ( ) ( ) ( ) ( ) ( ) ∑ + = Φ ∞ = Φ Φ Φ 1 0 1 2 , , , n n n n n n z y x z y x V t e z c y c x c F L L L L L L F t z y x V V V . Here ( )               + + − = 2 2 2 0 2 2 1 1 1 exp z y x n L L L t D n t e ρ ρ π , ( ) ( ) ( ) ( ) ∫ ∫ ∫ = x y z L L L n n n n u d v d w d w v u f v c v c u c F 0 0 0 , , ρ ρ , cn(χ) = cos (π n χ/Lχ). ( ) ( ) ( ) ( ) ∫ ∫ ∫ = x y z L L L n n n n u d v d w d w v u f v c v c u c F 0 0 0 , , ρ ρ , cn(χ) = cos (π n χ/Lχ). Approximations of the considered concentrations with higher orders (second, third, ...) have been calculated framework standard iterative procedure [13,14]. To use the procedure we shall replace the functions C(x,y,z,t), I(x,y,z,t), V(x,y,z,t), ΦI(x,y, z,t), ΦV(x,y,z,t) in the right sides of the Eqs. (1), (4) and (6) on the following sums αnρ +ρ n-1(x,y,z,t). 2. METHOD OF SOLUTION ( ) ( ) ( ) ( ) ( ) ∑ + = ∞ =1 0 1 2 , , , n nV n n n nC z y x z y x C t e z c y c x c F L L L L L L F t z y x V , ( ) ( ) ( ) ( ) ( ) ∑ + = Φ ∞ = Φ Φ Φ 1 0 1 2 , , , n n n n n n z y x z y x I t e z c y c x c F L L L L L L F t z y x I I I , ( ) ( ) ( ) ( ) ( ) ∑ + = Φ ∞ = Φ Φ Φ 1 0 1 2 , , , n n n n n n z y x z y x V t e z c y c x c F L L L L L L F t z y x V V V . Here ( )               + + − = 2 2 2 0 2 2 1 1 1 exp z y x n L L L t D n t e ρ ρ π , ( ) ( ) ( ) ( ) ∫ ∫ ∫ = x y z L L L n n n n u d v d w d w v u f v c v c u c F 0 0 0 , , ρ ρ , cn(χ) = cos (π n χ/Lχ). ( ) T z y x Pγ , , , 2. METHOD OF SOLUTION 4, No.2, June 2018 ( ) ( ) ( ) ( ) ( ) ∑ + = ∞ =1 0 1 2 , , , n nV n n n nC z y x z y x C t e z c y c x c F L L L L L L F t z y x V , ( ) ( ) ( ) ( ) ( ) ∑ + = ∞ =1 0 1 2 , , , n nV n n n nC z y x z y x C t e z c y c x c F L L L L L L F t z y x V , ( ) ( ) ( ) ( ) ( ) ∑ + = Φ ∞ = Φ Φ Φ 1 0 1 2 , , , n n n n n n z y x z y x I t e z c y c x c F L L L L L L F t z y x I I I , ( ) ( ) ( ) ( ) ( ) ∑ + = Φ ∞ Φ Φ Φ 0 1 2 , , , n n n n n V t e z c y c x c F L L L L L L F t z y x V V V . 2. METHOD OF SOLUTION The solutions could be written as ( ) ( ) ( ) ( ) ( ) ∑ + = ∞ =1 0 1 2 , , , n nC n n n nC z y x z y x C t e z c y c x c F L L L L L L F t z y x C , ( ) ( ) ( ) ( ) ( ) ∑ + = ∞ =1 0 1 2 , , , n nI n n n nI z y x z y x I t e z c y c x c F L L L L L L F t z y x I , ( ) ( ) ( ) ( ) ( ) ∑ + = ∞ =1 0 1 2 , , , n nC n n n nC z y x z y x C t e z c y c x c F L L L L L L F t z y x C , ( ) ( ) ( ) ( ) ( ) ∑ + = ∞ =1 0 1 2 , , , n nI n n n nI z y x z y x I t e z c y c x c F L L L L L L F t z y x I , 5 5 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 2. METHOD OF SOLUTION Equations for concentrations for considered concentrations for the second-order could be written as ( ) ( ) ( ) ( ) ( ) [ ] ( )     ×       + +         + + = ∗ ∗ T z y x P t z y x C V t z y x V V t z y x V x t t z y x C C , , , , , , 1 , , , , , , 1 , , , 1 2 2 2 2 1 2 γ γ α ξ ς ς ∂ ∂ ∂ ∂ ( ) ( ) ( ) ( ) ( ) ( )    ×     + + +    × ∗ ∗ 2 2 2 1 1 , , , , , , 1 , , , , , , , , , V t z y x V V t z y x V T z y x D y x t z y x C T z y x D L L ς ς ∂ ∂ ∂ ∂ ( ) ( ) ( ) ( ) ( ) [ ] ( )     ×       + +         + + = ∗ ∗ T z y x P t z y x C V t z y x V V t z y x V x t t z y x C C , , , , , , 1 , , , , , , 1 , , , 1 2 2 2 2 1 2 γ γ α ξ ς ς ∂ ∂ ∂ ∂     ( ) [ ] ( ) ( ) ( ) ( )    × +          + + × z t z y x C T z y x D z y t z y x C T z y x P t z y x C L C ∂ ∂ ∂ ∂ ∂ ∂ α ξ γ γ , , , , , , , , , , , , , , , 1 1 1 1 2 ( ) ( ) ( ) ( ) [ ] ( )          + +       + + × ∗ ∗ T z y x P t z y x C V t z y x V V t z y x V C , , , , , , 1 , , , , , , 1 1 2 2 2 2 1 γ γ α ξ ς ς (8) ( ) ( ) ( ) ( ) ( ) +     +     = y t z y x I T z y x D y x t z y x I T z y x D x t t z y x I I I ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , 1 1 2 ( ) [ ] ( ) ( ) ( ) ( )    × +          + + × z t z y x C T z y x D z y t z y x C T z y x P t z y x C L C ∂ ∂ ∂ ∂ ∂ ∂ α ξ γ γ , , , , , , , , , , , , , , , 1 1 1 1 2 ( ) [ ] ( ) ( ) ( ) ( )    × +          + + × z t z y x C T z y x D z y t z y x C T z y x P t z y x C L C ∂ ∂ ∂ ∂ ∂ ∂ α ξ γ γ , , , , , , , , , , , , , , , 1 1 1 1 2 ( ) ( ) ( ) [ ]    +   t z y x C t z y x V t z y x V 2 γ α ( ) ( ) ( ) ( ) [ ] ( )          + +       + + × ∗ ∗ T z y x P t z y x C V t z y x V V t z y x V C , , , , , , 1 , , , , , , 1 1 2 2 2 2 1 γ γ α ξ ς ς (8) ( ) ( ) ( ) ( ) ( ) +     +     = t z y x I T z y x D t z y x I T z y x D t z y x I I I ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , 1 1 2 ( ) ( ) ( ) ( ) [ ] ( )          + +       + + × ∗ ∗ T z y x P t z y x C V t z y x V V t z y x V C , , , , , , 1 , , , , , , 1 1 2 2 2 2 1 γ γ α ξ ς ς (8) ( ) ( ) ( ) ( ) [ ] ( )          + +       + + × ∗ ∗ T z y x P t z y x C V t z y x V V t z y x V C , , , , , , 1 , , , , , , 1 1 2 2 2 2 1 γ γ α ξ ς ς (8) ( ) ( ) ( ) ( ) ( ) +     +     = y t z y x I T z y x D y x t z y x I T z y x D x t t z y x I I I ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , 1 1 2 (8) ( ) ( ) ( ) ( ) ( ) +     +     = y t z y x I T z y x D y x t z y x I T z y x D x t t z y x I I I ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , 1 1 2 6 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 2. METHOD OF SOLUTION 4, No.2, June 2018 gy ( ), , , ( ) ( ) ( ) [ ] ( ) [ ] × + + −     + t z y x V t z y x I z t z y x I T z y x D z V I I , , , , , , , , , , , , 1 2 1 2 1 α α ∂ ∂ ∂ ∂ ( ) ( ) ( ) [ ] 2 1 2 , , , , , , , , , , , t z y x I T z y x k T z y x k I I I V I + − × α (9) ( ) ( ) ( ) ( ) ( ) +     +     = y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , 1 1 2 ( ) ( ) ( ) [ ] ( ) [ ] × + + −     + t z y x V t z y x I z t z y x V T z y x D z V I V , , , , , , , , , , , , 1 2 1 2 1 α α ∂ ∂ ∂ ∂ ( ) ( ) ( ) [ ] 2 V T k T k ( ) ( ) ( ) [ ] ( ) [ ] × + + −     + t z y x V t z y x I z t z y x I T z y x D z V I I , , , , , , , , , , , , 1 2 1 2 1 α α ∂ ∂ ∂ ∂ ( ) ( ) ( ) [ ] 2 1 2 , , , , , , , , , , , t z y x I T z y x k T z y x k I I I V I + − × α (9) ( ) ( ) ( ) ( ) ( ) +     +     = y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , 1 1 2 ( ) ( ) ( ) [ ] ( ) [ ] × + + −     t z y x V t z y x I z t z y x I T z y x D V I I , , , , , , , , , , , , 1 2 1 2 1 α α ∂ ∂ ( ) ( ) ( ) [ ] ( ) [ ] × + + −     + t z y x V t z y x I z t z y x I T z y x D z V I I , , , , , , , , , , , , 1 2 1 2 1 α α ∂ ∂ ∂ ∂ ( ) ( ) ( ) [ ] 2 1 2 , , , , , , , , , t z y x I T z y x k T z y x k I I I V I + − × α (9) ( ) ( ) ( ) [ ] 2 1 2 , , , , , , , , , , , t z y x I T z y x k T z y x k I I I V I + − × α (9) ( ) ( ) ( ) ( ) ( ) +     +     = y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , 1 1 2 ( ) ( ) ( ) [ ] 2 1 2 , , , , , , , , , , , t z y x I T z y x k T z y x k I I I V I + − × α (9) (9) ( ) ( ) ( ) ( ) ( ) +     +     = y t z y x V T z y x D y x t z y x V T z y x D x t t z y x V V V ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , , , , 1 1 2 ( ) ( ) ( ) ( ) +     +     y t z y x V T z y x D y x t z y x V T z y x D x V V ∂ ∂ ∂ ∂ ∂ ∂ ∂ , , , , , , , , , , , , 1 1     y y x x t ∂ ∂ ∂ ∂ ∂ ( ) ( ) ( ) [ ] ( ) [ ] × + + −     + t z y x V t z y x I z t z y x V T z y x D z V I V , , , , , , , , , , , , 1 2 1 2 1 α α ∂ ∂ ∂ ∂ ( ) ( ) ( ) [ ] ( ) [ ] × + + −     + t z y x V t z y x I z t z y x V T z y x D z V I V , , , , , , , , , , , , 1 2 1 2 1 α α ∂ ∂ ∂ ∂ ) ( ) ( ) [ ] ( ) [ ] × + + −   t z y x V t z y x I z t z y x V T V I , , , , , , , , , 1 2 1 2 1 α α ∂ ∂ ( ) ( ) ( ) [ ] 2 1 2 , , , , , , , , , , , t z y x V T z y x k T z y x k V V V V I + − × α ( ) ( ) ( ) ( )   × +     Φ = Φ Φ Φ T z y x D y x t z y x T z y x D x t t z y x I I I I , , , , , , , , , , , , 1 2 ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × +     Φ +   Φ Φ T z y x k z t z y x T z y x D z y t z y x I I I I I , , , , , , , , , , , , , 1 1 ∂ ∂ ∂ ∂ ∂ ∂ ( ) ( ) ( )t z y x I T z y x k t z y x I I , , , , , , , , , 2 − × (10) (10) ( ) ( ) ( ) ( )   × +     Φ = Φ Φ Φ T z y x D y x t z y x T z y x D x t t z y x V V V V , , , , , , , , , , , , 1 2 ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × +     Φ +   Φ × Φ T z y x k z t z y x T z y x D z y t z y x V V V I V , , , , , , , , , , , , , 1 1 ∂ ∂ ∂ ∂ ∂ ∂ ( ) ( ) ( ) ( )   × +     Φ = Φ Φ Φ T z y x D y x t z y x T z y x D x t t z y x V V V V , , , , , , , , , , , , 1 2 ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × +     Φ +   Φ Φ T z y x k z t z y x T z y x D z y t z y x V V V I V , , , , , , , , , , , , , 1 1 ∂ ∂ ∂ ∂ ∂ ∂ ( ) ( ) ( )t z y x V T z y x k t z y x V V , , , , , , , , , 2 − × . y ∂ 2. METHOD OF SOLUTION The second-order approximations of concentrations of dopant and radiation defects could be obtained in the final form by integration of the left and right sides of Eqs.(8)-(10) The second-order approximations of concentrations of dopant and radiation defects could be obtained in the final form by integration of the left and right sides of Eqs.(8)-(10) ( ) ( ) ( ) ( ) ( ) [ ] ( )     ∫ ×       + +         + + = ∗ ∗ t C T z y x P z y x C V z y x V V z y x V x t z y x C 0 1 2 2 2 2 1 2 , , , , , , 1 , , , , , , 1 , , , γ γ τ α ξ τ ς τ ς ∂ ∂ 7 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 2. METHOD OF SOLUTION 4, No.2, June 2018 ( ) ( ) ( ) ( ) × ∫ +     ∫ Φ +   Φ × Φ t I I t I I T z y x k d z z y x T z y x D z d y z y x I 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ( ) ( ) ( ) ( )z y x f d z y x I T z y x k d z y x I I t I , , , , , , , , , , , 0 2 Φ + ∫ − × τ τ τ τ (10a) ( ) ( ) ( ) ( ) × ∫ +     ∫ Φ +   Φ × Φ t I I t I I T z y x k d z z y x T z y x D z d y z y x I 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ t    z z y 0 0 ∂ ∂ ∂ ( ) ( ) ( ) ( )z y x f d z y x I T z y x k d z y x I I t I , , , , , , , , , , , 0 2 Φ + ∫ − × τ τ τ τ (10a) ( ) ( ) ( ) ( )z y x f d z y x I T z y x k d z y x I I t I , , , , , , , , , , , 0 2 Φ + ∫ − × τ τ τ τ (10a) ( ) ( ) ( ) ( )z y x f d z y x I T z y x k d z y x I I t I , , , , , , , , , , , 0 2 Φ + ∫ − × τ τ τ τ (10a) ( ) ( ) ( ) ( )  ∫ × +     ∫ Φ = Φ Φ Φ t t V V T z y x D y d x z y x T z y x D x t z y x V V 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × ∫ +     ∫ Φ +   Φ × Φ t V V t V I T z y x k d z z y x T z y x D z d y z y x V 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ( ) ( ) ( ) ( )z y x f d z y x V T z y x k d z y x V V t V , , , , , , , , , , , 0 2 Φ + ∫ − × τ τ τ τ ( ) ( ) ( ) ( )  ∫ × +     ∫ Φ = Φ Φ Φ t t V V T z y x D y d x z y x T z y x D x t z y x V V 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × ∫ +     ∫ Φ +   Φ t V V t V T z y x k d z z y x T z y x D z d y z y x V 0 , 0 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ( ) ( ) ( ) ( ) × ∫ +     ∫ Φ +   Φ × Φ t V V t V I T z y x k d z z y x T z y x D z d y z y x V 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ( ) ( ) ( ) ( ) f d V T k d V t 2 ∫ We calculate average values of the second-orders approximations of concentrations of dopant and radiation defects by using the following standard relations [13,14] We calculate average values of the second-orders approximations of concentrations of dopant and radiation defects by using the following standard relations [13,14] ( ) ( ) [ ] ∫∫∫∫ − Θ = Θ 0 0 0 0 1 2 2 , , , , , , 1 x y z L L L z y x t d x d y d z d t z y x t z y x L L L ρ ρ α ρ . 2. METHOD OF SOLUTION 4, No.2, June 2018 gy ( ) ( ) ( ) ( ) ( )     ∫ ×         + + +    × ∗ ∗ t L V z y x V V z y x V y d x z y x C T z y x D 0 2 2 2 1 1 , , , , , , 1 , , , , , , τ ς τ ς ∂ ∂ τ ∂ τ ∂ ( ) ( ) [ ] ( ) ( ) ( )  ∫ × +          + + × t L C L T z y x D z d y z y x C T z y x P z y x C T z y x D 0 1 1 2 , , , , , , , , , , , , 1 , , , ∂ ∂ τ ∂ τ ∂ τ α ξ γ γ ( ) ( ) ( ) ( ) [ ] ( ) ( ) +          + +       + + × ∗ ∗ τ ∂ τ ∂ τ α ξ τ ς τ ς γ γ d z z y x C T z y x P z y x C V z y x V V z y x V C , , , , , , , , , 1 , , , , , , 1 1 1 2 2 2 2 1 ( )z y x fC , , + (8a) ( ) ( ) ( ) ( )  ∫ × +     ∫ = t I t I T z y x D y d x z y x I T z y x D x t z y x I 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × ∫ −     ∫ +   × t I I t I T z y x k d z z y x I T z y x D z d y z y x I 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ( ) [ ] ( ) ( ) ( ) [ ] × ∫ + − + + × t I V I I I z y x I T z y x k z y x f d z y x I 0 1 2 , 2 1 2 , , , , , , , , , , , τ α τ τ α ( ) ( ) ( ) ( ) ( )     ∫ ×         + + +    × ∗ ∗ t L V z y x V V z y x V y d x z y x C T z y x D 0 2 2 2 1 1 , , , , , , 1 , , , , , , τ ς τ ς ∂ ∂ τ ∂ τ ∂ ( ) ( ) [ ] ( ) ( ) ( )  ∫ × +          + + × t L C L T z y x D z d y z y x C T z y x P z y x C T z y x D 0 1 1 2 , , , , , , , , , , , , 1 , , , ∂ ∂ τ ∂ τ ∂ τ α ξ γ γ ( ) ( ) ( ) ( ) [ ] ( ) ( ) +          + +       + + × ∗ ∗ τ ∂ τ ∂ τ α ξ τ ς τ ς γ γ d z z y x C T z y x P z y x C V z y x V V z y x V C , , , , , , , , , 1 , , , , , , 1 1 1 2 2 2 2 1 ( )z y x fC , , + (8a) (8a) ( ) ( ) ( ) ( )  ∫ × +     ∫ = t I t I T z y x D y d x z y x I T z y x D x t z y x I 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × ∫ −     ∫ +   × t I I t I T z y x k d z z y x I T z y x D z d y z y x I 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ) ( ) ( ) ( )  ∫ × +     ∫ = t I t I T z y x D y d x z y x I T z y x D x t z y x 0 0 1 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × ∫ −     ∫ +   × t I I t I T z y x k d z z y x I T z y x D z d y z y x I 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ( ) [ ] ( ) ( ) ( ) [ ] × ∫ + − + + × t I V I I I z y x I T z y x k z y x f d z y x I 0 1 2 , 2 1 2 , , , , , , , , , , , τ α τ τ α ( ) [ ] ( ) ( ) ( ) [ ] × ∫ + − + + × t I V I I I z y x I T z y x k z y x f d z y x I 0 1 2 , 2 1 2 , , , , , , , , , , , τ α τ τ α ( ) [ ] τ τ α d z y x V + × (9a) ( ) [ ] ( ) ( ) ( ) [ ] × ∫ + + + × I V I I I z y x I T z y x k z y x f d z y x I 0 1 2 , 1 2 , , , , , , , , , , , τ α τ τ α ( ) [ ] τ τ α d z y x V V , , , 1 2 + × (9a) ( ) [ ] τ τ α d z y x V V , , , 1 2 + × (9a) ( )] τ τ d z y x V , , , 1 + (9a) ( ) ( ) ( ) ( )  ∫ × +     ∫ = t V t V T z y x D y d x z y x V T z y x D x t z y x V 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × ∫ −     ∫ +   × t V V t V T z y x k d z z y x V T z y x D z d y z y x V 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ( ) [ ] ( ) ( ) ( ) [ ] × ∫ + − + + × t I V I V I z y x I T z y x k z y x f d z y x V 0 1 2 , 2 1 2 , , , , , , , , , , , τ α τ τ α ( ) [ ] τ τ α d z y x V V , , , 1 2 + × ( ) ( ) ( ) ( )  ∫ × +     ∫ = t V t V T z y x D y d x z y x V T z y x D x t z y x V 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( ) × ∫ −     ∫ +   × t V V t V T z y x k d z z y x V T z y x D z d y z y x V 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ( ) ( ) ( ) ( )  ∫ × +     ∫ = t V t V T z y x D y d x z y x V T z y x D x t z y x V 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( ) ∫   ∫  t t T k d z y x V T D d z y x V 1 1 , , , , , , τ ∂ ∂ τ ∂   y ( ) ( ) ( ) ( ) × ∫ −     ∫ +   × t V V t V T z y x k d z z y x V T z y x D z d y z y x V 0 , 0 1 1 , , , , , , , , , , , , τ ∂ τ ∂ ∂ ∂ τ ∂ τ ∂ ( ) [ ] ( ) ( ) ( ) [ ] × ∫ + − + + × t I V I V I z y x I T z y x k z y x f d z y x V 0 1 2 , 2 1 2 , , , , , , , , , , , τ α τ τ α ( ) [ ] τ τ α d z y x V V , , , 1 2 + × ( ) [ ] ( ) ( ) ( ) [ ] × ∫ + − + + × t I V I V I z y x I T z y x k z y x f d z y x V 0 1 2 , 2 1 2 , , , , , , , , , , , τ α τ τ α ( ) [ ] τ τ α d z y x V V , , , 1 2 + × ( ) ( ) ( ) ( )  ∫ × +     ∫ Φ = Φ Φ Φ t t I I T z y x D y d x z y x T z y x D x t z y x I I 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ ( ) ( ) ( ) ( )  ∫ × +     ∫ Φ = Φ Φ Φ t t I I T z y x D y d x z y x T z y x D x t z y x I I 0 0 1 2 , , , , , , , , , , , , ∂ ∂ τ ∂ τ ∂ ∂ ∂ 8 8 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. y ∂ 2. METHOD OF SOLUTION (11) (11) Using the relation (11) with account relations (8a)-(10a) leads to the following average values α 2ρ ( ) ∫∫∫ = x y z L L L C z y x С x d y d z d z y x f L L L 0 0 0 2 , , 1 α , (12) (12) ( ) [ { − + − − + + + = 11 20 10 2 00 2 00 2 10 01 00 2 4 1 2 1 IV II IV V II IV V II IV II I A A A A A A A A α α α ( ) 00 00 2 10 01 2 1 0 0 0 2 1 , , 1 II IV V II IV L L L I z y x A A A A x d y d z d z y x f L L L x y z α + + + −        ∫∫∫ − (13a) ( ) 4 3 1 3 4 2 3 4 2 4 4 4 2 1 B A B A B y B y B A B B V + −     − + − + = α . (13b) ( ) [ { − + − − + + + = 11 20 10 2 00 2 00 2 10 01 00 2 4 1 2 1 IV II IV V II IV V II IV II I A A A A A A A A α α α ( ) [ { − + − − + + + = 11 20 10 2 00 2 00 2 10 01 00 2 4 1 2 1 IV II IV V II IV V II IV II I A A A A A A A A α α α 2 1 1 1 L L L A A A x y z α + + +   (13b) 9 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 2. METHOD OF SOLUTION 4, No.2, June 2018 ( ) ( ) ( )     + − − + + + ∫∫∫ − 11 20 00 10 01 01 0 0 0 00 2 1 , , 2 2 IV VV II II IV IV L L L I z y x II A A A A A A x d y d z d z y x f L L L A x y z ( ) ( ) ( )     + − − + + + ∫∫∫ − 11 20 00 10 01 01 0 0 0 00 2 1 , , 2 2 IV VV II II IV IV L L L I z y x II A A A A A A x d y d z d z y x f L L L A x y z ( )] ( ) ( ) [ ] 00 01 00 10 10 10 01 00 10 01 01 2 1 2 1 2 1 IV IV II VV IV II IV IV II IV IV A A A A A A A A A A A + + + − + + + + + , ( ) ( + + +         − ∫∫∫ + = 10 01 2 01 11 0 0 0 20 2 01 00 0 , , 1 4 II IV IV IV L L L I z y x II IV II A A A A x d y d z d z y x f L L L A A A B x y z ) ( ) ( ) ( )     + − − + + + ∫∫∫ − + 11 20 00 10 01 01 0 0 0 00 2 2 1 , , 2 1 IV VV II II IV IV L L L V z y x II A A A A A A x d y d z d z y x f L L L A x y z ( )] 2 10 01 01 1 II IV IV A A A + + + , 6 2 3 3 2 3 3 2 B q p q q p q y + + + − − + = , ( )× − = 0 3 1 8 2 B B B q ) ( ) ( ) ( )     + − − + + + ∫∫∫ − + 11 20 00 10 01 01 0 0 0 00 2 2 1 , , 2 1 IV VV II II IV IV L L L V z y x II A A A A A A x d y d z d z y x f L L L A x y z ) ( ) ( ) ( )     + − − + + + ∫∫∫ − + 11 20 00 10 01 01 0 0 0 00 2 2 1 , , 2 1 IV VV II II IV IV L L L V z y x II A A A A A A x d y d z d z y x f L L L A x y z ( )] 2 10 01 01 1 II IV IV A A A + + + , 6 2 3 3 2 3 3 2 B q p q q p q y + + + − − + = , ( )× − = 0 3 1 8 2 B B B q ( )] 2 10 01 01 1 II IV IV A A A + + + , 6 2 3 3 2 3 3 2 B q p q q p q y + + + − − + = , ( ) 8 4 216 48 2 1 2 3 2 0 3 2 2 B B B B B B − − + + × , ( ) [ ] 72 2 8 2 3 2 2 0 3 1 B B B B p − − = , 2 2 3 4 8 B B y A − + = , ( ) 8 4 216 48 2 1 2 3 2 0 3 2 2 B B B B B B − − + + × , ( ) [ ] 72 2 8 2 3 2 2 0 3 1 B B B B p − − = , ( ) ( ) ( ) + ∫ ∫∫∫ − Θ Θ − = Θ Φ 0 0 0 0 20 2 , , , , , , 1 x y z I L L L I z y x II t d x d y d z d t z y x I T z y x k t L L L A α ( ) ( ) ( ) + ∫ ∫∫∫ − Θ Θ − = Θ Φ 0 0 0 0 20 2 , , , , , , 1 x y z I L L L I z y x II t d x d y d z d t z y x I T z y x k t L L L A α ( ) ( ) ( ) + ∫ ∫∫∫ − Θ Θ − = Θ Φ 0 0 0 0 20 2 , , , , , , 1 x y z I L L L I z y x II t d x d y d z d t z y x I T z y x k t L L L A α ( ) ∫∫∫ + Φ x y z L L L I z y x x d y d z d z y x f L L L 0 0 0 , , 1 (14) ( ) ( ) ( ) + ∫ ∫∫∫ − Θ Θ − = Θ Φ 0 0 0 0 20 2 , , , , , , 1 x y z V L L L V z y x VV t d x d y d z d t z y x V T z y x k t L L L A α ( ) ∫∫∫ + Φ x y z L L L I z y x x d y d z d z y x f L L L 0 0 0 , , 1 (14) ( ) ∫∫∫ + Φ x y z L L L I z y x x d y d z d z y x f L L L 0 0 0 , , 1 (14) ( ) ∫∫∫ + Φ x y z L L L I z y x x d y d z d z y x f L L L 0 0 0 , , 1 (14) ( ) ( ) ( ) + ∫ ∫∫∫ − Θ Θ − = Θ Φ 0 0 0 0 20 2 , , , , , , 1 x y z V L L L V z y x VV t d x d y d z d t z y x V T z y x k t L L L A α ( ) ∫∫∫ + Φ x y z L L L V z y x x d y d z d z y x f L L L 0 0 0 , , 1 . 2. METHOD OF SOLUTION 4, No.2, June 2018 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 2. METHOD OF SOLUTION 4, No.2, June 2018 Here Here ( ) ( ) ( ) ( ) ∫ ∫∫∫ − Θ Θ = Θ 0 0 0 0 1 1 , , , , , , , , , , 1 x y z L L L j i b a z y x abij t d x d y d z d t z y x V t z y x I T z y x k t L L L A , ( ) 2 00 00 2 00 2 00 2 00 4 2 VV II IV IV IV A A A A A B − − = , − + + = 2 00 10 00 3 00 01 2 00 00 3 IV II IV IV IV IV IV A A A A A A A B ( ) ( ) ( ) ( ) ∫ ∫∫∫ − Θ Θ = Θ 0 0 0 0 1 1 , , , , , , , , , , 1 x y z L L L j i b a z y x abij t d x d y d z d t z y x V t z y x I T z y x k t L L L A , ( ) ( ) ( ) ( ) ∫ ∫∫∫ − Θ Θ = Θ 0 0 0 0 1 1 , , , , , , , , , , 1 x y z L L L j i b a z y x abij t d x d y d z d t z y x V t z y x I T z y x k t L L L A , ( ) 2 00 00 2 00 2 00 2 00 4 2 VV II IV IV IV A A A A A B − − = , ( ) 2 00 00 2 00 2 00 2 00 4 2 VV II IV IV IV A A A A A B − − = , − + + = 2 00 10 00 3 00 01 2 00 00 3 IV II IV IV IV IV IV A A A A A A A B ( ) ( ) ( ) [ ]− + + − + + + − − 1 2 1 2 2 4 10 10 00 10 01 00 00 01 00 00 2 00 VV IV II II IV IV IV IV VV II IV A A A A A A A A A A A 2 00 01 00 2 00 00 10 2 4 IV IV IV IV II IV A A A A A A + − , ( ) ( ) ( ) [ ]− + + − + + + − − 1 2 1 2 2 4 10 10 00 10 01 00 00 01 00 00 2 00 VV IV II II IV IV IV IV VV II IV A A A A A A A A A A A 2 00 01 00 2 00 00 10 2 4 IV IV IV IV II IV A A A A A A + − , ( ) { × + + + + = 00 2 01 2 00 2 10 01 2 00 2 1 IV IV IV II IV IV A A A A A A B ( ) { × + + + + = 00 2 01 2 00 2 10 01 2 00 2 1 IV IV IV II IV IV A A A A A A B ( )     × − − − − + + × z y x II IV II II IV II IV IV IV IV IV L L L A A A A A A A A A A A 1 4 4 2 20 11 00 00 10 10 00 01 00 00 00 ( ) ( ) ( [{ + + − + + +      ∫∫∫ × 1 2 1 2 2 , , 10 00 10 01 00 00 01 0 0 0 IV II II IV IV IV IV L L L I A A A A A A A x d y d z d z y x f x y z ( ) ( ) ( [{ + + − + + +      ∫∫∫ × 1 2 1 2 2 , , 10 00 10 01 00 00 01 0 0 0 IV II II IV IV IV IV L L L I A A A A A A A x d y d z d z y x f x y z )] ( ) ( ) (     − − ∫∫∫ + + + + + 20 00 0 0 0 10 01 01 2 10 2 , , 2 1 2 VV II L L L V z y x II IV IV VV A A x d y d z d z y x f L L L A A A A x y z ) ( )] ( ) ( [ + + − + + + + + + − 1 2 2 1 2 1 10 00 00 01 10 01 00 10 01 01 11 IV II IV IV II IV IV II IV IV IV A A A A A A A A A A A )]} 10 VV A + , ( ) ( )   × ∫∫∫ − − + + = x y z L L L I x IV II IV IV IV x d y d z d z y x f L A A A A A B 0 0 0 11 2 10 01 01 00 1 , , 1 8 1 2 ) ( )] ( ) ( [ + + − + + + + + + − 1 2 2 1 2 1 10 00 00 01 10 01 00 10 01 01 11 IV II IV IV II IV IV II IV IV IV A A A A A A A A A A A )]} 10 VV A + , ) ( )] ( ) ( [ + + − + + + + + + − 1 2 2 1 2 1 10 00 00 01 10 01 00 10 01 01 11 IV II IV IV II IV IV II IV IV IV A A A A A A A A A A A )]} 10 VV A + ,  L )]} 10 VV A + , ( ) ( )   × ∫∫∫ − − + + = x y z L L L I x IV II IV IV IV x d y d z d z y x f L A A A A A B 0 0 0 11 2 10 01 01 00 1 , , 1 8 1 2 ( )− − + + + +     − × 00 10 10 00 01 00 00 2 01 00 01 00 20 4 1 II IV II IV IV IV IV IV II IV IV II z y A A A A A A A A A A A A L L ( ) ( )   × ∫∫∫ − − + + = x y z L L L I x IV II IV IV IV x d y d z d z y x f L A A A A A B 0 0 0 11 2 10 01 01 00 1 , , 1 8 1 2 ( )− − + + + +     − × 00 10 10 00 01 00 00 2 01 00 01 00 20 4 1 II IV II IV IV IV IV IV II IV IV II z y A A A A A A A A A A A A L L ( )− − + + + +     − × 00 10 10 00 01 00 00 2 01 00 01 00 20 4 1 II IV II IV IV IV IV IV II IV IV II z y A A A A A A A A A A A A L L 10 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 2 2 3 4 8 B B y A − + = , 2. METHOD OF SOLUTION ( ) ( ) ( ) + ∫ ∫∫∫ − Θ Θ − = Θ Φ 0 0 0 0 20 2 , , , , , , 1 x y z V L L L V z y x VV t d x d y d z d t z y x V T z y x k t L L L A α ( ) ( ) ( ) + ∫ ∫∫∫ − Θ Θ − = Θ Φ 0 0 0 0 20 2 , , , , , , 1 x y z V L L L V z y x VV t d x d y d z d t z y x V T z y x k t L L L A α ( ) ( ) ( ) + ∫ ∫∫∫ − Θ Θ − = Θ Φ 0 0 0 0 20 2 , , , , , , 1 x y z V L L L V z y x VV t d x d y d z d t z y x V T z y x k t L L L A α ( ) ∫∫∫ + Φ x y z L L L V z y x x d y d z d z y x f L L L 0 0 0 , , 1 . ( ) ∫∫∫ + Φ x y z L L L V z y x x d y d z d z y x f L L L 0 0 0 , , 1 . ( ) ∫∫∫ + Φ x y z L L L V z y x x d y d z d z y x f L L L 0 0 0 , , 1 . 11 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Now one can obtain equation for parameter α2C. Form of the equation depends on of parameter γ. Analysis of distributions of concentrations of dopant and radiation defects in space and time has been done by using the second-order approximations by using of modified method of averaging of function corrections (with decreased quantity of iterative steps) of these concentrations. The considered approximation is usually enough good approximation to analyze qualitatively the considered concentration and obtain some quantitative results. 2. METHOD OF SOLUTION The analytical calculation results have been checked comparison of the analytical results with results of numerical simulation. 3. DISCUSSION Main aim of the section is analysis of distribution of concentration of dopant in space and time in the considered heterostructure during annealing. Figs. 2 shows distributions of concentrations of dopants in space, which where infused or implanted in the considered epitaxial layer (see Fig. 2a and Fig. 2b, respectively) at the same values of annealing time (the same framework each figure). Larger numbers of curves corresponds to larger difference between dopant diffusion coefficients in epitaxial layer and substrate. The figures show that inhomogeneity could leads to increasing of absolute value of gradient of concentration of dopant (i.e. one can obtain increasing sharpness of the end of doped structure). The increasing leads to decreasing dimensions of the considered transistors and increasing of homogeneity of concentration of dopant in doped areas. We choose optimal value annealing time as compromise between increasing of homogeneity of dopant concentration in doped area and increasing of sharpness of the concentration in neighborhood of interface between substrate and epitaxial layer (see Fig. 3a for diffusion doping of materials and Fig. 3b for ion doping of materials) [15-20]. Framework the criteria one shall approximate real distributions of concentration of dopant by ideal step-wise distribution ψ (x,y,z). compromise value of annealing time by minimization of the following mean-squared error. Fig.2a. Distributions of infused dopant concentration in the considered heterostructure. Larger numbers of curves corresponds to larger difference between dopant diffusion coefficients in epitaxial layer and substrate Fig.2a. Distributions of infused dopant concentration in the considered heterostructure. Larger numbers of curves corresponds to larger difference between dopant diffusion coefficients in epitaxial layer and substrate 12 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Fig.2b. Distributions of implanted dopant concentration in the considered heterostructure. Larger numbers of curves corresponds to larger difference between dopant diffusion coefficients in epitaxial layer and substrate Fi 2b Di t ib ti f i l t d d t t ti i th id d h t t t L b Fig.2b. Distributions of implanted dopant concentration in the considered heterostructure. Larger numbers of curves corresponds to larger difference between dopant diffusion coefficients in epitaxial layer and substrate Fig. 3a. Dependences of distributions of concentration of infused dopant on coordinate. Curve 1 is idealized distribution of dopant. 3. DISCUSSION Curves 2-4 are real dependences of concentrations dopant on coordinate for different values of annealing time. Increasing of number of curve corresponds to increasing of annealing time Fig. 3a. Dependences of distributions of concentration of infused dopant on coordinate. Curve 1 is idealized distribution of dopant. Curves 2-4 are real dependences of concentrations dopant on coordinate for different values of annealing time. Increasing of number of curve corresponds to increasing of annealing time 13 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 Fig.3b. Dependences of distributions of concentration of implanted dopant on coordinate. Curve 1 is idealized distribution of dopant. Curves 2-4 are real dependences of concentrations dopant on coordinate for different values of annealing time. Increasing of number of curve corresponds to increasing of annealing time Fig.3b. Dependences of distributions of concentration of implanted dopant on coordinate. Curve 1 is idealized distribution of dopant. Curves 2-4 are real dependences of concentrations dopant on coordinate for different values of annealing time. Increasing of number of curve corresponds to increasing of annealing time Fig.4a. Dependences of dimensionless optimized annealing time of infused dopant on several parameters. Curve 1 describes dependence of dimensionless optimized annealing time on the value a/L and ξ = γ = 0 for equal to each other values of dopant diffusion coefficient in all parts of heterostructure. Curve 2 is the dependence of dimensionless optimal annealing time on value of parameter ε for a/L=1/2 and ξ = γ = 0. Curve 3 is the dependence of dimensionless optimal annealing time on value of parameter ξ for a/L =1/2 and ε = γ = 0. Curve 4 is the dependence of dimensionless optimal annealing time on value of parameter γ for a/L=1/2 and ε = ξ = 0 Fig.4a. Dependences of dimensionless optimized annealing time of infused dopant on several parameters. Curve 1 describes dependence of dimensionless optimized annealing time on the value a/L and ξ = γ = 0 for equal to each other values of dopant diffusion coefficient in all parts of heterostructure. Curve 2 is the dependence of dimensionless optimal annealing time on value of parameter ε for a/L=1/2 and ξ = γ = 0. Curve 3 is the dependence of dimensionless optimal annealing time on value of parameter ξ for a/L =1/2 and ε = γ = 0. 3. DISCUSSION Curve 4 is the dependence of dimensionless optimal annealing time on value of parameter γ for a/L=1/2 and ε = ξ = 0 14 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 ( ) ( ) [ ] ∫∫∫ − Θ = x y z L L L z y x x d y d z d z y x z y x C L L L U 0 0 0 , , , , , 1 ψ . (15) (15) (15) Fig.4b. Dependences of dimensionless optimized annealing time of implanted dopant on several parameters. Curve 1 describes dependence of dimensionless optimized annealing time on the value a/L and ξ = γ = 0 for equal to each other values of dopant diffusion coefficient in all parts of heterostructure.Curve 2 is the dependence of dimensionless optimal annealing time on value of parameter ε for a/L=1/2 and ξ = γ = 0. Curve 3 is the dependence of dimensionless optimal annealing time on value of parameter ξ for a/L =1/2 and ε = γ = 0. Curve 4 is the dependence of dimensionless optimal annealing time on value of parameter γ for a/L=1/2 and ε = ξ = 0 ependences of dimensionless optimized annealing time of implanted dopant on several s. Curve 1 describes dependence of dimensionless optimized annealing time on the value a/L and Fig.4b. Dependences of dimensionless optimized annealing time of implanted dopant on several parameters. Curve 1 describes dependence of dimensionless optimized annealing time on the value a/L and ξ = γ = 0 for equal to each other values of dopant diffusion coefficient in all parts of heterostructure.Curve 2 is the dependence of dimensionless optimal annealing time on value of parameter ε for a/L=1/2 and ξ = γ = 0. Curve 3 is the dependence of dimensionless optimal annealing time on value of parameter ξ for a/L =1/2 and ε = γ = 0. Curve 4 is the dependence of dimensionless optimal annealing time on value of parameter γ for a/L=1/2 and ε = ξ = 0 We present optimized annealing time on Figs. 4 (Fig. 4a for diffusion type of doping and Fig. 4b for ion type of doping). It is known, that radiation defects should be annealed after ion implantation. The annealing leads to spreading of distribution of concentration of dopant. 3. DISCUSSION The ideal spreading leads to achievement of nearest interfaces between materials of heterostructure. If dopant has no time for the achievement during the annealing, it is attracted an interest additional annealing of dopant. The situation leads to smaller value of optimal annealing time of implanted dopant in comparison with diffusion one. At the same time it should be noted, that ion type of doping leads to decreasing of mismatch-induced stress in heterostructure [21]. 4. CONCLUSION We consider an approach for increasing density of field-effect heterotransistors in a single-stage multi-path operational amplifier. At the same time one can obtain decreasing of dimensions of the above transistors. Dimensions of the elements could be decreased by manufacturing of these elements in a heterostructure with specific structure. The manufacturing is doing by doping of required areas of the heterostructure by diffusion or ion implantation with future optimization of annealing of dopant and/or radiation defects. 15 Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 REFERENCES Advanced Nanoscience and Technology: An International Journal (ANTJ), Vol. 4, No.2, June 2018 REFERENCES REFERENCES [1] G. Volovich. Modern Electronics. Issue 2. P. 10-17 (2006). [1] G. Volovich. Modern Electronics. Issue 2. P. 10-17 (2006). [2] A. Kerentsev, V. Lanin, Power Electronics. Issue 1. P. 34 (2008). [3] A.O. Ageev, A.E. Belyaev, N.S. Boltovets, V.N. Ivanov, R.V. Konakova, Ya.Ya. Kudrik, P.M. Litvin, V.V. Milenin, A.V. Sachenko. Semiconductors. Vol. 43 (7). P. 897-903 (2009). [4] N.I. Volokobinskaya, I.N. Komarov, T.V. Matioukhina, V.I. Rechetniko, A.A. Rush, I.V. Falina, A.S. Yastrebov. Semiconductors. Vol. 35 (8). P. 1013-1017 (2001). [5] K.K. Ong, K.L. Pey, P.S. Lee, A.T.S. Wee, X.C. Wang, Y.F. Chong, Appl. Phys. Lett. 89 (17), 172111-172114 (2006). [6] H.T. Wang, L.S. Tan, E. F. Chor. J. Appl. Phys. 98 (9), 094901-094905 (2006). [7] Yu.V. Bykov, A.G. Yeremeev, N.A. Zharova, I.V. Plotnikov, K.I. Rybakov, M.N. Drozdov, Yu.N. Drozdov, V.D. Skupov. Radiophysics and Quantum Electronics. Vol. 43 (3). P. 836-843 (2003). [8] V.V. Kozlivsky. Modification of semiconductors by proton beams (Nauka, Sant-Peterburg, 2003, in Russian). [9] V.L. Vinetskiy, G.A. Kholodar', Radiative physics of semiconductors. ("Naukova Dumka", Kiev, 1979, in Russian). [10] M. Yavari, T. Moosazadeh. Analog. Integr. Circ. Sig. Process. Vol. 79. P. 589-598 (2014). [11] Z.Yu. Gotra. Technology of microelectronic devices (Radio and communication, Moscow, 1991). [12] P.M. Fahey, P.B. Griffin, J.D. Plummer. Rev. Mod. Phys. 1989. V. 61. № 2. P. 289-388. [13] Yu.D. Sokolov. Applied Mechanics. Vol. 1 (1). P. 23-35 (1955). [14] E.L. Pankratov. The European Physical Journal B. 2007. V. 57, №3. P. 251-256. [15] E.L. Pankratov. Russian Microelectronics. 2007. V.36 (1). P. 33-39. [16] E.L. Pankratov. Int. J. Nanoscience. Vol. 7 (4-5). P. 187–197 (2008). [17] E.L. Pankratov. J. Comp. Theor. Nanoscience. Vol. 7 (1). P. 289-295 (2010). [18] E.L. Pankratov, E.A. Bulaeva. J. Comp. Theor. Nanoscience. Vol. 10 (4). P. 888-893 (2013). [19] E.L. Pankratov, E.A. Bulaeva. Int. J. Micro-Nano Scale Transp. Vol. 4 (1). P. 17-31 (2014). [20] E.L. Pankratov, E.A. Bulaeva. Int. J. Nanoscience. Vol. 11 (5). P. 1250028-1250035 (2012). [21] E.L. Pankratov, E.A. Bulaeva. J. Comp. Theor. Nanoscience. Vol. 11 (1). P. 91-101 (2014). 16
https://openalex.org/W2810229072	https://europepmc.org/articles/pmc6028423?pdf=render	English	null	Synthetic molecular evolution of hybrid cell penetrating peptides	Nature communications	2,018	cc-by	13,008	ARTICLE OPEN ARTICLE ARTICLE Results b Library construction. To evolve gain-of-function sequences from the known pTat48–60 (tat) and pAntp43–68 (penetratin) sequences, we created a peptide library of 8192 tat/penetratin hybrid sequences of 13–16 residues (Fig. 1). When aligned, the 13- residue tat sequence and 16-residue penetratin sequence share a lysine at position 4 and an arginine at position 10. We added a hydrophobic leucine option at position 10 to increase library diversity. Lys4 remains common to all sequences. This alignment creates a library with one cationic and one non-cationic residue possible at most positions. The three additional C-terminal resi- dues of penetratin, Trp–Lys–Lys, were randomly present or absent as a cassette, resulting in 13 variable positions in peptides of 13 or 16 residues (Fig. 1c). SME is used here to identify CPP sequences capable of efﬁ- ciently delivering PNA, peptides, and other cargoes to living cells. PNAs are synthetic nucleic acid analogs possessing a peptide bond linked N-(2-aminoethyl) glycine backbone with the nucleobase (A/T/C/G) attached via methylene carbonyl bonds19. These molecules are resistant to enzymatic degradation, stably bind complimentary nucleic acids with high speciﬁcity and high afﬁnity using Watson–Crick base pairing, and are easily con- jugated to amino acid sequences because they share backbone chemistry. As antisense gene therapy agents, PNAs can modulate transcription, translation, splicing, and replication20–27. In this work, we screened for CPPs capable of nuclear delivery of PNA705, an 18-residue PNA sequence that sterically blocks an aberrant splice site in an engineered luciferase transgene con- taining an intronic insert with a T705G mutation28. Steric blockage of the aberrant splice site drives the use of a cryptic site which produces functional luciferase. The splice correcting PNA705 sequence makes an ideal cargo for this study because PNAs are not efﬁciently delivered to cells by classical CPPs. Further, once a delivery peptide is identiﬁed, PNA sequences can likely be changed to target other DNA/RNA sequences without signiﬁcantly changing the physicochemical properties of the cargo or the efﬁciency with which it is delivered by a PNA delivery peptide (PDEP). g We used a split and recombine, one-bead one-sequence solid phase peptide synthesis strategy33 to synthesize the CPP–PNA library on TentaGel Megabeads. C-terminal photo-labile linkers were coupled to each synthesis bead, followed by the 18-residue PNA705 sequence, followed by an AEAA PNA spacer, and then the N-terminal library member (Fig. 1a). High-pressure liquid chromatography (HPLC) and mass spectrometry on multiple individual beads showed that the synthesis was successful. Synthetic molecular evolution of hybrid cell penetrating peptides W. Berkeley Kauffman1, Shantanu Guha1 & William C. Wimley1 Peptides and analogs such as peptide nucleic acids (PNA) are promising tools and therapeutics, but the cell membrane remains a barrier to intracellular targets. Conjugation to classical cell penetrating peptides (CPPs) such as pTat48–60 (tat) and pAntp43–68 (penetratin) facilitates delivery; however, efﬁciencies are low. Lack of explicit design principles hinders rational improvement. Here, we use synthetic molecular evolution (SME) to identify gain- of-function CPPs with dramatically improved ability to deliver cargoes to cells at low concentration. A CPP library containing 8192 tat/penetratin hybrid peptides coupled to an 18-residue PNA is screened using the HeLa pTRE-LucIVS2 splice correction reporter system. The daughter CPPs identiﬁed are one to two orders of magnitude more efﬁcient than the parent sequences at delivery of PNA, and also deliver a dye cargo and an anionic peptide cargo. The signiﬁcant increase in performance following a single iteration of SME demonstrates the power of this approach to peptide sequence optimization. 1 Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112, USA. Correspondence and requests for materials should be addressed to W.C.W. (email: wwimley@tulane.edu) NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 P e i T P eptides and peptide-like molecules are generating increas- ing interest as biotech tools and therapeutic agents1, 2. There are currently 60+ FDA-approved peptide drugs in the market with another 140+ in clinical trials and 500+ in pre-clinical development. The majority of approved peptides have extracellular targets because the cell membrane represents a barrier to intracellular targeting3. Similarly, antisense DNA analogs, including peptide nucleic acids (PNA) and phosphor- odiamidate morpholino oligomers (PMOs), are generating growing excitement3–5, but have yet to fully overcome limitations in the efﬁciency of delivery to the nuclei of the desired cells. Toward solving the delivery problem inherent to peptide, PNA, and PMO cargoes, cell penetrating peptides (CPPs) have shown promise as vehicles capable of transporting such cell-impermeant cargo to cytosolic or nuclear targets. However, there remains a need to identify CPPs with higher efﬁciencies, lower effective treatment concentrations, decreased cytotoxicity, and alternative mechanisms of action2, 6–8. mechanistic space. At low concentrations (<10 µM), the cationic guanidinium-rich tat and its analogs, including nona-arginine (Arg9), enter cells mostly by endocytosis31. Synthetic molecular evolution of hybrid cell penetrating peptides At higher con- centrations, a mostly energy-independent mechanism of entry dominates as the peptide enters cells directly, perhaps after accumulation at ceramide-rich nucleation zones on the plasma membrane8. Penetratin is an amphipathic CPP that is capable of either direct translocation through the plasma membrane or translocation via the formation of a transient membrane structure32. In this work, the hybrid library was screened for PNA delivery efﬁciency, and PDEP daughter sequences are identiﬁed that deliver PNA with greatly improved efﬁciency at low concentra- tion, and that signiﬁcantly outperform both parent sequences. PDEPs conjugated to peptides, PNAs, PMOs, or other cargoes may represent powerful biotechnological tools. They may also comprise therapeutic delivery strategies that are fast and efﬁcient, function at low micromolar concentrations in a variety of cell types, and have low cytotoxicity. More broadly, SME is shown here again to be a highly efﬁcient approach toward the targeted optimization of peptide sequences. Despite the need for improved CPPs, rational design is chal- lenging due to the lack of explicit sequence–structure–function relationship rules9. In this work, we identify gain-of- function CPPs with useful properties using synthetic molecular evolution (SME). SME is an iterative process of designing rational combinatorial libraries that explore the sequence space around known templates, and screening such iterative libraries, ortho- gonally, to ﬁnd members that display gain-of-function. It enables the utilization of known information, and the simultaneous testing of multiple hypotheses by rationally introducing constrained amino acid variability at speciﬁc locations throughout a template sequence. Previously, we have used SME to identify potent β-sheet pore-forming peptides10–12, enhancers of receptor tyrosine kinase activation13, spontaneous membrane translocating pep- tides14, gain-of-function and loss-of-function pore-forming peptides15, 16, pH-triggered pore-forming peptides17, and anti- microbial peptides18. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Basic amino acids are in blue and polar amino acids are in orange. d Plot of relative luminescent units vs. µg protein from the screen shows the distribution of library members, blank controls, PDEP-positive sequences, and four less active positive sequences. Peptides were photocleaved from the support resin, dissolved in ddH2O, and added to cells in serum-free media. After 1 h, the cells were incubated in full media for 24 h before being analyzed by luciferase and BCA assays luciferase for P11-PNA705, P17-PNA705, and P40-PNA705, while it had little effect on the delivery of P14-PNA705 suggesting moderate and sequence-speciﬁc levels of endosomal entrapment. In addition to comparing PDEPs to the classical CPPs tat, penetrating and Arg9, we also tested a peptide called Peptide B which has recently been shown to drive efﬁcient delivery of splice correcting PMOs in vivo4, 34, 35. PepB is (RXRRBR)2 which has eight arginines and four linear linkers, X is β-alanine, and B is 6-amino-hexanoic acid. In our test system, peptide B has relatively low PNA delivery efﬁciency; higher than that of tat, but less than penetratin and Arg9 (Fig. 2a). luciferase for P11-PNA705, P17-PNA705, and P40-PNA705, while it had little effect on the delivery of P14-PNA705 suggesting moderate and sequence-speciﬁc levels of endosomal entrapment. level in HeLa pLuc705 cells that had been treated with a 2× serial dilution of CPP-PNA705, starting at 5 µM. This concentration range is lower than the range at which CPPs are usually tested in vitro, providing a stringent test of delivery efﬁciency. PDEP–PNA conjugates are not toxic at this concentration range (Supplementary Figure 1). Cells were treated for 30 min in serum- free media at 37 °C, and then allowed to recover for 24 h in complete media. To assess the relative impact of endosomal uptake, which results in release to the cytosol, recycling, or lysosomal degradation, some cells were co-treated with the endosomal acidiﬁcation inhibitor chloroquine (CQ) (120 µM), which promotes release over degradation. After 24 h, cell lysates were assayed for luciferase by luminescence (Fig. 2a) and for splice corrected mRNA by quantitative PCR (Fig. 2b). To control for total cell density, lysates were also analyzed for total protein by the BCA assay. p g g To quantify splice correction at the mRNA level, we designed qRT-PCR primers to target the splice junction of the properly spliced mRNA transcript. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Peptide N-Terminus C-Terminus # Da ΔG +/– 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Tat G R K K R R Q R R R P P Q – – – 1718 Penetratin R Q I K I W F Q N R R M K W K K 16 Variable – – – – – – – – – – – – – – – L P17 G R K K R W F R R R R M K W K K 16 2303 4.8 +12 P14 – R K K R W F R R R R P K W K K 15 2212 5.4 +12 P11 – R I K R R F R R L R P K W K K 15 2124 5.4 +11 P40 R R K K I W F R R L R M K – – – 13 1873 2.9 +9 Lucife- -rase T705G Cryptic Lucife- -rase Luciferase PNA705 No treatment Aberrant splicing Correct splicing a c +9 13 8.91 2245 4.31 +8 N AEAA spacer S CCTCTTACCTCAGTTACA C UV light PNA705 Photo linker Megabead Peptide 13–16 amino acid CPP N 100,000 10,000 RLU P40 P17 P14 P11 1000 100 10 1 10 100 μg protein Library members Blanks Positives False positives b d Fig. 1 Selection of positive PNA delivery peptides (PDEPs). a Strategy for solid phase synthesis of photo-cleavable PDEP–PNA705 sequences onto TentaGel-S-NH2 megabeads. b Screening system. HeLa pTRE-Luc IVS2-705 cells possess luciferase transgenes with mutant human β-globin IVS2 inserts that introduce an aberrant splice site at position 705 resulting in non-functional luciferase. Binding of PNA705 to the pre-mRNA in the nucleus blocks this splice site, enabling utilization of a cryptic splice site that restores functional luciferase production. c Split and combine synthesis outline. Each library bead contains ~0.5 nmol of one peptide sequence, with randomly determined residues either from the sequence of tat or penetratin. Residues 14–16 were randomly absent or present. At position 10, a hydrophobic leucine option was available. PDEP-positive sequences are in green. Basic amino acids are in blue and polar amino acids are in orange. d Plot of relative luminescent units vs. µg protein from the screen shows the distribution of library members, blank controls, PDEP-positive sequences, and four less active positive sequences. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Peptides were photocleaved from the support resin, dissolved in ddH2O, and added to cells in serum-free media. After 1 h, the cells were incubated in full media for 24 h before being analyzed by luciferase and BCA assays a N AEAA spacer S CCTCTTACCTCAGTTACA C UV light PNA705 Photo linker Megabead Peptide 13–16 amino acid CPP N Lucife- -rase T705G Cryptic Lucife- -rase Luciferase PNA705 No treatment Aberrant splicing Correct splicing b b a 100,000 10,000 RLU P40 P17 P14 P11 1000 100 10 1 10 100 μg protein Library members Blanks Positives False positives d Peptide N-Terminus C-Terminus # Da ΔG +/– 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Tat G R K K R R Q R R R P P Q – – – 1718 Penetratin R Q I K I W F Q N R R M K W K K 16 Variable – – – – – – – – – – – – – – – L P17 G R K K R W F R R R R M K W K K 16 2303 4.8 +12 P14 – R K K R W F R R R R P K W K K 15 2212 5.4 +12 P11 – R I K R R F R R L R P K W K K 15 2124 5.4 +11 P40 R R K K I W F R R L R M K – – – 13 1873 2.9 +9 c +9 13 8.91 2245 4.31 +8 d c Fig. 1 Selection of positive PNA delivery peptides (PDEPs). a Strategy for solid phase synthesis of photo-cleavable PDEP–PNA705 sequences onto TentaGel-S-NH2 megabeads. b Screening system. HeLa pTRE-Luc IVS2-705 cells possess luciferase transgenes with mutant human β-globin IVS2 inserts that introduce an aberrant splice site at position 705 resulting in non-functional luciferase. Binding of PNA705 to the pre-mRNA in the nucleus blocks this splice site, enabling utilization of a cryptic splice site that restores functional luciferase production. c Split and combine synthesis outline. Each library bead contains ~0.5 nmol of one peptide sequence, with randomly determined residues either from the sequence of tat or penetratin. Residues 14–16 were randomly absent or present. At position 10, a hydrophobic leucine option was available. PDEP-positive sequences are in green. Results b After side-chain deprotection, beads were segregated into 96-well plates. PDEP–PNA705 constructs were cleaved from the resin with UV light, extracted into buffer, and incubated with HeLa pLuc705 cells in serum-free media in 96-well plate format. Cells were incubated with peptide-PNA solution for 1 h and then in full media for 24 h before being analyzed by luciferase and bicinchoninic acid (BCA) assays. The initial PDEP–PNA705 concentration in each well was about 7.5 μM. After 24 h, cells were lysed and analyzed for functional luciferase and total protein. Positive PDEP sequences were deﬁned as those that gave relative luminescence units (RLU) µg−1 protein values that were at least three standard deviations above the plate mean (Fig. 1d). Beads from which positive sequences were extracted contained residual peptide that was sequenced by Edman degradation. Following a preliminary assessment of eight potential positives, we selected four, called P11, P14, P17, and P40, for subsequent detailed analysis (Fig. 1d). Two of the best known, classical CPPs are pTat48–60 (tat) and pAntp43–68 (penetratin). Although they can deliver some cargoes to cells under some conditions29, 30, their ability to deliver PNA at low concentration is poor, as we show below. Here, we created a hybrid library from the aligned sequences of tat and penetratin. While each of the parent sequences is a CPP, their mechanisms of action differ, enabling the hybrid library to explore a broad PDEP–PNA705 mediated splice correction. To assess the efﬁ- ciency of nuclear PNA705 delivery and splice correction, we quantiﬁed luciferase production at both the protein and mRNA NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 PDEPs labeled with TAMRA cause diffuse cytosolic staining of many cells within 15 min of incubations at any temperature (Fig. 3), suggesting an energy independent mechanism of entry directly through the plasma membrane. This is consistent with the fact that similar delivery efﬁciency was measured at 37 °C, permissive for endocytosis, and at 4 °C and 21 °C, conditions under which endocytosis is inhibited. Cytotoxicity assays show no acute lactate dehydrogenase (LDH) leakage and no reduction metabolic potential or total protein in 24 h at concentrations ≤40 μM (Supplementary Figure 2). Inclusion of SYTOX Green in ﬂow cytometry and confocal microscopy show that PDEP-TA conjugates enter cells without any membrane permeabilization. PDEPs labeled with TAMRA cause diffuse cytosolic staining of many cells within 15 min of incubations at any temperature (Fig. 3), suggesting an energy independent mechanism of entry directly through the plasma membrane. This is consistent with the fact that similar delivery efﬁciency was measured at 37 °C, permissive for endocytosis, and at 4 °C and 21 °C, conditions under which endocytosis is inhibited. Cytotoxicity assays show no acute lactate dehydrogenase (LDH) leakage and no reduction metabolic potential or total protein in 24 h at concentrations ≤40 μM (Supplementary Figure 2). Inclusion of SYTOX Green in ﬂow cytometry and confocal microscopy show that PDEP-TA conjugates enter cells without any membrane permeabilization. While there was variability between cell types and incubation temperatures, daughter PDEPs consistently delivered much more TAMRA to a larger percentage of cells than either parent sequence or Arg9. For example, P17 delivered TAMRA to >98% of all four cell types, while tat and penetratin delivered TAMRA to less than 10% of cells in three of the four cell types. Furthermore, at all temperatures, the average intensity of intracellular TAMRA ﬂuorescence is much higher for PDEPs across four different cell types than for the parent sequences (Fig. 3). For example, in HeLa cells at 21 °C, P14 and P17 deliver TAMRA to average intensities 10–20-fold higher than tat and Unlike the luciferase splice correction assay where a single corrected mRNA transcript serves as template for many molecules of functional luciferase, the split GFP assay is stoichiometric; a single GFP11 peptide can bind to a single GFP1–10 protein producing a small, ﬁnite ﬂuorescent response. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 At both temperatures, individual cells rapidly and stochastically show the appearance of TAMRA with a diffuse cytosolic distribution in addition to bright puncta. All PDEPs behave similarly and maximum intensity is reached by 30 min at all temperatures. Cell surface-bound peptide is not observed under ﬂow cytometry conditions (Fig. 3a). To test the efﬁciency under stringent conditions, we used peptide concentrations of only 5 μM total containing 0.5 μM dye-labeled peptide. Peptides were incubated with HeLa, RAW264.7, HepG2, and MCF-7 cells for 30 min at 4 °C, 21 °C, or 37 °C, followed by trypsinization to detach cells for counting and to digest any externally bound peptide. PDEP-TA conjugates are not toxic at this concentration range (Supplementary Figure 2). Flow cyto- metric analysis (Fig. 3 and Supplementary Figure 4) was used to count TAMRA-positive cells and to measure TAMRA intensity distributions. Confocal microscopy was used to visualize peptide distribution in cells for each cell type (Fig. 3). Time series con- focal images of PDEP P17-TA incubated HeLa cells at 21 °C and 37 °C (Fig. 3a) show immediate punctate membrane association. At 37 °C, but not at 21 °C, uptake of dye in punctate structures is observed. At both temperatures, individual cells rapidly and stochastically show the appearance of TAMRA with a diffuse cytosolic distribution in addition to bright puncta. All PDEPs behave similarly and maximum intensity is reached by 30 min at all temperatures. Cell surface-bound peptide is not observed under ﬂow cytometry conditions (Fig. 3a). PDEP delivery of GFP11 peptide. To test the ability of PDEPs to deliver a protease-sensitive peptide cargo, we modiﬁed the well- known split-green ﬂuorescent protein (GFP) assay36 so that CPPs could be tested for the ability to deliver the polar 16-residue GFP11 peptide across the membrane barrier. The GFP11 peptide rescues the ﬂuorescence of cytosolic, non-ﬂuorescent GFP1–10 protein which is missing the GFP11 strand. Here, we stably transfected HeLa cells with a plasmid containing the full length mCherry fused to the non-ﬂuorescent GFP1–10. Following incubation with PDEP-GFP11 constructs at multiple concentra- tions and temperatures for 30 min in serum-free media, the cells were analyzed by ﬂow cytometry for mCherry and GFP ﬂuores- cence. Live single cells expressing mCherry (~50% of the total cells) were gated (Supplementary Figure 5) and their GFP ﬂuorescence was analyzed (Fig. 4). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Error bars are standard deviations of the data, which are shown as open circles, with 3–6 data points, from independent experiments, per average Fig. 2 PDEP-mediated delivery of splice correcting PNA. a Luciferase expression. HeLa pTRE-LucIVS2-705 cells were incubated with varying concentrations of PDEP-705 in serum-free media for 30 min and then incubated for 24 h in complete media. Cell lysates were analyzed for luminescence and standardized by total protein content. The ratios of the RLU μg−1 protein for treated samples divided by untreated samples are plotted. To examine the degree of endosomal entrapment, cells were co-incubated with 120 μM of the endosomolytic agent chloroquine. b mRNA correction. RNA was puriﬁed from lysates of cells treated as above and analyzed by qRT-PCR to determine the fold increase in correctly spliced luciferase mRNA standardized to ACTB1. The relative quantiﬁcation of correctly spliced mRNA over untreated cells was calculated using the Pfafﬂmodel56. Error bars are standard deviations of the data, which are shown as open circles, with 3–6 data points, from independent experiments, per average ecting PNA. a Luciferase expression. HeLa pTRE-LucIVS2-705 cells were incubated with varying concentrations n and then incubated for 24 h in complete media. Cell lysates were analyzed for luminescence and standardized penetratin, and 4–6-fold higher than Arg9. In most experiments, P17 >>P11 = P14 > P40 = Arg9 > tat = penetratin which is simi- lar to the efﬁciency of PNA705 delivery. To test the efﬁciency under stringent conditions, we used peptide concentrations of only 5 μM total containing 0.5 μM dye-labeled peptide. Peptides were incubated with HeLa, RAW264.7, HepG2, and MCF-7 cells for 30 min at 4 °C, 21 °C, or 37 °C, followed by trypsinization to detach cells for counting and to digest any externally bound peptide. PDEP-TA conjugates are not toxic at this concentration range (Supplementary Figure 2). Flow cyto- metric analysis (Fig. 3 and Supplementary Figure 4) was used to count TAMRA-positive cells and to measure TAMRA intensity distributions. Confocal microscopy was used to visualize peptide distribution in cells for each cell type (Fig. 3). Time series con- focal images of PDEP P17-TA incubated HeLa cells at 21 °C and 37 °C (Fig. 3a) show immediate punctate membrane association. At 37 °C, but not at 21 °C, uptake of dye in punctate structures is observed. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 We measured the levels of corrected luciferase mRNA after 30-min incubations followed by a 24-h recovery period with 5 µM, 2.5 µM, and 1.25 µM PDEP-705 (Fig. 2d). These data validate the luciferase assay results. We observe a >100 fold increase in properly spliced luciferase mRNA in cells treated with PDEP–PNA705 relative to control. At 5 μM, the most efﬁcient chimera, P14-PNA705, is 30-fold better than tat, 7-fold better than penetratin, and 4-fold better than Arg9. Even at 1.25 µM P14-PNA705, the increase in corrected mRNA is 2–3 fold, while tat and penetratin and Arg9 are not measurably higher than control. y y At 5 μM chimera, the parents do not efﬁciently deliver PNA705. Tat–PNA705 produced a slight 3-fold increase in luminescence over the background of untreated cells, while penetratin–PNA705 delivery produced a 15-fold increase over background (Fig. 2a). All four of the evolved PDEP daughter sequences (Fig. 1c) deliver PNA705 much more efﬁciently than the parent sequences, with P14 ≈P11 > P17 > P40 >>pen > tat. The top-performing daughter sequences, P11 and P14, increased luciferase over background by more than 400-fold, which is more than 100 times better than tat and 30 times better than penetratin. Even at 1.25 μM, PDEP–PNA705 chimeras increase the splice corrected mRNA and functional protein over background by several fold. Interestingly, the canonical tat analog Arg9, which was not a member of the library, delivered PNA705 much better than tat itself. Yet, PDEPs P11, P14, and P17 outperformed Arg9 by 2–3 fold at 5 μM. CQ treatment moderately increased PDEP-TAMRA delivery to multiple cell types. Parent and daughter sequences lacking the PNA cargo were characterized to determine the effect of the cargo on delivery. Sequences were labeled with the ﬂuorophore tetramethylrhodamine (TAMRA) on a C-terminal cysteine residue (PDEP-TA) and their delivery to multiple human cell types was measured by ﬂow cytometry. NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 tat Pen Arg9 P11 P14 P17 P40 PNA705 0 40 80 120 160 200 240 Fold increase in corrected mRNA 1.25 μM 2.5 μM 5.0 μM tat Pen Arg9 PepB P11 P14 P17 P40 PNA705 0 10 100 200 300 400 500 600 700 800 1.25 μM 2.5 μM 5.0 μM 5.0 μM + CQ RLU/mg protein (treated/untreated) a b Fig. 2 PDEP-mediated delivery of splice correcting PNA. a Luciferase expression. HeLa pTRE-LucIVS2-705 cells were incubated with varying concentrations of PDEP-705 in serum-free media for 30 min and then incubated for 24 h in complete media. Cell lysates were analyzed for luminescence and standardized by total protein content. The ratios of the RLU μg−1 protein for treated samples divided by untreated samples are plotted. To examine the degree of endosomal entrapment, cells were co-incubated with 120 μM of the endosomolytic agent chloroquine. b mRNA correction. RNA was puriﬁed from lysates of cells treated as above and analyzed by qRT-PCR to determine the fold increase in correctly spliced luciferase mRNA standardized to ACTB1. The relative quantiﬁcation of correctly spliced mRNA over untreated cells was calculated using the Pfafﬂmodel56. Error bars are standard deviations of the data, which are shown as open circles, with 3–6 data points, from independent experiments, per average tat Pen Arg9 PepB P11 P14 P17 P40 PNA705 0 10 100 200 300 400 500 600 700 800 1.25 μM 2.5 μM 5.0 μM 5.0 μM + CQ RLU/mg protein (treated/untreated) a tat Pen Arg9 P11 P14 P17 P40 PNA705 0 40 80 120 160 200 240 Fold increase in corrected mRNA 1.25 μM 2.5 μM 5.0 μM b b a a Fig. 2 PDEP-mediated delivery of splice correcting PNA. a Luciferase expression. HeLa pTRE-LucIVS2-705 cells were incubated with varying concentrations of PDEP-705 in serum-free media for 30 min and then incubated for 24 h in complete media. Cell lysates were analyzed for luminescence and standardized by total protein content. The ratios of the RLU μg−1 protein for treated samples divided by untreated samples are plotted. To examine the degree of endosomal entrapment, cells were co-incubated with 120 μM of the endosomolytic agent chloroquine. b mRNA correction. RNA was puriﬁed from lysates of cells treated as above and analyzed by qRT-PCR to determine the fold increase in correctly spliced luciferase mRNA standardized to ACTB1. The relative quantiﬁcation of correctly spliced mRNA over untreated cells was calculated using the Pfafﬂmodel56. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Error bars are standard deviations of the data, wh shown as open circles, with 3–4 data points, from independent experiments, per average 1 min a 7 min 13 min 19 min 25 min 30 min 37 °C 21 °C 1 min a b Tat-TA Pen-TA Arg9-TA P11-TA P14-TA P17-TA P40-TA TAMRA 12.2% 20.3% 0.71% 74.0% 98.7% 1.99% 19.0% 98.4% 7 min 13 min 19 min 25 min 30 min 37 °C 21 °C a b c Tamra 2 4 6 8 10 12 14 16 24 8.1 Fold increase over TAMRA only 4.5 4.9 73.4 80.2 99.6 %+ 1.6 2.8 61.7 86.3 98.3 %+ HeLa 4 °C 21 °C 37 °C 0 MCF-7 2 4 6 8 10 Fold increase over TAMRA only 4 °C 21 °C 37 °C Tat Pen Arg9 P11 P14 P17 P40 Tamra Tat Pen Arg9 P11 P14 P17 P40 17.2 0.0 0.5 81.0 14.4 RAW HEPG2 0 4 8 12 16 20 24 28 60 64.5 Fold increase over TAMRA only 15.5 69.9 83.3 97.6 %+ 4 8 12 16 20 16.6 Fold increase over TAMRA only 6.3 16.5 84.6 99.5 %+ 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C Tamra Tat Pen Arg9 P11 P14 P17 P40 Tamra Tat Pen Arg9 P11 P14 P17 P40 42.8 45.2 6.90 6.8 42.8 0.0 c Fig. 3 PDEP-mediated delivery of the ﬂuorescent dye TAMRA. a Confocal microscopy images of HeLa cells incubated for 30-min with 4.5 µM P17-C + 0.5 μM P17-C-TA at either 37 °C or at room temperature (21 °C). The cytolysis-sensitive dye SYTOX Green was also added. Scale bar = 10 μM. b Example ﬂow cytometry results for peptide-TA conjugates. Cells were trypsinized to remove externally bound peptides and subjected to ﬂow cytometry. Cells were gated by scatter to select single viable cells. The very small fraction of SYTOX Green positive cells were excluded. The threshold for TAMRA positive intensity was set to be just above that of cells treated with free TAMRA dye, which does not enter cells. c For the four cell types shown, the averages of TAMRA ﬂuorescence of live, SYTOX Green-negative cells were measured after treatment as above at 4 °C, 21 °C, and 37 °C. Averages are plotted as a ratio of the intensity of peptide-TA-treated cells to the intensity of cells treated with free TAMRA. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Due to the decreased sensitivity for this system, we did not observe a measurable signal at 5 μM PDEP-GFP11, so we tested all peptides at 10–40 μM. Cytotoxicity assays show no LDH leakage, reduction in metabolic potential, or reduction in total protein at concentrations ≤160 μM except for P14D-GFP11, which showed a reduction in metabolic activity determined by alamarBlue assay at concentrations ≥20 μM despite also testing negative for cytotoxicity in either the LDH leakage or BCA protein assays (Supplementary Figure 3). j g y p While there was variability between cell types and incubation temperatures, daughter PDEPs consistently delivered much more TAMRA to a larger percentage of cells than either parent sequence or Arg9. For example, P17 delivered TAMRA to >98% of all four cell types, while tat and penetratin delivered TAMRA to less than 10% of cells in three of the four cell types. Furthermore, at all temperatures, the average intensity of intracellular TAMRA ﬂuorescence is much higher for PDEPs across four different cell types than for the parent sequences (Fig. 3). For example, in HeLa cells at 21 °C, P14 and P17 deliver TAMRA to average intensities 10–20-fold higher than tat and At 40 μM, the parent peptide tat delivers GFP11 to 21% of mCherry-positive cells at 37 °C, and to 7% of such cells at 21 °C (Fig. 4a). Penetratin does not measurably deliver GFP11 under any condition studied. The daughter PDEPs P11, P14, and P17 deliver GFP11 to more cells than the parents, at all concentra- tions, and at both temperatures. P14-GFP11 delivered the peptide to 59% and 46% of cells at 37°C and 21 °C, respectively. Even at 20 and 10 μM, where both parent peptides are essentially inactive, PDEPs have a measurable GFP11 delivery capability (Fig. 4a). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Error bars are standard deviations of the data, which are shown as open circles, with 3–4 data points, from independent experiments, per average In addition to delivering peptide to more cells, ﬂow cytometric data show that the PDEPs deliver more peptide to each cell (Fig. 4b). The average GFP ﬂuorescence in PDEP-GFP11 treated cells is 3–5-fold higher than background, while tat-GFP11 and pen-GFP11 are similar to background even at 40 μM. At 20 µM, P14-GFP11 and P17-GFP1 are up to 3-fold higher than background at 37 °C and at 21 °C. At 10 µM, where most conjugates exhibit background ﬂuorescence levels, P14-GFP11 remains 44% above background at 37 °C and 72% above background at room temperature. P14 is most efﬁcient at GFP11 delivery, followed by P11 and P17. Interestingly, like penetratin, the PDEP P40 does not deliver GFP11 signiﬁcantly, although it successfully delivers PNA705 (Fig. 2). amount, indicating that proteolytic sensitivity has some effect on delivery efﬁciency, although not a dominant one. At 40 µM, tat (D)-GFP11 was delivered to more cells and at a higher concentration that tat-GFP11 at both 37 °C and 21 °C. GFP ﬂuorescence just above background was observed at lower concentrations for tat(D)-GFP11 but not for tat-GFP11. Simi- larly, P14(D)-GFP11 outperformed P14-GFP11 under all other conditions (Fig. 4). In fact, P14(D)-GFP11 was the only peptide tested that had measurable activity at 10 µM, with a >2-fold ﬂuorescence increase, over background at both temperatures. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 1 min RAW HEPG2 a b c Tat-TA Pen-TA Arg9-TA P11-TA P14-TA P17-TA P40-TA TAMRA 12.2% 20.3% 0.71% 74.0% 98.7% 1.99% 19.0% 98.4% Tamra 2 4 6 8 10 12 14 16 24 8.1 Fold increase over TAMRA only 4.5 4.9 73.4 80.2 99.6 %+ 1.6 2.8 61.7 86.3 98.3 %+ 0 4 8 12 16 20 24 28 60 64.5 Fold increase over TAMRA only 15.5 69.9 83.3 97.6 %+ 4 8 12 16 20 16.6 Fold increase over TAMRA only 6.3 16.5 84.6 99.5 %+ 7 min 13 min 19 min 25 min 30 min 37 °C 21 °C HeLa 4 °C 21 °C 37 °C 0 MCF-7 2 4 6 8 10 Fold increase over TAMRA only 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C Tat Pen Arg9 P11 P14 P17 P40 Tamra Tat Pen Arg9 P11 P14 P17 P40 Tamra Tat Pen Arg9 P11 P14 P17 P40 Tamra Tat Pen Arg9 P11 P14 P17 P40 17.2 0.0 42.8 45.2 6.90 0.5 81.0 14.4 6.8 42.8 0.0 Fig. 3 PDEP-mediated delivery of the ﬂuorescent dye TAMRA. a Confocal microscopy images of HeLa cells incubated for 30-min with 4.5 µM P17-C μM P17-C-TA at either 37 °C or at room temperature (21 °C). The cytolysis-sensitive dye SYTOX Green was also added. Scale bar = 10 μM. b Examp cytometry results for peptide-TA conjugates. Cells were trypsinized to remove externally bound peptides and subjected to ﬂow cytometry. Cells gated by scatter to select single viable cells. The very small fraction of SYTOX Green positive cells were excluded. The threshold for TAMRA po intensity was set to be just above that of cells treated with free TAMRA dye, which does not enter cells. c For the four cell types shown, the aver TAMRA ﬂuorescence of live, SYTOX Green-negative cells were measured after treatment as above at 4 °C, 21 °C, and 37 °C. Averages are plott ratio of the intensity of peptide-TA-treated cells to the intensity of cells treated with free TAMRA. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 21 °C 37 °C a c b tat tat(D) Pen Arg9 P11 P14 P14(D) P17 P40 GFP11 0 2 4 6 8 10 12 Fold increase over GFP11 only 10 μM 20 μM 40 μM GFP11 0 2 4 6 Fold increase over GFP11 only 10 μM 20 μM 40 μM Tat-GFP11 GFP+ 0.10 GFP+ 1.40 GFP+ 0.23 GFP+ 0.23 GFP+ 2.10 GFP+ 1.43 GFP+ 9.38 GFP+ 25.2 GFP+ 65.9 GFP+ 7.27 GFP+ 31.6 GFP+ 82.3 GFP+ 32.8 GFP+ 18.2 GFP+ 6.45 GFP+ 17.7 GFP+ 4.99 GFP+ 4.21 GFP+ 0.21 GFP+ 0.23 GFP+ 1.10 GFP+ 0.17 GFP+ 0.15 GFP+ 5.20 GFP+ 0.60 GFP+ 0.23 GFP+ 0.05 GFP+ 0.10 GFP+ 0.20 GFP+ 0.17 GFP+ 0.05 GFP+ 0.24 GFP+ 0.10 GFP+ 0.05 GFP+ 0.13 GFP+ 0.36 GFP+ 12.5 GFP+ 45.5 GFP+ 0.78 GFP+ 10.7 GFP+ 59.1 GFP+ 2.42 GFP+ 31.1 GFP+ 2.88 GFP+ 3.12 GFP+ 47.2 GFP+ 2.99 GFP+ 30.5 GFP+ 0.38 GFP+ 4.69 GFP+ 47.8 GFP+ 0.22 GFP+ 0.93 GFP+ 0.45 GFP+ 0.54 GFP+ 1.88 GFP+ 2.41 GFP+ 36.1 GFP+ 0.79 GFP+ 2.29 GFP+ 43.9 GFP+ 0.13 GFP+ 7.45 GFP+ 0.10 GFP+ 0.25 GFP+ 21.2 Tat(D)-GFP11 Pen-GFP11 Arg9-GFP11 P11-GFP11 P14-GFP11 P14(D)-GFP11 P17-GFP11 P40-GFP11 GFP11 H2O 10 μM 20 μM 40 μM 10 μM 20 μM 40 μM 21 °C 37 °C tat tat(D) Pen Arg9 P11 P14 P14(D) P17 P40 ted delivery of an anionic peptide cargo. a Cells were transfected with a plasmid encoding for intact m e incubated, either at room temperature or at 37 °C, with varying concentrations of Peptide-GFP11 for 3 gle, mCherry-positive cells were ﬁrst gated. Within this population, mCherry and GFP intensities were me he percentage of GFP-positive cells shown was determined by gating just above background GFP levels. Discussion g y g To test the potential effect of CPP proteolysis on delivery efﬁciency, we included in this assay D-amino acid versions of both tat and P14, coupled to L-amino acid GFP11. In both cases, the D-form CPPs outperformed the L-form CPPs by a moderate The global market for transfection reagents and equipment is forecast to reach $1.02B in 2021, up from $715.4M in 2016 according to Markets Research global forecast report37. Interest in lipidic and peptidic delivery strategies are expected to grow as NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 5 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 C (b) and at 37 °C (c) with CPP-GFP11 constructs Averages are divided by the average intensity of cells t NATURE COMMUNICATIONS \| DOI: 1 a Tat-GFP11 GFP+ 0.10 GFP+ 1.40 GFP+ 0.23 GFP+ 0.23 GFP+ 2.10 GFP+ 1.43 GFP+ 9.38 GFP+ 25.2 GFP+ 65.9 GFP+ 7.27 GFP+ 31.6 GFP+ 82.3 GFP+ 32.8 GFP+ 18.2 GFP+ 6.45 GFP+ 17.7 GFP+ 4.99 GFP+ 4.21 GFP+ 0.21 GFP+ 0.23 GFP+ 1.10 GFP+ 0.17 GFP+ 0.15 GFP+ 5.20 GFP+ 0.60 GFP+ 0.23 GFP+ 0.05 GFP+ 0.10 GFP+ 0.20 GFP+ 0.17 GFP+ 0.05 GFP+ 0.24 GFP+ 0.10 GFP+ 0.05 GFP+ 0.13 GFP+ 0.36 GFP+ 12.5 GFP+ 45.5 GFP+ 0.78 GFP+ 10.7 GFP+ 59.1 GFP+ 2.42 GFP+ 31.1 GFP+ 2.88 GFP+ 3.12 GFP+ 47.2 GFP+ 2.99 GFP+ 30.5 GFP+ 0.38 GFP+ 4.69 GFP+ 47.8 GFP+ 0.22 GFP+ 0.93 GFP+ 0.45 GFP+ 0.54 GFP+ 1.88 GFP+ 2.41 GFP+ 36.1 GFP+ 0.79 GFP+ 2.29 GFP+ 43.9 GFP+ 0.13 GFP+ 7.45 GFP+ 0.10 GFP+ 0.25 GFP+ 21.2 Tat(D)-GFP11 Pen-GFP11 Arg9-GFP11 P11-GFP11 P14-GFP11 P14(D)-GFP11 P17-GFP11 P40-GFP11 GFP11 H2O 10 μM 20 μM 40 μM 10 μM 20 μM 40 μM 21 °C 37 °C a Tat-GFP11 GFP+ 0.10 GFP+ 1.40 GFP+ 0.23 GFP+ 0.23 GFP+ 2.10 GFP+ 1.43 GFP+ 9.38 GFP+ 25.2 GFP+ 65.9 GFP+ 17.7 GFP+ 4.99 GFP+ 4.21 GFP+ 0.21 GFP+ 0.23 GFP+ 1.10 GFP+ 0.10 GFP+ 0.20 GFP+ 0.17 GFP+ 0.05 GFP+ 0.24 GFP+ 0.10 GFP+ 12.5 GFP+ 45.5 GFP+ 2.42 GFP+ 31.1 GFP+ 2.99 GFP+ 30.5 GFP+ 0.22 GFP+ 0.93 GFP+ 2.41 GFP+ 36.1 GFP+ 0.13 GFP+ 7.45 Tat(D)-GFP11 Pen-GFP11 Arg9-GFP11 P11-GFP11 P14-GFP11 P14(D)-GFP11 P17-GFP11 P40-GFP11 GFP11 H2O 10 μM 20 μM 40 μM 21 °C a 21 °C 37 °C c b tat tat(D) Pen Arg9 P11 P14 P14(D) P17 P40 GFP11 0 2 4 6 8 10 12 Fold increase over GFP11 only 10 μM 20 μM 40 μM GFP11 0 2 4 6 Fold increase over GFP11 only 10 μM 20 μM 40 μM GFP+ 0.23 GFP+ 0.23 GFP+ 2.10 GFP+ 1.43 GFP+ 9.38 GFP+ 25.2 GFP+ 65.9 GFP+ 7.27 GFP+ 31.6 GFP+ 82.3 GFP+ 32.8 GFP+ 18.2 GFP+ 6.45 GFP+ 17.7 GFP+ 4.99 GFP+ 4.21 GFP+ 0.21 GFP+ 0.23 GFP+ 1.10 GFP+ 0.17 GFP+ 0.15 GFP+ 5.20 GFP+ 0.60 GFP+ 0.23 GFP+ 0.05 GFP+ 0.10 GFP+ 0.20 GFP+ 0.17 GFP+ 0.05 GFP+ 0.24 GFP+ 0.10 GFP+ 0.05 GFP+ 0.13 GFP+ 0.36 GFP+ 12.5 GFP+ 45.5 GFP+ 0.78 GFP+ 10.7 GFP+ 59.1 GFP+ 2.42 GFP+ 31.1 GFP+ 2.88 GFP+ 3.12 GFP+ 47.2 GFP+ 2.99 GFP+ 30.5 GFP+ 0.38 GFP+ 4.69 GFP+ 47.8 GFP+ 0.22 GFP+ 0.93 GFP+ 0.45 GFP+ 0.54 GFP+ 1.88 Pen-GFP11 Arg9-GFP11 P11-GFP11 P14-GFP11 P14(D)-GFP11 P17-GFP11 P40-GFP11 GFP11 H2O tat tat(D) Pen Arg9 P11 P14 P14(D) P17 P40 ed delivery of an anionic peptide cargo. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 a Cells were transfected with a plasmid encoding for intact mCherry and non-ﬂuorescent incubated, either at room temperature or at 37 °C, with varying concentrations of Peptide-GFP11 for 30 min and analyzed by ﬂow gle, mCherry-positive cells were ﬁrst gated. Within this population, mCherry and GFP intensities were measured for about 10,000 cells e percentage of GFP-positive cells shown was determined by gating just above background GFP levels. b, c Average GFP intensity for C (b) and at 37 °C (c) with CPP-GFP11 constructs. Averages are divided by the average intensity of cells treated with the GFP11 peptide ns of tat and P14 conjugated to an L-form GFP11 peptide were also analyzed. Error bars are standard deviations of the normalized data, open circles, with three data points, from independent experiments, per average 21 °C 37 °C c b tat tat(D) Pen Arg9 P11 P14 P14(D) P17 P40 GFP11 0 2 4 6 8 10 12 Fold increase over GFP11 only 10 μM 20 μM 40 μM GFP11 0 2 4 6 Fold increase over GFP11 only 10 μM 20 μM 40 μM 0.23 GFP+ 0.23 GFP+ 2.10 GFP+ 1.43 GFP+ 9.38 GFP+ 25.2 GFP+ 65.9 GFP+ 7.27 GFP+ 31.6 GFP+ 82.3 GFP+ 32.8 GFP+ 18.2 GFP+ 6.45 GFP+ 17.7 GFP+ 4.99 GFP+ 4.21 GFP+ 0.21 GFP+ 0.23 GFP+ 1.10 GFP+ 0.17 GFP+ 0.15 GFP+ 5.20 GFP+ 0.60 GFP+ 0.23 GFP+ 0.05 GFP+ 0.10 GFP+ 0.20 GFP+ 0.17 GFP+ 0.05 GFP+ 0.24 GFP+ 0.10 GFP+ 0.05 GFP+ 0.13 GFP+ 0.36 GFP+ 12.5 GFP+ 45.5 GFP+ 0.78 GFP+ 10.7 GFP+ 59.1 GFP+ 2.42 GFP+ 31.1 GFP+ 2.88 GFP+ 3.12 GFP+ 47.2 GFP+ 2.99 GFP+ 30.5 GFP+ 0.38 GFP+ 4.69 GFP+ 47.8 0.22 0.93 0.45 0.54 1.88 Pen GFP11 Arg9-GFP11 P11-GFP11 P14-GFP11 P14(D)-GFP11 P17-GFP11 P40-GFP11 GFP11 H2O tat tat(D) Pen Arg9 P11 P14 P14(D) P17 P40 Fig. 4 PDEP-mediated delivery of an anionic peptide cargo. a Cells were transfected with a plasmid encoding for intact mChe GFP1–10. Cells were incubated, either at room temperature or at 37 °C, with varying concentrations of Peptide-GFP11 for 30 m cytometry. Live, single, mCherry-positive cells were ﬁrst gated. Within this population, mCherry and GFP intensities were measu for each sample. The percentage of GFP-positive cells shown was determined by gating just above background GFP levels. b, c cells treated at 21 °C (b) and at 37 °C (c) with CPP-GFP11 constructs. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 A relationship likely exists between hydrophobicity and amphipathicity, driven by the number and location of Trp in the peptide sequence, with increasing hydro- phobicity being associated with more efﬁcient membrane trans- location and amphipathicity with increasing cytotoxicity50. Taken together, these observations suggest the possibility for further ﬁne-tuning of the cargo-delivery capabilities of CPPs by allowing the location and number of Trp residues in the sequence to vary in a new round of molecular evolution. efﬁciencies improve and costs decrease. Similarly, the peptide therapeutics market was valued at $17.5B in 2015 and is expected to increase at a compound annual growth rate of ~10% through 2025 as researchers and drug companies look to bypass impedi- ments that arise with small-molecule therapeutics from off-target interactions, low response rates, and drug resistance2, 38. While antisense oligomers have not yet reached the therapeutic mar- ketplace, the potential for a substantial market is clear3, 39. The development of peptide, PNA, PMO, or other polymer drugs targeting intracellular ligands requires that they efﬁciently enter cells. While peptide and polymer conjugation, micro-reservoir delivery systems, and cell type-speciﬁc targeting moieties exist and address some pharmacokinetic impediments to such drugs40, effective strategies and new modalities for efﬁcient intracellular delivery of macromolecules are still needed to advance the ﬁelds. 41 42 CPPs as biotechnological tools41 and in therapeutic delivery42 constitute a valuable and growing strategy toward solving the problem of intracellular delivery of membrane impermeant car- goes. There are examples of successful delivery of some cargo types, especially ﬂuorescent dyes that are covalently attached to CPPs9, and oligonucleotide cargoes that are non-covalently complexed with CPPs41. However, delivery of other classes of cargoes, including proteins, peptides, PNA, PMO, and others will beneﬁt from improvements in CPP efﬁciency in vitro and in vivo. Further, the ﬁeld will beneﬁt from new processes for rapidly improving upon known CPPs, building on some successes in screening and algorithm-aided design43, 44. The PNA705 model ultimately requires nuclear delivery of the 18-residue PNA to target pre-splice luciferase mRNA (Fig. 1b). Cytosolic delivery is achieved via a combination of direct plasma membrane translocation and endocytosis and escape, while nuclear delivery from the cytosol is likely achieved via passive diffusion through the nuclear pore, and perhaps some nuclear localization signal-like activity encoded in the cationic CPP sequence. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 While the magnitude of the PDEP improvement over the commonly used tat and penetratin parent sequences observed at 5 µM is dramatic, perhaps even more promising is that we observe increases in corrected luciferase mRNA for 1.25 μM PDEP–PNA705 (Fig. 2d), a concentration at which few CPPs, including the parent sequences, deliver any cargo measurably. While this level of response means incorrectly spliced mRNA is still dominant, in many diseases, including cystic ﬁbrosis, hemophilia B, F7 deﬁciency, Fanconi anemia, Bardet-Biedl syn- drome, propionic acidemia, retinitis pigmentosa, and Duchenne muscular dystrophy, the production of even a small amount of functional protein has the potential to dramatically improve the phenotype3. The emerging consensus on the efﬁciency and mechanism of action of classical CPPs such as tat and penetratin9, 45, 46 is that direct translocation and endocytosis-dependent uptake and escape both occur in parallel, but at rates that depend on CPP sequence and concentration, cell type, cargo properties, buffer, temperature, and other experimental variables. Efﬁcient delivery of cargo with these classical CPPs in vitro often requires high concentration that leads to direct translocation across the mem- brane, but simultaneously can also cause membrane disruption and toxicity47. The lack of simple sequence–function relationship rules has impeded the engineering and design of CPPs with higher efﬁciencies, or with altered reliance on speciﬁc mechan- isms of entry. With a few exceptions, discovery of new CPPs is driven by trial and error experimentation. We anticipate that the PDEP–PNA constructs developed in this work may help to catalyze, for PNA, additional progress like that made in the application of CPP–PMO chimeras toward the correction of splice defects in vivo4, 39, 51, 52. In the PMO ﬁeld, development of some CPPs, mostly by trial and error, has resulted in CPP–PMO chimeras that correct splice defects in small animal models of diseases such as Duchenne muscular dystrophy35, 53–55. In the absence of explicit sequence–structure–function rela- tionships to guide rational improvements, we reasoned here that SME would be a powerful strategy for systematically discovering more efﬁcient CPPs. We thus synthetically evolved a family of peptides for efﬁcient delivery of an 18-residue PNA to cells. PNA is a class of non-natural antisense oligomer that binds with high sequence speciﬁcity to DNA and RNA and can modulate tran- scription, translation, splicing, and replication20–27 and can have such activity in vivo26, 27. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Averages are divided by the average intensity of cells trea alone. D-form versions of tat and P14 conjugated to an L-form GFP11 peptide were also analyzed. Error bars are standard deviatio which are shown as open circles, with three data points, from independent experiments, per average 6 NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecomm 21 °C b GFP11 0 2 4 6 Fold increase over GFP11 only 10 μM 20 μM 40 μM tat tat(D) Pen Arg9 P11 P14 P14(D) P17 P40 b b 37 °C c tat tat(D) Pen Arg9 P11 P14 P14(D) P17 P40 GFP11 0 2 4 6 8 10 12 Fold increase over GFP11 only 10 μM 20 μM 40 μM c Fig. 4 PDEP-mediated delivery of an anionic peptide cargo. a Cells were transfected with a plasmid encoding for intact mCherry and non-ﬂuorescent GFP1–10. Cells were incubated, either at room temperature or at 37 °C, with varying concentrations of Peptide-GFP11 for 30 min and analyzed by ﬂow cytometry. Live, single, mCherry-positive cells were ﬁrst gated. Within this population, mCherry and GFP intensities were measured for about 10,000 cells for each sample. The percentage of GFP-positive cells shown was determined by gating just above background GFP levels. b, c Average GFP intensity for cells treated at 21 °C (b) and at 37 °C (c) with CPP-GFP11 constructs. Averages are divided by the average intensity of cells treated with the GFP11 peptide alone. D-form versions of tat and P14 conjugated to an L-form GFP11 peptide were also analyzed. Error bars are standard deviations of the normalized data, which are shown as open circles, with three data points, from independent experiments, per average NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 present. The third most active PDEP, P11, has only the Trp containing terminal cassette with an Arg at Pos6, but is otherwise similar to P14. P40 consistently outperforms the parent sequences but underperforms the other PDEP positives. It contains the Trp at Pos6 but lacks the Trp containing cassette at Pos14–16. None of the less active positive sequences rejected for further testing (Fig. 1) contained a Trp in either position 6 or the cassette. In synthetic vesicles, the tryptophans in penetratin have been shown to insert themselves into the membrane and their degree of evolutionary conservation among homeoproteins suggests func- tional importance48, 49. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Speciﬁcally, the insights gained in this study will inform the design of iterative libraries to generate PDEPs with additional desirable traits such as cell and tissue targeting, resistance to serum inhibition, increased circulation, and more as we continue to work toward the dual goals of developing (i) useful tools for the biotechnology lab, and (ii) useful delivery vehicles for systemic therapeutics. Generation of HeLa p_mCherry-GFP1–10 cells. HeLa cells were transfected with 2500 ng of linearized (BamH1) mCherry-GFP1–10 (Addgene Plasmid #78591) using Lipofectamine 3000 following standard protocols and maintained under G418 selection at 900 µg ml−1; 106 cells were analyzed by ﬂow cytometry using a BD FACSARIA III and individual cells expressing high mCherry concentrations were sorted one cell per well into 96-well plates. Stable clones were selected, expanded, and frozen. Cell culture. HeLa pTRE-LucIVS2-705 cells in this study were generously donated by Dr. Rudolf Juliano (University of North Carolina, Chapel Hill). HeLa pTRE- LucIVS2-705, HeLa p_mCherry-GFP1–10, HeLa, RAW264.7, HepG2, and MCF-7 cells were obtained from ATCC. Cells were cultured at 37 °C with 5% CO2 in Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% antibiotic–antimycotic (Gibco), and 1% non- essential amino acids (Gibco). Cells were passaged 1:5 at 90% conﬂuency. All assays were conducted on cells passaged fewer than 10×. Cell culture. HeLa pTRE-LucIVS2-705 cells in this study were generously donated by Dr. Rudolf Juliano (University of North Carolina, Chapel Hill). HeLa pTRE- LucIVS2-705, HeLa p_mCherry-GFP1–10, HeLa, RAW264.7, HepG2, and MCF-7 cells were obtained from ATCC. Cells were cultured at 37 °C with 5% CO2 in Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% antibiotic–antimycotic (Gibco), and 1% non- essential amino acids (Gibco). Cells were passaged 1:5 at 90% conﬂuency. All assays were conducted on cells passaged fewer than 10×. PNA705 mediated luciferase splice correction. HeLa pTRE-LucIVS2-705 cells were seeded on ﬂat-bottom 96-well plates at 10,000 cells per well in 200 µl complete DMEM. The following day, cells were washed in phosphate buffered saline and topped with premixed 50 µl 2× serum/phenol red-free DMEM (Gibco) + 50 µl peptide cleavage solution (ddH2O + peptide). Cells were transfected for 30 min at 37 °C with 5% CO2. The transfection solution was removed, cells were topped with 100 µl complete DMEM. The next day (24-h recovery) cells were washed in PBS. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 The power of the technique is derived from the ability to design a screen to best select the desired traits and from the ability of the library to reﬂect current knowledge such that variations in the library can represent testable hypotheses about the mechan- ism of action. By identifying optimal sequences to overcome speciﬁc delivery challenges, SME has the potential to redeﬁne the way CPPs are generated, optimized, and used in both the laboratory and the clinic moving forward. High-pressure liquid chromatography. All PNA and peptide–PNA constructs were analyzed and puriﬁed by reversed-phase chromatography. The stationary phase was a 100 mm × 4.6 mm C-18 column from Kromasil. The mobile phase was composed of a gradient of distilled water (0.1% triﬂuoroacetic acid) and acetonitrile (0.1% triﬂuoroacetic acid) with a ﬂow rate of 1 ml min−1. Where possible, peptide and peptide fragments were analyzed using tryptophan ﬂuorescence (285ex/ 340em) and PNA-containing compounds were analyzed using absorbance at 260 nm. In the absence of tryptophan or PNA residues, peptide was analyzed by absorbance at 220 nm. MALDI mass spectrometry. PNA and peptide–PNA constructs were mass veriﬁed using a Bruker Autoﬂex III MALDI–TOF mass spectrometer (Bruker Daltonics). Mass spectra data were collected in both linear and reﬂector mode with positive ion detection. Typical sample preparation for MALDI–TOF data was performed by making stock solutions of 70% acetonitrile: H2O + 30% H2O with 0.1% triﬂuoroacetic acid saturated with α-Cyano-4-hydroxycinnamic acid matrix (20 mg ml−1). Ten microliters of the stock solution was mixed with 1 μl PNA/peptide–PNA solution at 10–1000 µM, deposited onto the MALDI target plate and allowed to evaporate via the dried droplet method. As macromolecular tools and therapeutics become more pre- valent and the demand for intracellular delivery of macro- molecules increases, the need for efﬁcient delivery strategies is becoming more pressing. Natural selection has produced CPP sequences, such as tat and penetratin, which function to deliver particular cargoes to cells. Yet, they are often not efﬁcient enough to be useful as generic delivery vehicles in the lab or clinic. Here, we have used SME to identify gain-of-function CPPs with dra- matically improved ability to deliver PNA, peptide, and other cargoes to cells in culture. Thus, SME is shown to be a powerful technique for rapidly improving upon existing functional peptide sequences, not only in the ﬁeld of CPPs and delivery, but in all ﬁelds in which bioactive peptides are studied. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 is supported by confocal microscopy measurements showing rapid appearance of diffuse cargo directly in the cytosol after peptide addition. Endocytosis-dependent uptake of CPP-cargo molecules is evidenced by some punctate intracellular ﬂuores- cence and by moderately increased delivery at 37 °C compared to lower temperatures. This is presumably observed because the relative contribution of endocytosis is increased at 37 °C, but perhaps also because the membrane physical properties become more permissive for translocation at 37 °C. Some contribution of endosomal uptake is also supported by the fact that the degree of PNA705 delivery is moderately enhanced, for some, by co- treatment with the endosomolytic agent CQ. Finally, D-amino acid tat and PDEP P14 both delivered protease-sensitive, L-amino acid GFP11 with somewhat increased efﬁciency, suggesting a role for endosomal, lysosomal, or cytosolic proteases. until completely dry. Cleaved peptide–PNA sequences were dissolved in 50 µl ddH2O and were added to cells that were used in subsequent luciferase/BCA assays. Peptide-PNA705 conjugates were synthesized as described above using Tentagel XV-NH2 resin (Rapp Polymere XV18130.002) starting from the C-terminal residue of the PNA. Conjugates were acid cleaved, ether precipitated, dissolved in glacial acetic acid and lyophilized. Purity was assessed on HPLC and mass was veriﬁed by MALDI mass spectrometry. TAMRA labeling. Amidated peptide sequences with a C-terminal cysteine were ordered from Biosynthesis (www.biosyn.com) and dissolved at 2 mg ml−1 in degassed PBS at pH 7. A 100× molar excess of tris-carboxyethylphosphine (TCEP) was added to ensure complete reduction of the C-terminal thiol group. Tetramethylrhodamine-5-Maleimide was dissolved in degassed dimethylforma- mide (DMF) and added to the peptide solution at 10× molar excess. The reaction mixture was topped with nitrogen gas and stored overnight at 4 °C. Excess dye was removed using cationic exchange resin (PolyLC TT1000CAT). Labeled peptide was eluted in 25% glacial acetic acid in ddH2O and lyophilized. Purity and mass were veriﬁed by HPLC and MALDI-TOF. Concentrations were determined by absor- bance at 556 nm using Ɛ = 89 L mmol−1 cm−1. y y p The SME screen in this study was speciﬁcally designed to identify sequences capable of delivering PNA cargo, although the sequences identiﬁed proved capable of delivering other cargoes. SME generates the most active sequences from the queried sequence space for satisfying the speciﬁc requirements of the screen. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Cells were lysed in 20 µl reporter lysis buffer (Promega E4030) following the manufacturer’s protocol. A volume of 10 μl of lysate from each well was transferred to a 96-well solid black plate; 100 μl of luciferase assay reagent from the Luciferase Assay System (Promega E1500) was added, the plate was agitated for 10 s, and luminescence was measured with a BioTek Synergy 2 plate reader with injector ports. Total protein was measured with a BCA assay (described below). Data were expressed as RLU per µg protein (RLU (µg protein)−1). During screening, positive wells were deﬁned as those whose RLU per µg protein values exceeded the average for the plate by at least three standard deviations. Subsequent PNA705 delivery experiments were conducted as described above except that volumes were scaled for 24-well plate format. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 While these properties make PNA an exciting candidate biotechnological tool and therapeutic, efﬁcient and routine intracellular delivery remains a challenge. y p y To assess the degree to which the improved performance of the PDEP daughter sequences over the parent sequences, and the mechanism of entry, was speciﬁc to the PNA cargo used in the screen, we tested delivery of peptides labeled with the dye TAMRA and peptides conjugated to the anionic GFP11 peptide. Daughter PDEPs effectively deliver GFP11 and TAMRA to multiple cell types at multiple temperatures with efﬁciencies that are much higher than the parent peptides. However, the rank order is not the same for all cargoes. For example, P17 is the most efﬁcient PDEP for TAMRA delivery, but is outperformed by P11 and P14 for PNA705 and GFP11 delivery. P40 delivers PNA705 efﬁciently, but does not measurably deliver GFP11. These observations of some cargo dependence on delivery efﬁciency, even among very similar CPPs is an argument for screening for delivery using the intended cargo, or a close physical chemical analog, rather than a surrogate cargo such as a ﬂuorescent dye. y g We simultaneously optimized for efﬁcient delivery of a polar PNA cargo of 5 kDa, low toxicity, and solubility of the CPP-PNA chimera using a hybrid library. The daughter PDEPs identiﬁed in the screen (Fig. 1d) are more than two orders of magnitude more active than the parent sequences at 5 μM, despite the fact that all daughter sequences are 50–60% identical to the parents. The daughters are more cationic than the parent CPPs, with charges ranging from +9 to +12. The polar residues Asn and Gln, pos- sible in ﬁve different positions in the library, are completely excluded from the positive sequences in favor of basic residues Lys and Arg, or the non-polar Phe. The two top-performing sequences selected from the library only differ at Pos12, Pro/Met, and Pos1-/Gly. In both sequences, Trp was selected over Arg at Pos 6 and the Trp-containing terminal cassette at Pos14–16 is PDEP enter cells by multiple mechanisms simultaneously. Direct plasma membrane translocation of PDEPs with cargo is evidenced by efﬁcient delivery at 21 °C (room temperature) and at 4 °C, where energy-dependent endocytosis is slow or absent, and NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 7 7 ARTICLE ¼ PDEP Control treated LDH Activity ð Þ ðSpontaneous LDH ActivityÞ Maximum LDH Activity ð Þ ðSpontaneous LDH ActivityÞ ´ 100: 139, 937–945 (2016). Confocal microscopy. Cells were treated as described above for the TAMRA delivery assay and immediately imaged to avoid artifacts resulting from ﬁxation. The distribution of TAMRA and Sytox Green was analyzed using a confocal scanning Nikon Eclipse Ti2 inverted microscope using a 40× oil-immersion objective. Sytox Green was excited using the 488 nM laser and TAMRA was excited using the 543 nM laser. 18. Rathinakumar, R. & Wimley, W. C. High-throughput discovery of broad- spectrum peptide antibiotics. FASEB J. 24, 3232–3238 (2010). 19. Nielsen, P. E., Egholm, M., Berg, R. H. & Buchardt, O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497–1500 (1991). p y 20. Cutrona, G. et al. Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat. Biotechnol. 18, 300–303 (2000). Split GFP assay. HeLa cells were transfected with p_mCherry-GFP1–10(29) (Addgene plasmid #78591) using Lipofectamine 3000 (ThermoFisher). Single mCherry-positive cells were sorted into 96-well plates using a BD FacsAria cell sorter and colonies were maintained under G418 selection at 600 nM. Stably transfected cells were plated in 12-well plates at 100,000 cells/well and grown overnight in complete DMEM. Cells were washed in PBS and incubated in 500 µl of PDEP-GFP11 solution at variable concentrations at 23 °C and 37 °C for 30 min. The PDEP-GFP11 solution was aspirated and 100 µl of 0.025% trypsin solution was added to each well. After detachment, cells were suspended in 500 µl of PBS + 2% FBS and 20 mM HEPES. Cells were analyzed using a BD LSRII ﬂow cytometer. Cells displaying a normal morphology were gated and ﬁrst analyzed for mCherry ﬂuorescence using the 543 nM laser. mCherry+ cells were used to generate a histogram of GFP ﬂuorescence measured with the 488 nM laser. The calculated mean of this distribution and the percentage of cells exhibiting GFP ﬂuorescence values higher than 103 were recorded. 21. Chen, J., Peterson, K. R., Iancu-Rubin, C. & Bieker, J. J. Design of embedded chimeric peptide nucleic acids that efﬁciently enter and accurately reactivate gene expression in vivo. Proc. Natl Acad. Sci. USA 107, 16846–16851 (2010). 22. Pooga, M. et al. Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16, 857–861 (1998). 23. Muratovska, A. et al. ¼ PDEP Control treated LDH Activity ð Þ ðSpontaneous LDH ActivityÞ Maximum LDH Activity ð Þ ðSpontaneous LDH ActivityÞ ´ 100: ¼ PDEP Control treated LDH Activity ð Þ ðSpontaneous LDH ActivityÞ Maximum LDH Activity ð Þ ðSpontaneous LDH ActivityÞ ´ 100: py y p 4. Moulton, H. M. In vivo delivery of morpholino oligos by cell-penetrating peptides. Curr. Pharm. Des. 19, 2963–2969 (2013). 5. Nielsen, P. E. & Shiraishi, T. Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers. Artif. DNA PNA XNA 2, 90–99 (2011). ð2Þ g f 6. Dissanayake, S., Denny, W. A., Gamage, S. & Sarojini, V. Recent developments in anticancer drug delivery using cell penetrating and tumor targeting peptides. J. Control Release 250, 62–76 (2017). AlamarBlue assay. A total of 24 h after PDEP treatment, HeLa cells in 100 μl media were treated with 10 μl of 10× alamarBlue reagent and incubated for 2 h at 37 °C. Fluorescence at ex570/em585 was measured and standardized to untreated wells. 7. Kristensen, M. & Nielsen, H. M. Cell-penetrating peptides as tools to enhance non-injectable delivery of biopharmaceuticals. Tissue Barriers 4, e1178369 (2016). 8. Wallbrecher, R. et al. Membrane permeation of arginine-rich cell-penetrating peptides independent of transmembrane potential as a function of lipid composition and membrane ﬂuidity. J. Control Release 256, 68–78 (2017). qRT-PCR. HeLa pTRE-LucIVS2 cells were treated as described above with PDEP–PNA705 constructs at varying concentrations. RNA was extracted using the Direct-ZolTM RNA puriﬁcation system (Zymo Research) and reverse transcribed with the iScript cDNA synthesis kit (Bio-Rad), and mRNA splice correction was detected using PowerUp SYBR Green master mix (ThermoFisher) on an Applied Biosystems QuantStudio 6 using the following primers sets: 9. Kauffman, W. B., Fuselier, T., He, J. & Wimley, W. C. Mechanism matters: a taxonomy of cell penetrating peptides. Trends Biochem. Sci. 40, 739–764 (2015). 10. Rausch, J. M., Marks, J. R., Rathinakumar, R. & Wimley, W. C. Beta-sheet pore-forming peptides selected from a rational combinatorial library: mechanism of pore formation in lipid vesicles and activity in biological membranes. Biochemistry 46, 12124–12139 (2007). y g g ACTB1: 5′-CCTTGCACATGCCGGAG-3′ ACTB2: 5′-ACAGAGCCTCGCCTTTG-3′ RE-Luc IVS2 Downstream: 5′- TCAATCAGAGTGCTTTTGGCG pTRE-Luc IVS2 Downstream: 5′- TCAATCAGAGTGCTTTTGGCG-3′ pTRE-Luc IVS2-Bridge: 5′-TTACGATCCCTTCAGGATTACAA-3′ pTRE-Luc IVS2 Downstream: 5′- TCAATCAGAGTGCTTTTGGCG-3′ pTRE-Luc IVS2-Bridge: 5′-TTACGATCCCTTCAGGATTACAA-3′ p pTRE-Luc IVS2-Bridge: 5′-TTACGATCCCTTCAGGATTACAA-3′ pTRE-Luc IVS2-Bridge: 5′-TTACGATCCCTTCAGGATTACAA-3′ The relative quantiﬁcation of correctly spliced mRNA (90 bp amplicon) over background was calculated using the Pfafﬂmodel56. 11. Rathinakumar, R. & Wimley, W. C. ¼ PDEP Control treated LDH Activity ð Þ ðSpontaneous LDH ActivityÞ Maximum LDH Activity ð Þ ðSpontaneous LDH ActivityÞ ´ 100: Biomolecular engineering by combinatorial design and high-throughput screening: small, soluble peptides that permeabilize membranes. J. Am. Chem. Soc. 130, 9849–9858 (2008). The relative quantiﬁcation of correctly spliced mRNA (90 bp amplicon) over background was calculated using the Pfafﬂmodel56. The relative quantiﬁcation of correctly spliced mRNA (90 bp amplicon) over background was calculated using the Pfafﬂmodel56. 12. Krauson, A. J., He, J., Hoffmann, A. R., Wimley, A. W. & Wimley, W. C. Synthetic molecular evolution of pore-forming peptides by Iterative combinatorial library screening. ACS Chem. Biol. 8, 823–831 (2013). TAMRA delivery assay. HeLa, RAW264.7, HCT116, HepG2, and MCF-7 cells were plated in 12-well plates at 100,000 cells/well. After 24 h, cells were gently washed in PBS, topped with 500 µl of PDEP-TA solution containing 0.5 µM PDEP-TA + 4.5 µM PDEP-C in sfDMEM without phenol red, and incubated for 30 min at either 4 °C, 23 °C, or 37 °C. The incubation solution was aspirated and cells were treated with 100 µl 0.025% Trypsin for 3 min at 23 °C; 500 µl DMEM containing 2% FBS, 125 nM Sytox Green, and 20 mM HEPES was used to suspend the cells. Cells were transferred to a ﬁlter-topped ﬂow cytometry tube and analyzed on a BD LSR II ﬂow cytometer. Cells displaying a normal mor- phology were gated and ﬁrst analyzed for GFP ﬂuorescence using the 488 nM laser. GFP-negative cells were used to generate a histogram of TAMRA ﬂuorescence, measured with the 543 nM laser. The calculated mean of this distribution and the percentage of cells exhibiting TAMRA ﬂuorescence values higher than 103 were recorded. 13. He, L., Hoffmann, A. R., Serrano, C., Hristova, K. & Wimley, W. C. High- throughput selection of transmembrane sequences that enhance receptor tyrosine kinase activation. J. Mol. Biol. 412, 43–54 (2011). y 14. Marks, J. R., Placone, J., Hristova, K. & Wimley, W. C. Spontaneous membrane-translocating peptides by orthogonal high-throughput screening. J. Am. Chem. Soc. 133, 8995–9004 (2011). 15. Krauson, A. J. et al. Conformational ﬁne-tuning of pore-forming peptide potency and selectivity. J. Am. Chem. Soc. 137, 16144–16152 (2015). 16. Krauson, A. J., He, J. & Wimley, W. C. Gain-of-function analogues of the pore-forming peptide melittin selected by orthogonal high-throughput screening. J. Am. Chem. Soc. 134, 12732–12741 (2012). 17. Wiedman, G., Kim, S. Y., Zapata-Mercado, E., Wimley, W. C. & Hristova, K. PH-triggered, macromolecule-sized poration of lipid bilayers by synthetically evolved peptides. J. Am. Chem. Soc. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 Data availability. All data are available from the corresponding author upon request. manufacturer’s protocol. Absorbance at 562 nm was measured using a measured with a BioTek Synergy 2 plate reader. Received: 16 December 2017 Accepted: 24 May 2018 LDH assay. HeLa cells were treated for 30 min at 37 °C with 100 μl of a 2× serial dilution of PDEP constructs ranging from 160 μM to 1.25 μM in serum-free DMEM. As a positive control for LDH release, cells were treated with lysis buffer. Cells incubated in serum-free DMEM lacking peptide were used as a negative control, 50 μl of the incubation solution was transferred to a separate 96-well plate, 50 μl of the reaction mixture was added to each well and incubated for 30 min at room temperature in the dark, and 50 μl of stop solution was added to each well and absorbance was read at 490 and 680 nm. % cytotoxicity was calculated as follows: References 1. Ellert-Miklaszewska, A., Poleszak, K. & Kaminska, B. Short peptides interfering with signaling pathways as new therapeutic tools for cancer treatment. Future Med. Chem. 9, 199–221 (2017). LDH@490 nm ð Þ LDH@680 nm ð Þ ½ ¼ LDH Activity; ð1Þ Methods PDEP–PNA PDEP–PNA synthesis. PDEP–PNA library synthesis was performed using a split and combine synthesis strategy on TentaGel MB NH2 resin (MB300_002) loaded with Fmoc-photolabile linker (Advanced ChemTech RT1095) using standard Solid Phase Peptide Synthesis (SPPS) protocols. FMOC protected peptide (Advanced ChemTech)/PNA monomers (PNABio FMA001, FMT001, FMG001, FMC001, FMO001) were dissolved in DMF at 3× molar excess relative to the manufacturer’s stated loading capacity. The reaction was catalyzed by the addition of 0.9× molar HBTU/HOBt or 0.9× molar HATU for peptides and PNAs respectively. All bases were double coupled (2×20-min reactions) and reaction completion was demon- strated with the Kaiser test for amines. Following the addition of each base, remaining reactive sites were capped with 50× molar acetic anhydride and DIPEA. Between the peptide and the PNA, two [2-(2-(Fmoc-amino) ethoxy) ethoxy] acetic acid spacer moieties were added. Following acid deprotection in Reagent B (88% v/ v triﬂuoroacetic acid, 5% v/v phenol, 5% v/v ddH2O, 2% v/v triisopropylsilane) supplemented with 2.5% v/v m-cresol, beads were washed thoroughly in dime- thylformamide and dichloromethane and dry-cleaved from the solid support under UV light for 4 h to produce amidated sequences. Synthesis quality was veriﬁed by performing HPLC, mass spectrometry, and Edman sequencing on the peptide released from individual beads. Individual beads were placed in 96-well plates, suspended in 200 μl hexaﬂuoro-2-propanol, and placed under UV light at 365 nm BCA assay. The remaining 10 µl of lysate from the splice correction assay was combined with 100 µl of the BCA reagents and incubated for 1–4 h at 37 °C per the NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 8 ARTICLE LDH@490 nm ð Þ LDH@680 nm ð Þ ½ ¼ LDH Activity; 2. Fosgerau, K. & Hoffmann, T. Peptide therapeutics: current status and future directions. Drug Discov. Today 20, 122–128 (2015). 3. Havens, M. A., Duelli, D. M. & Hastings, M. L. Targeting RNA splicing for disease therapy. Wiley Interdiscip. Rev. RNA 4, 247–266 (2013). NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications LDH@490 nm ð Þ LDH@680 nm ð Þ ½ ¼ LDH Activity; ð1Þ ð1Þ NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 (MEL) cells. Int. J. Mol. Med. 34, https://doi.org/10.3892/ijmm.2014.2005 (2014). 49. Christiaens, B. et al. Tryptophan ﬂuorescence study of the interaction of penetratin peptides with model membranes. Eur. J. Biochem. 269, 2918–2926 (2002). 49. Christiaens, B. et al. Tryptophan ﬂuorescence study of the interaction of penetratin peptides with model membranes. Eur. J. Biochem. 269, 2918–2926 (2002). (MEL) cells. Int. J. Mol. Med. 34, https://doi.org/10.3892/ijmm.2014.2005 (2014). 25. Shiraishi, T., Eysturskarth, J. & Nielsen, P. E. Modulation of mdm2 pre- mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions. BMC Cancer 10, 342 (2010). 50. Jobin, M. L. et al. The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides. Biochim. Biophys. Acta 1848, 593–602 (2015). g g j 26. Ivanova, G. D. et al. Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle. Nucleic Acids Res. 36, 6418–6428 (2008). p y 51. Boisguarin, P. et al. Delivery of therapeutic oligonucleotides with cell penetrating peptides. Adv. Drug Deliv. Rev. 87, 52–67 (2015). 27. Gao, X. et al. Peptide nucleic acid promotes systemic dystrophin expression and functional rescue in dystrophin-deﬁcient mdx mice. Mol. Ther. Nucleic Acids 4, e255 (2015). 52. Nizzardo, M. et al. Morpholino-mediated SOD1 reduction ameliorates an amyotrophic lateral sclerosis disease phenotype. Sci. Rep. 6, https://doi.org/ 10.1038/srep21301 (2016). 28. Kang, S. H., Cho, M. J. & Kole, R. Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development. Biochemistry 37, 6235–6239 (1998). p 53. Shabanpoor, F. et al. Identiﬁcation of a peptide for systemic brain delivery of a morpholino oligonucleotide in mouse models of spinal muscular atrophy. Nucleic Acid. Ther. 27, 130–143 (2017). 54. Betts, C. et al. Pip6-PMO, a new generation of peptide-oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment. Mol. Ther. Nucleic Acids 1, e38 (2012). 29. Dupont, E., Prochiantz, A. & Joliot, A. Penetratin story: an overview. Methods Mol. Biol. 683, 21–29 (2011). 30. Rizzuti, M., Nizzardo, M., Zanetta, C., Ramirez, A. & Corti, S. Therapeutic applications of the cell-penetrating HIV-1 Tat peptide. Drug Discov. Today 20, 76–85 (2015). 55. Gao, X. et al. Effective dystrophin restoration by a novel muscle-homing peptide-morpholino conjugate in dystrophin-deﬁcient mdx mice. Mol. Ther. 22, 1333–1341 (2014). 31. Wender, P., Galliher, W. C., Goun, E., Jones, L. R. & Pillow, T. H. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-04874-6 The design of guanidinium-rich transporters and their internalization mechanisms. Adv. Drug Deliv. Rev. 60, 452–472 (2008). 56. Pfafﬂ, M. W. A new mathematical model for relative quantiﬁcation in real- time RT-PCR. Nucleic Acids Res. 29, e45 (2001). 32. Guo, Z., Peng, H., Kang, J. & Sun, D. Cell-penetrating peptides: possible transduction mechanisms and therapeutic applications. Biomed. Rep. 4, 528–534 (2016). Acknowledgements The authors wish to thank Rudolf Juliano at the University of North Carolina for kindly providing HeLa pLuc705 cells. We also thank Scott Grayson for assistance with MALDI mass spectrometry, and Dorota Wyczechowska at the Louisiana State University core facility for assistance with ﬂow cytometry. Funded by NIH NIGMS R01GM111824 and by the Louisiana Board of Regents Support Fund. 33. Lam, K. S. et al. Synthesis and screening of “one-bead one-compound” combinatorial peptide libraries. Methods Enzymol. 369, 298–322 (2003). 34. Yin, H. et al. A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deﬁcient mdx mice. Hum. Mol. Genet. 18, 4405–4414 (2009). 35. Du, L. et al. Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum. Hum. Mol. Genet. 20, 3151–3160 (2011). Author contributions W.B.K. designed and performed most of the experiments. S.G. designed and performed the experiments. W.B.K., S.G., and W.C.W. analyzed the data. W.B.K. drafted the manuscript which was edited and revised with W.C.W. and S.G. W.B.K. designed and performed most of the experiments. S.G. designed and performed the experiments. W.B.K., S.G., and W.C.W. analyzed the data. W.B.K. drafted the manuscript which was edited and revised with W.C.W. and S.G. 36. Milech, N. et al. GFP-complementation assay to detect functional CPP and protein delivery into living cells. Sci. Rep. 5, https://doi.org/10.1038/srep18329 (2015). 37. Markets and Markets. Transfection Reagents and Equipment Market by Method (Biochemical (Calcium Phosphate, Lipofection, Dendrimers), Physical (Electroporation, Nucleofection), Viral)), Application (Biomedical, Protein Production), End User - Global Forecast to 2021. https://www. marketsandmarkets.com/Market-Reports/transfection-reagents-equipment- market-139141146.html (2018). ARTICLE ARTICLE Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-04874-6. marketsandmarkets.com/Market-Reports/transfection-reagents-equipment- market-139141146.html (2018). Competing interests: W.B.K. and W.C.W., together with Tulane University, have applied for a patent based on the intellectual property contained herein. The remaining authors declare no competing interests. 38. Daliri, E. B., Lee, B. H. & Oh, D. H. Current trends and perspectives of bioactive peptides. Crit. Rev. Food Sci. Nutr. 12, 1–12 (2017). 39. Moulton, H. M. Cell-penetrating peptides enhance systemic delivery of antisense morpholino oligomers. Methods Mol. Biol. 867, 407–414 (2012). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ ¼ PDEP Control treated LDH Activity ð Þ ðSpontaneous LDH ActivityÞ Maximum LDH Activity ð Þ ðSpontaneous LDH ActivityÞ ´ 100: Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease. Nucleic Acids Res. 29, 1852–1863 (2001). 24. Montagner, G. et al. Peptide nucleic acids targeting the marine beta-globin mRNAs selectively inhibit hemoglobin production in Marine Erythroleukemia 24. Montagner, G. et al. Peptide nucleic acids targeting the marine beta-globin mRNAs selectively inhibit hemoglobin production in Marine Erythroleukemia URE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications 9 © The Author(s) 2018 Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 40. Torchilin, V. P. & Lukyanov, A. N. Peptide and protein drug delivery to and into tumors: challenges and solutions. Drug Discov. Today 8, 259–266 (2003). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 41. Ezzat, K. et al. PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation. Nucleic Acids Res. 39, 5284–5298 (2011). 42. Nasrollahi, S. A., Taghibiglou, C., Azizi, E. & Farboud, E. S. Cell-penetrating peptides as a novel transdermal drug delivery system. Chem. Biol. Drug Des. 80, 639–646 (2012). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 43. Lin, Y. C. et al. Multidimensional design of anticancer peptides. Angew. Chem. Int. Ed. Engl. 54, 10370–10374 (2015). g 44. Wolfe, J. M. et al. Machine learning to predict cell-penetrating peptides for antisense delivery. ACS Cent. Sci. 4, 512–520 (2018). 45. Takeuchi, T. & Futaki, S. Current understanding of direct translocation of arginine-rich cell-penetrating peptides and its internalization mechanisms. Chem. Pharm. Bull. (Tokyo) 64, 1431–1437 (2016). 46. Mager, I., Langel, K., Lehto, T., Eiriksdottir, E. & Langel, U. The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides. Biochim. Biophys. Acta 1818, 502–511 (2012). 47. Saar, K. et al. Cell-penetrating peptides: a comparative membrane toxicity study. Anal. Biochem. 345, 55–65 (2005). 48. Gehring, W. J., Affolter, M. & Burglin, T. Homeodomain proteins. Annu. Rev Biochem. 63, 487–526 (1994). 10 NATURE COMMUNICATIONS \| (2018) 9:2568 \| DOI: 10.1038/s41467-018-04874-6 \| www.nature.com/naturecommunications
https://openalex.org/W2740301202	https://pustabiblia.iainsalatiga.ac.id/index.php/pustabiblia/article/download/954/679	English	null	Coping with Library Anxiety Using Interactive Technology	Pustabiblia	2,017	cc-by	2,070	Abstract A library has been viewed as a place for learning outside of the classroom. The informa tion technology that has resulted in digital library does not replace a library as a place. It enhances the library’s role as a learning place. However, students often experience what is called as “library anxiety” when visiting a library, especially when they visit it for the first time. Adding more technological applications will make current users get better information. And it will also help cope with any library anxiety. Keywords: coping, library anxiety, interactive technology PUSTABIBLIA: Journal of Library and Information Science; Vol. 1 No. 1, Juni 2017 DOI: http://dx.doi.org/10.18326/pustabiblia.v1i1.1-6 PUSTABIBLIA: Journal of Library and Information Science; Vol. 1 No. 1, Juni 2017 DOI: http://dx.doi.org/10.18326/pustabiblia.v1i1.1-6 Coping with Library Anxiety Using Interactive Technology Ida F Priyanto Universitas Gadjah Mada Introduction Technology has brought changes to the ways libraries serve users. It also affects the way librarians provide instruction to users. The libraries began their big change with the use of computerized catalogs in the 1980s-1990s. This change affected the way libraries provided services. Library automation has changed the way a library serves the users. In addition, the presence of Internet in the libraries also affects the way libraries provide services. Currently information technology has become an important part of a library. Libraries have continuously developed their online services and resulted in the existence of digital libraries. However, digital libraries do not make the library space unimportant. Lippincott (2005) said that physical spaces are still important in higher education including libraries. She Vol. 1 No. 1, Juni 2017 1 1 Ida F Priyanto further stated that libraries could be viewed as learning spaces outside of the classrooms where students could study both individually and together with other students. The emergence of bookless libraries nowadays also emphasizes the importance of library space in the real world besides virtual one. Library Anxiety: Past and Present Matthews (2013) identified that library users have three potential reasons for using a library or information service, they go to the library to do their assignments or projects, for personal reasons, to fulfill their information needs or for doing any activities. Therefore, it is understandable that academic libraries need to learn the characteristics of the users and what and how to fulfill their needs. This is emphasized by Fidishun (2007) who viewed the importance of librarians to understand why and how users come to the library; while Bolan, Canada, and Cullin, (2007) suggested that “librarians have to listen more, trust more, and be willing to relinquish some control to allow customers of all ages and backgrounds to have the best library experience possible” (p.41). In this way, librarians may learn the views of their libraries from the perspective of their users and the users’ ideas of a good library. There are students who do not know anything about library services and when they have to go to the library, they will feel so worried. This behavior was studied by Constance Mellon (1986) who found out that four causes of the students’ uncomfortable feelings, i.e. “(1) library size, (2) lack of knowledge about location of things in the library, (3) how to begin a library works, and (4) what to do in the library” (p. 162). Mellon’s study shows that the first two causes are from the physical space of the library; while the other two causes are from research process. In other words, being in a library without knowing the location of things will result in lack of confidence; so are lack of understanding of what to do and how to begin in the library. The uncomfortable feeling appears when students have to visit the library or when they have assignments relating to library services. Jiao and Onwuegbuzie (2004) mentioned that library physical comfort related PUSTABIBLIA: Journal of Library and Information Science 2 Coping with Library Anxiety Using Interactive Technology to students’ comfort with the library and satisfaction of space access. New students have to familiarize themselves with the library environment. They should learn how they can benefit from the library services. Robertson and Jones (2007) stated that “walking into a physical library for the first time and attempting to locate a particular area within the facility can potentially be a frustrating experience” (p.60). Library Anxiety: Past and Present Swigon (2011) emphasized that library anxiety “certainly exists and should be recognized as a potential barrier to access to information in libraries” (p. 149). Lee (2012) stated that students “were not aware of what the academic library had to offer them and were provided limited or no opportunities to learn” (p. 84). There is another consideration about users. Krotoski (2010) mentioned the importance of understanding current library users. He stated that “library visitors of the future will be demanding. They will expect interactive catalogues to contain every permutation of possible data, and for it to be accessible on multiple devices at any time” (p. 633). Shafique, Shafiq- ur-Rehman, & Mahmood (2012) also stated that “information needs and expectations are continuously changing in the rapidly changing information scenario. Libraries need to re-orient their collections, services, and facilities to keep pace with these advancements” (p. 3). Technology has been with the new generation of students and they can adapt with it easily. By implementing technology to guide and help them when they visit the library, they will have less anxiety when visiting the library. Library and Current Users Current academic library users sometimes still experience library anxiety due to the library size and lack of knowledge about the library facilities, besides not knowing what to do and how to begin in the library. Therefore, the best way of helping and attracting current students who are digital natives is by applying technology in the library in new and innovative ways. Technology will enhance library instruction and information. Technology can be a good medium to share information with students nowadays because they are familiar with technology and therefore the library information will Vol. 1 No. 1, Juni 2017 3 Ida F Priyanto be more engaging and entertaining for them. Beard and Bawden (2012) stated that the library of the future must be a place of study that is equipped with excellent computing facilities, common spaces for collaborative work and adequate silent space; while Niegaard (2011) said that the library should be an “intelligent space” (p. 180). Lippincott (2005) emphasized that “the library offers a venue where academic work can be carried out in a social context. As libraries renovate facilities to incorporate technology, they are also making them more suitable for student group work, informal socializing, and ubiquitous computing” (p. 56). Some libraries in Asia have even developed entertainment facilities that are enjoyed by users and the libraries are equipped with gaming areas, gym rooms, and XD theaters. Some people also use a library as a place for their pre-wedding photo taking. Today’s libraries will be appropriate if they implement technology for various services and information for users. Niegaard (2011) mentioned three conditions to consider in relation to the implementation of technology for library facilities and information: (1) users who have new media habits and changes in behavior; (2) changes in the library’s resources and tasks; and (3) the growth in self-service facilities. Library space for users is more important than that for collections nowadays. Haas and Stillwell (2010) stated that one of the major concerns in the library is “Space, students want more room to work on their projects” (p. 163). Rasul and Sahu (2011) considered the use of IT has become imperative for the efficient management of modern libraries. In other words, collaboration among librarians, IT people, and architects will help formulate the needs of today’s users. Niegard (2011) stated the importance of librarians to work with architects “to accommodate new digital technologies (p, 175). Library and Current Users In this case, the library functions and design and the IT placement are better accommodated and managed. Application of Interactive Technology to Enhance Library Information Based on the need to help and fulfill the needs of library users, the library can enhance its services by developing interactive and real-time information about the library where users can check the number of discussion PUSTABIBLIA: Journal of Library and Information Science 4 Coping with Library Anxiety Using Interactive Technology rooms available and can then book the room from the website or their smartphones. This will help students plan their visit to the library. As library seats and computer terminals are limited, real-time information about the number and location of available seats and computer terminals is important. Students can check how many and which seats and computer terminals are available from the interactive screen in the library lobby or via smartphones and tablets. This is useful when the library has many floors. Users can check the available seats and computer terminals before going up and downstairs of the library to look for the free seats and computers. An interactive and directive catalog will save time too. The library catalog is added with an application that provides shelf information where users can get the collection. Users can check the catalog about the availability of books and other printed materials and the interactive computer screen will show the collection’s location. When this is connected with smart shelves, not only the map that a user may get, but the shelf will also notify the user by sound or light. This sort of technology is appropriate for libraries in a developing countries. Some libraries in China have already implemented this technology. It is hoped that the application of such simple technology will help library users in coping with their confusion when visiting the library and save their time by knowing the location of the room or seat to sit; to find library collection, and possibly to cope with disaster. Conclusion Library as a place still plays an important role. Library users make use of the library to study, to have discussion with others, and to enjoy other library services and activities. The application of such simple technology will definitely help users to save time and cope with their anxiety when they visit the library, especially when they visit the library for the first time. Indeed libraries should bear in mind that the focus of the library is not on the library collection anymore, but “on the user’s stay in the library and on the user having access to both physical and digital resources” (Niegaard, 2011, p. 174). Vol. 1 No. 1, Juni 2017 5 Ida F Priyanto PUSTABIBLIA: Journal of Library and Information Science References Beard, C. & Bawden, D. (2012). University libraries and the postgraduate student: physical and virtual spaces. New Library World, 113(9/10), 439-447. Bolan, K. Canada, M, and Cullin, R. (2007). Web, library, and teen Services 2.0. Young Adult Library Services. 40-43. Fidishun, D. (2007). Women and the Public Library: Using Technology, Using the Library. Library Trends, 56(2), 328-343. Haas, L. & Stillwell, A. (2010). The library–information technology partnership: Challenges and solutions. Journal of Library Administration, 50, 51–66.h Jiao, Q. and Onwuegbuzie, A. (2004). The impact of information technology on Library Anxiety: The role of computer attitudes. Information Technology and Libraries, 23(4), 138-144. Krotoski, A. (2010). Libraries of the future. Nature, 468, 633. Lee, S. (2012). An Exploratory study of library anxiety in developmental education students. Community & Junior College Libraries, 18, 67–87. Lippincott, J. (2005). Net generation students and libraries. Educause. Retrieved from http://www.educause.edu/educatingthenetgen/. Matthews, R. (2013). Valuing Information, Information Services, and the Library: Possibilities and realities. Portal: Libraries and the Academy, 13(1), 91–112. Mellon, C. (1986). Library anxiety: A grounded theory and its development. College and Research Libraries, 47(2), 160-165. Niegaard, H. (2011). Library Space and Digital Challenges. Library Trends, 60(1), 174-189. Rasul, G. & Sahu,A. (2011). Use of IT and Its Impact onService Quality in an AcademicLibrary. Library Philosophy and Practice. Retrieved from http:// unllib.unl.edu/LPP/ Roberson, J. & Jones, J.G. (2009). Exploring academic library users’ preferences of delivery methods for library instruction: Webpage, digital Game, and other modalities. Reference & User Services Quarterly, 48(3), 259–269.ii Shafique, F., Shafiq-ur-Rehman, & Mahmood, K. (2012). A Macro Sketch of Users’ Needs, Satisfaction, and Library Performance: A Survey of University Libraries in Pakistan. Library Philosophy and Practice. Retrieved from http://digitalcommons.unl.edu/libphilprac/ Swigon, M. (2011). Library anxiety among Polish students: Development and validation of the Polish library anxiety scale. Library & Information Science Research, 33, 144–150 PUSTABIBLIA: Journal of Library and Information Science 6
https://openalex.org/W4238141591	https://www.researchsquare.com/article/rs-648838/latest.pdf	English	null	Experiences and Perception Towards Reproductive Health Education Among Secondary School Teachers in South India	Research Square (Research Square)	2,021	cc-by	6,705	Experiences and Perception Towards Reproductive Health Education Among Secondary School Teachers in South India Nitin Joseph (  drnitinjoseph@gmail.com ) Kasturba Medical College Mangalore Experiences and Perception Towards Reproductive Health Education Among Secondary School Teachers in South India Experiences and Perception Towards Reproductive Health Education Among Secondary School Teachers in South India Nitin Joseph (  drnitinjoseph@gmail.com ) Kasturba Medical College Mangalore Vaibhav Mahato Kasturba Medical College Mangalore Akhil Pandey Kasturba Medical College Mangalore Shikha Mishra Kasturba Medical College Mangalore Garima Prakash Kasturba Medical College Mangalore Rishika Gandhi Kasturba Medical College Mangalore Experiences and Perception Towards Reproductive Health Education Among Secondary School Teachers in South India Nitin Joseph (  drnitinjoseph@gmail.com ) Experiences and Perception Towards Reproductive Health Education Among Secondary School Teachers in South India Nitin Joseph (  drnitinjoseph@gmail.com ) Research Keywords: Experiences, Perception, Reproductive health education, Secondary school teachers, Urban area DOI: https://doi.org/10.21203/rs.3.rs-648838/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Reproductive Health on August 26th, 2021. See the published version at https://doi.org/10.1186/s12978-021-01224-6. Page 1/14 Abstract Background: Reproductive health education (RHE) is an important component of school curricula. It helps students in the decision-making process regarding several issues concerning reproductive health. However delivering RHE at schools is a difficult task for the teachers. Methods: This study was conducted to assess the experiences and perceptions towards reproductive health education (RHE) among 236 secondary school teachers in January 2019. Data were collected using a self-administered questionnaire. Results: Only 21(8.9%) were trained in RHE. Majority [179(75.8%)] identified cultural barriers as the major challenge involved in its implementation. 95(40.3%) teachers felt that the provision of sexual education as a part of RHE will promote pre-marital sexual activity among the students. Of the total, 185 (78.4%) had average while 51 (21.6%) participants had a good perception towards RHE. It was taught in only 3(16.7%) out of the 18 schools surveyed. Only 11(4.7%) participants felt that the availability of teaching aids to conduct RHE classes at their schools was adequate. Hardly 14(5.9%) teachers had taken RHE classes for students. Among the rest, 135(60.8%) expressed their willingness to take RHE classes with appropriate training. In multi variable analysis, participants aged ≤40 years (p=0.031), those belonging to nuclear families (p=0.013), and those who had taken classes in RHE (p=0.037) had significantly good perception level towards RHE. Conclusions: Teachers therefore need to be trained and given more opportunities to take RHE sessions which will help improve their perception towards RHE. Schools need to be better equipped with resources and various perceived barriers need to be overcome before RHE can be successfully implemented. Plain English Summary This study was conducted to assess the experiences and perceptions towards reproductive health education (RHE) secondary school teachers. The participants provided the required information by filling a questionnaire. Hardly one in ten of them had prior training in RHE and one in twenty had taken RHE classes at schools. More than three-fourth of them felt that cultural barriers could pose problems in its implementation at schools. One in four teachers had good perception towards RHE and two in three teachers expressed their willingness to take RHE classes with appropriate training. Favourable perception towards RHE were expressed by teachers who were young, from small families and those who had taken RHE classes before. Introduction Reproductive health education (RHE) is an important component of school curricula. It helps students in the decision-making process regarding several issues concerning reproductive health [1, 2]. The international community has always lent its support for the implementation of RHE at schools thereby protecting the rights of the adolescent population [3]. The importance of RHE has been acknowledged in the Sustainable Development Goals Agenda so as to ensure that the necessary knowledge and skills in this area are acquired by all learners. This would support the efforts aimed at ending all forms of violence against girls and women everywhere [4]. The scenario of its implementation in schools in developing countries like India has not been fully explored within academic literature. The Adolescence Education Program (AEP) in India was launched in 2005 to cover all secondary schools [5]. However, several political, religious leaders and teachers themselves opined that the AEP was against Indian cultural and moral values. Critics also felt that its introduction might encourage sexual activity among adolescent population. It was therefore banned across several states in India, including the state of Karnataka in 2007 [6–8]. The current state of RHE in schools across India appears to be in a disorganized manner. Most teachers do not teach RHE due to reasons such as embarrassment, or it not being part of the curriculum [9]. Studies have also observed that most parents are hesitant to discuss reproductive health-related issues with their children [10]. Moreover the information on these matters obtained from mass media and society, although easily accessible, are not always accurate and reliable. Family, society and schools all have a responsibility in providing RHE to the adolescent population. Since teachers spend a considerable amount of time with the students, it is easier for them to implement RHE in the teaching curriculum. Hence, schools become an ideal and reliable setting to offer RHE for the young population [11]. For this to materialize, the teachers need to be first equipped with the necessary knowledge, skills and comfort level to effectively deliver RHE. For effective implementation of sessions on any sensitive topics such as RHE, the concerns and the expectations desired by teachers for teaching RHE in the classroom set-up, needs to be well understood. This study was hence designed to study the experiences and perceptions towards RHE among secondary school teachers. Methods This included various aspects like their perceptions on the necessity of RHE at schools, regarding topics to be covered under RHE, right class to introduce RHE, whether these classes need to be taken separately for boys and girls, need of permanent personnel at schools to teach RHE, barriers involved in teaching RHE at schools and likely problems if there were no RHE for school students. Additionally, teachers’ perception towards a sensitive question, namely, whether the provision of sexual education as a part of RHE at the school level would promote early sexual activity among the learners, was also enquired under this section. Section B focused on implementation of RHE at the surveyed schools. Teachers in this section were enquired about, the details of personnel taking RHE classes, the availability of teaching aids to conduct RHE and the adequacy of the content related to RHE taught at their schools. Section C consisted of questions to teachers to enquire their experiences of teaching RHE, the comfort level experienced by them whilst teaching, incidents of any disruptive behavior by students during sessions and any report of objections by the parents for teaching RHE at schools. The various topics under RHE for which the teachers felt that they needed more training were also enquired. For those teachers who had not taken any RHE classes so far, their willingness to teach RHE, if the required training was offered to them, was enquired from them. The questionnaires took approximately 20 minutes for each respondent to fill. The investigators were present at the venue to respond to any clarifications from the study participants during the data collection phase. Perception level towards RHE was assessed based on the responses to seven questions designed in a five-point Likert scale which comprised of five positively worded questions: whether teachers felt RHE is necessary for school students, should RHE be placed as a separate chapter in science textbooks, should RHE classes be taken separately for boys and girls, whether there is a need for permanent personnel to be employed at schools to exclusively deal with problems on reproductive health among students and whether they were willing to take classes on RHE with appropriate training. Five points were awarded for a “strongly agree ”, 4 for “agree”, 3 for “neutral”, 2 for “disagree” and 1 for “strongly disagree” response. Methods This cross-sectional study was conducted in Mangalore city situated on the western coast of South India in January 2019. The Institutional Ethics Committee granted ethical clearance. Adopting a simple random sampling technique, six secondary schools (8th to 10th standard) each from government, aided (institute owned by private management but receives aid from the government) and private schools situated within the city limits were chosen for this study. The permission to conduct the study at the government and the aided schools was taken from the Block Education Officer of Dakshina Kannada District. Further, permission to conduct the study at the school was taken from the respective school principals. Later the school teachers were approached and were informed of the nature and the purpose of the study. The school principals and teachers were assured complete anonymity of the information which were to be collected. Informed consent for participation was taken in writing from all the consenting teachers. Page 2/14 Page 2/14 Based on the findings of a previous study done in Chandigarh, India [12] where 88% of school teachers were reported to have favorable perception towards RHE; the sample size using the formula Zα 2pq/d2 at 95% confidence intervals, 5% relative precision and adding a non-response rate of 10%, was calculated as 231. Teachers were approached in their waiting rooms at schools. They were enrolled using the convenience sampling method. Teachers with a minimum of one- year teaching experience and those consenting for participation were included in this study. Data were collected using a self-administered questionnaire. The questionnaire was content validated with the help of subject experts. In the government and aided schools, the Kannada version of the questionnaire was used. It was language validated by translation and back translation with the help of language experts. Pre-testing of the questionnaire was done based on the responses of 10 teachers chosen non-randomly from a private school which was not included in the main study. Cronbach’s alpha value for the reliability of the questionnaire (after excluding the sociodemographic information of the teachers) was 0.88. The questionnaire was semi-structured with both open and closed-ended questions. The initial part of the questionnaire was designed to obtain sociodemographic information and details of prior training in RHE amongst the teachers. Section A consisted of questions assessing the views and opinions of teachers regarding RHE. Methods In lieu of the last question, for those teachers who had already taken RHE classes earlier, 5 points meant for the “strongly agree” response, were awarded to each of them. For the other two questions which were negatively worded: whether RHE classes should be taken by same-gender teachers to the same-gender students and whether sexual education will promote early sexual activity among students, reverse scoring was done. The minimum attainable score based on the responses to these seven questions was 7 and the maximum was 35. Therefore, scores ranging from 7 to 16 were considered as poor, 17 to 26 as average and 27 to 35 as good perception level towards RHE among the teachers. herefore, scores ranging from 7 to 16 were considered as poor, 17 to 26 as average and 27 to 35 as good perceptio e considered as poor, 17 to 26 as average and 27 to 35 as good perception level towards RHE among the teachers Data entry and analysis were done using IBM SPSS for Windows version 25.0, Armonk, New York. Chi-square test and Binary logistic regression analysis were used to determine the variables associated with good perception towards RHE among school teachers. The p < 0.05 was taken as the cut-off for statistical significance. Only 21 (8.9%) teachers were trained in RHE. Overall, the training in RHE was observed to be inadequate among the participants. (Table 3) Results A total of 257 teachers were eligible to take part in this study. However, only 236 (91.8%) of them returned satisfactorily filled questionnaires. The mean age of the teachers was 40.3 ± 9.5 years. (Table 1) Only 60 (25.4%) taught science-related subjects while the rest taught other subjects. The mean years of teaching experience among the participants were 11.9 ± 7.1 years. The years of teaching experience ranged from 1 to 33 years. (Table 2) Page 3/14 Page 3/14 Table 1 Socio demographic distribution of school teachers (n = 236). Characteristics Number Percentage Age (years) ≤ 25 7 3.0 26–30 39 16.5 31–35 37 15.7 36–40 40 16.9 41–45 46 19.5 46–50 29 12.3 51–55 14 5.9 > 55 24 10.2 Gender Males 88 37.3 Females 148 62.7 Type of family Nuclear 138 58.5 Joint 98 41.5 Native place Urban 189 80.1 Rural 47 19.9 Total 236 100.0 Table 1 Table 1 Socio demographic distribution of school teachers (n = 236). Characteristics Number Percentage Page 4/14 Table 2 Table 2 Distribution of teachers based upon school related characteristics (n = 236). Characteristics Number Percentage Type of school (based on ownership) Government 80 33.9 Aided 81 34.3 Private 75 31.8 Type of school (based on co-education status) Co-educational school 227 96.2 All-boys school 3 1.3 All-girls school 6 2.5 Educational background Science 93 39.4 Arts 143 60.6 Subjects taught at school Science related 60 25.4 Others 176 74.6 Teaching experience (years) 1–5 53 22.5 6–10 71 30.1 11–15 46 19.5 16–20 35 14.8 21–25 21 8.9 > 25 10 4.2 Total 236 100.0 Page 5/14 Table 3 Characteristics related to reproductive health education (RHE) training among school teachers. As many as 215 (91.1%) of the total participants agreed/strongly agreed that there was a necessity for RHE for school students. Overall, the teachers had a favorable perception of RHE. (Table 4) Results Characteristics Number Percentage Trained in RHE (n = 236) Yes 21 8.9 No 215 91.1 Number of training sessions attended (n = 21) 1 8 38.1 2 10 47.6 3 3 14.3 The time gap between the most recent training session with the present time (n = 21) ≤ 2 years 4 19.1 2.1-3 years 5 23.8 3.1-5 years 3 14.2 5.1–10 years 4 19.1 > 10 years 5 23.8 Personnel who conducted the most recent training (n = 21) Medical professionals 15 71.4 Teachers 6 28.6 The venue of training (n = 21) At the school 21 100.0 Certified training (n = 21) Yes 2 9.5 No 19 90.5 Other sources of information about RHE (n = 236)† Textbooks 183 77.5 Internet 141 59.7 Television 137 58.0 Colleagues 119 50.4 †Multiple responses 1%) f th t t l ti i t d/ t l d th t th it f RHE f h l t d t O ll th As many as 215 (91.1%) of the total participants agreed/strongly agreed that there was a necessity for RHE for school students. Overall, the teachers had a favorable perception of RHE. (Table 4) Page 6/14 Page 6/14 Page 6/14 Perception regarding reproductive health education among school teachers. Results The other personnel identified by participants suitable for this task were student counsellors [88 (37.3%)], obstetricians [83 (35.2%)], any trained personnel [83 (35.2%)], medical officers [66 (28%)], teachers [47 (19.9%)], pediatricians [41 (17.4%)], parents [38 (16.1%)], class teachers [29 (12.3%)], senior teachers [10 (4.2%)] and school principals [10 (4.2%)]. Reasons for these preferences were: due to their proficiency in knowledge regarding reproductive health [223 (94.5%)], their accessibility [32 (13.6%)] and familiarity [25 (10.6%)] with the students, as stated by the participants. The common topics under RHE to be covered at schools as opined by the participants were concepts of puberty [209 (88.6%)], awareness of good/bad touch [177 (75%)], menstrual hygiene [174 (73.7%)], information on sexually transmitted diseases (STDs) [159 (67.4%)], description and functions of reproductive organs [143 (60.6%)], benefits of ideal family size [140 (59.3%)], information about right age at marriage [139 (58.9%)], concept of menarche [136 (57.6%)], information about right age at pregnancy [134 (56.8%)], sexual abuse/harassment [133 (56.4%)] and about contraceptives [38 (16.1%)]. Topics under RHE which the participants specifically suggested to be introduced before secondary school were: awareness of good/bad touch [55 (23.3%)], about concepts of puberty [20 (8.5%)], description and functions of reproductive organs [13 (5.5%)] and menstrual hygiene [6 (2.5%)]. Topics under RHE which the participants specifically suggested to be introduced after secondary school were: issues concerning teenage pregnancies [48 (20.3%)], about contraceptives [33 (14%)], information on STDs [5 (2.1%)] and description and functions of reproductive organs [5 (2.1%)]. The common challenges involved in teaching RHE in schools as opined by teachers were: cultural barriers [179 (75.8%)], parental objections [94 (39.8%)], lack of a standardized teaching module [61 (25.8%)], teachers not being trained in RHE [52 (22%)], school administrators not recognizing the importance of RHE [15 (6.4%)] and unavailability of sufficient resource materials at schools to conduct RHE sessions [(11 (4.7%)]. The common problems that would be encountered if there were no RHE at schools as perceived by the participants were: students ending up acquiring incorrect information about reproductive health from various informal sources [109 (46.2%)], students ending up in an anxious state when they encounter issues concerning reproductive health [102 (43.2%)], greater risk of teenage pregnancies [45 (19.1%)], more chances of premarital sexual experiences [39 (16.5%)], more instances of abortions [16 (6.8%)] and risk of having an unsuccessful marital life in future [15 (6.4%)]. Results Characteristics Number Percentage The necessity of RHE for school students Strongly agree 67 28.4 Agree 148 62.7 Neutral 14 5.9 Disagree/strongly disagree 7 3.0 When should it be introduced for school children (n = 215) 1st to 5th standard 23 10.7 6th to 7th standard 88 40.9 8th to 10th standard 104 48.4 When should it be introduced if not during schooling years (n = 7) During pre-university course 6 85.7 Before marriage 1 14.3 RHE should be introduced to which gender Both boys and girls 222 94.1 Only girls 14 5.9 Should RHE sessions be taken separately for boys and girls Strongly agree 82 34.8 Agree 92 39.0 Neutral 14 5.9 Disagree 42 17.8 Strongly disagree 6 2.5 The same gender teacher should teach RHE to the same gender students Strongly agree 13 5.5 Agree 65 27.5 Neutral 70 29.7 Disagree 73 30.9 Strongly disagree 15 6.4 Suitable teaching aids to conduct RHE at schools† Posters 130 55.1 Flip charts 112 47.5 Video films 91 38.6 Models 66 28.0 RHE should be a separate chapter in science textbooks Strongly agree 48 20.3 Agree 108 45.8 Neutral 48 20.3 Disagree 31 13.2 Strongly disagree 1 0.4 RHE classes to be taught after usual teaching hours at schools Page 7/14 Page 7/14 Characteristics Number Percentage No 142 60.2 Not sure 65 27.5 Should curriculum makers take teacher’s suggestions while preparing a RHE module Yes 230 97.5 No 6 2.5 Reasons for the same (n = 230) Teachers directly deal with students 69 30.0 Teachers understand students the best 7 3.0 Need for permanent personnel at schools to exclusively deal with reproductive health-related problems among students Strongly agree 15 6.4 Agree 106 44.9 Neutral 98 41.5 Disagree 12 5.1 Strongly disagree 5 2.1 Provision of sexual education as a part of RHE will promote premarital sexual activity among the students Strongly agree 7 3.0 Agree 88 37.3 Neutral 72 30.5 Disagree 67 28.4 Strongly disagree 2 0.8 Total 236 100.0 †Multiple responses When the teachers were asked regarding who they felt were the right persons to teach RHE to the students, the majority [156 (66.1%)] stated biology teachers. A total of 14 (5.9%) teachers had taken classes on RHE. Overall, the majority of the participants felt that the resource materials for conducting RHE classes at the surveyed schools were not adequate. (Table 5) Results Out of the 215 teachers who agreed/strongly agreed with the introduction of RHE in schools, 76 (35.3%) felt that it would help students to understand more about themselves and 29 (13.5%) felt that it would benefit students in getting all their misconceptions cleared regarding this topic. Among the 14 teachers who felt that RHE is to be introduced only for the girls, 6 (42.9%) thought so because girls need to be aware of consequences following sexual misadventures. Page 8/14 Page 8/14 The cumulative perception scores of the participants ranged from 17 to 30. Among them, 185 (78.4%) had average while 51 (21.6%) had a good perception towards RHE. Implementation of RHE at schools was done in only 3 (16.7%) out of the 18 schools. Implementation of RHE was observed in a government, aided and private school. All these were co-educational schools. Formal RHE classes were given only in the private school and it was for students from 6th to 10th standard. Sessions were taken by both teachers from the same institute and by teachers from other institutes. In the other two schools, RHE sessions were offered informally for only 10th standard students and the resource persons were teachers from the same institution. A total of 14 (5.9%) teachers had taken classes on RHE. Overall, the majority of the participants felt that the resource materials for conducting RHE classes at the surveyed schools were not adequate. (Table 5) Page 9/14 Table 5 Table 5 Experiences of teachers with reproductive health education sessions at the surveyed schools. In multivariable analysis, participants aged ≤ 40 years (p = 0.031), those belonging to nuclear families (p = 0.013) and those who had taken classes in RHE (p = 0.037), had significantly good perception level towards RHE as compared to others (Table 7). Results Characteristics Number Percentage Content of RHE delivered at the school Adequate 18 7.6 Inadequate 88 37.3 Not sure 130 55.1 Availability of teaching aids at schools to conduct RHE Adequate 11 4.7 Inadequate 90 38.1 Not sure 135 57.2 Taken classes on RHE Yes 14 5.9 No 222 94.1 If not, willingness to take with appropriate training (n = 222) Agree 135 60.8 Neutral 54 24.3 Disagree 12 5.4 Strongly disagree 21 9.5 Topics under RHE for which additional training is required† Sexually transmitted diseases 56 23.7 Counseling children with issues related to RHE 53 22.5 Physiology of menstruation 21 8.9 Feeling of uneasiness while taking classes on RHE (n = 14) Neutral 1 7.1 Disagree 7 50.0 Strongly disagree 6 42.9 Feeling of uneasiness while taking classes on RHE to the opposite gender (n = 14) Neutral 3 21.4 Disagree 7 50.0 Strongly disagree 4 28.6 Disruptive behavior by students during RHE sessions (n = 14) Yes 2 14.3 No 12 85.7 Parental objection for taking classes on RHE (n = 14) Yes 4 28.6 No 10 71.4 Total 236 100.0 †Multiple responses who underwent training in RHE in the past, 3 (14.3%) had taken classes on RHE for the students. Among the 215 te aining in RHE, 11 (5.1%) had taken classes on RHE for the students (p = 0.116). Out of the 14 teachers who had tak Out of the 21 teachers who underwent training in RHE in the past, 3 (14.3%) had taken classes on RHE for the students. Among the 215 teachers who did not undergo any form of training in RHE, 11 (5.1%) had taken classes on RHE for the students (p = 0.116). Out of the 14 teachers who had taken classes in RHE, 11 (78.6%) did not undergo any form of training in RHE. Some of the open suggestions/observations given by teachers regarding reproductive health were: school students often find it uncomfortable in accepting their bodily changes during puberty (5), the current teaching of RHE at schools is inadequate (3), provision of sexual education as a part of RHE may promote Page 10/14 Page 10/14 Page 10/14 premarital sexual activity among the students (3), the mass media have promoted obscenity leading to promiscuous behavior among the students (1) and that there are several misconceptions present regarding menstruation among girls (1). Discussion The proportion of teachers who underwent training in RHE was only 8.9% in the present study in comparison to 25.8–70% reported in previous studies [2, 3, 13, 14]. Considering the multidisciplinary nature of RHE, all teachers during their preservice and in-service training years need to be given training in reproductive health. Close to three-fourth of teachers in the present study felt that RHE sessions need to be taken separately for boys and girls. In co-education RHE sessions, students might experience discomfort as learners of one gender may feel embarrassed to discuss with their teachers, certain topics under reproductive health in the presence of students of the opposite gender. More than three-fourth of teachers in this study felt that the cultural barriers were the main challenges involved in teaching RHE at schools. In previous studies, teachers listed religion, culture, restrictive policies, inadequate time being allotted, untrained teachers, lack of confidence among teachers, lack of infrastructure, lack of teaching aids, lack of support from teachers, objections raised by students, parents or school administrators, as the potential barriers for the same [2, 14–20]. A total of 40.3% teachers in this study and 39–71.7% in previous studies felt that sex education as a part of RHE would promote early sexual activity among the students [2, 3, 21]. These misconceptions need to be addressed in training sessions for teachers in order to bring a more favorable perception towards RHE. Implementation of RHE was seen merely among three of the surveyed schools in the present study. Moreover, just one out of these three schools had sessions taken by teachers from other institutions. In the South African study, it was observed that outside personnel like the Department of Health Officials and school nurses were occasionally invited to take RHE classes for the students [14]. Teachers at these schools felt that this initiative made RHE classes more effective because learners could relate and discuss sensitive issues concerning reproductive health better with outsiders than with their educators [14]. In this study, hardly 8% of respondents felt that the content of RHE sessions and hardly 5% felt that the teaching aids to conduct these sessions were adequate at their schools. Similarly, in the South African study, hardly 10% of teachers felt that the schools had adequate resources to enable them to take RHE classes [14]. In this study, hardly 6% of teachers had taken classes in RHE. Results Participants aged ≤ 40 years, females, those belonging to nuclear families, those who underwent training in RHE and those who had taken classes in RHE, had significantly good perception level towards RHE as compared to the rest (Table 6). Participants aged ≤ 40 years, females, those belonging to nuclear families, those who underwent training in RHE and those who had taken classes in RHE, had significantly good perception level towards RHE as compared to the rest (Table 6). Table 6 Association between determinants and perception level towards reproductive health education among school teachers. Characteristics Perception level towards RHE Total Good No. (%) Average No. (%) Age group ≤ 40 years 36(29.3) 87(70.7) 123 > 40 years 15(13.3) 98(86.7) 113 X2 = 8.893, p = 0.003 Gender Male 12(13.6) 76(86.4) 88 Female 39(26.4) 109(73.6) 148 X2 = 5.267, p = 0.022 Type of family Nuclear 40(29) 98(71) 138 Joint 11(11.2) 87(88.8) 98 X2 = 10.671, p = 0.001 Underwent training in RHE Yes 9(42.9) 12(57.1) 21 No 42(19.5) 173(80.5) 215 X2 = 6.143 p = 0.013 Taken classes on RHE Yes 8(57.1) 6(42.9) 14 No 43(19.4) 179(80.6) 222 X2 = 11.092, p = 0.001 Total 51 185 236 rticipants aged ≤ 40 years (p = 0.031), those belonging to nuclear families (p = 0.013) and those who had taken class Page 11/14 Page 11/14 Table 7 Table 7 Binary logistic regression analysis of variables associated with good perception level towards reproductive health education among the school teachers (n = 236). Characteristics Unadjusted OR 95% CI for unadjusted OR P value Adjusted OR 95% CI for adjusted OR P value Lower Upper Lower Upper Age (years) ≤ 40 2.703 1.386 5.272 0.003 2.147 1.073 4.299 0.031 > 40 1 1 Gender Males 1 1 Females 2.266 1.114 4.61 0.022 1.014 0.429 2.396 0.975 Type of family Nuclear 3.228 1.56 6.679 0.001 2.579 1.219 5.459 0.013 Joint 1 1 Underwent training in RHE Yes 3.089 1.222 7.812 0.013 2.163 0.813 5.751 0.122 No 1 1 Taken classes in RHE Yes 5.55 1.83 16.835 0.001 3.4 1.08 10.709 0.037 No 1 1 Funding: No funding was acquired for this study. Limitations There is a possibility of under reporting of information on this sensitive topic by participants of this study. Availability of data and materials: The SPSS spread sheet containing the data of this research study has been uploaded as a supplementary file. Authors’ contributions: NJ: guarantor of this research work, concept, design, literature search, proforma preparation, data collection, data entry, data analysis, manuscript preparation, revising the work critically for important intellectual content VM: literature search, manuscript preparation, interpretation of data, revising the work critically for important intellectual content, manuscript editing AP d i i f d i i h k i i ll f i i ll l i di i VM: literature search, manuscript preparation, interpretation of data, revising the work critically for important intellectual content, manuscript editing AP: data entry, interpretation of data, revising the work critically for important intellectual content, manuscript editing SM: literature search, interpretation of data, revising the work critically for important intellectual content, manuscript editing GP: interpretation of data, revising the work critically for important intellectual content, manuscript editing RG: interpretation of data, revising the work critically for important intellectual content, manuscript editing All authors approved the final manuscript before submission VM: literature search, manuscript preparation, interpretation of data, revising the work critically for important intellectual content, manuscript editing AP: data entry, interpretation of data, revising the work critically for important intellectual content, manuscript editing SM: literature search, interpretation of data, revising the work critically for important intellectual content, manuscript editing VM: literature search, manuscript preparation, interpretation of data, revising the work critically for important intellectual content, manuscript editing AP: data entry, interpretation of data, revising the work critically for important intellectual content, manuscript editing All authors approved the final manuscript before submission Consent for publication: This was taken from the co-investigators of this study. Discussion Among them, most were not even trained in RHE. This dismal picture was also reported in a study done in four countries of pacific islands where a significant number of teachers taught RHE without any training [2]. Prior research has shown that training has a major influence on the confidence levels of teachers teaching sensitive topics like RHE. Lack of the required training was found to negatively impact their quality of teaching [18, 19]. Among the teachers who had taken RHE classes for students in this study, more than one-fourth of them reported parental objection to the conduct of these classes. In the South African study, 90% of teachers reported a lack of support from parents [14]. Parental counseling on the importance of RHE might help in addressing these issues before initiating RHE sessions at schools. Page 12/14 Certain variables were found to be associated with good perception level towards RHE among participants in this study. In other studies, teachers with more than four years of teaching experience [22], science teachers [22], the gender of the teacher [23], trained teachers [23] and teachers teaching RHE [23] had a more positive attitude towards the importance of sex education at schools. Declarations Ethics approval: The study protocol was approved by the Institutional Ethics Committee. The ethics approval number was IECKMCMLR/012/2019 on 14th January 2019. Conclusions It is evident from the findings of this study that few teachers were trained in RHE. Similarly, few teachers had taken classes on RHE. Teachers therefore need to be trained during the pre and in-service training programs and need to be given more opportunities to take RHE sessions. This would benefit them in having a good perception of RHE, as supported by the observations of this study. Schools need to be better equipped with resources for the successful implementation of RHE. The authors declare that they have no competing interests. The authors declare that they have no competing interests. References Perception of Parent’s And Teacher’s Towards Introduction Of Sex Education In Senior Secondary Schools I Bauchi State, Nigeria. IOSR JRME. 2018;8:20–5. 21. Mohammed M, Sadiq AM, Mohammed K. Perception of Parent’s And Teacher’s Towards Introduction Of Sex Education In Senior Secondary Schools In Bauchi State, Nigeria. IOSR JRME. 2018;8:20–5. 22. Fentahun N, Assefa T, Alemseged F, Ambaw F. Parents’ perception, students’ and teachers’ attitude towards school sex education. Ethiop J Health Sci. 2012;22:99–106. 23. Esimai A. Introduction of sex education into Nigerian schools: the Parents’, Teachers’ and students' perspectives [Dissertation]. Ile-Ife: Obafemi Awolowo University;2003. Acknowledgements: We authors of this study thank the Block Education Officer of Dakshina Kannada District for permitting us to do the study at the various Government and Aided schools at the setting. We also thank the Principals of the respective schools for granting us the permission. Finally we thank all the teachers who enthusiastically took part in this study. Page 13/14 SchooldataRHE.sav References Emerging Evidence from National Surveys. PLoS ONE. 2013;8:e71584. 8. Tripathi N, Sekher TV. Youth in India Ready for Sex Education? Emerging Evidence from National Surveys. PLoS 9. Thirunavukarasu A, Simkiss D. Developments in Reproductive Health Education in India. J Trop Pediatr. 2013;59:255–7. 9. Thirunavukarasu A, Simkiss D. Developments in Reproductive Health Education in India. J Trop Pediatr. 201 0. Lebese RT, Davhana-Maselesele M, Obi CL. Sexual health dialogue between parents and teenagers: an imperat 2010;33:33–42. ; 11. Wilson SF, Strohsnitter W, Baecher-Lind L. Practices and perceptions among pediatricians regarding adolescent contraception with emphasis on intrauterine contraception. J Pediatr Adolesc Gynecol. 2013;26:281–4. 1. Wilson SF, Strohsnitter W, Baecher-Lind L. Practices and perceptions among pediatricians regarding adolescent intrauterine contraception. J Pediatr Adolesc Gynecol. 2013;26:281–4. 12. Parwej S, Kumar R, Walia I, Aggarwal AK. Reproductive health education intervention trial. Indian J Pediatr. 2005;72:287–91. 2. Parwej S, Kumar R, Walia I, Aggarwal AK. Reproductive health education intervention trial. Indian J Pediatr. 200 13. Ramiro L, Reis M, de Matos MG. Health and Sexuality Education in Portugal: Principal’s, Teacher’s, Parent’s and Student’s Perception. J Healthc Commun. 2015;1:1–10. 13. Ramiro L, Reis M, de Matos MG. Health and Sexuality Education in Portugal: Principal’s, Teacher’s, Parent’s and Student’s Perception. J Healthc Commun. 2015;1:1–10. 3. Ramiro L, Reis M, de Matos MG. Health and Sexuality Education in Portugal: Principal’s, Teacher’s, Parent’s and 2015;1:1–10. 14. Mchunu NJ. Teachers' perceptions of the teaching of sexuality education in secondary schools in the Pinet of KwaZulu-Natal; 2007. 14. Mchunu NJ. Teachers' perceptions of the teaching of sexuality education in secondary schools in the Pinetown district [Dissertation]. Pinetown: University of KwaZulu-Natal; 2007. 14. Mchunu NJ. Teachers' perceptions of the teaching of sexuality education in secondary schools in the Pinetown district [Dissertation]. Pinetown: University of KwaZulu-Natal; 2007. 15. United Nations Educational, Scientific, and Cultural Organization. Levers of Success: Case studies of Natio UNESCO; 2010. Available from: http://unesdoc.unesco.org/images/0018/001884/188495e.pdf. 15. United Nations Educational, Scientific, and Cultural Organization. Levers of Success: Case studies of National Sexuality Education Programmes. Paris: UNESCO; 2010. Available from: http://unesdoc.unesco.org/images/0018/001884/188495e.pdf. 15. United Nations Educational, Scientific, and Cultural Organization. Levers of Success: Case studies of National Sexuality Education Programmes. Paris: UNESCO; 2010. Available from: http://unesdoc.unesco.org/images/0018/001884/188495e.pdf. Kumi-Kyereme A, Awusabo-Asare K, Stillman M. Perceptions about sexuality education in Ghana. 2017. Available 16. Esia-Donkoh K, Kumi-Kyereme A, Awusabo-Asare K, Stillman M. Perceptions about sexuality education in G https://iussp.confex.com/iussp/ipc2017/mediafile/Presentation/Paper2807/Perceptions%20about%20sex 16. Esia-Donkoh K, Kumi-Kyereme A, Awusabo-Asare K, Stillman M. References 1. Breuner CC, Mattson G, AAP Committee on Adolescence, AAP Committee on Psychosocial Aspects of Child and Family Health. Sexuality Education for Children Adolescents Pediatrics. 2016;138:e20161348. 2. United Nations Educational Scientific and Cultural Organization. Attitudinal Survey Report on the Delivery of HIV and Sexual Reproductive Health Education in School Settings in Nauru, Niue, Palau and Samoa. Paris: UNESCO; 2015. 3. United Nations Educational, Scientific, and Cultural Organization. Implementation of sexuality education in middle schools in China. Paris: UNESCO; 2018. Educational, Scientific, and Cultural Organization. Implementation of sexuality education in middle schools in Chin 3. United Nations Educational, Scientific, and Cultural Organization. Implementation of sexuality education in 4. United Nations. Transforming Our World: The 2030 Agenda for Sustainable Development. A/RES/70/1. UN; 20 https://sustainabledevelopment.un.org/content/documents/21252030%20Agenda%20for%20Sustainable%20 ns. Transforming Our World: The 2030 Agenda for Sustainable Development. A/RES/70/1. UN; 2015. Available at 4. United Nations. Transforming Our World: The 2030 Agenda for Sustainable Development. A/RES/70/1. UN; 2015. Available at: https://sustainabledevelopment.un.org/content/documents/21252030%20Agenda%20for%20Sustainable%20Development%20web.pdf. 4. United Nations. Transforming Our World: The 2030 Agenda for Sustainable Development. A/RES/70/1. UN; 2015. Available at: https://sustainabledevelopment.un.org/content/documents/21252030%20Agenda%20for%20Sustainable%20Development%20web.pdf. ainabledevelopment.un.org/content/documents/21252030%20Agenda%20for%20Sustainable%20Development% 5. National AIDS Control Organization. Adolescent Education Programme. Life Skills Development. Available from: http://164.100.130.11:8091/webarsh/Resources%20on%20ARSH/Adolescent%20Education%20Programme%20Facilitators%20Guide.pdf Accessed July 27 2020. S Control Organization. Adolescent Education Programme. Life Skills Development. Available from: http://164.100.130.11:8091/webarsh/Resources%20on%20ARSH/Adolescent%20Education%20Programme%20Facilitators%20Guide.pdf Accessed July 27 2020. 6. Balasubramanian P, Prakash RR, Srilakshmi N. Religious Fundamentalism and Comprehensive Sexuality Education in South India. Building New Constituencies for Women’s Sexual and Reproductive Health (SRHR) and Rights: Interlinkages Between Religion and SRHR Karumarapakkam Village: 6. Balasubramanian P, Prakash RR, Srilakshmi N. Religious Fundamentalism and Comprehensive Sexuality Education in South India. Building New Constituencies for Women’s Sexual and Reproductive Health (SRHR) and Rights: Interlinkages Between Religion and SRHR. Karumarapakkam Village: Rural Women’s Social Education Centre; 2016. 6. Balasubramanian P, Prakash RR, Srilakshmi N. Religious Fundamentalism and Comprehensive Sexuality Educa 6. Balasubramanian P, Prakash RR, Srilakshmi N. Religious Fundamentalism and Comprehensive Sexuality Educa Constituencies for Women’s Sexual and Reproductive Health (SRHR) and Rights: Interlinkages Between Religio Constituencies for Women’s Sexual and Reproductive Health (SRHR) and Rights: Interlinkages Between Religion an Rural Women’s Social Education Centre; 2016. 7. No sex education in Karnataka schools now: Minister. OneIndia. 2007 April 18 Available from: https://www.oneindia.com/2007/04/18/no-sex-education- in-karnataka-schools-now-minister-1176893759.html Accessed July 29 2020. 7. No sex education in Karnataka schools now: Minister. OneIndia. 2007 April 18 Available from: https://www.one in-karnataka-schools-now-minister-1176893759.html Accessed July 29 2020. 8. Tripathi N, Sekher TV. Youth in India Ready for Sex Education? References Perceptions about sexuality education in Ghana. 2017. Available at: https://iussp.confex.com/iussp/ipc2017/mediafile/Presentation/Paper2807/Perceptions%20about%20sexuality%20education%20in%20Ghana_extended p.confex.com/iussp/ipc2017/mediafile/Presentation/Paper2807/Perceptions%20about%20sexuality%20educatio 7. Yusran S, Sabilu Y, Yuniar N, Hanafi H, Badara H. The Needs of Sexual and Reproductive Health Education for S Southeast Sulawesi, Indonesia. Indian J Sci Technol. 2018;11:1–9. 17. Yusran S, Sabilu Y, Yuniar N, Hanafi H, Badara H. The Needs of Sexual and Reproductive Health Education for Secondary School in Kendari City, Southeast Sulawesi, Indonesia. Indian J Sci Technol. 2018;11:1–9. 17. Yusran S, Sabilu Y, Yuniar N, Hanafi H, Badara H. The Needs of Sexual and Reproductive Health Education for Secondary School in Kendari City, Southeast Sulawesi, Indonesia. Indian J Sci Technol. 2018;11:1–9. 18. Eisenberg ME, Madsen N, Oliphant JA, Sieving RE. Barriers to Providing the Sexuality Education That Teachers Believe Students Need. J Sch Health. 2013;83:335–42. 18. Eisenberg ME, Madsen N, Oliphant JA, Sieving RE. Barriers to Providing the Sexuality Education That Teachers Believe Students Need. J Sch Health. 2013;83:335–42. 19. Kibombo R, Neema S, Moore AM, Ahmed FH. Adults’ Perceptions of Adolescents’ Sexual and Reproductive Health: Qualitative Evidence from Uganda, (Occasional Report No. 35). New York: Guttmacher Institute; 2008. 19. Kibombo R, Neema S, Moore AM, Ahmed FH. Adults’ Perceptions of Adolescents’ Sexual and Reproductive Health: Qualitative Evidence from Uganda, (Occasional Report No. 35). New York: Guttmacher Institute; 2008. 9. Kibombo R, Neema S, Moore AM, Ahmed FH. Adults’ Perceptions of Adolescents’ Sexual and Reproductive Hea (Occasional Report No. 35). New York: Guttmacher Institute; 2008. 20. Herman L, Ovuga E, Mshilla M, Ojara S, Kimbugwe G, Adrawa AP, Mahuro N. Knowledge. Perceptions and Acceptability to Strengthening Adolescents’ Sexual and Reproductive Health Education amongst Secondary Schools in Gulu District. World Acad Sci Eng Technol. 2013;7:1787–802. 0. Herman L, Ovuga E, Mshilla M, Ojara S, Kimbugwe G, Adrawa AP, Mahuro N. Knowledge. Perceptions and Accep Sexual and Reproductive Health Education amongst Secondary Schools in Gulu District. World Acad Sci Eng T 20. Herman L, Ovuga E, Mshilla M, Ojara S, Kimbugwe G, Adrawa AP, Mahuro N. Knowledge. Perceptions and Acceptability to Strengthening Adolescents’ Sexual and Reproductive Health Education amongst Secondary Schools in Gulu District. World Acad Sci Eng Technol. 2013;7:1787–802. 20. Herman L, Ovuga E, Mshilla M, Ojara S, Kimbugwe G, Adrawa AP, Mahuro N. Knowledge. Perceptions and Acceptability to Strengthening Adolescents Sexual and Reproductive Health Education amongst Secondary Schools in Gulu District. World Acad Sci Eng Technol. 2013;7:1787–802. 21. Mohammed M, Sadiq AM, Mohammed K. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 14/14 Page 14/14 Page 14/14
W4392466173.txt	https://www.degruyter.com/document/doi/10.1515/9783111027159-003/pdf	de		1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt: Theorien und Diskurse	De Gruyter eBooks	2,024	cc-by	35,301	1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt: Theorien und Diskurse Die Geschichte der (lebenden) Skulptur im 20. und 21. Jahrhundert Begriffe und Kanon Vor einer Darstellung der Geschichte der Bildhauerei im 20. und 21. Jahrhundert ist die terminologische Unterscheidung von ‚Skulptur‘/‚skulptural‘ und ‚Plastik‘/‚plastisch‘ sowie deren Diskursgeschichte zu klären. Während die beiden Begriffe ursprünglich zwei Herstellungsweisen bezeichnen, nämlich für die Plastik (griech. platto = formen, bilden) eine additive Akkumulation von Material, wohingegen die Skulptur (sculpere = schneiden, stechen, schnitzen) ein subtraktives Verfahren verlangt, ist diese Differenzierung heute obsolet, weil beide Termini meistens synonym verwendet werden.1 1958 verweist Werner Hofmann in Die Plastik des 20. Jahrhunderts auf Plinius d. Ä., der „statuarius“ den Künstler nenne, der in Metall gießt, und „sculptor“ den Arbeiter mit dem Meißel.2 Letzterer sei der eigentliche Bildhauer bzw. Bildschnitzer, je nachdem ob er in Stein und Marmor oder in Holz arbeitet. Auf einer theoretischen Ebene sei, so Hofmann, die begriffliche Differenzierung durchaus anzuwenden, jedoch entstehen spätestens dann Probleme, wenn Künstlerinnen beide Verfahren nutzen.3 In seiner Publikation habe er sich mehrheitlich für ‚Plastik‘ entschieden, „weil es im Deutschen bildkräftiger wirkt als die ‚Skulptur‘ und weil es überdies das Merkmal der Dreidimensionalität, das allem plastischen Gestalten gemeinsam ist, besonders sinn 1 2 3 Vgl. u.a. Stefanie Heckmann, Figur – Struktur – Index: Zur Modernität des Steins in der Skulptur der Gegenwart, Freiburg im Breisgau: Rombach 1999, S. 15: „Da eine eindeutige Definition aufgrund der gewachsenen und etablierten Bedeutungsdoppelung kaum mehr möglich ist, kann dies nur bedeuten, auf das Problem selbst hinzuweisen.“ Werner Hofmann, Die Plastik des 20. Jahrhunderts, Frankfurt am Main: Fischer 1958, S. 18. „Im Deutschen spricht man ohne eindeutige Unterscheidung vom Bildhauer und vom Plastiker, von Skulptur und Plastik. Die bedenkenlose Vertauschung dieser Ausdrücke ist so lange unvermeidlich, als man übersieht, daß diese ursprünglich Verschiedenes bezeichnen sollten.“ (Ebd.) Ebd., S. 18f. Open Access. © 2024 bei der Autorin, publiziert von der Walter de Gruyter GmbH, Berlin/Boston. Dieses Werk ist lizenziert unter der Creative Commons Namensnennung 4.0 International Lizenz. https://doi.org/10.1515/9783111027159-002 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt fällig zum Ausdruck bringt.“4 Angesichts der Entgrenzung des Mediums Skulptur und eines fehlenden „gemeinsamen Dachbegriff[s]“ schlägt er „plastisches Gebilde“ vor, „da damit sowohl die menschliche Gestalt als auch die sogenannte ‚gegenstandslose‘ Formerfindung bezeichnet ist.“5 Seiner Ansicht nach ist es widersinnig, von gegenstandsloser Plastik zu sprechen, da Skulpturen immer einen physischen Gegenstand beinhalten, d.h. „neue drei dimensionale Wirklichkeiten“.6 Zur physischen Faktizität zitiert er Charles Baudelaire, der der Bildhauerei eine „brutale Tatsächlichkeit“ mit seinem (oftmals Ad Reinhardt zugeschriebenen) Ausspruch attestiert habe, wonach Skulptur das sei, woran man sich stoße, wenn man zurücktrete, um ein Gemälde zu betrachten.7 Kurt Badt weist in Wesen der Plastik (1962) auf die doppelte Bedeutung von ‚plastisch‘ hin, zum einen als Verfahren, zum anderen als gattungsübergreifende Eigenschaft – wie bei Heinrich Wölfflin – in Musik, Malerei und Literatur und damit als positive „Wertbezeichnung“, wenn etwas plastisch hervortritt.8 Erstgenanntes gehe auf Albertis Traktat De Statua (1464) zurück, der zwischen per via di porre und per via di levare unterscheidet und auf die Gleichwertigkeit beider Verfahren pocht.9 Sein epochenübergreifendes Verständnis des „plastischen Verfahrens“ im Sinne einer organisch-evolutionären Oberflächengestaltung veranschaulicht Badt an der „Künstlern vertrauten“ Natur: Nicht leicht wird etwas eine Mohnkapsel, eine Melone, eine Zwiebel, eine geschlossene Tulpenblüte an Plastizität überbieten, woran sich zeigt, daß die geschlossene, sich nach außen vorwölbende, die konvex begrenzte Masse das Charakteristische der Plastizität ausmacht.10 4 5 6 7 8 9 10 Ebd., S. 19. Ebd., S. 25. „Alle diese Vorstöße in Neuland lassen es ratsam erscheinen, die künstlerischen Ergebnisse des Suchens unserer Gegenwart möglichst umfassend als ‚plastische Gebilde‘ zu umschreiben, da damit sowohl die menschliche Gestalt als auch die sogenannte ‚gegenstandslose‘ Formerfindung bezeichnet ist. […] Im Augenblick besitzt die Plastik das Selbstbewußtsein einer reifen Geschichtsstunde, und sie weiß, ihre Chance zu nützen.“ (Ebd., S. 161.) Auch gebe es eine Gebrauchsplastik, etwa Keramiken. Ebd., S. 26. Ebd.; vgl. John Thompson, „New times, new thoughts, new sculpture“, in: Marianne Ryan (Hg.), Gravity and Grace. The Changing Condition of Sculpture, 1965–1975, Ausst.-Kat. Hayward Gallery London, London: The South Bank Centre 1993, S. 11–34, S. 11: „There is a very amusing apocryphal story which has it that when asked to define sculpture, the French Symbolist poet Charles Baudelaire replied by saying that ‚it was something that you fell over when you stepped back to look at a painting‘.“ Ein Nachweis mit Zuschreibung zu Reinhardt findet sich als Anfangszitat bei Lucy R. Lippard, „‚As Painting is to Sculpture: A Changing Ratio‘“, in: Maurice Tuchman (Hg.), American Sculpture of the Sixties, Ausst.-Kat. Los Angeles County Museum of Arts, Philadelphia Museum of Art, Los Angeles: Anderson, Ritchie & Simon 1967, S. 31–34, S. 31: „Ad Reinhardt: ‚A definition of sculpture: something you bump into when you back up to look at a painting.‘“ Kurt Badt, Raumphantasien und Raumillusionen, Köln: DuMont Schauberg 1963, S. 135. Ursprünglich handelt es sich um einen Vortrag 1940 am Warburg Institute London. „Plastisch – als Werturteil – weist auf ein beschlossenes Gebilde der Fülle, lebendig aus einer seine Materie voll durchwirkenden Kraft, die in das Gebilde bis zu dessen äußersten Grenzen, diese erfüllend und spannend, hinaussteht, sei diese Materie Wort oder Ton, Farbzusammenstellung oder selbst ein Körper, derart, daß die Kraft den Stoff für den Verstehenden seiner schweren und bloßen Materialität beraubt und ihn zum Ausdrucksträger ihrer eigenen Gewalt macht.“ (Ebd., S. 136.) Ebd., S. 138. Ebd., S. 136. 20 Begriffe und Kanon Plastizität formuliert sich Badt zufolge in der „Eindringlichkeit“, im „Auf-uns-Eindringen“ der Erscheinungen, in ihrem „Von-innen-nach-außen-Drängen“.11 Er resümiert: „Plastisch nennt man also eine Form, in welcher sich Leben körperbildend wahrnehmbar anzeigt“, d.h. es sind Gebilde, deren Kraft sich an den Grenzen ihrer Erscheinung ausdrückt – Plastizität als ein Merkmal des Körperlichen mit Wachstumsprozessen.12 Durch die wachsende Bedeutung angelsächsischer Forschung seit Ende der 1950er-Jahre verlagert sich der Fokus erneut auf den Terminus ‚Skulptur‘/‚sculpture‘. Während in der deutschen und englischen Version von Carola Giedion-Welckers erstem Band Moderne Plastik. Elemente der Wirklichkeit, Masse und Auflockerung (1937), der direkt übersetzt wurde als Modern Plastic Art. Elements of Reality, Volume and Disintegration (1937), von ‚Plastik‘ bzw. ‚plastic art‘ die Rede ist, tragen ihre Nachfolgepublikationen den Titel Plastik des XX. Jahrhunderts. Volumen- und Raumgestaltung (1955) und in der Übersetzung Contemporary Sculpture. An Evolution in Volume and Space (1955).13 Eine Differenzierung fehlt, da in der englischen Einführung von Contemporary Sculpture mehrfach „plastic art“ bzw. „plastic medium“ oder „plastic principle“, aber auch „Age of Sculpture“ auftaucht.14 Bei Michel Seuphor, einem belgisch-französischen Kunstkritiker und Künstler, steht ‚Sculpture‘ im Titel der französischen Erstausgabe und ‚Plastik‘ in der deutschen Übersetzung.15 Udo Kultermann bemerkt 1967 in Neue Dimensionen der Plastik, dass „die spitzfindigen Definitionen der Skulptur und der Plastik von der Namensgenese oder von der Technik her […] illusorisch geworden“ seien.16 Gemeint sei die Gesamtheit der mit Massen im Raum arbeitenden Verfahren.17 Zahlreiche Malerinnen bedienten sich heute plastischer Wirkungselemente. Die Plastik sei eine künstlerische Sprache, die Skulptur oder Bildnerei mittels „Masse, Hohlraum, Linie und Farbe“ hervorbringe, d.h. mit „vorfixierten gebrauchten oder neuen Gegenständen“, und die seit den 1960er-Jahren an Bedeutung gewinne.18 Im „Streben nach größerer Authentizität“ greift Seuphor den von Johann Gottfried Herder und Adolf von Hildebrand mit der Aufwertung des Tastsinns untermauerten Topos des Wahrhaftigen der Skulptur auf.19 Seine Einteilung erfolgt nach der klassischen Gattungshierarchie: Mensch, 11 12 13 14 15 16 17 18 19 Ebd., Herv.i.Orig. Ebd., S. 137, Herv.i.Orig. Carola Giedion-Welcker, Moderne Plastik. Elemente der Wirklichkeit, Masse und Auflockerung, Zürich: Girsberger 1937; dies., Plastik des XX. Jahrhunderts. Volumen- und Raumgestaltung, Stuttgart: Gerd Hatje 1955 (= 1955a); dies., Contemporary Sculpture. An Evolution in Volume and Space, London: Faber and Faber 1955 (= 1955b). Eine überarbeitete und erweiterte Ausgabe veröffentlicht sie dort 1961. Giedion-Welcker 1955a, S. XIf., XIV, XXIII. Michel Seuphor, La sculpture de ce siècle. Dictionnaire de la sculpture moderne, Neuchâtel: Édition du Griffon 1959. Deutsche Übersetzung: Die Plastik unseres Jahrhunderts. Wörterbuch der modernen Plastik, übers. von Henri Holz-Fay, Köln: M. Dumont Schauberg 1959. Udo Kultermann, Neue Dimensionen der Plastik, Tübingen: Wasmuth 1967, S. 5. Dieser hier zum Ausdruck kommenden begrifflichen Problematik soll nachgegangen werden. Ferner stellen für diese und weitere Fragen die Kataloge der Skulptur Projekte Münster (1977–2017) eine wichtige Quelle dar. Kultermann 1967, S. 5. Ebd. 21 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt Tier, Pflanze, Stillleben, Gebrauchsgegenstände etc. Althergebrachte Techniken werden ergänzt durch „hinzugetretene Mittel (Collage, Körperabguß, Formung von Kunststoffen, Magnetismus, Elektrizität, Luftdruckunterschiede)“.20 Seuphors Ziel besteht darin, über die abstrakte Plastik hinauszugehen, um die Bandbreite der künstlerischen Möglichkeiten zu demonstrieren. Er hebt – das ist für den vorliegenden Kontext wichtig – die Entgrenzung des Mediums durch eine enge Bindung an Musik, Tanz, Theater und Happening und die Verzeitlichung der ‚neuen‘ Plastik hervor: „Man versucht, Lebendiges, Prozesse in die Arbeit einzubeziehen, und nicht mehr allein mit den Mitteln der Illusion.“21 Die „heilsame Offenheit und Freiheit“ anerkennend, fährt er fort: Idole der Höhlenmenschen, Fetische aus Afrika und Polynesien, Skulpturen der Antike und der Renaissance, aber auch Warenhausreklame und Fernseharrangements, Industrieprodukte und Autofriedhöfe – alles ist Stoff für den Bildhauer unserer Zeit geworden, sei es im Sinne der Übernahme des Materials, der Paraphrase, der Anähnlichung der Form oder der zitathaften oder vergewaltigenden Aneignung.22 Auch Eduard Trier differenziert zwischen beiden Verfahren. Da der „Bild-Hauer“ Material weghauen müsse, sei für das Ergebnis dieses Tuns der Terminus ‚Skulptur‘ besser geeignet.23 Ulrich Gertz spricht in Plastik der Gegenwart (1964) die begriffliche Pluralität an: „Plastik, Skulptur, Bildhauerei […], Bezeichnungen, die in unserer Zeit nicht mehr den Wirklichkeiten gerecht werden. Und es ist mehr eine Gewohnheit, wenn diese Begriffe weiterhin angewendet werden.“24 Auch wenn skulpturale Praktiken und verwendete Materialien seit den 1960er-Jahren expandierten, finden in der Forschungsliteratur die Termini ‚Skulptur‘ und ‚Plastik‘ weiterhin synonym Verwendung. Meines Erachtens bietet sich diese Differenzierung in erster Linie für historische Werke an.25 Rosalind Krauss verwendet „sculptural“ im Kontext von „sculptural production“, „sculptural project“ und „sculptural art“, ohne dies terminologisch zu reflektieren oder zu präzisieren.26 Die beiden Pole ihres poststrukturalistischen Diagramms benennt 20 21 22 23 24 25 26 Ebd. Ebd., S. 6. Ebd. Eduard Trier, Moderne Plastik von Rodin bis Marini, Berlin: Gebr. Mann 1954, S. 10. Ulrich Gertz, Plastik der Gegenwart. Zweite Folge, Berlin: Rembrandt 1964, S. 6. Vgl. auch Heckmann 1999, S. 17f.: „Obwohl immer präzisere, den Arbeitsprozeß berücksichtigende Bezeichnungen mit den neuen künstlerischen Ausdrucksformen entstanden – wie zum Beispiel Assemblage, Materialcollage, Objekt oder Konstruktion –, fehlte nach wie vor ein plausibler Oberbegriff, um die entstandene Vielfalt der dreidimensionalen Ausdrucksformen zusammenzufassen. Statt dessen behielt man Skulptur und Plastik bei, die nun – ihrer ursprünglichen Bedeutung enthoben, da jede Rücksicht auf ihre Etymologie angesichts der neuen Verfahren sinnlos geworden war – nur mehr als gleichwertige Gattungsbezeichnungen weiterexistierten.“ Krauss 1979, S. 34, 37. Krauss begegnet 1979 dieser inflationären Nutzung mit ihrem Diagramm „Sculpture in the Expanded Field“, dessen Struktur und Rezeption im Abschnitt „Von der Skulptur zum Skulpturalen: Rosalind Krauss“ noch ausführlicher besprochen werden. 22 Skulpturhistoriographie sie: „Landscape and architecture – terms that could function to define the sculptural […] only in their negative or neuter condition.“27 Skulpturhistoriographie Die Geschichte der Bildhauerei des 20. und 21. Jahrhunderts wird oft als teleologisches modernistisches Narrativ anhand eines europäischen und nordamerikanischen Kanons vorwiegend männlicher, weißer Künstler erzählt. Die Gliederung erfolgt nach Herkunfts ländern – noch immer werden in der Regel Geburts- und Arbeitsort/-land von Künstlerinnen angegeben –, wodurch die Werke geographisch verortet werden und die Rezeption direktiv in eine Perspektive gelenkt wird. Ausgehend von den großen Macht- und Kunstzentren Berlin, London, New York und Paris richtet sich bis heute oftmals die Aufmerksamkeit auf eine wiederholt rezipierte Selektion an Werken, die die Infrastrukturen der Moderne entscheidend mitbestimmen. Selten werden bestehende Verflechtungen kritisch reflektiert; ein forschender Blick hinter die etablierten Positionen fehlt. Ein in Paris angesiedeltes Forschungsprojekt namens AWARE Archives of Women Artists, Research and Exhibitions informiert in einer online frei zugänglichen Datenbank über unbekannte, in Forschung, Museum und Kunstmarkt marginalisierte Künstlerinnen des 20. Jahrhunderts aus einer transnationalen Perspektive.28 Auch zu queeren Bildhauerinnen fehlen bislang umfassende Datenbanken und Überblickswerke. Ansätze finden sich u.a. bei Terry Berlier, Queer Art History und Queerbio.com, im GLBTQ Encyclopedia Project, bei Queerate Tate und The Museum of Transgender History & Art (MOTHA).29 Ähnlich wie bei den Bildhauerinnen richten Forschende und Ausstellungen den Blick auf Einzelphänomene, etwa Cassils, Felix Gonzalez- Torres, George Segal und Renée Sintenis.30 Oftmals werden die Bildhauerinnen und ihre Werke unabhängig von genderbezogenen Ausrichtungen und Fragestellungen diskutiert und in den Kanon eingereiht. Oder aber die genderbezogene Individualität blieb aufgrund 27 28 29 30 Ebd., S. 38. Siehe auch Dobbe/Ströbele 2020. Https://awarewomenartists.com/en/artistes_femmes/ (19.8.2019). Siehe auch das Ad Hoc Woman Artist Committee, das u.a. Lucy R. Lippard 1970 mitbegründete; die Mitglieder brachten das Whitney Museum dazu, mehr Frauen in Ausstellungen zu integrieren. Https://exploreintrosems.stanford.edu/news/artstudi-150n-queer-sculpture; https://www.queerarthistory. com/tag/sculpture/; https://queerbio.com/wiki/index.php/LGBTQ_Sculptors; http://www.glbtqarchive.com; https://www.tate.org.uk/art/queerate-tate; https://www.motha.net/about (7.2.2023). Sofia Gonzalez- Rodriguez, „Clayman Institute spotlights Terry Berlier’s wordlessly eloquent conceptual sculpture“, in: The Stanford Daily, 9.2.2022, https://stanforddaily.com/2022/02/09/clayman-institute-spotlights-terryberliers-wordlessly-eloquent-conceptual-sculpture/ (10.2.2023). David Getsy, Abstract bodies. Sixties sculpture in the expanded field of gender, New Haven: Yale University Press 2015; https://www.mmk.art/de/whats-on/felix-gonzalez-torres/ (7.2.2023); Joseph Disponzio, „George Segal’s Sculpture on a Theme of Gay Liberation and the Sexual-political Equivocation of Public Consciousness“, in: Harriet Senie/Sally Webster (Hg.), Critical Issues in Public Art: Content, Context, and Controversy, New York: Oxford University Press 1992, S. 199–214; Alexandra Demberger/ Julia Wallner (Hg.), Zwischen Freiheit und Moderne. Die Bildhauerin Renée Sintenis, Ausst.-Kat. Kunstforum Ostdeutsche Galerie Regensburg, Georg Kolbe Museum Berlin, Berlin: Edition Cantz 2019. 23 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt von intersektionalen Restriktionen im Hintergrund. Zunehmend wird dem zwischenzeitlich gleichfalls ‚kanonisierten‘ Titel des Essays von Linda Nochlin „Why Have There Been No Great Women Artists?“ (1971), in dem sie institutionellen Widerständen und Hindernissen nachgeht, produktiv begegnet.31 Ausgehend von diesen Überlegungen intendiert das Forschungsprojekt zum Thema Que(e)rschnittsgeschichte, Revision des Skulpturenkanons und Erweiterung medienspezifischer Termini eine andere ,Erzählung‘, d.h. eine Transformation des Kanons und vernetzt auf Basis genderübergreifender, skulpturtheoretischer und künstlerischer Begriffscluster die Künstlerinnen, die in der tradierten Skulpturgeschichte niemals aufeinander treffen.32 Im Kontext der beiden Künstler Hans Haacke und Pierre Huyghe tritt durch künstlerische Gesten der Domestizierung, Zähmung und Unterwerfung nicht-menschlicher Lebewesen und damit durch ein erweitertes Verhältnis zwischen ‚Material‘ und Autor eine Spannung auf, die werkspezifisch zu reflektieren ist. Beide rekurrieren auf ein westlich geprägtes Konzept von Skulptur, daher sind zunächst dessen wesentliche Narrative und Paradigmen im Hinblick auf zeitbasierte lebende skulpturale Arbeiten zu skizzieren. Überblickswerke zur Geschichte der Bildhauerei beginnen ihre Darstellung größtenteils mit den ‚impressionistischen‘ Skulpturen Auguste Rodins als ‚Vater der Moderne‘ und setzen sie bis zur abstrakten Plastik der 1950er- und 1960er-Jahre fort – die faschistische Skulptur ausklammernd.33 Alex Potts versucht in The Sculptural Imagination. Figurative, Modernist, 31 32 33 Linda Nochlin, „Why Have There Been No Great Women Artists?“ (1971), in: dies., Women, Art, and Power, New York: Harper & Row 1988, S. 145–178. Zwei exemplarisch genannte Ausstellungen mit begleitenden Publikationen im Berliner Georg Kolbe Museum Die erste Generation. Bildhauerinnen der Berliner Moderne (2018) und dem Bremer Gerhard-Marcks-Haus Bildhauerinnen in Deutschland (2019) widmen sich im deutschsprachigen Raum weiblichen Künstlerinnen und der Aufarbeitung dieses Desiderats. Vgl. auch frühere Ausstellungen der 1970er-Jahre, etwa: California Girls (Richmond Art Center 1971); 26 Contemporary Women Artists (kuratiert von Lucy R. Lippard, Aldrich Museum of Contemporary Art, Ridgefield, Connecticut 1971); American Woman Artist Show (kuratiert von Sybille Niester, Hamburger Kunsthaus 1972). Außerdem erschienen Publikationen wie: Renate Kroll (Hg.), Künstlerinnen schreiben. Ausgewählte Texte zur Kunsttheorie aus drei Jahrhunderten, Berlin: Reimer 2018; Anja Cherdron-Modig, ‚Prometheus war nicht ihr Ahne‘. Berliner Bildhauerinnen der Weimarer Republik, Marburg: Jonas 2000. Im Kunstmagazin Avalanche (Winter 1971, S. 5) findet sich ein kurzer Abschnitt zum Thema Frauenausstellungen bzw. eine Ankündigung von California Girls, kuratiert von Lippard. Hier ist interessant zu sehen, dass Frauenkunst, wenn überhaupt, insbesondere mit einem feministischen Hintergrund der 1970er-Jahre, separat als eigenes ‚Phänomen‘ gezeigt wurde und Genderfragen dabei programmatisch verhandelt wurden. Seit einigen Jahren werden erneut bisher marginalisierte weibliche Positionen ausgestellt, im Versuch, den Kanon zu hinterfragen bzw. zu erweitern. Es handelt sich um ein neues Forschungsprojekt https://www.zikg.eu/forschung/projekte/projekte-zi/ skulptur-1900-2000 (16.5.2023). Siehe u.a. William Tucker, The Language of Sculpture, London: Thames and Hudson 1977; Rosalind Krauss, Passages in Modern Sculpture, New York: Viking Press 1977 (im Folgenden zit. n. der 1981 bei MIT Press erschienenen Ausgabe); Andrew Carnduff Ritchie, Sculpture of the Twentieth Century, Ausst.-Kat. MoMA New York 1952. Hier findet sich auch wieder die bekannte Gliederung: „The object in relation to light: Rodin and his influence, The object idealized: Maillol and related sculptures, The object purified: Brancusi and organic abstraction“. Eine Ausnahme bildet Penelope Curtis, die sich diesem Phänomen aus der Perspektive des „celebratory body“ und der „statuemania“ widmet (Penelope Curtis, Sculpture 1900–1945: After Rodin, Oxford: Oxford University Press 1999, S. 238, 38). 24 Skulpturhistoriographie Minimalist (2000), die Hegemonie Rodins zu relativieren. Er verweist auf die ausgestellten Antikenfunde fragmentierter Körper, die bereits vor Rodin eine Exponierung des Torso provozierten, während Rodins Werke (Homme qui marche, 1907) im Vergleich zu den ‚Objekten‘ Constantin Brancusis aus teils gefundenen Materialien (Torso of a young man, 1916) deutlich einer figurativen Formensprache verhaftet bleiben.34 In seiner epochenübergreifenden Argumentation führt Potts vor, wie ein Rückgriff auf die klassizistische Skulptur für ein Verständnis moderner Skulptur fruchtbar gemacht werden kann, und stellt Antonio Canova Robert Morris, einem Künstler des 20. Jahrhunderts, gegenüber, indem er ihr „play of surface“, d.h. ihren Umgang mit Oberflächen und Texturen sowie ihre wirkungsästhetische Konzeption in der Nah- und Fernsicht vergleicht.35 Auswahlkriterien der Überblickswerke für die Gliederung und formal-analytische Perspektiven sind material-, länder- oder technikbezogene Kapitel, skulpturspezifische Parameter wie Volumen, Platzierung im Raum und Oberflächenbearbeitung oder eine motivische Unterteilung ähnlich der klassischen Gattungshierarchie Mensch, Tier, Pflanze und Gebrauchsgegenstände.36 Der Vergleich mit der Malerei und erneut bemühte Paragone- Argumente führen in diesen Texten häufig zu einer zu engen, starren Definition von Skulptur.37 Ein wesentliches Narrativ beschreibt die Entwicklung moderner Skulptur in zwei Hauptsträngen: erstens zur Öffnung der Form bzw. Aufsplitterung in konstruktivistische Materialassemblagen, deren entmaterialisierte, bewegliche und transparente Objekte mit virtuellem Volumen (László Moholy-Nagy, Naum Gabo, Antoine Pevsner, Alexander Calder) in den Raum ausgreifen und die plastische Masse auflösen; zweitens zu einer abstrahierten kompakten plastischen Masse, etwa bei Aristide Maillol oder Henry Moore.38 Die Einbeziehung des lebenden Körpers und organischen Materials ab den 1960er-Jahren leitet einen Paradigmenwechsel ein, der bis heute wirkt. Neue Materialien wie Kunststoffe oder Gene finden Verwendung. Skulptur vereint unterschiedlichste Kunstformen und erlebt eine zunehmende Entgrenzung, die aktuell in neuen Technologien wie Virtual Reality, Augmented Reality und 3D-Druck andauert. Und doch fehlt bislang ein Überblicksband zur (Non-Human) Living Sculpture und zur skulpturalen Ästhetik des Lebendigen. Der Fokus der vorliegenden Untersuchung auf zeitbasierten, lebenden Skulpturen, insbesondere system ästhetischen und situationsästhetischen Konzepten bestimmt die Auswahl an Publikationen, die nun in einem vergleichenden Close-Reading und einer detaillierten Literaturexegese in 34 35 36 37 38 Alex Potts, The Sculptural Imagination. Figurative, Modernist, Minimalist, New Haven [u.a.]: Yale University Press 2000, S. 5. Ebd., S. 13. Kultermann 1967. Seuphor 1959, u.a. S. 219–222. Weitere deutschsprachige Publikationen, die für den Kanon der Bildhauerei der Moderne maßgeblich waren, seien hier nur kurz genannt: Hans Platte, Die Kunst des 20. Jahrhunderts. Plastik (Bd. 2), Hamburg: Standard 1957; Franz Roh, Deutsche Plastik von 1900 bis heute mit Selbstbekenntnissen der Bildhauer, München: Bruckmann 1963; Hanns Theodor Flemming, Figur und Raum in der Plastik der Gegenwart, Bremen: Angelsachsen-Verlag 1964; Abraham Marie Hammacher, Die Entwicklung der modernen Skulptur. Tradition und Erneuerung, Berlin: Propyläen 1973. 25 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt den Blick genommen werden, um die Spuren zu verfolgen, die zu einer skulpturalen Ästhetik des Lebendigen führen. Moderne Plastik als Volumen- und Raumorchestrierung: Carola Giedion-Welcker Das 1937 im Girsberger Verlag erschienene Buch Moderne Plastik. Elemente der Wirklichkeit, Masse und Auflockerung von Carola Giedion-Welcker steht exemplarisch am Anfang der modernen Skulpturgeschichte. Die Autorin denkt Plastik „sichtbar und abtastbar“ ausgehend von der „Gestaltung realer Körper“.39 Mit Beginn der Renaissance konstatiert sie das „künstliche Piedestal“, das sich zwischen Kunst und Leben geschoben habe bis zur „völligen Lebensentfremdung“ in der Kunst des 19. Jahrhunderts, wohingegen das 20. Jahrhundert eine „veränderte (plastische) Sprache“ präge, deren „große, durchgehende und bindende Linien“ und historische, teils außereuropäische Verankerung Giedion- Welcker nachzuzeichnen versucht.40 Für sie bilden Körperlichkeit und physische Faktizität ein entscheidendes Merkmal plastischer Kunst, die damit der Lebenswelt näherkomme als andere Kunstformen: Schon die körperliche Realität des Menschen lebt aktiv im Raum, in einer Welt von statistischen und dynamischen Körpern. Aus der Bewegung mit ihnen, mit dem lebendig Wachsenden eines Baumstammes oder künstlich Hergestellten eines Verkehrszeichens bis zum alltäglichen Umgang mit Teller, Tasse, Frucht, Ei etc. entstehen ununterbrochen Beziehungen, die auch zum Wesenskern der plastischen Gestaltung gehören. Statik und Dynamik, Volumen in Raum und Zeit, Masse und ihre Auflösung – von allem literarischen und psychologischen Ballast befreit – erhalten nun eine neue und direkte Bedeutung. Die heutige Plastik geht von diesen elementaren Emotionen aus. Es sind ihre unverhüllten Fundamente. Das bedeutet eine bewußte Verwurzelung in dem Boden unserer realen Existenz, gleichzeitig eine Betonung des Allgemeinen und Objektiven.41 Damit begründet Giedion-Welcker ihre durch das Studium bei Heinrich Wölfflin geprägte formal-gestalterische Perspektive auf die sogenannten geometrischen und organischen künstlerischen Ausdrucksmittel. Ihre Geschichte beginnt mit Aristide Maillol, führt über Auguste Rodin, Honoré Daumier, Edgar Degas und Henri Matisse zu den Avantgarden (Kubismus, Futurismus, Dadaismus, Surrealismus, Neoplastizismus, Suprematismus, Konstruktivismus), unterbrochen von einzelnen Abschnitten zu Hans Arp und Constantin Brancusi. Das Abbildungsverzeichnis setzt mit Daumiers Ratapoil (1848) ein, dessen expressive Silhouette und Körperhaltung sie als Beispiel des frühen Impressionismus in der Skulptur anführt. Sie ordnet die Protagonisten in das dualistische Modell Barock („barocke Welle“ mit Daumier, Rodin und Degas) versus Klassik („klassische Tradition“ bei Boccioni und Maillol) ein.42 Skulptur spezifische und entwicklungsgeschichtliche Aspekte wie Bewegungs- und Lichtprobleme, Innenmodellierung, Masse-Raumbeziehungen, Vereinfachung des Volumens, Auflockerung des Massivs sowie „Simultanisierung (Durchdringung) der verschiedenen Raumqualitäten“ 39 40 41 42 Giedion-Welcker 1937, S. 5. Ebd., S. 5f. Ebd., S. 6, Herv.i.Orig. Ebd., S. 7f. 26 Skulpturhistoriographie und Verzeitlichung der Plastik zählen zu den stilistischen Merkmalen, die die Autorin heraus arbeitet.43 Mit dem Kubismus, darunter Duchamp-Villon, verändere sich das ‚statische Volumen‘ sukzessive zu einem ‚kinetischen Volumen‘ zugunsten der plastisch-kinetischen Substanz.44 Die entmaterialisierten, beweglichen und transparenten Objekte mit teils virtuellem Volumen u.a. von László Moholy-Nagy, Naum Gabo, Antoine Pevsner, Alexander Michailowitsch Rodschenko, Julio González und Alexander Calder greifen anschließend, so die teleologisch ausgerichtete Erzählung Giedion-Welckers, in den Raum aus, beziehen Luftvolumen ein und lösen die plastische Masse auf. Die Suggestion von Lebendigkeit erfolgt nun über die Abstraktion, vermittelt in organistisch-vitalistischen bzw. tektonisch-architektonischen Metaphern der Autorin, so in ihren Notizen zu Arps „Naturverdichtungen“: Weniger erstaunt wäre man, würde man diesen ungewöhnlichen Körperballungen Arps in einsamen Gegenden begegnen, völlig in sich versponnenen und gleichzeitig doch verwachsen mit dem Naturganzen. So scheinen sie auch ihre plastische Prägung eher von einem jahrtausend alten Gletscherschliff als von einer menschlichen Hand empfangen zu haben.45 Die ‚unterbewussten, direkten Naturbeziehungen‘ der Künstlerinnen durchziehen den gesamten Band, sei es bei einem inmitten der Landschaft platzierten Werk von Lipchitz oder in der Abbildung prähistorischer, ritueller Steinfragmente aus Cornwall (Men-an-Tol) bzw. „plastischer Schneebildungen“, die gleichberechtigt neben afrikanischen oder frühchristlichen Skulpturen in die Bilderfolge eingereiht sind, dabei den lebensweltlichen Bezug unterstreichen sollen.46 Zwar kommuniziert Giedion-Welcker die Naturphänomene nicht als eigenständige, quasi lebende Kunstwerke einer ‚bildhauernden‘ Natur, doch verweisen sie auf deren formende und gestaltende Kraft (natura naturans), die die Künstlerinnen nachzuahmen suchen. In der Ausgabe von 1937 sind winterliche Schneeformationen in einem Flussbett der Concrétion Humaine (1933) Hans Arps auf einer Doppelseite gegenübergestellt (Abb. 3).47 Eine Abbildung aus dem Gletschergarten Luzern platziert Giedion- Welcker neben Arps Configurations (1932) (Abb. 4).48 Natur dient als Vorbild, hier aus 43 44 45 46 47 48 Ebd., S. 8, Herv.i.Orig. Ebd., S. 52. Ebd., S. 10. Ebd., S. 81, 90. Diese sind ohne die Angabe von Künstlerinnennamen abgebildet. Ebd., S. 90f. Ebd., S. 88f. Ein Vergleich der beiden Abbildungsverzeichnisse (1937 und 1955) zeigt, dass sie größtenteils dieselben Werke aufnimmt, 1955 zusätzliche Fotografien anderer Perspektiven und Werke eines Künstlers integriert und angesichts des späteren Erscheinungsdatums Skulpturen Ende der 1940er- bis Mitte der 1950er-Jahre ergänzt. Medardo Rosso, Antoine Bourdelle, Karl Burckhardt, Wilhelm Lehmbruck und Otto Freundlich zählen u.a. zu den Künstlern, die erst in der Ausgabe von 1955 erscheinen. Zu Giedion-Welcker und ihrem Verhältnis zur Skulptur siehe in jüngster Zeit u.a. Nikola Doll, „Carola Giedion-Welcker (1893–1979), Moderne Plastik: Elemente der Wirklichkeit, Masse und Auflockerung, Zürich: H. Girsberger, 1937“, in: K. Lee Chichester/Brigitte Sölch (Hg.), Kunsthistorikerinnen 1910–1980, Berlin: Reimer 2021, S. 168–187; Megan R. Luke, „Formlinge. Carola Giedion-Welcker, Hans Arp, and the prehistory of modern sculpture“, in: Elisa Tamaschke/Jana Teuscher/Loretta Würtenberger (Hg.), 27 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 3 Carola Giedion-Welcker, Moderne Plastik, Zürich: Girsberger 1937, S. 90f. 4 Carola Giedion-Welcker, Moderne Plastik, 1937, S. 88f. 28 Skulpturhistoriographie einer vitalistischen Tradition heraus, so will der bildimmanente Vergleich zeigen. Ab den 1960er-Jahren hinterlässt die Land Art von Agnes Denes, Mary Miss oder Richard Long und später Andy Goldsworthy ihre plastischen Spuren direkt in der Landschaft oder erfasst diese ephemeren, skulpturalen Formationen selbst fotografisch und überführt sie in den White Cube. Auch Haacke stellt physikalische und biologische Naturphänomene nach bzw. rahmt diese, erfasst sie anschließend fotografisch, doch ist eine Distanz zur vitalistisch-organistischen Herkunft deutlich erkennbar. In der erweiterten Publikation Plastik des XX. Jahrhunderts. Volumen- und Raumgestaltung von 1955 mit der Einleitung der früheren Ausgabe kontrastiert Giedion-Welcker die Schneeformationen mit Arps Konfiguration (1932) (Abb. 5): Drei kleinere organische Gebilde aus Gips liegen auf einer größeren, durch sanfte Rundungen bestimmten Form und können bewegt werden, wodurch Zeitlichkeit und Performativität in den Vordergrund rücken.49 Ergänzt ist der begleitende Text durch Zitate des Künstlers, die sein Werk in der „großen Werkstatt der Natur“ verorten.50 Sein Wolkenhirte (1949–1953) wird auf der folgenden Doppelseite von der Fotografie eines in seinem Nest schlafenden Schwans begleitet (Abb. 6): „Der ruhende, schlummernde Ausdruck aneinandergeschmiegter Formen in der Natur findet bei Arp sein künstlerisches Echo in den ‚Concrétions Humaines‘.“51 Dem Formenrepertoire der Natur versucht Giedion-Welcker mit der Kamera nachzugehen, so auch bei Frucht eines schwarzen Steins, deren wellenförmige Binnenmodellierung ihr in einer beleuchteten, dadurch plastisch erscheinenden Schneedüne begegnet.52 Ähnlich kontrastiert sie – in der Ausgabe von 1937 – Brancusis Poisson mit den Dolmen des Marchands in der Bretagne (Abb. 7), um auf die „gemeinsame Wirkung dieser großen Monolithe und ihrer ruhigen Proportionen in der Natur“ hinzuweisen.53 Brancusis Werk hebe sich durch die spezifische Oberflächenbehandlung und „handwerkliche Vollendung“ von den ungeschliffenen Felsen des Hünengrabs ab, womit die Autorin den Topos künstlerischer Virtuosität als legitimatorisches Argument erneut aufruft.54 Die Monolithen aus Stonehenge und der Bretagne „im Zusammenhang mit Kult, Raum, Natur“ konterkarieren diese Leichtigkeit und Auflösung der Masse, wirken abrupt, beenden den ersten Band zur Bildhauerei im 20. Jahrhundert und schließen den Kreis zu den Anfängen plastischen Gestaltens bzw. verweisen indirekt auf zukünftige Tendenzen der Kunst. 49 50 51 52 53 54 Hans Arp and other masters of 20th century sculpture (Stiftung Arp e.V. papers, 3), Remagen 2020, S. 54–67. Giedion-Welcker 1955a, S. 100f. Ebd., S. 100. Ebd., S. 102f. Ebd., S. 106f. Giedion-Welcker 1937, S. 102f. Im ersten Buch sind mit Katharina Kobro und Barbara Hepworth lediglich zwei Frauen vertreten, während 1955 neun Künstlerinnen hinzukommen. Die Construction dans l’espace (1933) von Kobro kommentiert eine Fotografie Palucca Sprung im Raum – eine in die Luft springende Frau mit ausgestreckten Gliedmaßen –, wodurch Bewegungs- und Richtungsdimension der gebogenen, nach außen geöffneten Plastiken markiert werden. 29 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 5 Carola Giedion-Welcker, Plastik des XX. Jahrhunderts, Stuttgart: Gerd Hatje 1955, S. 100f. 6 Carola Giedion-Welcker, Plastik des XX. Jahrhunderts, 1955, S. 102f. 30 Skulpturhistoriographie 7 Carola Giedion-Welcker, Moderne Plastik, 1937, S. 102f. In ihrem Resümee vor dem Bildteil fasst Giedion-Welcker die Gemeinsamkeiten der heterogenen plastischen Tendenzen der ersten drei Jahrzehnte zusammen, indem sie ihre formal-essenzialistische Perspektive auf die Gattung Skulptur fortsetzt, die „Grundlagen und Gesetze des eigenen Ausdrucksgebietes“ im Sinne einer Volumengestaltung im und durch Raum erörtert, ferner eine Abkehr von „illusionistischen und imitativen Bestrebungen“ zugunsten einer eigenen plastischen Realität und Verzeitlichung vermerkt sowie die Beziehung der Skulptur zu anderen Disziplinen wie Architektur, Dichtung, Malerei, Musik, Philosophie, Physik, ferner zur „modernen Civilisation“, zum „Prähistorischen, Archaischen und Primitiven“ anspricht.55 Im Annex der Einleitung von Plastik des XX. Jahrhunderts, die Abbildungen von Werken im Außenraum innerhalb einer landschaftlichen Umgebung enthält, beschreibt Giedion- Welcker die „heutige Situation“ und stellt eine positive Verschiebung zur Plastik, gar „ein plastisches Zeitalter“ fest, bestimmt durch Entmaterialisierung und Berücksichtigung der Eigengesetze des Materials, durch dynamische Räumlichkeit und Öffnung des skulpturalen Korpus nach außen („Volumen- und Raumorchestrierung“).56 Sie vergleicht wiederholt die „lebendige Gegenwartsplastik“ mit Sprache,57 wodurch die Verbundenheit von Betrachterin 55 56 57 Ebd., S. 17. Giedion-Welcker 1955a, S. XXIII, XXIV, XXVI. Auch William Tucker betitelt sein 1977 erschienenes Buch: The Language of Sculpture. 31 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt und Kunst als Spiegelbild eines dynamischen Weltbilds deutlich hervortrete.58 Metaphernreich beschreibt Giedion-Welcker die Formentwicklungen der Plastik, d.h. die Raumkon struktionen Pevsners und Gabos als „vibrierendes Leben eines nervösen Blattgeäders“, und integriert Marcel Duchamp, der sich mit seiner Stimulierung durch die Technik in eine „Symbiose des Biologisch-Wachsenden und Technisch-Konstruierten“ begebe.59 Figuren im Raum: Eduard Trier Eduard Trier veröffentlichte mehrere gattungsbezogene Bücher (und Aufsätze) und verfasste 1959 als Ausstellungskurator der Documenta 2 unter Arnold Bode einen Einleitungstext für den Katalog zur Skulptur, die in einer eigenen Ausstellung in der Orangerie „kein Lücken büßer zwischen Gemälden“ mehr sein musste.60 In seinen Publikationen Moderne Plastik von Rodin bis Marini (1954), Figur und Raum. Die Skulptur des XX. Jahrhunderts (1960) und Bildhauertheorien im 20. Jahrhundert (1971) versammelt Trier für eine medienspezifische Theorie wegweisende Ansätze. Demzufolge gipfelt die Entwicklungsgeschichte der Bild hauerei in einer Auflösung des Körperlichen und der Verzeitlichung als Grundlage, die für die heterogenen künstlerischen Praktiken einer „Skulptur im erweiterten Feld“ (Krauss 1979) und darüber hinaus für die Gegenwartskunst gelten kann. In Moderne Plastik von Rodin bis Marini (1954) erzählt Trier (s)eine Geschichte der Bildhauerei von der Naturdarstellung/Mimesis, d.h. in einem dichotomen Modell von der Figuration zur Abstraktion – ähnlich wie Giedion-Welcker – aus der Perspektive formal- kompositorischer Gestaltung und ihrer Beziehung zur Natur. Gegliedert ist sein Überblick chronologisch, sortiert nach Ländern mit einem Schwerpunkt auf Deutschland.61 Plastik versteht er als Körperkunst, allgemein ein dreidimensionales, geformtes Ding, d.h. etwas Gemachtes, Gefertigtes. Dies inkludiert Artefakte unterschiedlicher Art, nicht Gefundenes im Sinne eines Objet Trouvé, das er neben Duchamps Readymade später allerdings aufnimmt. Die Adressierung des Tastsinns und die von Herder formulierte Umkehrung der Gattungshierarchie schildert Trier zu Beginn seines Textes in einer Anekdote über den Besuch einer Henry-Moore-Ausstellung durch eine „Blindengruppe“, die das „Ertasten als menschliches Grundbedürfnis“ sowie „das plastische Empfinden“ nahelegt.62 Doch fügt er ergänzend hinzu: „Plastik ist auch für die Augen da. Unsere optischen Erfahrungen ersetzen dann das Tastgefühl.“63 Plastik sei eine Besitzergreifung des Raums, beinhalte die Konstruktion eines Dings 58 59 60 61 62 63 Giedion-Welcker 1955a, S. XXVI. Historisch geht sie hier weiter zurück als im ersten Buch. Ebd., S. XXXIX, XXXI. Die Plastik des Faschismus klammert sie aus. Eduard Trier/Arnold Bode (Hg.), Skulptur. Orangerie, Ausst.-Kat. Documenta 2, Kassel 1959, S. 10. Trier 1954. Bei ihm liegt der (formal-analytische) Fokus auf deutscher bzw. europäischer Bildhauerei, mehr als bei Giedion-Welcker. Ein Beispiel für Triers formale Beschreibungen: „Zueinander von gebauten Formen in architektonischen Innenräumen; Figur statisch in einem Netz räumlicher Beziehungen“ (ebd., S. 52). Ebd., S. 7. Ebd., S. 8. 32 Skulpturhistoriographie mittels Hohlräumen und Volumen, Masse und Leere, ferner deren Änderung, Kontraste, ständige und wechselseitige Spannung und Gleichgewicht.64 In Triers Werkbeschreibungen stehen architektonisch-gebaut-tektonische und organisch- fließend-gewachsene Formen dialektisch gegenüber. Das bildhauerische Paradigma Materialgerechtigkeit begreift er trotz Verwendung neuer Stoffe wie Kunstharzen, Eisen, Aluminium und Plexiglas weiterhin als vorherrschend, da das Material auch hier über seine Eigenschaften den Gestaltungsprozess vorgebe. Form und Material stehen, so Trier, in einer direkten Wechselbeziehung. Eine skulpturtheoretische Ästhetik des Lebendigen hingegen, wie sie hier erörtert wird, relativiert dieses seit dem Neoplatonismus vorherrschende und einseitig gerichtete Verhältnis zwischen Idee und Material, indem Pflanzen und Tiere als Ko-Akteure die Gestaltung des Werks mitbestimmen können (agency). Trier und Giedion-Welcker skizzieren die Entwicklung der Skulptur als Befreiung von ihrer Masse und Schwerkraft, als Emanzipation von Trägergrund und Verzeitlichung. Diese Perspektive durchzieht die kunstgeschichtliche und -wissenschaftliche Literatur als roter Faden, etwa nachfolgend bei Krauss in Passages in Modern Sculpture (1977).65 Einen entscheidenden Schritt vollziehen Pevsner, Gabo und Calder, indem sie Luft als Gestaltungselement integrieren, so Trier, und sich vom Körpervolumen zum Raumvolumen wenden: „Wir betrachten Raum als neues, absolut skulpturales Element, […] für uns ist er kein Abstraktum.“66 Auch wenn es sich noch um deutlich konturierte Objekte handelt, kann diese Aufnahme des Raums als wegweisend für erweiterte Konzepte des Skulpturalen seit der Post-Minimal Art gelten – so Environment und Land Art mit ihren skulpturalen Situationen und relationale Konzepte von Skulptur, die sich über die Einbeziehung der Umgebung schon andeuten. In seinem sechs Jahre später veröffentlichten Band Figur und Raum. Die Skulptur des XX. Jahrhunderts (1960) setzt Trier seine formale Untersuchung zur Typologie neuer ‚Skulptur‘ – nun verwendet er diesen Begriff im Titel – fort und gliedert diese in die dreifache Frage nach Form, Bedeutung (oder Sinn) und Aufgabe.67 Hier versteht er den Leerraum als ‚skulpturales Material‘, spricht von Lochplastik bzw. transparenter Plastik seit Alexander Archipenko und Jacques Lipchitz, von „immateriellen Raumzeichen“ bei González bis zu einem „auf ein Minimum eingeengten Körpervolumen“ und konstatiert statt formaler Konzentration nun Exzentrik68: „Die Plastik ‚entäußert‘ sich im wahrsten Sinne des Wortes ihrer Körperlichkeit, um sich in den Raum zu projizieren.“69 Licht avanciert mit Nicolas Schöffer zum neuen skulpturalen Material, so auch Burnham 1968 in Beyond Modern Sculpture. Trier berücksichtigt neueste Tendenzen, die kinetische Plastik Jean Tinguelys ebenso wie die „plastischen Situationen“ der magnetischen Arbeiten Takis’ und sogenannte „Konglomerat volumen“, d.h. die „‚Ansammlung‘ gefundener Dinge“ wie Jean Dubuffets Der Zauberer 64 65 66 67 68 69 Ebd., S. 9. Trier 1954, u.a. S. 70. Siehe auch Krauss 1981. Zit. n. Trier 1954, S. 73. Eduard Trier, Figur und Raum. Die Skulptur des XX. Jahrhunderts, Berlin: Gebr. Mann 1960. Ebd., S. 13, 12, Herv.i.Orig. Ebd., S. 12. 33 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 8 Jean Dubuffet, Der Zauberer, 1954, Lavaschlacke, Wurzelholz, H. 110cm (1954) – eine Figur aus Lavaschlacke und Wurzelholz (Abb. 8)70: „Sein bildnerischer Akt besteht nur darin, daß er die vorhandene unerkannte Form erkennt und zur Plastik erklärt, außerdem andere gefundene Formen hinzufügt.“71 Auch Dubuffets ‚Schwammfiguren‘ (Saimiri, 1954) demonstrieren dieses Konzept von Skulptur als entscheidenden Schritt zum Umgang mit lebendem, organischen Material und damit zu einer skulpturalen Ästhetik des Lebendigen. Tiere treten bei Trier (noch) als dargestellte Sujets auf, doch erwähnt er am Schluss seiner Geschichte den Garten Isamu Noguchis vor dem UNESCO-Gebäude (1958), „in dem skulptierte und natürliche Steinformen mit Wasserläufen, modelliertem Gelände, 70 71 Ebd., S. 16, 18, Herv.i.Orig. Ebd., S. 19, Herv.i.Orig. Dubuffet locke die immanenten Formwerte durch ‚assemblage‘ hervor. Zu den Schwammfiguren: Jean Dubuffet, Heulsuse, 1960, Schwamm und Stein, 41 x 15 x 16 cm, Sammlung Michael und Illeana Sonnabend, New York, Abbildung in Margit Rowell (Hg.), Skulptur im 20. Jahrhundert. Figur – Raumkonstruktion – Prozeß, Ausst.-Kat. Centre Georges Pompidou, Musée National d’Art Moderne, Paris 1986, München: Prestel 1986, S. 189. 34 Skulpturhistoriographie 9 Isamu Noguchi, Japanischer Garten, 1958, UNESCO-Gebäude, Paris Pfaden und Pflanzen zu einer künstlerischen Einheit arrangiert werden […].“72 (Abb. 9) Das Werk könne folglich als begehbare skulpturale Situation betrachtet werden, die mit Bäumen, Gräsern und Wasser eine Synthese mit der benachbarten Architektur eingeht: In der phantasievollen Leichtigkeit der Formen und Bewegung kontrastiert der Garten mit dem strengen Grundmuster der Architektur; er nimmt den Menschen auf, um ihm beim Wandel und Betrachten seine plastisch-räumlichen Formen zum Erlebnis werden zu lassen –, also Plastik im weitesten Sinne als ein Umwelterlebnis des Menschen.73 Auch wenn kanonische, größtenteils anthropomorphe, figurative Werke dominieren, führt Trier weitere Untergattungen von Skulptur ein, etwa „Plastik als Landschaft“, die er mit barocken Attributen wie Gianlorenzo Berninis Wolken- und Felsendarstellungen historisch 72 73 Trier 1960, S. 58. Ebd., S. 58f., Herv.i.Orig. 35 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 10 Bernard Rosenthal, Riverrun, 1959 verankert.74 In der Moderne avancierten diese Attribute eines Stilllebens isoliert zum eigenständigen Bildthema.75 Es handle sich um ‚Naturelemente‘, beispielsweise eine abstrahierte „Vogelschau der fruchtbaren oder in Trockenheit zerrissenen Erde“ wie Bernard Rosenthals Relief Riverrun (1959) (Abb. 10). In Triers Bildhauertheorien im 20. Jahrhundert (1971) kommen wiederum die bereits diskutierten Künstler zu Wort.76 Um ein eigenes Kapitel hat er mit Kinetischer Plastik, Objet Trouvé und Readymade seinen Kanon erweitert. Unter „Formen veränderter stofflicher Konsistenz“ 74 75 76 Ebd., S. 52. Ebd. Trier 1971a. Ebenfalls 1971 veröffentlicht Trier in einer Ausgabe der Zeitschrift für Ästhetik und Allgemeine Kunstwissenschaft, die sich schwerpunktmäßig „einem in der Kunsttheorie weithin vernachlässigten Thema: der Frage nach dem Wesen und den Möglichkeiten der Plastik“ widmet, einen an seine Publikation anknüpfenden umfassenden Beitrag (Eduard Trier, „Formprobleme der Plastik im 20. Jahrhundert in Selbstzeugnissen der Künstler“, in: Zeitschrift für Ästhetik und Allgemeine Kunstwissenschaft, 16, 2, 1971, S. 135–212 (= 1971b)). Ursula Aldinger konstatiert in ihrem dort ebenfalls erschienenen Text „Neue Materialien in der Plastik des 20. Jahrhunderts. Zum Problem: Werkstoff und Kunstwerk“ die ‚notwendige‘ Abkehr von obsolet gewordenen „ästhetischen Bestimmungen“, bevor neue Materialien in der Skulptur verwendet wurden, darunter „Altmaterialien“, alltägliche Gegen stände und Materialien der Architektur und Technik (ebd., S. 242–259). Doch auch sie geht nicht auf lebende Skulpturen ein. 36 Skulpturhistoriographie erlangen auch Haacke, David Medalla und Les Levine Gehör. Im Kontext pneumatischer Plastik, die Trier als Teilbereich zur kinetischen Kunst zählt und deren unvorhersehbare Formdynamik er unterstreicht, zitiert er Haacke und Jack Burnham, bevor er zu Medallas ‚Plastik‘ aus Seifenschaum übergeht. ‚Plastik‘ setzt er in Anführungszeichen, vermutlich um zu untermauern, dass der Begriff die Tragweite dieser Entgrenzung nur unzureichend erfasst. Die Auflösung einer konturierten Gestalt belegt Trier mit Les Levine und schließt den Abschnitt mit den raucherfüllten Räumen Preston McClanahams, die sich einem klassischen Konzept von Bildhauerei diametral entgegenstellen. Interessanterweise nennt Trier im Unterkapitel „Die Identität von Urheber und Bildwerk“ das Magnetische Manifest von Takis und zitiert aus diesem Text: Am 29. November 1960 ließ der griechische Bildhauer den südafrikanischen Dichter der Beat Generation Sinclair Beiles in der Pariser Galerie Iris Clert kurzfristig in der Luft ‚hängend‘ sein Magnetisches Manifest rezitieren: L’impossible: l’homme dans l’espace. Der Dichter verwandelt sich in eine sprechende Living Sculpture. Literatur in Form eines künstlerischen Manifests dient als selbsterklärender Kommentar „Ich bin eine Skulptur“ mit einer Kritik an Atomwaffen in Zeiten militärischer Aufrüstung. Der scheinbar schwebende, der Schwerkraft entfliehende, mit einem Schutzhelm ausgestattete Poet bringt Takis’ Faszination für das Unsichtbare zum Ausdruck und ermöglicht den Betrachterinnen neue skulpturspezifische, gesellschaftspolitische Reflexionen.77 In der nachträglichen Rezeption erfolgt das Zusammenspiel von Bild und Text sukzessive – es existieren wenige Schwarz-Weiß-Aufnahmen, u.a. von Hans Haacke, durch die die Performance nur annähernd rekonstruiert werden kann, und das Manifest. Trier benennt die ‚Verwandlung‘ des Dichters in eine lebende Skulptur nicht explizit. Elf Jahre nach Figur und Raum entwickelt Trier eine Geschichte der Bildhauerkunst auf Basis einer Zitatsammlung, die er aus Publikationen, Broschüren und Zeitschriften zusammengetragen habe, um der Gefahr eines Authentizitätsverlustes durch „bestellte Theorien“ seitens der Künstlerinnen zu entgehen.78 Als „bestellte Theorien“ betrachtet Trier Wortmeldungen, die für einen bestimmten Anlass, sei es Interview oder Katalogtext, eingeholt werden, folglich inszeniert und konstruiert seien. Doch sind die gesammelten Beiträge meist in ähnlichen Situationen entstanden, weshalb ihre Authentizität und die inszenatorischen 77 78 Der Manifesttext lautet wie folgt: „I am a sculpture. There are other sculptures like me. The main difference is that they cannot speak. When some of the sculptures try to speak they explode. They cause death. When I speak ‚Bomb‘ by Gregory Corso I am speaking life and death and no one is getting hurt. Two kinds of images can be made of me. One has already been made. This is the nuclear bomb. The other is a long-lived machine man, sub-human or super-human I cannot say. Super-humans would be difficult to conceive since we humans of flesh are super-mirrors for all organic life. I am a sculpture. I am here to be bought. I hope that the person who buys me will transport me to Hiroshima to witness Takis making another sculpture from the actual mechanism of a hydrogen bomb. I would like to see all the nuclear bombs on earth turned into sculptures. Guns and bayonets? … Stage stuff for history pageants played by the armies of the world.“ (Takis, „Magnetic Manifesto“, in: Signals, 1, 3–4, 1964, S. 6.) Trier 1971a, S. 1. 37 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt Strategien quellenkritisch beleuchtet werden müssten.79 Trotz der Problematik der Verlässlichkeit einer Künstleraussage sei jedoch gerade ihre Subjektivität geeignet, so Trier, daraus ‚Bildhauertheorien‘ abzuleiten. Dieser Plural zeigt Vielfalt und Heterogenität der ‚theoretischen‘ Anschauungen, die der Autor kommentiert, aber nicht kritisch reflektiert, und zu einem skulpturtheoretischen Konzept zusammenfasst. Im ersten Kapitel verwendet er noch den Singular: „Zur Theorie der Bildhauerkunst im 20. Jahrhundert“. Nach einem Einstieg über Herder, Goethe, Schmarsow, Sedlmayr und Hildebrand gliedert er das ‚Textmaterial‘ in weitere neun Kapitel, die neben Vorbereitung und Entstehung, Material und Technik sowie Inhalt auch die Plastik und ihr Gegenüber, das Verhältnis zu den anderen bildenden Künsten und zur Architektur sowie Plastik als öffentliche Kunst thematisieren. Haacke wird ein zweites Mal im Abschnitt über die Plastik und ihr Gegenüber zitiert, da seine frühen Objekte mit Flüssigkeiten auf aktive Rezipientinnen angewiesen seien:80 Ich liebe dieses körperliche Beteiligtsein. Es stellt eine Interdependenz zwischen Betrachter und Objekt her. In größeren Arbeiten, die eine Handhabung nicht zulassen, würde ich Fotozellen nutzen, um diese intime Beziehung herzustellen.81 Der seit den Ursprüngen der Bildhauerei wiederholt bemühte Topos der Verlebendigung besteht auch im 20. Jahrhundert, so belegen Zitate in Triers ‚Lesebuch‘. „Natur als Thema“ und Formenrepertoire diskutiert er in einem Unterkapitel und konstatiert eine fehlende Definition von Natur.82 Die dort versammelten künstlerischen Aussagen verbindet ein ähn liches Naturverständnis: Arp vergleicht den künstlerischen Akt mit natürlichem Wachstum, versteht Kunst analog zur Natur und kehrt einem mimetischen Ideal den Rücken. Während zur modernen Plastik „das Zufällige, das Viel- oder Mehrbedeutsame und die Variabilität der Materialien“ gehöre, so Calders Mobile, die Leben darstellen, indem sie sich bewegen, und unterschiedliche Bildhauer, etwa Ossip Zadkine, das Eigenleben der Materie als „wesentliche Bedeutung des Plastischen“ berücksichtigen, ist dieses Paradigma in der lebenden Skulptur ab den 1960er-Jahren vollständig umgesetzt.83 Neue Dimensionen der Skulptur: Michel Seuphor, Ulrich Gertz, Herbert Read, Udo Kultermann, Hans Joachim Albrecht, Lucie Schauer Nach dem Zweiten Weltkrieg entstanden zahlreiche Übersichtsdarstellungen zur Bildhaue rei in der Kunstgeschichte. Die Erweiterung des Mediums und die Bandbreite an technischen Möglichkeiten sowie die Präsenz der Skulptur im Öffentlichen Raum mögen das rege Interesse der Kunstwissenschaft an diesem Gegenstand erklären, wie die im Folgenden ge79 80 81 82 83 Auch in der hier vorgenommenen Untersuchung zu Hans Haacke und Pierre Huyghe fließen Aussagen der beiden Künstler ein, doch sind sie methoden- und quellenkritisch genau zu befragen. Trier 1971a, S. 176. Ebd., zit. n. Jack Burnham, „Hans Haacke. Wind and water sculpture“, in: Tri-Quarterly Supplement, 1, 1967, S. 1–24, S. 8. Trier 1971a, S. 138–144. Ebd., S. 207, 225. 38 Skulpturhistoriographie nannten Autorinnen demonstrieren. So veröffentlichte Michel Seuphor 1959 La sculpture de ce siècle, das den Untertitel Dictionnaire de la sculpture moderne trägt.84 Sein ‚Lexikon‘ ist, wie oft üblich, nach Namen und Ländern mit eigenen Kapiteln zur aktuellen Situation in Frankreich, Italien und England gegliedert; deutsche Künstlerinnen werden marginal, zusammengefasst mit Österreich, Schweiz und Jugoslawien erwähnt. Sein innovativer Ansatz zeigt sich unter anderem darin, dass mit 24 Bildhauerinnen von über zweihundert künstlerischen Positionen sein ‚Kanon‘ deutlich mehr weibliche Positionen (darunter einige südamerikanische Bildhauerinnen) als die Darstellungen von Trier und Giedion-Welcker enthält. Seuphor kritisiert das bipolare Verhältnis von abstrakt und gegenständlich und begründet dies mit dem veränderten Naturverständnis seiner Zeit: „Die Ausdrücke abstrakt und gegenständlich werden allmählich ihre gegensätzliche Bedeutung verlieren. Seit langem ist die Natur durch das Mikroskop und das Teleskop abstrakt geworden.“85 Das klassische Narrativ der Skulpturhistoriographie einer teleologisch bedingten Entwicklung von der Figuration zur Abstraktion greife zu kurz, denn mit dem dialektischen Begriffspaar richte sich der Blick primär auf die Naturnachahmung, auf variierende Grade der Verfremdung und den (historischen) Topos der Mimesis. Ulrich Gertz publizierte 1953 Plastik der Gegenwart; eine zweite Folge erschien 1964. Ein eigenes Kapitel widmet er in der Erstausgabe „plastischen Mitteln der Tiergestaltung“, wohingegen er später Tierplastik und Menschendarstellungen unter „das Geistige und das Kreatürliche“ zusammenfasst.86 Seine Geschichte der Bildhauerei folgt einem ähnlichen Narrativ wie Giedion-Welcker und Trier (die er beide erwähnt): Das künstlerische Medium strebe im Verlauf des 20. Jahrhunderts zu Verräumlichung, Entkörperlichung und Entgrenzung. Gertz formuliert sein Interesse für die „Zwittersituation der Künste“ und konstatiert eine „Tendenz zum Gesamtkunstwerk“.87 Von besonderer Relevanz ist angesichts heutiger digitaler Medienpräsenz von Kunstwerken in sozialen Netzwerken und Online-Ausstellungen sein Credo: Die Aufgabe eines Kunstwerks liege nicht darin, ein fotogenes Objekt zu sein.88 Herbert Read gliedert seine Concise History of Modern Sculpture (1964) nach Stilen, die im 20. Jahrhundert zu einer „diffusion of style“ ohne „coherent movement“ führen.89 Sein Skulpturenverständnis, das er zwischen „mannerism“ und „romanticism“ ansiedelt, bleibt 84 85 86 87 88 89 Sein Autorenname ist ein Pseudonym als Anagramm von ‚Orpheus‘. Seuphor 1959, S. 57. Seuphor verweist auf Calders Atelier, in dem lauter Pflanzen standen: „Das eine ist voller lebender Pflanzen. Sie kriechen an der [sic!] Mauern hoch, hängen von den Glasscheiben herab, begraben unter sich das Klavier und verschonen nur die Feuerstelle, wo das Holzfeuer in so lebhaftem Rot brennt, dass es in all diesem Grün aussieht, als sei es künstlich“ (ebd., S. 89). Ulrich Gertz, Plastik der Gegenwart, Berlin: Rembrandt 1953, Vorwort n.p. Er nennt Platte, Giedion- Welcker, Hofmann und Trier. Ders. 1964, S. 12–24. Ebd., S. 242. Ebd., S. 126. Herbert Read, A Concise History of Modern Sculpture, London: Thames and Hudson 1964. 1954 veröffentlichte er The Art of Sculpture (The A.W. Mellon Lectures in the Fine Arts, National Gallery of Art, Washington), London: Faber and Faber limited 1954. Hier zeigt er Degas’ Tänzerin, aber auch chinesische und andere außereuropäische Skulpturen. Er geht bis in die Antike zurück, integriert afrikanische Masken und Statuetten aus Ägypten, von den Azteken oder aus Mesopotamien mit 39 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt dem dichotomen Modell verankert, indem er das virtuose Können einer skulpturalen Objekt ästhetik verfolgt:90 The sculptor cannot paraphrase Pollock’s well-known statement and say: ‚When I am in my sculpture, I am not aware of what I’m doing.‘ The sculptor (despite some brave attempts by Étienne- Martin) must inevitably remain outside his sculpture, a conscious craftsman.91 Innerhalb seiner positivistischen Argumentation interessiert sich Read für Metall- und Eisen skulpturen aufgrund der Ubiquität des Materials (geeignet für die skulpturspezifische Dauer haftigkeit) als Spiegel eines ‚New Iron Age‘. Ein wichtiger Vertreter sei Eduardo Paolozzi, der technoide Wesen entworfen habe, auch wenn diese Annäherung an das kommende Computerzeitalter (nur) motivisch erfolgt.92 Read fragt nach dem ontologischen Status gegenwärtiger Skulptur angesichts der Aufgabe einer geschlossenen Form und Masse zugunsten von Offenheit und Dynamik: One must then ask a devastating question: to what extent does the art remain in any traditional (or semantic) sense sculpture? […] What has been gained, and what has been lost, by this transition to a linear sculpture?93 Während Read organische, vitalistische Skulpturen favorisiert, sieht er mit den konstruktivisti schen Tendenzen linearer, transparenter und offener Gebilde das Fortbestehen der Skulptur in Gefahr.94 Dass in seiner Darstellung Living Sculptures fehlen, ist dem frühen Erscheinen seines Buches geschuldet. Udo Kultermanns Neue Dimensionen der Plastik (1967) sei für den vorliegenden Kontext der Living Sculpture ebenso genannt, da er sich im Abschnitt „Elementare Medien“ ephemeren, zeitbasierten, kinetischen Plastiken, Wasser-, Luft- und Licht-Skulpturen teils aus organischen Materialien widmet. Auf einer Doppelseite zeigt er Hans Haackes (Blaues) Segel (Abb. 11) neben Keith Sonniers Untitled (1966) und Andy Warhols Silver Clouds aus demselben Jahr, ferner Haackes Brett Klar/Klar (1965) u.a. neben Yutaka Ohashis Rain Box (1964) und Pino Pascalis An der Wand: 1 Kubikmeter Erde, 2 Kubikmeter Erde; auf der Erde: 12 Kubikmeter Pfützen (1967).95 Medalla hat mit The First Sand Machine (1964) und Cloud Canyons no. 2 (1964) sowie Alain Jacquet mit seinem kaum konturierbaren, alles andere als 90 91 92 93 94 95 egenüberstellungen verschiedener Epochen (etwa Tierplastik von Balla und Duchamp mit zwei Reliefs G aus Mesopotamien). Ebd., S. 232. „What is distinctive about the post-war situation, particularly in sculpture, is a determination to belong to no movement, an artistic ‚free thinking‘“ (ebd., S. 230); „complexity of the modern scene is partly due to the co-existence of such movements and counter-movements“ (ebd.). Ebd., S. 229. Das trifft für die entgrenzte Skulptur nicht mehr zu. Ebd., S. 234. Zur Metallskulptur: „the prevalence of metalwork“ (ebd., S. 237), „metal is so docile that it will submit to any formal conception the sculptor may have“ (ebd., S. 247). Ebd., S. 250, Herv.i.Orig. Ebd., S. 250–255. Causey zufolge habe sich Read auf seine drei Freunde Hepworth, Moore und Gabo konzentriert (Andrew Causey, Sculpture since 1945, Oxford [u.a.]: Oxford University Press 1998, S. 63). Kultermann 1967, S. 176f., 180f. 40 Skulpturhistoriographie 11 Hans Haacke, Blaues Segel, 1966, in: Udo Kultermann, Neue Dimensionen des Plastischen, Tübingen: Wasmuth 1967, S. 180f. objekthaften Grey Smoke (1967) Eingang in den Kanon der Skulptur gefunden. Wasser als skulpturales Material, das Haacke seit seinem Condensation Cube nutzt, bringt Kultermann, der 1968 seine Dissertationsschrift zum Barockbildhauer Gabriel Grupello verfasste,96 mit „barocken Gestaltungsformen“ in Verbindung.97 Die Integration lebender Tiere in die Kunst führt er auf die barocken Kunst- und Raritätenkabinette des 16. und 17. Jahrhunderts zurück, „eine erneute Hinwendung zur barocken Universalität“.98 Kultermann konstatiert, dass „die schwimmende Plastik“ noch in ihren Anfängen stecke, „obgleich gerade hier viele unausgeschöpfte Möglichkeiten liegen“.99 Neben Haackes Segel trage auch Piero Manzoni mit Fiato d’artista dazu bei, dass „sich die Skulptur vom Erdboden löste“. Kultermann verfolgt diese Entwicklung bis zu Charles Fraziers „flying sculptures“ und den Aktionen von Gutai.100 Seine Geschichte der Skulptur mündet in skulpturale Räume, Happenings und Environments. Er sieht darin die größten Innovationen seit 1960, „eine immer stärker werdende Tendenz zum Plastisch-Räumlichen beherrscht unsere Kultur“.101 Zu 96 Udo Kultermann, Gabriel Grupello, Berlin: Deutscher Verein für Kunstwissenschaft 1968. 97 Kultermann 1967, S. 173–201, S. 173. 98 Ebd., S. 62f. 99 Ebd., S. 175. 100 Ebd. 101 Ebd., S. 5. 41 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt bemerken sei überdies die „Neigung zur Synthese“.102 In ihrer Annäherung an die Wirklichkeit versuchten Künstlerinnen, Lebendiges, Prozesse einzubeziehen.103 Sein Buch erschien, als er bereits in die USA gezogen war und nach seiner Tätigkeit als Direktor des Leverkusener Museum Schloss Morsbroich seit 1967 eine Professur für Kunstgeschichte und Architekturtheorie an der Washington University in St. Louis innehatte, woraus sich sein transnationaler Fokus begründet. Im selben Jahr wie Krauss’ Passages in Modern Sculpture erschien 1977 von Hans Joachim Albrecht Skulptur im 20. Jahrhundert. Raumbewußtsein und künstlerische Gestaltung. In seiner wahrnehmungsbezogenen und phänomenologischen Abhandlung konstatiert er eine Veränderung der dreidimensionalen Gestaltung zugunsten einer Überwindung der Schwerkraft und Auflösung des „Massenvolumens“.104 Er rekurriert wie Gertz auf die kanonischen Beobachtungen Giedion-Welckers und Triers und kritisiert den häufig verwendeten Begriff der Entmaterialisierung.105 Vielmehr sei, so Albrecht, selbst Künstler, die „ausschließliche Herrschaft des ‚festen‘ Körpers zu Ende gegangen“, wozu Licht und Luft als skulpturale Materialien entschieden beigetragen hätten, wie die kinetische Kunst mit ihrer verselbstständigten Bewegung demonstriere.106 Sein „Bildteil: Beispiele skulpturaler Kunst“ enthält die reflektierte Vermittlung einer (bestimmten) Seherfahrung von Skulptur durch Fotografie, indem er von Julio González’ Palettentänzerin (1933) eine Abfolge von zwölf Bildern aus unterschiedlichen Ansichten integriert und Ausleuchtung, Blickwinkel, Entfernung, Perspektive und Hintergrund befragt.107 Seine Lektüre von Burnhams Beyond Modern Sculpture spiegelt sich in der Nennung von Kybernetik und Biotechnologie als skulpturale Orientierung, seinem Schwerpunkt auf kinetischer („motorisierter“) Plastik und skulpturaler Lichtkunst. Er konstatiert die fehlende Tragweite des Begriffspaars Skulptur – Plastik und schlägt den Terminus „dreidimensional bildende Kunst“ vor.108 Einen umfassenden Anspruch erhebt der Katalog Bildhauertechniken. Dimensionen des Plastischen (1981): In ihrem Einführungstext erläutert Lucie Schauer die Intention der Ausstellung im Neuen Berliner Kunstverein, „den Gesamthabitus plastischer Arbeiten“ und drei- 102 Ebd., S. 211. Kultermanns Darstellung beinhaltet eine größere Anzahl an Frauen als Seuphors; Kultermann erwähnt allerdings nicht die amerikanischen (Post-)Minimal-Künstlerinnen. 1966 veröffentlicht er einen kurzen Aufsatz „Junge deutsche Bildhauerinnen“ in: Das Kunstwerk, 19, S. 14–18. 103 Kultermann 1967, S. 6: „Nach der zeitweiligen Verengung der Horizonte im Bereich der von doktrinären Ideologien beherrschten abstrakten Plastik hat sich heute eine Freiheit künstlerischer Möglichkeiten eröffnet […].“ 104 Hans Joachim Albrecht, Skulptur im 20. Jahrhundert. Raumbewußtsein und künstlerische Gestaltung. Köln: DuMont 1977, S. 102. 105 Ebd., S. 103. Albrecht favorisiert den Terminus ‚Skulptur‘ (ebd., S. 9), möchte keine Entwicklung zeitgenössischer Skulptur skizzieren, sondern den Fokus auf Einzelwerke legen. Am Ende des 19. Jahrhunderts habe es zwei differierende Auffassungen des skulpturalen Raums gegeben, deren Vertreter Rodin und Hildebrandt seien (ebd., S. 12f.). 106 Ebd., S. 103, 124. Zur plastisch-räumlichen Funktion von Licht und als eigenständige, raum-zeitliche Qualität siehe ebd., S. 124–129. 107 Ebd., S. 147f. 108 Ebd., S. 10. 42 Skulpturhistoriographie 12 Gilbert & George, The Singing Sculpture, 1970 dimensionale Gestaltungsmöglichkeiten zu thematisieren.109 Ausgehend von M aterialien und Techniken ist der Band in elf Kapitel gegliedert: Stein, Terrakotta, Holz, Bronze, Eisen, Kunststoffe, Fundstücke, Lichtkinetik, sogenannte Nichttraditionelle Materialien, Land Art und Konzeptuelle Plastik. Mit Verweis auf Seuphor und den Futuristen Filippo Tommaso Marinetti stellt Schauer die Signifikanz der vierten Dimension Zeit und die Integration des Luftraums als mitgestaltendes Moment heraus, die eine „gewaltige Explosion der gestalterischen Möglichkeiten innerhalb der Moderne“ bewirken.110 Neben der Nutzung „nichttraditioneller Materialien“ belegt der Band die Erweiterung der „plastischen Dimension“ zugunsten von Prozessualität und Performativität. Räumliche, plastische Situationen entstehen, in denen Betrachterinnen eigene Erfahrungen machen, etwa wenn César Polyurethanschaum ausströmen lässt, dessen amorphe Expansionen nicht planbar sind.111 Im Zusammenhang mit Barry Flanagan, Robert Morris und Reiner Ruthenbeck thematisiert Jürgen Schilling, einer der Autorinnen, das Eigenleben des Werks, auch wenn die von ihm genannten Künstler keine lebenden, organischen Materialien nutzen. Unter die 109 Lucie Schauer, „Kunst dreidimensional – Die Eroberung des Raumes“, in: dies. (Hg.), Bildhauertechniken. Dimensionen des Plastischen, Ausst.-Kat. Neuer Berliner Kunstverein e.V. in der Staatlichen Kunsthalle, Berlin: Frölich & Kaufmann 1981, S. 6–10, S. 6. 110 Ebd., S. 7. 111 Jürgen Schilling, „Nichttraditionelle Materialien – der erweiterte Spielraum“, in: Schauer 1981, S. 138– 151, S. 138, 140. 43 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 13 Haackes Condensation Cube, 1963–1965, Acrylglas, Wasser, 30 x 30 x 30 cm 14 Hans Haacke, Eisring, 1970, Kühlmotor, Wasser, Plexiglas, 20 x 120 x 200 cm nichttraditionellen Materialien fasst Schilling die Living Sculpture von Gilbert & George (Abb. 12): […] ja selbst der menschliche Körper, wie im Falle von Gilbert & George, die sich als Human Sculpture präsentieren und sämtliche, noch so alltäglich erscheinende Aktivitäten als Teil dieser Skulptur verstehen, also Kunst und Leben in ihrem Auftritt miteinander verschmelzen.112 112 Ebd., S. 141f. „Ein entscheidendes Moment liegt in der Prozeßhaftigkeit und Instabilität nichttraditioneller Materialien, Zeit und Raum wachsen Bedeutung zu und der künstlerische Akt beruht häufig darin, durch minimale Eingriffe Bewußtwerdungsprozesse nicht nur ästhetischer Art in Gang zu setzen.“ (Ebd., S. 142). 44 Zur skulpturalen Ästhetik des Lebendigen Unter dem Titel Human Sculpture adressierte das britische Künstlerduo eine Postkarte an den Autor „A small sculpture for you“, auf deren Vorderseite die beiden in einem Garten posieren. „Nichttraditionell“ impliziert das Neue, Ungewohnte. Auch die biologischen, physikalischen und sozialen Realzeitsysteme Haackes finden Erwähnung, sein Condensation Cube (Abb. 13) und Eisring (Abb. 14). Betont wird die charakteristische wesenskonstituierende, dynamische Veränderung des Aggregatzustandes dieser ‚Objekte‘. Zwar deutet Schilling auf die rationale Nachvollziehbarkeit der technischen, physikalischen Vorgänge wie Kühlung und Gefrieren, doch fehlt eine Erklärung, welche „weiterreichenden Erlebnisse“ diese „medialen Objekte“ vermitteln.113 Die Hinwendung zur Natur belegt der Abschnitt zur „Land Art. Vorstoß in andere Dimensionen“ von Rolf Wedewer.114 Bereits Krauss situierte die plastische Dimension dieser Kunstrichtung in ihrem grundlegenden Diagramm zur Skulptur im erweiterten Feld. Zur skulpturalen Ästhetik des Lebendigen Von der Objektästhetik zur Systemästhetik: Jack Burnham In seinem Buch Beyond Modern Sculpture. The effects of science and technology on the sculpture of this century (1968) bezieht sich Jack Burnham auf skulpturale Randphänomene und zielt auf die Verbindung von Skulptur und Technik im Zuge einer Lösung vom Anthro pomorphismus.115 Eine detaillierte Untersuchung seiner Schrift im Kontext der Skulpturforschung blieb bisher aus und soll hier nun erfolgen. Wenngleich auch er kaum organische Werke einschließt, versteht er Skulptur als Spiegel des jeweiligen Kenntnisstands in der Biologie (und Technik) mit der Option, Leben simulierende Systeme in der Kunst zu entwerfen. Burnhams teleologische Entwicklungsgeschichte, die für ihn in einem produktiven Austausch mit den zeittypischen technologisch-naturwissenschaftlichen Errungenschaften gipfelt, nimmt Positio nen jenseits des Kanons in den Blick: Gliederpuppen, Automaten, Kinetik, Lichtskulpturen und kybernetische Kunst. Krauss reagiert in Passages in Modern Sculptures (1977) auf Burnhams 113 Ebd., S. 142. Eine maßgebliche Rolle für den Umgang mit „nichttraditionellen“ Materialien spielt Joseph Beuys mit seiner Sozialen Plastik und dem ‚Prozess des pädagogischen und sozialen Modellierens‘, s.u. den Exkurs „Joseph Beuys – Soziale Plastik“. 114 Rolf Wedewer, „Land Art. Vorstoß in andere Dimensionen“, in: Schauer 1981, S. 152–159. Skulpturale Qualitäten in Form von Markierungen und Einschneidungen erläutert Wedewer exemplarisch an Jan Dibbets, Michael Heizer und Richard Long. Ähnlich wie in Burnhams Beyond Modern Sculpture (1968) bildet das Kapitel zur „Lichtkinetik – das dynamische Raumzeitkontinuum“ von Hannah Weitemeier ein eigenes Thema (in: Schauer 1981, S. 127–137). 115 Rezensionen etwa bei Norbert Lynton, „Rezension zu Jack Burnham, Beyond Modern Sculpture, New York: Braziller 1968“, in: Leonardo, 3, 1970, S. 108f.; Jonathan Benthall, „Whose move?“, in: Studio International, 177, 1969, S. 148–150. Beide merken die programmatische, manifestartige Rhetorik der Schrift an, loben aber die Auseinandersetzung mit dieser relevanten, vernachlässigten Thematik. Burnham nennt nur wenige skulptural arbeitende Künstlerinnen, darunter Barbara Hepworth, Yayoi Kusama und Germaine Richier, zudem einige, die über den klassischen Kanon hinausgehen, namentlich Martha Boto, Chryssa, Deborah de Moulpied, Anne Truitt und Grazia Varisco. 45 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt Argumentation, der vom October-Kreis kritisiert, von Krauss und Benjamin Buchloh als technokratisch, von Thierry de Duve als kiffender „eco-sensitive enthusiast of technology“ und „utopianist of art’s dissolution into life“ degradiert wurde.116 Burnham ist den technischen Möglichkeiten gegenüber aufgeschlossen, doch wäre es verfehlt, ihm blinde Technikeuphorie zu attestieren, die jegliche ideologische Gefahren ausblendet. Seine Schrift ist durch kunsthistorische Ansätze etwa von Alois Riegl, Gottfried Semper, Wilhelm Worringer und Herbert Read bis zu philosophischen, soziologischen, naturwissenschaftlichen, informations-technologischen, biologischen und psychologischen Theorien geprägt. Er verfasste sein Buch bereits 1967, als wesentliche Meilensteine einer „Sculpture in the Expanded Field“ wie Richard Longs Line made by walking, Richard Serras Splashings und Robert Morris’ Felt Pieces noch nicht realisiert waren und Burnham vermutlich die Arte Povera und erste Ansätze einer digi talen Kunst, beispielsweise Herbert W. Frankes Überlegungen zur virtuellen Skulptur, nicht kannte.117 Mit seiner Erweiterung des Kanons und seiner Forderung einer Ästhetik künstlicher Intelligenz bewegt sich Burnham nah an heute relevanten Themen. Ähnlich wie nach ihm Krauss diagnostiziert er einen Identifikationsverlust des ontologischen Status von Skulptur und präzisiert das titelgebende „beyond“ als Fortsetzung: Already as a means of description, the term ‚sculpture‘ has lost its identity; it has become a misnomer for an art once concerned with carving and modeling for the purpose of simulating biological appearances, but which now generically designates all three-dimension art construction. […] It 116 Thierry de Duve, Kant after Duchamp, Cambridge, Mass: MIT Press 1996, S. 285f. Erst Burnhams Schriften ab den 1970er-Jahren mit seiner Hinwendung zur Kabbala muten kryptisch an. Für Beyond Modern Sculpture macht György Kepes am MIT Werbung. Zur Rezeption siehe u.a. Luke Skrebowski, „The Artist as Homo Arbiter Formae: Art and Interaction in Jack Burnham’s Systems Aesthetics“, in: Samuel Bianchini/Erik Verhagen (Hg.), Practicable: From Participation to Interaction in Contemporary Art, Cambridge, Mass.: MIT Press 2016, S. 39–54. 117 Vgl. u.a. Herbert W. Franke, „Virtual Sculptures“, in: Michele Emmer (Hg.), Mathematics and Culture II, Berlin & Heidelberg: Springer 2005, S. 145–149, http://www.zi.biologie.uni-muenchen.de/~franke/ VirtS1.html (17.9.2019). Siehe auch Simon Penny, „Systems Aesthetics + Cyborg Art: The Legacy of Jack Burnham“, in: Sculpture Magazine, 18, 1, 1999, https://sculpturemagazine.art/systems-aestheticscyborg-art-the-legacy-of-jack-burnham/ (5.3.2023). Neben seiner kritischen Lektüre Burnhams fragt Penny nach den skulpturalen Möglichkeiten im Zeitalter des Digitalen. „The problematic discontinuity between the tangibility of sculpture and sculptural practice and the ephemeral temporality of informatics is a case study in the cultural phasetransition of our times. […] Although he sensed the force of systems theory, Burnham, perhaps limited by his fixation on sculpture, was unable to foresee many dimensions of the integration of sculpture with electronic and digital media. […] Not only has CAD revolutionized drawing and modeling, but the utilization of computer controlled milling, stereolithography, and so forth has changed the actual creation of conventional sculpture. More importantly, microprocessors have transformed the language of spatial art practice into a temporal and interactive practice. While Burnham wrote of body art and environmental and site-specific working styles, we now witness the disembodiment and deterritorialization of virtualized and Net-based practice. Whereas video remained primarily an image medium, albeit technologized, cyber art is concerned with simulated behavior and the building of virtual machines as artworks. ‚Code‘ is the ephemeral structuring system of the work. Code is an enigmatic and paradoxical phenomenon: a text that is simultaneously a (virtual) machine is a long step from the pragmatic materiality of sculpture.“ Trotz dieser neuen, „numerous explorations into virtual sculpture“ sei daraus bisher keine neue Ästhetik des Digitalen entstanden. 46 Zur skulpturalen Ästhetik des Lebendigen becomes important, therefore, that we look upon sculpture as an indication of man’s changing conception of biology […] and especially as a form of biological activity itself.118 Burnham beschreibt den Weg vom Anthropomorphen zum Verdinglichten und Modellhaften, ferner die Hinwendung zum Material zugunsten eines technikinspirierten Artefakts. Aus der Sackgasse des Vitalismus und Formalismus führe nur die sogenannte post-formalistische Kunst, die er als systemästhetische Auffassung von Skulptur zu etablieren sucht. So besteht der Band aus den Abschnitten „Sculpture as Object“ und „Sculpture as System“ mit insgesamt acht Kapiteln. Im ersten Kapitel befasst er sich mit den Funktionen (etwa Verbildlichung) und dem Verschwinden des Sockels in der Moderne zugunsten neuer Präsentationsformen und differenziert zwischen floor-bound sculpture, ceiling-bound sculpture, floating, flying und air-borne sculpture (u.a. Haackes Sphere in Oblique Air Jet, 1967). Das zweite Kapitel „The Biotic Sources of Modern Sculpture“ thematisiert den Einfluss des Vitalismus und Neo-Vitalismus (u.a. Henri Bergson, Herbert Read, Henri Focillon) auf die Skulptur, den pygmalionischen Topos von Lebensnähe durch Virtuosität und Adaption von Naturformen und wie sich dieses Bestreben im Zeitalter zunehmender Technologisierung und neuer Erkenntnisse in Chemie, Medizin und Biologie (Ernst Haeckel, Rudolf Virchow, Friedrich Wöhler etc.) ändert. Aus dem naturwissenschaftlichen Verständnis eines Organismus als „network of inter related systems“ leitet Burnham seine Systemästhetik ab; der Vitalismus fungiere (nur) als Zwischenschritt vom unbelebten Objekt zum System.119 Charakteristisch für Burnhams Herangehensweise ist seine Herleitung der (abstrakten) Kunsttendenzen aus technischen Entwicklungen: „The neo-vitalist aesthetics, as embodied in Bergson’s élan vital, was ready- made for a sculpture that seemed not to be carved but to grow from an inner direction.“120 Über seine Herbert-Read-Lektüre verweist er auf die Polarität von organisch (bzw. repräsentativ/amorph) versus geometrisch (bzw. linear/abstrakt): „the apparent schism between organic and constructive (i.e., geometric) sculpture“.121 Dieses „Schisma“ relativiere sich in der Moderne dank Kybernetik, Informationstheorie und Systemanalyse, präge aber weiterhin die Überblickswerke zur Skulptur. Dem „anachronistischen“ Vitalismus – das ist für die system ästhetische Living Sculpture wichtig – setzt Burnham die „aesthetic of true organicism“ entgegen, d.h. die „organization of processes and interacting systems“, und problematisiert im Hinblick auf Henry Moores Plastik die Heterogenität des Naturbegriffs („‚Natural‘ is a diabolic term.“).122 118 Burnham 1968, S. 5f. 119 Ebd., S. 65f., 76. Burnham bezieht sich hier u.a. auf den Physiologen Claude Bernard, der einen Organismus als Netzwerk miteinander agierender Systeme beschrieb (ebd., S. 61). 120 Ebd., S. 68, Herv.i.Orig. 121 Ebd., S. 74. 122 Ebd., S. 94, 96. Die Kinetik führe zu einer veränderten Haltung gegenüber unbeweglichen Objekten: „Increasingly we will regard the traits of vitalistic sculpture with the same amused tolerance that we now reserve for obvious antiquing nineteenth-century Neoclassical sculpture.“ (Ebd., S. 96) Zwar gesteht er dem Vitalismus eine Grundvoraussetzung für die Avantgarde-Skulptur zu, doch rechnet er 47 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt Im dritten Kapitel skizziert Burnham die naturwissenschaftlichen und kunsthistorischen Hintergründe des Formalismus in der Skulptur. Bereits hier verweist Burnham auf sein bis Anfang der 1970er-Jahre relevantes systemtheoretisches Denken. Mit zunehmender Prägung durch die Technik entfalte sich eine systemästhetische Konzeption in der Skulptur, die das konturierte, singuläre Objekt ablöse.123 An künstlerischen Beispielen (u.a. De Stijl, Konstruktivismus) belegt Burnham die „love-hate relationship with science“, die zu einer der Geometrie entlehnten Formensprache, zur visuellen Anknüpfung an wissenschaftliche Modelle und industrielle Materialien, zur „open-sculpture“, Oberflächen ohne individuellen Duktus und zur Öffnung der Gestalt (Gabo, Pevsner, Archipenko) führe, aber nie als „scientific art“ zu bezeichnen sei, sondern ihren ontologischen Status gerade ihrer ‚kreativen Freiheit‘ verdanke.124 So avancierte die Topologie zur Grundlage des neuen skulpturalen Formalismus.125 Die Skulpturen von Max Bill demonstrieren, wie das Möbiusband in unterschiedlichem Material, Positionierung und Format aufgegriffen wird, weshalb die Skulpturen an die seriellen Objekte der Minimal Art erinnern, an die monumentalen topologischen Skulpturen Serras, in denen der Raum zur plastischen Kategorie mutiert – anders als bei den im Inneren offenen Skulpturen Archipenkos („penetration“) und Barbara Hepworths.126 Der Formalismus als „proto-mechanization“ führe zu einer Optiziät der Skulptur.127 Das vierte Kapitel „Form Exhaustion and the Rise of Phenomenalism“ thematisiert die Rezeption der 1962 auf Englisch erschienenen Phänomenologie der Wahrnehmung Maurice Merlau-Pontys und leitet zum Systembegriff im zweiten Teil über. In der Moderne oszilliere die Skulptur zwischen Statuarik und Bewegung/Vergänglichkeit mit zunehmender Räumlichkeit.128 Das Objekt verlange, so Burnham, eine veränderte Form des Sehens, wofür die Phänomenologie mit ihrer leiblichen Wahrnehmung einstehe. Zugleich rechnet er mit dem Biomorphismus in der Skulptur ab, der ähnlich wie der Formalismus in eine künstlerische Sackgasse geführt habe. gleichermaßen mit dieser „metaphysischen Doktrin“ ab (ebd., S. 100): „Vitalism, based as it is on nonphysical substances and states of life, is a metaphysical doctrine concerned with the irreducible effects and manifestations of living things.“ (Ebd., S. 94.) Er fragt: „Why, if vitalism played such a reactionary role in the biological sciences, did it provide some of the most advanced justifications for avant-garde sculpture?“ (Ebd., S. 56.) 123 Ebd., S. 114: „The demands of technology are such that separate, unique entities are a cardinal sin against the technological strategy of duplication and interchangeability.“ 124 Ebd., S. 116, 128. „The sculptor […] was fascinated by the model because it seemed to represent the creation of form purely through mental activity with no reference to visible reality.“ (Ebd., S. 126.) 125 Ebd., S. 142. 126 Ebd., S. 150. Burnham resümiert: „Sculpture, moreover, has always been dominated by image makers – and the more immaterial sculpture became, the more important seemed the concept of the iconic ‚image‘.“ (Ebd., S. 152.) Siehe auch Eric de Bruyn, „Topologische Wege des Post-Minimalismus“, in: Wolfram Pichler/Ralph Ubl (Hg.), Topologie. Falten, Knoten, Netze, Stülpungen in Kunst und Theorie, Wien: Turia & Kant 2009, S. 361–404. 127 Burnham 1968, S. 218. 128 Die Parameter klassischer Skulptur seien überholt, der Objektbegriff (weight, mass, form) passe nicht mehr (ebd., S. 167). 48 Zur skulpturalen Ästhetik des Lebendigen Kapitel 5 „Sculpture and Automata“ beschreibt die ‚Subskulptur‘ der Automaten und Gliederpuppen als Vorgeschichte kinetischer Skulptur und, so ließe sich fragen, als Vorläufer der Living Sculpture?129 In diesem Kapitel holt Burnham weit aus, wenn er die Geschichte der Zeitmessung, Descartes’ mechanisches Körperbild, Vaucansons komplexe Automaten aus dem 18. Jahrhundert bis zu den ersten Robotern des 20. Jahrhunderts, Oskar Schlemmers roboterhafte Kostüme oder Hans Bellmers Dolls resümiert.130 Im sechsten, ausführlich und reich bebilderten Kapitel rückt er die Kinetische Kunst als ‚unerwiderte‘, vernachlässigte („unrequited“) Kunstform ins Zentrum, die sich durch Realzeit, Bewegung (als „passion of twentieth-century artists“) und Ungegenständlichkeit auszeichne, auf keinem traditionellen Handwerk wie noch die vorangegangenen Automaten basiere, sondern auf einer ‚Abhängigkeit‘ von der Technik, die deren „relative aesthetic failure“ begründe.131 Der Autor skizziert die Entwicklungs- und Rezeptionsgeschichte, die aufgrund technischer, finanzieller und legitimatorischer Schwierigkeiten bis Anfang der 1960er-Jahre gebremst und damit verspätet einsetzte, obgleich mit Marcel Duchamp, Umberto Boccionis Technisches Manifest der futuristischen Plastik (1912) und Naum Gabos Das Realistische Manifest (1920) deutlich früher entscheidende Fundamente gelegt worden waren.132 Neben der Technisierung der Skulptur beschreibt Burnham skulpturale Positionen, die, wie Calder, externe Bewegung, etwa einen Luftzug in das Werk integrieren, bis zu Victor Vasarely, Lucio Fontana, Yves Klein und Jean Tinguely, der die Maschine nahezu vermenschlichte.133 Burnham führt diesen Werdegang auf technologische und naturwissen schaftliche Neuerungen zurück. Insbesondere die physikalischen Feldtheorien prägten eine relational, systemästhetisch ausgelegte Kunst. Die kinetische Kunst stellt für ihn den Übergang von einer Objektästhetik zu einer Systemästhetik dar, weshalb er ihr einen großen Stellenwert einräumt. Auch Haackes Water Boxes mit ihrem „natural movement“ integriert Burnham in die Kinetik, die er als „radical departure from all past forms of sculpture“ klassifiziert, weshalb neue Begrifflichkeiten notwendig seien.134 Der Verwendung neuer Materialien und den Immaterialisierungstendenzen begegnet Burnham im siebten Kapitel „Light as Sculpture Medium“. Dessen räumliche, plastische Quali tät sah bereits Moholy-Nagy, indem er die nächtliche Stadt als pulsierende Lichtskulptur 129 Ebd., S. 186. „But suppose automata had been raised to the level of a fine art thousands of years ago, would we today have an aesthetic of moving statuary?“ (Ebd., S. 187.) 130 Ebd., S. 213. Einen indirekten Hinweis auf die lebende Skulptur findet sich in Burnhams Kommentar zu Oskar Schlemmer: „In fact, Schlemmer found his real medium for sculpture only when he was able to create choreography and costumes for his dancers.“ (Ebd., S. 214.) 131 Ebd., S. 219. „Kinetic artists, until recently, have not considered the construction of machines by themselves as representing sculpture.“ (Ebd., S. 224.) 132 Umberto Boccioni, „Technisches Manifest der futuristischen Plastik“ (1912), in: ders., Futuristische Malerei und Plastik (Bildnerischer Dynamismus), hg. von Astrit Schmidt-Burkhardt, Dresden: Verlag der Kunst 2002, S. 237–249; Naum Gabo, „The Realistic Manifesto“, in: Martina Hammer/Christina Lodder (Hg.), Gabo on Gabo. Texts and Interviews, Forest Row: Artists Bookworks 2000, S. 20–35. Das Manifest wurde von Gabos Bruder, später bekannt als Antoine Pevsner, mitsigniert. 133 Burnham 1968, S. 245. 134 Ebd., S. 279, 280. 49 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt betrachtete.135 Burnham greift erneut auf Light-Space Modulator als wichtigen Prototyp für Lichtskulptur zurück. Bei Otto Piene, Nicolas Schöffer, Frank Malina, Group de Recherche d’Art Visuel und Dan Flavin macht Burnham in seiner Europa und die USA umfassenden Auflistung (die ihn als Künstler inkludiert) einen Trend zur schwindenden Haptizität („intangibility“) aus. Das letzte Kapitel nimmt die „Robot and Cyborg Art“ in den Blick, die mit dem „cybernetic changeover“ (vgl. Norbert Wiener, Ludwig von Bertalanffy, Ross Ashby) einhergehe und eine bidirektionale Kommunikation zwischen Werk und Betrachterin anstrebe, wie sie auch für die Living Sculpture virulent wird.136 Cyborg Art vermutet Burnham als letzten konsequenten Schritt in der Entwicklung der Skulptur und bindet sie an den historischen Topos des sculptor deus und das Faustische Drama des 20. Jahrhunderts zurück. Burnhams Konzept deutet auf ein realzeitliches System, wie er es in der Folge in seinen im Artforum publizierten Aufsätzen genauer erläutert. Mit der Verzeitlichung der Skulptur gehe eine be grenzte Lebensdauer einher, die die traditionelle memoriale Funktion und Überzeitlichkeit des Kunstwerks konterkariert. Legitimation erfährt Burnhams systemtheoretische, kybernetische Herangehensweise durch die gesellschaftliche Ubiquität von Systemen bis zu künst lichen, intelligenten Lebensformen: „everyone lives intermeshed daily with a hierarchy of synthetic systems dominating the quality of life. This in effect is the new biological reality which both man and art must abide by.“137 Burnhams Fortschrittsglaube und seine Technikeuphorie spiegeln sich in der Entwicklungsgeschichte des Roboters, „to interact convincingly and intelligently with human beings – not to frighten them as do apparitions of the Frankenstein variety.“138 Erneut sei das Ziel der Bildhauerinnen die menschliche Figur. Mehrfach klingt die Benachteiligung der nicht über dasselbe technische Know-How wie Ingenieurinnen und Programmiererinnen verfügenden Bildhauerinnen an, die in schwächere Nachahmungen münde: Variationen von ‚Subskulptur‘ wie Puppen als Fetischobjekte, Wachsfiguren, Schaufensterpuppen, hyperrealistische Figuren (u.a. George Segal, Edward Kienholz, Frank Gallo) – ‚Protoroboter‘.139 In der Gegenwartskunst spielt die Verschaltung von Maschine und Körper, die Kreation von hybriden Wesen eine große Rolle.140 Eine von Burnhams (nicht ganz neuen) Thesen besteht im fortlaufenden Anthropomorphismus der Skulptur. Neu sei an der Cyborg Art und Post-Kinetic Art der Versuch, die Struktur des Lebens zu simulieren oder wie Schöffer mit CYSP I (1956) quasi handelnde/reagierende Skulpturen zu entwerfen.141 In der 135 Ebd., S. 290. 136 Ebd., S. 312. 137 Ebd., S. 320. 138 Ebd., S. 324. 139 Ebd., S. 327. 140 Vgl. Donna Haraway, „Ein Manifest für Cyborgs. Feminismus im Streit mit den Techno-Wissenschaften“ (1985), in: dies., Die Neuerfindung der Natur. Primaten, Cyborgs und Frauen, hg. von Carmen Hammer und Emmanuel Stieß, übers. von B. Ege, D. Fink, H. Kelle, C. Hammer, A. Scheidhauer, Immanuel Stieß, F. Wolf, Frankfurt am Main & New York: Springer 1995, S. 33–72. 141 Burnham 1968, S. 332f. „The term cyborg refers to both electromechanical systems with lifelike behavior and man-made machine systems which parallel (through feedback) some of the properties of single 50 Zur skulpturalen Ästhetik des Lebendigen 15 Hans Haacke, Ant Coop, 1969, Ameisen, Acrylglas, Sand, zerkleinerte Getreidekörner Living Sculpture entfällt diese Simulation; das Leben wird selbst ins Werk geholt. Burnham stellt die entscheidende Frage nach der Autorschaft, d.h. wo endet die Kreativität der Maschine und wo beginnt die menschliche Erfindung? Für die vorliegende Untersuchung sind die von Burnham in diesem Zusammenhang aufgelisteten Projektideen Medallas mit lebenden Organismen wie Schnecken, Shrimps und Ameisen interessant: (1) ‚Collapsible Sculptures‘ or forms […]. Snails would move over touch-sensitive plates setting off different tones; or shrimps could be induced to perform an underwater ballet; or ants could be made part of a magnified pattern of shapes in a glass farm; (2) ‚Hydrophonic Rooms‘ would be rooms with walls and ceilings of special porous rock on which thousands of edible mushrooms could be grown; (3) ‚Transparent Sculptures that Sweat and Perspire‘; these would be heatand light-sensitive masses whose shape and pulsation could be controlled by the environment; (4) ‚Radio-controlled Flying Sculptures‘ would consist of objects which could fly from a ‚hive‘ to all parts of a city, returning with various non-valuable objects.142 Sie demonstrieren ein Weiterdenken von Skulptur und Ansätze einer Non-Human Living Sculpture avant la lettre. Haackes Ant Coop (Abb. 15) wird bereits als Idee genannt; Burnham hebt dieses neue Level der systemischen Environmental Art hervor. Haackes physikalische Arbeiten folgen in Burnhams Auflistung; „living system“ nennt er dessen Grass biological organisms.“ (Ebd., S. 333.) Das Kapitel enthält eine umfassende Bibliographie zur Kybernetik. 142 Ebd., S. 346. 51 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt ube.143 Dem Vorwurf des Didaktischen entgegne Haacke mit der Verflochtenheit seiner C Werke mit größeren Systemen außerhalb des White Cube als Teil einer „environmental systems philosophy“.144 In seinem vorletzten Abschnitt über die Zukunft responsiver Systeme in der Kunst und sogenannte „systems sculptures“ nennt Burnham E.A.T. und prophezeit, „that electronical technicians will become regular members of museum staffs.“145 Die von Fried kritisierte Theatralität (Art and Objecthood, 1967) sieht er als Schritt zur Systemkunst.146 Selbst der Mensch stehe durch die Bakterien seines Organismus mit seiner Umgebung im Austausch und sei Teil eines größeren Systems – ein Thema, das beispielsweise Annika Yi in der Gegenwartskunst skulptural umsetzt.147 Beyond Modern Sculpture endet mit einer (teleologischen) Theorie moderner Skulptur und dem konstatierten Verlust des Objekts.148 Die ‚Skulpturbildnerei‘ sieht Burnham als anthropologische Grundkonstante, indem er sie parallel zu technologischen, naturwissenschaftlichen und psychologischen Entwicklungen erfasst. Während die klassische Skulptur durch einen Ortsbezug, die Einbindung in ein bauliches Ensemble und ihre rituelle Funktion charakterisiert sei, kennzeichne seine eigene Gegenwart das Systemische, das die vormals starre Werk-Betrachterinnen-Dichotomie in ein interaktives Gefüge einbindet. Es werden sein naturwissenschaftliches, hier explizit neurowissenschaftliches, molekularbiologisches Interesse und sein Glauben an Evolutions geschichte und Fortschritt deutlich; er gesteht der Skulptur eine aufklärerische Schlüsselfunktion zu, da diese in ihrer engen Anbindung an technologische Neuerungen einem ‚Frühwarnsystem‘ ähnlich fungiere.149 143 Ebd., S. 347. 144 Ebd., S. 349. 145 Ebd., S. 363. Ebenso hellsichtig urteilt Burnham über die Auswirkungen des technischen Fortschritts: „So far the motive forces behind technology have made life comfortable for a relatively few humans while they have unintentionally but progressively destroyed the biosphere, that thin film of organic life covering the earth.“ (Ebd., S. 371.) 146 Michael Fried, „Kunst und Objekthaftigkeit“ (1967), in: Gregor Stemmrich (Hg.), Minimal Art. Eine kritische Retrospektive, Dresden: Verlag der Kunst 1998, S. 334–374; ders., Art and Objecthood. Essays and Reviews, Chicago & London: Chicago University Press 1997. 147 Vgl. https://www.anickayistudio.biz/series/bacteria-cultures (25.2.2023). 148 Dabei denkt Burnham über gesellschaftliche Zukunftsszenarien („a world controlled by superintelligent automata“) und das Wesen des Menschen nach (Burnham 1968, S. 375). 149 Ebd., S. 371, 376. Die freistehende Rundplastik habe sich in Griechenland auch parallel zum Aufkommen der Wissenschaft herausgebildet, so Burnham, und er fragt, ob Kunst nicht eine Art biologisches Signal sei (ebd., S. 374). Siehe auch ders., „Some thoughts on systems methodology applied to art“ – ein Text, den er mit einem Brief (datiert 17.11.1967) an György Kepes schickt (Archiv Center for Advanced Visual Studies, Special Collection, J_0012, MIT, Program in Art, Culture & Technology, Cambridge, Mass., S. 1–9: „The system approach does not retard creativity in any way – once the artist has an idea. What it can provide is a method for developing an idea into alternative physical systems. It defines known and unknown factors, constraints, boundaries, desired outputs, and the means for locating optimum solutions. Moreover, the systems approach allows the artist to consider viewer interaction and environmental conditions within the scope of his thinking. It is a way of thinking logically which the artist will evaluate intuitively.“ (Ebd., S. 4, Herv.i.Orig.)) Trotzdem sei nie der gesamte Entstehungsprozess von Kunst zu verstehen; wichtig ist Burnham dabei die Integration der Betrachtenden und der Umgebung. „Ecological systems are prime open systems, as are all individual forms of plant and animal 52 Zur skulpturalen Ästhetik des Lebendigen 16 Pierre Huyghe, Untilled, 2011–2012, Lebewesen und unbelebte Dinge, gemacht und nicht gemacht Burnhams Abkehr von einem objektästhetischen zugunsten eines systemästhetischen Ansatzes betont die Relationalität einer entgrenzten, aus mehreren Elementen bestehenden Skulptur, „an order that cannot be described in terms of length, width, and height“, so Hans Haacke 1967.150 Neben dem Potenzial eines systemischen Verständnisses, das im zweiten Kapitel der vorliegenden Untersuchung an Haackes Franziskanischer Serie erörtert wird, rückt die Frage in den Blick, inwiefern Skulptur zu einer Situation wird, wenn sie im Beziehungsgefüge mehrerer Objekte und Ko-Akteure (drittes Kapitel Pierre Huyghe) (Abb. 16) entsteht. Wird Skulptur damit selbst zum Handlungsfeld? life. Unlike closed systems (i.e. art objects) which demand empathetic appreciation, open systems as works of art can be enjoyed on the level of constant interaction. This is very much the potential of computer art. […] Artistic objectives can never be set down with any serious precision. Neither should the artist plan his art system in terms of some desired human response. Interesting systems always evolve multi-layered and unexpected responses.“ (Ebd., S. 5f.) Und doch solle der Künstler die technologischen Potenziale nicht mit der „rigor of an engineer“, sondern mit der „naturalness of instinctive attitude“ verfolgen (ebd., S. 9). 150 Hans Haacke, Untitled Statement, „For some years …“ (1967), in: Alexander Alberro (Hg.), Working Conditions. The Writings of Hans Haacke, Cambridge, Mass.: MIT Press 2016, S. 10. 53 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt Willoughby Sharp, der Haacke zu einigen Ausstellungen einlud, greift diesen Gedanken eines systemästhetischen Konzepts in seinem Ausstellungskatalog Air Art (1968) auf, indem er einen Abschnitt mit „Sculpture as System“ betitelt und die Obsoletheit des klassischen Skulpturverständnisses deklariert. Für die von ihm ausgestellte zeitbasierte kinetische Kunst sei der Terminus ‚Skulptur‘ unzureichend – hier ist er mit Burnham einer Meinung: „Kinetic works are more accurately described as systems.“151 Wenige Jahre vor Burnhams Buch war 1962 Umberto Ecos Opera Aperta (Das offene Kunstwerk) erschienen, das zu einem veränderten relationalen Verständnis von Kunst beitrug.152 Von der Skulptur zum Skulpturalen: Rosalind Krauss Rosalind Krauss hat entscheidende Schriften zur Skulptur vorgelegt, die für die vorliegende Untersuchung von zentraler Bedeutung sind und daher im Hinblick auf eine skulpturale Ästhetik des Lebendigen nun genauer beleuchtet werden. Seit ihrer Dissertation zu David Smith (1969/1971) befasst sich die amerikanische Kunstkritikerin und -theoretikerin mit Skulptur.153 1973 veröffentlicht sie im Artforum den Beitrag „Sense and Sensibility – Reflections on Post ’60s Sculpture“.154 Zwar reflektiert sie keineswegs, wie der Titel vermuten ließe, die Kategorie Skulptur in ihrer ontologischen Ausprägung, sondern widmet sich dem Bezug zur Geschichte der Kunst der 1960er-Jahre am Beispiel der proklamierten Differenz bzw. konzeptuellen Markierung zwischen Minimalismus und Post-Minimalismus sowie Lippards Credo der Dematerialisierung.155 Bereits hier zeichnen sich Krauss’ phänomenologisch und strukturalistisch geprägte Herangehensweise und ihr Umgang mit begrifflichen Negationen ab, wie sie dies in „Sculpture in the Expanded Field“ (1979) fortsetzt.156 Das Präfix ‚Post-‘ impliziere einen „unterstellten historischen Zeitsprung mit Veränderung der Sensibilität“, wobei ihre Bezugnahme auf Jean-Luc Godard und dessen Ablehnung von Geschichte als erzählter Vergangenheit auf ihre eigene Kritik an einem teleologischen Geschichtsverständnis deutet. Doch wie begründet sie ihre Ablehnung einer „hermetischen Logik der Vaterschaft“, wenn sie sich wenige Jahre später in ihrem poststrukturalistischen Diagramm einer ‚Skulptur im erweiterten Feld‘ trotzdem auf einen historisch determinierten Begriff stützt – zweifach im Titel des Essays und des Diagramms und 151 Willoughby Sharp (Hg.), Air Art, Ausst.-Kat. Arts Council Philadelphia, New York: Kineticism Press 1968, S. 6. 152 Umberto Eco, Das offene Kunstwerk, Frankfurt am Main: Suhrkamp 1977, u.a. S. 154. Statt eines eindimensionalen Beziehungsverhältnisses ruft Eco in seiner Betrachtung abstrakter Kunst (u.a. Calder, Dubuffet) ein vielschichtiges Feld auf, das, so könnte man argumentieren, bei Burnham zum System und schließlich zu einem die Skulptur bis heute prägenden relationalen Gefüge ausgedehnt wird. 153 Vgl. Rosalind Krauss, „The Essential David Smith, Part I“, in: Artforum, 7, 6, 1969, S. 43–49; dies., „The Essential David Smith, Part II“, in: Artforum, 7, 8, 1969, S. 34–41. 154 Im Folgenden zit. n. der deutschen Version: Rosalind Krauss, „Sinn und Sinnlichkeit. Reflexionen über die nachsechziger Skulptur“, in: Stemmrich 1998, S. 471–497. 155 Ebd., S. 476f. 156 Krauss 1979. Im Folgenden wird vor allem die deutsche Übersetzung zitiert: Rosalind E. Krauss, „Skulptur im erweiterten Feld“, in: dies., Die Originalität der Avantgarde und andere Mythen der Moderne, hg. von Herta Wolf, Dresden: Verlag der Kunst 2000, S. 331–346. 54 Zur skulpturalen Ästhetik des Lebendigen in diesem selbst als Verortung an der Peripherie zwischen (Nicht-)Landschaft und (Nicht-) Architektur?157 Erst auf Seite 18 von „Sinn und Sinnlichkeit“ fällt der Begriff ‚Skulptur‘, wenn Krauss retrospektiv auf Konzeptkünstler wie Mel Bochner, Douglas Huebner, Joseph Kosuth und Robert Barry blickend schreibt: „Während der gleichen Zeitspanne gibt es ein paralleles Projekt in den Arbeiten anderer Skulpturkünstler: die Entdeckung des Körpers als eine vollständige Veräußerlichung des Selbst.“158 Ihr einerseits von Merleau-Ponty, andererseits von Sprachtheorien und der Wittgenstein’schen Philosophie geprägter methodischer Hinter grund führt dazu, dass Krauss die Subjektivität der Wahrnehmung für die Kunst im Sinne der ‚erfahrenen Form‘ unterstreicht. Sinneseindrücke seien subjektiv; das Kunstwerk bedürfe einer künstlerischen Intention, um sich als „Index eines Schaffensaktes“ realiter zu manifestieren.159 Der künstlerischen Idee, weniger der materiellen Ausprägung gebührt eindeutig Vorrang. „Sinn und Sinnlichkeit“ beleuchtet die Aufgabe des Illusionismus und damit einen erweiterten Sinnzusammenhang im Werk wie in der aktiven, individualisierten Rezeption, die eine kontextabhängige gesamtkörperliche Erfahrung verlange und eine Reflexion über die Prozesshaftigkeit des Materials forciere. 1977 gibt Krauss David Smiths Catalogue Raisonné heraus und veröffentlicht Passages in Modern Sculpture, worin sie die Entwicklung der Skulptur von der Denkmalplastik des 19. Jahrhunderts bis zu den performativ erfahrbaren Werken der Minimal Art und Land Art als modernistische, formalästhetische Geschichte einer zunehmenden Verzeitlichung des Mediums, das auf ein aktives Publikum angewiesen ist, beschreibt.160 Vor dem Hintergrund der Lessing’schen Trennung zwischen Raum- und Zeitkünsten und der Vernachlässigung des Temporalen in der Geschichte moderner Bildhauerei bei Giedion-Welcker (Jack Burnham nennt Krauss in ihrer Einführung nicht) kritisiert sie deren Fokus auf Räumlichkeit und Materialbehandlung und plädiert für eine enge Verzahnung beider Pole.161 Skulptur beinhalte die Verbindung von „stillness“ und „motion“.162 Rodins Werk bezeichnet auch für sie den Beginn der Moderne, da beim Höllentor eine kohärente narrative Sequenz im Bildfeld und klassische Figur-Grund-Trennung fehlen, es mit der dreifachen Figur des Schatten eine 157 Krauss 1998, S. 473. 158 Ebd., S. 488, Herv.i.Orig. 159 Ebd., S. 479. 160 Rosalind E. Krauss, The Sculpture of David Smith: A Catalogue Raisonné, New York: Garland 1977; dies. 1981. Ihre entwicklungsgeschichtliche Argumentation von Passages in Modern Sculpture scheint damit fast im Widerspruch zu „Sinn und Sinnlichkeit“ zu stehen. Als modernistisch versteht Krauss vor allem die Kunstauffassungen im Umfeld von Clement Greenberg, insbesondere die Kunst von der historischen Moderne bis zu den 1960er-Jahren. Vgl. Clement Greenberg, „Modernistische Malerei“ (1960), in: Karlheinz Lüdeking (Hg.), Clement Greenberg. Die Essenz der Moderne. Ausgewählte Essays und Kritiken, Dresden: Verlag der Kunst 1997, S. 265–278. Vgl. ebenso seine Texte „Die neue Skulptur“ (1949), „Skulptur in unserer Zeit“ (1958) und „Neuerdings die Skulptur“ (1967). Auch Krauss listet nur wenige Bildhauerinnen auf, namentlich Louise Bourgeois, Eva Hesse, Barbara Hepworth und Louise Nevelson. 161 Krauss 1981, S. 3–5. „Into any spatial organization there will be folded an implicit statement about the nature of temporal experience.“ (Ebd., S. 4.) 162 Ebd., S. 5. 55 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 17 Auguste Rodin, Höllentor, 1880–1917 serielle Aneinanderreihung (Abb. 17) gebe, das Tor jeglicher Funktion entbehre und zur ortsungebundenen Plastik avanciere, der Bildhauer explizit Spuren seiner Bearbeitung auf der Oberfläche sichtbar lasse. In Anlehnung an literarische Konzepte (u.a. Raymond Roussel) differenziert Krauss unterschiedliche Auffassungen von Zeit, doch bleibt sie dem Narrativ moderner Skulptur verhaftet: Nach einem Kapitel zum Futurismus und Konstruktivismus folgen Duchamp und Brancusi als Vertreter des Readymade, Skulptur im Surrealismus, die neue Eisen- und Metallskulptur („welded image“), ein Kapitel zu „Light, motion, theater“, das Kinetik, ZERO, Happening, Performance und Haackes physikalisches System wie erneut dessen Condensation Cube enthält. Das Schlusskapitel versammelt zeitgenössische (US-amerikanische) Positionen, darunter Land Art, Pop Art, Abstrakter Expressionismus, Post-Minimal Art. Ein Beispiel für die reale Erlebniszeit und Aktivierung der Betrachterinnen seien Gia comettis skulpturale Spielbretter, Tischskulpturen und Käfige, da sie ihr Gegenüber zur Handlung („physical immediacy“) einladen, eine reale Bewegung synchron zur individuellen Erfahrung stattfinde, ähnlich bei Arps Concrétions und ab Mitte der 1960er-Jahre im Ersten Werksatz Franz Erhard Walthers (Abb. 18), den Krauss jedoch nicht nennt.163 Ein dialektischer 163 Krauss 1981, S. 118, 120, 138f. 56 Zur skulpturalen Ästhetik des Lebendigen 18 Franz Erhard Walther, Elfmeterbahn, 1964, Vorführung in der Aula der Kunstakademie Düsseldorf mit Sigmar Polke im Mai 1967 Dualismus spiegelt sich in der die Skulpturgeschichte der Moderne durchziehenden Gegen überstellung von Gabo („virtual volume“) bzw. Pevsner und Moore: Erstere konstruierten ein Volumen, Letzterer modellierte einen Körper („carver’s instinct“).164 Mit „Tanktotem: welded images“ widmet Krauss der Eisen- und Stahlplastik seit den 1950er-Jahren ein eigenes Kapitel, insbesondere Smith. An Caros Early One Morning (Abb. 19) beschreibt sie die piktoriale Erfahrung, die die auf zwei Wahrnehmungsperspektiven – frontal, d.h. flächig/piktorial und seitlich, d.h. räumlich-physisch – angelegte, farbig gefasste Skulptur bietet. Das sechste Kapitel nimmt die skulpturalen Erweiterungen der Minimal Art in Richtung Theater, Tanz und Performance beginnend mit Morris’ Aufführung Columns (1961) (Abb. 20), die damit verbundene Verzeitlichung der Skulptur und Frieds Kritik an der Theatralisierung zum Ausgangspunkt. Theatralität fungiere, so Krauss, als Dachbegriff für eine Kritik an einem neuen Skulpturverständnis und vereine künstlerische Ausprägungen wie Kinetik, Licht- und Klangarbeiten, Performance, Happening, „environmental and tableau sculpture“.165 Zur historischen Kontextualisierung einer „theatrical sculpture“ zieht sie frühe Beispiele des Theatralen und Kinetischen heran, wie Francis Picabias Bühnensetting Relâche 164 Ebd., S. 134, 143. 165 Ebd., S. 204. 57 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 19 Anthony Caro, Early One Morning, 1962, lackierter Stahl, Aluminium, 289,6 x 619,8 x 335,3 cm, Tate Gallery, London 20 Robert Morris, Columns, 1961, Sperrholz, 5,33 x 5,33 x 5,33 cm und Moholy-Nagys Light-Space Modulator (1923–1930), der wie ein mechanischer Akteur Licht als zeitbasiertes, räumliches Medium nutze. Mit einem Verweis auf Pierre-Jacques Droz’ mechanische Puppen des 18. Jahrhunderts führt Krauss ihre Kritik an Burnhams Fokus auf das mimetische Bestreben in der Bildhauerei, „the imitation, simulation, and non-biological 58 Zur skulpturalen Ästhetik des Lebendigen re-creation of life“, ein.166 Allerdings analysiert auch Burnham keineswegs nur figurative Werke, so dass Krauss, wenn sie ihren Fokus auf die ungegenständliche Skulptur des Mini malismus und Post-Minimalismus legt, das Feld des Nicht-Anthropomorphen zu Unrecht von Burnham vernachlässigt sieht. Sie und Burnham vergleichen ähnliche Objekte, etwa Moholy-Nagys Light-Space Modulator, Calders Bicycle und Morris’ Columns. Ihr Skulpturenkanon unterscheidet sich (bis auf den Surrealisten-Teil in den Passages) weniger, als Krauss es vermutlich favorisieren würde. Sie unterstellt Burnham einen technokratischen Ansatz, mechanistische Weltsicht und Vernachlässigung der ideologischen Kontexte, doch attestiert sie seinem Buch, „one of the most extensively and closely argued presentations of sculpture“ zu sein.167 Im Vergleich zu ihren Passages ist Burnhams Beyond Modern Sculpture theoretischer mit einer Vielzahl an interdisziplinären Referenzen. Einige bildhauerische Positionen werden von beiden behandelt, wie Nicolas Schöffer, der ‚animierte‘ Skulpturen mit computergesteuerten Interfaces konzipiert; die mechanisierte kinetische Plastik der 1960er-Jahre „is meant to ‚enact‘ itself“ und übernehme die Rolle von quasi selbst handelnden Ko-Akteuren. Zu fragen wäre, ob es sich hierbei um eine Vorform der Living Sculpture handelt.168 Haackes Condensation Cube ist auf einer Doppelseite mit Tinguelys Homage to New York und Pol Burys 18 Superimposed Balls abgebildet (Abb. 21), findet jedoch im Text keine Erwähnung.169 Die Bildzusammenstellung belegt den Kontext der Kinetik, in dem die Autorin Haacke verankert sieht, ohne den nicht-mechanischen Charakter des Werks zu berücksichtigen. Claes Oldenburg zieht Krauss heran, um anhand von „gigantism“ und „softness“ die Theatralisierung der Umgebung durch das Objekt zu erläutern: Seine Soft Sculptures, die überproportional groß Objekte aus der Konsum- und Alltagskultur wie Hamburger, Tortenstück, Sandwichtoaster oder Toilette darstellen, affizieren ihr Gegenüber durch einen Wiedererkennungseffekt und vermitteln über das Weiche, Nachgebende eine Nähe zur eigenen Körperlichkeit.170 Oldenburgs Soft Sculptures dienen ihr ähnlich wie Morris’ Columns und dessen Verbindung zum Judson Church Dance Theater als Beispiel für eine Art Anthropomorphisierung bzw. Verlebendigung der Skulptur und deren Affinität zum Theater. Eine Aktivierung erfolgt auf beiden Seiten: beim Objekt und bei den Rezipientinnen, erkennbar am Beispiel von Bruce Naumans Corridor (1968–1970), der auch Krauss’ Cover ziert und in dem die Körper der durch die ‚Passage‘ Schreitenden über die Abbildung auf zwei Bildschirmen doppelt integriert werden.171 166 Ebd., S. 211. Krauss spricht von Vaucanson, zeigt aber Bilder von Droz. 167 Ebd., S. 212. 168 Ebd., S. 220. 169 Ebd., S. 224f. Vgl. Burnham 1968, S. 272f. 170 „These objects, staged, like lugubrious obstructions in our space, do theatralize their environment, do render us participants or actors in the drama of their presentation.“ (Krauss 1981, S. 229.) 171 Ebd., S. 242. „[…] yet another attack on the convention of sculpture […] essential here is that kind of theatricality one finds in the work of Oldenburg, Morris, and Nauman is central to the reformulation of the sculptural enterprise […].“ 59 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 21 Jean Tinguely, Pol Bury und Hans Haacke in: Rosalind Krauss, Passages in Modern Sculpture, Cambridge, Mass.: MIT Press 1981, S. 224f. Das letzte Kapitel „The Double Negative: a new syntax for sculpture“ widmet sich der Minimal Art und Post-Minimal Art, etwa Wiederholung als Kompositionsprinzip in Form serieller Reihung bei Judd, Serra und Morris, Negation von Bedeutung als Abkehr vom abstrakten Expressionismus und Illusionismus sowie von der Logik des organischen Wachstums, wie es für die moderne Skulptur Moores, Arps oder Hepworths charakteristisch war. Im russischen Konstruktivismus sieht Krauss – auch das ist ein sich fortsetzender Topos, der die politischen, ideologischen Hintergründe ausklammert – einen Vorläufer für die amerikanischen Künstlerinnen der 1960er- und 1970er-Jahre, da Tatlin, Gabo und Pevsner präfabrizierte Materialien (Plastik und Plexiglas) nutzten, die additive, konstruktive Herangehensweise der Ablösung des hermetisch geschlossenen, monolithischen Körpers proklamierten und der Bedeutungsgenerierung aus dem Inneren heraus eine Absage erteilten. Die Bedeutung der L-Beams von Morris liege nicht mehr in einer manuell bearbeiteten Oberflächenstruktur wie bei Rodin, sondern verlagere sich vom individuellen Gestus ins Öffentliche, in den Raum und zu den Betrachtenden. Durch die variierende Positionierung dreier identischer weiß lackierter Balken treten die Betrachterinnen in eine Beziehung zum skulpturalen Objekt: 60 Zur skulpturalen Ästhetik des Lebendigen „This difference is their sculptural meaning.“172 Signifikant ist Krauss’ Sprachmetaphorik, die das Prozessuale und Handlungsbezogene spiegelt und auf Serras Verb List (1967) („list of behavioral attitudes“) bzw. seinen Film Hand Catching Lead (1969) anspielt.173 Dass der Körperbezug trotz Abstraktion und einer kontinuierlichen Dezentralisierung in der Skulptur bestehen bleibe, zeigt sie abschließend in einer phänomenologischen, psychologischen Lesart an Michael Heizers Double Negative (1969), Smithsons abschreitbarer Spiral Jetty (1970) und Serras Landschaftsskulptur Shift (1970), womit die Monumentalität der drei dimensionalen, den Körper in sich aufnehmenden Form (Passage) die Betrachterinnen auf sich selbst zurückwirft, eine Außenperspektive ausschließt und Skulptur zum temporalen Medium avanciert. Diese Prozessästhetik – in Abgrenzung zur Objektästhetik – impliziert eine rezeptionsbezogene Form von Zeitlichkeit, die sich erst im Dialog zwischen Artefakt und Rezipientin entfaltet.174 Auf der Produktionsebene prägen die von Krauss und Burnham genannten frühen Rotoreliefs Duchamps (1935) und seiner Nachfolger aus der kinetischen und kybernetischen Kunst sowie auto-generative Werke das vom Topos der fixierten statua befreite skulpturale Erscheinungsbild. Temporalität meint hier eine Abkehr von der traditionellen durata.175 Die von Krauss vorgenommene Situierung einer ‚Skulptur im erweiterten Feld‘ zwischen Architektur – Landschaft und deren Negation leistet einen grundlegenden Beitrag für die Ortsspezifik und das Raum-Zeit-Verhältnis des Post-Minimalismus. Der Aufsatz erschien 1979 in der achten Ausgabe der von Krauss mitbegründeten Zeitschrift October und erfuhr eine vielfache Rezeption. Ihr in Skulpturtheoriekreisen ‚berühmtes‘ Diagramm (Abb. 22) wird hier im Hinblick auf eine skulpturale Ästhetik des Lebendigen befragt. Angesichts der inflationären Verwendung des Begriffs ‚Skulptur‘ strebt Krauss eine terminologische Neu definition dieses „kategorialen Niemandslands“ an.176 Krauss begreift Skulptur als „historisch determinierte“, nicht „universelle Kategorie“ und wendet sich in ihrer Kritik an Clement Greenbergs essenzialistischem, medienspezifischem Konzept von Skulptur einem strukturellen Verständnis von Skulptur, genauer dem Skulpturalen zu.177 Das Skulpturale ist nicht „die Definition eines bestimmten Mediums auf 172 Ebd., S. 267. 173 Ebd., S. 275f. 174 Vgl. auch Gerhard Graulich, Die leibliche Selbsterfahrung des Rezipienten – ein Thema transmodernen Kunstwollens, Essen: Blaue Eule 1989. Eine Dynamisierung und Verzeitlichung der Form strebten bereits die Bildhauer des Futurismus und Kubismus an. Vgl. Umberto Boccioni: „In der Plastik wie in der Malerei kann man nichts erneuern, wenn man nicht den Stil der Bewegung sucht […]. Werfen wir also alles über den Haufen und proklamieren wir die absolute und vollständige Abschaffung der Linie und der in sich geschlossenen Statue. Reißen wir die Figur auf, und schließen wir die Umgebung in sie ein.“ (Boccioni 2002, S. 243f.) 175 Siehe auch u.a. Naum Gabos kinetische Plastiken der 1920er-Jahre und Alexander Rodtschenkos Hängekonstruktion (1920). 176 Sie ist nicht die Einzige, vgl. u.a. André Bloc 1956, zit. n. Trier 1971a, S. 67: „Man verwendet immer noch das ‚Wort‘ Skulptur, um die verschiedenartigen Erzeugnisse abstrakter Künstler zu bezeichnen. […] [W]ir schlagen […] vor: ‚Die Kunst, Raum einzunehmen‘“. 177 Krauss 2000, S. 334, 345. Vgl. auch Dobbe/Ströbele 2020, S. 3. 61 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 22 Rosalind Krauss, Sculpture in the Expanded Field, 1979 der Basis des Materials“ oder dessen Wahrnehmung, sondern fungiert als Herangehens weise, die einen bestimmten Umgang mit den Dingen benennt und Plastizität, Räumlichkeit, Ikonizität und Zeitlichkeit heraushebt.178 Damit richtet Krauss sich gegen ihre frühere essenzialistische Auffassung von Skulptur, ebenso gegen ihre entwicklungsgeschichtlich geprägte Argumentation in Passages in Modern Sculpture, d.h. gegen eine historistische Einbettung von Skulptur allgemein. Rodin fungiert bei Krauss als Repräsentant des Übergangs zur Moderne, die neben einer verzeitlichten Skulptur durch einen Verlust der Memorial- und Repräsentationsfunktion sowie einen Ortsverlust aufgrund von Selbstreferenzialität (Denkmalsplastik, etwa Höllentor und Balzac) charakterisiert ist. Das von Krauss skizzierte Feld ist nicht nur ein Diskursfeld, sondern konkret als räumliche Erweiterung zu verstehen, insofern mit der Skulptur der 1960er- und 1970er-Jahre ein Wandel einsetzt, diese sich in der Landschaft entfaltet, neue Räume zwischen Kultur und Natur, zwischen Gebautem und Nicht-Gebautem in einem relationalen Gefüge von Betrachterin, Ort und Werk eröffnet und die site-specificity der Land Art und Post-Minimal Art virulent wird. Zwischen Architektur – Landschaft und deren Negation eröffnen sich vier neue Unterkategorien von Skulptur:179 1. „Orts-Konstruktion“ als Verbindung von Landschaft und Architektur, beispielsweise Smithsons Partially Buried Woodshed (1970) oder Morris’ Observatory (1977); 2. „markierte Orte“ zwischen Landschaft und Nicht-Landschaft, etwa Christo & Jeanne-Claudes Running Fence (1972/76) bzw. 178 Vgl. Ausrichtung des Wissenschaftlichen Netzwerks Theorie der Skulptur: http://theoriederskulptur.de/ projekt/ (19.9.2019). 179 Marta Pan verortet die Bildhauerei gleichfalls zwischen Natur und Architektur (1961), siehe Trier 1971a, S. 195. 62 Zur skulpturalen Ästhetik des Lebendigen Smithsons Spiral Jetty; 3. „axiomatische Strukturen“ als Verbindung von Architektur und Nicht-Architektur, Werke, die architektonische Erfahrungen ermöglichen, etwa Naumans Green Light Corridor (1970) oder Serras raumumspannende, begehbare Werke. Die vierte, an die Peripherie gerückte Kategorie bildet die Skulptur in einer doppelten Negativität von Nicht-Landschaft und Nicht-Architektur, u.a. Morris’ grau lackierte geometrische Sperrholzobjekte, eine „quasi architektonische Einheit“, und seine Mirrored Boxes (1965), die die umgebende Landschaft spiegeln, aber selbst nicht Teil der Landschaft sind.180 Damit hatte die Skulptur „den vollständigen Zustand ihrer inversiven Logik erreicht und war reine Negativität geworden […] eine ontologische Abwesenheit“.181 Sie tritt nun zweifach auf, als Oberbegriff im Titel und innerhalb des erweiterten Felds als eigene Kategorie.182 Die Originalität dieses Ansatzes liege Krauss zufolge darin, „logische Operationen mit einer Reihe kultureller Begriffe, für die jedes Medium […] verwendet werden kann“, materialunabhängig zu nutzen, so dass sich das Feld für diverse Techniken und Stofflichkeiten – ephemere, immaterielle Phänomene inbegriffen – öffnet: „Somit sorgt das Feld […] für eine erweiterte, aber endliche Reihe miteinander verbundener Positionen, die ein bestimmter Künstler besetzen und erforschen kann“.183 Krauss’ Schwerpunkte, die auf der Logik des Denkmals basieren, führen aber dazu, dass der lebende Körper, d.h. die Human und Non-Human Living Sculpture und die Soziale Plastik vernachlässigt werden, obgleich sich Walther zwischen 1967 und 1971, Rebecca Horn zwischen 1972 und 1981 in New York aufhielten und Gilbert & George 1970 ihre Singing Sculpture vorstellten. Diese Versuche, den Körper als plastisches Material einzusetzen, lassen sich kaum zwischen Landschaft und Architektur bzw. ihrer inversiven Umkehrung verorten. Die einzige Möglichkeit besteht in einer Zuordnung zum Bereich der ‚klassischen‘ Skulptur als Nicht-Landschaft und Nicht-Architektur zusammen mit Morris’ L-Beams oder Judds Specific Objects. Führt dies aber nicht zu einer erneut inflationären Verwendung des Begriffs ‚Skulptur‘? Benjamin Buchloh bemerkt in einer Paneldiskussion 2007 zu Krauss’ Skulpturfeld, dass Raum dort eine abstrakte Kategorie bleibe und nicht den sozialen Raum mitdenke.184 Hal Foster unterstreicht: Artists like Serra do not simply privilege the tectonic in a reactive mode. They are also concerned about recovering a bodily experience, which cannot be fitted into the diagram. We have mentioned the commodity, but where is the body?185 180 Krauss 2000, S. 337f. 181 Ebd., S. 338. 182 Auch Michael Lüthy hat darauf verwiesen: Michael Lüthy, „Expanded Field/Rosalind Krauss“, in: Brigitte Franzen/Kasper König/Carina Plath (Hg.), skulptur projekte münster 07, Köln: Walther König 2007, S. 356f. 183 Krauss 2000, S. 345. Siehe auch Dobbe/Ströbele 2020, S. 4. 184 Benjamin Buchloh, in: „The Expanded Field Then: A Roundtable Conversation“, in: Spyros Papapetros/ Julian Rose (Hg.), Retracing the Expanded Field. Encounters between Art and Architecture, Cambridge, Mass.: MIT Press 2014, S. 1–46, S. 13. 185 Hal Foster, in: ebd., S. 27, Herv.i.Orig. 63 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 23 Claire Bischop & Joe Scanlan, WANDERERS in the Expanded Field, 2014 Über eine Erweiterung des Diagramms nachdenkend, fragt Yve-Alain Bois: „The notnot-body and the not-person?“186 Unterschiedliche Raumkonzepte werden in Krauss’ Diagramm relativiert, weshalb Philip Ursprung Smithsons site/non-site und Serra’s site-specificity fruchtbarer findet.187 Einen Versuch der Erweiterung unternimmt der Künstler Joe Scanlan zusammen mit Claire Bishop, indem sie ein eigenes Strukturmodell zum ‚Gehen‘ als künstlerische Praxis entwickeln (Abb. 23): At first glance, the expanded field doesn’t account for the artist’s body as sculptural site. However, on closer examination, the diagram would seem to posit all artists as sculptures, since their bodies are neither landscape nor architecture. If the artist’s body can explicitely be a site marker (Mendieta), a constructed site (Oiticica), or an axomatic structure (Nauman), then the body of every artist must implicitly be a sculpture.188 186 Yve-Alain Bois, in: ebd., S. 125. Buchloh ergänzt: „I think there is an axis from Graham to Nauman that is important to recognize.“ (Ebd., Herv.i.Orig.) 187 „In her diagram, there is no place for the performative, or for video work. […] Performance, moving images, the ephemeral – in other words, the issues of temporality – are not part of the picture.“ (Ebd., S. 194.) 188 In Kooperation mit Claire Bishop, anlässlich der Ausstellung zum Thema 2009, in: ebd., S. 226, Herv.i.Orig. Wie berücksichtigt Krauss den Körper in Bewegung? Sie zeigt dokumentarische Fotos des Gehens in Bezug auf das erweiterte Feld, aber sie erwähnt nicht, wie mit dem Akt des Gehens an sich umzugehen sei. 64 Zur skulpturalen Ästhetik des Lebendigen 24 Franz Erhard Walther, Speier (Versuch, eine Skulptur zu sein), 1958, Wasser, Milch, Backpulver, Blechschüssel Droht auch hier eine inflationäre Zuschreibung zur (lebenden) Skulptur? Scanlans und Bishops Interesse liegt in den „wandering artists“ und einer Integration von „uncertainty“ und „doubt“, zwischen den vier Polen „Hunter/Gatherers“ (Thoreau), „Cyborgs“ (The Terminator), „Flaneurs“ (Baudelaire) und „Pilgrims“ (Jesus) verortet.189 Die einzelnen Künstlerinnen scheinen innerhalb des Feldes gemäß ihren Bewegungsabsichten und Gehverhalten zugeordnet zu werden. Valie Export beispielsweise findet in der rechten äußeren Hälfte nahe „Cyborg“ und neben Janet Cardiff Erwähnung. Wie aber könnte ein Körper als plastisches Material in anderen Bewegungsmodi als dem Gehen oder im Moment des Stillstands, wie Walthers Versuch, eine Skulptur zu sein (1958) (Abb. 24), graphisch veranschaulicht werden? Bietet es sich an, Joseph Beuys’ Konzept der sozialen Plastik einzubeziehen? Bereits Duchamp verwandelte sich zur Skulptur, als er in Monte Carlo Bond 1924 sein Gesicht mit Schaum bedeckte und sich selbst ausstellte (Medallic sculpture).190 In Walthers Blindobjekt (1964) (Abb. 25) agiert ebenfalls eine Person als Skulptur: Verhüllt, blind tastend, unterliegt sie einer physisch-haptischen Erfahrung, mit der eine veränderte Raum-Zeit-Vorstellung einhergeht.191 Miwon Kwon resümiert: „It is actually quite partial – as it tells only one story about sculpture among many others that could be told […] along the lines of commodity culture, or the body, or the phenomenological, for instance.“192 Ausgehend von Krauss’ Überlegungen könnte neben der Integration des lebenden Körpers eine Erweiterung bzw. Neuformulierung für digitale, virtuelle Skulpturen entwickelt werden.193 189 Ebd.. 190 Abb. in: Sharp 1968a, S. 5. 191 Siehe Ursula Ströbele, „OBJEKTE, benutzen. Angewandte(s in der) Kunst seit Franz Erhard Walther und Hans Haacke“, in: Hans Körner/Manja Wilkens (Hg.), Angewandte Kunst und Bild, München: Morisel 2018, S. 134–151. 192 Miwon Kwon, in: Papapetros/Rose 2014, S. 33. 193 Einen ersten Versuch siehe Ursula Ströbele/Andreas Greiner, „Notes on Sculpture“, in: dies./Jan-Philipp Sexauer (Hg.), 24 h Skulptur. Notes on Time Sculptures, Berlin: Distanz 2015, S. 16–36. 65 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 25 Franz Erhard Walther, Blindobjekt, 1966, Filz Auch in ihrem Essay „A Voyage on the North Sea. Art in the Age of the Post-Medium Condition“ wendet sich Krauss vom Primat der Medienspezifik und einem essenzialistischen Kunstbegriff ab.194 Am Beispiel der Konzeptkunst, Marcel Broodthaers und seinen ‚Mixed-Media Installationen‘, etwa seinem Musée d’Art Moderne, Département des Aigles, erörtert sie das Ende der Medienspezifik und unser Zeitalter einer „Post-Medium Condition“, das zum einen durch die Absage der Postmoderne an eine „purity of an artistic medium“ herbeigeführt wurde, zum anderen durch die Verwendung der Handkamera im Medium Video, die in Kunst bzw. Fernsehen Optizität erzeugte:195 „in the ‚60s‘ ‚opticality‘ was also serving as more than just a feature of art; it had become a medium of art.“196 Es bleibt zu fragen, inwiefern Krauss’ Konzept eines erweiterten skulpturalen Feldes, das verschiedene Formen von Materialität und Medien inkludiert, wie bei Smithsons Spiral Jetty, die neben der Arbeit im Salzsee Karten, Fotografien, Film und Text vereint, sich mit der Post-Medium Condition in Einklang bringen lässt. 194 Rosalind Krauss, A Voyage on the North Sea. Art in the Age of the Post-Medium Condition, London & New York: Thames & Hudson 2000. 195 Ebd., S. 12, 32f. 196 Ebd., S. 30. „The specificity of mediums, even modernist ones, must be understood as differential, self-differing, and thus as a layering of conventions never simply collapsed into the physicality of their support.“ (Ebd., S. 53, Herv.i.Orig.) 66 Zur skulpturalen Ästhetik des Lebendigen Wenn Krauss Broodthaers als Gewährsmann für Intermedia-Kunst betrachtet, erinnert dies an Dick Higgins’ gleichnamigen Text „Intermedia“, der Mitte der 1960er-Jahre die bestehenden Gattungsgrenzen kritisierte und die Kunst von Claes Oldenburg, Robert Rauschenberg, Allan Kaprow und Wolf Vostell, wie es später Juliane Rebentisch formulierte, „neuartig hybriden Bereichen zwischen den Künsten“ zuschreibbar sieht.197 Die Verwendung ungewöhnlicher Materialien, die räumliche Erweiterung und kulturelle Veränderungen lösten den bestehenden Kanon ab, wie Higgins vorschlägt: „I would like to suggest that the use of intermedia is more or less universal throughout the fine arts, since continuity rather than categorization is the hallmark of our new mentality.“198 Er unterscheidet ‚mixed-media‘, das ihn weniger interessiert. Das Happening sei ein Beispiel für „intermedium“ zwischen Collage, Musik und Theater. Intermedia behält gedanklich die einzelnen Medien bei, während Post-Medium Condition temporal, entwicklungsgeschichtlich zu verstehen ist und bei Mixed-Media oder Multi-Media verschiedene Medien zusammengesetzt werden. Reben tisch verweist auf die Emanzipation der Kunst von den traditionellen Gattungsgrenzen, die erst eine Reflexion der Medienspezifika ermöglichte und die von Greenberg mit einer essenzialistischen Kunst in eine bestimmte Richtung gelenkt wurde.199 Bishop sieht die Installation als ein Beispiel von „post-medium specific art“, die verschiedene Medien in sich vereine, aus mehreren Elementen bestehe und die Betrachterinnen immersiv in sich aufnehme: „Installation art creates a situation into which the viewer physically enters, and insists that you regard this as a singular totality.“200 Ähnlich einer Installation adressiert auch die erweiterte Skulptur multisensorische Ebenen und geht von einem aktivierten, dezentrierten „embodied viewer“ aus, die/der das Werk komplettiert.201 In seinem 2001 publizierten Aufsatz „Installation and Sculpture“ versucht Alex Potts eine konzeptuelle, historische und terminologische Differenzierung zwischen den beiden Kunstformen. Er betont strukturelle Affinitäten zwischen der Barockplastik etwa in Sakral ensembles und klassizistischen Skulpturen Canovas auf der einen Seite sowie Serras und Andres’ begehbaren Objekten und Installationskunst auf der anderen Seite im Hinblick auf die Integration des Umraums, Aktivierung des Publikums und skulpturales „staging“: „the idea of a non-installation art would be something of an oxymoron.“202 Dabei insistiert Potts 197 Ebd., S. 45, Herv.i.Orig. Dick Higgins, „Intermedia“ (1966), in: ders., Horizons. The Poetics and Theory of the Intermedia, Carbondale [u.a.]: Southern Illinois University Press 1984, S. 18–28; Juliane Rebentisch, Ästhetik der Installation, Frankfurt am Main: Suhrkamp 2003, insbesondere Kapitel II. „Inter medialität“, S. 79–231. 198 Higgins 1984, S. 22f. 199 Rebentisch 2003, S. 81. 200 Claire Bishop, Installation Art. A Critical History, London: Tate Publications 2005, S. 6. Nicht ganz nachvollziehbar ist ihre Differenzierung: „Installation art therefore differs from traditional media (sculpture, painting, photography, video) in that it addresses the viewer directly as a literal presence in the space.“ (Ebd.) 201 Ebd., S. 6, 11, Herv.i.Orig. Die Entstehung der Installationskunst verortet Bishop zwischen 1965 und 1975. 202 Alex Potts, „Installation and Sculpture“, in: The Oxford Art Journal, 24, 2001, 2, S. 5–24, S. 7f. 67 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt auch auf die Affinitäten zwischen Installation und Skulptur. So habe die Installationskunst keineswegs zur „dissolution of the sculptural object“ geführt; neu sei ab den 1960er-Jahren „a more cinematic, way of staging work“.203 Benjamin Buchloh bezeichnet Hans Haackes Germania (1993) im deutschen Pavillon der Biennale di Venezia sogar als „Installations skulptur“, um den raumzeitlichen und performativen Charakter zu unterstreichen.204 Insbesondere die realzeitlichen Systeme Hans Haackes oder die situationsästhetischen Skulpturen Pierre Huyghes verkörpern, so ließe sich ergänzen, eine strukturelle Nähe zu raumspannenden Werken der Installationskunst und zur erweiterten Modalität des Kinematographischen, „which encloses rather than faces the viewer.“205 In der vorliegenden Untersuchung werden Skulptur und das Skulpturale als ein methodischer Ansatz erörtert, auf den einerseits die hier analysierten Künstlerinnen explizit rekurrieren, der andererseits in den skizzierten Theo rien verhandelt wird und historisch auf den traditionellen Gattungskategorien und deren Grenzen fußt. Skulptur nach 1945 Andrew Causey, Richard J. Williams, Alex Potts Der folgende Abschnitt widmet sich zum Abschluss des Überblicks über die Konzepte und Historiographie von Skulptur drei Bänden aus dem angelsächsischen Raum, die als gemeinsamen Schwerpunkt die Skulptur nach 1945 im Hinblick auf deren Entgrenzung und ontologischen Status beleuchten: Andrew Causeys Sculpture since 1945 (1998), Richard J. Williams’ After Modern Sculpture. Art in the United States and Europe 1965–1970 (2000) und Alex Potts’ The Sculptural Imagination. Figurative, Modernist, Minimalist (2000).206 Während der britische Kunsthistoriker Andrew Causey, der als Doktorvater die Schrift von Williams betreute, das Phänomen des lebenden Körpers in der Skulptur am Beispiel von Robert Rauschenberg und Gilbert & George erörtert, finden Werke mit lebenden Organismen bei Richard J. Williams und Alex Potts nur marginal Erwähnung. Causey unterrichtete von 1953 bis 1967 an der Londoner St. Martins School of Art, bevor er als Professor for Modern Art History an der University of Manchester lehrte. Er war daher mit den Aktionen von Gilbert & George sowie mit den Walking Sculptures von Richard Long vertraut. Alle drei Künstler absolvierten an der St. Martins School ihr Studium – vermutlich ein Grund, warum Causey „the body as sculpture“ einen eigenen Abschnitt widmet, gleichermaßen 203 Ebd., S. 1, 8. „We could think of installation as sculpture that has now been fully absorbed within the modern, or post-modern, society of the spectacle.“ (Ebd., S. 19.) 204 Benjamin H.D. Buchloh, „Hans Haacke. Von der faktographischen Skulptur zum Gegendenkmal“, in: Matthias Flügge/Robert Fleck (Hg.), Hans Haacke. Wirklich. Werke 1959–2006, Berlin: Akademie der Künste, Ausst.-Kat. Deichtorhallen Hamburg, Akademie der Künste Berlin, 2006, S. 42–59, S. 57. 205 Ebd., S. 16. 206 Fast zeitgleich erschien auch: Thomas McEvilley, Sculpture in the age of doubt, New York: Allworth Press 1999. Etwas früher mit einem spezifischen Fokus auf weiche, nachgebende Materialien in der Skulptur: Maurice Fréchuret, Le mou et ses formes. Essai sur quelques catégories de la sculpture du XXe siècle, Paris: École Nationale Supérieure des Beaux-Arts 1993. 68 Zur skulpturalen Ästhetik des Lebendigen die zeitbasierten, teils ephemeren Werke von Piero Manzoni, Klein, Nauman, Horns Körper prothesen und die frühen skulpturalen Choreografien Rauschenbergs mit lebenden Tieren (Kuh, Schildkröten) – exemplarisch Elgin Tie (1964) und Pelican (1963) – sowie Beuys verhandelt.207 Er greift nicht auf den Terminus Living Sculpture zurück, auch nicht, wenn er Manzonis Aktion des Signierens seiner sculture vivente erwähnt, spricht stattdessen von Body Art „as sculptural category of that moment“.208 Die Integration des lebenden Körpers, das zeigt Causey anschaulich, erfolgt über die Verbindung Bildender Künstler wie Robert Morris und Robert Rauschenberg mit dem Judson Church Dance Theatre, respektive Tänzerinnen wie Yvonne Rainer, Carolee Schneemann und Simone Forti – eine Verbindung, die auch Potts für seine „sculptural imagination“ seit Mitte der 1960er-Jahre fruchtbar macht.209 Causey erläutert: The change in the 1960s in performance/dance, and Rainer’s equation of body and object brought the practice closer to sculpture, especially in the work of artists like Gilbert & George’s The Singing Sculpture, where movement is ritualized and common style of dress reduces the separatedness of the two artists’ identities.210 In seiner Werkbeschreibung der als Rebellion gegen das traditionelle Skulpturverständnis des zwischen 1953 und 1981 an der St. Martins School lehrenden Anthony Caro programmatischen Arbeit ruft Causey skulpturspezifische Parameter auf, wenn er den Tisch als (notwendiges) Podium bzw. Sockel deklariert, die mechanisch wiederholten Handlungen als Geste der Objekt-Werdung oder ihre Stasis als Mittel „to enforce the image that they are not people but sculptures, not private but public“ versteht.211 Gleichzeitig gehe das Skulpturale mit einer Einbettung des Körpers in soziale Räume und gesellschaftspolitische Kontexte einher. Allerdings nimmt Causey keine werkspezifische skulpturtheoretische Perspektive ein. Zwar integriert er ein Kapitel „Natural Materials“, doch präsentiert er, wie so häufig in den wiederkehrenden Narrativen der Bildhauereigeschichte, keine Non-Human Living Sculptures, sondern Land Art- und Earth Art-Künstlerinnen, etwa den eher selten genannten Briten Andy Goldsworthy. Trotzdem begreift Causey, wie er an Morris’ Continuous Project Altered Daily (1969) (Abb. 26) belegt, die Erweiterung von Skulptur und deren Verzeit lichung durchaus in diesem Sinne: 207 Causey 1998, S. 99f. Benjamin Buchlohs Schriften zur Skulptur werden im nachfolgenden Kapitel zu Hans Haacke rezipiert. 208 Ebd., S. 133. Causey erwähnt die Arbeit Manzonis mit der Signatur lebender Körper, verwendet aber den Skulpturenbegriff hierfür nicht. Auch Manzonis Magic Base nennt er ohne den expliziten Kontext der Living Sculpture: „[…] plinths for non-existant statues bear the idea of the monumental, and of the heroic status of the artist.“ (Ebd., S. 91.) 209 Siehe zuletzt die Ausstellung Judson Dance Theater. The Work Is Never Done (MoMA New York 2019), https://www.moma.org/calendar/exhibitions/3927 (17.5.2023). 210 Causey 1998, S. 156. 211 Ebd., S. 162. Causey vergleicht Gilbert & George mit Marilyn Monroe, von der wir eigentlich nur Bilder kennen; sie seien ihr eigenes Simulakrum. „Their work comments on the accessibility of reality and the real individual in a world saturated by the media with secondary imagery.“ 69 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 26 Robert Morris, Continuous Project Altered Daily, 1969, Erde, Ton, Baumwolle, Wasser, Fett, Kunststoff, Filz, Holz, Fadenabfall, Licht, Fotografien, Tonbandgerät, Leo Castelli Warehouse, New York At the extreme, late 1960s sculptures could be made from earth and sand, growing plants, live birds and animals, fabric, classical fragments, architectural structures, neon tubes, light beams, and the human body itself. There were hard and soft components; materials like sand, liquids, and air that need containment to give them form; living materials, which relate a sculpture to the time-scales of the organic world […]. The consensus that sculpture was generally made from a single material collapsed.212 Die Nennung von lebenden ‚Vögeln‘ als plastischem Material mag auf Jannis Kounellis’ Senza Titolo (1967) zurückgehen, das Causey wie später auch John Thompson im Kontext der Arte Povera mit einer Farbabbildung in sein Buch aufnimmt und das neben einer Ansammlung von Kakteen einen Papagei enthält.213 Zwei Jahre später erschien Richard J. Williams’ After Modern Sculpture. Art in the United States and Europe 1965–70. Williams grenzt den Untersuchungszeitraum auf fünf Jahre ein mit der Begründung: „This book is about the end of modern sculpture.“214 Seine Geschichte 212 Ebd., S. 131, Herv.i.Orig. Veränderung habe es seit den 1960er-Jahren auch im skulpturalen Vokabular gegeben, nun existieren Environment, Happening, Performance und Installation, „groups of objects in real space“ (ebd., S. 9). „In so far that a performance or happening can be regarded as sculpture, sculpture might now have a short life and, therefore, exist in the same time-frame as the audience.“ 213 Ebd., S. 152. 214 Richard J. Williams, After Modern Sculpture. Art in the United States and Europe 1965–70, Manchester [u.a.]: Manchester University Press 2000, S. 1. 70 Zur skulpturalen Ästhetik des Lebendigen 27 Robert Morris, Untitled (Dirt), 1968, Erde, Fett, Torfmoos, Ziegel, Stahl, Kupfer, Aluminium, Messing, Zink, Filz nachmoderner bzw. postmodernistischer Skulptur beginnt mit einer Gegenüberstellung von US-amerikanischer Post-Minimal Art und italienischer Arte Povera. Dass er den bestehenden künstlerischen Kanon fortschreibt, zeigt sich an seiner Lektüre etablierter Autorinnen aus dem Umkreis der Zeitschriften October und Artforum.215 Ähnlich wie bei Causey fehlen Burnhams Beyond Modern Sculpture (1968) und seine systemästhetischen Schriften zur Skulptur. Williams’ Moderneverständnis beinhaltet die Skulptur vor der Minimal Art von kanonisierten Künstlern wie Caro und Smith und die Minimal Art selbst, vertreten durch Judd und den frühen Morris. Williams’ Fokus liegt auf der zeitgenössischen Kunstkritik, insbesondere einer Rezeptions-, Institutions-, Diskurs- und Ausstellungsgeschichte der (vorwiegend) in den USA zwischen 1965 und 1970 produzierten und präsentierten skulpturalen Praktiken ausgehend von Maurice Tuchmans kontrovers debattierter Schau Sculpture of the Sixties (1967 LACMA Los Angeles) bis zur Amsterdamer Ausstellung Op Losse Schroeven: Situaties en cryptostructures (1969). Lebendes Material in Form von Ko-Akteuren taucht bei Williams nur peripher und unkommentiert auf, so in seiner Erörterung der Earthworks-Ausstellung bei Virginia Dawn 1968 am Beispiel von Morris’ Dirt (Abb. 27), das der Künstler selbst als „amorphous sculpture“ kategorisierte: Ein Erdhaufen auf Plastikfolie mit abfallartigen Residuen und Moos sowie eine Pflanze, ungeplant aus dem Erdboden sprießend. In dieselbe Kategorie fällt Oldenburgs Worm Earth Piece, dessen kriechende Protagonisten in ihrem Plexiglaskasten nicht überlebten.216 Ein übergreifendes Narrativ der Skulpturgeschichte seit den 1960er-Jahren betrifft die Abkehr vom singulären, konturierten, monolithischen Objekt, die parallel mit Environment, Performance, Happening und Installation, aber auch in der „negated presence of sculpture“ bzw. einem „undoing of sculpture as plastic shape“ etwa bei Eva Hesse stattfindet.217 Dass die gattungsspezifischen Grenzen fließend sind und Zuordnungen variieren, zeigt sich bei 215 „I have not, for example, discovered new artists who were somehow omitted from earlier versions of the story.“ (Ebd., S. 2.) 216 Ebd., S. 128. Williams weist zu Recht darauf hin, dass bei Morris’ Dirt Piece Plastikfolie unter dem Haufen hervorlugt, die Trennung zwischen Kunst- und Betrachterinnensphäre dadurch zusätzlich betont wird. Anders verhält sich dies bei Haacke, der Bowery Seeds (1970) direkt auf seinem Atelierdach installierte. 217 Potts 2000, Kapitel 9, S. 311–356; Einleitung S. 8. 71 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt Williams, wenn er Allan Kaprows Yard (1961) in seine Sammlung aufnimmt, ohne die skulpturalen Qualitäten dieser Arbeit und des Happenings zu reflektieren. Zu den Themen Verzeitlichung, Ephemeralisierung von Skulptur und die Involvierung der Betrachterinnen liegt inzwischen Forschungsliteratur vor.218 Alex Potts rekurriert in The Sculptural Imagination auf diese Phänomene, indem er unterschiedliche Formen des „staging“ und „modes of viewing“ problematisiert – Skulptur, die begehbar ist oder sich verändert, wie Continuous Project Altered Daily zeigt, dessen Materialzusammenstellung und Gestalt Morris täglich veränderte, sowie Skulptur, die sich erst über ihre physische Faktizität („akwardness“) in der Interaktion mit den Rezipientinnen konstituiert.219 Potts’ epochenübergreifende, phänomenologische Herangehensweise, die die jeweilige Ausstellungsgeschichte mit ihren Displays integriert, ist für die vorliegende Untersuchung von Interesse, da er skulpturale/plastische Probleme verhandelt, die in der zunehmend unabhängig von architektonischen Rahmen, Park- oder Kirchenensembles agierenden Bildhauerei des späten 18. Jahrhunderts (Canova) und in der Moderne (Rodin, Brancusi) sowie in der (Post-) Minimal Art (Andre, Morris, Hesse) virulent sind. Zudem benennt Potts skulpturtheoretische 218 Krauss 1981. Mittlerweile ist die Verbindung von Skulptur und Zeit ein etabliertes Thema in der Forschungsliteratur, siehe zuletzt u.a. Paul-Louis Rinuy, La sculpture contemporaine, Saint-Denis: Presses Universitaires de Vincennes 2016, Kapitel 3 „Espace, temps, mouvement“, S. 66–88. Siehe auch Guido Reuter/Ursula Ströbele (Hg.), Skulptur und Zeit im 20. und 21. Jahrhundert, Köln: Böhlau 2017; Martina Dobbe, „Das Ephemere und das Skulpturale“, in: Petra Maria Meyer (Hg.), Ephemer, Paderborn: Wilhelm Fink 2020, S. 225–250. 219 Potts 2000, S. 4. Zwar konstatiert Alex Potts die Bedeutung von Künstlerinnen, doch integriert er in sein Buch nur wenige Beispiele, die bereits dem Kanon angehören, wie Louise Bourgeois und Eva Hesse: „Nowadays, not only are many if not most of the more significant artists working in three dimensions female, but the general character of ambitious sculpture no longer has the masculine resonances it retained as late as the 1960s when making a serious piece of sculpture was still generally considered man’s work.“ (Ebd., S. XIf.) Siehe auch die konzisen Rezensionen von Stefan Neuner, in: Kritische Berichte, 30, 3, 2002, S. 86–91 und Pamela M. Lee, „Rezension von Alex Potts, The sculptural imagination: figurative, modernist, minimalist. – New Haven: Yale University Press 2001“, in: The Art Bulletin, 84, 2002, S. 392–396. Ausgehend von Lippard und Donald Judd verfolgt Jo Applin mit ihrer Methodik die Analyse skulpturaler Objekte, die nicht der vorherrschenden minimalistischen Ästhetik der 1960er-Jahre entsprachen. Sowohl Lippard als auch Judd interessierten sich für ähnliche Künstlerinnen, die den modernistischen Grundsätzen in der Bildhauerei zuwiderliefen. In Eccentric Objects: Rethinking Sculpture in 1960s America (2012) erläutert Jo Applin „this new and eccentric mode of thinking about sculpture“, ihren ‘unsicheren Zustand‘ und ihre Destabilisierung am Beispiel des „in-between status“ von Lee Bontecous Wandreliefs, Claes Oldenburgs Soft Sculptures, die sich auf die Pop Art beziehen und eine körper liche Identifikation anstreben, Lucas Samaras‘ „materialised secrecy“ seiner Boxen, H. C. Westermanns Bricolage in seinen autobiografischen Skulpturen und Bruce Naumanns Verweise auf die Werke von Kollegen wie Henry Moore und Constantin Brancusi. Wie Applin zeigt, sind ihre Werke von Ambivalenz und Brüchen geprägt. Dabei verweist sie auf die Beziehungen und Allianzen zwischen den Künstlerinnen, die in den 1960er-Jahren die klassische Objektästhetik in Frage stellten. In ihrer Analyse des Spezifischen, Exzentrischen, Erotischen, Körperlichen und Feierlichen problematisiert Applin darüber hinaus die einschränkende Fokussierung der Kunstliteratur auf einige wenige, kanonisierte Namen, indem sie einen Blick auf marginalisierte Positionen wirft und die Linearität des modernistischen Diskurses verweigert. (Jo Applin, Eccentric Objects: Rethinking Sculpture in 1960s America, New Haven, Conn.: Yale Univ. Press 2012, S. 5–7, S. 139–140, S. 11) 72 Die (Human) Living Sculpture Schriften, die den Beginn einer skulpturalen Ästhetik im 18. Jahrhundert untermauern.220 Seine gattungsspezifische historische Rückbindung an Canova, an die skulpturtheoretischen Abhandlungen wie Herders Traktat Plastik, Hildebrands Problem der Form in der Bildenden Kunst und an Rilkes Texte zu Rodin resultieren aus seiner Forschung zum Neoklassizismus und Winckelmann.221 Potts’ gewinnbringende Lektüre der Schriften Merleau-Pontys aus skulpturtheoretischer Sicht und die Miteinbeziehung von Künstlerinnentexten eröffnen Perspektiven auf physische, sensuelle und affektive Dimensionen in der Begegnung mit Skulptur und deren Fokus auf die leibliche, kinästhetische Wahrnehmung, folglich keinen „disembodied gaze“. Doch bleibt auch Potts einer Medienspezifik von Skulptur im Vergleich zur Malerei sowie modernistischen Narrativen (Schwerpunkt Minimal Art), ihrer Rezeption inklusive Formalismusdebatte und dem October-Kreis verbunden, ohne die außerhalb der USA, zum Beispiel in Deutschland erschienene Forschung umfassend zu berücksichtigen.222 Ähnlich wie Krauss schätzt er die Schriften zur Skulptur von Burnham als reinen „techno-utopianism“ mit einer „intringuely eccentric analysis“ ein.223 Seine Gegenüberstellung von Herbert Read und Jack Burnham fällt kurz aus, ihre Gemeinsamkeit liege in ihrer „fascination with sculpture as concretised fantasy“.224 Auch Potts konstatiert mit Arte Povera, Minimalismus und Neo-Dada eine Vorherrschaft dreidimensionaler Kunst seit den 1960er-Jahren. Die Skizzierung ausgewählter Positionen der Skulpturhistoriographie hat gezeigt, dass eine skulpturale Ästhetik des Lebendigen in diesen Abhandlungen nur peripher berücksichtigt wurde und keine umfassende Reflexion erfuhr, eine einheitliche Terminologie und umfassende ontologische Analyse fehlen. Erste künstlerische Ansätze finden sich etwa in der Nutzung organischer Materialien und der Konzeption kinetischer Objekte. Die (Human) Living Sculpture Eine Ästhetik des Lebendigen beschreibt den Umgang mit Menschen, Pflanzen und Tieren als lebendes ‚Material‘ bzw. als Ko-Akteure von künstlerischen Arbeiten. Der folgende Abschnitt, dessen Titel der gleichnamigen Publikation von Cord Riechelmann und Brigitte Oetker (2015) entlehnt ist, erörtert materialhistoriographisch, -ikonographisch und typo 220 Potts würdigt Krauss’ Ansatz und betont in diesem Zusammenhang seine eigene historische Rückführung bis Canova (Potts 2000, S. 12). 221 Vgl. Alex Potts, Flesh and the Ideal. Winckelmann and the Origins of Art History, New Haven: Yale University Press 1994. 222 Potts 2000, S. 3f. Mehrfach betont er die räumliche, kinästhetische und intellektuelle Involvierung der Betrachtenden durch Skulptur. 223 Ebd., S. 147. 224 Ebd. „In making this unlikely juxtaposition between Read’s lumbering plastic forms and Burnham’s sci-fi automata, I was also intrigued by the possibility of evoking two very different variants of modernist sculptural nightmare, with Moore’s wombless, faceless megalithis mothers stirring into life and confronting the relentless drive of some terminator cyborgs.“ (Ebd., S. 148.) 73 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt logisch Geschichte und Vielfalt lebender Skulpturen.225 Historischer Vorläufer für die Integration lebender organischer Entitäten ist die Gartenkunst, damit verbunden die Grotten- und Brunnenplastik sowie Bosquette und Labyrinthe, wie sie aus dem Park von Versailles bekannt sind und in Renaissance und Barock wesentliche Ausstattungsensembles höfischer Machtdemonstration darstellen.226 In multisensorischen Inszenierungen ergeben Pflanzen, Tiere, Wasser, Muscheln, Felsgestein, Marmor- und Bronzefiguren meist mythologischer Herkunft eine skulpturale Situation, deren evozierte Metamorphose durch die transformierende Kraft des bewegten Wassers und die Eigenzeitlichkeit des Naturrahmens hervorgehoben wird. Dynamik und Veränderung des lebenden Stoffes sind Bestandteile des jeweiligen Konzepts. Anders als im 16. bis 18. Jahrhundert geht es im 20. Jahrhundert um die Ausweitung des Kunstbegriffs, die von den Surrealisten in polysensuellen, raumumfassenden Ausstellungen angedacht und materialiter umgesetzt wurde, etwa mit der Internationalen Surrealistenausstellung 1938 in der Pariser Galerie Beaux-Arts.227 Eine Inkorporation lebenden organischen ‚Materials‘ als Objet Trouvé bzw. Naturobjekt erfolgte im Taxi Pluvieux (Regentaxi) (Abb. 28) von Salvador Dalí, das mit Flechten und anderen Pflanzen innen ausgekleidet und mit zwei verfremdeten Schaufensterpuppen, künstlichem Regen und kriechenden Weinberg schnecken ausstaffiert war.228 Eine weitere Inszenierung bestand aus e inem kleinen Teich 225 Cord Riechelmann/Brigitte Oetker (Hg.), Toward an aesthetics of living beings. Zu einer Ästhetik des Lebendigen, Berlin: Sternberg Press 2015. Bei dem Buch handelt es sich um eine Textanthologie mit Abbildungen zu einzelnen künstlerischen Beispielen, etwa Pierre Huyghe. Was genau eine Ästhetik des Lebendigen charakterisiert, wird eher zwischen den Zeilen deutlich; thematisiert werden u.a. das Verhältnis Tier – Mensch, die Darstellung von Tieren in der Kunst, die Problematik taxonomischer Zuordnungen, Fragen der Evolution, Wachstumsprozesse. In der Ästhetik des Lebendigen, wie es auch in der vorliegenden Untersuchung verstanden wird, geht es um die Art des Aufeinandertreffens und den Rhythmus, der die Wechselbeziehung zwischen den Lebewesen und ihrer Umwelt kennzeichnet. 226 Siehe hierzu die Forschung von Jürgen Wiener, u.a. „Natur als Skulpturenrahmen, Skulptur als Naturrahmen, Rahmen als Naturskulptur“, in: Hans Körner/Karl Möseneder (Hg.), Rahmenphänomene in der Gartenplastik und das Labyrinth von Versailles, Berlin: Reimer 2010, S. 31–68; ders., „Die Tränen der Verliebten. Wasser und andere mimetische Materialien in der frühneuzeitlichen Gartenskulptur“, in: Andrea von Hülsen-Esch (Hg.), Ephemere Materialien, Düsseldorf: Düsseldorf University Press 2015, S. 79–144; ders., „In der Natur versteckt: Dissimulative Strategien in der frühneuzeitlichen Gartenskulptur“, in: Astrid Lang/Wiebke Windorf (Hg.), Blickränder. Grenzen, Schwellen und ästhetische Randphänomene in den Künsten. Liber amicorum für Hans Körner, Berlin: Lukas 2017, S. 417–451. Vgl. auch Stephanie Hank, „‚Ars‘ und ‚natura‘ in Genueser Grotten des 16. bis 20. Jahrhunderts. Beobachtungen zu Materialität und Raumstruktur“, in: Uta Hassler (Hg.), Felsengärten, Gartengrotten, Kunstberge. Motive der Natur in Architektur und Garten, München: Hirmer 2014, S. 222–243; dies., „Water in baroque garden“, in: John D. Lyons (Hg.), The Oxford Handbook of the Baroque, Oxford: Oxford University Press 2019, S. 88–118. 227 Auch Schwitters beherbergte in seinem Merzbau Meerschweinchen, wie überlieferte Schilderungen und ein Foto preisgeben. Alles konnte bei ihm zur Kunst werden, auch plante er von Mäusen bewohnte konstruierte Merzbilder. Vgl. Sabine Bartelsheim, Pflanzenkunstwerke. Lebende Pflanzen in der Kunst des 20. Jahrhunderts, München: Silke Schreiber 2001, S. 22–24. 228 Ebd., S. 25f. Eine Beschreibung des Kunstwerks in eigenwilliger Rechtschreibung von Dalí selbst findet sich bei Georges Hugnet, Pleins & Déliés. Souvenirs et Témoignages 1926–1972, La Chapelle-sur- Loîre: Guy Authier 1972, S. 339: „Commisariat General de l’imagination publique. Le ‚Taxi pluvieux‘ pour dame esnob et surréaliste comportera: de ‚l’obscurité bégétale‘, instalation de plui intériere, 74 Die (Human) Living Sculpture 28 Salvador Dali, Taxi Pluvieux, 1938, Exposition Internationale du Surréalisme, Galerie Beaux-Arts, Paris und Wasserlilien (Abb. 29), umgeben von Schilf, Moos, Farn und Rosen, neben Betten, einer Schale mit Kohleglut und an der Wand hängenden Bildern, der Boden ausgelegt mit getrocknetem Laub, das unter den Füßen der Besucherinnen raschelte und die Schritte dämpfte – intendiert als Verschmelzung von Kunst und Leben.229 Während die Pflanzen eine surrealistische Traumwelt evozieren, Natur als dunkle, dämonische, mythisch-unbewusste Instanz fungiert, nimmt seit Mitte der 1960er-Jahre mit der wachsenden Bedeutung ‚lebender Kunstwerke‘ und dem Rückgriff auf ‚Naturmaterialien‘ der ökologische Impetus zu.230 Wie die Analysen zu Hans Haacke und Pierre Huyghe zeigen, können auch Pflanzen 200 escargots de bourgogne vibants, 12 grenoilles lilputicienes, portan chacune delles une très fine courone d’or agripé sur la tête. Le chofeur portera un casque construit avec une machoire de roquin. La d’Ame s’habillera de préférence avec une cretone sordide ou sera imprimé l’estigmate de l’Angélus de Millet et de ces sansationnelles glaneuses. Bon pour toute l’Ane 1938. – Salvador Dali.“ Das finale Kunstwerk enthielt allerdings keine Frösche. 229 Eine Abbildung und Schilderung findet sich etwa in Bishop 2005, S. 20–22. Sie schreibt den ‚Teich‘ Dalí zu. 230 Siehe auch Bartelsheim 2001, S. 28f., zu Robert Rauschenbergs Growing Painting (1953) und Dirt Painting (for John Cage) mit Erde, Flechten und Schimmelpilzen. Während der Ausstellung habe der Künstler sein Werk regelmäßig bewässert. Bartelsheim bezeichnet es als „living painting“. 75 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 29 Rauminstallation mit Teich, 1938, Exposition Internationale du Surréalisme, Galerie Beaux-Arts, Paris oder Tiere ein ‚präformiertes Fundstück‘, d.h. ein „(un)assisted biological ready-made“ sein.231 „Originalität beruhte nicht mehr auf einer spezifischen Bearbeitung des Materials, sondern auf der Herstellung des Beziehungsgefüges“, konstatiert Sabine Bartelsheim in Pflanzenkunstwerke (2001).232 Auch wenn Bartelsheim nicht dezidiert auf das Skulpturale und die Living Sculpture rekurriert, versammelt ihr Buch eine gewinnbringende Zusammenstellung pflanzenbasierter künstlerischer Arbeiten im 20. Jahrhundert. Passend zur wachsenden Ökologiebewegung seit den 1960er-Jahren offeriert Haackes Rheinwasseraufbereitungsanlage (Abb. 30) ein skulpturales Lösungsmodell, das die Entgrenzung der Kunst (Skulptur) zugunsten einer Ästhetik des Lebendigen demonstriert. Das Interesse an Verzeitlichung, Prozessualität und Wachstumsprozessen mündet in Formen der Bio Art oder Transgenen Kunst, indem Veränderungen auf genetischer Ebene erfolgen oder aber in gemeinsamen Baumpflanzaktionen bei Antje Majewski. Urban-Gardening-Strategien, Indoor-Gardening bzw. hydroponische Strukturen („Utility sculptures“) leben nach einer ‚Blüte‘ in den 1970er-Jahren (Helen 231 Vgl. Edward Fry, „Hans Haacke – Realzeitsysteme“, in: ders. (Hg.), Hans Haacke. Werkmonographie, Köln: Dumont 1972, S. 8–22, S. 14–16. 232 Ebd., S. 10. Bartelsheim verhandelt diese Werke nicht aus der Perspektive des Skulpturalen, versammelt aber in ihrer – wie sie es versteht – Grundlagenforschung eine Vielzahl unterschiedlicher Pflanzenkunstwerke und bietet einen historischen Überblick. Haacke nennt sie auch mit Grass Grows und Bowery Seeds. 76 Die (Human) Living Sculpture 30 Hans Haacke, Rheinwasseraufbereitungsanlage, 1972, Glas- und Acrylbehälter, Pumpe, Rheinwasser, Schläuche, Filter, Chemikalien und Goldfische, Installationsansicht, Museum Haus Lange, Krefeld Mayer Harrison and Newton Harrison, Portable Orchard, 1972–1973) aktuell wieder auf, so in Nick Laessings Plant Orbiter (2017) (Abb. 31). Die lebenden Kunstwerke variieren durch den Einsatz unterschiedlicher Lebewesen und sozialgesellschaftlicher, ökologischer Hintergründe, die in einem systemästhetischen Geflecht wie bei Haacke oder Huyghe zusammen auftreten, weshalb eine taxonomische Unterscheidung nach Spezies entfällt. Das breite Spektrum spiegelt sich in der terminologischen, historisch, konzeptuell, geographisch und ideologisch zu differenzierenden Vielfalt wider: Pflanzenkunst firmiert unter Plant Art und Vegetal Art, Kunstwerke mit Tieren werden als Animal Art bezeichnet und im Rahmen der Animal Studies wissenschaftlich untersucht. Weitere gebräuchliche Begriffe für lebende Kunstwerke sind u.a. Environmental Art, Eco Art, Organic Art, Life Art, Live Art, Natur-Kunst, Living Painting bzw. Living Sculpture.233 Auch Land 233 Siehe u.a. Barbara Nemitz (Hg.), trans plant. Living vegetation in contemporary art, Ostfildern-Ruit: Hatje Cantz 2000. Die Künstlerin hat das Projekt KünstlerGärten Weimar initiiert (https://www.barbaranemitz. de/static/kuenstlergaerten/index.html, 5.10.2019); Prudence Gibson, The Plant Contract. Art’s Return to Vegetal Life, Leiden & Boston: Brill Rodopi 2018; Wodek Majewski, De l’animal et du végétal dans l’art belge contemporain, Ausst.-Kat. Atelier 340, Brüssel 1985; Jessica Ullrich (Hg.), Tiere erzählen (Tierstudien, 15), Berlin: Neofelis 2019; Anna Barcz (Hg.), Animals and their people connecting East and West in cultural animal studies, Leiden & Boston: Brill 2019; Linda Kalof (Hg.), The Oxford handbook of animal studies, Oxford: Oxford University Press 2017; Reingard Spannring et al. (Hg.), Disziplinierte 77 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 31 Nick Laessing, Plant Orbiter, 2017, 133 x 82 x 185 cm Art oder Earth Art greifen auf lebende Organismen zurück und modellieren die Landschaft.234 Aus den vorherrschenden Narrativen und der im ersten Teil des Kapitels skizzierten modernistischen Erzählung von Skulptur lassen sich entwicklungsgeschichtlich – so die These – für eine Ästhetik des Lebendigen drei Hauptstränge herauskristallisieren: T iere? Perspektiven der Human-Animal Studies für die wissenschaftlichen Disziplinen, Bielefeld: transcript 2015; Linda Weintraub, To Life! Eco art in pursuit of a sustainable planet, B erkeley [u.a.]: University of California Press 2012; Viktorija Vesna Bulajić, „Mel Chin. Provocative eco-art in action“, in: Art Journal, 65, 1, 2006, S. 63f.; Jeffrey Kastner, Land and environmental art, London: Phaidon 1998; Marc A. Cheetham, Landscape into eco art: articulations of nature since the ’60s, Pennsylvania: The Pennsylvania State University Press 2018; T. J. Demos, Decolonizing Nature. Contemporary Art and the Politics of Ecology, Berlin: Sternberg Press 2016; Timothy Morton, Ecology Without Nature: Rethinking Environmental Aesthetics, Havard: Harvard University Press 2007; James Nisbet, Ecologies, environments, and energy systems in art of the 1960s and 1970s, Cambridge, Mass.: MIT Press 2014; Malcolm Miles, Eco-Aesthetics (Radical Aesthetics – Radical Art), London & New York: Bloomsbury 2014; Philippe Descola, Jenseits von Natur und Kultur, Frankfurt am Main: Suhrkamp 2011; ders., Die Ökologie der Anderen, Berlin: Matthes & Seitz 2014; Louis Bec, „Life Art“, in: Kac 2007, S. 83–92; Cindy Nemser, „The Alchemist and the Phenomenologist“, in: Art in America, 59, 1971, S. 100–103. 234 Siehe u.a. Burcu Dogramaci, Fotografie der Performance. Live Art im Zeitalter ihrer Reproduzierbarkeit, Paderborn: Wilhelm Fink 2018; Anne Hoormann, „Von der Landschaft zum öffentlichen Raum. Natur- Kunst in der Stadt“, in: Falko Herlemann/Michael Kade (Hg.), Kunst in der Öffentlichkeit. Ästhetisierung, Historisierung, Medialisierung, Frankfurt am Main [u.a.]: Lang 1996, S. 199–219; dies., Land Art. 78 Die (Human) Living Sculpture 32 Senga Nengudi, Studio performance with R.S.V.P., 1976 1. Materialhistoriographische Ebene: Die Verwendung von Naturmaterial wie Holz, Felsgestein und Erde, amorphe Figurationen u.a. im Biomorphismus Hans Arps, Louise Stomps oder Maria Papa Rostkowskas, Taxidermie, organische Fundstücke im Umfeld des Surrealismus oder bei Jean Dubuffet, der an Fantasiewesen erinnernde Naturspolien wie Schwämme einsetzt. Die Abkehr vom Topos der durata, von Härte und Dauerhaftigkeit des Materials hin zu nachgebenden, elastischen Arbeiten, etwa Claes Oldenburgs Soft Sculptures oder Magdalena Abakanowiczs Textilarbeiten bzw. Mária Bartuszovás Objekte aus Gips, Holz und Garn. 2. Technologische Ebene: Faktische Bewegung und Zeitlichkeit über technische Apparate und eine kinetische, teils mechanische Plastik bzw. Mobiles von Jean Tinguely, Alexander Calder, Alicia Penalba und Louise Bourgeois bzw. modularisierte, form- und gestaltbare, Kunstprojekte zwischen Landschaft und öffentlichem Raum, Berlin: Reimer 1996; Sandra Shapshay/ Levi Tenen (Hg.), The Good, the Beautiful, the Green: Environmentalism and Aesthetics (Themenheft). The journal of aesthetics and art criticism, 76, 4, 2018; Philip Kaiser/Miwon Kwon (Hg.), Ends of the Earth. Land Art to 1974, Ausst.-Kat. The Museum of Contemporary Art, Los Angeles, The Geffen Contemporary, MOCA, Haus der Kunst München, München: Prestel 2012; Udo Weilacher, Zwischen Landschaftsarchitektur und Land Art, Basel [u.a.]: Birkhäuser 1996; Amanda Boetzkes, The Ethics of Earth Art, Minneapolis, MN [u.a.]: University of Minnesota Press 2010; John Beardsly, Earthworks and beyond. Contemporary art in the landscape, New York: Abbeville Press 1984; Barbara C. Matilsky, Fragile ecologies. Contemporary artists’ interpretations and solutions, Ausst.-Kat. The Queens Museum, New York: Rizzoli 1992. 79 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 33 Rosemary Mayer, Shekinah, 1973, Stoffe, Kupfer, Holz, Schnur, 213,4 × 518,2 × 365,8 cm Rezipientinnen aktivierende Skulpturen, so William Turnbulls Permutation Sculpture (1956) oder Wolf Vostells ‚Erlebnis-Plastiken‘. 3. Diskursanalytische Ebene: Die Expansion des Skulpturbegriffs seit den 1960er-Jahren mit einer Prozess- oder Systemästhetik, die fest konturierte Gefüge durch zunehmende Ephemerisierung auflöste, etwa Robert Morris’ Begriff Anti-Form, Fujiko Nakayas Nebel skulpturen der 1960er-Jahre, performative Kunstpraktiken (Performing the making) sowie die Verbindung skulpturaler Praktiken mit Performance, Tanz und Happening, etwa Senga Nengudis Objekte aus Strumpfhosen der 1970er-Jahre, die sie in tänzerischen Choreografien formt (Abb. 32), dehnt und zieht, oder Rosemary Mayers Textilskulpturen wie Hroswitha (1973) und Shekinah (1973) (Abb. 33). Die Entwicklung vollzieht sich von der Skulptur in der Landschaft wie Henry Moores Sheep Piece (1971/72) – Skulptur mit darunter stehenden Schafen – bis zur Verwendung von unbearbeiteten Felsbrocken, die Morris 1977 in der Kasseler Karlsaue für die Documenta 6 platzierte (Abb. 34), zugunsten eines relationalen Charakters von Skulptur, wie er für Haacke und Huyghe signifikant ist. Es ging nicht mehr um eine mimetisch Leben und Bewegung nachahmende Figur, sondern um eine per se lebendige Skulptur mit prozessualer Plastizität. Mit der Integration diverser Lebewesen hielt das ‚Echte‘, ‚Unverfälschte‘, Unmittelbare, Natürliche, ‚Wahrhaftige‘ erneut Einzug in Praxis und Theorie des Skulpturalen und knüpft an das traditionelle Bildhauereiparadigma seit dem Paragone und Herders Umkehrung der Sinneshierarchie zugunsten von Haptizität an. Seine Betonung der Faktizität und Dreidimensionalität räumt der (in Anlehnung an Winckelmann anthropomorphen) Skulptur einen Vorrang ein, da „das Gesicht uns nur Gestalten, das Gefühl allein, Körper zeige.“235 235 Herder 1969, S. 34, Herv.i.Orig. 80 Die (Human) Living Sculpture 34 Robert Morris, Untitled, 1977, Feldsteine, 140 x 110 x 2 m, Documenta 6, Kassel „Der Körper, den das Auge sieht, ist nur Fläche; die Fläche, die die Hand tastet, ist Körper.“236 Dieses historische Ideal eines durch die modellierende Kraft der Bildhauerinnen beseelten Bildwerks bzw. Bildsäule ist für die lebendige Skulptur relevant, da hier nun ein faktisch atmender Körper eines (menschlichen) Wesens zum Kunstwerk erklärt wird. Die Menschen, Tiere oder Pflanzen – organische Materialien – machen per se noch nicht das Werk aus. Ihre Bedeutung ergibt sich aus dem jeweiligen Kontext, der Differenz von Natur und Kunst im Werk beziehungsweise aus der Verbindung beider Ebenen. Als natürliche Readymades wie bei Hans Haacke werden die Naturmaterialien zu selbstreferenziellen Zeichen und Versatzstücken einer gesellschaftspolitischen Realität. Sie sind Natur und verweisen auf ein Naturphänomen oder Umweltproblem. Materialikonographische und -ästhetische Fragestellungen sind für die vorliegende Untersuchung relevant, da sie für die skulpturale Problematisierung von Plastizität und Räumlichkeit Gültigkeit besitzen. Die Rückbindung an die Materialität, wie sie in der Forschung u.a. von Ann-Sophie Lehmann, Dietmar Rübel und Monika Wagner erörtert wurde, ermöglicht eine historische Kontextualisierung und ästhetische Erfahrungen in der Auseinandersetzung mit immersiven, die Betrachterinnen involvierenden, sich prozessual entfaltenden Formen.237 Sie macht eine künstlerische Entscheidung 236 Ebd., S. 37. 237 Vgl. Monika Wagner, „Gras, Steine, Erde. Naturspolien in der zeitgenössischen Kunst“, in: Museumskunde, 61, 1, 1996, S. 26–36; dies., Das Material der Kunst. Eine andere Geschichte der Moderne (2001), München: Beck 2013; Rübel 2012; Ann-Sophie Lehmann, „Taking fingerprints. The indexical affordances of artworks’ material surfaces“, in: Magdalena Bushardt/Henrike Haug (Hg.), Spur der Arbeit, Köln: Böhlau 2018, S. 199–218; dies., „The matter of the medium. Some tools for an art-theoretical interpretation of materials“, in: Christy Anderson/Anne Dunlop/Pamela H. Smith (Hg.), The matter of art. Materials, practices, cultural logics, c. 1250–1750, Manchester: Manchester University Press 2015, S. 21–41. 81 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt sinnfällig. Doch verlangen lebende ‚Materialien‘ eine Berücksichtigung ihrer agency, wie sie im New Materialism von Karen Barad, Rosi Braidotti oder auch Susanne Witzgall und Kerstin Stakemeier thematisiert wird,238 jedoch ohne ein Formverständnis, das sich kontinuierlich (nur) positiv weiterschreibt, wie es Catherine Malabou mit ihrem Plastizitätskonzept unterstreicht.239 Neuer Materialismus und Agentieller Realismus gestehen dem Material per se eine gewisse agency, das heißt Wirkmächtigkeit zu. Karen Barads aus der Physik stammende Erweiterung des Materialbegriffs hin zu Transmaterialitäten umfasst beispielsweise die experimentelle agency und Bewegung von Materie, „promiscuous and inventive in its agential wanderings“ mit „capacities for imaginative, desiring, and affectively charged forms of bodily engagements“.240 Übertragen auf die Bildhauerei erlaubt dieses Materialverständnis einer „ongoing transformation“ ein fruchtbares Weiterdenken von Materie als Kondensation einer „(entangled) respons-ability“: „Materiality in its entangled psychic and physical manifestations is always already a patchwork, a suturing of disparate parts.“241 Barads Konzept der Intra-Aktion eröffnet somit neue erkenntnistheoretische Perspektiven, indem Materielles – hier Bronze, Stein, Werkzeuge – und Diskurse verflochten, bedeutungsgenerierend und nicht als a priori existente, voneinander getrennte Entitäten aufgefasst werden. Das plastische Material ist ohne die Künstlerinnenhand, die historischen und soziopolitischen Voraussetzungen seiner Entstehung und Produktion, das korrelierende Werkzeug und die jeweilige Umgebung inklusive Rezipientinnen nicht zu denken. Barad betont, dass Apparate und Werkzeuge, hier unter anderem Meißel und Gussofen, keine „passiven Beobachtungsinstrumente“ seien. Im Gegenteil, so Barad, bringen sie die Phänomene hervor und seien Teil derselben.242 In diesem Sinn erlaubt das feministisch beziehungsweise posthumanistisch erweiterte Materialverständnis eine andere Sichtweise auf das Medium Skulptur. 238 Siehe etwa Karen Barad, Agentieller Realismus. Über die Bedeutung materiell-diskursiver Praktiken, Berlin: Suhrkamp 2012; Rosi Braidotti, Nomadic Subjects. Embodiment and sexual difference in contemporary feminist theory, New York: Columbia University Press 1994; Susanne Witzgall/Kerstin Stakemeier (Hg.), Macht des Materials, Politik der Materialität, Zürich: Diaphanes 2014; Friedrich Weltzien, „‚Material Agency‘ und die Lebendigkeit der Dinge“, in: ders./Martin Scholz (Hg.), Die Sprachen des Materials. Narrative – Theorien – Strategien, Berlin: Reimer 2016, S. 229–242. Weltzien verhandelt die Konsequenzen einer ‚material agency‘ für Designpraktiken und Entwurfsstrategien und fragt nach den Herausforderungen, d.h. wie sei die agency eines Materials produktiv zu machen, wenn es nicht instru mentalistisch genutzt wird? Wie lasse sich die Widerständigkeit des Materials zeigen? „Gäbe es das Produkt, das Artefakt, das gemachte Werk nicht – wir wären gar nicht im Stande, Material als solches zu erkennen. Daraus lässt sich der Schluss ziehen, dass nicht das Ding aus einem Material hergestellt wird, sondern dass umgekehrt Design das Material des Dinges erst kenntlich macht.“ (Ebd., S. 232.) 239 Catherine Malabou, Ontologie des Akzidentiellen. Über die zerstörerische Plastizität des Gehirns, hg. von Frank Ruda/Jan Völker, Berlin: Merve 2011; dies., „Plastizität des Leibes. Eine zweifache Annäherung“, in: Kirsten Maar/Frank Ruda/Jan Völker (Hg.), Generische Formen. Dynamische Konstellationen zwischen den Künsten, Paderborn: Wilhelm Fink 2017, S. 211–224. 240 Karen Barad, „TransMaterialities. Trans/Matter/Realities and Queer Political Imaginings“, in: GLQ, 21, 2–3, 2015, S. 387–422, S. 387f. 241 Ebd., S. 411. 242 Dies., Agentieller Realismus. Über die Bedeutung materiell-diskursiver Praktiken, Berlin: Suhrkamp, 2012, S. 24. 82 Die (Human) Living Sculpture Im Fokus der vorliegenden Untersuchung steht die Analyse der Materialität (und Media lität) lebender Skulpturen und wie sich der ontologische Charakter solch organischer, teils computergestützter Werke definiert. Dies schließt die Frage ein, wie sich das Verhältnis zwischen Faktizität als dem tatsächlich Gegebenen und Faktualität im Sinne einer Aktualisierung des Faktischen im Rezeptionsprozess und auf produktionsästhetischer Seite beschreiben lässt. Diskursanalytisch befragt wird Burnhams Zeitlichkeitskonzept von ‚Realzeit‘, insbesondere sein Bezug zur ‚Presentness‘ der Minimal Art, zu computerbasierten Echtzeitprozessen und zur Produktions- bzw. Rezeptionszeit von Skulptur.243 Günter Bandmann betont in seinen Überlegungen zu einer Ikonologie des Materials am Beispiel frühneuzeitlicher und barocker Ausstattungs- und Sakralensembles deren sinnliche Qualitäten, semantische Zuschreibungen und stilbildende Eigenschaften und fragt, welche Bedeutung das Material eines Werks besitzt.244 Die Auffassung von Material in seiner F unktion als Informationsträger und zugleich Medium, das seit Niklas Luhmanns soziologischer System theorie und dessen Verständnis von Kommunikation als Trias Information – Mitteilung – Verstehen seine Neutralität verloren hat, ist für die Analyse skulpturaler Situationen und systemästhetischer Konzepte eine gewinnbringende Theorie.245 Auch wenn es bei tierlichen und pflanzlichen Ko-Akteuren schwerfällt, von Material oder Stoff zu sprechen, spiegelt es einerseits die Tatsache, dass sie Veränderungen bzw. einer ‚Bearbeitung‘ unterliegen, damit historisch an die Tradition der Bildhauerei anknüpfen, andererseits die Machtrelation zwischen Künstlerin und Material, explizit die Frage nach der Materialgerechtigkeit. Ausgehend von einer idealistisch geprägten Ästhetik und dem Primat der idea mit ihrer Materialsublimierung bzw. -negierung rückt das Paradigma der Materialgerechtigkeit seit dem 19. Jahrhundert im Zuge der Industrialisierung des Kunsthandwerks in den Fokus der Bildenden Künste; Material werden stilbildende Qualitäten zugesprochen: Das Material trägt aufgrund seiner spezifischen natürlichen oder auch zugeschriebenen Qualitäten, manchmal aber auch nur durch Unterscheidung vom benachbarten Material etwas zur Bedeutung des Bildes bei. Insofern ist Material ikonologisch aussagefähig, es kann Informationsträger sein.246 243 Fried 1995. Vgl. auch Martina Dobbe, „Presentness, presence, present continuous past(s) und Gegenwartsspitzen: Zeitformen von ‚Skulptur im erweiterten Feld‘“, in: Reuter/Ströbele 2017, S. 65–83. 244 Günter Bandmann, „Bemerkungen zu einer Ikonologie des Materials“, in: Städel-Jahrbuch, N.F. 2, 1969, S. 75–100. „Solange man das Wesen der Kunst in der Idee, im Disegno und Concetto begründet sah, bezeichnete das verwendete Material nur das Medium, dessen die nach Anschaulichkeit drängende Idee bedurfte.“ (Ebd., S. 75.) Zu seinen Beispielen gehören die Aachener Pfalzkapelle, barocke Sakralräume, u.a. die Klosterkirche Birnau am Bodensee. Das Material signalisiere eine bestimmte Wertigkeit und besitze eine Realitätsbereiche verwandelnde Kraft (ebd., S. 82). 245 Siehe u.a. Niklas Luhmann, Soziale Systeme. Grundriß einer allgemeinen Theorie, Frankfurt am Main: Suhrkamp 1984; ders., Die Kunst der Gesellschaft, Frankfurt am Main: Suhrkamp 1995. Für einen system theoretischen Ansatz siehe auch Hans Dieter Huber, System und Wirkung: Rauschenberg, Twombly, Baruchello. Fragen der Interpretation und Bedeutung zeitgenössischer Kunst; ein systemtheoretischer Ansatz, München: Fink 1989. 246 Bandmann 1969, S. 77. Siehe auch ders., „Der Wandel der Materialbewertung in der Kunsttheorie des 19. Jahrhunderts“, in: Helmut Koopmann/J. Adolf Schmoll, gen. Eisenwerth (Hg.), Beiträge zur Theorie 83 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt Bandmann verweist auf die semantische Nähe zwischen „Gerechtigkeit“ und „Angemessenheit“.247 Perspektivwechsel erfolgten, so Bandmann und Nadine Rottau, durch John Ruskin („truth to material“), die Arts & Craft-Bewegung und Gottfried Sempers materialistisch- positivistische Überlegungen.248 In den Materialien selbst sei Geschichte gespeichert, so Ruskin, der für eine traditionelle Materialverarbeitung plädierte und die Imitation mit Ersatzmaterialien ablehnte.249 Hofmann betont, dass am Ende des 19. Jahrhunderts die Forderung nach Materialgerechtigkeit für die Skulptur erhoben worden sei, als handwerkliche Kenntnisse sich „in oberflächlicher Virtuosität verloren“ hatten.250 Sein traditionelles Künstlerbild zeigt sich daran, dass er Rodin dafür kritisiert, seine Bronzeplastiken von Handwerkern in Marmor übertragen zu lassen und mechanische Vergrößerungsverfahren zu nutzen.251 Henry Moore ist der Bildhauer des 20. Jahrhunderts, der zum Thema Materialgerechtigkeit wiederholt zitiert wurde, u.a. von Burnham und Krauss. In seiner Abhandlung über den Vitalismus kommt Burnham auf die Beziehung zwischen Natur, Form und Material bei Moore zu sprechen: „Truth to material. Every material has its own individual qualities.“252 Entscheidend sei, dass die Künstlerinnen in „an active relationship“ mit dem Material arbeiten, denn nur so könne dieses an der Ideenfindung („shaping of an idea“) beteiligt sein.253 Es geht um Härte, Nachgiebigkeit, Dehnbarkeit und Oberflächentexturen. In der Natur gebe es, so Moore, eine Vielfalt an Formen und Rhythmen, deren Reichtum, erweitert durch Erfindungen wie Mikroskop und Teleskop (das ist für Burnham wichtig), zu nutzen und zu erhalten sei. Burnham selbst, der Moore nach Herbert Read zitiert, steht dem Konzept Materialgerechtigkeit skeptisch gegenüber und betont dessen Ambivalenz: „Any forming or shaping must take advantage of the plasticity of each material, and, more importantly no material will do what it is not meant to do. […] The attraction is […] its ring of moral equilibrium and natural propriety.“254 Krauss bezeichnet dies als „alert responsiveness“ und schränkt die Bedeutung des Materials berechtigterweise mit dem Hinweis ein, dass die Idee nicht von den Künstlerinnen zu trennen sei.255 Zugleich stellt sich die für lebende Skulpturen essenzielle ethische Frage nach Art-‚Gerechtigkeit‘ und Autorschaft: „after all, who is to be the master?“256 Für die Skulptur der Künste im 19. Jahrhundert, Bd. 1, Frankfurt am Main: Klostermann 1971, S. 129–157. Siehe auch Raff 1994, insbesondere S. 18–32, sowie die Textsammlung in: Dietmar Rübel/Monika Wagner/Vera Wolff (Hg.), Materialästhetik. Quellentexte zu Kunst, Design und Architektur, Berlin: Reimer 2005. 247 Bandmann 1971, S. 138f., 148f., 153. 248 Nadine Rottau, Materialgerechtigkeit. Ästhetik im 19. Jahrhundert, Aachen: Shaker 2012. So lobte Semper in Der Stil (1860) die „absolute Gefügigkeit des Materials“ bei Kautschuk (ebd., S. 15); zu Ruskin siehe ebd., S. 17–32. 249 Ebd. 250 Hofmann 1958, S. 19–23, S. 21. 251 Ebd. 252 Burnham 1968, S. 95f., Herv.i.Orig. 253 Ebd. 254 Ebd., S. 96. 255 Krauss 1981, S. 143f. Sie zitiert denselben Passus von Moore. 256 Burnham 1968, S. 96. Siehe auch ebd., S. 155: „‚Truth to material‘, more than being an honest and analytical approach to the use of sculpture materials, was an overreaction to earlier excesses“ – auch wenn gerade bei computergestützten Werken diese Frage nach wie vor entscheidend ist. 84 Die (Human) Living Sculpture des 21. Jahrhunderts sind die wachsende Bedeutung des 3D-Drucks mit seinem Simulations potenzial, Virtual Reality und damit verbundene Auslagerungen des Produktions- und Rezeptionsprozesses auf die Maschine sowie die Materialgerechtigkeit digitaler Skulpturen zu untersuchen. Während bei Morris Materialgerechtigkeit in seinen Felt Pieces und Stacks, bei Senga Negudi in ihren Skulpturen aus gedehnten Strumpfhosen (inklusive Materialermüdung) erprobt wird (Swing Low, 1976/2014), schließt dies bei künstlerischen Arbeiten mit lebendem Material, die eine eigene agency prägen, neben ethischen auch moralische und saisonalrhythmisch bzw. organisch-lebenszyklisch bedingte Fragen mit ein.257 Greifen Künstlerinnen wie Dieter Roth, Michael Badura (Eingeweckte Welt, 1964) und H. A. Schult mit seiner Biokinetik (1969) oder Tissue Culture Art Project mit ihren Semi-Living Sculptures (2001) auf Mikroorganismen, Bakterien, Zell- und Pilzkulturen zurück, handelt es sich eher um amorphe Gebilde einer bewegten, wachsenden, sich verändernden Biomasse mit eigenzeitlicher Dynamik denn um individuelle Gegenüber, wie sie bei Haacke oder Huyghe anzutreffen sind oder bei Sommerer & Mignonneaus Interactive Plant Growing (1992) (Abb. 35), wenn Pflanzen aktiv werden, als Interface die elektrische Spannung der Userinnen aufnehmen und durch die Interaktion auf einer Projektionsfläche virtuelle Pendants entstehen.258 Dass die Living Sculpture in der Fachliteratur nur einen marginalen Stellenwert besitzt und skulpturtheoretisch bisher nicht hinreichend untersucht wurde, stellt ein Desiderat dar.259 257 Monika Wagner führt den Begriff ‚Materialgerechtigkeit‘ auf Goethes Plädoyer (1778) für die gequälten Steine des Mailänder Doms und seiner Forderung nach einer Einfühlung in das Material zurück: Monika Wagner, „‚Materialgerechtigkeit‘. Debatten um Werkstoffe in der Architektur des 19. und frühen 20. Jahrhunderts“, in: Jürgen Pursche (Hg.), Historische Architekturoberflächen. Kalk – Putz – Farbe/ Historical Architectural Surfaces. Lime – Plaster – Colour (ICOMOS, Hefte des Deutschen National komitees, XXXIX), München 2003, S. 135–138, S. 135; dies., „Vom Ende der materialgerechten Form. Kunst im Plastikzeitalter“, in: Barbara Naumann/Thomas Strässle/Caroline Torra-Mattenklott (Hg.), Stoffe. Zur Geschichte der Materialität in Künsten und Wissenschaften, Zürich: vdf Hochschulverlag AG an der ETH 2006, S. 229–246. Siehe auch Wolfgang Kemp, „Material der bildenden Kunst. Zu einem ungelösten Problem in der Kunstwissenschaft“, in: Prisma 9, 1975, S. 25–34. Siehe auch Monika Wagner, „Material“, in: Karl-Heinz Barck et al. (Hg.), Ästhetische Grundbegriffe, Bd. 3, Stuttgart und Weimar: Metzler 2001, S. 866–882. Wagner geht hier nicht auf Moore und die Bildhauerei ein. 258 Zu Bio Art in der Gegenwartskunst und anderen künstlerischen Formen von non-human agency siehe u.a. die Ausstellungs- und Rechercheinstitution Art Laboratory Berlin, https://artlaboratory-berlin.org/ de/ (24.2.2023). 259 Siehe Rowell 1986. Auch hier gibt es kein eigenes Kapitel zur Living Sculpture; Kounellis’ unbetitelte Arbeit mit Kakteen wird genannt; als Beispiel für eine maximale Erweiterung der Skulptur dient etwa Lawrence Weiner mit seinen konzeptuellen schriftbasierten Arbeiten. Seit 2014 gibt Claire Maingon bei Presses Universitaires de Rouen et du Havre jährlich die Zeitschrift Sculptures. Études sur la sculpture (XIXe–XXIe siècle) heraus. Während die erste Ausgabe mit Sculpture et Performance betitelt ist und einen Text von Erik Verhagen zu Franz Erhard Walther enthält, bringt die vierte Ausgabe La sculpture et le vivant (2017) nur in einem Text ein Beispiel des Lebendigen, namentlich Valie Export („La sculpture vivante comme expression carnavalesque chez VALIE EXPORT“ von Juliette Bertron), wohingegen die anderen Beiträge sich dem Lebendigen eher in einer motivischen Dimension bzw. im Kontext der Mimesis oder skulpturalen Phänomenen an der Schnittstelle zur Pantomime und Kinetik widmen. In seiner knappen Einführung skizziert Thierry Dufrêne ausgehend vom Pygmalion-Topos die Singing Sculpture des Künstlerduos Gilbert & George, nennt kurz ikonische 85 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 35 Sommerer & Mignonneau, Interactive Plant Growing, 1992, 5 Sockel & Interfaces, Pflanzentöpfe mit lebenden Pflanzen, Computer, Software, Spots, Projektor, Pflanzenlampe Ausgewählte Beispiele sogenannter Human Living Sculpture und Non-Human Living Sculpture werden im Folgenden erörtert, um das Spektrum dieses skulpturalen Phänomens zu beleuchten, bevor einzelne umfassende Werkanalysen sich anschließen.260 I dream of a day when I shall create sculptures that breathe, perspire, cough, laugh, yawn, smirk, wink, pant, dance, walk, crawl, … and move among people as shadows move among people. …261 In seinem 1965 veröffentlichten MMMMMMM….Manifesto beschwört der philippinische Künstler David Medalla den Traum der Verlebendigung einer Statue, wie sie durch Pygmalion aus der antiken Mythologie bekannt ist. Dieser verliebte sich in das von ihm geschaffene anmutige Standbild aus Elfenbein: Galathea, die durch Venus’ Gnade zu atmen begann und von ihrem Sockel stieg.262 Auch wenn in Medallas Manifest dieser Topos noch anklingt, wirkt die Vorstellung seiner Living Sculpture mit ihren alltäglichen Körperreaktionen und Gefühlsregungen weniger romantisch. Die Faszination des Menschen, sein Ebenbild zu erschaffen, gipfelt in künstlicher Intelligenz, automatisierten Apparaten oder Robotern wie in der Erzählung Der Sandmann von E.T.A. Hoffmann (1816) oder auch Science-Fiction Filmen wie Ex Machina (2015). Beispiele des Lebendigen wie Anselmo, Beuys, Haacke, Kac, Kounellis, Kowalski und Penone, ohne dabei Begriffsgeschichte und mögliche Formen der Transformation einer skulpturalen Ästhetik des Lebendigen zu problematisieren. Auch verweist er nicht auf Burnham trotz seiner Bezugnahme auf die Kinetik, Automaten und Cyborg Art (ebd., S. 3–6). 260 Die Übersicht beansprucht keine Vollständigkeit. 261 David Medalla, „MMMMMMM….Manifesto“ (1965), in: Guy Brett (Hg.), Exploding Galaxies. The Art of David Medalla, London: Kala Press 1995. Zuerst veröffentlicht in Signals Newsbulletin, 1, 8, 1965. 262 Vgl. Ovid, Metamorphosen, übers. und hg. von Erich Rösch/Niklas Holzberg, München: Deutscher Taschenbuchverlag 1999, X. Buch, 243–294. 86 Die (Human) Living Sculpture Was ist eine lebende Skulptur? Durch welche Merkmale zeichnet sich diese „Metamorphose“ aus? Inwiefern greifen die Künstlerinnen auf Räumlichkeit, Unbeweglichkeit und Plastizität zurück, wo brechen bzw. erweitern sie sie? Franz Erhard Walther gehört mit seinem Versuch, eine Skulptur zu sein (1958) (Abb. 24) zu den frühen Beispielen einer Kunstgeschichte des ‚lebenden Artefakts‘.263 Mit 19 Jahren ließ er sich in einer Serie absurd-humorvoll anmutender Handlungen fotografieren. Speier zeigt den jungen Künstler im Schneidersitz auf dem Boden seines Fuldaer Ateliers, die Hände in den Schoß gelegt, mit Backpulver und Milch vermischtes Wasser im hohen Bogen ausspeiend. Vor ihm steht eine Blechschüssel. Walthers Blick ist nach oben gerichtet, die Augen sind weit geöffnet. Er trägt unauffällige Arbeitskleidung; den Hintergrund bildet eine Art weiße, gerahmte Projektionsleinwand, auf der sich durch den starken Schlagschatten die Silhouette vergrößert abzeichnet. Die von Walther um den temporalen und partizipatorischen Aspekt erweiterte Definition von Skulptur erlangt vor dem Hintergrund klassischer (Bildhauerei-)Diskurse besondere Signifikanz. In einem Interview äußerte er sein Bestreben, die ursprünglich statuarische Gattung Skulptur unter die Herrschaft des Zeitlichen zu stellen. Ich benutze mit Absicht diesen traditionellen Begriff, weil das, was ich tue, wenig mit traditionellen Vorstellungen zu tun hat. […] die Vorstellungen, die in diesem Raum, den ich meine, ent stehen, können sich an diesem traditionellen Begriff ansammeln als Kristallisationspunkt. […] Was ich unter Zeit verstehe, das ist kein Abstraktum, es ist ein Stoff, mit dem ich forme. Ganz einfach, es kommen Zeitdehnungen vor, […] Zeitzusammenziehungen [, …] Zeitsegmentierungen […].264 Der Künstler selbst avanciert zu einer verlebendigten Statue. Vor der Kamera vollzieht sich die Objektwerdung des Körpers. Bezogen auf traditionelles Bildhauereirepertoire lassen die zwischen Pose und Aktion oszillierenden skulpturalen Inszenierungen Walthers an steinerne Personifikationen denken, die mit spezifischen Attributen versehen Denkmäler flankieren, Fassaden dekorieren und Raumensembles bevölkern. Ist in diesem Kontext (Abb. 36) die Wagenleuchte ein Zeichen der lichtbringenden Veritas, der Stierschädel Walthers gar ein Vanitas-Symbol (Abb. 37) und die Schüssel eine entfernte (humorvolle) Reminiszenz an das Füllhorn der Abundantia?265 263 Vgl. Ursula Ströbele, „Mensch Tier Pflanze. Human and Non-human Living Sculptures“, in: Stefan Vicedom (Hg.), Andreas Greiner. Anatomy of a Fairy Tale, Wien: Verlag für Moderne Kunst 2016, S. 80–108. 264 Franz Erhard Walther, „Der andere Werkbegriff“, in: Kunstforum International, 29, 5, 1978, S. 102– 104, S. 104. 265 Für weitere Ausführungen zu Walthers Serie und dem Begriff der Pose sowie Craig Owens Fototheorie vgl. Ursula Ströbele, „Performing the making – die Eigenzeit der ‚lebenden Skulptur‘ zwischen Dauer und Augenblick“, in: Reuter/Ströbele 2017, S. 143–160. „Ich dachte darüber nach, ob man einer Skulptur auch Tempo geben könnte. Zeitlichkeit. Etwas Fließendes. Holz oder Stein kamen nicht infrage. Auch die Skulpturenhandlungen nachzuzeichnen, das war es nicht. Also bat ich einen Bekannten, mich in meinem Atelierraum zu fotografieren.“ (Franz Erhard Walther im Interview mit Kolja Reichert, „Skulpturen ohne Ende“, in: FRIEZE.de, September– November 2014, https://koljareichert.de/artikel/skulpturen-ohne-ende/, 15.3.2023.) 87 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 36 Franz Erhard Walther, Versuch, eine S kulptur zu sein, 1958, Wagenlampe, Stierschädel, Blechschüssel, Wasser 37 Franz Erhard Walther, Versuch, eine Skulptur zu sein, 1958, Stierschädel, Stuhl 38 Josef Bauer, Ulli, Körperskulptur, 1972 88 Die (Human) Living Sculpture Erst Walthers Vorführungen des 1. Werksatzes 1969 finden als intersubjektive Begegnungen vor einer kunstaffinen Öffentlichkeit statt. Heute bleiben nur die das Ereignis dokumentierenden Fotografien, die einzelne Momente der Aktion festhalten, ähnlich wie beim österreichischen Konzeptkünstler Josef Bauer, der Anfang der 1970er-Jahre neben seiner „Taktilen Poesie“ auch Ulli, Körperskulptur (1972) (Abb. 38) realisierte. Wir erleben die Aktion mit einer zeitlichen Verzögerung, vergegenwärtigen sie uns, während wir das Foto betrachten. Die Handlung ist nur der Vorläufer, d.h. Vorgeschichte und Voraussetzung von der Bewegung zur Momentaufnahme. Dieses Innehalten unterstreicht den skulpturalen Aspekt der durata bei gleichzeitiger Transformation des Dreidimensionalen in die piktoriale Räumlichkeit des zweidimensionalen Bildes. Die Fotografie bannt die Momentaufnahme zum Stillstand, wodurch der temporale Fokus erneut reduziert wird. Sukzession wird Dauer; Haptik und Plastizität werden auf die Imagination verlagert, nur mit dem inneren Auge der Betrachterinnen erfahren. In ihrer frühen Serie Corps-Sculptures ließ sich auch ORLAN zwischen 1964 und 1967 als lebende Skulptur fotografieren. Die Arbeit wurde in der Forschung bislang wenig beachtet, ein weiteres Beispiel für die kritisch zu hinterfragenden Kriterien einer kunsthistorischen Kanonbildung.266 Die Schwarz-Weiß-Fotografien zeigen die 1947 in Frankreich als Mireille Suzanne Francette Port geborene Künstlerin als junge Frau, unbekleidet, das Gesicht auf einigen Bildern durch eine Maske verborgen. Auf drei Aufnahmen versucht sie, aus einem ovalen Bilderrahmen zu steigen. Sie trägt ihr langes braunes Haar offen, die Nägel lackiert, im Versuch, sich mit dem rechten Arm aus dem prunkvollen Rahmen zu lehnen, den Kopf nach rechts gewandt, mit wachem, neugierigen Blick, schräg nach oben sehend. Auf den anderen beiden Bildern variiert ihre Pose jeweils; die Beine hat sie aus dem einzwängenden Rahmen befreit bzw. sie streckt den Kopf heraus (Abb. 39). Die japanisch anmutende Maske ist männlich und verdeckt ihre Gesichtszüge, konterkariert dabei ihre weibliche Körperlichkeit. Kunsthistorische Referenzen finden sich u.a. in den Reliefs Gianlorenzo Berninis, etwa bei der Büste Gabriele Fonsecas (1668–1673, San Lorenzo in Lucina, Rom) oder den der Verzückung der Hl. Theresa (1647–1652, Cornaro-Kapelle, Santa Maria della Vittoria, Rom) beiwohnenden Gästen, auch wenn der Kontext ein gänzlich anderer ist. Der römische Barockbildhauer schuf diese buchstäblich rahmensprengenden Figuren als Zuschauerinnen an einem sakralen Ereignis bzw. als Teil eines Grabensembles. Doch auch hier sind die skulptierten Gestalten bemüht, die ästhetische Grenze zu überwinden, um sich der Betrachterinnensphäre zu nähern, eine gesteigerte Mimesis und emotionale Anteilnahme am Geschehen, das sich vor ihren Augen in der Kapelle abspielt.267 Auch ORLAN nutzt vergleichbar Walthers fotografischer Serie Versuch, eine Skulptur zu sein das Medium Fotografie, um den Körper in eine Skulptur zu transformieren. Beide 266 Erst in jüngster Zeit erlangte diese frühe Arbeit im Kontext der Skulptur größere Aufmerksamkeit, siehe u.a. Quentin Petit dit Duhal, „Devenir-sculpture. La métamorphose photographique de l’identité. Étude des Corps-sculptures d’ORLAN“, in: Sculptures, 7, 2020, S. 103–108. 267 Zur Ästhetischen Grenze siehe Ernst Michalski, Die Bedeutung der Ästhetischen Grenze für die Methode der Kunstgeschichte (1932), Berlin: Mann 1996. 89 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 39 ORLAN, CORPS-SCULPTURES, Tentative de sortir du cadre fond noir avec masque n. 2, 1965 „erstarren“ in Posen, die wiederum skulpturale Referenzen beinhalten – bei Walther Personifikationen und Bauplastik, bei ORLAN Bezüge zur italienischen Barockplastik. ORLAN entscheidet sich für einen dekontextualisierten Raum ohne sichtbare architektonische Gegebenheiten und greift auf klassische Displays wie Rahmen und Sockel zurück, aber auch auf Attribute wie Flagge, ihre Haare und Schlange. Walther situiert seine Aktion im Atelier vor einer Leinwand. Beide nutzen einen starken Licht-Schatten-Kontrast, der die Plastizität und damit Skulpturalität des Körpers unterstreicht, Theatralität evoziert. ORLAN thematisiert diesen Tanz mit ihrem Schatten in einer gleichnamigen Serie Corps-sculpture sans visage en mouvement dansant avec ombre (1965) (Abb. 40). Beide lassen sich alleine ohne Publikum im Atelier aufnehmen. Für die Rezeption des Werks steht die Fotografie. In den Bildern nimmt ORLAN rebellische, selbstbewusst erscheinende, männlich dominant wirkende oder unterwürfige und Verletzlichkeit ausstrahlende Haltungen ein. Überhaupt oszillieren diese zwischen der Exponierung des weiblichen Körpers als Lustobjekt, das zum Voyeurismus einlädt, und dessen Verhüllen bzw. Verbergen, indem sie maskiert mit ihren beiden Händen ihre Scham bedeckt (Abb. 41). Mal ist sie in sich zusammengekauert, mal greifen ihre Gliedmaßen weit in den Raum. Mal wirkt ihr Körper fast schmerzhaft verdreht, mal zeigt sie sich entspannt liegend. Damit konterkariert sie klassische Posen, wie sie für die künstlerische Ausbildung üblich waren. In einem Bild inszeniert sie eine fiktive Geburt, aus der ein Mannequin hervorgeht, dessen Kunstkörper als ihr Double fungiert. Seit Ende der 1970er-Jahre setzt ORLAN ihren eigenen Körper als skulpturales Material ein, indem sie ihn wiederholt chirurgischen Eingriffen unterzog und seine Gestalt modellierte (Carnal Art).268 Dabei greift sie auf ikonische Vorbilder und Figuren der Kunstgeschichte zurück, 268 Vgl. https://www.orlan.eu/bibliography/carnal-art/ (11.2.2023). 90 Die (Human) Living Sculpture 40 ORLAN, CORPS- SCULPTURES, ORLAN danse avec son ombre, CORPS-SCULPTURE dansant avec son ombre dit «en mouvement», 1965 41 ORLAN, CORPS-SCULPTURES, Masque à genoux mains sur sexe, 1965 insbesondere berühmte Frauengestalten. Sie besteht auf dem Recht zur Veränderung des eigenen Körpers, insofern die technologischen Veränderungen der Gesellschaft Möglichkeiten bereitstellen, stereotype weibliche Schönheitsideale zu hinterfragen. Damit greift sie gesellschaftliche Tabuthemen auf ähnlich wie dieder queere Performance-Künstlerin und Bodybilderin (Heather) Cassils, dieder den Körper als „Soziale Skulptur“ begreift und 91 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 42 Cassils, Cuts: A Traditional Sculpture: Time Lapse (Front), 2011, archivalischer Pigmentdruck, 152,4 x 101,06 cm 92 Die (Human) Living Sculpture inäre Geschlechterkonzepte durchkreuzt.269 Mit Cuts: A Traditional Sculpture (2011–2013) b (Abb. 42) rekurriert Cassils auf Eleanor Antins Carving: A Traditional Sculpture (1972). Die US-amerikanische Künstlerin unterzog sich zwischen dem 15. Juli und 21. August 1972 einer Diät und dokumentierte die physischen Veränderungen jeden Tag fotografisch von vorn, hinten, links und rechts: „Technically it is carried out in what can be considered the matter of archaic and classical Greek sculpture (peeling small layers off an overall body image until the image is gradually refined to the point of aesthetic satisfaction).“270 Serielle Fotografien greifen, ebenso wie bei Cassils, naturwissenschaftlich anmutende historische Körper- und Bewegungsanalysen auf.271 Konträr zu Antin unterzog Cassils den Körper über den Zeitraum von 23 Wochen einem regelmäßigen Krafttraining und Ernährungsprogramm, um Muskelmasse zu generieren. Cuts entstammt der Terminologie des Bodybuildings und bezieht sich auf die einander abwechselnden Phasen des Aufbaus und der Definition. Die daraus entstandene Fotoserie Time Lapse spielt visuell auf eine Geschlechtsumwandlung an, indem Cassils den ursprünglich weiblichen Körper in einen männlich konnotierten Körper transformiert; die Bildästhetik erinnert an Eadweard Muybridge und sexualmedizinische Aufnahmen zu Beginn des 20. Jahrhunderts.272 Auch bei Lorenza Böttner klingen Topoi der Mimesis und Queerness an. Von 1978 bis 1984 studierte die als Ernst Lorenz Böttner geborene deutsch-chilenische Künstlerin (1959– 1994) an der Gesamthochschule Kassel (heute Kunsthochschule Kassel) bei Harry Kramer, selbst Bildhauer und Tänzer. Dies war die Zeit, in der sie ihren Namen in Lorenza änderte und sich selbst als weiblich transgender definierte. Sie malte, skulptierte und zeichnete mit den Füßen und arbeitete als Straßenkünstlerin. Mit ihrer Performance Venus von Milo (1986) (Abb. 43) imitierte sie mit ihrem eigenen armlosen, mit einer feinen Gipsschicht umhüllten Körper den antiken Torso als Living Sculpture, befragte etablierte Idealisierungen weiblicher Schönheit und gesellschaftlich normierte Körpervorstellungen. Diese queerte sie zugleich, da sie hierfür ihren männlichen Körper einsetzte.273 Auf einem mobilen Podium stehend wurde Lorenza Böttner als Venus auf die Bühne geschoben, öffnete ihre Augen und fing zu sprechen an: „Was würdest du denken, wenn Kunst zum Leben erwacht?“, trat vom Podium herunter und tanzte vor dem Publikum. Warum, so wird explizit gefragt, definieren 269 Https://www.cassils.net/cassils-about-the-artist (12.2.2023). 270 Eleanor Antin zit. n. https://webarchive.henry-moore.org/hmi/exhibitions/eleanor-antin-carving-a-traditionalsculpture (13.2.2023). 271 Vgl. Getsy 2015, S. 269; Lena Nievers, „Körpertransformationen“, in: Julia Brennacher/dies./Jürgen Tabor (Hg.), Mapping the body. Der Körper in der heutigen Lebenswelt, Ausst.-Kat. Galerie im Taxis palais, Wien: Verlag für moderne Kunst 2016, S. 56–60, S. 56; https://www.cassils.net/cassils-artworkcuts (13.2.2023). 272 Nievers 2016, S. 56. 273 Siehe Paul B. Preciado, Lorenza Böttner. Requiem für die Norm/Requiem for the Norm, hg. vom Württembergischen Kunstverein Stuttgart 2019, https://www.wkv-stuttgart.de/uploads/media/Lorenza_ Boettner_Booklet_final_kl.pdf (12.12.2021). Die Performance wurde 1987 in der Alabama-Halle in München und 1988 beim Tonight Performance Festival in der Künstlerwerkstatt in der Lothringer straße, in München, aufgeführt (ebd.). 93 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 43 Lorenza Böttner, Ohne Titel, Venus von Milo, 1987, Videodokumentation einer Performance, Combinale, Alabama Halle München, Farbe, Ton 22:22‘ wir die armlose Skulptur der antiken Venus von Milo als Schönheitsideal, nicht aber einen solchen Körper wie den Lorenzas? Fungiert die Anbindung an die beauté idéale als Nobilitierung des eigenen, gesellschaftlichen Exklusionsmechanismen unterworfenen, scheinbar deformierten Trans-Körpers des Andersartigen? Erst in den letzten Jahren folgten mehrere Ausstellungen, darunter 2017 auf der Documenta, 2018–2019 in La Virreina, Centre de la Image in Barcelona, 2019 im Württembergischen Kunstverein in Stuttgart und anschließend im Museum der Universität von Toronto 2020, kuratiert von Paul B. Preciado, Transgenderaktivist und Philosoph. Fluide Genderkonzepte verkörpern Eva & Adele, die seit den 1990er-Jahren im jeweils neuen Zwillingslook auf Ausstellungseröffnungen und anderen Kunstveranstaltungen auftreten, weiblich geschminkt, kahl rasiert und identisch gekleidet. Meist tragen sie aufwändige Kostüme in Pink- oder Rosatönen, hochhackige Schuhe und Handtaschen, c harakterisieren 94 Die (Human) Living Sculpture 44 Eva & Adele, The Dress, 2015–2016 sich selbst als Hermaphroditen.274 Ihrem eigenen Motto folgend – „Wherever we are is the museum.“ – stellt sich das symbiotische Doppelwesen in einer Dauerperformance als quasi Living Sculpture selbst aus, ebenso Attribute wie ihr pinkfarbenes Campingmobil oder abgelegte Kleidung, die sie als „biographische Skulpturen“ bezeichnen (The Dress, 2015–2016) (Abb. 44). Selbstporträts dokumentieren ihre Auftritte (Polaroid-Diary, 1991–2005). Marcus Steinweg attestiert ihnen eine „complex sculptural presence“ und die Konstituierung ihrer „living sculpture“ in der Wiederholung.275 Ab 1959 – nach Walthers skulpturalen Posen – beginnt Ben Vautier, Menschen seines Umfelds als lebende Skulpturen zu deklarieren: Sculptures Vivantes. Er stellt atmende Körper aus, um seinem Ideal von ‚Wahrhaftigkeit‘, Authentizität und Lebensnähe zu entsprechen: „J’en vins donc en 1959 à exposer l’individu lui-même car il ne peut y avoir plus de ressemblance à un corps qu’un corps lui-même.“276 Anders als Piero Manzoni signiert Vautier die Körper nicht, sondern hält ihren Status vertraglich fest. So übertrug Gaston Gabriel Melidonian am 18. Juli 1961 dem Künstler „le droit de considérer mon propre corps comme 274 Vgl. Sebastian Preuss, „Eva & Adele: Radikal vereint“, in: Zeit Online, 27..4.2018, https://www.zeit. de/kultur/kunst/2018-04/eva-adele-kuenstlerpaar-ausstellungen-berlin-weltkunst (14.2.2023); You are my biggest inspiration. Eva & Adele, Ausst.-Kat. Musée d’Art Moderne de la Ville de Paris, München: Hirmer 2016; Nicole Gnesa (Hg.), Eva & Adele. Keep the rosy wing strong, Ausst.-Kat. Nicole Gnesa Galerie München, München: Hirmer 2021. 275 Marcus Steinweg, „Für Eva & Adele“, in: You are my biggest inspiration 2016, S. 112–121, S. 113, 121. 276 Ben Vautier zit. n. Barbara Roosen, Ben Vautier. Einführung in das Werk und Überlegungen zur autothematischen Reflexion des künstlerischen Selbstverständnisses (= Dissertation Universität Bonn 2007), Bonn 2007, S. 88, https://hdl.handle.net/20.500.11811/2764 (13.3.2023). 95 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 45 Timm Ulrichs, Erstes lebendes Kunstwerk (Selbstausstellung), 1961, Galerie Patio, Frankfurt am Main, Remake 1966 s culpture vivante, dans tous ses instants, et tous ses gestes“.277 Durch die Adelung zur Skulptur, vertragliche Vereinbarung und Kaufpreis adaptiert Vautier die Gepflogenheiten des Kunstbetriebs und den Warencharakter von Kunst – eine Implikation, die bei Walthers Versuch, eine Skulptur zu sein keine Rolle spielt. Vautiers Inbesitznahme und Zuschreibung als Teil seines Œuvres ist bei den männlichen Künstlern signifikant, wenn sie Menschen, Tiere und Pflanzen (teils auch humorvoll) als Ko-Akteure in ihr skulpturales Werk inkorporieren und damit eine Geste der Macht formulieren. Das aufkeimende Interesse an einer Exponierung des menschlichen Körpers und dessen Verrückung in den Kunstkontext zeigte sich 1961 in Erstes lebendes Kunstwerk (Selbstausstellung) von Timm Ulrichs (Abb. 45).278 Die Privatsphäre seines Wohnraums verschmilzt mit dem öffentlichen Raum, indem er sich selbst zum künstlerischen Exponat erklärt. Ähnlich wie bei Ulrichs, der sich seine Signatur auf den Oberarm tätowieren ließ, werden bei Ben Vautier und Piero Manzoni (Abb. 46) die sculture vivente durch Signatur und Echtheitszertifikat als Skulpturen deklariert: „Es wird bestätigt, dass ___ [Name wird handschriftlich eingetragen] durch meine Hand signiert wurde und daher ab sofort als echtes und wahres Kunstwerk anzusehen ist.“279 Diejenigen, die sich auf Manzonis Basi Magici – Sockel aus Sperrholz 277 Ebd., S. 90. 278 Siehe auch: Peter Greenaway, 100 Objects to represent the world/100 Objekte zeigen die Welt, Ausst.- Kat. Akademie der Bildenden Künste Wien, Stuttgart: Hatje 1992. In seiner Schau stellte Greenaway auch „lebende Objekte“ aus, beispielsweise ein Paar, das Adam und Eva repräsentierte und auf einem Sockel posierte. 279 Das Konzept wurde 1961 im Kontext der „Zimmergalerie Timm Ulrichs“ formuliert, doch erst 1966 in einer Galerie realisiert. Siehe u.a. Birgit Jooss, „Die Erstarrung des Körpers zum Tableau. Lebende Bilder 96 Die (Human) Living Sculpture 46 Piero Manzoni, Sculture Viventi, 13. Januar 1961 mit zwei Filzsohlen zur Positionierung der Füße – stellten, avancierten in einer konzeptuellen Weiterführung selbst zum plastischen Objekt, ohne der Unterschrift Manzonis zu bedürfen. Ortswechsel: Ende der 1960er-Jahre stellte sich der kalifornische Konzeptkünstler Robert Kinmont in einer Serie von 8 Natural Handstands auf seine Hände, um die Welt aus umgekehrter Perspektive an unterschiedlichen Orten in Augenschein zu nehmen. Dieser skulpturale Akt, wie er ihn nannte, wurde in quadratischen Schwarz-Weiß-Fotografien dokumentiert.280 Lucy R. Lippard bildet in Six Years: The dematerialization of the art object from 1966 to 1972 den ersten seiner Handstände ab, den Kinmont in einer dramatisch wirkenden Pose in schwindelnder Höhe auf einem Felsvorsprung vollführte (Abb. 47).281 Stillstand ist Illusion. Alles ist in Bewegung. Es gilt, die Balance zu halten, kleine Abweichungen sofort auszugleichen. Erst die Fotografie hält den Augenblick fest. Ähnlich wie bei Walther in Performances“, in: Christian Janecke (Hg.), Performance und Bild – Performance als Bild, Berlin: Philo & Philo Fine Arts 2004, S. 272–303, S. 276–278. Siehe Martin Engler (Hg.), Piero Manzoni. Als Körper Kunst wurden, Ausst.-Kat. Städel Museum Frankfurt am Main, Bielefeld: Kerber 2013, S. 204f. Abb. 202. Die Dokumente sind zweisprachig (englisch, französisch) und fortlaufend nummeriert. In der Originalversion heißt es: „On certifie que … a été signé(e) par ma main et pourtant est consideré(e) de la date ci-dessous œuvre d’art authentique et véritable.“ (Ebd., S. 205.) 280 Diese die acht Handlungen dokumentarisch erfassenden Fotografien begreift Kinmont „structurally meaningful as conceptual sculpture“ (Julie Ault, In Step with the Desert: The Morphology of Robert Kinmont, Alexander and Bonin Gallery New York 2009, n.p.). 281 Lucy R. Lippard, Six Years: The dematerialization of the art object from 1966 to 1972, Berkely [u.a.]: University of California Press 1997 (zuerst erschienen 1973), S. 70. Siehe auch Ursula Ströbele, „Attention! Skulpturale Formen des Stillstands in living sculpture und Fotografie“, in: Barbara Gronau (Hg.), Künste des Anhaltens, Berlin: neofelis 2019, S. 96–112. 97 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 47 Robert Kinmont, 8 Natural Handstands, 1969/2009, 9 Silbergelantineabzüge, je 21,5 x 21,5 cm f unktioniert diese Übertragung bei Robert Kinmont, wenn er seine Handstände in freier Landschaft als skulpturalen Akt versteht und sich im entscheidenden Augenblick der Stasis ablichten lässt. Auch Jannis Kounellis suggeriert mit seinen skulpturalen Posen und Apollo-Maske vor dem Gesicht, Fragmenten antiker Statuen und Flötenspieler (1973) eine wechselseitige Spannung zwischen der Statuarik traditioneller Statuen und einer verlebendigten Skulptur. Ein Beispiel für die Verbindung lebender Skulptur mit elektronischen Geräten und Musik ist Charlotte Moormans und Nam June Paiks TV-Bra for Living Sculpture (Abb. 48), u.a. in der Kölner Ausstellung Happening & Fluxus (1970).282 Die Arbeit besteht aus zwei kleinen TV-Bildröhren, die sich Moorman vor ihre Brust schnallt, um anschließend mit ihrem Cello zu performen. In einer Kooperation mit Otto Piene steigt Moorman als ‚fliegende‘ Skulptur mit ihrem Cello in den Himmel. Ungefähr zeitgleich posierten Gilbert & George (Abb. 12) singend, bemalt mit Metallic farben und ausgestattet mit Anzug, Hut, Handschuh und Stock auf Tischen. Während Walthers Versuch, eine Skulptur zu sein nur über das Foto rezipiert werden kann, wir die Handlung als Vorläufer und Voraussetzung für die Skulptur nacherleben, finden die Live- Auftritte des britischen Künstlerduos in der Realzeit statt. In seiner Theorie des Bildakts erläutert Horst Bredekamp: 282 Vgl. u.a. https://walkerart.org/collections/artworks/tv-bra-for-living-sculpture (21.8.2019). Siehe auch Rinuy 2016, S. 96f. 98 Die (Human) Living Sculpture 48 Charlotte Moorman performt Nam June Paiks TV-Bra for Living Sculpture (1969) auf dem Dach ihres Lofts, Pearl Street 62, New York, 30.7.1982 Sich selbst als living sculpture definierend, haben sie die Frage nach der Lebendigkeit des Werks durch deren permanente Bestätigung obsolet gemacht. […] Gilbert und George sind das Werk als lebendes Bild, und damit ahmen sie sich selbst in Echtzeit nach, so dass die Unterscheidung von Bild, Nachbild und Leben schwindet. Die Künstler präsentieren sich selbst als Bild und hierin sind sie Realsymbole des schematischen Bildakts.283 Anlässlich der von Jean-Christophe Ammann kuratierten Wanderausstellung Europe in the seventies. Aspects of recent art (1977–1979) verweist David Brown in seinem Katalogtext auf die Problematik einer Zuordnung zur Gattung Skulptur. Richard Longs und Hamish Fultons Entgrenzungen unterscheiden sich deutlich von Anthony Caros und William Tuckers Skulpturalität. Das Werk von Gilbert & George gilt als Skulptur, weil die Künstler selbst auf den Begriff rekurrieren: Gilbert and George’s work would not be sculpture at all, despite an insistence on their part – no less great than that of Tucker in respect of his own work – that in all their work they are employing this mode. Their single work of sculpture is themselves and their lives. Parts of this works are presented to the public in the form of events or tangible objects. Whatever the forum is, it is described as sculpture, including interview sculptures, singing sculptures, drinking sculptures, eating sculptures, book sculptures, drawing sculptures, painting sculptures, photo sculptures, postcard sculptures and video sculptures; all presented in an exemplary fashion with a nicely judged feeling for the rightness of form.284 283 Horst Bredekamp, Theorie des Bildakts, Berlin: Suhrkamp 2010, S. 119f., Herv.i.Orig. 284 Brown 1977, S. 19. Siehe auch Lippard 1997, S. XVI: „The emphasis on process also led to art-as-life, life-as-art pieces, like Lozano’s Piper’s, and Gilbert & George’s living sculptures, and especially Mierle Ladermann Ukeles’s ‚Maintenance Art‘ series, which began in 1969.“ 99 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt Beatrice von Bismarck versteht die Kategorisierung als Skulptur durch Gilbert & George folgerichtig als „strategische Setzung“, mit der sich das Künstlerduo neben Bruce McLean – Stephen Cripps wäre zu ergänzen – und John Latham im britischen Kunstfeld, explizit an der St. Martins School, gegen eine formalistische Tradition und Bildhauerheroen wie Moore oder Caro, Philipp King oder Tim Scott positionierte.285 Einerseits übten sie damit Kritik an der Medienspezifik, andererseits am traditionellen Begriff der Skulptur und standen für dessen programmatische Erweiterung ein, wobei meines Erachtens diese „Setzung“ nur über die Anknüpfung an die nobilitierte Gattung Skulptur und Einreihung in deren Tradition erfolgreich war. Durch die inflationäre Verwendung von ‚Skulptur‘ für Collagen, Postkarten, Gemälde etc. verweisen Gilbert & George auf die gattungsübergreifende bzw. materialnegierende Konzeptkunst ihrer Zeit. Dieser Angriff gegen die vermeintliche Virtuosität im Umgang mit Material und einen damit verbundenen Geniekult, der das Material zum Ausdrucksträger eines geistreichen Künstlerprotagonisten degradierte, sowie die Infragestellung überlieferter Materialwertigkeiten sind möglicherweise auch für Ulrichs und Manzoni ausschlaggebend. Die Auseinandersetzung mit Duchamps Readymade, die aufkommende Konzeptkunst, Performance Art und die Infragestellung der Greenberg’schen Medienspezifik sind für die Entwicklung der Living Sculpture prägend gewesen. Die Geschichte der Human Living Sculpture – eine mögliche Bezeichnung – bzw. der Body Art, wie sie oftmals beschrieben wird, weist einen großen Facettenreichtum auf. Bei einigen Positionen verschmelzen Künstlerinnenkörper und Werk, bei anderen ist es der Körper einer/s Akteurin oder zufällig anwesender Rezipientinnen, die in einer Art Metamorphose temporär zum skulpturalen ‚Artefakt‘ mutieren. Mit seinen One Minute Sculptures kreiert Erwin Wurm lebende Artefakte und orientiert sich am traditionellen Skulpturenvokabular.286 Den Ausstellungsbesucherinnen, die seinen Handlungsanweisungen folgen, bietet er weiße Podeste und Attribute zur Ausführung der jeweiligen „Zeitskulptur“ an.287 Kleine Zeichnungen und kurze Kommentare ergänzen die Anleitung. Die lebendige Statue macht die kunstvolle Meißeltechnik Pygmalions überflüssig. Valie Export persifliert mit ihrem Tapp- 285 Beatrice von Bismarck, „Zwischen Revoltieren und Legitimieren – Aufführungen des Bildes. Zur Singing Sculpture von Gilbert & George“, in: Janecke 2004, S. 247–271, S. 254. Siehe auch Rinuy 2016, S. 99 (Gilbert & George im Kapitel zu akustischer Skulptur, „départ d’une carrière de sculpteurs humaines“ [ebd., S. 100]). Eine Liste der Living Sculpture Presentations bis 1991 findet sich bei: Robert Violette/ Hans-Ulrich Obrist (Hg.), The Words of Gilbert & George. With Portraits of the Artists from 1968 to 1997, London: Thames & Hudson, Violette Editions 1997, S. 305f. Die Wirkung des Künstlerduos auf Rezipientinnen beschreibt Daniel Thomas: „Most visitors were previously unfamiliar with Gilbert & George and came perhaps to scoff; all left filled with admiration for the immaculate perfection of what was being done (small children mostly took if for granted that they were triumphs of technology, not real people) and for the fascinating beauty of it.“ (Daniel Thomas, „Gilbert & George“, in: ART and Australia, Oktober–Dezember 1973, S. 135.) 286 Vgl. u.a. Erwin Wurm, One Minute Sculptures, 1988–1998, Werkverzeichnis, hg. von Kunsthaus Bregenz, Fonds Régional d’Art Contemporain de Bourgogne Dijon, CAN Centre d’Art Neuchâtel, Ostfildern 1999. 287 Vgl. Ursula Ströbele, „Erwin Wurms One Minute Sculptures im Kontext der lebenden Plastik“, in: Erwin Wurm, Ausst.-Kat. Berlinische Galerie, Berlin: Prestel 2016, S. 7–11. 100 Die (Human) Living Sculpture 49 Valie Export, Körperkonfigurationen, 1982 50 Rebecca Horn, Messkasten, 1970, schwarzer Lack, Aluminium, Stahl, 200 x 90 x 90 cm und Tastkino die der Plastik eigene Haptik, Vanessa Beecroft mit ihren skulptural anmutenden Performances weiblicher Akteure, die stundenlang bis zur physischen Erschöpfung stillstehen, die der Skulptur eigene Stasis. Die Koreanerin Kim Sooja erstarrt in ihrer Serie A Needle Woman (1999–2001) zum eigenen Standbild inmitten turbulenten Großstadtgewühls. Stillstand im Sinne eines temporären „Denkmals“ als subversive Reaktion auf die Unruhe unserer Zeit? Doch bleiben diese Werke einer erweiterten Objektästhetik verhaftet – mit Ausnahme von Valie Export: Mit ihren Körperkonfigurationen (Abb. 49) scheint sie selbst zum plastischen Bauschmuck zu mutieren, indem sie sich an Balustraden und Treppenstufen schmiegt, Wandvorsprünge oder Nischen ausfüllt, diese mit ihrer femininen Figur nachzeichnet. Ihr weiblicher Körper unterwirft sich der machtausstrahlenden Architektur; das Organische, Weiche und Biegsame eines lebenden Menschen kontrastiert mit der Starrheit und Härte von Stein.288 Auch Rebecca Horn unterwirft ihren Körper diversen Experimenten, etwa in ihren ab 1968 entstandenen prothesenhaften Körperextensionen Arm-Extensionen 288 Siehe u.a. Mechthild Widrich, Performative Monuments. The rematerialization of public art, Manchester [u.a.]: Manchester University Press 2014, S. 73–80. Widrich erörtert Valie Exports körperliche Auseinandersetzung mit Architektur und öffentlichem Raum, verhandelt deren Körperkonfigurationen allerdings nicht aus einer skulpturtheoretischen Perspektive, vielmehr betrachte die Künstlerin Architektur wie eine Art zweite Haut. Siehe auch Juliette Bertron, „La sculpture vivante comme expression carnavalesque chez VALIE EXPORT“, in: Sculptures, 4, 2017, S. 39–43. Valie Export bezeichnet diese Werkgruppe 101 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt (1968), Einhorn (1970), Weisser Körperfächer (1972) und Handschuhfinger (1972).289 In Messkasten (1970) (Abb. 50) zwängt sie sich in ein mit Stangen durchsetztes rechteckiges Gehäuse, das wie ein Folterinstrument ihren Körper aufzuspießen scheint, dem Titel gemäß eine physisches Volumen erfassende Maßvorrichtung darstellt. Alexandra Pirici lässt ihre Performerinnen Monumente und Pressebilder nachstellen.290 Die rumänische Künstlerin und Choreografin ist dafür bekannt, dass sie historische Denk mäler im öffentlichen Raum kritisch kommentiert, dabei künstlerische Methoden wie ephemere skulpturale Ergänzungen einsetzt und so tableaux-vivants-ähnliche Körperbilder schafft, um Statuen kritisch zu reflektieren und temporär auf performative Weise neu zu codieren. Für Soft Power. Sculptural Additions to Petersburg Monuments, das im Rahmen der Manifesta 10 für zeitgenössische Kunst im Jahr 2014 in St. Petersburg präsentiert wurde, wählte Pirici die Statuen von Wladimir Lenin, Katharina der Großen und Peter dem Großen, um die Konstruktion von Geschichte, die Verherrlichung von Denkmälern und die damit verbundene offensichtliche Machtdemonstration zu hinterfragen.291 Den Bereich um die monumentale Statue von Peter dem Großen und deren Sockel (Abb. 51) okkupierten die Performerinnen, um in der Sonne zu liegen, zu lesen oder Musik zu hören, und verwandelten diesen Umraum so in einen allgemein zugänglichen Freizeit- und Erholungsbereich. Das inhärente, historisch determinierte Machtverhältnis kommt hier weniger in dessen Überwindung, vielmehr in der Umdeutung, d.h. in der Besetzung des Raums der Statue, einer gegen wirkenden Soft Power zum Ausdruck. Das Denkmal Wladimir Lenins mit seiner massiven Sockelstruktur wurde in einem choreografierten Tableau vivant imitiert (Abb. 52), wodurch sich der Kontrast zwischen den Proportionen der menschlichen Körper und der überlebensgroßen Figur, zwischen Fleisch und Bronze zusätzlich steigerte. Pirici übersetzte die figurierte Ohnmacht des Individuums in eine selbstermächtigende Geste. Ist diese Form der temporären Neukodierung als ephemerer ikonoklastischer Akt zu betrachten?292 Anstatt Statuen selbst als lebende Skulptur (siehe u.a. VALIE EXPORT, Ausst.-Kat. Centre National de la Photographie Paris, Montreuil: Éditions de l’Oeil 2003, S. 157). 289 Siehe u.a. Sandra Beate Reimann (Hg.), Rebecca Horn. Body Fantasies, Ausst.-Kat. Museum Tinguely Basel, Wien: Verlag für moderne Kunst 2019; Bettina Busse (Hg.), Rebecca Horn. Concert for anarchy, Ausst.-Kat. Bank Austria Kunstforum Wien, Berlin: Hatje Cantz 2021. 290 Siehe u.a. Alexandra Pirici, Leaking Territories (2017), in: König/Peters/Wagner 2017, S. 244–248. 291 Http://manifesta10.org/en/artists/alexandra-pirici/ (12.1.2023). Siehe auch Mélanie Boucher, „Soft Power ou Les corps-monuments d’Alexandra Pirici“, in: Espace, 112, 2016, S. 26–35; Ursula Ströbele, „Toppling things: Artistic Approaches and Media Strategies in Dealing with Monuments – Alexandra Pirici, Morehshin Allahyari, Julius von Bismarck & Julian Charrière“, in: Nausikaä El-Mecky, Tomas Macsotay (Hg.), Toppling Things. The Visuality, Space and Affect of Monument Removal, Series Thamyris-Intersecting: Place, Sex, and Race, Leiden: Brill (= erscheint 2024). 292 Wie Gerhard Paul belegt, ist das Anbringen von temporären Kommentaren an öffentlichen Statuen eine gängige Praxis. Als historisches Beispiel führt er die DDR an, wo Lenin-Statuen nach dem Fall der Berliner Mauer mit ironischen Inschriften auf Spruchbändern geschmückt wurden. So nahm der polnische Künstler Krzysztof Wodiczko 1990 eine Neuinterpretation vor, als er eine Lenin-Statue mithilfe einer Projektion in einen Einkaufswagen voller billiger Elektronikartikel verwandelte. Gerhard Paul, „Good Bye Lenin! Bildersturm und Denkmalsturz im 20. Jahrhundert“, in: ders. (Hg.), BilderMACHT. Studien zur Visual History des 20. und 21. Jahrhunderts, Göttingen: Wallstein 2013, S. 539–566, S. 559. 102 Die (Human) Living Sculpture 51 Alexandra Pirici, Soft Power, 2014, sculptural addition to the Bronze Horseman public monument, St. Petersburg, Manifesta 10, 2014 52 Alexandra Pirici, Soft Power, 2014, sculptural addition to/ enactment of the statue of Lenin in Finland Square, St. Petersburg, Manifesta 10, 2014 103 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt physisch zu zerstören oder zu stürzen, entwirft Pirici eine spezifische Form der skulpturalen Gegen-Monumentalität und De-Monumentalisierung. Hierfür ist es gewinnbringend, auf das 1992 von James Edward Young geprägte Konzept „counter-monument“ zu verweisen.293 Young nutzt den Begriff, um die Werke einiger westdeutscher Künstlerinnen zu beschreiben, die in den 1980er-Jahren die Erinnerung an den Holocaust repräsentierten, wobei er das Denkmal gegen den Faschismus in Harburg (1989) von Esther Shalev-Gerz und Jochen Gerz als ein Beispiel anführt. Diese Gegenmonumente lehnten die traditionellen Formen und Gründe für öffentliche Gedenkkunst ab, einschließlich ihrer Stasis und Überzeitlichkeit. Young zufolge besteht ihr Hauptziel darin, dem Publikum seine Selbstgefälligkeit zu nehmen, es dazu zu bringen, seine Annahmen zu hinterfragen und Denkmäler nicht länger zu ermutigen, „to do our memory-work for us“.294 Das Harburger Denkmal gegen den Faschismus stellt den ontologischen Status von Denkmälern und ihre didaktische Funktion infrage. Konkret aktivierte es die Schaulustigen, indem sie ihre Namen auf eine Säule schreiben konnten, die langsam in den Boden gesenkt wurde, bis sie verschwand.295 Young argumentiert überzeugend, dass das Denkmal gegen den Faschismus Vergänglichkeit, konzeptionelle Selbstzerstörung und die Negation einer festen Form als Hauptmerkmale eines Gegen-Denkmals verkörpert. Pirici erweitert das Verständnis des Young’schen Gegenmonuments und entwickelt zugleich eine zweite Strategie, d.h. die Strategie der De-Monumentalisierung. Gegenmonumentalität bezieht sich hier auf die temporäre, performative Geste, die das historische Monument mit einer Tableau-Vivant-Performance konfrontiert und eine kollektive Living Sculpture schafft, die nur in individuellen Nachbildern und fotografischen Dokumentationen überlebt. Andererseits funktioniert die Entmonumentalisierung als künstlerische Strategie, sich mimetischen Forderungen zu entledigen: Die Aneignung des Sockels und Umraums der Statue Peters des Großen ignoriert ihr historisches Emblem der Macht und ihre Überzeitlichkeit. Beide Herangehensweisen Piricis kreieren eine Gegenerzählung zur Monumentalität, indem sie etablierte Erinnerungskonzepte neu verhandeln und die (Idee der) Neutralität des öffentlichen Raums infrage stellen. Exkurs: Joseph Beuys – Soziale Plastik296 Joseph Beuys trug maßgeblich zur Erweiterung des Kunstbegriffs bei, weshalb sein Skulpturverständnis und die Soziale Plastik als wegweisender Schritt zu einer skulpturalen Ästhetik des Lebenden beleuchtet werden. 1961 wurde er auf den Lehrstuhl für Monumentale Bild293 James Edward Young, „The Counter-Monument: Memory against Itself in Germany Today“, in: Critical Inquiry, 18, 2, 1992, S. 267–296; Nora Sternfeld, „Münsters Gegen-Monumente: Ästhetiken der Erinnerung im öffentlichen Raum“, in: Public Matters. Debatten & Dokumente aus dem Skulptur Projekte Archiv, Köln: Verlag der Buchhandlung Walther König 2019, S. 213–228. 294 Young 1992, S. 273. 295 Um die Erinnerung lebendig zu halten, müsse das Gegenmonument auf die Betrachterinnen zurückwirken und ihnen die Erinnerungsfunktion/-aufgabe übertragen (ebd., S. 294). 296 Der folgende Abschnitt ist einem Text der Autorin für das von Timo Skrandies und Bettina Paust herausgegebene Joseph Beuys-Handbuch. Leben – Werk – Wirkung (Stuttgart: J.B. Metzler 2021, S. 100–105) entlehnt. 104 Die (Human) Living Sculpture 53 Josef Beuys, Der Chef – The Chief (Fluxus-Gesang), 1964, Galerie René Block, Berlin hauerei an die Kunstakademie Düsseldorf berufen. In die Anfangszeit seiner Lehrtätigkeit fällt die Auseinandersetzung mit Fluxus; 1963 führte er eine Aktion mit Fett in der Kölner Galerie Zwirner anlässlich eines Vortrags von Allan Kaprow auf. Seitdem mehrten sich die Arbeiten mit Fett und Filz als identitätsstiftenden Materialien. 1964 realisierte Beuys seinen Fettstuhl; im selben Jahr führte er in Kopenhagen und Berlin die skulptural anmutende Aktion Der Chef – The Chief (Fluxus-Gesang) (Abb. 53) auf, während der er mehrere Stunden isoliert in eine Filzdecke gehüllt auf dem Boden liegt und ihm zu Kopf und Füßen zwei tote Hasen als Verlängerung seines Körpers liegen.297 Durch ein Mikrofon unter der Decke und im Raum angebrachte Lautsprecher waren außen Gurgellaute, Atmen, Röcheln, Zischen und Husten zu vernehmen; Haarbüschel und Fingernägel, verschiedene Formationen von Fett und zwei Kupferstäbe ergänzten die Szenerie. Charakteristisch für die plastische Situation von Der Chef ist die statuarische Selbstpräsentation, die den Künstlerkörper zur Living Sculpture, zu einem textilen, die Silhouette nahezu verbergenden Objekt werden lässt. Vor der Tür der Berliner Galerie René Block ließ Beuys ein Schild mit der Aufschrift „Der Chef“ 297 Vgl. Uwe M. Schneede, Joseph Beuys, die Aktionen. Kommentiertes Werkverzeichnis mit fotografischen Dokumentationen, Ostfildern-Ruit: Hatje Cantz 1994, S. 68–75; Sven Lindholm, Inszenierte Metamorphosen. Beuys’ Aktionen vor dem Hintergrund von Goethes Gestalttheorie, Freiburg: Rombach [u.a.] 2008, S. 230–236. 105 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt anbringen. Diesen Hinweis integrierte er in sein gleichnamiges Plastisches Bild, wodurch sich die enge Verzahnung zwischen skulpturalen Objekten, Requisiten, Multiples, Aktionen, Zeichnungen und Diskussionen erklärt, die alle seinen erweiterten Kunstbegriff verkörpern. Geplant war ursprünglich eine Parallelaktion mit Robert Morris in New York. Der Galerist Schmela hatte Morris für eine Ausstellung nach Düsseldorf eingeladen, wo sich beide Künstler kennenlernten, Beuys mit ihm und Yvonne Rainer einen Tanzabend an der Akademie organisierte.298 Seit den frühen 1960er-Jahren führte Morris skulpturale Performances wie Columns (1961) und in Düsseldorf 1964 Site mit Yvonne Rainer auf.299 Die von Beuys vorgesehene Aktion in New York realisierte Morris allerdings nicht.300 1968 trafen die beiden jeweils auf unterschiedliche Weise nach einer Neudefinition von Skulptur strebenden Künstler erneut aufeinander, als deren Ausstellungen im Van Abbemuseum in Eindhoven sich überschnitten und Beuys den Filzraum von Morris sah, woraus kritische Fragen nach Einfluss und Vorbildern erwuchsen, insbesondere als Morris die neuen Kunsttendenzen auf die USA rückbezog.301 Im selben Jahr lehnte Beuys eine Einladung von Morris für 9 at Leo Castelli in New York ab und begründete dies mit Differenzen im metaphorischen Gehalt der Arbeiten.302 Trotz Burnhams differierenden konzeptuellen und ideengeschichtlichen Hintergrunds ist auch bei Beuys die Skulptur ein Bestandteil eines größeren Systems, d.h. ein Requisit oder von einer Aktion geformtes Objekt.303 Wie Hans Körner und Reinhard Steiner bemerken, bedürfen die Arbeiten der Rückbindung an die Person Beuys, dessen Materialverständnis und -ästhetik, weshalb sie „unselbständige Teile einer als Ganzheit verstandenen ‚Parallel aktion‘“ sind.304 Die von Michael Fried kritisierte Theatralität kommt in den Aktionen von Beuys zum Höhepunkt. Anleihen aus dem Theater, dessen Handlungsdynamiken und Aufführungspraktiken sind deutlich, wie seine soziale Plastik unterstreicht.305 Er bezeichnet sich 298 Dirk Luckow, Joseph Beuys und die amerikanische Anti Form-Kunst. Einfluß und Wechselwirkung zwischen Beuys und Morris, Hesse, Nauman, Serra, Berlin: Mann 1998, S. 43. 299 Ebd., S. 42, 54f. 300 Luckow 1998, S. 58–60, 65–68; Schneede 1994, S. 69. 301 Siehe u.a. Caroline Tisdall, Joseph Beuys, London: Thames and Hudson 1979, S. 190; Dirk Luckow, „Unerwartete Parallelen. Joseph Beuys und die amerikanische Anti-Form-Kunst“, in: Ulrich Müller (Hg.), Joseph Beuys – Parallelprozesse, München: Hirmer 2012, S. 98–115, S. 101f. 302 Vgl. Causey 1998, S. 139. 303 Volker Harlan/Rainer Rappmann/Peter Schata (Hg.), Soziale Plastik. Materialien zu Joseph Beuys, Achberg: Achberger Verlag 1984, S. 110 (= 3., erweiterte und ergänzte Auflage). Beuys resümiert: „Die Objekte sind nur verständlich im Zusammenhang mit meinen Ideen.“ 304 Beuys zit. n. Hans Körner/Reinhard Steiner, „‚Plastische Selbstbestimmung?‘ Ein kritischer Versuch über Joseph Beuys“, in: Das Kunstwerk, 35, 3, 1982, S. 32–41, S. 35. Eine absolutistische, subjektistische Haltung, d.h. die „imaginäre, fiktive Erweiterung des individuellen Subjekts zu einem Groß-Subjekt“ unterstellen ihm die beiden Autoren und kritisieren zu Recht, dass er Machtfragen verschweigt: „Wenn jemand meine Sachen sieht, dann trete ich in Erscheinung“ (ebd., S. 38). Zum Materialspektrum zählen neben Filz und Fett Metalle wie Kupfer und Eisen, tote Tiere, Schiefer, Honig, Objects Trouvés, ferner Materialien, die Assoziationen an Schmerz und Krankheit wecken, darunter Mullbinden, Gaze, Nadeln, Röntgenplatten und Knochen. 305 Vgl. McEvilley 1999, S. 189; Barbara Gronau, „‚Man muss … eine Art ständiges Theater spielen‘: Performativität und Aufführung bei Joseph Beuys“, in: Müller 2012, S. 116–127. 106 Die (Human) Living Sculpture in seiner Selbstinszenierung als Kunstwerk, doch nicht als Living Sculpture. In den Überblickswerken zur Skulptur des 20. Jahrhunderts der 1970er- bis 1990er-Jahre werden seine Arbeiten nur peripher genannt, vermutlich weil die traditionellen skulpturalen Parameter versagen – trotz vitalistischer Ausrichtung und Interesse an plastisch arbeitenden Künstlern wie Wilhelm Lehmbruck, Ewald Mataré, Pablo Picasso und Marcel Duchamp sowie seiner Würdigung antiker griechischer Plastik als Ausdruck des ‚ganzen Menschen‘. Mit der sozialen Plastik, wie Beuys sie verstand, wollte er eine „ideale Kultur der Vereinigung“ erreichen. Seine plastische Theorie sei eine „Grundlagenforschung“, die eine gesamtgesellschaftliche Transformation begründet und tradierte Gattungskategorien überschreitet bzw. obsolet werden lässt. Mit seinem Slogan „Jeder Mensch ist ein Künstler“ verlagert er das plastische Arbeiten aus der Bildenden Kunst in eine anthropologische, universale Grundbestimmung, der zufolge bereits das Denken und das Handeln Formbildungsprozesse, genuin kreative künstlerische Vorgänge, seien. Beim „Hineindrücken einer Tat, in die Materie“ verlässt das Plastische den institutionalisierten Bereich; das Modellieren ist übertragen als pädagogisch-sozialer Prozess zu verstehen:306 „Eine Gesellschaftsordnung wie eine Plastik zu formen, das ist meine und die Aufgabe der Kunst.“307 Seit 1967 nehmen Vorträge, Organisationen und Gespräche als Teil von Beuys’ Kunst zu – insbesondere nach seiner Entlassung aus der Akademie 1972 –, um Meinungsbildungsprozesse zu initiieren. Dabei greift Beuys neben der Vielzahl der Rollenformate mit identifikatorischer Kleidung (Filzanzug, Anglerweste, Hut etc.) weiterhin darstellungsbezogene Kunstformen auf. Lebenslauf und Werklauf sind proklamatorisch verbunden.308 Jede Plastik sei eine statische Aktion, d.h. ein „Kraftzentrum“, das auf die Betrachterinnen wirke. Jede Aktion sei eine bewegte Skulptur oder soziale Plastik. So schließt er auch organisch-physiologische Körperprozesse wie die embryonale Entwicklung oder Verdauung ein, etwa wenn er in der Aktion Hauptstrom (1967) Butterstücke aß, das plastische Material quasi verinnerlichte und mit seinem Körper verband. Die Nähe zu Wolf Vostell wird deutlich, da dieser Aktionen in seine „Ereignis-Plastik“ inkludierte.309 Auch Haacke stellte die Geburtsurkunde seines Sohnes aus, dessen embryonale Entwicklung und Heranwachsen er als skulpturales Realzeit-System auffasste (Abb. 54). Eine therapeutische Aufgabe komme laut Beuys den Künsten zu, da sie der am Materialis mus krankenden Gesellschaft zu einer neuen sozialen, demokratischen Lebensform verhelfen.310 Auch pflanzliche Wachstumsprozesse gehören zur prozessualen, polysensuellen 306 Beuys zit. n. Harlan/Rappmann/Schata 1984, S. 125. 307 Beuys zit. n. Hiltrud Oman, Joseph Beuys. Die Kunst auf dem Weg zum Leben, München: Heyne 1998, S. 7. Diese radikale Umdeutung wird häufig als einer seiner wichtigsten Beiträge zur Neo-Avantgarde gesehen (Gronau 2012, S. 118). 308 Wulf Herzogenrath (Hg.), Selbstdarstellung. Künstler über sich, Düsseldorf: Droste 1973, S. 22; Melitta Kliege, „Vom Prinzip Plastik zur Sozialen Plastik. Immaterielle Formfindungsprozesse als Sinnbild der Kunst“, in: Müller 2012, S. 82–97, S. 92. 309 Wolf Vostell. Das plastische Werk 1953–87, Mailand: Mult(h)ipla Edizioni 1988, S. 291. „Ein Ereignis ist Plastik = ist Information = ist Dokumentation.“ 310 Siehe Beuys, in: Harlan/Rappmann/Schata 1984, S. 121. 107 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 54 Hans Haacke, Carl Samuel Sélavy Haacke, Geburtsregistrierungsschein, New York University Hospital, 13.1.1969 lastik: 1977 pflanzte er Kartoffeln in den Vorgarten der Galerie René Block und erntete P diese im Herbst. An der Biene erläutert Beuys die Polarität zwischen Plastik und Bildhauerei (vgl. Bienenkönigin): Während die Weiselzelle der Bienenkönigin amorph ist, weisen die Normalzellen eine kristalline geometrische Struktur auf: Das sind Polaritäten, die mich immer interessiert haben, auch schon allein im Hinblick auf die Theorie zwischen Bildhauerei und Plastik. Denn Plastik bezieht sich mehr auf die Möglichkeit, sich darin zu bewegen, während Bildhauerei immer praktisch geometrisch ist […] man muss beides sein, genau wie die Biene beides ist.311 1977 stellte der Künstler anlässlich der Skulptur Projekte Münster einen monumentalen Wachskeil aus, der während der Ausstellung nicht erkaltete und erst danach als Unschlitt/Tallow (Wärmeskulptur auf Zeit hin angelegt) gezeigt werden konnte – die Eigenzeitlichkeit des Materials bestimmt die Werkgenese. Insgesamt zeigt die kunsthistorische 311 Beuys zit. n. Helmut Friedel/Lothar Schirmer, Joseph Beuys im Lenbachhaus und Schenkung Lothar Schirmer, München: Schirmer/Mosel 2013, S. 127. 108 Die Non-Human Living Sculpture ontextualisierung seiner skulpturalen Arbeiten, dass Beuys, durch die Rezeption oftmals K zum ‚Solitär‘ stilisiert, durchaus mit Positionen der Post-Minimal Art, einem erweiterten, partizipativen Skulpturverständnis und der künstlerischen Integration organischer Materialien in Zusammenhang zu bringen ist und damit auch als ein Wegbereiter für systemästhetische und situationsästhetische Konzepte des Skulpturalen gelten darf. Die Non-Human Living Sculpture Pflanzen, Tiere und Mikroorganismen, biolumineszente Algen und Bakterienkulturen, Kleinstlebewesen wie Insekten und Spinnen, auch (domestizierte) Tiere der alltäglichen Umgebung dienen als lebende Ko-Akteure oder – wie Dieter Roth sie nennt – „Mitarbeiter“ in skulpturalen Werken, weshalb ich den Begriff der Non-Human Living Sculpture vorschlage.312 Die lebenden Protagonisten fungieren auf unterschiedliche Weise als plastischer Stoff in den Händen der Leben spendenden bzw. verändernden Künstlerinnen, die wiederum als Gärtnerinnen, Entomologinnen, Experimentatorinnen und Züchterinnen agieren, in ihrem organischen ‚Artefakt‘ einen evolutionären Prozess anstoßen oder zur Disposition stellen. Das Material ist lebendig und lässt sich als eigenwilliger Ko-Akteur nur bedingt zähmen, worin eine werkspezifische Spannung und ein damit verbundenes Machtgefüge liegt. Inwiefern begegnen die Künstlerinnen ihrem Material antwortend und gestehen ihm eine gewisse Eigenzeitlichkeit zu? Am Beispiel von Hans Haacke, Pierre Huyghe und anderen Künstlerinnen wird dieser Aspekt eingehend erörtert. Mit einem Blick auf die Diskurse einer Anthropozentrismus-Kritik mag beim Begriff Non-Human Living Sculpture das Problem der Subsumption einer heterogenen Vielzahl von Entitäten und Lebewesen hervortreten, da ein kategorialer Dualismus fortgesetzt wird: menschlich/human und nicht-menschlich/non-human.313 Bedingt durch die historische Kontextualisierung von Human Living Sculptures der Künstlerinnen wie Gilbert & George, ORLAN oder Ben Vautier, die sich einer der Hauptaufgaben traditioneller Bildhauerei, anthropomorphe Figuren zu zeigen, stellen, und durch künstlerische Positionen wie Pierre Huyghe, die Tiere und Pflanzen in ihren skulpturalen Situationen einsetzen, erscheint diese Differenzierung zwischen human und non-human für die Werkanalyse trotzdem geeignet.314 312 Dieter Roth zit. n. Theodora Vischer/Bernadette Walter (Hg.), Roth Zeit. Eine Dieter Roth Retrospektive, Ausst.-Kat. Schaulager Basel, Baden: Lars Müller Publishers 2003, S. 95. 313 Vgl. Bruno Latour, Das Parlament der Dinge. Für eine politische Ökologie, Frankfurt am Main: Suhrkamp 2012. Latour beschreibt mögliche Assoziationen von Menschen und nicht-menschlichen Wesen und plädiert für eine Aufhebung der dichotomen Trennung. „Die Begriffe Subjekt und Objekt verweisen gerade nicht auf isolierte Realitätsbereiche, die anschließend durch ein überlegenes Bewusstsein verbunden oder durch eine dialektische Bewegung ‚aufgehoben‘ werden müssten, sondern haben einzig den Zweck, den Ball jeweils ins andere Lager zurückzuschlagen und diese ständig auf dem ‚Quivive‘ zu halten.“ (Ebd., S. 105.) 314 Der Begriff impliziert keinen Absolutheitsanspruch; vielmehr könnten auch andere Begriffe bzw. Unter kategorien, die weiterführend sind, Verwendung finden. Ein weiterer Vorschlag wäre „more-than human“. 109 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt In der vorliegenden Untersuchung liegt der Fokus auf systemästhetischen und situationsästhetischen Konzepten des Skulpturalen. Der Einsatz lebender Tiere und Pflanzen in der Bildenden Kunst nimmt im 20. Jahrhundert seit den 1960er-Jahren vielfältige Formen an und kann aufgrund seiner Diversität nur exemplarisch verhandelt werden. Ethische und tierrechtliche Fragen werden eher selten, inzwischen von den Animal Studies gestellt und bedürfen einer eigenen Untersuchung.315 Vielleicht sind es ‚Experimentierfreude‘, Hinterfragung der Gattungsgrenzen und Interesse an (Eigen-)Zeitlichkeit, Prozessualität und Performativität, die Künstlerinnen dazu anregen, unterschiedliche Lebewesen (oftmals bedenkenlos) in die künstlerische Arbeit einzubinden – auch Richard Serra, der (nur) in seinem Frühwerk daran anknüpfte, weshalb diese „student works“ in seinem Catalogue Raisonné fehlen.316 Aus einem skulptural-architektonischen Konzept heraus entwirft der junge, mit einem Fulbright-Stipendium in Italien weilende Bildhauer für seine erste Einzelausstellung Animal habitats live and stuffed (1965–1966) in der römischen Galerie La Salita Gehäuse und Ställe für verschiedene Tiere, darunter: Hase, Huhn und eine Muttersau nebst einzelnen Taxidermien (Abb. 55). In der Literatur wird der künstlerische Beitrag von Nancy Graves, damalige Ehefrau Serras und Dermoplastikerin, selten genannt, obgleich sie als Künstlerin heute mit ihrem eigenen Œuvre durchaus bekannt ist.317 Eine Publikation mit Werkabbildungen, kurzem Künstlerstatement, das die „projected sexual metaphor“ der Arbeiten betont, und knapper Biographie erschien anlässlich dieser Ausstellung, deren künstlerische Stoßrichtung das Times Magazin lapidar kommentierte: 315 Vgl. u.a. die Forschungen von Jessica Ullrich und die von ihr herausgegebene Zeitschrift Tierstudien (seit 2012, Berlin: Neofelis Verlag). 316 Ernst-Gerhard Güse (Hg.), Richard Serra, Ausst.-Kat. Westfälisches Landesmuseum für Kunst und Kulturgeschichte, Münster 1987 [u.a.], Stuttgart: Hatje 1987, S. 364. 317 Petra Lange-Berndt, Animal Art. Präparierte Tiere in der Kunst 1850–2000, München: Silke Schreiber 2009, S. 134, 274. Sie gibt an, dass die Werke der Ausstellung vom Künstlerpaar anschließend zerstört worden seien. Viele der Objekte spielten auf die weiblichen Genitalien an, weshalb die Schau zur Vernissage fast geschlossen wurde. Siehe auch Bettina Ruhrberg, Arte Povera. Geschichte, Theorie und Werke einer künstlerischen Bewegung in Italien (= Dissertation Rheinische Friedrich-Wilhelm-Universität Bonn 1991), Bonn 1992, S. 245. Auch sie erwähnt Nancy Graves nicht, erläutert aber den Eindruck der Serra-Ausstellung auf die italienischen Arte-Povera-Künstler, u.a. Pino Pascali, dem eine Art Netzskulptur mit lebenden Affen vorschwebte (ebd., S. 271). Dirk Luckow bespricht Serras Ausstellung, erwähnt Graves jedoch nicht (Luckow 1998, S. 251–254). Auch Serra selbst äußerte sich nicht zu den Taxidermien von Graves. Stattdessen seien seine Arbeiten „inspired by Clemente Susini’s stuffed cadavers in La Specola, a zoological museum in Florence.“ (Bernice Rose [Hg.], Richard Serra – drawing. A retrospective, New Haven [u.a.]: Yale University Press 2011, S. 207.) Zudem sei er von Velásquez inspiriert gewesen, ein Künstler, „who made me see that my way of dealing with painting was limited to looking at something inside a frame … That’s when I decided to make cages, to stuff them with material, to use life animals, to do anything to get away from my education, from all of it.“ (Ebd.) In einem Gespräch mit Hal Foster ergänzt er: „At the same time I became interested in zoos (Florence had the first zoos). So I got the idea of stuffing animals and presenting assemblages, and I used cages, stacking them one on top of the other à la Brancusi.“ (Richard Serra im Gespräch mit Hal Foster, „Sand dunes and still mills“, in: dies. [Hg.], Conversations about Sculpture, New Haven: Yale University Press 2018, S. 7–18, S. 16.) 110 Die Non-Human Living Sculpture 55 Richard Serra, Animal habitats live and stuffed, 1965–1966, Galerie La Salita, Rom „he is currently deep in his zoo period.“318 Seine Verwendung lebenden ‚Materials‘ begründet Serra mit seinem Interesse an „discarded material“ und damit, dass er lebende Tiere im Privatleben um sich hatte, deren Habitatvorlieben er in einem künstlerischen, zoologischen Experiment beobachtete, wie er rückblickend in einem Interview mit Kynaston McShine erläutert: I would give them different barnyard materials to see what kind of habitats they would make naturally. Then I would try to emulate what they were doing. It was all playful and experimental, and it led to this series of animal-cage pieces.319 Auch wenn dieser mimetisch motivierte Prozess nicht wörtlich zu nehmen ist (kein Kaninchen baut sich einen Stall wie den von Serra entworfenen), rekurriert diese Äußerung auf den Topos der Naturnachahmung bzw. -nachbildung. Dabei scheint Serra das Verhältnis des isolierten (lebenden oder ausgestopften) Tieres als skulpturales Element zu seinem Käfig zu interessieren. Krauss bezeichnet sein (neues) ‚Material‘ als „das ästhetisch ungeformte Medium biologischen Lebens“, mit dem er seine Ställe „füllte“.320 Diese Formulierung und Charakterisierung des ästhetisch Ungeformten wirft Fragen nach Macht, Dominanz und 318 „Exhibitions: Please don’t feed the sculpture“, in: Times, 10.6.1966, in: Richard Serra. Early works, Ausst.-Kat. David Zwirner, New York 2013, Göttingen: Steidl 2013, S. 36f. Hier wird Nancy Graves zumindest als „amateur taxidermist“ genannt. 319 Kynaston McShine/Lynne Cooke (Hg.), Richard Serra sculpture. Forty years, Ausst.-Kat. Museum of Modern Art, New York 2007, S. 20: „I certainly wasn’t consciously working out of Rauschenberg when I startet the Habitats. What led to them was my interest in using discarded material, and the fact that I was stuffing animals at the time and had various animals living with me. I had set up a kind of zoological experiment […]. It was student work and I did not pursue it any further after the show in Rome.“ 320 Rosalind E. Krauss, „Richard Serra. Skulptur“, in: Richard Serra. Props, Ausst.-Kat. Wilhelm Lehmbruck Museum, Duisburg 1994, S. 28–110, S. 41. 111 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 56 Jannis Kounelli, Senza Titolo (1967) mit Papagei und Kakteen, Installationsansicht Hayward Gallery, London, 1993 Instrumentalisierung des Tieres auf. Serras Ausstellung mit ihren Material-Assemblagen (Stroh, Zweige, Käfige, lebende und ausgestopfte Tiere und andere Fundstücke) wird in der Literatur zu Serra als prägend für die italienische Arte Povera verstanden, umgekehrt könnte er von der italienischen Avantgarde inspiriert worden sein.321 In Anlehnung an eine 1975 vom britischen Bildhauer William Tucker kuratierte Ausstellung The Condition of Sculpture kuratierte Marianne Ryan 1993 in der Londoner H ayward Gallery Gravity and Grace. The Changing Condition of Sculpture, 1965–1975.322 Im Unterschied zu Tuckers Auswahl zeigte sie Tiere und Pflanzen, darunter Serras frühe Life Animal Habitats (1965–1966) und Kounellis’ Senza Titolo (1967) mit lebendem Papagei und Kakteen (Abb. 56). Die Aufnahme dieser Werke belegt die wachsende Berücksichtigung sogenannter Non-Human Living Sculptures. Neben Serra nutzten auch andere Künstlerkolleginnen in den 1950er- und 1960er-Jahren Taxidermien für ihre plastischen Arbeiten, etwa Robert Rauschenberg (Monogram, 1955–1959), Nancy Graves (Camel Rug, 1968) oder Wolf Vostell (Chicken Box, 1966).323 Auch Joseph Beuys ‚konstruierte‘ wenige Jahre später ein Habitat in Form eines Mäusestalls aus einem Holzrahmen und Metallgitter (1968–1970) (Abb. 57), das er auf der Unterseite 321 Auch wenn Luckow zufolge die Schau von Serra in Europa und den USA wenig rezipiert worden sei (Luckow 1998, S. 253f.). 322 Ryan 1993. 323 Lange-Berndt 2009, S. 25f. Die Nähe der Dermoplastik zur Skulptur unterstreichen gegen Ende des 19. Jahrhunderts Präparatoren und der Direktor des New Yorker American Museum of National History Frederic A. Lucas in ihrer Forderung, der Dermoplastik denselben Status zuzuerkennen wie der Bildhauerei. 112 Die Non-Human Living Sculpture 57 Joseph Beuys, Mäusestall, 1968–1970, Holzstall mit Metall gitter, Nussschalen, Eierschalen, Getreidekörner, Kräutersträußchen, 53 x 104 x 49 cm des Stalldeckels signierte und das sich heute in der Sammlung des Münchner Lenbach hauses befindet.324 Ähnlich wie Serra hielt er das Tier zu Hause in seiner Düsseldorfer Wohnung am Drakeplatz. Auf dem Boden des Stalls befinden sich Eier- und Nussschalen, Speisereste, Zweige, Holz und Stroh, die auf den ehemaligen Lebensraum der Maus deuten, deren Verbleib zu erahnen ist. Beuys legte einen getrockneten Strauß auf das kleine Gehäuse, das der Maus als Rückzugsort diente. Ein klares Machtgefüge zwischen menschlichen Urheberinnen und tierlichen Protagonisten kommt in diesen ‚funktionalen‘ Skulpturen zum Ausdruck, da die Tiere gezwungenermaßen Bewohner dieser skulpturalen Artefakte wurden, der Anschauung dienen und den Blicken der Besucherinnen direkt ausgesetzt sind, wie John Berger in Why look at animals (1980) u.a. am Beispiel der Zootiere konstatiert.325 Vermutlich ist Serras zoologisches Interesse an diesen Cage Pieces vordergründig, und es geht ihm weniger um die Beobachtung eines kollektiven Verhaltens, wie Haacke dies für sein Ant Coop (1969) vorgibt, sondern mit der isolierten Haltung der Tiere um Größen- und Proportionsverhältnisse sowie um die Beziehung zwischen innen und außen, die in seinen späteren, begehbaren Skulpturen virulent werden. Bereits 1962–1963 integrierte der belgische Maler Roger Raveel in Käfigen lebende Vögel in seine Leinwandbilder (Abb. 58), wodurch sie entfernt an Robert Rauschenbergs Combine Paintings erinnern, eine Referenz, die auch Serra zugeschrieben wurde.326 324 Vgl. Friedel/Schirmer 2013, S. 76f. 325 Siehe dazu die Diskussion im dritten Kapitel zu Pierre Huyghes skulpturalen Situationen Untilled und After ALife Ahead. 326 Siehe Roger Raveel, Neerhof met levende duif, 1962/63, 150 x 440 cm; Het verschrikkelijke mooie leven, 1965, olieverf/doek+spiegel+vogelkooi, 150 x 440 cm, Privatsammlung Zwevegem. Abb. in: Eliane De Wilde/Rini Dippel (Hg.), Raveel, Ausst.-Kat. Stedelijk Museum, Amsterdam 1974, Abb. 13 und 23, n.p. 113 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 58 Roger Raveel, Neerhof met levende duif, 1963, Öl auf Leinwand, Vogelkäfig, Vogel, 150 x 440 cm 59 Münchener Gruppe EFFEKT, Mausbild, 1965, Holz, Glas, 15 weiße Mäuse, 90 x 90 x 30 cm E dward Kienholz realisierte 1964–1965 The Wait mit lebendem Kanarienvogel. Die Münchener Gruppe EFFEKT entwarf 1965 Mausbild (Abb. 59) – ein schwarzes Hochhaus mit acht Stockwerken, das von weißen Mäusen bewohnt wurde. Die Bewegungen in diesem kinetischen Objekt entstanden durch die Auftritte der Vierbeiner. Die gefiederten Tiere bei Raveel unterstreichen mit ihren kleinen, alles andere als ‚artgerechten‘ Käfigen den plasti schen Charakter des flachen Bildes; es handelt sich dabei jedoch nicht um Non-Human Living Sculptures: Die Singvögel Raveels muten wie lebendes ‚Zierwerk‘ an, das das Gemälde um eine akustische Ebene zu bereichern vermag. Jannis Kounellis platzierte 1967 in der römischen Galleria L’Attico seinen Papagei direkt auf einer Stange vor eine monochrome Wandfläche neben einem futuristisch, technoid anmutenden Kaktusfeld, einem Baumwoll-Stahl-Objekt und einem gesockelten Aquarium.327 In der Formensprache hallen wie 327 Campi (Kaktusfelder), Pappagallo (Papagei) und Cotoniera (Baumwollbehälter) sind 1967 für eine Einzel ausstellung in der Galleria L’Attico in Rom (November–Dezember) entstanden. Der Galerist Fabio 114 Die Non-Human Living Sculpture 60 Jannis Kounellis, Senza Titolo, 1967, Stoff auf Leinwand, 250 x 320 cm, 24 Vogelkäfige mit je einem lebenden Vogel, Galerie L‘Attico, Rom bei den physikalischen Arbeiten Haackes die industriell gefertigten Objekte der Minimal Art nach. In der vorangegangenen Ausstellung Il Giardino, I Giuochi (Der Garten, die Spiele, April–Mai 1967) hatte Kounellis bereits Vögel in sein Werk Senza Titolo einbezogen: Zebrafinken in kleinen, übereinander platzierten Käfigen flankierten dekorierend zu beiden Seiten ein weißes Rosenbild (Abb. 60). Im selben Jahr installierte Hélio Oiticica sein Environment Tropicália mit Pflanzen, Papageien und Sand im Museu de Arte Moderne in Rio de Janeiro.328 Auf die Evolutionsgeschichte des Vogels deutet Gloria Friedman, wenn sie einen Käfig mit Vogel neben die Abbildung eines Archeopteryx-Fossils hängt (1991).329 Heute sind aus tierrechtlichen Gründen in Käfigen gehaltene Vögel seltener Bestandteil eines Werks. So rief S argentini hatte Jannis Kounellis schon zum zweiten Mal in diesem Jahr eingeladen, seine Räume zu nutzen. 328 Abb. bei Bishop 2005, S. 62. 329 Abb. in: Thomas Zaunschirm, „Im Zoo der Kunst I“, in: Kunstforum International, 174, 2005, S. 36– 104, S. 77. 115 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 61 Céleste Boursier-Mougenot, from here to ear (Detail), 2007–2009 1993 die Ausstellung des Papageis von Kounellis in der Gruppenschau Gravity & Grace (Hayward Gallery London) kritische Pressestimmen hervor, die eine Entnahme des Vogels aus der Ausstellung f orderten.330 Weiterhin finden sich jedoch durchaus lebende Ko-Akteure innerhalb einer raumumfassenden Situation, so bei Marc Camille Chaimowicz Forty and Forty mit Klara Lidén und Manfred Pernice und vierzig durch den Galerieraum fliegenden Kanarienvögeln (2014), bei Mark Dions ortsspezifischen The Library for the Birds of Antwerp (1994) oder The Library for the Birds of London (2018) und Céleste Boursier-Mougenots from hear to ear (2007–2009) (Abb. 61).331 Dezidiert die Kategorie Skulptur ruft Jan Dibbets in Rotkehlchenterritorium/Skulptur (1969) auf, als er den Versuch unternahm, Flugbahn und Aufenthaltsort eines Rotkehlchens über die Versetzung von Stangen als Landeplatz innerhalb eines Areals in einem Amsterdamer Park zu kontrollieren und dieses Experiment mittels Fotografien und textuellen Notizen in einem Buch veröffentlichte.332 unkten Nach Yayoi Kusamas Horse Play (1967), in dem sie ein Pferd und sich selbst mit P überzog, folgt 1969 Kounellis’ Ausstellung Senza Titolo (12 Cavalli) (Abb. 62): Zwölf lebende Vierbeiner besetzten schnaubend und hufescharrend den White Cube und demonstrierten den seit Ende der 1960er-Jahre konstatierten Umbruch in der Werkauffassung zugunsten offener Experimente und Erweiterung tradierter Gattungsgrenzen mit multisensorischen 330 Siehe Scans des Presseechos bei https://artsandculture.google.com/exhibit/dgLS6HR1JTdQJQ (19.9.2019). In einem Artikel „Parrot stays in sculpture“ des Express + Star, Wolverhampton (23.9.1993) wird er als „part of a living sculpture“ beschrieben. 331 Galerie Neu Berlin. Jessica Ullrich bietet einen Überblick in „Kunst aus der Vogelperspektive. Zur Rolle von lebenden Vögeln in der Gegenwartskunst“, in: Gundel Mattenklott/Constanze Rora (Hg.), Das Reich der Vögel. Beiträge zur ästhetischen Bildung und zur Einübung in ästhetische Aufmerksamkeit, zur Kindheitsforschung, zur Didaktik und zum Unterricht (Themenheft). Zeitschrift Ästhetische Bildung, 8, 1, 2016, http://zaeb.net/wordpress/2018/05/27/das-reich-der-voegel/ (16.3.2023). 332 Jan Dibbets, Roodborst territorium, sculptuur 1969, Seth Siegelaub New York, Köln: Walther König 1970. 116 Die Non-Human Living Sculpture 62 Jannis Kounellis, Senza Titolo (12 Cavalli), 1969, Galerie L‘Attico, Rom Erlebnissen. Seine Geste, Reitpferde in der Galerie L’Attico auszustellen, verstand er gesellschaftspolitisch (und institutionskritisch): „I wasn’t trying to be sensational, I was only interested in mapping the space, in creating an image that would stand for change.“333 Wenn der Arte-Povera-Vertreter dennoch einiges Aufsehen erregte und mehr als nur die Räumlichkeiten der Galerie neu ‚vermaß‘, ebnete er rückblickend den Weg für die Animal Art, die in der gleichnamigen Ausstellung des Steirischen Herbst 1987 zum Thema avancierte und von Louis Bec als „the art of domestication“ bezeichnet wurde.334 Kounellis interessierte das lebende Bild, dessen einzelne Elemente er innerhalb der Galeriearchitektur readymade-artig komponierte. Die Kontextverschiebung, sein Rückgriff auf ein ursprünglich allseits genutztes Transportmittel riefen Irritationen hervor und erinnerten an Zeiten, in denen Pferde das Stadtbild prägten. Die Suche nach dem Echten und Authentischen, das sich in den Vögeln zeigt, spiegelt sich auch in der Ausstellungsbroschüre von Campi, Pappagallo, Cotoniera, in der Kounellis ein Gespräch mit Kindern vor den Werken und einige Kinderfotos abdrucken ließ.335 Haacke fotografierte Installationsansichten, in denen Kinder – als die ‚wahren‘, ehrlichen Rezipienten – mit seinen Skulpturen interagieren bzw. auf lebende Werke wie schlüpfende Küken reagieren (Abb. 63).336 Über den sprechenden Papagei und dessen Vereinzelung, die ihm 333 Michele Robecchi, „Jannis Kounellis: Non-Verbal Communication“, in: Artpulse, 2012, http://artpulse magazine.com/jannis-kounellis-non-verbal-communication (3.8.2019). 334 Bec 2007, S. 84. 335 Jannis Kounellis, L’Attico 67–68, Ausst.-Kat. Galleria L’Attico 1968, n.p. Germano Celant bezeichnet die Vögel als autonome Wesen, deren Verhalten frei und unkontrollierbar sei, was für besagtes Werk unzutreffend ist: „Therefore the work is alive within a visual world: the image of the roses, and the concrete reality of the live canaries create a contrast between real and imaginary elements which in his later works, such as Men and the Parrot, becomes even more pronounced.“ (Germano Celant, „Jannis Kounellis“, in: Studio International, 191, 1976, S. 36–41, S. 38.) 336 Siehe Archiv Hans Haacke, New York. 117 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 63 Mr. W. J. Withrow. Mr. And Mrs. Bovey, Mr. P. Gurvish viewing “Chickens Hatching”, September 26, 1969, at the exhibition, New Alchemy: Elements, Systems, Forces, Art Gallery of Ontario, September 27–October 26, 1969 64 Wolf Vostell, Endogene Depression, 1980, 7 Truthähne, 30 Fernsehergehäuse 118 Die Non-Human Living Sculpture 65 Nam June Paik, TV-Buddha für Enten, 1987, Bronze, Fernseher, Bakelit, Plexiglas, Skulptur Projekte Münster k einen Kontakt mit Artgenossen erlaubt, erfolgt bei Kounellis eine Anthropomorphisierung des Tiers, der durch seine Nachahmung der menschlichen Sprache eine Nähe zu den Menschen simuliert. Wenn Germano Celant bei Pappagallo Form-, Farb- und M aterialkontraste betont und fast nebenbei bemerkt, dass Kounellis einen natürlichen Prozess zeige, „such as the parrot’s behaviour or the growing of cactus“, klammert er die damalige Kritik an der Integration des Papageis aus.337 Kounellis befrage mit seinen Pferden, Papagei und Kakteen die wahrnehmungsbezogene Struktur von Kunst (Celant spricht nicht von Skulptur), indem er den anonymen und multisensorischen Galerieraum der lebendigen Kraft („violence“) der Tiere gegenüberstellt.338 Auch technologische Entwicklungen stellen Künstler*innen der Tierwelt gegenüber, etwa Wolf Vostell 1970 im Kölnischen Kunstverein anlässlich der von Szeemann kuratierten Schau Happening & Fluxus. Er zeigte dort TV-Ochsen; die kalbende Kuh begriff er als „skulpturellen Lebensvorgang“ oder setzte bei Endogene Depression (1975) sechs Truthähne einem Environment mit einbetonierten Fernsehern aus (Abb. 64): „Die Natur-Skulptur der Truthähne, die Schönheit der Tiere soll mit der Schönheit des Fernsehers verglichen werden, durch die Neben-Einander-Stellung.“339 Nam June Paik platzierte 1987 im Münsteraner Aasee seinen TV-Buddha für Enten (Abb. 65). In den drei 337 Celant 1976, S. 38. Gleichzeitig wähle er „live subjects“, um neue Beziehungen zu Dingen herzustellen. Kounellis interessiere sich nicht, so Celant weiter, für moralische und soziale Werte bzw. urteile nicht darüber. 338 Ebd., S. 39. 339 Wolf Vostell 1988, S. 291. „Die Geburt eines Kalbes soll als skulptureller Lebensvorgang bewußt gemacht werden, und zwar im Museum, statt im Stall. Neben der kalbenden Kuh ist ein Knochenberg aufgetürmt, der als plastische Materie vorwegnimmt, was mit Kuh und Kalb geschieht.“ (Ebd., S. 175, Herv.i.Orig.) Die Polizei intervenierte allerdings vor der geplanten Geburt des Kalbes. Lebende Kühe stellte Vostell bereits 1963 in Wuppertal, 1964 in Ulm aus. Siehe auch Bill Beckley Ululations mit einem Raben (Raven Recitations) und Papagei (Parrot Recitations); Rooster, Bed, Lying Piece mit einem Hahn, in: Avalanche, Fall, 1971, S. 18–21. 119 1. Bildsäule, Raumgestalterin, Readymade, Living Sculpture oder Biofakt 66 The Tissue Culture & Art Project (Oron Catts & Ionat Zurr), A Semi-Living Worry Doll H, 2000, McCoy Zell-Linie, biologisch resorbierbares Polymer und medizinische Fäden, 2 x 1,5 x 1 cm, Installations ansicht The Tissue Culture & Art(ificial) Wombs Installation, Ars Electronica 2000 letztgenannten Arbeiten liegt der Fokus neben der Integration lebenden ‚Materials‘ auf der Verbindung bzw. versuchten ‚Aussöhnung‘ von Natur und Technik/Medien/Konsumgut. Die Liste an künstlerischen Positionen, die mit Tieren und Pflanzen arbeiten, ließe sich fortsetzen. Jüngere Beispiele, darunter Carsten Höller & Rosemarie Trockel, Revital Cohen & Tuur van Balen, Damien Hirst, Tomas Sarraceno, Oron Catts & Ionat Zurr (Abb. 66) kennzeichnet einerseits ein systemästhetischer Charakter, andererseits eröffnen sie teils neue Felder wie Bio Art oder Transgenic Art, indem sie Pflanzen und Tiere ausstellen und genetisch in deren Wachstum eingreifen. Louis Bec fragte: „will people performing cloning operations become the sculptors of tomorrow?“340 Eduardo Kac zählt mit seinem fluoreszierenden Kaninchen zu den bekanntesten Beispielen. In Forschungszweigen wie den Animal Studies oder posthumanistischen Theorien werden diese Arbeiten aus ethischen, soziologischen, philosophischen und kulturanthropologischen Perspektiven diskutiert.341 In der Systemtheorie, wie sie Haacke und Burnham anfänglich verstanden, eröffnet sich eine Denkrichtung, die Polaritäten des ‚Natürlichen‘ und des ‚Sozialen‘ abzulegen, stattdessen multidirektional zu verflechten. Ist es ethisch vertretbar, Lebewesen zu einem Werkbestandteil zu machen? Zu 340 Bec 2007, S. 91. 341 Exemplarisch für dieses umfassende, zunehmend wachsende Forschungsfeld sei Folgendes genannt: Einen Überblick bietet Zaunschirm 2005; ders., „Im Zoo der Kunst II“, in: Kunstforum International, 175, 2005, S. 36–95. Vgl. auch Arnaud Gerspacher, „Animal Art (1987) and the Split Origins of Bioart“, in: Charissa N. Terranova/Meredith Tromble (Hg.), The Routledge Companion to Biology in Art and Architecture, New York & London: Routledge 2017, S. 317–335. Siehe u.a. Lange-Berndt 2009. 120 Die Non-Human Living Sculpture untersuchen wäre, inwiefern sich seit dem ersten Earth Day 1970 und der zunehmenden Umweltkrise der Umgang mit Tieren und Pflanzen ändert. Seit Ende der 1960er-Jahre entstehen zunehmend künstlerische Konzepte mit ökologischem Impetus – wie die Projekte von Helen Mayer Harrison und Newton Harrison (u.a. eine Auflistung gefährdeter Spezies weltweit für die Ausstellung Fur and Feathers im Museum of Crafts in New York 1971 oder Lagoon Cycle 1974–1986) –, die Pflanzen und Tiere unabhängig von ihren äußeren Merkmalen integrieren. 121
https://openalex.org/W3194660841	https://www.frontiersin.org/articles/10.3389/fpls.2021.715694/pdf	English	null	NRT1.1 Dual-Affinity Nitrate Transport/Signalling and its Roles in Plant Abiotic Stress Resistance	Frontiers in plant science	2,021	cc-by	12,688	REVIEW published: 23 August 2021 doi: 10.3389/fpls.2021.715694 NRT1.1 Dual-Affinity Nitrate Transport/Signalling and its Roles in Plant Abiotic Stress Resistance Xian Zhi Fang 1, Shu Qin Fang 1, Zheng Qian Ye 1, Dan Liu 1, Ke Li Zhao 1 and Chong Wei Jin 2 1 Key Laboratory of Soil Contamination Bioremediation of Zhejiang Province, State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Zhejiang, China, 2 State Key Laboratory of Plant Physiology and Biochemistry, College of Natural Resources and Environmental Science, Zhejiang University, Hangzhou, China NRT1.1 is the first nitrate transport protein cloned in plants and has both high- and low-affinity functions. It imports and senses nitrate, which is modulated by the phosphorylation on Thr101 (T101). Structural studies have revealed that the phosphorylation of T101 either induces dimer decoupling or increases structural flexibility within the membrane, thereby switching the NRT1.1 protein from a low- to high-affinity state. Further studies on the adaptive regulation of NRT1.1 in fluctuating nitrate conditions have shown that, at low nitrate concentrations, nitrate binding only at the high-affinity monomer initiates NRT1.1 dimer decoupling and priming of the T101 site for phosphorylation activated by CIPK23, which functions as a high-affinity nitrate transceptor. However, nitrate binding in both monomers retains the unmodified NRT1.1, maintaining the low-affinity mode. This NRT1.1-mediated nitrate signalling and transport may provide a key to improving the efficiency of plant nitrogen use. However, recent studies have revealed that NRT1.1 is extensively involved in plant tolerance of several adverse environmental conditions. In this context, we summarise the recent progress in the molecular mechanisms of NRT1.1 dual-affinity nitrate transport/signalling and focus on its expected and unexpected roles in plant abiotic stress resistance and their regulation processes. Edited by: Vicent Arbona, University of Jaume I, Spain Reviewed by: Amita Pandey, Shriram Institute for Industrial Research, India Mohsin Tanveer, University of Tasmania, Australia Correspondence: Xian Zhi Fang fangxz@zafu.edu.cn Edited by: Vicent Arbona, University of Jaume I, Spain Reviewed by: Amita Pandey, Shriram Institute for Industrial Research, India Mohsin Tanveer, University of Tasmania, Australia Correspondence: Xian Zhi Fang fangxz@zafu.edu.cn Correspondence: Xian Zhi Fang fangxz@zafu.edu.cn Keywords: abiotic stress, nitrate transporter 1.1, dual-affinity, nitrate transport, nitrate signalling Specialty section: This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science INTRODUCTION Nitrogen (N) is a primary constituent of proteins and nucleotides that are essential for life. Nitrogen accounts for approximately 2–5% of the total dry biomass of plants (Xu et al., 2012). Nitrate (NO3 −) is a major source of nitrogen in most plants grown in agricultural and natural systems (Wang et al., 2018). As plants have adapted to variable soil nitrate concentrations, sophisticated nitrate transporter systems have evolved. During the past two decades, four families of nitrate transport proteins, namely, nitrate transporter 1 (NRT1), nitrate transporter 2 (NRT2), chloride channel (CLC), and slow anion channel associated homologues (SLAC/SLAH), have been identified in higher plants (Krapp et al., 2014). Among these, NRT1.1, which has multiple functions, is one of the most well-studied. Initially, NRT1.1 was characterised as a dual-affinity nitrate transporter involved in nitrate uptake by roots, as well as root-to-shoot nitrate translocation in Arabidopsis (Liu et al., 1999; Léran et al., 2013). Independent of its transport function, Received: 27 May 2021 Accepted: 02 August 2021 Published: 23 August 2021 Keywords: abiotic stress, nitrate transporter 1.1, dual-affinity, nitrate transport, nitrate signalling Contribution of NRT1.1 Dual-Affinity to Nitrate Uptake in Plantsifi NRT1.1 was first characterised as a low-affinity nitrate transporter (LAT), as disruption of NRT1.1 function in nrt1.1 mutants led to a >80% decrease in nitrate uptake in sufficient nitrate (25 mm KNO3) growth medium compared with that of the wild-type plants (Huang et al., 1996). Consistent with this result, a recent study by Ye et al. (2019) reported that the nrt1.1 mutants showed approximately 50% less nitrate uptake than the wild type under 4 mm nitrate conditions, indicating that the contribution of LATS of NRT1.1 at high nitrate supply was at least 50%. However, when nitrate levels were below 0.25 mm, NRT1.1 was shown to act as a high-affinity nitrate transporter (HAT) and NRT1.1 was demonstrated to be responsible for approximately 75% of HATS in Arabidopsis (Liu et al., 1999). Subsequent analysis of nitrate uptake activity in plants and Xenopus oocytes revealed that NRT1.1 has a biphasic nitrate uptake kinetic feature, in which the affinity switch is regulated by the phosphorylation on the T101 residue of the NRT1.1 protein (Liu and Tsay, 2003; Rashid et al., 2018), and these findings provided the underlying operating mechanism of NRT1.1 dual-affinity activity. Notably, investigators in some later studies questioned the contribution of the HATS of NRT1.1 to nitrate uptake under low nitrate conditions (Glass and Kotur, 2013; Noguero et al., 2018); for example, functional disruption of NRT2.1 in plants resulted in a 96% reduction in the HATS influx of nitrate (Yong et al., 2010; Kotur et al., 2012), indicating that the contribution of the HATS of NRT1.1 at low nitrate supply was <4% of the wild-type uptake. Intriguingly, Ye et al. (2019) recently re-evaluated the role of NRT1.1 in nitrate uptake in Arabidopsis under low nitrate supply by generating a nrt1.1/2.1/2.2 triple mutant that could eliminate the contributions of NRT2.1 and NRT2.2 on the HATS influx of nitrate. The nrt1.1/2.1/2.2 triple mutant was found to have greater growth arrest and a lower rate of nitrate uptake than the nrt2.1/2.2 double mutants in 0.2 mm nitrate growth medium, suggesting that NRT1.1-mediated HATS is necessary for plant growth under low nitrate growth conditions. By subtracting the root nitrate uptake rate of the nrt1.1/2.1/2.2 mutants from those of the nrt2.1/2.2 mutants, the authors proposed that ~12% of the high-affinity nitrate uptake in plants was attributed to NRT1.1 in 0.2 mm nitrate growth medium (Ye et al., 2019). Contribution of NRT1.1 Dual-Affinity to Nitrate Uptake in Plantsifi Therefore, NRT1.1 is indispensable for maintaining plant growth under both high- and low nitrate growth conditions. Citation: Fang XZ, Fang SQ, Ye ZQ, Liu D, Zhao KL and Jin CW (2021) NRT1.1 Dual-Affinity Nitrate Transport/ Signalling and its Roles in Plant Abiotic Stress Resistance. Front. Plant Sci. 12:715694. doi: 10.3389/fpls.2021.715694 Front. Plant Sci. 12:715694. doi: 10.3389/fpls.2021.715694 August 2021 \| Volume 12 \| Article 715694 1 Frontiers in Plant Science \| www.frontiersin.org NRT1.1 Function in Abiotic Stress Fang et al. for nitrate (Liu and Tsay, 2003). Switching between high- and low affinity of NRT1.1 is mediated via phosphorylation modification on a key threonine residue, Thr101 (T101). Recent structural analysis revealed that the phosphorylation of T101 not only induces dimer decoupling, but also increases structural flexibility within the membrane, thereby switching the NRT1.1 protein from a low- to high-affinity state (Tsay, 2014). Further structural and biochemical modelling has uncovered a bistable control of NRT1.1-mediated nitrate signalling by activating its upstream CBL9-CIPK23 complex in response to a wide range of fluctuating soil nitrate conditions (Rashid et al., 2019). NRT1.1 was later shown to serve as a main nitrate sensor that regulates many aspects of physiological and developmental responses to nitrate, including regulating the expression levels of nitrate-related genes, modulating root system architecture, and relieving seed dormancy (Bouguyon et al., 2015). Moreover, NRT1.1 displays auxin transport activity, which relies largely on external nitrate availability in Arabidopsis (Maghiaoui et al., 2020a). In recent years, specific topics associated with the transport and sensing functions of NRT1.1 have been discussed in several excellent reviews (Sun and Zheng, 2015; Maghiaoui et al., 2020b; Vidal et al., 2020; Wang et al., 2020b). A series of studies on NRT1.1 have also provided new insights into its function in multiple abiotic stresses in plants. In this review, we briefly summarise the important milestones in the discovery process, dual-affinity features, and structural basis of the dual transport/sensing function of NRT1.1 in Arabidopsis. More importantly, we highlight the most recently characterised functions of NRT1.1 in plant abiotic stress resistance and the correlation between NRT1.1-mediated nitrate transport/signalling and different abiotic stresses, mainly in Arabidopsis (Table 1). Discovery of NRT1.1h y The active uptake of nitrate through membrane transporters via the roots is the first critical step in nitrogen acquisition. To date, many genes encoding nitrate transporters have been identified in higher plants. The first plant mutant defective in nitrate uptake, chl1-1, identified as early as 1978, showed impaired absorption of chlorate, a nitrate analogue that is toxic to plants (Doddema et al., 1978; Wen et al., 2017). However, these studies failed to isolate CHL1. In 1993, Tsay et al. successfully screened a new chlorate-resistant mutant that was an allele of chl1-1 among a pool of T-DNA-tagged transgenic plants. Further analysis of the genomic DNA flanking the T-DNA insert revealed that the target gene was mapped to the top of chromosome 1, where chl1-1 is located. Missing fragments of the CHL1 mutant were then isolated from wild- type Arabidopsis. Thus, the CHL1 gene was successfully cloned for the first time and had no significant identity to any other reported protein sequence until 1993. By comparing the predicted membrane topology with many other cotransporters in plants and animals, Tsay et al. (1993) proposed that CHL1 may encode a nitrate transporter. To further determine the function of the CHL1 protein, the authors engineered a CHL1-injected oocyte expression system which had a clear inward current of nitrate across the plasma membrane, especially at relatively low pH conditions (Tsay et al., 1993; Crawford and Glass, 1998). Therefore, this finding marks the first successful identification of the nitrate transport gene NRT1.1 (CHL1) in plants. Dual-Affinity Function of NRT1.1l PHO2 and NRT1.1 influence the transcript levels of each other NO3 − signalling Fe deficiency A lack of NRT1.1 enhanced plant tolerance to Fe deficiency; the reduced accumulation of internal nitrate in nrt1.1 mutants may impair the FIT-dependent Fe deficiency signalling pathway NO3 − signalling Structural Basis of NRT1.1 Dual-Affinity alpha helices (TMHs) that form a clearly defined cavity that TABLE 1 \| Summary of the regulatory mechanism of NRT1.1 in abiotic stress resistance. Abiotic stress types NRT1.1 Function The relation with NO3 − transport or signalling Reference H+ H+ toxicity induced NRT1.1-mediated H+-coupled NO3 − uptake, which in turn alleviated plant H+ stress by enhancing rhizosphere pH NO3 − uptake Fang et al. (2016) Na+ NRT1.1 intensified Na+ accumulation in plants grown with NO3 − but entrapped plants in a Cl−-excess status under NH4 + conditions NO3 − transport Álvarez-Aragón and Rodríguez-Navarro, (2017); Liu et al. (2020) Drought Disruption of NRT1.1 in plants reduced nitrate accumulation in guard cells and did not cause nitrate-induced membrane depolarisation, leading to smaller stomatal opening NO3 − transport Guo et al. (2003) Cd2+ Loss of NRT1.1 in plants led to decreased levels of Cd in NO3 −-containing medium; NRT1.1-mediated NO3 − allocation to roots by coordinating Cd2+ accumulation in root vacuoles, facilitating Cd2+ detoxification of the wild type NO3 − transport Mao et al. (2014); Jian et al. (2018) Zn2+ A lack of NRT1.1 function in plants led to the reduced accumulation of Zn in nrt1.1 mutants under Zn stress, thereby enhancing Zn tolerance NO3 − uptake Pan et al. (2020) Pb2+ The reduced Pb uptake in wild type was caused by the reduction of Pb bioavailability in the rhizosphere due to H+ consumption during NO3 − uptake of NRT1.1 NO3 − uptake Zhu et al. (2019) Low-K+ NRT1.1 participated in coordinating nitrate and potassium uptake and allocating plants under low-K+, which rely on the interactions between NRT1.1 and K+ channels/ transporters located in the root epidermis- cortex and central vasculature NO3 − transport Fang et al. (2020) NH4 + NH4 + toxicity was related to a nitrate- independent signalling function of NRT1.1 in Arabidopsis, characterised by reduced NH4 + accumulation and improved NH4 + metabolism, which may affect ethylene synthesis of nrt1.1 mutants NO3 − signalling Hachiya and Noguchi, (2011); Jian et al. Dual-Affinity Function of NRT1.1l (2018) P starvation PHO2 functioned as an integrator of the N availability into the PSR because the effect of N on PSR is significantly affected in PHO2 mutants. PHO2 and NRT1.1 influence the transcript levels of each other NO3 − signalling Medici et al. (2019) Fe deficiency A lack of NRT1.1 enhanced plant tolerance to Fe deficiency; the reduced accumulation of internal nitrate in nrt1.1 mutants may impair the FIT-dependent Fe deficiency signalling pathway NO3 − signalling Liu et al. (2015) TABLE 1 \| Summary of the regulatory mechanism of NRT1.1 in abiotic stress resistance. Abi ti t t NRT1 1 F ti Th l ti TABLE 1 \| Summary of the regulatory mechanism of NRT1.1 in abiotic stress resistance. Hachiya and Noguchi, (2011); Jian et al. (2018) Medici et al. (2019) Liu et al. (2015) Fe deficiency Structural Basis of NRT1.1 Dual-Affinity Dual-Affinity Function of NRT1.1l In response to fluctuations in external nitrate concentrations, two nitrate uptake systems have evolved in plants: a low-affinity transport system (LATS) and a high-affinity transport system (HATS), which are controlled through the NRT1 and NRT2 gene families, respectively (Wang et al., 2012). Interestingly, NRT1.1 is an exception, having both high- and low affinity August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 2 NRT1.1 Function in Abiotic Stress Fang et al. TABLE 1 \| Summary of the regulatory mechanism of NRT1.1 in abiotic stress resistance. Abiotic stress types NRT1.1 Function The relation with signalling H+ H+ toxicity induced NRT1.1-mediated H+-coupled NO3 − uptake, which in turn alleviated plant H+ stress by enhancing rhizosphere pH NO3 − uptake Na+ NRT1.1 intensified Na+ accumulation in plants grown with NO3 − but entrapped plants in a Cl−-excess status under NH4 + conditions NO3 − transport Drought Disruption of NRT1.1 in plants reduced nitrate accumulation in guard cells and did not cause nitrate-induced membrane depolarisation, leading to smaller stomatal opening NO3 − transport Cd2+ Loss of NRT1.1 in plants led to decreased levels of Cd in NO3 −-containing medium; NRT1.1-mediated NO3 − allocation to roots by coordinating Cd2+ accumulation in root vacuoles, facilitating Cd2+ detoxification of the wild type NO3 − transport Zn2+ A lack of NRT1.1 function in plants led to the reduced accumulation of Zn in nrt1.1 mutants under Zn stress, thereby enhancing Zn tolerance NO3 − uptake Pb2+ The reduced Pb uptake in wild type was caused by the reduction of Pb bioavailability in the rhizosphere due to H+ consumption during NO3 − uptake of NRT1.1 NO3 − uptake Low-K+ NRT1.1 participated in coordinating nitrate and potassium uptake and allocating plants under low-K+, which rely on the interactions between NRT1.1 and K+ channels/ transporters located in the root epidermis- cortex and central vasculature NO3 − transport NH4 + NH4 + toxicity was related to a nitrate- independent signalling function of NRT1.1 in Arabidopsis, characterised by reduced NH4 + accumulation and improved NH4 + metabolism, which may affect ethylene synthesis of nrt1.1 mutants NO3 − signalling P starvation PHO2 functioned as an integrator of the N availability into the PSR because the effect of N on PSR is significantly affected in PHO2 mutants. Structural Basis of NRT1.1 Dual-Affinity alpha helices (TMHs) that form a clearly defined cavity that opens towards the cytoplasmic side (Sun and Zheng, 2015; Rashid et al., 2019), within which the substrate can bind. Therefore, unmodified NRT1.1 has an inward-facing conformational state. In the crystal, the phosphorylation site, T101, is located at the N-terminal end of one TMH and is entirely buried in a hydrophobic pocket that is directly adjacent to the dimer interface. Based on data from several analyses, Sun et al. (2014) proposed that NRT1.1 adopts a dimer configuration and functions as a low-affinity transporter, whereas With the aim of further illustrating how T101 phosphorylation switches the transport affinity of NRT1.1, researchers in two independent studies revealed the crystal structure of Arabidopsis NRT1.1, suggesting a potential structural significance for phosphorylation (Parker and Newstead, 2014; Sun et al., 2014). The NRT1.1 protein crystallises with two monomers (A and B) in each asymmetric unit which are almost identical to each other and adopt the canonical major facilitator superfamily fold. Each monomer is comprised of 12 transmembrane spanning August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 3 NRT1.1 Function in Abiotic Stress Fang et al. phosphorylated NRT1.1 undergoes dimer decoupling and shows a high-affinity state. How the dimeric switch regulates the Michaelis constant (Km) of NRT1.1 remains unknown. sites. In silico mutational analyses in monomer A showed that the single amino acid mutant, Thr101Ala (which mimics the de-phosphorylated state of NRT1.1), breaks the rigid cluster that is responsible for allosteric communication into two distinct clusters, whereas the mutant Thr101Asp (which mimics the phosphorylated state of NRT1.1) maintains the intact allosteric rigid cluster. This finding is in parallel with the experimental result of Ho et al. (2009). Therefore, these results suggest that the priming of the T101 site in monomer A for the phosphorylation is allosterically triggered by the high-affinity nitrate binding, whereas in monomer B, such allosteric communication and priming are absent (Rashid et al., 2018, 2019).ht In addition to decoupling the dimer, phosphorylation of T101 can alter the localised structural properties of the dimer (Parker and Newstead, 2014). To investigate the function of T101 phosphorylation, Parker and Newstead generated a Thr101Asp mutant, which can mimic permanent phosphorylation of NRT1.1. As predicted, the Thr101Asp mutant, as NRT1.1- 101D, showed a lower melting temperature, indicating enhanced structural flexibility compared to the NRT1.1 protein of the wild type. Structural Basis of NRT1.1 Dual-Affinity Meanwhile, the nitrate transport rate of the Thr101Asp mutant was higher than that of the wild-type protein based on the liposome-based uptake assay. Thus, T101 phosphorylation increases the nitrate transport rate, which may result from the enhanced structural flexibility of the NRT1.1 protein. The seemingly contrasting conclusions of the two studies can, however, be reconciled—phosphorylation on T101 induces dimer decoupling, which might increase structural flexibility, thereby converting the low-affinity state of NRT1.1 to a high-affinity state (Figure 1; Tsay, 2014; Rashid et al., 2018). p g The NRT1.1 protein functions as a toggle shift via the phosphorylation/dephosphorylation of T101, a functional switch for regulating nitrate signalling and transport. Nitrate binding to NRT1.1 is responsible for generating special calcium waves through the action of phospholipase C, and blocking the induction of these waves could severely influence several nitrate- induced responses (Riveras et al., 2015; Armijo and Gutiérrez, 2017). For this phosphorylation, activities of the CBL9-CIPK23 complex towards NRT1.1 appear to be dependent on these calcium waves (Ho et al., 2009; Léran et al., 2015). More recently, the dimerization switch of NRT1.1 was confirmed to play an important role in creating cytoplasmic calcium waves sensed by CBL9, which activates the kinase, CIPK23, at low nitrate concentrations, which is inhibited at high nitrate concentrations (Rashid et al., 2018, 2019). Because dimerization itself can change the binding affinity of NRT1.1, the relative intermonomer dynamics were demonstrated to have strong connections with dimer coupling/decoupling. At low external nitrate concentrations, nitrate binds only to the high-affinity monomer A, which induces significant changes in collective atomic motions and causes the loss of interface area and priming dimer decoupling. The resulting conformational dynamics also reorient the nitrate-channelling helices, inhibiting nitrate binding at low-affinity monomer B. Altogether, binding of nitrate at the high-affinity monomer initiates NRT1.1 dimer decoupling and priming of the T101 site for phosphorylation activated by CIPK23 at low nitrate concentrations. This monomeric state of NRT1.1 acts as a high-affinity nitrate transceptor. However, when nitrate binds to both monomers, the dimeric state of NRT1.1 is maintained, with concurrent attenuation of CIPK23 activity, thereby regulating low-affinity nitrate signalling and transport (Figure 1). Nitrate Binding in NRT1.1 and its Biphasic Adaptive Activity p y A key question for the working mechanism of NRT1.1 is how can nitrate be recognised? The aforementioned studies on the NRT1.1 crystal implied that His356 is an important structural element for nitrate binding of NRT1.1, which was demonstrated by mutagenesis studies where H356A abolished nitrate uptake activity of NRT1.1 at high and low nitrate concentrations (Sun et al., 2014; Wen and Kaiser, 2018). Consistent with this finding, Rashid et al. (2018) carefully compared the nitrate-binding pocket composition of two monomers (A and B) in apo- and nitrate-bound crystal structures of NRT1.1, noting that nitrate binds to His356 and Thr360 through H-bonding in monomer A, and to His356 and Arg45 in monomer B. Compared with the apo-protein structure, in the nitrate-bounded protein structure, the T101 neighbourhood composition in monomer A differs by the residues Ala106 and Val163, and in protomer B, the composition differs by the residues Ala165. Furthermore, Ramachandran plot and electron density maps for NRT1.1 apo- and nitrate-bound protein showed that nitrate binding triggers large conformational changes of both the nitrate-binding residues and phosphorylation sites T101, enhancing asymmetries between the monomers, which bring a functional consequence that the affinity of monomer A has almost a 5-fold higher affinity than monomer B, indicating their differential roles in the nitrate binding of NRT1.1 (Pires and Ascher, 2016; Rashid et al., 2018). Further rigidity analysis of protein structure found that nitrate binding triggers more changes in chemical interactions in monomer A, resulting in the redistribution of rigid clusters of atoms, which form the largest rigid cluster (LRC) and interlink the nitrate-binding pocket and the phosphorylation site residues (Rashid et al., 2018, 2019). Such a rigid cluster has not been predicted in protomer B, indicating weak or absent allosteric communication between the binding and T101 Frontiers in Plant Science \| www.frontiersin.org Roles of NRT1.1 in Abiotic Stress and Their Relation to Nitrate Transporth The uptake, accumulation, and assimilation of nitrate have long been observed to be closely associated with abiotic stress (Guo et al., 2003; Luo et al., 2012; Zhang et al., 2018). As the most studied nitrate transporter, NRT1.1 has been revealed to be responsible for most of the nitrate uptake of plants via roots and root-to-shoot nitrate translocation as well as nitrate transport in guard cells (Léran et al., 2013; Zhang et al., 2018). NRT1.1-mediated nitrate transport in different tissues mainly contributes to plant growth; however, it may also hint at an August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 4 NRT1.1 Function in Abiotic Stress Fang et al. FIGURE 1 \| A model of NRT1.1-mediated biphasic control of nitrate signalling and transport. At low nitrate concentrations, nitrate binds only at the high-affinity site of monomer A, which induces asynchronous motions that initiate NRT1.1 dimer decoupling and priming of the Thr101 site for phosphorylation by the interactions with the CBL9-activated kinase, CIPK23. This phosphorylation eventually establishes a stable monomeric state of NRT1.1, which acts as a high-affinity nitrate transceptor. At high nitrate concentrations, nitrate binds to both monomers, which maintains synchronous motions that retain the dimeric state of NRT1.1 by attenuating the activity of the kinase, CIPK23, thereby regulating low-affinity nitrate signalling and transport. FIGURE 1 \| A model of NRT1.1-mediated biphasic control of nitrate signalling and transport. At low nitrate concentrations, nitrate binds only at the high-affinity site of monomer A, which induces asynchronous motions that initiate NRT1.1 dimer decoupling and priming of the Thr101 site for phosphorylation by the interactions with the CBL9-activated kinase, CIPK23. This phosphorylation eventually establishes a stable monomeric state of NRT1.1, which acts as a high-affinity nitrate transceptor. At high nitrate concentrations, nitrate binds to both monomers, which maintains synchronous motions that retain the dimeric state of NRT1.1 by attenuating the activity of the kinase, CIPK23, thereby regulating low-affinity nitrate signalling and transport. Feng et al., 2020). In conclusion, H+ in the rhizosphere induces H+-coupled NO3 − uptake by NRT1.1, thus altering the rhizosphere pH. Therefore, this function is largely attributable to the direct effect of NRT1.1 uptake activity. However, information on how plants perceive acid stress is still required in order to better understand the role of NRT1.1 in plant response to proton stress. evolutionary adaptation of plants to environmental changes. Roles of NRT1.1 in Abiotic Stress and Their Relation to Nitrate Transporth In recent years, increasing evidence has suggested that NRT1.1 is extensively involved in resolving adverse environmental conditions (Table 1). NRT1.1 has been reported to use different mechanisms to regulate plant resistance to different stresses, some of which seem to have a potential connection (Figure 2). Here, we summarise the expected and unexpected roles of NRT1.1 in plant resistance to abiotic stresses and further discuss the relationship between these regulatory mechanisms and nitrate transport mediated by NRT1.1. Drought and High Salt Stress g g Drought and high salt are two major abiotic stresses that retard plant growth and reduce crop yield. Plants grown in nature have developed unique and overlapping resistance mechanisms in response to drought and salt stress (Zhu, 2016). Although NRT1.1 has been reported to participate in plant resistance to these two types of stress, their control mechanisms seem to have no intersection. Drought stress is well known to trigger the production of abscisic acid (ABA), which in turn leads to stomatal closure and induces the expression of several stress- related genes to acquire drought resistance in plants (Mittler and Blumwald, 2015; Jogawat et al., 2021). Nevertheless, NRT1.1- regulated plant resistance to drought might not be associated with ABA, as exogenous ABA application to leaves caused no significant difference in stomatal apertures between wild-type plants and nrt1.1 mutants (Guo et al., 2003). NRT1.1 is also expressed in Arabidopsis guard cells. The nrt1.1 mutants were found to have smaller stomatal apertures and thus more drought tolerance than wild-type plants grown in the medium with nitrate, which might be due to a lack of NRT1.1 and decreased nitrate accumulation in guard cells and failed to show Proton Toxicity y NRT1.1 has been reported to contribute to the bulk of total nitrate uptake in roots via the mechanism of one nitrate ion and two protons symport across the plasmalemma (Huang et al., 1996; Wang et al., 2012). Recently, NRT1.1 was proposed to play an important role in plant tolerance to H+ toxicity. By examining the H+ tolerance of nrt1.1 knockout mutants, an uptake- and sensing-decoupled mutant, chl1-9 (which has reduced nitrate uptake but exhibits normal nitrate sensing activity; Ho et al., 2009), and wild-type plants, these nrt1.1 mutants were found to have reduced H+ tolerance compared with the wild type, indicating that nitrate uptake activity was required for the NRT1.1-conferred H+ tolerance. Further experiments in these plants also revealed that NRT1.1-conferred H+ tolerance of plants is closely related to the enhanced rhizosphere pH as a consequence of the increased nitrate absorption stimulated by H+ toxicity (Fang et al., 2016; August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 5 NRT1.1 Function in Abiotic Stress Fang et al. FIGURE 2 \| Schematic illustration of NRT1.1 nitrate transport in response to different stresses by mediating several transporters/channels. Proton toxicity (in red), lead stress (green), cadmium stress (orange), high external salt (purple), high external ammonium (blue), and low external potassium (pink). Arrows in solid lines and broken lines denote the demonstrated positive regulation and hypothetical regulation of transporters/channels by NRT1.1, respectively. Blunt arrows indicate negative regulation of targets by NRT1.1. FIGURE 2 \| Schematic illustration of NRT1.1 nitrate transport in response to different stresses by mediating several transporters/channels. Proton toxicity (in red), lead stress (green), cadmium stress (orange), high external salt (purple), high external ammonium (blue), and low external potassium (pink). Arrows in solid lines and broken lines denote the demonstrated positive regulation and hypothetical regulation of transporters/channels by NRT1.1, respectively. Blunt arrows indicate negative regulation of targets by NRT1.1. of NH4 + was abolished by the removal of Cl− but was not mitigated by Na+ removal, implying that excess Cl− rather than Na+ is responsible for NH4 +-conferred salt hypersensitivity. Because NRT1.1 also participates in root Cl− acquisition, NRT1.1 knockout in plants reduced their root Cl− uptake and alleviated NH4 +-aggravated salt stress in plants. Therefore, the potential mechanisms of NRT1.1-conferred salt stress in plants might be closely related to the form of nitrogen supplied to the growth medium. Proton Toxicity In brief, NRT1.1 intensifies Na+ accumulation in plants grown with NO3 − but entraps plants in a Cl−-excess status under NH4 + conditions. How NRT1.1 balances NO3 − and Cl− uptake in response to salt stress under conditions of different NO3 − and NH4 + levels still needs to be explored. nitrate-induced membrane depolarisation (Guo et al., 2003). This finding suggests that the inhibition of NRT1.1-mediated NO3 − transport into guard cells may enhance plant resistance to drought stress, but the mechanisms underlying this are still elusive. Notably, it was reported recently that ABA signalling negatively regulates nitrate acquisition via phosphorylation of NRT1.1 by SnRK2s in Arabidopsis under nitrogen deficiency (Su et al., 2021). Several researchers have also found that CIPK23 is involved in ABA responses (Léran et al., 2015; Reyes and Grégory, 2020; Su et al., 2021). Therefore, endogenous ABA might play an important role in modulating NRT1.1- mediated NO3 − transport during drought stress via two routes, including CIPK23 and SnRK2. Future work should concentrate on the molecular mechanisms connecting ABA to NRT1.1 under drought stress. Frontiers in Plant Science \| www.frontiersin.org Heavy Metals Stressf Although the two studies provide different, even partly conflicting, results regarding the role of NRT1.1 in mediating Cd stress response in Arabidopsis, both processes require the coordination of NO3 − transport. epidermis-cortex and central vasculature. NRT1.1-involved coordination of NO3 − and K+ uptake and allocation largely relied on the interactions between NRT1.1 and K+ channels/ transporters located in the root epidermis-cortex and central vasculature. Given that the uptake rates of NO3 − and K+ are often found to be positively correlated (Coskun et al., 2016), the activity of nitrate transporters in roots may be affected by K+, as evidenced by the observation that appropriate K+ supply clearly increased the expression of NRT1.1 in roots (Xu et al., 2020). Notably, Fang et al. (2020) revealed that these K+ uptake-related interactions are dependent on an H+- consuming mechanism associated with the H+/NO3 − symport facilitated by NRT1.1. Nevertheless, NRT1.5-involved K+ loading into the xylem was verified to be only associated with its role as a proton-coupled H+/K+ antiporter (Li et al., 2017), which is not associated with NO3 − transport. However, the detailed molecular mechanisms of such interactions in root K+ uptake, xylem K+ loading with NO3 −, and the involvement of NRT1.1 and K+ channels/transporters in this process are still unclear. p Similarly, the indirect effect of NRT1.1 nitrate transport activity was found to play a role in plant resistance to Zn stress. The lack of NRT1.1 function in nrt1.1 mutants led to reduced accumulation of Zn in both roots and shoots under Zn stress, suggesting that the modification of NRT1.1 activity might also enhance the Zn tolerance of plants in an NO3 − uptake-dependent manner (Pan et al., 2020). Notably, the mechanism by which NRT1.1 confers resistance to Pb stress in plants markedly differs from that of NRT1.1 in Cd and Zn stresses. Loss of NRT1.1 function in plants caused greater Pb toxicity and higher Pb accumulation in NO3 −- sufficient growth medium. The reduced Pb uptake in wild- type plants was further found to result from the reduction of Pb bioavailability in the rhizosphere due to H+ consumption during NO3 − uptake by NRT1.1 (Zhu et al., 2019). In addition, exogenous application of low Mo in plants has been shown to induce the transcript levels of NRT1.1 (Liu et al., 2017). Heavy Metals Stressf Heavy metals affect plant growth and development and lead to severe human health hazards through contaminated food chains. NRT1.1 has been reported to be involved in regulating plant resistance to several heavy metal stresses (Mao et al., 2014; Jian et al., 2018; Zhu et al., 2019; Pan et al., 2020). Mao et al. (2014) found that the loss of NRT1.1 in plants under Cd treatment increased biomass and caused less uptake of Cd in both roots and shoots in the presence of nitrate, whereas no difference was observed between the nrt1.1 mutants and wild-type plants in the absence of nitrate. This finding indicates that the functional disruption of NRT1.1 reduces Cd uptake, which enhances Cd tolerance based on NO3 − uptake activity. However, Jian et al. (2018) reported that wild-type plants are more Cd tolerant than the nrt1.1 mutants, as more g As the presence of nitrate enhances both root Na+ uptake and shoot Na+ accumulation in plants (Álvarez-Aragón et al., 2016), one or several nitrate transporters might modulate Na+ transport in plants. Although Na+ accumulation in the nrt1.1 mutants was significantly lower than that in wild-type plants, this difference was abolished when nitrate was removed (Álvarez- Aragón and Rodríguez-Navarro, 2017). This finding indicates that NRT1.1 either partly mediates or modulates NO3 −-dependent Na+ transport. However, a more recent study by Liu et al. (2020) proposed novel ideas of NRT1.1-conferred salt stress in plants. According to these researchers, several plant species fed NH4 + were more hypersensitive to NaCl stress and acquired more Cl− and less Na+ than those fed NO3 −. Further investigation of Arabidopsis showed that salt stress induced by the supply August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 6 NRT1.1 Function in Abiotic Stress Fang et al. Cd and nitrate are allocated to the vacuole of roots, which is correlated with transcript level repression of NRT1.5 but upregulation of NRT1.8. The distinct expression levels of NRT1.5 and NRT1.8 in the wild-type and nrt1.1 mutants also suggested that the expression of these two genes is regulated by NRT1.1 (Gojon and Gaymard, 2010). This discrepancy may be related to the variance of nitrate or iron concentrations in growth conditions between the two experiments, which are believed to markedly affect Cd uptake by roots in many studies (Yang et al., 2016; He et al., 2017; Zhu et al., 2020). Ammonium Toxicity Ammonium Toxicity Ammonium (NH4 +) can be utilised as a predominant nitrogen source in some plant ecosystems, but becomes toxic at high concentrations, especially when available as the sole nitrogen source (Gao et al., 2010; Ruan et al., 2016). The presence of an appropriate concentration of nitrate can clearly alleviate NH4 + toxicity in many plant species (Roosta and Schjoerring, 2007; Hachiya et al., 2011). However, NRT1.1-mediated nitrate uptake did not appear to play a positive role in plant tolerance to NH4 + toxicity, as the functional disruption of NRT1.1 in plants caused higher tolerance to high NH4 +, and the application of nitrate did not enhance the ammonium tolerance of nrt1.1 mutants (Hachiya and Noguchi, 2011). Therefore, a nitrate- independent function of NRT1.1 could exist. Jian et al. (2018) proposed that high NH4 + levels induced the activities of NADH- dependent glutamate dehydrogenase and glutamic-oxaloacetic transaminase in NRT1.1 knockout mutants chl1-1 and chl1-5, which reduced NH4 + accumulation and thus improved tolerance to NH4 + toxicity. Because the NRT1.1 P492L point mutant chl1-9 retains normal function in nitrate signalling, the similar sensitivity symptoms of chl1-9 and the wild type in response to high NH4 + indicate that the existence of the signalling function of NRT1.1 is sufficient to induce NH4 + toxicity. Given that the phosphorylation state and NRT1.1 protein levels in chl1-9 are similar to those of the wild type, the decreased assimilation rate of NH4 + in wild-type plants could also occur in chl1-9 mutants, which results in NH4 + toxicity (Hachiya and Noguchi, 2011; Jian et al., 2018). However, convincing Heavy Metals Stressf Collectively, these reports show that NRT1.1-associated strategies may be useful for manipulating the absorption and accumulation of heavy metals in plants; however, the chemical features of the heavy metals per se should be carefully considered. With respect to much of the progress concerning the molecular mechanisms of NRT1.1-regulated resistance to heavy metal stresses in Arabidopsis, the physiological relevance of these findings in crop species needs to be thoroughly studied. Roles of NRT1.1 in Abiotic Stress and Their Relation to Nitrate Signalling Despite the aforementioned abiotic stress, NRT1.1 also participates in a few other types of abiotic stress resistance, which may be related to nitrate signalling. However, the underlying mechanisms of the sensing function of NRT1.1, which confers resistance to abiotic stress, remain largely unclear. es of NRT1.1 in Abiotic Stress and r Relation to Nitrate Signalling g g Despite the aforementioned abiotic stress, NRT1.1 also participates in a few other types of abiotic stress resistance, which may be related to nitrate signalling. However, the underlying mechanisms of the sensing function of NRT1.1, which confers resistance to abiotic stress, remain largely unclear. Frontiers in Plant Science \| www.frontiersin.org P and Fe Deficiencyi Nutrient deficiency can seriously deter the normal growth of plants and consequently result in a reduction in crop yield (Shrestha et al., 2020). The mechanisms regulating plant responses to single nutrient stress have been documented over the past few decades (Wang and Wu, 2013; Tewari et al., 2021). However, much remains to be studied, especially if one specific component is selected as a molecular technique to improve the resistance of plants to different nutrient deficiency stresses. Interestingly, NRT1.1 has been shown to be involved not only in regulating the resistance of Arabidopsis to low-K+ stress, but also in responding to P and Fe nutrient deficiencies. In a study by Medici et al. (2015), an early nitrate-inducible transcription factor (TF), HRS1 and its close homologue HHO1, was reported to repress primary root growth caused by P deficiency, but only when nitrate is present, suggesting a complex regulation of N and P signals. In another recent study, Medici et al. (2019) found that the phosphate starvation response (PSR) can be actively controlled by N supply, and this process also relies on a combination of local and long-distance systemic nitrate signalling pathways. PHOSPHATE2 (PHO2) transcript accumulation is upregulated by nitrate depletion, which is dependent on NRT1.1. However, most PSR genes were not found to be regulated by nitrate in the PHO2 mutants, indicating that PHO2 integrates nitrate signals into the PSR. Furthermore, NRT1.1 was repressed by P starvation and PHO2 acted as a positive regulator of NRT1.1, as the transcript levels of NRT1.1 in the PHO2 mutant were lower than those in the wild type (Huang et al., 2013; Medici et al., 2015, 2019). These results p y g Liu et al. (2015) reported that the lack of NRT1.1 enhances plant tolerance to Fe deficiency stress; however, the expression of Fe acquisition related-genes FRO2, IRT1, and FIT was lower in the nrt1.1 mutants than in wild-type plants under Fe-deficient conditions, indicating that the FIT-dependent Fe deficiency signalling pathway was not involved in NRT1.1- regulated Fe deficiency responses. Because nitrate functions as a nutrient and a signalling molecule (Krouk, 2017), it is conceivable that the reduced accumulation of internal nitrate in nrt1.1 mutants may impair the FIT-dependent Fe deficiency signalling pathway. However, more detailed studies are needed to explore the mechanisms underlying the NRT1.1-regulated Fe deficiency responses. Low-K+ Stress In addition, a nitrate-inducible, GARP-type transcription repressor 1.2 (NIGT1.2) was found to modulate P and nitrate uptake in response to P starvation in Arabidopsis. Under P deficiency conditions, NIGT1.2 directly upregulated the expression of the phosphate transporter genes PHT1;1 and PHT1;4 and downregulated transcription of NRT1.1 via binding to cis-elements in their promoters. The authors also identified a similar regulatory pathway in maize (Wang et al., 2020a). Collectively, these findings highlight the complexity of the nitrate and phosphate responses, with NRT1.1 having a crucial conserved role in modulating the interaction. Further studies are needed to investigate the relevant downstream signal transduction pathways of this N–P integrator. provide important insights into the underlying molecular mechanism by which N and P signalling pathways interact. Recently, several studies have demonstrated that the dependence of PSR on nitrate availability is conserved across a wide range of plant species (Hu et al., 2019; Medici et al., 2019; Wang et al., 2020a). In rice, high nitrate supply increased P acquisition and induced the transcript levels of P transporter (PT) genes and P starvation-induced (PSI) genes, which correlates with an increase biomass of rice. However, this nitrate induction of PSI genes was found to be abolished in mutants of the OsNRT1.1B transporter, the orthologue of AtNRT1.1 in rice, indicating that the nitrate-triggered P response is dependent on OsNRT1.1B function (Hu et al., 2019). Hu et al. further found that nitrate-stimulated interaction of OsNRT1.1B with OsSPX4 facilitates the ubiquitination and degradation of the P signalling repressor protein OsSPX4, which allows the release of OsPHR2 (Zhou et al., 2008), a master TF of phosphate signalling, into the nucleus and activates the transcription of P utilization genes. Importantly, OsSPX4 was also shown to interact with and control the activity of the master TF of nitrate signalling, OsNLP3, in rice. Therefore, nitrate-stimulated degradation of OsSPX4 activates expression of phosphate and nitrate uptake genes, ensuring a coordinated utilization of N and P in plants (Hu et al., 2019; Poza-Carrión and Paz-Ares, 2019). In addition, a nitrate-inducible, GARP-type transcription repressor 1.2 (NIGT1.2) was found to modulate P and nitrate uptake in response to P starvation in Arabidopsis. Under P deficiency conditions, NIGT1.2 directly upregulated the expression of the phosphate transporter genes PHT1;1 and PHT1;4 and downregulated transcription of NRT1.1 via binding to cis-elements in their promoters. The authors also identified a similar regulatory pathway in maize (Wang et al., 2020a). Low-K+ Stress Collectively, these findings highlight the complexity of the nitrate and phosphate responses, with NRT1.1 having a crucial conserved role in modulating the interaction. Further studies are needed to investigate the relevant downstream signal transduction pathways of this N–P integrator. experimental data are still needed. Another plausible interpretation of the different tolerance to NH4 + toxicity in NRT1.1 knockout mutants chl1-1 and chl1-5 and NRT1.1 P492L point mutant chl1-9 is that they may show different capacities for NH4 + uptake. The existence of NRT1.1 plays a positive role in inducing the expression of AMT1s and NH4 + uptake (Jian et al., 2018). Although whether this mechanism is indeed involved in chl1-9 needs to be further confirmed by biological analyses, it is worth assuming that the NRT1.1 in chl1-9 is likely involved in NH4 + uptake. As a common component, CIPK23 was previously shown to directly interact with and phosphorylate the ammonium transporters AMT1; 1/2 and nitrate transporter NRT1.1, modulating their activity (Ho et al., 2009; Straub et al., 2017; Tian et al., 2021). It has been shown that the CBL9-CIPK23 complex is inhibited by NRT1.1 dimer (Rashid et al., 2018), which implies that the altered phosphorylation state of NRT1.1 in chl1-9 could affect the activity of AMT1 proteins under control of different nitrogen signals (Wu et al., 2019). Accordingly, the signalling function of NRT1.1 might play a positive role in mediating NH4 + uptake and accumulation. In addition, NRT1.1-related NH4 + toxicity has been shown to be associated with ethylene and auxin synthesis (Esteban et al., 2016; Jian et al., 2018). However, more studies are needed to elucidate how ethylene and auxin are involved in modulating the ammonium tolerance of nrt1.1 mutants. provide important insights into the underlying molecular mechanism by which N and P signalling pathways interact. Frontiers in Plant Science \| www.frontiersin.org Low-K+ Stress Low potassium (K+) concentrations in most soils often limit plant growth (Maathuis, 2009). Although many potassium channels and transporters have been identified over the past few decades (Wang and Wu, 2017). the molecular mechanisms underlying potassium transport and regulation in plants require a more complete understanding. Recently, nitrate transporter 1.5 (NRT1.5), initially characterised as a pH-dependent bidirectional nitrate transporter, has been shown to be involved in K+ allocation in plants (Drechsler et al., 2015; Li et al., 2017). Fang et al. (2020) also found that the loss of NRT1.1 in nrt1.1 mutants disturbs K+ uptake and root-to-shoot allocation, resulting in greater growth arrest under low K+ stress conditions. Further physiological and genetic evidence revealed that both the uptake and root-to-shoot allocation of K+ in wild-type plants require the expression of NRT1.1 in the root August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 7 NRT1.1 Function in Abiotic Stress Fang et al. provide important insights into the underlying molecular mechanism by which N and P signalling pathways interact. Recently, several studies have demonstrated that the dependence of PSR on nitrate availability is conserved across a wide range of plant species (Hu et al., 2019; Medici et al., 2019; Wang et al., 2020a). In rice, high nitrate supply increased P acquisition and induced the transcript levels of P transporter (PT) genes and P starvation-induced (PSI) genes, which correlates with an increase biomass of rice. However, this nitrate induction of PSI genes was found to be abolished in mutants of the OsNRT1.1B transporter, the orthologue of AtNRT1.1 in rice, indicating that the nitrate-triggered P response is dependent on OsNRT1.1B function (Hu et al., 2019). Hu et al. further found that nitrate-stimulated interaction of OsNRT1.1B with OsSPX4 facilitates the ubiquitination and degradation of the P signalling repressor protein OsSPX4, which allows the release of OsPHR2 (Zhou et al., 2008), a master TF of phosphate signalling, into the nucleus and activates the transcription of P utilization genes. Importantly, OsSPX4 was also shown to interact with and control the activity of the master TF of nitrate signalling, OsNLP3, in rice. Therefore, nitrate-stimulated degradation of OsSPX4 activates expression of phosphate and nitrate uptake genes, ensuring a coordinated utilization of N and P in plants (Hu et al., 2019; Poza-Carrión and Paz-Ares, 2019). PERSPECTIVE transporters/channels by the same kinase CIPK23 supports the aforementioned speculation that the interactions might be coordinated, or at least partially coordinated, at the molecular level. In addition, CIPK23 has also been shown to participate in the drought stress response and in the regulation of ABA responsiveness of guard cells during their closure and opening via phosphorylation and triggering the opening of the guard cell anion channels SLAC1/SLAH3 (Maierhofer et al., 2014; Reyes and Grégory, 2020). It has been reported that the CBL9-CIPK23 complex is inhibited by the dimer coupling state of NRT1.1 at high nitrate concentrations (Rashid et al., 2019), which means that it also influences the transport of other ions or the responses to certain stresses. However, much work is still needed, making use of biochemical and structural approaches to master the functional specificities that allow a single protein to regulate such diverse abiotic stresses. The dual-affinity mode of nitrate transport is one of the most outstanding functions of NRT1.1. As a result, considerable efforts have been made to characterise the structural mechanisms regulating the switch between the two states of the NRT1.1 protein. Through structural and biochemical modelling, the dimerisation state and/or structural flexibility of NRT1.1 have been proposed to play a key role in the phosphorylation- governed affinity switch. Remarkably, the sensor function of NRT1.1 also exhibits a biphasic manner, which is regulated by the phosphorylation of T101, which is controlled by the kinase CIPK23. However, many important questions remain to be addressed to further understand this unique protein. For example, with the fluctuation of nitrate concentrations in the external environment, the maintenance of dynamic balance and transition between the signalling and transport functions, and whether NRT1.1 can synchronously activate the signalling and transport functions should be addressed in future studies. As nitrate only binds to high-affinity monomer A, which initiates NRT1.1 dimer decoupling and priming of the T101 site for phosphorylation by CIPK23 in a low nitrate concentration (Rashid et al., 2018), the signalling and transport functions of both monomers in NRT1.1 at different monomeric and dimerisation states should be systematically characterised. By disrupting the dimer interface (Robertson et al., 2010), a phosphorylation-independent NRT1.1 monomer mutant may be obtained. Further structural analyses of such mutants could help to determine whether monomer B in phosphorylated NRT1.1 is functional and how the intermonomer allostery affects the levels of cytosolic calcium waves. PERSPECTIVE As the overlapping resistance processes of NRT1.1 in response to different stresses were found in different studies, the future efforts are needed to systematically investigate its detailed mechanisms in regulating a combination of two or more different abiotic stresses, which may be expected to enhance plant resistance to naturally occurring environmental conditions. Although NRT1.1 is believed to be preferentially responsible for nitrate transport and signalling, many extended roles that are involved in the regulation of diverse abiotic stresses have been determined. As previously mentioned, NRT1.1 plays a positive role in the resistance of Arabidopsis to H+, Pb2+, and low-K+ stress, and a negative role in modulating many types of stress, such as Cd2+, Zn2+, NH4 +, high-Na+, and drought stress (Figure 2). The reason why NRT1.1 can play multiple physiological roles and whether it simultaneously mediates these stress processes needs to be elucidated. The cation-anion balance process seems to be the most common mechanism whereby NRT1.1-mediated NO3 − transport modulates the synergetic transport of cations (such as H+, K+, Cd2+, Zn2+, and Na+), which theoretically might depend on the cooperation between anion transporters/channels and cation transporters/channels (Figure 2). However, there is as yet no molecular evidence for direct protein–protein interactions in this regard. Remarkably, a common signalling module, the CBL9-CIPK23 complex, has previously been shown to modulate the transport activities of AKT1, TPK (K+ channel), HAK5 (K+ transporter), IRT1 (Fe2+/Cd2+/Zn2+ transporter), AMT1.1/2 (NH4 + transporter), and NRT1.1 (NO3 − transporter), as well as the activity of FRO2 (ferric-chelate reductase), in several studies (Ragel et al., 2015; Tian et al., 2016; Straub et al., 2017; Dubeaux et al., 2018; Tang et al., 2020). Regulation of nitrate and cation To date, most advances in understanding the molecular mechanisms of NRT1.1, which regulates plant tolerance to abiotic stress, have been achieved in controlled unique laboratory conditions or a certain genotype of model plants. In rice and maize, homologues of NRT1.1 have been characterised and revealed to have nitrate transport activity, indicating a conserved function of NRT1.1 in nitrate transport across different plant species (Hu et al., 2015; Wen et al., 2017; Wang et al., 2020a). In future, the ideal NRT1.1-related traits identified in Arabidopsis will be expected to be transferred to crops and subsequently produced via myriad molecular biology methods. P and Fe Deficiencyi As previously mentioned, NRT1.1 plays a positive role in the resistance of Arabidopsis to H+, Pb2+, and low-K+ stress, and a negative role in modulating many types of stress, such as Cd2+, Zn2+, NH4 +, high-Na+, and drought stress (Figure 2). The reason why NRT1.1 can play multiple physiological roles and whether it simultaneously mediates these stress processes needs to be elucidated. The cation-anion balance process seems to be the most common mechanism whereby NRT1.1-mediated NO3 − transport modulates the synergetic transport of cations (such as H+, K+, Cd2+, Zn2+, and Na+), which theoretically might depend on the cooperation between anion transporters/channels and cation transporters/channels (Figure 2). However, there is as yet no molecular evidence for direct protein–protein interactions in this regard. Remarkably, a common signalling module, the CBL9-CIPK23 complex, has previously been shown to modulate the transport activities of AKT1, TPK (K+ channel), HAK5 (K+ transporter), IRT1 (Fe2+/Cd2+/Zn2+ transporter), AMT1.1/2 (NH4 + transporter), and NRT1.1 (NO3 − transporter), as well as the activity of FRO2 (ferric-chelate reductase), in several studies (Ragel et al., 2015; Tian et al., 2016; Straub et al., 2017; Dubeaux et al., 2018; Tang et al., 2020). Regulation of nitrate and cation AUTHOR CONTRIBUTIONS XF wrote the first draft and edited the manuscript. CJ added content and edited the manuscript. All authors contributed to the article and agreed to the submitted version. PERSPECTIVE Another equally important question that requires precise clarification is whether the nitrate perception site is the same as the transport site. NRT1.1 has been found to be expressed in the epidermis- cortex and central cylinder of mature roots as well as guard cells of shoots (Guo et al., 2003; Fang et al., 2020). Future studies should focus on the specific functions that have been ascribed to NRT1.1 in different tissues for the regulation of plant tolerance to certain environmental stresses. As NRT1.1 can act as a transceptor by sensing variations in extracellular nitrate concentrations to modulate its biphasic adaptive state (Rashid et al., 2019), it could also play a role in sensing nitrate concentrations in different organs. However, the signalling function of NRT1.1 in plant tissues in response to environmental changes remains unclear. As the overlapping resistance processes of NRT1.1 in response to different stresses were found in different studies, the future efforts are needed to systematically investigate its detailed mechanisms in regulating a combination of two or more different abiotic stresses, which may be expected to enhance plant resistance to naturally occurring environmental conditions. To date, most advances in understanding the molecular mechanisms of NRT1.1, which regulates plant tolerance to abiotic stress, have been achieved in controlled unique laboratory conditions or a certain genotype of model plants. In rice and maize, homologues of NRT1.1 have been characterised and revealed to have nitrate transport activity, indicating a conserved function of NRT1.1 in nitrate transport across different plant species (Hu et al., 2015; Wen et al., 2017; Wang et al., 2020a). In future, the ideal NRT1.1-related traits identified in Arabidopsis will be expected to be transferred to crops and subsequently produced via myriad molecular biology methods. NRT1.1 has been found to be expressed in the epidermis- cortex and central cylinder of mature roots as well as guard cells of shoots (Guo et al., 2003; Fang et al., 2020). Future studies should focus on the specific functions that have been ascribed to NRT1.1 in different tissues for the regulation of plant tolerance to certain environmental stresses. As NRT1.1 can act as a transceptor by sensing variations in extracellular nitrate concentrations to modulate its biphasic adaptive state (Rashid et al., 2019), it could also play a role in sensing nitrate concentrations in different organs. However, the signalling function of NRT1.1 in plant tissues in response to environmental changes remains unclear. P and Fe Deficiencyi Overall, a clear link was found between NO3 − and P, K, or Fe in the transport and signalling cascade (of NO3 −) coordinated via NRT1.1 in plants. However, an in-depth understanding of the effects of the crosstalk between nitrogen and one or more nutrients is still necessary, which is very important for understanding and engineering plant adaptive responses to a fluctuating nutritional environment. August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 8 NRT1.1 Function in Abiotic Stress Fang et al. PERSPECTIVE The dual-affinity mode of nitrate transport is one of the most outstanding functions of NRT1.1. As a result, considerable efforts have been made to characterise the structural mechanisms regulating the switch between the two states of the NRT1.1 protein. Through structural and biochemical modelling, the dimerisation state and/or structural flexibility of NRT1.1 have been proposed to play a key role in the phosphorylation- governed affinity switch. Remarkably, the sensor function of NRT1.1 also exhibits a biphasic manner, which is regulated by the phosphorylation of T101, which is controlled by the kinase CIPK23. However, many important questions remain to be addressed to further understand this unique protein. For example, with the fluctuation of nitrate concentrations in the external environment, the maintenance of dynamic balance and transition between the signalling and transport functions, and whether NRT1.1 can synchronously activate the signalling and transport functions should be addressed in future studies. As nitrate only binds to high-affinity monomer A, which initiates NRT1.1 dimer decoupling and priming of the T101 site for phosphorylation by CIPK23 in a low nitrate concentration (Rashid et al., 2018), the signalling and transport functions of both monomers in NRT1.1 at different monomeric and dimerisation states should be systematically characterised. By disrupting the dimer interface (Robertson et al., 2010), a phosphorylation-independent NRT1.1 monomer mutant may be obtained. Further structural analyses of such mutants could help to determine whether monomer B in phosphorylated NRT1.1 is functional and how the intermonomer allostery affects the levels of cytosolic calcium waves. Another equally important question that requires precise clarification is whether the nitrate perception site is the same as the transport site. Although NRT1.1 is believed to be preferentially responsible for nitrate transport and signalling, many extended roles that are involved in the regulation of diverse abiotic stresses have been determined. REFERENCES Alleviation of proton toxicity by nitrate uptake specifically depends on nitrate transporter 1.1 in Arabidopsis. New Phytol. 211, 149–158. doi: 10.1111/nph.13892 Léran, S., Muños, S., Brachet, C., Tillard, P., Gojon, A., and Lacombe, B. (2013). Arabidopsis NRT1.1 is a bidirectional transporter involved in root- to-shoot nitrate translocation. Mol. Plant 6, 1984–1987. doi: 10.1093/mp/ sst068 Feng, H. M., Fan, X. R., Miller, A. J., and Xu, G. H. (2020). Plant nitrogen uptake and assimilation: regulation of cellular pH homeostasis. J. Exp. Bot. 71, 4380–4392. doi: 10.1093/jxb/eraa150 Li, H., Yu, M., Du, X. Q., Wang, Z. F., Wu, W. H., Quintero, F. J., et al. (2017). NRT1.5/NPF7.3 functions as a proton-coupled H+/K+ antiporter for K+ loading into the xylem in Arabidopsis. Plant Cell 29, 2016–2026. doi: 10.1105/tpc.16.00972h Gao, Y., Li, Y., Yang, X., Li, H., Shen, Q., and Guo, S. (2010). Ammonium nutrition increases water absorption in rice seedlings (Oryza sativa L.) under water stress. Plant Soil 331, 193–201. doi: 10.1007/s11104-009-0245-1 Liu, X., Cui, H., Li, A., Zhang, M., and Teng, Y. (2015). The nitrate transporter NRT1.1 is involved in iron deficiency responses in Arabidopsis. J. Plant Nutr. Soil Sci. 178, 601–608. doi: 10.1002/jpln.201400480fi Glass, A. D., and Kotur, Z. (2013). A re-evaluation of the role of Arabidopsis NRT1.1 in high-affinity nitrate transport. Plant Physiol. 163, 1103–1106. doi: 10.1104/pp.113.229161 Liu, K. H., Huang, C. Y., and Tsay, Y. F. (1999). CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell 11, 865–874. doi: 10.1105/tpc.11.5.865 Gojon, A., and Gaymard, F. (2010). Keeping nitrate in the roots: an unexpected requirement for cadmium tolerance in plants. J. Mol. Cell Biol. 2, 299–301. doi: 10.1093/jmcb/mjq019h Liu, K. H., and Tsay, Y. F. (2003). Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO J. 22, 1005–1013. doi: 10.1093/emboj/cdg118f Guo, F. Q., Young, J., and Crawford, N. M. (2003). The nitrate transporter AtNRT1. 1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis. Plant Cell 15, 107–117. doi: 10.1105/ tpc.006312 Liu, L., Xiao, W., Li, L., Li, D. M., Gao, D. S., Zhu, C. Y., et al. (2017). Effect of exogenously applied molybdenum on its absorption and nitrate metabolism in strawberry seedlings. Plant Physiol. Biochem. 115, 200–211. doi: 10.1016/j. plaphy.2017.03.015 Hachiya, T., Mizokami, Y., Miyata, K., Tholen, D., Watanabe, C. K., and Noguchi, K. (2011). REFERENCES doi: 10.1104/pp.18.00410 Doddema, H., Hofstra, J. J., and Feenstra, W. J. (1978). Uptake of nitrate by mutants of Arabidopsis thaliana, disturbed in uptake or reduction of nitrate. I. Effect of nitrogen source during growth on uptake of nitrate and chlorate. Physiol. Plant. 43, 343–350. doi: 10.1111/j.1399-3054.1978.tb01592.x Jian, S., Luo, J., Liao, Q., Liu, Q., Guan, C., and Zhang, Z. (2019). NRT1.1 regulates nitrate allocation and cadmium tolerance in Arabidopsis. Front. Plant Sci. 10:384. doi: 10.3389/fpls.2019.00384 Jogawat, A., Yadav, B., and Chhaya., Lakra, N., and Narayan, O. P., (2021). Crosstalk between phytohormones and secondary metabolites in the drought stress tolerance of crop plants: a review. Physiol. Plant. 172, 1106–1132. doi: 10.1111/ppl.13328 Drechsler, N., Zheng, Y., Bohner, A., Nobmann, B., von Wirén, N., Kunze, R., et al. (2015). Nitrate-dependent control of shoot K homeostasis by the nitrate transporter1/peptide transporter family member NPF7.3/NRT1.5 and the stelar K+ outward rectifier SKOR in Arabidopsis. Plant Physiol. 169, 2832–2847. doi: 10.1104/pp.15.01152 Kotur, Z., Mackenzie, N., Ramesh, S., Tyerman, S. D., Kaiser, B. N., and Glass, A. D. (2012). Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1. New Phytol. 194, 724–731. doi: 10.1111/j.1469-8137.2012.04094.x Dubeaux, G., Neveu, J., Zelazny, E., and Vert, G. (2018). Metal sensing by the IRT1 transporter-receptor orchestrates its own degradation and plant metal nutrition. Mol. Cell 69, 953–964. doi: 10.1016/j.molcel.2018.02.009 Krapp, A., David, L. C., Chardin, C., Girin, T., Marmagne, A., Leprince, A. S., et al. (2014). Nitrate transport and signalling in Arabidopsis. J. Exp. Bot. 65, 789–798. doi: 10.1093/jxb/eru001 Esteban, R., Ariz, I., Cruz, C., and Moran, J. F. (2016). Mechanisms of ammonium toxicity and the quest for tolerance. Plant Sci. 248, 92–101. doi: 10.1016/j. plantsci.2016.04.008 Krouk, G. (2017). Nitrate signalling: calcium bridges the nitrate gap. Nat. Plants 3:17095. doi: 10.1038/nplants.2017.95 Fang, X. Z., Liu, X. X., Zhu, Y. X., Ye, J. Y., and Jin, C. W. (2020). The K+ and NO3 − interaction mediated by NITRATE TRANSPORTER1.1 ensures better plant growth under K+-limiting conditions. Plant Physiol. 184, 1900–1916. doi: 10.1104/pp.20.01229 Léran, S., Edel, K. H., Pervent, M., Hashimoto, K., Corratgé-Faillie, C., Offenborn, J. N., et al. (2015). Nitrate sensing and uptake in Arabidopsis are enhanced by ABI2, a phosphatase inactivated by the stress hormone abscisic acid. Sci. Signal. 8:ra43. doi: 10.1126/scisignal.aaa4829 Fang, X. Z., Tian, W. H., Liu, X. X., Lin, X. Y., Jin, C. W., and Zheng, S. J. (2016). REFERENCES Ho, C. H., Lin, S. H., Hu, H. C., and Tsay, Y. F. (2009). CHL1 functions as a nitrate sensor in plants. Cell 138, 1184–1194. doi: 10.1016/j.cell.2009.07.004 Álvarez-Aragón, R., Haro, R., Benito, B., and Rodríguez-Navarro, A. (2016). Salt intolerance in Arabidopsis: shoot and root sodium toxicity, and inhibition by sodium-plus-potassium overaccumulation. Planta 243, 97–114. doi: 10.1007/ s00425-015-2400-7 Hu, B., Jiang, Z., Wang, W., Qiu, Y., Zhang, Z., Liu, Y., et al. (2019). Nitrate– NRT1.1B–SPX4 cascade integrates nitrogen and phosphorus signalling networks h Hu, B., Jiang, Z., Wang, W., Qiu, Y., Zhang, Z., Liu, Y., et al. (2019). Nitrate– NRT1.1B–SPX4 cascade integrates nitrogen and phosphorus signalling networks in plants. Nat. Plan. Theory 5, 401–413. doi: 10.1038/s41477-019-0420-1 Álvarez-Aragón, R., and Rodríguez-Navarro, A. (2017). Nitrate-dependent shoot sodium accumulation and osmotic functions of sodium in Arabidopsis under saline conditions. Plant J. 91, 208–219. doi: 10.1111/tpj.13556 Hu, B., Wang, W., Ou, S., Tang, J., Li, H., Che, R., et al. (2015). Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nat. Genet. 47:834. doi: 10.1038/ng.3337 j Armijo, G., and Gutiérrez, R. A. (2017). Emerging players in the nitrate signalling pathway. Mol. Plant 10, 1019–1022. doi: 10.1016/j.molp.2017.07.006 Huang, N. C., Chiang, C. S., Crawford, N. M., and Tsay, Y. F. (1996). CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots. Plant Cell 8, 2183–2191. doi: 10.1105/tpc.8.12.2183 Bouguyon, E., Brun, F., Meynard, D., Kubeš, M., Pervent, M., Leran, S., et al. (2015). Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1. Nat. Plants 1:15015. doi: 10.1038/nplants.2015.15h Huang, T. K., Han, C. L., Lin, S. I., Chen, Y. J., Tsai, Y. C., Chen, Y. R., et al. (2013). Identification of downstream components of ubiquitin-conjugating enzyme PHOSPHATE2 by quantitative membrane proteomics in Arabidopsis roots. Plant Cell 25, 4044–4060. doi: 10.1105/tpc.113.115998 Coskun, D., Britto, D. T., and Kronzucker, H. J. (2016). The nitrogen–potassium intersection: membranes, metabolism, and mechanism. Plant Cell Environ. 10, 2029–2041. doi: 10.1111/pce.12671 ots. Plant Cell 25, 4044–4060. doi: 10.1105/tpc.113.115998 Crawford, N. M., and Glass, A. D. M. (1998). Molecular and physiological aspects of nitrate uptake in plants. Trends Plant Sci. 3, 389–395. doi: 10.1016/ S1360-1385(98)01311-9 Jian, S., Liao, Q., Song, H., Liu, Q., Lepo, J. E., Guan, C., et al. (2018). NRT1.1- related NH4 + toxicity is associated with a disturbed balance between NH4 + uptake and assimilation. Plant Physiol. 178, 1473–1488. FUNDING This work was financially supported by the Research and Development Fund of Zhejiang A&F University (203402009801). August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 9 NRT1.1 Function in Abiotic Stress Fang et al. expression and favoring competition between Fe and cd uptake. Plant Soil 416, 453–462. doi: 10.1007/s11104-017-3232-y expression and favoring competition between Fe and cd uptake. Plant Soil 416, 453–462. doi: 10.1007/s11104-017-3232-y REFERENCES R., Zagotta, W. N., and Zheng, N. (2014). Crystal structure of the plant dual-affinity nitrate transporter NRT1.1. Nature 507, 73–77. doi: 10.1038/nature13074 Maierhofer, T., Diekmann, M., Offenborn, J. N., Lind, C., Bauer, H., Hashimoto, K., et al. (2014). Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid. Sci. Signal. 7:ra86. doi: 10.1126/scisignal.2005703 Sun, J., and Zheng, N. (2015). Molecular mechanism underlying the plant NRT1.1 dual-affinity nitrate transporter. Front. Physiol. 6:386. doi: 10.3389/ fphys.2015.00386 Mao, Q. Q., Guan, M. Y., Lu, K. X., Du, S. T., Fan, S. K., Ye, Y. Q., et al. (2014). Inhibition of nitrate transporter 1.1-controlled nitrate uptake reduces cadmium uptake in Arabidopsis. Plant Physiol. 166, 934–944. doi: 10.1104/ pp.114.243766 Tang, R. J., Zhao, F. G., Yang, Y., Wang, C., Li, K., Kleist, T. J., et al. (2020). A calcium signalling network activates vacuolar K+ remobilization to enable plant adaptation to low-K environments. Nat. Plants 6, 384–393. doi: 10.1038/ s41477-020-0621-7 Medici, A., Marshall-Colon, A., Ronzier, E., Szponarski, W., Wang, R., Gojon, A., et al. (2015). AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip. Nat. Commun. 6:6274. doi: 10.1038/ncomms72i Tewari, R. K., Yadav, N., Gupta, R., and Kumar, P. (2021). Oxidative stress under macronutrient deficiency in plants. J. Soil Sci. Plant Nutr. 21, 832–859. doi: 10.1007/s42729-020-00405-9 Medici, A., Szponarski, W., Dangeville, P., Safi, A., Dissanayake, I. M., Saenchai, C., et al. (2019). Identification of molecular integrators shows that nitrogen actively controls the phosphate starvation response in plants. Plant Cell 31, 1171–1184. doi: 10.1105/tpc.18.00656 Tian, W. H., Ye, J. Y., Cui, M. Q., Chang, J. B., Liu, Y., Li, G. X., et al. (2021). A transcription factor STOP1-centered pathway coordinates ammonium and phosphate acquisition in Arabidopsis. Mol. Plant. doi: 10.1016/j.molp.2021.06.024 Mittler, R., and Blumwald, E. (2015). The roles of ROS and ABA in systemic acquired acclimation. Plant Cell 27, 64–70. doi: 10.1105/tpc.114.133090 Tian, Q. Y., Zhang, X. X., Yang, A., Wang, T. Z., and Zhang, W. H. (2016). CIPK23 is involved in iron acquisition of Arabidopsis by affecting ferric chelate reductase activity. Plant Sci. 246, 70–79. doi: 10.1016/j. plantsci.2016.01.010 Noguero, M., Leran, S., Bouguyon, E., Brachet, C., Tillard, P., Nacry, P., et al. (2018). Revisiting the functional properties of NPF6.3/NRT1.1/CHL1 in xenopus oocytes. BioRxiv [Preprint] hal-01777543. doi:10.1101/244467 Pan, W., You, Y., Weng, Y. N., Shentu, J. L., Lu, Q., Xu, Q. R., et al. (2020). REFERENCES Evidence for a nitrate-independent function of the nitrate sensor NRT1.1 in Arabidopsis thaliana. J. Plant Res. 124, 425–430. doi: 10.1007/s10265-010-0385-7 Liu, X. X., Zhu, Y. X., Fang, X. Z., Ye, J. Y., Du, W. X., Zhu, Q. Y., et al. (2020). Ammonium aggravates salt stress in plants by entrapping them in a chloride over-accumulation state in an NRT1.1-dependent manner. Sci. Total Environ. 746:141244. doi: 10.1016/j.scitotenv.2020.141244 Hachiya, T., and Noguchi, K. (2011). Mutation of NRT1.1 enhances ammonium/ low pH-tolerance in Arabidopsis thaliana. Plant Signal. Behav. 6, 706–708. doi: 10.4161/psb.6.5.15068 He, X. L., Fan, S. K., Zhu, J., Guan, M. Y., Liu, X. X., Zhang, Y. S., et al. (2017). Iron supply prevents cd uptake in Arabidopsis by inhibiting IRT1 Luo, B. F., Du, S. T., Lu, K. X., Liu, W. J., Lin, X. Y., and Jin, C. W. (2012). Iron uptake system mediates nitrate-facilitated cadmium accumulation in August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 10 Fang et al. NRT1.1 Function in Abiotic Stress tomato (Solanum lycopersicum) plants. J. Exp. Bot. 63, 3127–3136. doi: 10.1093/jxb/ers036 Shrestha, J., Kandel, M., Subedi, S., and Shah, K. K. (2020). Role of nutrients in rice (Oryza sativa L.): a review. Agri 9, 53–62. doi: 10.5958/2394-448X.2020.00008.5 Maathuis, F. J. M. (2009). Physiological functions of mineral macronutrients. Curr. Opin. Plant Biol. 12, 250–258. doi: 10.1016/j.pbi.2009.04.003 Straub, T., Ludewig, U., and Neuhaeuser, B. (2017). The kinase CIPK23 inhibits ammonium transport in Arabidopsis thaliana. Plant Cell 29, 409–422. doi: 10.1105/tpc.16.00806 Maghiaoui, A., Bouguyon, E., Cuesta, C., Perrine-Walker, F., Alcon, C., Krouk, G., et al. (2020a). The Arabidopsis NRT1.1 transceptor coordinately controls auxin biosynthesis and transport to regulate root branching in response to nitrate. J. Exp. Bot. 71, 4480–4494. doi: 10.1093/jxb/eraa242 Su, H., Wang, T., Ju, C. F., Deng, J. P., Zhang, T. Q., Li, M., et al. (2021). Abscisic acid signalling negatively regulates nitrate uptake via phosphorylation Su, H., Wang, T., Ju, C. F., Deng, J. P., Zhang, T. Q., Li, M., et al. (2021). Abscisic acid signalling negatively regulates nitrate uptake via phosphorylation of NRT1.1 by SnRK2s in Arabidopsis. J. Integr. Plant Biol. 63, 597–610. doi: 10.1111/jipb.13057 Maghiaoui, A., Gojon, A., and Bach, L. (2020b). NRT1.1-centered nitrate signaling in plants. J. Exp. Bot. 71, 6226–6237. doi: 10.1093/jxb/eraa361f of NRT1.1 by SnRK2s in Arabidopsis. J. Integr. Plant Biol. 63, 597–610. doi: 10.1111/jipb.13057 Sun, J., Bankston, J. R., Payandeh, J., Hinds, T. REFERENCES Zn stress facilitates nitrate transporter 1.1-mediated nitrate uptake aggravating Zn accumulation in Arabidopsis plants. Ecotoxicol. Environ. Saf. 190:110104. doi: 10.1016/j.ecoenv.2019.110104 Tsay, Y. F. (2014). Plant science: how to switch affinity. Nature 507, 44–45. doi: 10.1038/nature13063 Tsay, Y. F., Schroeder, J. I., Feldmann, K. A., and Crawford, N. M. (1993). The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate- inducible nitrate transporter. Cell 72, 705–713. doi: 10.1016/0092-8674(93)90399-B Parker, J. L., and Newstead, S. (2014). Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1. Nature 507, 68–72. doi: 10.1038/ nature13116 Vidal, E. A., Alvarez, J. M., Araus, V., Riveras, E., Brooks, M. D., Krouk, G., et al. (2020). Nitrate in 2020: thirty years from transport to signalling networks. Plant Cell 32, 2094–2119. doi: 10.1105/tpc.19.00748 Pires, D. E., and Ascher, D. B. (2016). CSM-lig: a web server for assessing and comparing protein-small molecule affinities. Nucleic Acids Res. 44, 557–561. doi: 10.1093/nar/gkw390 Wang, Y. Y., Cheng, Y. H., Chen, K. E., and Tsay, Y. F. (2018). Nitrate transport, signalling, and use efficiency. Annu. Rev. Plant Biol. 69, 85–122. doi: 10.1146/ annurev-arplant-042817-040056 Poza-Carrión, C., and Paz-Ares, J. (2019). When nitrate and phosphate sensors meet. Nat. Plants 5, 339–340. doi: 10.1038/s41477-019-0403-2 Wang, Y. Y., Hsu, P. K., and Tsay, Y. F. (2012). Uptake, allocation and signaling of nitrate. Trends Plant Sci. 17, 458–467. doi: 10.1016/j.tplants.2012.04.006 Ragel, P., Rodenas, R., Garcia-Martin, E., Andres, Z., Villalta, I., Nieves-Cordones, M., et al. (2015). The CBL-interacting protein kinase CIPK23 regulates HAK5-mediated high-affinity K+ uptake in Arabidopsis roots. Plant Physiol. 169, 2863–2873. doi: 10.1104/pp.15.01401 Wang, W., Hu, B., Li, A., and Chu, C. (2020b). NRT1.1s in plants: functions beyond nitrate transport. J. Exp. Bot. 71, 4373–4379. doi: 10.1093/jxb/erz554 Rashid, M., Bera, S., Banerjee, M., Medvinsky, A. B., Sun, G. Q., Li, B. L., et al. (2019). Feedforward control of plant nitrate transporter NRT1.1 biphasic adaptive activity. Biophys. J. 118, 898–908. doi: 10.1016/j. bpj.2019.10.018 Wang, X., Wang, H. F., Chen, Y., Sun, M. M., Wang, Y., and Chen, Y. F. (2020a). The transcription factor NIGT1.2 modulates both phosphate uptake and nitrate influx during phosphate starvation in Arabidopsis and maize. Plant Cell 32, 3519–3534. doi: 10.1105/tpc.20.00361 Rashid, M., Bera, S., Medvinsky, A. B., Sun, G. Q., Li, B. L., and Chakraborty, A. (2018). Adaptive regulation of nitrate transceptor NRT1.1 in fluctuating soil nitrate conditions. iScience 2, 41–50. doi: 10.1016/j.isci.2018.03.007 Wang, Y., and Wu, W. H. (2013). REFERENCES Potassium transport and signaling in higher plants. Annu. Rev. Plant Biol. 64, 451–476. doi: 10.1146/annurev- arplant-050312-120153 Wang, Y., and Wu, W. H. (2017). Regulation of potassium transport and signaling in plants. Curr. Opin. Plant Biol. 39, 123–128. doi: 10.1016/j.pbi.2017.06.006 Wen Z and Kaiser B N (2018) Unraveling the functional role of NPF6 Reyes, R., and Grégory, V. (2020). Regulation of root nutrient transporters by CIPK23: ‘one kinase to rule them all’. Plant Cell Physiol. pcaa156. doi: 10.1093/pcp/pcaa156 Wang, Y., and Wu, W. H. (2017). Regulation of potassium transport and signaling in plants. Curr. Opin. Plant Biol. 39, 123–128. doi: 10.1016/j.pbi.2017.06.006 Wen, Z., and Kaiser, B. N. (2018). Unraveling the functional role of NPF6 transporters. Front. Plant Sci. 9:973. doi: 10.3389/fpls.2018.00973 j Wen, Z., and Kaiser, B. N. (2018). Unraveling the functional role of NPF6 transporters. Front. Plant Sci. 9:973. doi: 10.3389/fpls.2018.00973 Riveras, E., Alvarez, J. M., Vidal, E. A., Oses, C., Vega, A., and Gutierrez, R. A. (2015). The calcium ion is a second messenger in the nitrate signalling pathway of Arabidopsis. Plant Physiol. 169, 1397–1404. doi: 10.1104/pp.15.00961 Wen, Z., Tyerman, S. D., Dechorgnat, J., Ovchinnikova, E., Dhugga, K. S., and Kaiser, B. N. (2017). Maize NPF6 proteins are homologs of Arabidopsis CHL1 that are selective for both nitrate and chloride. Plant Cell 29, 2581–2596. doi: 10.1105/tpc.16.00724 Robertson, J. L., Kolmakova-Partensky, L., and Miller, C. (2010). Design, function and structure of amonomeric ClC transporter. Nature 468, 844–847. doi: 10.1038/nature09556 Wu, X. Y., Liu, T., Zhang, Y. J., Duan, F. Y., Benjamin, N., Uwe, L., et al. (2019). Ammonium and nitrate regulate NH4 + uptake activity of Arabidopsis ammonium transporter AtAMT1;3 via phosphorylation at multiple C-terminal sites. J. Exp. Bot. 70, 4919–4930. doi: 10.1093/jxb/erz230 Roosta, H. R., and Schjoerring, J. K. (2007). Effects of ammonium toxicity on nitrogen metabolism and elemental profile of cucumber plants. J. Plant Nutr. 30, 1933–1951. doi: 10.1080/01904160701629211 Xu, X., Du, X., Wang, F., Sha, J., and Jiang, Y. (2020). Effects of potassium levels on plant growth, accumulation and distribution of carbon, and nitrate metabolism in apple dwarf rootstock seedlings. Front. Plant Sci. 11:904. doi: 10.3389/fpls.2020.00904 Ruan, L., Wei, K., Wang, L., Cheng, H., Zhang, F., Wu, L., et al. (2016). Characteristics of NH4 + and NO3 − fluxes in tea (Camellia sinensis) roots measured by scanning ion-selective electrode technique. Sci. Rep. 6:38370. REFERENCES doi: 10.1038/srep38370 August 2021 \| Volume 12 \| Article 715694 11 Frontiers in Plant Science \| www.frontiersin.org NRT1.1 Function in Abiotic Stress Fang et al. phytoremediation efficiency by improving iron status in plants. J. Hazard. Mater. 384:121473. doi: 10.1016/j.jhazmat.2019.121473 Xu, G. H., Fan, X., and Miller, A. J. (2012). Plant nitrogen assimilation and use efficiency. Annu. Rev. Plant Biol. 63, 153–182. doi: 10.1146/annurev- arplant-042811-105532 phytoremediation efficiency by improving iron status in plants. J. Hazard. Mater. 384:121473. doi: 10.1016/j.jhazmat.2019.121473 Zhu, J., Fang, X. Z., Dai, Y. J., Zhu, Y. X., Chen, H. S., Lin, X. Y., et al. (2019). Nitrate transporter 1.1 alleviates Pb toxicity in Arabidopsis by preventing rhizosphere acidification. J. Exp. Bot. 70, 6363–6374. doi: 10.1093/ jxb/erz374 Yang, Y., Xiong, J., Chen, R., Fu, G., Chen, T., and Tao, L. (2016). Excessive nitrate enhances cadmium (cd) uptake by up-regulating the expression of OsIRT1 in rice (Oryza sativa). Environ. Exp. Bot. 122, 141–149. doi: 10.1016/j. envexpbot.2015.10.001 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ye, J. Y., Tian, W. H., and Jin, C. W. (2019). A reevaluation of the contribution of NRT1.1 to nitrate uptake in Arabidopsis under low-nitrate supply. FEBS Lett. 593, 2051–2059. doi: 10.1002/1873–3468.13473 Yong, Z., Kotur, Z., and Glass, A. D. (2010). Characterization of an intact two-component high-affinity nitrate transporter from Arabidopsis roots. Plant J. 63, 739–748. doi: 10.1111/j.1365-313X.2010.04278.xh Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Zhang, G. B., Meng, S., and Gong, J. M. (2018). The expected and unexpected roles of nitrate transporters in plant abiotic stress resistance and their regulation. Int. J. Mol. Sci. 19:3535. doi: 10.3390/ijms19113535 Zhou, J., Jiao, F., Wu, Z., Li, Y., Wang, X., He, X., et al. (2ss008). OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants. Plant Physiol. 146, 1673–1686. doi: 10.1104/ pp.107.111443 Copyright © 2021 Fang, Fang, Ye, Liu, Zhao and Jin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Plant Science \| www.frontiersin.org August 2021 \| Volume 12 \| Article 715694 REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhu, J. K. (2016). Abiotic stress signaling and responses in plants. Cell 167, 313–324. doi: 10.1016/j.cell.2016.08.029 Zhu, Y. X., Du, W. X., Fang, X. Z., Zhang, L. L., and Jin, C. W. (2020). Knockdown of BTS may provide a new strategy to improve cadmium- August 2021 \| Volume 12 \| Article 715694 Frontiers in Plant Science \| www.frontiersin.org 12
https://openalex.org/W4391432123	https://www.qeios.com/read/ZD359H/pdf	English	null	Review of: "Post-Conflict Reconstruction: How Social Identity Change Informs our Understanding of the Ukrainian Experience of Forced Migration"	null	2,024	cc-by	317	Qeios, CC-BY 4.0 · Review, February 1, 2024 Review of: "Post-Conflict Reconstruction: How Social Identity Change Informs our Understanding of the Ukrainian Experience of Forced Migration" Sebastien Arcand1 1 École des Hautes Études Commerciales Sebastien Arcand1 Potential competing interests: No potential competing interests to declare. Qeios ID: ZD359H · https://doi.org/10.32388/ZD359H Potential competing interests: No potential competing interests to declare. The article Post-Conflict Reconstruction: How Social Identity Change Informs our Understanding of the Ukrainian Experience of Forced Migration explores identity change in 13 women who forcibly fled Ukraine in 2022. Taking a social identity approach to identity change in the context of migration, the author provides a solid conceptual framework for a clear and relevant analysis of the data collected. The literature review is fairly up-to-date, drawing on major works in the field of social identity. The methodological approach is well explained. However, the real contribution of the talking stone approach is questioned. Apart from what is said about what it can bring to the interviewees, we don't know the full extent of the contribution of this approach to the data specific to this research. The exploratory approach is well established, and the themes identified as a result of the analysis are relevant and original, yet aligned with the conceptual elements selected. It would have been interesting to have more analysis of the fact that the sample is made up of 13 women. Is it possible to add a gender reading to the analyses presented? How can the migratory experience and changes in identity be explained, or not, in part through these women's experiences? In closing, we'd like to reiterate that this is a very interesting article, both for its topicality and for the points it raises. With the increase in migratory flows, particularly of refugees fleeing war zones and/or natural disasters, it's clear that we need more studies like this one. Qeios ID: ZD359H · https://doi.org/10.32388/ZD359H 1/1
https://openalex.org/W3023454111	https://journal.unnes.ac.id/nju/index.php/INTUISI/article/download/23573/pdf	Indonesian	null	Penerapan Model Pembelajaran Picture and Picture Berbasis Keanekaragaman Hayati Dalam Pembentukan Empati Anak Usia Dini	Intuisi	2,020	cc-by	3,906	INTUISI JURNAL PSIKOLOGI ILMIAH http://journal.unnes.ac.id/nju/index.php/INTUISI Terindeks DOAJ: 2541-2965 INTUISI JURNAL PSIKOLOGI ILMIAH http://journal.unnes.ac.id/nju/index.php/INTUISI Terindeks DOAJ: 2541-2965 INTUISI JURNAL PSIKOLOGI ILMIAH http://journal.unnes.ac.id/nju/index.php/INTUISI Terindeks DOAJ: 2541-2965 Abstrak Data awal menunjukkan bahwa masih terdapat anak usia dini yang belum menunjukkan perilaku yang mengarah pada empati terhadap orang lain, sementara perilaku empati ini sangat penting untuk membina kehidupan sosial. Keberadaan binatang dan tumbuhan akan menjadi objek yang menyenangkan bagi anak sebagai pengenalan karakter dan penyampaian pesan. Anak tersebut meniru perilaku teman maupun perilaku yang sudah dibawa dari rumah. Salah satu upaya untuk mengembangkan kemampuan empati anak usia dini adalah melalui penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati. Tujuan penelitian ini adalah untuk mendapatkan fakta dan menjelaskan tentang perbedaan kemampuan empati anak usia dini berdasarkan pada penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati. Hipotesis dalam penelitian ini adalah terdapat perbedaan kemampuan empati pada 41 anak usia dini ditinjau dari penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati. Subjek penelitian menggunakan anak usia dini di TKB. Teknik sampling menggunakan sampel jenuh. Metode pengumpulan data menggunakan Skala Empati Anak Usia Dini, serta analisis data menggunakan Independent Samples t-Test. Hasil dari penelitian ini adalah terdapat perbedaan kemampuan empati anak usia dini melalui penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati, t=2,310 dengan taraf signifikansi 0,026. Kemampuan empati anak usia dini kelompok eksperimen lebih tinggi daripada kelompok kontrol. Sejarah Artikel: Disubmit 26 Februari 2019 Direvisi 21 Maret 2020 Diterima 30 Maret 2020 Keywords: Emphaty, Children, Picture INTUISI 12 (1) (2020) INTUISI 12 (1) (2020) Alamat korespondensi: Jurusan Psikologi, Fakultas Ilmu Pendidikan Universitas Negeri Semarang, Kampus Sekaran, Gunungpati, Semarang Indonesia hennypa@mail.unnes.ac.id Fakultas Ilmu Pendidikan, Universitas Negeri Semarang, Indonesia Fakultas Ilmu Pendidikan, Universitas Negeri Semarang, Indonesia Abstract Abstract Preliminary data showed that there are still young children who have not behaved that lead to empathy for others, while empathy behavior is very important to foster social relationships. The existence of animals and plants is a pleasant object for children and a means of character recognition and delivery of messages. Such as children imitate the behaviour of friends or behaviour that occurs at home. Efforts to improve children‟s application of picture and picture models. This study aims to prove and explain the differences in early childhood empathy abilities by using biodiversity based picture and picture learning model. The hypothesis of this study is that there is difference in early childhood empathy through biodiversity based picture and picture learning method. Research subjects used 41 early childhood from kindergarten group B. Sampling technique using saturated samples. The data collection method uses early childhood emphaty scales, while data analysis uses independent sample t-test. The result of the analysis showed that there was a difference in the ability of emphaty for early childhood through the application of a biodiversity picture and picture learning model, t=2.310, with a significance probability of 0.026. Emphaty ability of the early age experimental group was higher than the control group. © 2020 Universitas Negeri Semarang © 2020 Universitas Negeri Semarang p-ISSN 2086-0803 e-ISSN 2541-2965 Alamat korespondensi: Jurusan Psikologi, Fakultas Ilmu Pendidikan Universitas Negeri Semarang, Kampus Sekaran, Gunungpati, Semarang Indonesia hennypa@mail.unnes.ac.id p-ISSN 2086-0803 e-ISSN 2541-2965 66 pandang orang lain merupakan bekal dalam memposisikan dirinya pada posisi orang lain. Kemampuan ini sangat dibutuhkan dalam hubu ngan sosial. Anak turut merasakan apa yang dirasakan temannya. Imajinasi (fantasy) merupakan aspek empati yang dominan. Kemampuan untuk seolah-olah mempunyai karakter yang dimiliki oleh tokoh pada buku maupun film. Imajinasi positif akan mengarahkan pada munculnya perilaku empati dan menolong. Buku bergambar dan film ini memiliki alur dan tema yang dapat diikuti. Aspek selanjutnya adalah perhatian empatik (empatic concern). Perasaan simpatik, belas kasihan, dan peduli pada orang lain menjadi bagian dari perhatian empatik. Perhatian empatik dapat dilihat secara gender. Anak berjenis kelamin perempuan memiliki kemampuan sosial dan prososial lebih tinggi dibanding anak laki-laki (Altay & Gure, 2012). Anak perempuan mampu berbagi dan bekerjasama. Distress pribadi merupakan pembiaran terhadap ketidaknyamanan diri yang disebabkan oleh permasalahan orang lain. Hal ini dapat terwujud dalam bentuk ketakutan, kecemasan, kegelisahan, kecemasan, kebingungan, dan kuatir kalau tidak menolong. PENDAHULUAN Empati merupakan wujud dari kepedulian terhadap sesama. Terdapat banyak alasan untuk tanggap terhadap perasaan orang lain. Orang dewasa maupun anak usia dini dapat memiliki empati. Anak usia dini merupakan anak yang berusia 0-6 tahun (Astuti, 2013). Anak usia dini merupakan anak sejak lahir hingga berusia 6 tahun (Peraturan Menteri Pendidikan dan Kebudayaan RI No. 37 Tahun 2014 Tentang Standar Nasional Pendidikan Anak Usia Dini, 2014). Dalam usia ini, anak banyak membutuhkan pendampingan dari orangtua dan orang dewasa di sekitarnya. Pola perilaku yang terbentuk tidak lepas dari proses pembelajaran dari lingkungan. Empati merupakan suatu keterampilan yang dapat dipahami dan dipelajari anak. Anak mengamati perilaku tersebut dari orang dewasa maupun teman sebaya untuk dijadikan acuan dalam berperilaku di kemudian hari. Proses ini dapat tampak secara langsung maupun tidak. Empati menjadi kajian yang tidak dapat terlepas dari anak usia dini. Empati selalu mengikuti segala aktivitas anak. Empati merupakan kemampuan untuk menangkap dan memaknai perasaan orang lain dengan berbagai masalahnya, berpikir dari pesepsi orang lain, serta menghargai berbagai jenis perasaan (Iis, 2012). Ketika anak memunculkan empati dalam dirinya, berarti anak mulai mampu memposisikan perasaannya pada perasaan orang lain. Perilaku yang muncul setelahnya adalah peduli. Empati disebut sebagai pemahaman persepsi yang mengacu pada respon emosi dan reaksi orang lain. Penulis mengumpulkan data awal di salah satu TK (Taman Kanak-kanak) di Gunungpati. Tampak anak yang masih asyik dengan dirinya tanpa peduli dengan teman sekitar. Terlihat anak mentertawakan temannya yang jatuh dan berdarah, hal ini dianggap sebagai lelucon. Dalam pembelajaran di kelas pun, beberapa anak enggan untuk meminjamkan alat tulis pada temannya. Guru kelas menceritakan bahwa pola perilaku anak sebagian besar dibawa dari rumah. Perkataan tidak pantas pun sering ditujukan pada temannya. Orangtua yang sibuk cenderung memanjakan anaknya dengan cara menuruti segala permintaan anak. Bahkan terdapat orangtua yang tidak terima ketika anaknya dikritik. Orangtua menilai bahwa anaknya adalah anak penurut yang Aspek empati terdiri dari empati kognitif (pengambilan perspektif dan imajinasi) dan empati afektif (perhatian empatik dan distress pribadi) (Zoll & Enz, 2012). Pada pengambilan perspektif (perspective taking), kemampuan spontan anak dalam berpikir berdasarkan sudut 67 kebun binatang (Yamahashi, et al., 2015). Selain itu, binatang juga dapat diperkenalkan melalui cerita bergambar. Model pembelajaran ini bersifat aktif dengan menggunakan gambar berpasangan maupun berurutan. Contohnya menyusun gambar berurutan, menunjukkan gambar, memberi keterangan gambar, dan menjelaskan gambar. Gambar yang dipakai dapat berupa keanekaragaman hayati. Hal ini dimaksudkan agar anak dapat mengembangkan empati dalam kemasan keanekaragaman hayati. PENDAHULUAN Cinta terhadap lingkungan juga harus ditumbuhkan sejak dini. tidak mungkin melakukan hal yang tidak diinginkan oleh orang lain. Kegiatan parenting belum dapat dilakukan secara berkala oleh pihak sekolah, sehingga jika terdapat masalah, biasanya diselesaikan sendiri oleh guru. Penulis berharap anak usia dini memiliki rasa empati terhadap sesama. Empati ini akan menjadikan anak lebih peduli pada teman dan orang lain di sekitarnya. Empati merupakan suatu hal yang dapat dipelajari dan diterapkan oleh anak. Orang dewasa di sekitar anak diharapkan dapat memfasilitasi pengembangan empati ini. Penulis memilih picture and picture berbasis keanekaragaman hayati sebagai model pembelajaran. Anak akan menyukai kartu bergambar manusia, hewan, dan tumbuhan yang merupakan cerita seri. Anak diminta mengurutkan dan menceritakan apa yang tergambar dalam kartu tersebut. Kemudian peneliti mengajak diskusi tentang tema dalam cerita. Anak akan belajar hal baru tanpa merasa tertekan dan tersudut. Setelah mendapatkan data awal, penulis ingin mengetahui perbedaan empati anak usia dini berdasarkan pada penerapan media pembelajaran picture and picture berbasis keanekaragaman hayati. Jadi, terdapat kesenjangan antara harapan dan kenyataan secara nyata. Data awal menunjukkan bahwa anak usia dini di salah satu TK di Kecamatan Gunungpati belum memiliki tingkatan empati sesuai harapan penulis, yang mencakup empati kognitif (pengambilan perspektif dan imajinasi) dan empati afektif (perhatian empatik dan distress pribadi). Tujuan penelitian ini adalah untuk mendapatkan fakta dan menjelaskan tentang perbedaan kemampuan empati anak usia dini kelompok eksperimen dan kelompok kontrol berdasarkan pada penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati. Manfaat penelitian ini adalah untuk mendapatkan masukan secara empiris tentang perbedaan kemampuan empati anak usia dini berdasarkan pada penerapan metode picture and picture, sehingga dipakai sebagai dasar pemberian perlakuan dalam situasi pembelajaran di sekolah. Hipotesis yang diajukan adalah terdapat perbedaan empati anak usia dini kelompok eksperimen dan kelompok kontrol berdasarkan pada penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati. METODE Populasi merupakan wilayah generalisasi untuk diselidiki, dibatasi sebagai bagian yang setidaknya mempunyai satu sifat yang sama untuk dapat disimpulkan (Sudaryono, 2017). Populasi penelitian ini adalah anak kelompok TK B di salah satu TK di Gunungpati. Populasi berjumlah 41 anak. Sampel merupakan bagian dari populasi yang memiliki ciri-ciri sama. Sampel dalam penelitian ini adalah bagian dari populasi, yaitu anak kelompok TK B berjumlah 41 anak. Penulis memilih sampel Pembelajaran merupakan suatu proses dalam pengenalan pengetahuan baru yang merupakan hasil interaksi dari beberapa pihak. Terdapat banyak model pembelajaran yang diterapkan di Indonesia. Penerapan model pembelajaran ini juga berlaku untuk anak usia dini. Model pembelajaran picture and picture dapat diterapkan bagi anak. Pemerolehan pengetahuan dalam lingkungan informal sangat menguntungkan untuk menarik minat dan perhatian anak, misalnya mengunjungi 68 jenuh untuk teknik samplingnya. Ciri-ciri subjek pada penelitian ini adalah berusia 5-6 tahun, tinggal di Kecamatan Gunungpati, dan status siswa aktif. METODE Tabel 1 Data Demografi Subjek Penelitian Nama Usia Jenis Kelamin Agama AF 5 Tahun 3 Bulan Laki-laki Islam AB 6 Tahun Perempuan Islam AHL 5 Tahun 7 Bulan Laki-laki Islam AP 6 Tahun Laki-laki Islam AAC 5 Tahun 1 Bulan Perempuan Islam AG 5 Tahun 8 Bulan Laki-laki Islam APG 5 Tahun 4 Bulan Perempuan Islam ATF 5 Tahun 1 Bulan Laki-laki Islam BK 5 Tahun 3 Bulan Perempuan Kristen BS 5 Tahun 4 Bulan Laki-laki Islam BHL 6 Tahun Perempuan Islam BBN 5 Tahun 10 Bulan Perempuan Islam BP 5 Tahun 7 Bulan Laki-laki Islam CY 5 Tahun 1 Bulan Laki-laki Katholik CPK 5 Tahun 4 Bulan Laki-laki Islam CL 5 Tahun 9 Bulan Perempuan Islam CR 5 Tahun 3 Bulan Perempuan Islam DZ 5 Tahun Laki-laki Islam DPS 5 Tahun 4 Bulan Perempuan Islam DRT 5 Tahun 3 Bulan Perempuan Islam DW 5 Tahun 7 Bulan Perempuan Kristen ESD 5 Tahun 9 Bulan Perempuan Islam EP 5 Tahun 3 Bulan Laki-laki Islam ELK 5 Tahun 3 Bulan Perempuan Islam GD 5 Tahun 7 Bulan Laki-laki Islam HP 6 Tahun Laki-laki Islam HJD 5 Tahun 8 Bulan Laki-laki Kristen HBM 5 Tahun 2 Bulan Perempuan Islam HM 5 Tahun 10 Bulan Perempuan Islam IJG 5 Tahun 6 Bulan Laki-laki Islam JK 5 Tahun 11 Bulan Perempuan Islam KGD 5 Tahun 7 Bulan Laki-laki Islam KB 5 Tahun 3 Bulan Perempuan Islam LS 5 Tahun 4 Bulan Perempuan Islam MM 5 Tahun 5 Bulan Laki-laki Islam MD 5 Tahun 5 Bulan Perempuan Islam PDL 5 Tahun 1 Bulan Perempuan Islam RSN 5 Tahun 9 Bulan Laki-laki Islam RD 5 Tahun 4 Bulan Perempuan Islam ZM 5 Tahun Perempuan Islam ZLW 5 Tahun 8 Bulan Perempuan Islam Tabel 1 Data Demografi Subjek Penelitian 69 Berdasarkan data demografi table, maka dapat dijelaskan bahwa partisipan berjumlah 41 anak, berusia antara 5-6 tahun, berjenis kelamin laki-laki sebesar 18 anak (43,90 %) dan perempuan 23 anak (56.10 %), serta beragama Islam 37 anak (90.24 %), Kristen 3 anak (7.32 %), dan Katholik 1 anak (2.44 %). Selama kegiatan penelitian, semua partisipan dapat berpartisipasi secara aktif. empati terdiri dari empati kognitif (pengambilan perspektif dan imajinasi) dan empati afektif (perhatian empatik dan distress pribadi). Variabel dalam penelitian ini adalah empati anak usia dini sebagai variabel dependen dan model pembelajaran picture and picture berbasis keanekaragaman hayati sebagai variabel independen. METODE Metode pengumpulan data untuk mengetahui kemampuan empati anak di bawah 6 tahun dengan menggunakan Skala Empati AUD yang berjumlah 32 item. Skala Empati Anak Usia Dini merupakan skala yang disusun oleh penulis. Tinggi rendahnya empati dibuktikan dengan skor yang diperoleh. Semakin tinggi skor yang diperoleh berarti semakin tinggi empati anak usia dini, dan semakin rendah skor yang diperoleh berarti semakin rendah empati anak usia dini. Skala Empati Anak Usia Dini yang digunakan terbagi ke dalam empat alternatif jawaban, yaitu Sangat Sesuai (SS), Sesuai (S), Tidak Sesuai (TS), dan Sangat Tidak Sesuai (STS). Berdasarkan empat alternatif jawaban tersebut, maka pemberian skor pada item-item favourable bergerak dari 4-1 (dari SS sampai STS) dan untuk item-item unfavourable bergerak dari 1-4 (dari SS sampai STS). Penulis menggunakan Independent Samples t-Test untuk mengetahui perbedaan empati anak usia dini berdasarkan pada penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati. Independent Samples t-Test merupakan uji statistik parametrik untuk mengukur perbedaan mean dua kelompok data yang independen. Desain eksperimen dalam penelitian ini adalah Intact Group Comparison. X O1 O2 Gambar 1. Desain Eksperimen Intact Group Comparison Ket: O1=Hasil pengukuran setengah kelompok yang diberikan perlakuan (X). O2=Hasil pengukuran setengah kelompok yang tidak diberikan perlakuan (X). Desain eksperimen ini menggunakan kelompok eksperimen (dikenakan perlakuan) dan kelompok kontrol (tidak dikenakan perlakuan) (Sudaryono, 2017). Model pembelajaran picture and picture berbasis keanekaragaman hayati adalah model pembelajaran aktif melalui penyajian gambar makhluk hidup (manusia, hewan, dan tumbuhan) dan dipasangkan atau diurutkan secara sistematis. Perlakuan dalam penelitian ini dengan mengurutkan gambar, menunjukkan gambar, memberikan informasi tentang gambar tersebut, dan menjelaskannya. Gambar yang dipakai adalah gambar keanekaragaman hayati. Perlakuan diberikan sebanyak 15 kali, yaitu seminggu 3 kali. Hal ini dimaksudkan agar anak dapat mengembangkan empati dalam kemasan keanekaragaman hayati. Empati anak usia dini adalah kemampuan anak di bawah 6 tahun untuk menangkap dan memaknai perasaan orang lain dengan berbagai masalahnya, berpikir dari pesepsi orang lain, serta menghargai berbagai jenis perasaan. Aspek Penulis berusaha menegakkan etika penelitian, dari perizinan sampai dengan berakhirnya penelitian. Selain perizinan kepada lembaga yang terkait, penulis juga melakukan perizinan kepada orangtua, mengingat partisipan masih berusia dini. Peneliti juga tidak mengambil gambar wajah partisipan, tetapi lebih kepada proses kegiatan eksperimennya. Hasil penelitian pun 70 bersifat privasi, terutama yang menyangkut individu partisipan. bersifat privasi, terutama yang menyangkut individu partisipan. METODE Skor empati AUD yang diperoleh adalah 7 anak (35 %) berada dalam kategori rendah, 11 anak (55 %) berada dalam kategori sedang, dan 2 anak (10 %) berada dalam kategori tinggi. Apabila dilihat secara keseluruhan, maka dapat disimpulkan bahwa skor empati AUD kelas kontrol yang diperoleh subjek penelitian berada dalam kategori sedang. HASIL DAN PEMBAHASAN Deskripsi statistik variabel penelitian pada Skala Empati AUD adalah sebagai berikut: Tabel 2 Mean Empati AUD Berdasarkan Kelompok No. Empati AUD Mean Min Maks 1. Kelompok Eksperimen 87.81 55 119 2. Kelompok Kontrol 73.80 50 112 Mean Empati AUD Berdasarkan Kelompok No. Empati AUD Mean Min Maks 1. Kelompok Eksperimen 87.81 55 119 2. Kelompok Kontrol 73.80 50 112 Langkah selanjutnya adalah melakukan analisis data untuk menjawab hipotesis penelitian, yaitu Independent Sample t-Test dengan menggunakan software pengolah data statistik. Sebelumnya dilakukan uji normalitas dan uji homogenitas. Uji asumsi normalitas menggunakan Kolmogrov- Smirnov. Selanjutnya dilakukan pengelompokan pada Skala Empati AUD. Peneliti menggunakan kategorisasi sebagai berikut, rendah (x = m + -1 SD), sedang (m + -1 SD < x = m + 1 SD), dan tinggi (x > m + -1 SD). Kategori subjek digunakan untuk mengelompokkan skor dari Variabel Skala Empati AUD sebagai berikut: Tabel 3 Uji Normalitas Data Penelitian No Variabel Empati AUD Koef. Normalitas P Ket. 1. Kelompok Eksperimen 0.153 0.200 Normal 2. Kelompok Kontrol 0.152 0.200 Normal Uji Normalitas Data Penelitian Kategorisasi Skor Empati AUD Kelompok Kategorisasi Skor Empati AUD Kelompok Eksperimen (N=41) Kategorisasi Skor Empati AUD Kelompok Eksperimen (N=41) p ( ) No. Skor Kategori Frekuensi Persentase 1. x ≤ 63 Rendah 5 23.81 % 2. 64 < x ≤ 96 Sedang 6 28.57 % 3. x ≥ 97 Tinggi 10 47.62 % Uji normalitas di atas menghasilkan nilai Z sebesar 0.153 dengan p > 0.05 untuk kelompok eksperimen dan 0.152 dengan p > 0.05 untuk kelompok kontrol. Berdasarkan hasil tersebut, maka dapat dikatakan bahwa sebaran skor data tersebut normal. Skor empati AUD yang diperoleh adalah 5 anak (23.81 %) berada dalam kategori rendah, 6 anak (28.57 %) berada dalam kategori sedang, dan 10 anak (47.62 %) berada dalam kategori tinggi. Apabila dilihat secara keseluruhan, dapat disimpulkan bahwa skor empati AUD kelas eksperimen yang diperoleh subjek penelitian berada dalam kategori tinggi. Tabel 6 Uji Homogenitas Data Penelitian No. Variabel Koef. F Lavene P Ket. 1. Empati AUD 2.797 0.102 Homogen Uji homogenitas di atas menghasilkan nilai F Lavene sebesar 0.656 dengan p > 0,05. Berdasarkan hasil tersebut, maka dapat dikatakan bahwa varian dari dua atau lebih kelompok data adalah sama. Kategorisasi Skor Empati AUD Kelompok Kategorisasi Skor Empati AUD Kelompok Kontrol Kategorisasi Skor Empati AUD Kelompok Kontrol No. Skor Kategori Frekuensi Persentase 1. x ≤ 63 Rendah 7 35 % 2. HASIL DAN PEMBAHASAN 64 < x ≤ 96 Sedang 11 55 % 3. x ≥ 97 Tinggi 2 10 % Hasil uji normalitas dan homogenitas menunjukkan bahwa data yang terkumpul memenuhi syarat untuk analisis selanjutnya, 71 yaitu menggunakan Independent Sample t- Test. Hasil pengujian hipotesis dalam penelitian ini sebagai berikut: Tabel 7 Uji Beda Data Penelitian No. Variabel T P Ket 1. Empati AUD 2.310 0.026 Terdapat Perbedaan yaitu menggunakan Independent Sample t- Test. Hasil pengujian hipotesis dalam penelitian ini sebagai berikut: Tabel 7 langsung memahami makna empati melalui penyajian gambar bertema yang memuat pesan empati. Pesan tersebut akan dimaknai dan tersimpan untuk pemecahan masalah di kemudian hari. Bukan hanya orangtua, lingkungan sekitar juga turut bertanggung jawab terhadap tumbuh kembang anak, termasuk guru. (Widyasari & Zainuddin, 2018) menambahkan bahwa usia 5-7 tahun merupakan usia kritis. Guru harus memiliki persepsi yang tepat untuk perkembangan emosinya dan memberikan stimulasi yang tepat. Guru dipandang sebagai individu yang mampu memberikan pengaruh luar biasa pada anak. Berbagai model dapat guru pilih sebagai upaya mendapatkan perkembangan emosi anak yang optimal, termasuk empati. Penulis menerapkan model pembelajaran picture and picture berbasis keanekaragaman hayati untuk mengasah kemampuan empati anak usia dini. Kegemaran anak pada binatang maupun tumbuhan dapat digunakan untuk menstimulasi perkembangannya. Gambar cerita dapat membentuk konsep lingkungan yang sesungguhnya. Anak akan dibawa kepada pemahaman kondisi yang sesungguhnya (Gonen & Guler, 2011). Uji hipotesis di atas menghasilkan nilai t sebesar 2.310 dengan p < 0,05. Berdasarkan hasil tersebut, maka dapat dikatakan bahwa terdapat perbedaan yang signifikan empati anak usia dini ditinjau dari penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati. Empati anak usia dini pada kelompok eksperimen lebih tinggi daripada kelompok kontrol. Hipotesis dalam penelitian ini diterima. Sejalan dengan pendapat (Iis, 2012), empati merupakan kemampuan untuk menangkap dan memaknai perasaan orang lain dengan berbagai masalahnya, berpikir dari pesepsi orang lain, serta menghargai berbagai jenis perasaan. Ketika anak memunculkan empati dalam dirinya, berarti anak mulai mampu memposisikan perasaannya pada perasaan orang lain. Kemampuan empati tidak dengan sendirinya dimiliki oleh anak, tetapi empati merupakan suatu kemampuan yang harus dibentuk oleh lingkungan. Hal ini menjadi tugas orang dewasa yang berada di sekitar anak. Penelitian dilakukan pada orangtua dalam menstimulasi anak dengan memberikan training tentang tumbuh kembang anak (Tarnoto, Tentama, & Pranungsari, 2017). Hasilnya orangtua yang mendapatkan training tentang tumbuh kembang mempunyai kemampuan lebih dalam melakukan stimulasi anak. HASIL DAN PEMBAHASAN Berbeda dengan penelitian Tarnoto, Tentama, dan Pranungsari yang menggunakan perlakuan training tumbuh kembang pada orangtua, penelitian ini menggunakan model pembelajaran yang langsung diberikan pada anak. Anak secara Aspek empati terdiri dari empati kognitif (pengambilan perspektif dan imajinasi) dan empati afektif (perhatian empatik dan distress pribadi) (Zoll & Enz, 2012). Sepakat jika cerita bergambar dapat meningkatkan empati, imajinasi, kejujuran, solidaritas, dan mengatasi stress (Cengiz & Duran, 2017). Anak mampu mengembangan perilaku empati dengan meniru perilaku tokoh dalam cerita. Hal ini membuktikan bahwa keberadaan tokoh dan keteladanannya sangat diperlukan untuk mendampingi anak dalam mencapai kemampuan empati. Seluruh aspek ini dapat dikemas secara utuh dalam tampilan gambar yang memiliki tema dan pesan yang tersampaikan. 72 Pembelajaran dapat dilaksanakan dengan cara bekerja sama (Huda, 2011). Picture and picture adalah model pembelajaran kerjasama aktif yang menggunakan gambar berpasangan atau urutan sistematis, seperti urutan gambar, menunjukkan gambar, informasi gambar, dan menjelaskannya. Gambar yang dipakai dapat berupa keanekaragaman hayati. Hal ini dimaksudkan agar anak dapat mengembangkan empati dalam kemasan keanekaragaman hayati. Cinta terhadap lingkungan juga harus ditumbuhkan sejak dini. Anak berjenis kelamin perempuan lebih dominan memiliki kepedulian terhadap lingkungan daripada anak laki-laki, diperlukan upaya lebih untuk membuat anak laki-laki peduli pada lingkungan (Akyol, Sali, & Korukcu, 2011). SIMPULAN Pada penelitian ini terdapat perbedaan empati anak usia dini ditinjau dari penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati. Kemampuan empati anak usia dini pada kelompok eksperimen lebih tinggi daripada kelompok kontrol. Model pembelajaran ini juga mengangkat binatang dan tumbuhan sebagai objek stimulasi. Selain manusia, binatang dan tumbuhan merupakan objek yang sangat digemari oleh anak, sehingga mempermudah masuknya pesan yang disampaikan dalam cerita bergambar tersebut. Model pembelajaran picture and picture berbasis keanekaragaman hayati secara nyata dapat membuat kemampuan empati anak usia dini sesuai dengan yang diharapkan. Kemampuan empati yang terbentuk sejak dini akan menetap dan dikembangkan sampai usia dewasa nanti. Saran penulis bagi guru dan orangtua diharapkan dapat memberikan model, kesempatan dan pendampingan pada anak untuk mengembangkan kemampuan empati, serta bagi penulis selanjutnya untuk dapat memodifikasi model pembelajaran ini dalam rangka meningkatkan empati anak usia dini. Perbedaan kemampuan empati anak usia dini dapat dilihat dari mean, mean empati kelas eksperimen adalah 87.81 dan kelompok kontrol 73.80. Empati kelompok eksperimen memiliki mean lebih tinggi daripada empati kelompok kontrol. Penerapan model pembelajaran picture and picture berbasis keanekaragaman hayati efektif untuk mendapatkan kemampuan empati anak usia dini secara optimal. Kelebihan penelitian ini adalah model pembelajaran yang sangat menyenangkan, sehingga mengundang ketertarikan anak, yaitu model pembelajaran picture and picture berbasis keanekaragaman hayati. Pesan yang disampaikan lewat gambar akan sampai kepada anak. Penelitian ini juga memiliki keterbatasan, mengingat partisipan adalah anak usia dini, maka perlakuan tidak semudah yang dibayangkan. Penulis harus mengondisikan anak semaksimal mungkin, masih terdapat beberapa anak yang asyik bermain sendiri, meskipun akhirnya semua anak berhasil menyelesaikan tugas dengan tuntas. Astuti, H. P. (2013). Perkembangan Anak Usia Dini 1. Yogyakarta: Deepublish. DAFTAR PUSTAKA Akyol, A. K., Sali, G., & Korukcu, O. (2011). Children's Emphatic Tendencies with Respect to Their Gender and Grade Level. The 2011 Barcelona European Academic Conference, (pp. 1045- 1051). Barcelona. Altay, F. B., & Gure, A. (2012). Relationship among the Parenting Styles and the Social Competence and Prosocial Behaviors of the Children Who are Attending to State and Private Preschools. Educational Sciences: Theory & Practice, 2712-2718. Astuti, H. P. (2013). Perkembangan Anak Usia Dini 1. Yogyakarta: Deepublish. 73 Cengiz, S., & Duran, E. (2017). Analysis of Values on Preschool Period Children Story and Tale Books. Education and Science, 42(191), 205-233, doi: 10.15390/EB.2017.6945. Tarnoto, N., Tentama, F., & Pranungsari, D. (2017). Intervention Model of Children Growth and Development to Improve Stimulation Skills of Parent with Early Child. 3rd Asean Conference on Psychology, Counseling and Humanities (AC-PCH 2017) (pp. 218-222). Malang: Atlantis Press. Gonen, M., & Guler, T. (2011). The Environment and Its Place in Children's Picture Story Books. Procedia Social and Behavioral Sciences, 3633-3639, doi: 10.1016/j.sbspro.2011.04.347. Widyasari, C., & Zainuddin, A. (2018). Emotion Development Stimulation Method in Children 5-7 Years Old. The 3rd Progressive and Fun Education (pp. 79-83). Surabaya: Asosiasi LPTK Perguruan Tinggi Muhammadiyah (ALPTK-PTM). Huda, M. (2011). Cooperative Learning Metode, Teknik, Struktur, dan Model Penerapan. Yogyakarta: Pustaka Pelajar. Iis, N. (2012). Pengembangan Empati Anak Usia Dini melalui Mendongeng di Taman Kanak-kanak Asyiyah Pariaman. Pesona PAUD, 1(1), 1-11. Yamahashi, C., Yamaguchi, E., Inagaki, S., Okuyama, H., Tajima, J., Akiko, H., . Bando, G. (2015). Supporting Zoo Visitors' Scientific Observations through the Picture-story Show. Procedia-Social and Behavioral Sciences, 85-90, doi: 10.1016/j.sbspro.2014.12.647. Peraturan Menteri Pendidikan dan Kebudayaan RI. (2014). Peraturan Menteri Pendidikan dan Kebudayaan RI No. 37 Tahun 2014 Tentang Standar Nasional Pendidikan Anak Usia Dini. Jakarta: Kemendikbud. Zoll, C., & Enz, S. (2012). A Questionnaire to Assess Affective and Cognitive Empathy in Children. Bamberg: OPUS Publications Server. Sudaryono. (2017). Metode Penelitian. Jakarta: Rajawali Pers. 74
https://openalex.org/W3042614562	https://www.mdpi.com/2076-3425/10/7/451/pdf?version=1594779285	English	null	Deciphering the Invdupdel(8p) Genotype–Phenotype Correlation: Our Opinion	Brain sciences	2,020	cc-by	7,000	Received: 24 June 2020; Accepted: 10 July 2020; Published: 15 July 2020 Abstract: The 8p inverted duplication/deletion is a rare chromosomal rearrangement clinically featuring neurodevelopmental delay, mild to severe cognitive impairment, heart congenital defects and brain abnormalities. Patients affected also present typical facial dysmorphisms and skeletal malformations, and it is thought that the composite clinical picture may fall into the chromosomal rearrangement architecture. With the major aim of better framing its related clinical and diagnostic paths, we describe a patient carrying a de novo invdupde[8p] whose clinical features have not been described so far. Hence, through an extensive genotype–phenotype correlation analysis and by reviewing the dedicated scientific literature, we compared our patient’s features with those reported in other patients, which allows us to place our proband’s expressiveness in an intermediate area, widening the scope of the already known invdupde[8p] genotype–phenotype relationship. Keywords: invdupdel(8p); 8p23.1 sub-band; chromosome 8; genomic rearrangement; inversion; deletion; duplication; CGH-array; FISH brain sciences brain sciences Deciphering the Invdupdel(8p) Genotype–Phenotype Correlation: Our Opinion Manuela Lo Bianco 1,* , Davide Vecchio 2 , Tiziana A. Timpanaro 3, Alessia Arena 3, Marina Macchiaiolo 2, Andrea Bartuli 2, Laura Sciuto 1, Santiago Presti 1 , Sarah Sciuto 1 , Annamaria Sapuppo 1, Agata Fiumara 3, Lidia Marino 1, Giulia Messina 1 and Piero Pavone 3,* 1 Postgraduate Training Program in Pediatrics, Department of Clinical and Experimental Medicine, University of Catania, 95100 Catania, Italy; laurasciuto93@gmail.com (L.S.); santiagopresti@gmail.com (S.P.); Manuela Lo Bianco 1,* , Davide Vecchio 2 , Tiziana A. Timpanaro 3, Alessia Arena 3, Marina Macchiaiolo 2, Andrea Bartuli 2, Laura Sciuto 1, Santiago Presti 1 , Sarah Sciuto 1 , Annamaria Sapuppo 1, Agata Fiumara 3, Lidia Marino 1, Giulia Messina 1 and Piero Pavone 3,* 1 Postgraduate Training Program in Pediatrics, Department of Clinical and Experimental Medicine, University of Catania, 95100 Catania, Italy; laurasciuto93@gmail.com (L.S.); santiagopresti@gmail.com (S.P.); sarah.sciuto@gmail.com (S.S.); annamaria.pan@hotmail.it (A.S.); lidia.m@hotmail.it (L.M.); giuliamessina@hotmail.it (G.M.) 2 Manuela Lo Bianco 1,* , Davide Vecchio 2 , Tiziana A. Timpanaro 3, Alessia Arena 3, Marina Macchiaiolo 2, Andrea Bartuli 2, Laura Sciuto 1, Santiago Presti 1 , Sarah Sciuto 1 , Annamaria Sapuppo 1, Agata Fiumara 3, Lidia Marino 1, Giulia Messina 1 and Piero Pavone 3,* 1 Postgraduate Training Program in Pediatrics, Department of Clinical and Experimental Medicine, University of Catania, 95100 Catania, Italy; laurasciuto93@gmail.com (L.S.); santiagopresti@gmail.com (S.P.); sarah.sciuto@gmail.com (S.S.); annamaria.pan@hotmail.it (A.S.); lidia.m@hotmail.it (L.M.); giuliamessina@hotmail.it (G.M.) 1 Postgraduate Training Program in Pediatrics, Department of Clinical and Experimental Medicine, University of Catania, 95100 Catania, Italy; laurasciuto93@gmail.com (L.S.); santiagopresti@gmail.com (S.P.); sarah.sciuto@gmail.com (S.S.); annamaria.pan@hotmail.it (A.S.); lidia.m@hotmail.it (L.M.); giuliamessina@hotmail.it (G.M.) 2 Rare Disease and Medical Genetics, Academic Department of Pediatrics, Bambino Gesù Children’s Hospital, 00146 Rome, Italy; davide.vecchio@opbg.net (D.V.); marina.macchiaiolo@opbg.net (M.M.); andrea.bartuli@opbg.net (A.B.) 3 Department of Clinical and Experimental Medicine, University of Catania, 95100 Catania, Italy; timpanarotiziana@yahoo.it (T.A.T.); alessia.arena@gmail.com (A.A.); agataﬁumara@yahoo.it (A.F.) 3 Department of Clinical and Experimental Medicine, University of Catania, 95100 Catania, Italy; timpanarotiziana@yahoo.it (T.A.T.); alessia.arena@gmail.com (A.A.); agataﬁumara@yahoo.it (A.F.) * Correspondence: lobianco.manuela@gmail.com (M.L.B.); ppavone@unict.it (P.P.); Tel : +39-3401841225 (M L B ); +39-0953781193 (PP) timpanarotiziana@yahoo.it (T.A.T.); alessia.arena@gmail.com (A.A.); agataﬁumara@yahoo.it (A.F.) * Correspondence: lobianco.manuela@gmail.com (M.L.B.); ppavone@unict.it (P.P.); Tel.: +39-3401841225 (M.L.B.); +39-0953781193 (P.P.) * Correspondence: lobianco.manuela@gmail.com (M.L.B.); ppavone@unict.it (P.P.); Tel.: +39-3401841225 (M.L.B.); +39-0953781193 (P.P.) 2. Case Report The proband was a female admitted in our pediatric unit at the age of 8 years old. The patient is the second-born of non-consanguineous parents, both aﬀected by glaucoma while hypothyroidism was diagnosed in the mother when she was 33 years old. The family history was not informative for any genetic conditions except for a proband’s ﬁrst cousin aﬀected by Turner syndrome. She was delivered at 39 weeks of gestation (WG) by cesarean section, electively performed due to the known maternal glaucoma, and after a pregnancy complicated by multiple abortion threats and placental abruption mostly occurring during the ﬁrst trimester and dealt with using isoxsuprine hydrochloride and tranexamic acid. At the birth, her parameters were: weight 2640 g (5 ◦C), length 46 cm (<3 ◦C), head circumference 36 cm (95 ◦C); APGAR score was 9 at 1′, 10:5′. In early infancy her clinical history was characterized by growth delay and the diagnosis of hypothyroidism at the age of 2 months old when she started therapy with levotiroxine, which is still ongoing. Neurodevelopmental milestones were reached with delay; she maintained an erect position at 2 years old, reaching an autonomous deambulation at the age of 3 years old. Up to the age of 4 years old she said only a few words, but the support of logopedic rehabilitation was in the long run ineﬀective since she currently utters about 10 words and is still not able to elaborate complete meaningful sentences. At the last physical examination, her parameters were: weight 18.9 kg (<3◦pc), height 121 cm (10◦pc), head circumference 51 cm (50◦pc); skin and annexes were characterized by transient marbling cutaneous reaction with generalized hypertrichosis and signs of a previous sacrococcygeal ﬁstula on the back. While her muscular mass was hypotrophic and hypotonic, lacking in subcutaneous tissue, the skeletal examination showed an extra-rotation of the lower limbs, mainly on the left, varus position of both the knees, ﬂat foot with a pronation tendency, and hypo-eligible osteotendinous reﬂexes. Moreover, bilateral cutaneous dimples were visible on both elbows and knees, together with a shield chest with inverted nipples and winged shoulder blades. We noted several facial dysmorphisms, as follows: prominent forehead, arched eyebrow, thin nose with rounded tip and anteverse nostrils, ﬂat ﬁlter, thin downturned lips, slight micrognathia, and low-set posteriorly rotated ears. 1. Introduction The interstitial inverted duplication of the short arm of chromosome 8, associated with its terminal deletion (invdupdel[8p]), is estimated to affect 1 in 10,000 to 1 in 30,000 newborns [1]. Although few patients have been so far reported, their clinical pictures reveal a homogeneous pattern of features mostly characterized by neurodevelopmental delays/intellectual disabilities, congenital heart defects, agenesis of the corpus callosum and other forms of brain involvement (i.e., atrophy), skeletal malformations, and facial dysmorphisms. Moreover, it has been also reported that the above-mentioned severity of clinical manifestations may strictly depend on the rearrangement size due to the final expression–dosage effect of those genes harbored within [1]. By reviewing the main dedicated scientific literature, we herein describe an 8-year-old girl showing a peculiar phenotype which widens the scope of the already known invdupdel[8p] genotype–phenotype relationship. Brain Sci. 2020, 10, 451; doi:10.3390/brainsci10070451 www.mdpi.com/journal/brainsci www.mdpi.com/journal/brainsci Brain Sci. 2020, 10, 451 2 of 11 2. Case Report Finally, a single palmar crease on the right hand and a bilateral clinodactyly of the IV and V ﬁngers completed her phenotype picture. During the visit, while she had a discrete environmental involvement, she displayed emotiveness and impulsiveness, and a decreased attention span. Blood analyses tested normal except for IgE levels of 522 IU/mL, (range 0–200 UI/mL) and eosinophils 10.5% (range 0.5–5%). Regarding her neurophenotype, the brain Magnetic Resonance Imaging (MRI) performed through T1, T2, and Fluid-Attenuated Inversion Recovery (FLAIR) sequences, revealed a dysmorphic cranial conformation mostly characterized by an incomplete encephalic myelinization, slight dilatation of lateral ventricles with enhancement of liquoral spaces (Figure 1a–c), and a pineal gland’s small ectasia likely due to a partially cystic aspect (Figure 2a,b). The posterior fossa anatomy also showed a moderate cystic cisterna magna’s ectasia (with no bulk-up effect on the subtentorial structures) and a retrocerebellar cystic ectasia (Figure 3). Thus, we also performed a cine MRI which showed a liquor hydrodynamic involvement due to flow turbulence throughout III and IV ventricles (Figure 4). However, since the stroke volume value on the aqueduct of Sylvius was normal, she did not require any peritoneal ventricle derivation. Moreover, no signs of endocranial hypertension were also found at eye examination which instead revealed a pale papilla with clear boundaries and peri-papillar pigmentary ring, in addition to a global chorio-retinic dystrophia. During her follow-up she undertook several electroencephalograms (EEGs), with features that were constantly within the normal range, as were the cardiological examinations performed (echocardiographic examination included). Finally, since at a phoniatric evaluation sialorrhea and extravelic palatin tonsils were noted, she underwent a rhinofibroscopic examination which detected an isolated ogival palate and an audiometric evaluation showing a type C tympanogram with the absence of stapedial reflex on the left. 3 of 11 3 o Brain Sci. 2020, 10, 451 , Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (a), axial T2 FLAIR sequence (b), and coronal multi planar rendering (c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLA sequence (1b), and coronal multi planar rendering (1c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). 2. Case Report Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. a b Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (a), axial T2 FLAIR sequence (b), and coronal multi planar rendering (c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLA sequence (1b), and coronal multi planar rendering (1c). Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. a b Figure 2. Poly-lobed cystic pineal gland in sagittal (a) and coronal (b) T2 FLAIR sections. Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. 2a 2b Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (a), axial T2 FLAIR sequence (b), and coronal multi planar rendering (c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLA sequence (1b), and coronal multi planar rendering (1c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. a b Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (a), axial T2 FLAIR sequence (b), and coronal multi planar rendering (c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLA sequence (1b), and coronal multi planar rendering (1c). Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. a b Figure 2. Poly-lobed cystic pineal gland in sagittal (a) and coronal (b) T2 FLAIR sections. 2. Case Report Human Agilent CGH microarray kit 8 × 60 K showed a complex rearrangement on the short arm of the chromosome 8 characterized by a 6.7 Mb terminal deletion (ranged between 221,611 to 6,914,076 nucleotides) at the 8p23.3p23.1 sub-band in addition to an interstitial inverted duplication spanning the 8p23.1p12 sub-bands of 19.8 Mb that ranged between 12,583,259 to 32,380,292 nucleotides. These rearrangements and their orientations were subsequentially confirmed through Fluorescent in situ hybridization (FISH) analysis by using RP11-45O16 and RP11-139G9 locus-specific probes. Thus, since the proband’s constitutional karyotype was 46,XX,der(8)del(8)(p23.1)invdup(p12p23.1) (Table S1) and her molecular karyotype was arr8p23.2p23 (221,611-6,914,076)×1, 8p23.1p12 (12,583,259-32,380,292)×3, a final diagnosis of invdupdel[8p] syndrome was made. Figure 4. Cine MRI showing a ﬂow turbulence throughout III and IV ventricles. Human Agilent CGH microarray kit 8 × 60 K showed a complex rearrangement on the short rm of the chromosome 8 characterized by a 6.7 Mb terminal deletion (ranged between 221,611 to 914,076 nucleotides) at the 8p23.3p23.1 sub-band in addition to an interstitial inverted duplication panning the 8p23.1p12 sub-bands of 19.8 Mb that ranged between 12,583,259 to 32,380,292 nucleotides. hese rearrangements and their orientations were subsequentially confirmed through Fluorescent in situ ybridization (FISH) analysis by using RP11-45O16 and RP11-139G9 locus-specific probes. Thus, since the roband’s constitutional karyotype was 46,XX,der(8)del(8)(p23.1)invdup(p12p23.1) (Table S1) and her molecular karyotype was arr8p23.2p23 (221,611-6,914,076)×1, 8p23.1p12 (12,583,259-32,380,292)×3, a final iagnosis of invdupdel[8p] syndrome was made. Figure 4. Cine MRI showing a flow turbulence throughout III and IV ventricles. Figure 4. Cine MRI showing a ﬂow turbulence throughout III and IV ventricles. Figure 4. Cine MRI showing a flow turbulence throughout III and IV ventricles. Figure 4. Cine MRI showing a ﬂow turbulence throughout III and IV ventricles. Human Agilent CGH microarray kit 8 × 60 K showed a complex rearrangement on the short arm of the chromosome 8 characterized by a 6.7 Mb terminal deletion (ranged between 221,611 to 6,914,076 nucleotides) at the 8p23.3p23.1 sub-band in addition to an interstitial inverted duplication spanning the 8p23.1p12 sub-bands of 19.8 Mb that ranged between 12,583,259 to 32,380,292 nucleotides. These rearrangements and their orientations were subsequentially confirmed through Fluorescent in situ hybridization (FISH) analysis by using RP11-45O16 and RP11-139G9 locus-specific probes. Thus, since the proband’s constitutional karyotype was 46,XX,der(8)del(8)(p23.1)invdup(p12p23.1) (Table S1) and her molecular karyotype was arr8p23.2p23 (221,611-6,914,076)×1, 8p23.1p12 (12,583,259-32,380,292)×3, a final diagnosis of invdupdel[8p] syndrome was made. 2. Case Report Human Agilent CGH microarray kit 8 × 60 K showed a complex rearrangement on the short arm of the chromosome 8 characterized by a 6.7 Mb terminal deletion (ranged between 221,611 to 6,914,076 nucleotides) at the 8p23.3p23.1 sub-band in addition to an interstitial inverted duplication spanning the 8p23.1p12 sub-bands of 19.8 Mb that ranged between 12,583,259 to 32,380,292 nucleotides. These rearrangements and their orientations were subsequentially confirmed through Fluorescent in situ hybridization (FISH) analysis by using RP11-45O16 and RP11-139G9 locus-specific probes. Thus, since the proband’s constitutional karyotype was 46,XX,der(8)del(8)(p23.1)invdup(p12p23.1) (Table S1) and her molecular karyotype was arr8p23.2p23 (221,611-6,914,076)×1, 8p23.1p12 (12,583,259-32,380,292)×3, a final diagnosis of invdupdel[8p] syndrome was made. Human Agilent CGH microarray kit 8 × 60 K showed a complex rearrangement on the short arm of the chromosome 8 characterized by a 6.7 Mb terminal deletion (ranged between 221,611 to 6,914,076 nucleotides) at the 8p23.3p23.1 sub-band in addition to an interstitial inverted duplication spanning the 8p23.1p12 sub-bands of 19.8 Mb that ranged between 12,583,259 to 32,380,292 nucleotides. These rearrangements and their orientations were subsequentially confirmed through Fluorescent in situ hybridization (FISH) analysis by using RP11-45O16 and RP11-139G9 locus-specific probes. Thus, since the proband’s constitutional karyotype was 46,XX,der(8)del(8)(p23.1)invdup(p12p23.1) (Table S1) and her molecular karyotype was arr8p23.2p23 (221,611-6,914,076)×1, 8p23.1p12 (12,583,259-32,380,292)×3, a final diagnosis of invdupdel[8p] syndrome was made. Human Agilent CGH microarray kit 8 × 60 K showed a complex rearrangement on the short arm of the chromosome 8 characterized by a 6.7 Mb terminal deletion (ranged between 221,611 to 6,914,076 nucleotides) at the 8p23.3p23.1 sub-band in addition to an interstitial inverted duplication spanning the 8p23.1p12 sub-bands of 19.8 Mb that ranged between 12,583,259 to 32,380,292 nucleotides. These rearrangements and their orientations were subsequentially confirmed through Fluorescent in situ hybridization (FISH) analysis by using RP11-45O16 and RP11-139G9 locus-specific probes. Thus, since the proband’s constitutional karyotype was 46,XX,der(8)del(8)(p23.1)invdup(p12p23.1) (Table S1) and her molecular karyotype was arr8p23.2p23 (221,611-6,914,076)×1, 8p23.1p12 (12,583,259-32,380,292)×3, a final diagnosis of invdupdel[8p] syndrome was made. 2. Case Report Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. 2a 2b Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (a), axial T2 FLAIR sequence (b), and coronal multi planar rendering (c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FL sequence (1b), and coronal multi planar rendering (1c). Figure 1. Ventricular asymmetry (left > right) in axial T2 weighted sequence (1a), axial T2 FLAIR sequence (1b), and coronal multi planar rendering (1c). Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. a b Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. a b Figure 2. Poly-lobed cystic pineal gland in sagittal (a) and coronal (b) T2 FLAIR sections. Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. a 2b al (2a b a Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sec Figure 2. Poly-lobed cystic pineal gland in sagittal (a) and coronal (b) T2 FLAIR sections. Figure 2. Poly-lobed cystic pineal gland in sagittal (2a) and coronal (2b) T2 FLAIR sections. Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted secti Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted sec Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. Figure 3. Retrocerebellar cystic ectasia in axial T2 weighted section. 4 of 11 Brain Sci. 2020, 10, 451 Figure 4. Cine MRI showing a flow turbulence throughout III and IV ventricles. 3. Discussi 3. Discussion Inverted duplication deletion of 8p (invdupdel[8p]) is an uncommon chromosome 8 rearrangement with a rated prevalence of 1:10,000–30,000 newborns [1]. Floridia et al. proposed the generation of invdup(8) as a result of an abnormal pairing of chromosomes 8 at the time of maternal meiosis I, followed by an unequal crossover at anaphase I [2]. This mispairing through a subsequential asymmetric breakage of the dicentric chromosome can explain at the same time the observed inverted duplication and its terminal deletion onset [3]. More recently, Giglio et al. demonstrated that the unbalanced cross-over is likely due to the presence of some gene clusters (named ORDRs) spanning the 8p arm which, serving as breakpoints, are thought to be responsible for the generation of different and recurrent chromosome 8p rearrangements, as well as the supernumerary marker chromosome +der(8)(8p23.1pter) and some submicroscopic inversion polymorphisms [4,5]. The counterevidence of this origin comes from the evidence that ORDRs gene clusters are even present on 4p16, where similar inversion polymorphisms have been documented Inverted duplication deletion of 8p (invdupdel[8p]) is an uncommon chromosome 8 rearrangement with a rated prevalence of 1:10,000–30,000 newborns [1]. Floridia et al. proposed the generation of invdup(8) as a result of an abnormal pairing of chromosomes 8 at the time of maternal meiosis I, followed by an unequal crossover at anaphase I [2]. This mispairing through a subsequential asymmetric breakage of the dicentric chromosome can explain at the same time the observed inverted duplication and its terminal deletion onset [3]. More recently, Giglio et al. demonstrated that the unbalanced cross-over is likely due to the presence of some gene clusters (named ORDRs) spanning the 8p arm which, serving as breakpoints, are thought to be responsible for the generation of different and recurrent chromosome 8p rearrangements, as well as the supernumerary marker chromosome +der(8)(8p23.1pter) and some submicroscopic inversion polymorphisms [4,5]. The counterevidence of this origin comes from the evidence that ORDRs gene clusters are even present on 4p16, where similar inversion polymorphisms have been documented [6,7]. In Table 1 we report the karyotype/CGH-arrays, FISH analysis results and related phenotypes from different cases found in literature compared to our patient. 5 of 11 Brain Sci. 2020, 10, 451 Table 1. Karyotype/CGH-arrays, FISH analysis results and phenotypes reported in previous studies and in our patient. +, analysis performed; - analysis not performed; nr, data not reported. Table 1. 3. Discussi 3. Discussion Karyotype/CGH-arrays, FISH analysis results and phenotypes reported in previous studies and in our patient. +, analysis performed; - analysis not performed; nr, data not reported. Study Karyotype/CGH-Arrays FISH Phenotype Fan et al. karyotype of 46,XY,add(8)(p23) 46,XY,der(8)(qter→ q24.13::p21.3→p23.3::p23.3→qter) + Global developmental delay. Marked hypotonia, weak low cry. Bitemporal low set ears, upslanting palpable ﬁssures, wide nasal bridge, right cleft lip, micrognathia, excess nuchal skin, hypoplastic and widely spaced nipples. Left testis in the inguinal canal. Atrial septal defect, membranous ventricular septal defect, patent ductus arteriosus with a parachute mitral valve. Right pelvic dysplastic kidney and left hydronephrosis. Partial agenesis of the corpus callosum, communicating hydrocephalus, Dandy Walker malformation, intramedullary cord defect. Masuda et al. der(8) (qter→p23.1::p23.1→p12:) + Severe motor delay and mental impairment. Hypotonia. Prominent forehead, posteriorly angulated ears, broad nose with depressed nasal bridge, wide mouth, high-arched palate and downward slanting eyes. Tetralogy of Fallot (TOF). Agenesis of the corpus callosum. Vermeesch et al. 46,XX,del(8)(p23.3) inv dup(8)(p21.1p23.2)/46,XX,del(8)(p21.1) + Delayed psychomotor development. Axial hypotonia. Upward slanting palpebral ﬁssures, synophys, and left preauricular tag, low set thumbs with hypotrophic thenars, bilateral clinodactyly of the ﬁfth ﬁngers. Linear areas of depigmentation with bordering areas of hyperpigmentation on the lumbar and presacral region and on both legs. Feeding problems with gastro-esophageal reﬂux [8]. Ciccone et al. 46,XX,psu dic(8)(p23.2)/46,XX,del(8)(p23.1) + Severe mental impairment. Asymmetrical face with the left eye lower than the right, left palpebral ptosis, dental malocclusion, zygomatic arch hypoplasia, low set ears, and a short neck with webbing. Kyphoscoliosis, globous abdomen, short upper and lower limbs, premature grey hair. Cooke et al. 46,XX,der(8)dir dup(8)(p21p23.1) del(8)(p23.1p- ter).ish der(8)dir dup(8)(p21p23.1)del(8)(p23.1pter) (wcp8þ,pter -) + Global developmental delays. No meaningful speech. Poor auditory attention, impulsiveness and decreased attention span. Upward slanting palpebral ﬁssures, epicanthal folds, low columella with hypoplastic alae nasi, smooth philtrum, thin vermilion to the upper lip, high arched palate, bilateral clinodactyly. Partial complex seizures. Recurrent upper and lower respiratory tract infections. Mild degree of brain atrophy and evidence of a Dandy–Walker variant in the posterior fossa. Caglayan et al. Del 8p23.1: 6.99 Mb;Dup 8p11.2→8p23.1: 31.51 Mb nr Severe cognitive delay. Microcephaly, frontal bossing, malformed ears, thin vermilion of upper lip, abnormal maxilla and mandible, strabismus, coloboma. Corpus callosum agenesis. Buysse et al. 46,XY,der(8)(qter→q24.13::p21.3→ p23.3::p23.3→qter) Del 8p23.1l: 6.9 Mb; Dup 8p22: 3.4 Mb;Dup 8qter→24.13: 20.9 Mb + Global developmental delay. Knijnenburg et al. 46 XY 3. Discussi 3. Discussion Hypertelorism, intermittent strabismus of the left eye, hetero-chromia iridis of the right eye, upslanting palpebral ﬁssures, blue sclerae, slight retrognathia, ears posteriorly rotated with a preauricular tag on the left side. Intergluteal hairy dimple. Supravalvular pulmonary stenosis. Bilateral decreased vision with astigmatism and hypermetropia. Hand et al. Del 8p23.1: 6.8 Mb;Mosaic Del 8p21.2: 1.7Mb; Mosaic Dup p21.2→p23.1:11Mb - Cognitive, speech and motor delays. Hypotonia. Bilateral single palmar creases, no clinodactyly. Skin pigmentary abnormalities (faint lines of hyperpigmentation on the backs of the both legs). No evidence of facial dysmorphisms. Cheerful disposition, eager to please. 6 of 11 Brain Sci. 2020, 10, 451 Table 1. Cont. Table 1. Cont. Study Karyotype/CGH-Arrays FISH Phenotype Ergun et al. Del 8p23.1: 6.71 Mb;Dup 8p11.2!8p23.1: 29.26 Mb nr Absent nasal bone and clenched left hand. Enlarged thickened heart walls along with polyvalvular dysplasia. Dilatation of the main pulmonary artery and branches. History of necrotizing enterocolitis. Agenesis of the corpus callosum, enlarged third ventricle and cerebellar hypoplasia. Fisch et al. 1. arr 8p23.3p23.1(90,616-6,913,476)X1 dn,8p23.1p11.1(12,547,803–43,647,263)X23 dn,8p11.23p11.22(39,356,395– 39,505,456)X0 2. arr 8p23.3p23.1(166,252–6,913,476)X1 dn,8p23.1p11.1(12,547,803–37,028,346)X23 dn, 8p11.23p11.22(39,356,395– 39,505,456)X1 3. arr 8p23.1p21.3(8,117,071–22,366,537)X23 dn,8p11 .23p11.22(39,356,395–39,505,456)X0 4. arr 8p23.3p23.1(166,252–6,913,476)X1 dn,8p23.1p21.3(12,511,655–21,726,774)X25 dn, 8p11 .23p11.22(39,356,395– 39,505,456)X0 nr 1. Long face, wide open eyes. Lack of expressive speech and language. Mild-to-moderate autism. Hyperactivity, restlessness and impulsivity. 2. Large head and prominent forehead. Lethargic, no eye contact. Extremely limited speech/language. Lower than adequate levels of adaptive behavior. Subclinical thought problems and signiﬁcant anxious/withdrawn behaviors, psychosomatic problems and emotional lability. 3. Severely developmentally and intellectually disabled. Lower than adequate levels of adaptive behavior. Severe autism. Severe thought, withdrawn, social problems. Garcìa-Santiago et al. 1. Del 8p23.1(330,897–6,420,809): 6.09 Mb; Dup 8p12– > 8p21 (28,529,348–39,899,187): 11.37 Mb;mosaic dup 8p11.218q11 (41,348,847–48,885,448): 7.54 Mb; dup 8q24.3 (143,626,319–146,157,954): 2.53 Mb 2. Del 8p23.1 (1–6,901,486): 6.90 Mb;Dup 8p12- > 8p23.1 (12,627,630–36,027,465): 23.40 Mb 3. Del 8p23.1 (1–7,233,949): 7.3 Mb;Dup 8p12- > 8p23.1 (12,554,743–34,577,042): 22.03 Mb 4. Del 8p23.1 (1–6,925,869): 6.94 Mb;Dup 8p11.1- > 8p23.1 (12,554,743–41,232,360): 28.76 Mb 5. Del 8p23.1 (1–6,900,000): 6.90 Mb;Dup 8p12- > 8p23.1 (12,296,000–32,800,000): 20.5 Mb 6. Del 8p23.1 (1–6,900,000): 6.90 Mb;Dup 8p11.2- > 8p23.1 (12,296,000–43,700,000): 31 Mb 7. 46,XX,del(8)(p23.3) invdup(8) p21.1p23.2) 1. Mild delayed Speech development. Attention deﬁcit, hyperactivity disorder. Small pointed chin, wide nasal bridge and thick vermilion of the lower lip. Cutis marmorata. Asymmetry of lower limbs. 1. Mild delayed Speech development. Attention deﬁcit, hyperactivity disorder. Small pointed chin, wide nasal bridge and thick vermilion of the lower lip. Moderate intellectual disability. Flat occiput, epicanthal folds, downturned corners of the mouth, broad based nose, broad hands with tapering ﬁngers and mild 2-3 toe syndactyly. Atrial septal defect. Obesity. Occasionally aggressive outbursts. 3. Discussi 3. Discussion Extra-rotation of the lower limbs, varus position of both the knees, ﬂat feet. Bilateral cutaneous dimples on both elbows and knees, shield chest, inverted nipples, winged shoulder blades. Emotiveness, impulsiveness, decreased attention span. Dilatation of lateral ventricles, pineal gland’s small ectasia, moderate cystic cisterna magna’s ectasia, retrocerebellar cystic ectasia. Global chorio-retinic dystrophia, pale papilla with clear boundaries, peri-papillar pigmentary ring. Sialorrhea and extravelic palatin tonsils, ogival palate, type C tympanogram with absent stapedial reﬂex on the left. Regarding to the invdupdel[8p] phenotype, which is usually characterized by facial dysmorphisms and a wide range (to a usually severe degree) of neurodevelopmental delays, several congenital malformations have been described over time as part of its clinical spectrum such as heart defects, facial dysmorphism, skeletal abnormalities and brain anomalies [8–10]. By comparing our proband’s features with a 13 patient series reported in the main scientific literature from 2001 to 2018 (Tables 1 and 2), in this study we review the already known [inv dup del(8p)] genotype–phenotype relationship in order to: (i) better frame the overall clinical picture associated, (ii) highlight some specific features of this rare complex genetic disorder that are helpful in the clinical diagnostic arena, and (iii) improve the knowledge for its management and follow-up. In this view, our proband’s overall clinical presentation falls in a milder phenotype since most of the severe manifestations were not present such as heart congenital defects (HCD) and corpus callosum agenesis (CCa) which are commonly described in these patients. This milder phenotype, compared to other patients, could be determined due a smaller duplication size or a highly suggestive positional effect gained from the inversion [10]. In particular, referring to the heart congenital defects, the most common anomalies reported are patent ductus arteriosus (PDA), ventricular septal defects (VSD) and atrial septal defect (ASD, especially of type II) [1,11,12]. Moreover, while pulmonary stenosis has been described by Hand et al. and by Buysse et al. [13,14], Ergun et al. described a dextrocardia complex case [11] of HCDs, referring to a case of double right ventricle outlet and two tetralogy of Fallot (TOF) cases that were also respectively reported by Gargìa-Santiago et al. [1] and Masuda et al. [12]. Concerning our patient, she only presented a right bundle branch focal block on the ECG. Brain imaging from MRI/TC frequently revealed corpus callosum agenesis (CCA) as a typical anomaly sign, ranging from total to partial agenesis [1,11,12,14–16]. 3. Discussi 3. Discussion Cutis marmorata. Asymmetry of lower limbs. 2. Mild intellectual delay. Hypotonia. Prominent forehead, cupped simple ears, smooth philtrum. Bilateral ﬁfth ﬁnger clinodactyly. Fronto-parietal brain atrophy. 2. Del 8p23.1 (1–6,901,486): 6.90 Mb;Dup 8p12- > 8p23.1 (12,627,630–36,027,465): 23.40 Mb 3. Del 8p23.1 (1–7,233,949): 7.3 Mb;Dup 8p12- > 8p23.1 (12,554,743–34,577,042): 22.03 Mb 3. Moderate intellectual delay. Hypotonia. Protruding tongue in the absence of macroglossia. Patent Ductus Arteriosus (PDA). Brachydactyly. Thinning of the corpus callosum. 4. Del 8p23.1 (1–6,925,869): 6.94 Mb;Dup 8p11.1- > 8p23.1 (12,554,743–41,232,360): 28.76 Mb p 4. Severe intellectual delay. Hypotonia. Macrocephaly, narrow and small forehead, facial asymmetry, micrognathia, ptosis, smooth ﬁltrum, macroglossia, spaced teeth, narrow palate, tendency to open mouth and large ears. Corpus callosum agenesis. Valgus feet. 5. Del 8p23.1 (1–6,900,000): 6.90 Mb;Dup 8p12- > 8p23.1 (12,296,000–32,800,000): 20.5 Mb 6. Del 8p23.1 (1–6,900,000): 6.90 Mb;Dup 8p11.2- > 8p23.1 (12,296,000–43,700,000): 31 Mb 7. 46,XX,del(8)(p23.3) invdup(8) p21.1p23.2) 5. Severe intellectual delay. Hypotonia. Brachycephaly, broad forehead with bitemporal narrowing, facial asymmetry, short palpebral ﬁssures, straight and narrow nose, low-set, posteriorly rotated ears, and large mouth. Bilateral ﬁfth ﬁngers clinodactlyly. Ventricular septal defects (VSD). Corpus callosum agenesis. 6. Mild to moderate Intellectual delay. Hypotonia. Protruding ears, straight nose with bulbous tip. High palate, thick vermilion of lips and micrognathia. 7. Double outlet right ventricle (DORV), ventricular septal defects (VSD). Corpus callosum agenesis. Moderate intellectual disability. Flat occiput, epicanthal folds, downturned corners of the mouth, broad based nose, broad hands with tapering ﬁngers and mild 2-3 toe syndactyly. Atrial septal defect. Obesity. Occasionally aggressive outbursts. 7 of 11 Brain Sci. 2020, 10, 451 Table 1. Cont. Table 1. Cont. Study Karyotype/CGH-Arrays FISH Phenotype Kumar et al. 6.7- Mb deletion on chromosome 8p23.3p23.1 and a 31-Mb interstitial duplication on chromosome 8p23.1p11.1. nr Global developmental delay. Generalized hypotonia. Broad forehead, low set ears, thick lips, prominent philtrum. Harrison sulcus. History of generalized seizures. Large doubly committed ventricular septal defect (VSD) with left to right shunt and severe hyperkinetic pulmonary artery hypertension. Colpocephaly with complete absence of corpus callosum, prominent ventricles. Our patient 46, XX, der(8)del(8)(p23.1)invdup(p12p23.1) + Developmental and speech delay. No meaningful sentences. Hypotonia. Hypothyroidism. Prominent forehead, arched eyebrow, thin nose with rounded tip and anteverse nostrils, ﬂat ﬁlter, thin down-turned lips, slight micrognathia, low-set posteriorly rotated ears. Single palmar crease on the right hand and bilateral IV-V ﬁngers clinodactyly. Hypertrichosis, previous sacrococcygeal ﬁstula sign. 3. Discussi 3. Discussion However, mild to severe degree brain atrophy is also reported [1,12,17] and a Dandy–Walker variant of the posterior fossa defines some cases [15,17]. Dysmorphic cranial conformation like colpocephaly or flat occiput are also described [15,18]. Moreover, Fan et al. illustrated a case of communicating hydrocephalus and intramedullary cord defects never reported before [15]. In this context, even if our patient’s brain MRI did not show corpus callosum anomalies (which are the main 8 of 11 Brain Sci. 2020, 10, 451 brain defect associated with invdupdel[8p]) or other relevant anomalies, some elements such as a mild ventricular dilatation not related to a significant stroke volume value on cine-MRI sequence integration were highlighted as well as those retrieved in other patients from the presented series analysis (Table 1). Furthermore, regarding the neurobehavioral phenotype, Fisch et al. first described four children with mild to severe cognitive impairment and a significantly lower level of adaptive behavior [19]. By using CARS scores, they also outlined autism or autistic-like features in three out of four children in this series. Three of them also satisfied the DSM-IV-TR diagnosis of Attention Deficit Hyperactivity Disorder (ADHD), showing co-morbid characteristics of hyperactivity and attention deficits [19]. This, in accordance with the above-mentioned literature, can confirm a common neurobehavioral phenotype since our patient was also diagnosed with severe Developmental Delay/Intellectual Disability (DD/ID), ADHD, and speech delay. Among skeletal anomalies, the most frequently represented in literature are kyphoscoliosis, winged shoulders, shield chest, clinodactyly of IV–V fingers and flat feet. A case of syndactyly of II-III fingers was also reported by Knijnenburg et al. [20]. The extra-rotation of lower limbs and varus position of the knees were also remarkable in our patient. In regards to the less frequent abdominal anomalies, we remark on the presence of a case of congenital diaphragmatic hernia [5] and pelvic dysplastic kidneys and hydronephrosis, which have also been reported [15], but these were not detected among our proband’s features. Finally, according to the presented report, we recommend that some ancillary aspects should not be neglected in these patients such as the phoniatric evaluation, since bad occlusion, sialorrhea and hypotonia could affect the severity of the clinical picture. References 1. García-Santiago, F.A.; Martínez-Glez, V.; Santos, F.; García-Miñaur, S.; Mansilla, E.; Meneses, A.G.; Rosell, J.; Granero, Á.P.; Vallespín, E.; Fernández, L.; et al. Analysis of invdupdel(8p) rearrangement: Clinical, cytogenetic and molecular characterization. Am. J. Med. Genet. A 2015, 167, 1018–1025. [CrossRef] [PubMed] 2. Floridia, G.; Piantanida, M.; Minelli, A.; Dellavecchia, C.; Bonaglia, C.; Rossi, E.; Gimelli, G.; Croci, G.; Franchi, F.; Gilgenkrantz, S.; et al. The same molecular mechanism at the maternal meiosis I produces mono- and dicentric 8p duplications. Am. J. Hum. Genet. 1996, 58, 785–796. 2. Floridia, G.; Piantanida, M.; Minelli, A.; Dellavecchia, C.; Bonaglia, C.; Rossi, E.; Gimelli, G.; Croci, G.; Franchi, F.; Gilgenkrantz, S.; et al. The same molecular mechanism at the maternal meiosis I produces mono- and dicentric 8p duplications. Am. J. Hum. Genet. 1996, 58, 785–796. 3. Ciccone, R.; Mattina, T.; Giorda, R.; Bonaglia, M.C.; Rocchi, M.; Pramparo, T.; Zuﬀardi, O. Inversion polymorphisms and non-contiguous terminal deletions: The cause and the (unpredicted) eﬀect of our genome architecture. J. Med. Genet. 2006, 43, e19. [CrossRef] [PubMed] 3. Ciccone, R.; Mattina, T.; Giorda, R.; Bonaglia, M.C.; Rocchi, M.; Pramparo, T.; Zuﬀardi, O. Inversion polymorphisms and non-contiguous terminal deletions: The cause and the (unpredicted) eﬀect of our genome architecture. J. Med. Genet. 2006, 43, e19. [CrossRef] [PubMed] 4. Giglio, S.; Broman, K.W.; Matsumoto, N.; Calvari, V.; Gimelli, G.; Neumann, T.; Ohashi, H.; Voullaire, L.; Larizza, D.; Giorda, R.; et al. Olfactory receptor-gene clusters, genomic-inversion polymorphisms, and common chromosome rearrangements. Am. J. Hum. Genet. 2001, 68, 874–883. [CrossRef] [PubMed] 4. Giglio, S.; Broman, K.W.; Matsumoto, N.; Calvari, V.; Gimelli, G.; Neumann, T.; Ohashi, H.; Voullaire, L.; Larizza, D.; Giorda, R.; et al. Olfactory receptor-gene clusters, genomic-inversion polymorphisms, and common chromosome rearrangements. Am. J. Hum. Genet. 2001, 68, 874–883. [CrossRef] [PubMed] 5. Shimokawa, O.; Kurosawa, K.; Ida, T.; Harada, N.; Kondoh, T.; Miyake, N.; Yoshiura, K.; Kishino, T.; Ohta, T.; Niikawa, N.; et al. Molecular characterization of inv dup del(8p): Analysis of ﬁve cases. Am. J. Med. Genet. A 2004, 128, 133–137. [CrossRef] [PubMed] 5. Shimokawa, O.; Kurosawa, K.; Ida, T.; Harada, N.; Kondoh, T.; Miyake, N.; Yoshiura, K.; Kishino, T.; Ohta, T.; Niikawa, N.; et al. Molecular characterization of inv dup del(8p): Analysis of ﬁve cases. Am. J. Med. Genet. A 2004, 128, 133–137. [CrossRef] [PubMed] 6. 3. Discussi 3. Discussion Thus, an audiometric evaluation for the occasional reported neurosensorial hypoacusia [12], and a whole eye examination because of refractory disorders, should be offered both at the diagnosis as well as during the follow-up for the often underrated and occasionally reported ophthalmologic problems such as strabismus [14,18] and/or the chorioretinic dystrophia which was first documented in our patient. Brain Sci. 2020, 10, 451 9 of 11 Table 2. Extensive invdupdel[8p] genotype–phenotype correlation analysis and scientiﬁc literature review broken out per article, author, year, number of patients described, patients’ sex, and clinical/instrumental reported anomalies. +, retrieved feature; −, not retrieved feature; nr, not reported. Article Year No. of Patients Sex Dysmorphisms Intellectual Disabilities/Behavioural Disorders Brain MRI Anomalies Congenital Heart Defects Abdominal Anomalies Skeletal Anomalies Fan et al. 2001 1 M + + + + + − Masuda et al. 2002 2 1F/1M + + + + − − Vermeesch et al. 2003 1 F + + nr − − − Ciccone et al. 2006 1 F + + nr − − + Cooke et al. 2008 1 F + + + − − + Caglayan et al. 2009 1 ? + + + − − − Buysse et al. 2009 1 F + + nr + − − Hand et al. 2010 1 F − + nr + − − Ergun et al. 2010 1 F + + + + − − Fisch et al. 2011 4 2F/2M + + nr − − − Garcìa−Santiago et al. 2014 7 4F/3M + + + + − + Knijnenburg et al. 2017 1 M + + nr + − + Kumar et al. 2018 1 M + + + + − + Our patient 2020 1 F + + + − − + Table 2. Extensive invdupdel[8p] genotype–phenotype correlation analysis and scientiﬁc literature review broken out per article, author, year, number of patients described, patients’ sex, and clinical/instrumental reported anomalies. +, retrieved feature; −, not retrieved feature; nr, not reported. Brain Sci. 2020, 10, 451 10 of 11 10 of 11 4. Conclusions In conclusion, comprehensive cytogenetic and molecular analyses of the standard karyotype, CGH-array and FISH are mandatory to gain a diagnosis in patients showing a complex phenotype which includes dysmorphic features, neurodevelopmental delay, and major or minor congenital anomalies [21]. In these patients, invdupdel[8p] should be considered in the differential diagnosis [17], as further shown in this study where we highlighted this rare complex genetic disorder’s features whose proper framework may be helpful both on the clinical diagnostic arena as well as to improve patient management and follow-up. Finally, further studies are needed to completely evaluate its genotype–phenotype relationship, especially to widen the scope on the genes–dosage impairment-driven role that we propose once again could be determined by several factors such as breakage sites, genomic size and orientation of the rearrangements involved. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3425/10/7/451/s1, Table S1: Full name, OMIM code and function of genes whose loci are harbored within the 8p23.3p23.1 sub-bands, between nucleotidis 221,611 and 6,914,076 of around. Author Contributions: Conceptualization, M.L.B., D.V. and P.P.; methodology, A.A., A.F.; investigation, L.S., A.B.; data curation, G.M.; L.M., S.S.; writing—original draft preparation, P.P., D.V., M.M., M.L.B.; writing—review and editing, T.A.T., A.S., S.P., G.M., L.M.; supervision, A.A., D.V. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The document has been edited for proper English language, grammar, punctuation, typos error and spelling by AME (American manuscript editors USA. Certiﬁcate Veriﬁcation Key: 234-589-107-043-904: Project Number: 72815). Acknowledgments: The document has been edited for proper English language, grammar, punctuation, typos error and spelling by AME (American manuscript editors USA. Certiﬁcate Veriﬁcation Key: 234-589-107-043-904: Project Number: 72815). Conﬂicts of Interest: No competing interest to declare. Conﬂicts of Interest: No competing interest to declare. References Giglio, S.; Calvari, V.; Gregato, G.; Gimelli, G.; Camanini, S.; Giorda, R.; Ragusa, A.; Guerneri, S.; Selicorni, A.; Stumm, M.; et al. Heterozygous Submicroscopic Inversions Involving Olfactory Receptor–Gene Clusters Mediate the Recurrent t(4;8)(p16;p23) Translocation. Am. J. Hum. Genet. 2002, 71, 276–285. [CrossRef] 7. Piccione, M.; Salzano, E.; Vecchio, D.; Ferrara, D.; Malacarne, M.; Pierluigi, M.; Ferrara, I.; Corsello, G. 4p16.1-p15.31 duplication and 4p terminal deletion in a 3-years old Chinese girl: Array-CGH, genotype- phenotype and neurological characterization. Eur. J. Paediatr. Neurol. 2015, 19, 477–483. [CrossRef] 8. Vermeesch, J.; Thoelen, R.; Salden, I.; Raes, M.; Matthijs, G.; Fryns, J. Mosaicism del(8p)/inv dup(8p) in a dysmorphic female infant: A mosaic formed by a meiotic error at the 8p OR gene and an independent terminal deletion event. J. Med. Genet. 2003, 40, e93. [CrossRef] 11 of 11 Brain Sci. 2020, 10, 451 9. de Die-Smulders, C.E.; Engelen, J.J.; Schrander-Stumpel, C.T.; Govaerts, L.C.; de Vries, B.; Vles, J.S.; Wagemans, A.; Schijns-Fleuren, S.; Gillessen-Kaesbach, G.; Fryns, J.P. Inversion duplication of the short arm of chromosome 8: Clinical data on seven patients and review of the literature. Am. J. Med. Genet. 1995, 59, 369–374. [CrossRef] 10. Devriendt, K.; Matthijs, G.; Van Dael, R.; Gewillig, M.; Eyskens, B.; Hjalgrim, H.; Dolmer, B.; McGaughran, J.; Bröndum-Nielsen, K.; Marynen, P.; et al. Delineation of the critical deletion region for congenital heart defects, on chromosome 8p23.1. Am. J. Hum. Genet. 1999, 64, 1119–1126. [CrossRef] p 11. Ergün, M.A.; Kula, S.; Karaer, K.; Perçin, E.F. A case with de novo inv dup del(8p) associated with dextrocardia and corpus callosum agenesis. Pediatr. Int. 2010, 52, 845–846. [CrossRef] 12. Masuda, K.; Nomura, Y.; Yoshinaga, M.; Nakamura, M.; Matsuda, Y.; Oku, S.; Miyata, K. Inverted duplication/deletion of the short arm of chromosome 8 in two patients with tetralogy of Fallot. Pediatr. Int. 2002, 44, 534–536. [CrossRef] [PubMed] 13. Hand, M.; Gray, C.; Glew, G.; Tsuchiya, K.D. Mild phenotype in a patient with mosaic del(8p)/inv dup del(8p). Am. J. Med. Genet. A 2010, 152, 2827–2831. [CrossRef] [PubMed] 14. Buysse, K.; Antonacci, F.; Callewaert, B.; Loeys, B.; Fränkel, U.; Siu, V.; Mortier, G.; Speleman, F.; Menten, B. Unusual 8p inverted duplication deletion with telomere capture from 8q. Eur. J. Med. Genet. 2009, 52, 31–36. [CrossRef] [PubMed] 15. Fan, Y.S.; Siu, V.M. Molecular cytogenetic characterization of a derivative chromosome 8 with an inverted duplication of 8p21.3–>p23.3 and a rearranged duplication of 8q24.13–>qter. Am. J. Med. Genet. References 2001, 102, 266–271. [CrossRef] [PubMed] 16. Kumar, V.; Roy, S.; Kumar, G. An Interesting and Unique Case of 8p23.3p23.1 Deletion and 8p23.1p11.1 Interstitial Duplication Syndrome. J. Pediatr. Genet. 2018, 7, 125–129. [CrossRef] 17. Cooke, S.L.; Northup, J.K.; Champaige, N.L.; Zinser, W.; Edwards, P.A.W.; Lockhart, L.H.; Velagaleti, G.V.N. Molecular cytogenetic characterization of a unique and complex de novo 8p rearrangement. Am. J. Med. Genet. Part A 2008, 146, 1166–1172. [CrossRef] 18. Caglayan, A.O.; Engelen, J.J.M.; Ghesquiere, S.; Alofs, M.; Saatci, C.; Dundar, M. Fluorescence in situ hybridization and single nucleotide polymorphism of a new case with inv dup del(8p). Genet. Couns. 2009, 20, 333–340. [PubMed] 19. Fisch, G.S.; Davis, R.; Youngblom, J.; Gregg, J. Genotype-phenotype association studies of chromosome 8p inverted duplication deletion syndrome. Behav. Genet. 2011, 41, 373–380. [CrossRef] 20. Knijnenburg, J.; Uytdewilligen, M.E.W.; van Hassel, D.A.C.M.; Oostenbrink, R.; Eussen, B.H.J.; de Klein, A.; Brooks, A.S.; van Zutven, L.J.C.M. Postzygotic telomere capture causes segmental UPD, duplication and deletion of chromosome 8p in a patient with intellectual disability and obesity. Eur. J. Med. Genet. 2017, 60, 445–450. [CrossRef] 21. Piro, E.; Consiglio, V.; Agrifoglio, M.; Sireci, F.; Ballacchino, A.; Salvago, P.; Martines, F.; Graziano, F.; Busè, M.; Sanﬁlippo, C.; et al. Diagnosis and follow-up of complex congenital malformations/mental retardation (MRA/MR). Acta Med. Mediterr. 2013, 29, 321–325. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3108068865	https://www.mdpi.com/2504-3900/60/1/39/pdf?version=1609293940	English	null	Preliminary Study on Electrochemical Ion Imprinted Polymeric Film in Sensor Development for Cd(II) Ions Determination in Water	null	2,020	cc-by	4,226	Sabrina Di Masi 1,, Antonio Pennetta 2 and Cosimino Malitesta 1 Keywords: ion imprinted polymer; 4-APA; electrochemical sensor; Cd(II) ions; electropolymerisation Keywords: ion imprinted polymer; 4-APA; electrochemical sensor; Cd(II) ions; electropolymerisation Sabrina Di Masi 1,, Antonio Pennetta 2 and Cosimino Malitesta 1 Sabrina Di Masi 1,, Antonio Pennetta 2 and Cosimino Malitesta 1 1 Laboratorio di Chimica Analitica, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Monteroni, 73100 Lecce, Italia; cosimino.malitesta@unisalento.it 2 Laboratorio di Spettrometria di Massa Analitica e Isotopica, Dipartimento di Beni Culturali, Università del Salento, Via Monteroni, 73100 Lecce, Italia; antonio.pennetta@unisalento.it Correspondence: sabrina dimasi@unisalento it; Tel : +39-0832-299-457 1 Laboratorio di Chimica Analitica, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Monteroni, 73100 Lecce, Italia; cosimino.malitesta@unisalento.it 2 Laboratorio di Spettrometria di Massa Analitica e Isotopica, Dipartimento di Beni Culturali, Università del Salento, Via Monteroni, 73100 Lecce, Italia; antonio.pennetta@unisalento.it * Correspondence: sabrina.dimasi@unisalento.it; Tel.: +39-0832-299-457 † Presented at the 1st International Electronic Conference on Biosensors, 2–17 November 2020; Available online: https://iecb2020.sciforum.net/. 1 Laboratorio di Chimica Analitica, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Monteroni, 73100 Lecce, Italia; cosimino.malitesta@unisalento.it 2 Laboratorio di Spettrometria di Massa Analitica e Isotopica, Dipartimento di Beni Culturali, Università del Salento, Via Monteroni, 73100 Lecce, Italia; antonio.pennetta@unisalento.it * Correspondence: sabrina.dimasi@unisalento.it; Tel.: +39-0832-299-457 † Presented at the 1st International Electronic Conference on Biosensors, 2–17 November 2020; Available online: https://iecb2020.sciforum.net/. Published: 2 November 2020 Published: 2 November 2020 Published: 2 November 2020 Abstract: Preliminary results on an electrosynthesized ion imprinted polymeric film (IIP-film) for Cd(II) ions determination in sensor development are here reported. The sensor was prepared by electropolymerization of 4-aminophenylacetic acid (4-APA) monomer in presence of Cd(II) ions, which acts as the template. The screen-printed carbon electrodes (SPCE) were used as transducer during sensor development, whereas the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were selected as the electrochemical methods for the synthesis and Cd(II) ions sensing, respectively. The incubation of the developed sensor in NaOH 250 mM involved into remove the template and the formation of specific recognition cavities into the polymer. A multivariate optimization based on central composite design (CCD) was employed to study the effect of three independent parameters on electrochemical performances of the sensor. The electrochemical characterization of sensors was performed in ferrocyanide-ferricyanide redox couple and in KCl 0.1 M, the latter revealing redox properties from the polymeric film. The performances of sensors and the control (non-imprinted film, NIP) was observed in sodium acetate buffer (100 mM, pH = 5) over the Cd(II) concentration range 0.1–10 µM. Proceedings Proceedings 2020, 60, 39; doi:10.3390/IECB2020-07037 1. Introduction Heavy metals pollution refers to a global issue, due to the high toxicity and dangerous effects on environment and human health. Among heavy metals, cadmium is one of the most toxic. Main sources of this ion in environment is industrial wastewater, fertilizers and so on. Currently, the most analytical method for cadmium detection is represented by atomic adsorption spectroscopy (AAS), inductively coupled plasma-mass spectroscopy (ICP-MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES). Those techniques are sensitive, accurate but also expensive, and on-site determination of targets is not so suitable. Due to the complexity of those instrumentation, there is the need to point different methods to be available for on-site determination. Electrochemical methods can be used for that. Moreover, diverse electrochemical methods are explored today for the determination of heavy metals in water environment [1]. Proceedings 2020, 60, 39; doi:10.3390/IECB2020-07037 www.mdpi.com/journal/proceedings 2 of 8 Proceedings 2020, 60, 39 Imprinted polymers define robust and artificial materials able to mimic recognition processes of such analytes, such as proteins, small molecules, or ions [2]. The process results in the selective formation of ion-sized imprinted cavities, which are complementary to a specific template in terms of its functional groups. These materials can be easily applied to identify, monitor and remove the target ions in water environment [3]. In this view, the ion imprinted polymers (IIPs) can be described. Their synthesis can be carried out both chemically and electrochemically. The latter leads to the preparation of imprinted films, which are compatible in conjunction with transducers in sensor development [4,5]. Very few works report the electrochemical synthesis of ion imprinted polymers and their application as sensors for metal ion detection [6,7]. With this regard, we propose the synthesis, optimization, characterization and subsequent application of an electrosynthesized IIPs for the electrochemical detection of cadmium (II) in water. The proposed sensor was prepared by electropolymerization of 4-aminophenylacetic acid (4-APA) monomer in presence of Cd(II) ions, which acts as the template. The screen-printed carbon electrodes (SPCE) were used as transducer during sensor development, whereas the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were selected as the electrochemical methods for the synthesis and Cd(II) ions sensing, respectively. The incubation of the developed sensor in NaOH 250 mM involved the removal of the template and the formation of specific recognition cavities into the polymer. 1. Introduction A multivariate optimization was employed for studying the effect of three independent parameters on electrochemical performances of the sensor. The electrochemical characterization of sensors was performed in ferrocyanide-ferricyanide redox couple and in KCl 0.1 M, the latter revealing redox properties from the polymeric film. The performances of sensors and the control (non-imprinted polymer, NIP) were observed in sodium acetate buffer (100 mM, pH = 5) over the Cd(II) concentration range of 0.1–10 µM. 2.1. Materials Acetic acid, 4-Aminophenylacetic acid (4-APA, 98%), sodium acetate trihydrate, cadmium nitrate tetrahydrate (98%), and ethylenediaminetetraacetic acid (99%) were purchased from Sigma- Aldrich (Italy). Sulphuric acid and sodium hydroxide solutions were commercially available as analytical reagent grade. All reagents were used without further purification. MilliQ water was used for washing the polymeric film after the preparation. Sodium acetate buffer (100 mM, pH = 5). 2.2. Apparatus CV and DPV measurements were performed using a PalmSens potentiostat equipped with a cable connector (DropSens, Milano, Italy) for screen-printed electrodes. PSTrace was the software to control the instrument and data acquisition. The polymeric film was deposited on screen-printed carbon electrode (SPCE). The SPCEs were composed of three-electrode configuration on a planar ceramic support (3.3 × 1 cm) and they consisted of a carbon disk-shaped working electrode (4 mm diameter), a platinum electrode as counter electrode and a pseudo Ag/AgCl paste electrode as reference electrode. SPCE were commercially available (Metrohm, Milano, Italy). 2.3. Preparation of Electrosynthesized Ion Imprinted Polymer and Non-Imprinted Polymer The preparation of ion imprinted polymer (IIP) based on poly-4-aminophenylacetic (poly-4- APA) films was performed by cyclic voltammetry (CV) in a potential range between −0.2 and 1.2 V vs. pseudo Ag/AgCl, at a scan rate of 50 mV s−1 for 40 cycles in a solution of H2SO4 0.5 M containing 1 mM of Cd2+ ions. The porogen was chosen based on previous works about the electrosynthesis of poly-4-APA on SPE [8]. After the electropolymerization, the electrode was rinsed with MilliQ water and incubated in different solvent (EDTA 100 mM and 250 mM, H2SO4 500 mM, NaOH 100 mM and 250 mM) to remove the target. The preparation of the control (non-imprinted polymer, NIP) was obtained with the same protocol, but without adding the template into the polymerization mixture. Proceedings 2020, 60, 39 3 of 8 The treatment in NaOH 250 mM was also performed on NIP. All prepared sensors were taken in air when not in use. The treatment in NaOH 250 mM was also performed on NIP. All prepared sensors were taken in air when not in use. 2.4. Cd2+ Ion Sensing The electrochemical responses of IIP and NIP films towards Cd2+ ions were recorded using DPV measurements in the potential range of −0.2 to + 0.4 V, modulation amplitude of 50 mV, step potential of 4.95 mV, and equilibration time of 2 s. Cd2+ ions interacted with the imprinted film by drop-casting on the electrode surface an appropriate amount (100 µL) of a solution of sodium acetate buffer (100 mM, pH = 5) containing different concentration of Cd2+ ions (0.1–10 µM), by leaving the drop on the electrode for 10 min. After each measurement, the electrode surface was gently washed with sodium acetate buffer for 2 min. 2.5. Experimental Design in Optimization Studies Multivariate optimization was conducted with the light to optimize the development of IIPs and NIP. The selected optimization model was the central composite design (CCD), which allowed the selection of main three factors affecting the development of the sensors, such as (i) the monomer concentration, (ii) the rate between template-monomer (mainly affecting the number of cavities on the polymeric network) and (iii) the number of CV cycles during the electrosynthesis. MODDE® Software (Umetrics, version 12, https://www.sartorius.com/en/products/process-analytical- technology/data-analytics-software/doe-software/modde) was used for design, mathematical modelling and optimization. The levels of studied independent variables are listed in Table 1. Table 1. Levels of independent variables considered in this work. Variable Low High Monomer concentration (X1) 0.5 5 Rate Cd2+/monomer (X2) 1 3 Number of CV cycles (X3) 10 40 Table 1. Levels of independent variables considered in this work. The response was the difference of current (Δi, µA) recorded in ferrocyanide-ferricyanide redox probe before and after the electropolymerization of the different imprinted sensors. Based on CCD principle, the design consisted of 2k fractional factorial points plus 2k axial points and 1 center point, where k defines the number of central points (in this case, k = 3). Eighteen experiment runs were conducted, and the second-order polynomial equation consisted of linear, quadratic, and first-order interaction terms is shown below (Equation (1)): Y = 𝛽଴+ ∑ 𝛽௜𝑋௜+ ∑ 𝛽௜௜𝑋௜ ଶ+ ∑ ∑ 𝛽௜௝𝑋௜𝑋௝ ௞ ௝(ஷ௜) + 𝜀 ௞ ௜ୀଵ ௞ ௜ୀଵ ௞ ௜ୀଵ , (1) (1) where Y is the response variables, Xi represent the dependent variables, 𝛽଴, 𝛽௜, 𝛽௜௜, 𝛽௜௝ were the regression coefficient for intercept, linear, quadratic and interaction terms, respectively. 3.1. Preparation of Electrosynthesized IIP and NIP Films Figure 1a presented a typical cyclic voltammetry recorded during the electropolymerization of 2.1 mM 4-APA in the presence of 2.1 mM Cd2+ ions in 0.5 M H2SO4 on a screen-printed carbon electrode. Figure 1b shows the electropolymerization of 4-APA on SPCE, without the template (NIP). -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -10 0 10 20 30 40 50 60 70 Potential (V) Current (μA) -0.2 0.0 0.2 0.4 -10 -8 -6 -4 -2 0 2 4 (a) -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -10 0 10 20 30 40 50 60 70 Current (μA) Potential (V) (b) Figure 1. (a) The electropolymerization of 2.1 mM 4-APA in the presence of 2.1 mM Cd2+ in 0.5 M H2SO4. Inset: Focused cyclic voltammetry for (black) 1st, (pink) 2nd (blue), and 17th cycle; (b) the electropolymerization of 2.1 mM 4-APA in 0.5 M H2SO4. Voltammetric condition: (i) Potential range: −0.2 to + 1.2 V; (ii) scan rate: 50 mV s−1; (iii) CV cycles: 40. -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -10 0 10 20 30 40 50 60 70 Current (μA) Potential (V) (b) -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -10 0 10 20 30 40 50 60 70 Potential (V) Current (μA) -0.2 0.0 0.2 0.4 -10 -8 -6 -4 -2 0 2 4 Potential (V) Potential (V) (b) (a) Figure 1. (a) The electropolymerization of 2.1 mM 4-APA in the presence of 2.1 mM Cd2+ in 0.5 M H2SO4. Inset: Focused cyclic voltammetry for (black) 1st, (pink) 2nd (blue), and 17th cycle; (b) the electropolymerization of 2.1 mM 4-APA in 0.5 M H2SO4. Voltammetric condition: (i) Potential range: −0.2 to + 1.2 V; (ii) scan rate: 50 mV s−1; (iii) CV cycles: 40. During the CV, the first peak at +0.70 V indicated the formation of cation radicals that promoted the polymerization process, once to the oxidation of 4-APA. Further peaks at +0.19 V and at −0.025 V are related to the reduction of the polymer film on the SPCE surface. Following the second potential cycle, two oxidation waves appeared at the potentials of +0.040 and + 0.305 V, corresponding to the oxidation of the formed polymeric film. 3.1. Preparation of Electrosynthesized IIP and NIP Films After around 17 cycles of polymerization, a decrease in the anodic peaks current was notable, indicating the subsequent formation of the polymer film (see Inset of Figure 1a). Finally, the formation of the film produced a partial blockage of the electrode surface. The electropolymerization of NIP (Figure 1b) followed the same interpretation of the process, with differences in terms of current appeared along the second cycle of CV. 3. Results and Discussions The electropolymerization of 4-APA in presence of Cd2+ produced a sensitive polymeric imprinted film for that template, showing superior characteristics against its control. The optimal condition of synthesis was established by employing a multivariate experimental design, and this approach has recently gained interest from scientists regarding optimized sensors and biomimetic sensors. The advantage of using the produced IIPs consisted of revealing a redox property of the polymer, which directly addresses the interaction between imprinted cavities and template. The interaction was visible, close to + 0 V (see related DPV measurements), of which potential is higher than normally observed for the electroactivity of Cd2+ in solution. Proceedings 2020, 60, 39 4 of 8 3.1. Preparation of Electrosynthesized IIP and NIP Films 3.1. Preparation of Electrosynthesized IIP and NIP Films 3.2. Optimization of Sensor Performances by Experimental Design The optimization of performances was possible by a multivariate approach, that considered all factors together, including linear, quadratic and interaction terms in the model. All the selected factors were related to the electrosynthesis process. Among them, with the emphasis to develop imprinted materials, the relationship between all reagent should be described. Preliminary results have shown the factor’s importance on responses were the initial concentration of functional monomer (X1) and the number of CV cycles during the electrosynthesis (X3). Figure 2 shows the significant coefficients related to factors. 5 of 8 Proceedings 2020, 60, 39 9 5 of 8 Figure 2. Plot of the significant coefficients obtained from model. Figure 2. Plot of the significant coefficients obtained from model. The regression equation for the achieved responses, including significant factors, is reported (Equation (2)). Y = 14.86 + 4.08𝑋ଵ−1.78𝑋ଶ+ 3.80𝑋ଷ−3.30𝑋ଵ𝑋ଵ, (2) (2) After evaluation of Equation (2), it appears as though the monomer concentration is in strong correlation with the other factors. In particular the ratio of monomer/Cd2+ should be regulated to assume a correct orientation of cavities on the polymer network. The factor related to the growth of the electrosynthesized imprinted film was also significant, confirming that the deposition of the film on electrode surface is involved in the difference of currents recorded by the electrochemical probe. In light of maximizing the responses, the experimental conditions used for further measurements were (i) 2.1 mM 4-APA, (ii) 2.1 mM Cd2+ (ratio 1:1), and (iii) 40 CV cycles during the electrosynthesis. 3.3. Electrochemical Characterization of IIP and NIP Films Electrochemical Characterization of IIP and NIP Films The prepared sensors were first subjected to electrochemical characterization in ferrocyanide- ferricyanide redox probe and in KCl 0.1 M, by applying a CV measurement for bare SPCE, IIP film and NIP film after the electrodeposition (Figure 3a,b). -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 -100 -80 -60 -40 -20 0 20 40 60 80 2 3 Current (μA) Potential (V) 1 (a) -0.2 0.0 0.2 0.4 0.6 0.8 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 3 2 1 Potential (V) Current (μA) (b) Figure 3. (a) Electrochemical characterization by CV (fifth cycle) in ferrocyanide-ferricyanide redox probe for (1) bare screen-printed carbon electrode; (2) Cd2+-ion imprinted polymer film/screen-printed carbon electrode (SPCE) and (3) non-imprinted polymer (NIP) film/SPCE after polymerization. Voltammetric condition: (i) Potential range: −0.5 to + 0.8 V; (ii) scan rate: 50 mV s−1; (iii) CV cycles: 5; (b) electrochemical characterization by CV (fifth cycle) in 0.1 M KCl for (1) bare screen-printed carbon electrode; (2) Cd2+-IIP film/SPCE; and (3) NIP film/SPCE after polymerization. Voltammetric condition: (i) Potential range: −0.2 to + 0.8 V; (ii) scan rate: 50 mV s−1; (iii) CV cycles: 5. -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 -100 -80 -60 -40 -20 0 20 40 60 80 2 3 Current (μA) Potential (V) 1 (a) -0.2 0.0 0.2 0.4 0.6 0.8 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 3 2 1 Potential (V) Current (μA) (b) (a) (b) Figure 3. (a) Electrochemical characterization by CV (fifth cycle) in ferrocyanide-ferricyanide redox probe for (1) bare screen-printed carbon electrode; (2) Cd2+-ion imprinted polymer film/screen-printed carbon electrode (SPCE) and (3) non-imprinted polymer (NIP) film/SPCE after polymerization. Voltammetric condition: (i) Potential range: −0.5 to + 0.8 V; (ii) scan rate: 50 mV s−1; (iii) CV cycles: 5; (b) electrochemical characterization by CV (fifth cycle) in 0.1 M KCl for (1) bare screen-printed carbon electrode; (2) Cd2+-IIP film/SPCE; and (3) NIP film/SPCE after polymerization. Voltammetric condition: (i) Potential range: −0.2 to + 0.8 V; (ii) scan rate: 50 mV s−1; (iii) CV cycles: 5. Both electrochemical characterizations revealed higher electroactivity of the imprinted polymer when compared to NIP film. In addition, as shown in Figure 2b, no signals were obtained for bare SPCE. 3.3. Electrochemical Characterization of IIP and NIP Films The electroactivity of IIP film than NIP suggest the imprinting effect of the polymer, where Proceedings 2020, 60, 39 Proceedings 2020, 60, 39 6 of 8 possibly Cd2+ ions are possibly able to enhance the overall electrochemical process during polymerization. The removal of the template ion—to obtain the imprinted cavities—was carried out by exposure of the sensor to different solutions, such as EDTA 100 mM and 250 mM, H2SO4 500 mM, NaOH 100 mM, and 250 mM. In all cases, different times of elution were tested, in a range between 1 and 15 min (1, 3, 5, 10, 15 min, respectively). As the most effective method, NaOH 250 mM incubated for 3 min was used. CV markable differences recorded in KCl 0.1 M for NIP and IIPs treated with NaOH 250 mM were visible (Figure 4), confirming the elution of Cd2+ ions from the imprinted cavities. -0.2 0.0 0.2 0.4 0.6 0.8 -1.5 -1.0 -0.5 0.0 0.5 1.0 2 3 1 Figure 4. Electrochemical characterization by CV (fifth cycle) in 0.1 M KCl for (1) bare screen-printed carbon electrode; (2) Cd2+-IIP film/SPCE; and (3) NIP film/SPCE after treatment in 250 mM NaOH for 3 min. Voltammetric condition: (i) Potential range: −0.2 to + 0.8 V; (ii) scan rate: 50 mV s−1; (iii) CV cycles: 5. Figure 4. Electrochemical characterization by CV (fifth cycle) in 0.1 M KCl for (1) bare screen-printed carbon electrode; (2) Cd2+-IIP film/SPCE; and (3) NIP film/SPCE after treatment in 250 mM NaOH for 3 min. Voltammetric condition: (i) Potential range: −0.2 to + 0.8 V; (ii) scan rate: 50 mV s−1; (iii) CV cycles: 5. 3.4. Electrochemical Performances of IIP and NIP Film The electrochemical sensing of Cd2+ ions was performed by DPV measurements on NIP and Cd2+-IIP film: 100 mM sodium acetate buffer (pH = 5) was selected as the electrolyte solution for the determination of Cu2+ ions. DPV measurements recorded for Cd2+-IIP film are shown in Figure 5, and related calibration curves are also reported (Figure 5b). -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 l a Potential (V) Current (μA) (a) 0 2 4 6 8 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055 Cd 2+-IIP film NIP film Cd 2+ concentration (μM) Current (μA) (b) 0 2 4 6 8 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055 Cd 2+-IIP film NIP film Cd 2+ concentration (μM) Current (μA) -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 l a Potential (V) Current (μA) Current (μA) Potential (V) (a) (b) 7 of 8 Proceedings 2020, 60, 39 g , , 0.0 0.2 0.4 0.6 0.8 1.0 0.015 0.020 0.025 0.030 0.035 0.040 y = 0.003x + 0.016 R 2 = 0.854 y = 0.0163x + 0.0185 R 2 = 0.990 Cd 2+-IIP film NIP film Cd 2+ concentration (μM) Current (μA) (c) Figure 5. (a) Differential pulse voltammograms recorded for Cd2+-IIP film after the exposure to (a) blank (sodium acetate buffer), (b) 0.1, (c) 0.2, (d) 0.4, (e) 0.6, (f) 0.8, (g) 1.0, (h) 2.0, (i) 4.0, (j) 6.0, (k) 8.0, (l) 10 µM of Cd2+ ions in the presence of sodium acetate buffer; (b) comparison of the electrochemical responses between Cd2+-IIP and NIP films along all tested Cd2+ concentration; (c) comparison between responses from Cd2+-IIP and NIP film in the linear range revealed between 0.1 and 1 µM Cd2+ ions. 0.0 0.2 0.4 0.6 0.8 1.0 0.015 0.020 0.025 0.030 0.035 0.040 y = 0.003x + 0.016 R 2 = 0.854 y = 0.0163x + 0.0185 R 2 = 0.990 Cd 2+-IIP film NIP film Cd 2+ concentration (μM) Current (μA) (c) (c) Figure 5. 3.4. Electrochemical Performances of IIP and NIP Film (a) Differential pulse voltammograms recorded for Cd2+-IIP film after the exposure to (a) blank (sodium acetate buffer), (b) 0.1, (c) 0.2, (d) 0.4, (e) 0.6, (f) 0.8, (g) 1.0, (h) 2.0, (i) 4.0, (j) 6.0, (k) 8.0, (l) 10 µM of Cd2+ ions in the presence of sodium acetate buffer; (b) comparison of the electrochemical responses between Cd2+-IIP and NIP films along all tested Cd2+ concentration; (c) comparison between responses from Cd2+-IIP and NIP film in the linear range revealed between 0.1 and 1 µM Cd2+ ions. As shown from Figure 5a, the current responses value increased within the tested Cd2+ ion concentration. However, saturation reached value upper than 2 µM, due to the occupancy of cavities. Notably, the imprinted sensor shows high affinity and specificity towards Cd2+ ions compared to that obtained for NIP films, confirming the imprinting effect on this polymer. The linear regression was established between 0.1 and 1 µM, with a sensitivity of 0.0163 µA µM−1. In addition, it was possible to evaluate the imprinted factor as 6.86, highly indicating the specific recognition of template from imprinted cavities on Cd2+-IIP films. The proposed imprinted sensor shows high sensitivity and possesses superior specific properties towards Cd2+ ions. These preliminary results are currently encouraging us to perform further experiments in regard to selectivity properties of the imprinted polymer against NIP and its application to real water matrices, of which discussion will be presented soon. 4. Conclusions Preliminary study on electrosynthesis of ion imprinted polymeric sensor on SPCE transducer for Cd2+ ion determination in water is reported here. The electrosynthesis of the imprinted cavities revealed the newly approach to produce highly sensitive films towards environmental targets. In this light, the developed imprinted polymeric film shows greater sensitivity than NIP film, with an imprinting factor of 6.86. Those achieved preliminary results open the possibly to employ this sensor for quantitative determination of Cd2+ ions in water. Further experiments to evaluate more properties of the sensor are currently under study. Funding: This research was funded by the project “CASCADE” (014-2020 Interreg V-A IT-HR CBC “Strategic” project ID 10255941). Acknowledgments: Authors would like to thank Marco Costa for his helping during experiments. Conflicts of Interest: The authors declare no conflict of interest. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References 1. Wong, A.; A. Ferreira, P.; Santos, A.M.; Cincotto, F.H.; Silva, R.A.B.; Sotomayor, M.D.P.T. A new electrochemical sensor based on eco-friendly chemistry for the simultaneous determination of toxic trace elements. Microchem. J. 2020, 158, 105292, doi:10.1016/j.microc.2020.105292. 1. Wong, A.; A. Ferreira, P.; Santos, A.M.; Cincotto, F.H.; Silva, R.A.B.; Sotomayor, M.D.P.T. A new electrochemical sensor based on eco-friendly chemistry for the simultaneous determination of toxic trace elements. Microchem. J. 2020, 158, 105292, doi:10.1016/j.microc.2020.105292. 2. Li, N.; Yang, H. Construction of natural polymeric imprinted materials and their applications in water treatment: A review. J. Hazard. Mater. 2021, 403, doi:10.1016/j.jhazmat.2020.123643. 2. Li, N.; Yang, H. Construction of natural polymeric imprinted materials and their applications in water treatment: A review. J. Hazard. Mater. 2021, 403, doi:10.1016/j.jhazmat.2020.123643. 8 of 8 Proceedings 2020, 60, 39 3. Jinadasa, K.K.; Peña-Vázquez, E.; Bermejo-Barrera, P.; Moreda-Piñeiro, A. New adsorbents based on imprinted polymers and composite nanomaterials for arsenic and mercury screening/speciation: A review. Microchem. J. 2020, 156, 104886, doi:10.1016/j.microc.2020.104886. 4. Crapnell, R.D.; Hudson, A.; Foster, C.W.; Eersels, K.; van Grinsven, B.; Cleij, T.J.; Banks, C.E.; Peeters, M. Recent advances in electrosynthesized molecularly imprinted polymer sensing platforms for bioanalyte detection. Sensors 2019, 19, 1204, doi:10.3390/s19051204. 5. Sharma, P.S.; Pietrzyk-Le, A.; D’Souza, F.; Kutner, W. Electrochemically synthesized polymers in molecular imprinting for chemical sensing. Anal. Bioanal. Chem. 2012, 402, 3177–3204, doi:10.1007/s00216-011-5696-6. 6. Sharma, G.; Kandasubramanian, B. Molecularly Imprinted Polymers for Selective Recognition and Extraction of Heavy Metal Ions and Toxic Dyes. J. Chem. Eng. Data 2020, 65, 396–418, doi:10.1021/acs.jced.9b00953. 7. Di Masi, S.; Pennetta, A.; Guerreiro, A.; Canfarotta, F.; Egidio, G.; Benedetto, D.; Malitesta, C. Sensor based on electrosynthesised imprinted polymeric film for rapid and trace detection of copper (II) ions. Sensors Actuators B. Chem. 2020, 307, 127648, doi:10.1016/j.snb.2019.127648. 8. Pimenta, T.C.; Santos, C.d.C.; Thomasini, R.L.; Ferreira, L.F. Impedimetric immunosensor for dengue diagnosis using graphite screen-printed electrodes coated with poly(4-aminophenylacetic acid). Biomed. Microdevices 2018, 20, 1–9, doi:10.1007/s10544-018-0324-2. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2890662600	https://www.research-collection.ethz.ch/bitstream/20.500.11850/291154/2/s41467-018-06080-w.pdf	English	null	Deglacial mobilization of pre-aged terrestrial carbon from degrading permafrost	Nature communications	2,018	cc-by	14,759	ETH Library Publication date: 2018-09-10 Rights / license: g Creative Commons Attribution 4.0 International Author(s): Winterfeld, Maria; Mollenhauer, Gesine; Dummann, Wolf; Köhler, Peter; Lembke-Jene, Lester; Meyer, Vera D.; Hefter, Jens; McIntyre, Cameron; Wacker, Lukas; Kokfelt, Ulla; Tiedemann, Ralf Journal Article Author(s): Winterfeld, Maria; Mollenhauer, Gesine; Dummann, Wolf; Köhler, Peter; Lembke-Jene, Lester; Meyer, Vera D.; Hefter, Jens; McIntyre, Cameron; Wacker, Lukas; Kokfelt, Ulla; Tiedemann, Ralf Author(s): Winterfeld, Maria; Mollenhauer, Gesine; Dummann, Wolf; Köhler, Peter; Lembke-Jene, Lester; Meyer, Vera D.; Hefter, Jens; McIntyre, Cameron; Wacker, Lukas; Kokfelt, Ulla; Tiedemann, Ralf Originally published in: Originally published in: Nature Communications 9, https://doi.org/10.1038/s41467-018-06080-w g y p Nature Communications 9, https://doi.org/10.1038/s41467-018-06080-w This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. ARTICLE Deglacial mobilization of pre-aged terrestrial carbon from degrading permafrost Maria Winterfeld1,2, Gesine Mollenhauer 1,2,3, Wolf Dummann2,7, Peter Köhler 1, Lester Lembke-Jene 1 Vera D. Meyer1, Jens Hefter1, Cameron McIntyre 4,8, Lukas Wacker5, Ulla Kokfelt6 & Ralf Tiedemann1,2 Maria Winterfeld1,2, Gesine Mollenhauer 1,2,3, Wolf Dummann2,7, Peter Köhler 1, Lester Lembke-Jene 1 Vera D. Meyer1, Jens Hefter1, Cameron McIntyre 4,8, Lukas Wacker5, Ulla Kokfelt6 & Ralf Tiedemann1,2 The mobilization of glacial permafrost carbon during the last glacial–interglacial transition has been suggested by indirect evidence to be an additional and signiﬁcant source of greenhouse gases to the atmosphere, especially at times of rapid sea-level rise. Here we present the ﬁrst direct evidence for the release of ancient carbon from degrading permafrost in East Asia during the last 17 kyrs, using biomarkers and radiocarbon dating of terrigenous material found in two sediment cores from the Okhotsk Sea. Upscaling our results to the whole Arctic shelf area, we show by carbon cycle simulations that deglacial permafrost-carbon release through sea-level rise likely contributed signiﬁcantly to the changes in atmospheric CO2 around 14.6 and 11.5 kyrs BP. 1 Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar-und Meeresforschung (AWI), 27570 Bremerhaven, Germany. 2 Department of Geosciences, University of Bremen, 28359 Bremen, Germany. 3 MARUM—Center for Marine Environmental Sciences, University of Bremen, 28359 Bremen, Germany. 4 Department of Earth Sciences, Geological Institute, ETH Zürich, 8092 Zürich, Switzerland. 5 Department of Physics, Laboratory for Ion Beam Physics, ETH Zürich, 8093 Zürich, Switzerland. 6 Geological Survey of Denmark and Greenland, 1350 Copenhagen, Denmark. 7Present address: Institute of Geology and Mineralogy, University of Cologne, 50674 Cologne, Germany. 8Present address: Scottish Universities Environmental Research Centre, East Kilbride G75 0QF, UK. Correspondence and requests for materials should be addressed to G.M. (email: gesine.mollenhauer@awi.de) 1 NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w T T he carbon reservoir in northern high-latitude permafrost regions is substantially larger than the carbon content of the atmosphere today, and has been estimated to be approximately 1.5 times larger than its modern size during the Last Glacial Maximum (LGM)1,2. Consequently, permafrost degradation and rapid respiration of previously frozen organic matter during the last deglaciation potentially had profound effects on the global carbon cycle via positive feedbacks. Growing evidence suggests that large quantities of pre-aged carbon released from degrading permafrost contributed to the deglacial rise in atmospheric carbon dioxide and methane concentrations, thereby amplifying warming1,3,4. Results Accumulation rates and radiocarbon ages of terrige- nous carbon in sediments adjacent to areas of permafrost degradation thus allow a data-based evaluation of the permafrost carbon remobilization and associated carbon-climate feedback, including its contribution to deglacial CO2 rise. g g 2 To provide more insights into past East Asian permafrost dynamics, we here present a reconstruction of vegetation devel- opment in, and terrestrial carbon accumulation off of, the Amur River catchment over the past 17 kyrs. We analysed terrestrial organic compounds and their radiocarbon ages deposited in marine sediments off the mouth of the Amur River in the Okhotsk Sea, a marginal sea of the North Paciﬁc Ocean. The Amur River basin is the largest catchment in East Asia. The region was completely covered by permafrost during the LGM8 and is, as a result of deglacial permafrost mobilization, almost entirely permafrost-free today (Fig. 1)9. The East Sakhalin margin near the mouth of the Amur River is a location of high sediment accumulation, where river-discharged material, together with terrigenous particles entrained in ocean currents and derived from the shelf areas of the northwestern Okhotsk Sea, accumu- lates10 (see Methods). We studied material from two sediment cores, LV28-4-4 (51°08.475′ N, 145°18.582′ E; 674 m water depth) and SO178-13-6 (52°43.881′N, 144°42.647′E; 713 m water depth), retrieved from this zone of high glacial to Holocene sedimenta- tion. We analysed concentrations of two terrigenous biomarker groups (high molecular weight (HMW) n-alkanes, branched glycerol dialkyl glycerol tetraethers (brGDGTs)) and quantiﬁed the age of the deposited terrigenous material by compound- speciﬁc radiocarbon dating of HMW n-alkanoic acids. In our analysis, we compare our accumulation rates of terrigenous bio- markers with rates of global sea level rise, wetland development The deglacial warming also led to permafrost degradation within the Amur catchment, likely resulting in the formation of thermokarst lakes and wetland expansion, followed by drying and further thawing18. Analogous to today, permafrost-derived carbon likely has been released via emissions of CO2 and CH4 into the atmosphere6,7,19, and via dissolved and particulate organic matter through rivers to the ocean20. Being the dominant aquatic carbon export pathway, dissolved organic matter derived from thawing permafrost today is rapidly oxidized in Arctic rivers21 and signiﬁcantly contributes to greenhouse gas emissions. The hydrological evolution and resulting vegetation development is thus tightly linked to the process of ongoing permafrost retreat. Results Net losses of carbon from degrading permafrost, which has been photosynthetically ﬁxed millennia before mobilization and thus is depleted in radiocarbon, will result in export of pre-aged carbon. Permafrost degradation processes include thawing and deepening of the active layer, extension of wetlands, development of thermokarst resulting in subsidence, lake development and land-sliding, and coastal erosion. Until now, assessments of the susceptibility of permafrost to degradation and assessments of future and past feedbacks rely mainly on simulation scenarios, including assumptions based only on indirect estimates of the age of permafrost-derived old carbon, but physical data are so far lacking3,6. Thus, the age, timing and quantity of terrigenous carbon remobilized during the last deglaciation is largely unknown. Terrigenous material accumulation in Okhotsk Sea sediments. Highest accumulation rates of HMW long-chain (C27–C33) n- alkanes derived from terrestrial higher plants, and soil microbial brGDGTs are observed during the deglaciation between 17 and 10 kyrs BP, decreasing towards the Holocene and reaching low and rather constant values after 8 kyrs BP (Fig. 2c, d). Two dis- tinct maxima are observed in both biomarker records, namely between 14.5 and 13 kyrs BP during the Bølling-Allerød (B/A), and between 11.5 and 10 kyrs BP during the Pre-Boreal periods. Furthermore, a less-pronounced local maximum exists between 17 and 15.5 kyrs BP during Heinrich Stadial 1 (HS1). These records of terrigenous material accumulation reach their highest values during times of rapid sea-level rise (Fig. 2b)14, connected with the global melt water pulses (MWP) centred around 11 and 14 kyrs BP raising sea-level by a total of approximately 80 m. We interpret this temporal coincidence as an indication that coastal erosion was the main cause for this permafrost carbon mobilization. The abrupt increase in biomar- ker accumulation at these times implies that erosion of coastal permafrost happened relatively fast, which is plausible, as today coastal erosion of permafrost deposits occurs at extremely rapid rates15 and results in failure of coastal bluffs, supplying large amounts of particulate organic matter directly to the ocean16. In a next step, this material is then distributed, further degraded, and re-buried in marine sediments5,17. In contrast to dissolved organic matter, particulate organic matter is the only fraction of exported material that can be pre- served in sediments and records past climate and environmental conditions. Deglacial mobilization of pre-aged terrestrial carbon from degrading permafrost The deglacial processes might serve as modern analogues, as positive climate feedbacks from coastal erosion and hinterland thawing of carbon-rich permafrost soil deposits are also expected under future warming5,6. The rates and pathways of carbon release from permafrost are highly uncertain, but crucial to understand how strongly, and over which time- scales, these feedbacks may affect climate7. and changes in Amur river discharge, and the pre-depositional age of terrigenous biomarkers with deglacial records of the carbon cycle from the literature11–13. Thereby, we investigated mobili- zation of pre-aged terrestrial carbon by coastal erosion and thermal permafrost degradation in the hinterland and subsequent ﬂuvial export. By upscaling and applying our new ﬁndings as boundary conditions to a carbon cycle model, we simulate how this mobilization of permafrost carbon might have inﬂuenced the levels of atmospheric CO2 and Δ14C. We ﬁnd that about 50% of the abrupt rises in atmospheric CO2 found in ice cores at 14.6 and 11.5 kyrs BP can be explained by this mobilization of pre-aged terrestrial carbon via coastal erosion in the Arctic Ocean fol- lowing sea-level rise. However, the long-term contributions of this process to the CO2 rise and Δ14C decline across Termination I are small. Results The Amur river discharge as reconstructed from the accumula- tion rate of freshwater algae (Fig. 2f, Methods), was considerably higher during the deglaciation than it is today. It reached its maximum around 10 kyrs BP (Fig. 2f) likely exporting vast amounts of dissolved and particulate organic matter during this time interval, which is indicated by relatively higher biomarker accumulation rates between 10 and 8 kyrs BP compared to the modern situation (Fig. 2c, d). However, because of the different timing of the Amur discharge maximum and the peaks in biomarker accumulation rates centred at ~14 and 11 kyrs BP, especially during the Pre-Boreal, we conclude that river-derived NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w Compound-speciﬁc radio- carbon analyses (CSRA; Methods) of HMW n-alkanoic acids (C26:0 and C28:0) from selected intervals (six from SO178-13-6 and four from LV28-4-4) reveal that during the deglaciation terrigenous biomarkers were strongly pre-aged by 5 to 10 kyrs at the time of deposition and became progressively younger after the Fig. 1 Permafrost distribution in East Asia. Okhotsk Sea study area with locations of the investigated cores (red circles) and a core referenced in this study26. The Amur River basin is outlined in black. a Modern permafrost extent66 is indicated in blue (dark: continuous permafrost, light: discontinuous and sporadic permafrost) and wetlands67 in green. Red arrows represent the surface water circulation with the East Sakhalin Current (ESC) and the East Kamchatka Current (EKG), and blue arrows represent the Dense Shelf Water (DSW) pathways. b Permafrost extent8 and exposed shelf areas (132 m isobaths) during the last glacial (~21 kyrs BP). The modern coastline is indicated by the dashed line. Maps are created using GMT (Generic Mapping Tools, http://gmt.soest.hawaii.edu/) software NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications 3 Fig. 1 Permafrost distribution in East Asia. Okhotsk Sea study area with locations of the investigated cores (red circles) and a core referenced in this study26. The Amur River basin is outlined in black. a Modern permafrost extent66 is indicated in blue (dark: continuous permafrost, light: discontinuous and sporadic permafrost) and wetlands67 in green. Red arrows represent the surface water circulation with the East Sakhalin Current (ESC) and the East Kamchatka Current (EKG), and blue arrows represent the Dense Shelf Water (DSW) pathways. b Permafrost extent8 and exposed shelf areas (132 m isobaths) during the last glacial (~21 kyrs BP). The modern coastline is indicated by the dashed line. Maps are created using GMT (Generic Mapping Tools, http://gmt.soest.hawaii.edu/) software organic matter has only partially contributed to these peaks. Since the Amur discharge is largely controlled by precipitation intensity in East Asia, i.e., the East Asian summer monsoon (Fig. 2g)22, we ﬁnd a general synchronicity of river discharge and monsoonal precipitation, which follows local summer insolation, as recorded in speleothems23 (Fig. 2g) in the early Holocene. Age of terrigenous organic matter. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w s only partially contributed to these peaks. Since ge is largely controlled by precipitation intensity he East Asian summer monsoon (Fig. 2g)22, we nchronicity of river discharge and monsoonal Age of terrigenous organic matter. Compoun carbon analyses (CSRA; Methods) of HMW (C26:0 and C28:0) from selected intervals (six f and four from LV28-4-4) reveal that during N 105° 105° 110° 110° 115° 115° 120° 120° 125° 125° 130° 130° 135° 135° 140° 140° 145° 145° 150° 150° 155° 155° 160° 160° 165° 165° 35° 35° 40° 40° 45° 45° 50° 50° 55° 55° 60° 60° 65° 65° SO178−13−6 LV28−4 XP07−C9 500 km N 105° 105° 110° 110° 115° 115° 120° 120° 125° 125° 130° 130° 135° 135° 140° 140° 145° 145° 150° 150° 155° 155° 160° 160° 165° 165° 35° 35° 40° 40° 45° 45° 50° 50° 55° 55° 60° 60° 65° 65° SO178−13−6 LV28−4 XP07−C9 a b EKC ESC DSW tribution in East Asia. Okhotsk Sea study area with locations of the investigated cores (red circles) and a core iver basin is outlined in black. a Modern permafrost extent66 is indicated in blue (dark: continuous permafrost, ligh and wetlands67 in green. Red arrows represent the surface water circulation with the East Sakhalin Current (E (EKG), and blue arrows represent the Dense Shelf Water (DSW) pathways. b Permafrost extent8 and exposed ast glacial (~21 kyrs BP). The modern coastline is indicated by the dashed line. Maps are created using GMT (Gen waii.edu/) software N 105° 110° 115° 120° 125° 130° 135° 140° 145° 150° 155° 160° 165° 35° 35° 40° 40° 45° 45° 50° 50° 55° 55° 60° 60° 65° 65° SO178−13−6 LV28−4 XP07−C9 a EKC ESC DSW 500 km N 105° 110° 115° 120° 125° 130° 135° 140° 145° 150° 155° 160° 165° 35° 35° 40° 40° 45° 45° 50° 50° 55° 55° 60° 60° 65° 65° SO178−13−6 LV28−4 XP07−C9 b organic matter has only partially contributed to these peaks. Since the Amur discharge is largely controlled by precipitation intensity in East Asia, i.e., the East Asian summer monsoon (Fig. 2g)22, we ﬁnd a general synchronicity of river discharge and monsoonal precipitation, which follows local summer insolation, as recorded in speleothems23 (Fig. 2g) in the early Holocene. Age of terrigenous organic matter. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w 2 Proxies for terrigenous organic matter mobilization compared with records of deglacial environmental changes. a Greenland NGRIP δ18O68; b rate of global sea-level change14; biomarker records obtained from sediment core SO178-13-6, mass accumulation rate (MAR) of c branched glycerol dialkyl glycerol tetraethers (brGDGTs) and d high molecular weight (HMW) n-alkanes (C27, C29, C31 and C33); e Paq ratio25 from nearby core XP07-C926; f accumulation rate (AR) of chlorophycean freshwater algae Pediastrum spp. from core LV28-4-4, note the logarithmic scale; g speleothem δ18O record from Dongge cave as indicator for East Asian summer monsoon intensity, which controls precipitation in the Amur Basin23; h summer insolation at 50°N. Circles at the bottom show age control points (AMS 14C dates) with 2σ uncertainties for cores LV28-4-4 (brown) and SO178-13-6 (green)46. Orange boxes highlight the warm phases Bølling-Allerød (B/A) and Pre-Boreal (PB), the Younger Dryas cold spell (YD) and Heinrich Stadial 1 (HS1) are marked in blue; grey boxes mark the periods of melt water pulse 1 A (MWP-1A) and MWP-1B Fig. 2 Proxies for terrigenous organic matter mobilization compared with records of deglacial environmental changes. a Greenland NGRIP δ18O68; b rate of global sea-level change14; biomarker records obtained from sediment core SO178-13-6, mass accumulation rate (MAR) of c branched glycerol dialkyl glycerol tetraethers (brGDGTs) and d high molecular weight (HMW) n-alkanes (C27, C29, C31 and C33); e Paq ratio25 from nearby core XP07-C926; f accumulation rate (AR) of chlorophycean freshwater algae Pediastrum spp. from core LV28-4-4, note the logarithmic scale; g speleothem δ18O record from Dongge cave as indicator for East Asian summer monsoon intensity, which controls precipitation in the Amur Basin23; h summer insolation at 50°N. Circles at the bottom show age control points (AMS 14C dates) with 2σ uncertainties for cores LV28-4-4 (brown) and SO178-13-6 (green)46. Orange boxes highlight the warm phases Bølling-Allerød (B/A) and Pre-Boreal (PB), the Younger Dryas cold spell (YD) and Heinrich Stadial 1 (HS1) are marked in blue; grey boxes mark the periods of melt water pulse 1 A (MWP-1A) and MWP-1B Pre-Boreal towards the late Holocene (Table 1, Fig. 3c; late Holocene dates are not shown in Figure). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w 0 2 4 6 8 10 12 14 16 18 Age (cal kyrs BP) 480 490 500 510 520 530 Summer insolation 50°N (W m–2) –4 –5 –6 –7 –8 –9 –10 100 1000 10,000 100,000 Pediastrum spp. (μg cm–2 yr–1) 0.24 0.28 0.32 0.36 0.4 0.44 Paq 0 0.04 0.08 0.12 0.16 MAR (μg cm–2 yr–1) brGDGTs/HMW n-alkanes 0 0.4 0.8 1.2 1.6 –20 –10 0 10 20 30 40 Rate of sea-level change (m kyr–1) –48 –44 –40 –36 –32 NGRIP δ18O (‰) Dongge cave δ18O (‰ VPDB) 0 2 4 6 8 10 12 14 16 18 Age (cal kyrs BP) a c/d e f g MWP 1B MWP 1A Age control points HS1 YD B/A PB Holocene b Core SO178-13-6 Core LV28-4-4 Wetland contribution + – h Amur discharge + – Fig. 2 Proxies for terrigenous organic matter mobilization compared with records of deglacial environmental changes. a Greenland NGRIP δ18O68; b rate of global sea-level change14; biomarker records obtained from sediment core SO178-13-6, mass accumulation rate (MAR) of c branched glycerol dialkyl ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w 0 2 4 6 8 10 12 14 16 18 Age (cal kyrs BP) 480 490 500 510 520 530 Summer insolation 50°N (W m–2) –4 –5 –6 –7 –8 –9 –10 100 1000 10,000 100,000 Pediastrum spp. (μg cm–2 yr–1) 0.24 0.28 0.32 0.36 0.4 0.44 Paq 0 0.04 0.08 0.12 0.16 MAR (μg cm–2 yr–1) brGDGTs/HMW n-alkanes 0 0.4 0.8 1.2 1.6 –20 –10 0 10 20 30 40 Rate of sea-level change (m kyr–1) –48 –44 –40 –36 –32 NGRIP δ18O (‰) Dongge cave δ18O (‰ VPDB) 0 2 4 6 8 10 12 14 16 18 Age (cal kyrs BP) a c/d e f g MWP 1B MWP 1A Age control points HS1 YD B/A PB Holocene b Core SO178-13-6 Core LV28-4-4 Wetland contribution + – h Amur discharge + – Fig. 2 Proxies for terrigenous organic matter mobilization compared with records of deglacial environmental changes. a Greenland NGRIP δ18O68; b global sea-level change14; biomarker records obtained from sediment core SO178-13-6, mass accumulation rate (MAR) of c branched glycerol d l l t t th (b GDGT ) d d hi h l l i ht (HMW) lk (C C C d C ) P ti 25 f b XP07 C Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w Compound-speciﬁc radio- carbon analyses (CSRA; Methods) of HMW n-alkanoic acids (C26:0 and C28:0) from selected intervals (six from SO178-13-6 and four from LV28-4-4) reveal that during the deglaciation terrigenous biomarkers were strongly pre-aged by 5 to 10 kyrs at the time of deposition and became progressively younger after the Age of terrigenous organic matter. Compound-speciﬁc radio- carbon analyses (CSRA; Methods) of HMW n-alkanoic acids (C26:0 and C28:0) from selected intervals (six from SO178-13-6 and four from LV28-4-4) reveal that during the deglaciation terrigenous biomarkers were strongly pre-aged by 5 to 10 kyrs at the time of deposition and became progressively younger after the NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w These pre-depositional ages indicate that terrestrial deposits, which were eroded during deglacial sea-level rise and exported from degrading permafrost soils within the Amur catchment, contained large amounts of several millennia-old carbon, a situation which is comparable to modern-day organic matter-rich permafrost deposits, particularly the ice-rich Yedoma found as glacial relict primarily in eastern Siberia, Alaska, and parts of Canada (ref. 24 and references therein). Such types of deposit likely covered large areas of the glacial Asian continent and adjacent exposed shelves18, and its degradation and erosion led to massive terrestrial organic carbon remobilization and partial burial in marine sediments. Today, Pre-Boreal towards the late Holocene (Table 1, Fig. 3c; late Holocene dates are not shown in Figure). These pre-depositional ages indicate that terrestrial deposits, which were eroded during deglacial sea-level rise and exported from degrading permafrost soils within the Amur catchment, contained large amounts of several millennia-old carbon, a situation which is comparable to modern-day organic matter-rich permafrost deposits, particularly the ice-rich Yedoma found as glacial relict primarily in eastern Siberia, Alaska, and parts of Canada (ref. 24 and references therein). Such types of deposit likely covered large areas of the glacial Asian continent and adjacent exposed shelves18, and its degradation and erosion led to massive terrestrial organic carbon remobilization and partial burial in marine sediments. Today, erosion of Yedoma along the coasts results in complete failure of the coastal bluffs16, and there is no evidence for Yedoma pre- served subsea. The material eroded from Yedoma settles rapidly, resulting in high burial rates of strongly pre-aged terrestrial car- bon in near-coast and shelf sediments5,17. Terrigenous HMW n- alkanoic acids deposited during peak discharge of the Amur at around 10 kyrs BP and also later at around 8 kyrs BP are likewise pre-aged, ~8–10 kyrs old at the time of deposition (Fig. 3c), indicating that also the terrestrial carbon transported from the thawing hinterland permafrost by the Amur river to the Okhotsk Sea contained several millennia old carbon and thus contributed to the accumulation of pre-aged terrestrial carbon in the sediment. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w Table 1 Compound-speciﬁc radiocarbon data ± propagated errors (σ) of long-chain n-alkanoic acids for cores SO178-13-6 and LV28-4-4 Sample depth (cm below surface) Deposition age (mid-point) [cal kyrs BP] n-alkanoic acid Corrected F14C ± σF14Ca Δ14C ± σ14C [‰] Age at deposition ± 1σ [yr] Core SO178-13-6 55–65 0.76 n-C26:0 0.6465 ± 0.0064 −358.5 ± 6.1 3065 ± 123 695–705 5.92 n-C26:0 0.3078 ± 0.0041 −694.6 ± 4.0 4790 ± 120 1435–1445 10.02 n-C26:0b 0.1567 ± 0.0036 −844.5 ± 3.4 8060 ± 210 1435–1445 10.02 n-C28:0b 0.1514 ± 0.0035 −849.7 ± 3.3 8410 ± 205 1805–1815 11.97 n-C26:0 0.1762 ± 0.0046 −825.2 ± 4.5 4980 ± 325 2033–2041 14.16 n-C26:0b 0.1152 ± 0.0031 −885.7 ± 2.9 6780 ± 290 2335–2342 17.26 n-C26:0 0.0683 ± 0.0030 −932.2 ± 2.9 8600 ± 260 Core LV28-4-4 54–56 0.71 n-C26:0 0.5943 ± 0.0084 −411.1 ± 8.1 4115 ± 50 54–56 0.71 n-C28:0 0.5692 ± 0.0102 −435.2 ± 9.8 4580 ± 190 751–753 8.29 n-C28:0 0.1586 ± 0.0041 −842.6 ± 4.0 9690 ± 240 860–862 11.78 n-C26:0 0.1045 ± 0.0036 −896.3 ± 3.5 10,160 ± 345 860–862 11.78 n-C28:0 0.1100 ± 0.0046 −890.8 ± 4.4 9690 ± 445 926–928 16.0 n-C26:0 0.0973 ± 0.0039 −736.0 ± 4.2 6530 ± 340 aCorrected for blank contribution and methylation; ±propagated error (see Methods) b66% split of sample 4 6 8 10 12 14 16 18 Age (cal kyrs BP) 0 40 80 120 160 200 240 MAR brGDGTs (μg cm–2 kyr–1) 2000 4000 6000 8000 10,000 12,000 HMW n-alkanoic acid age at deposition (yr) 0 100 200 300 400 500 Δ14C (‰) 4 6 8 10 12 14 16 18 Age (cal kyrs BP) 180 200 220 240 260 280 Atmospheric CO2 (ppm) LV28-4-4 C26:0 C28:0 C26:0 C28:0 52 54 56 Latitute (°N) Botovskaya cave Okhotnichya cave a c d MWP 1B MWP 1A HS1 YD B/A PB Holocene b e SO178-13-6 Fig. 3 Proxies used to reconstruct permafrost dynamics and carbon mobilization. a Atmospheric CO2 mixing ratio from ice cores12, 13 (data as in ref. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w 69); b atmospheric Δ14C (‰) as reconstructed in IntCAL1311; c age at deposition of higher plant derived C26:0 and C28:0 n-alkanoic acids from SO178-13-6 (red triangles) and LV28-4-4 (pink triangles) derived from compound-speciﬁc radiocarbon dates of the respective biomarkers with horizontal error bars representing 2σ age uncertainties of the closest age control point (see Fig. 2) and vertical error bars representing the propagated age uncertainties after blank correction (see Methods), please note that the age scale goes from 4 to 18 kyrs BP here and that additional ages of n-alkanoic acids deposited before 4 kyrs BP can be found in Table 1; d mass accumulation rates (MAR) of branched glycerol dialkyl glycerol tetraethers (brGDGTs) of core SO178-13-6 representative of MAR of all terrigenous biomarkers; e periods of vadose speleothem growth linked to permafrost thaw and/or absence at the Botovskaya cave and the Okhotnichya cave9. Orange boxes highlight the warm phases Bølling-Allerød (B/A) and Pre-Boreal (PB), the Younger Dryas cold spell (YD) and Heinrich Stadial 1 (HS1) are marked in blue; grey boxes mark the periods of melt water pulse 1a (MWP-1A) and the putative MWP-1B NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications 5 4 6 8 10 12 14 16 18 Age (cal kyrs BP) 0 40 80 120 160 200 240 MAR brGDGTs (μg cm–2 kyr–1) 2000 4000 6000 8000 10,000 12,000 HMW n-alkanoic acid age at deposition (yr) 0 100 200 300 400 500 Δ14C (‰) 4 6 8 10 12 14 16 18 Age (cal kyrs BP) 180 200 220 240 260 280 Atmospheric CO2 (ppm) LV28-4-4 C26:0 C28:0 C26:0 C28:0 52 54 56 Latitute (°N) Botovskaya cave Okhotnichya cave a c d MWP 1B MWP 1A HS1 YD B/A PB Holocene b e SO178-13-6 Fig. 3 Proxies used to reconstruct permafrost dynamics and carbon mobilization. a Atmospheric CO2 mixing ratio from ice cores12, 13 (data as in ref. 69); b atmospheric Δ14C (‰) as reconstructed in IntCAL1311; c age at deposition of higher plant derived C26:0 and C28:0 n-alkanoic acids from SO178-13-6 (red triangles) and LV28-4-4 (pink triangles) derived from compound-speciﬁc radiocarbon dates of the respective biomarkers with horizontal error bars representing 2σ age uncertainties of the closest age control point (see Fig. ARTICLE This degradation of permafrost and associated wetland development during the B/A, and particularly the Pre-Boreal warm phases continuing until ~8 kyrs BP, is supported by the Paq-record25, representing leaf-wax lipids ascribed to aquatic plants versus lipids derived from higher vascular plants, from a nearby core (Figs. 1 and 2e)26. Further- more, the generally wetter conditions in the Amur catchment during the Pre-Boreal are in agreement with prior studies of pollen and biomarker records in peatlands27,28. These wetlands located at the southern boundary of the boreal region are potential source areas of CH4, which could have contributed to the rapid deglacial increases in atmospheric methane29. By the onset of the Holocene the continuous permafrost boundary almost reached its modern position leaving most of the Amur catchment permafrost-free, which is indicated by speleothem growth initiation in two caves near Lake Baikal on the north- eastern boundary of the Amur catchment (Fig. 3e)9. depleted CO2. The contribution from degrading hinterland per- mafrost as a result of thermokarst lake and wetland development as well as soil destabilization and erosion are spatially hetero- geneous processes and therefore difﬁcult to extrapolate to the wider permafrost region. In our extrapolation, we therefore focus in the following on the potential contribution from sea-level- induced coastal erosion. Deglacial sea-level rise eventually ﬂooded 1.9 million km2 of the Arctic shelf area24,30. About 80% of the exposed shelf is assumed to have been covered by Yedoma during the glacial sea-level lowstand18. A recent study estimated that 259 PgC were lost from the total Yedoma domain during the tran- sition between the last glacial maximum and the Holocene23 providing an estimate of glacial permafrost carbon that might have been deposited on the Arctic shelves and was remobilized during their ﬂooding. Between 18 and 11 kyrs global sea level rose from 130 to 50 m below present14. Considering the regional bathymetry, we cal- culated consistently from two different approaches (similarly as in either ref. 3 or ref. 30), that about 50% of the Arctic shelf area was ﬂooded during this time period by this 80 m rise in global sea level. We therefore take a similar fraction of the proposed per- mafrost carbon deposited on the Arctic shelves (i.e.,129.5 PgC) into account that could have been released during this time interval. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w Timing of terrigenous material accumulation peaks. Our records of accumulation rates of terrigenous biomarkers and their age at the time of deposition (Fig. 3c) illustrate the relative timing and interplay of two deglacial processes—coastal erosion and hinterland thermal degradation. At approximately 17.25 kyrs BP, when the rate of sea-level rise was low, HMW n-alkanoic acids were about 8.6 kyrs old at the time of deposition. Compound- speciﬁc radiocarbon ages of HMW n-alkanoic acids in modern core-top sediments of the East Siberian Shelf reach values of up to 18 kyrs at locations of active coastal erosion of glacial deposits17. Thus, the reconstructed pre-depositional age of 8.6 kyrs is com- parable to the modern situation when accounting for the addi- tional ageing of >10 kyrs since the organic matter was buried at our core locations during the last deglaciation. During the period starting slightly before and encompassing MWP 1A, which was also characterized by slightly increasing river discharge, HMW n- alkanoic acids were about 6.5 kyrs in age at the time of deposition, and terrigenous biomarkers accumulated at high rates. These deposits might have been partly derived from coastal erosion and from inland material, the latter of which is found to be between 3 and 5 kyrs old in the Arctic Lena river delta today17. Synchronous to the phase of lowest deglacial sea-level rise, HMW n-alkanoic acids of at least 5 kyrs in age were deposited during the Younger Dryas cold period. During the Pre-Boreal, a second peak in ter- rigenous biomarker accumulation occurred, and HMW n-alka- noic acids deposited before this peak were ~10 kyrs old at deposition. Likely, this event was again primarily related to sea- level rise induced erosion, as the onset in our MAR is delayed from the abrupt rise in global sea level by only a few centuries (Fig. 2a–d), which might be due to regional differences in sea- level rise within the Okhotsk Sea as well as age model uncer- tainties of the investigated sediment core. The maximum peak in Amur river discharge (Fig. 2f) occurred later at around 10 kyrs BP, when slightly younger but still strongly pre-aged HMW n- alkanoic acids (~8 kyrs in age) were deposited, presumably con- taining a substantial fraction of material from degrading inland permafrost exported through the Amur river. ARTICLE At 14.6 kyrs BP we simulate a CO2 peak of 6 ppm (Fig. 5a) together with a drop in Δ14C of 6 or 8 ‰ (Fig. 5b) depending on whether a pre- depositional carbon age of either 5 or 10 kyrs was assumed. For this event, permafrost thawing has already been suggested as a possible cause3, but assuming greater carbon release of 125 PgC y g Using the global carbon cycle model BICYCLE3, we simulated how the ﬂooding of the Arctic shelves during the last deglaciation may have contributed to changes in atmospheric carbon records. In our model simulation, CO2 release from permafrost, derived from the assumption that 66% of mobilized permafrost carbon was respired, was restricted to a time window of 200 years similar to a previous study3. Both the release length and the pinning of its onset to sea level changes was assumed to be identical for the two other events at 16.5 and 11.5 kyrs, while the annual release rates (0.17 or 0.09 PgC per year) were derived from the total carbon amount that was assumed to be remineralized approximately scaled to the amplitudes of our biomarker MAR records. As a result, our model simulated a permafrost carbon release of 34 PgC at 11.5 and 14.6 kyrs BP and of 17 PgC at 16.5 kyrs BP (Fig. 4a). In an alternative scenario, the gradual release of the 85 PgC was simulated with a constant release rate across the last deglaciation. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w 2) and vertical error bars representing the propagated age uncertainties after blank correction (see Methods), please note that the age scale goes from 4 to 18 kyrs BP here and that additional ages of n-alkanoic acids deposited before 4 kyrs BP can be found in Table 1; d mass accumulation rates (MAR) of branched glycerol dialkyl glycerol tetraethers (brGDGTs) of core SO178-13-6 representative of MAR of all terrigenous biomarkers; e periods of vadose speleothem growth linked to permafrost thaw and/or absence at the Botovskaya cave and the Okhotnichya cave9. Orange boxes highlight the warm phases Bølling-Allerød (B/A) and Pre-Boreal (PB), the Younger Dryas cold spell (YD) and Heinrich Stadial 1 (HS1) are marked in blue; grey boxes mark the periods of melt water pulse 1a (MWP-1A) and the putative MWP-1B 5 ARTICLE For our simulation of the potential impact of permafrost carbon release, we assume, based on a recent estimate of remi- neralization rate of eroded Yedoma at an Arctic coast5, that 66% of this organic matter (i.e., ~85 PgC) was oxidized to CO2 and released to the atmosphere. Since our biomarker-MAR records show three distinct peaks (Fig. 2c, d) which are related to sea level rise, we focus our simulations on the assumption that the proposed 85 PgC have been released by these three events. Furthermore, we rely on an earlier simulation study3 discussing the 14.6 kyr-event, that clearly showed (based on U/Th-dated atmospheric 14C anomalies from Tahiti corals)31 that a sea level-related carbon release started no later than 14.6 kyrs BP. This information pins the start of our carbon release from ﬂooded shelf permafrost to the onset of the sea level rise (Fig. 4a), while the peak in our biomarker MARs occurred some centuries later, potentially delayed by some transformation processes32. However, the apparent delay might solely be related to age model uncertainties. solely be related to age model uncertainties. Using the global carbon cycle model BICYCLE3, we simulated how the ﬂooding of the Arctic shelves during the last deglaciation may have contributed to changes in atmospheric carbon records. In our model simulation, CO2 release from permafrost, derived from the assumption that 66% of mobilized permafrost carbon was respired, was restricted to a time window of 200 years similar to a previous study3. Both the release length and the pinning of its onset to sea level changes was assumed to be identical for the two other events at 16.5 and 11.5 kyrs, while the annual release rates (0.17 or 0.09 PgC per year) were derived from the total carbon amount that was assumed to be remineralized approximately scaled to the amplitudes of our biomarker MAR records. As a result, our model simulated a permafrost carbon release of 34 PgC at 11.5 and 14.6 kyrs BP and of 17 PgC at 16.5 kyrs BP (Fig. 4a). In an alternative scenario, the gradual release of the 85 PgC was simulated with a constant release rate across the last deglaciation. Our results show that the sea-level rise-induced rapid mobili- zation of old permafrost-derived carbon likely coincided with the abrupt rises in the atmospheric CO2 record12 at 14.6 and 11.5 kyrs BP, but not at 16.5 kyrs BP (Fig. 5a, c, d). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w The full CO2 amplitude of the 14.6 kyr-event in WAIS Divide ice core was calculated to be 12 ± 1 ppm when averaging data points before and after the rise12, but might actually be around 15 ppm when calculating the peak-to-peak-difference (Fig. 5a). Such a CO2 peak would be explained in our model by the release of the entire estimate of 85 PgC within only two centuries. This amount of carbon is signiﬁcantly lower than in the initial proposal3, but if solely based on the radiocarbon-depleted carbon from permafrost thawing, might still explain the Δ14C anomaly in the Tahiti corals (Fig. 5b). Terrestrial biomarker records in a sediment core retrieved in the Black Sea34 also point towards the degradation of permafrost in Eastern Europe at the onset of the Northern Hemisphere warming into the Bølling-Allerød, potentially con- tributing to the rapid CO2 rise at 14.6 kyrs BP. 10 Age (cal. kyr BP) –20 –10 0 10 20 30 40 Δ (sea level) (m kyr–1) 0.0 0.05 0.1 0.15 0.2 0.25 C release (PgC yr–1) C release: Gradual 3 peaks a 0.0 0.5 1.0 1.5 2.0 MAR (μg cm–2 yr–1) MAR: HMW n-alkanes 10 × brGDGTs 12 14 16 18 Age (cal. kyr BP) 180 190 200 210 220 230 240 250 260 270 280 b 0 10 20 30 40 50 60 70 80 90 100 CO2 data Simulated Δ(CO2): Permafrost thaw, gradual Permafrost thaw, 3 peaks atm. CO2 (ppm) atm. Δ(CO2) (ppm) 10 12 14 16 18 b The simulated amplitudes for both the 11.5 and 14.6 kyr-event are similar, since they are based on identical carbon release rates, and would explain a substantial part of the CO2 rise found in the WAIS Divide ice core12, which has been quantiﬁed to 13 ± 1 ppm at 11.5 kyrs BP (Fig. 5c). In line with our baseline assumptions, evidence for permafrost carbon mobilization during this time period has also been found on the Laptev Sea shelf20. At 16.5 kyrs BP, we simulated a rise in CO2 of only 3 ppm and a decline in Δ14C of 4–6‰ (Fig. 4). Here, the simulated CO2 rise occurs 300 years earlier than the abrupt rise seen in the WAIS Divide ice core and is only a quarter of the amplitude in the ice core data (Fig. 5d). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w ARTICLE 10 Age (cal. kyr BP) Age (cal. kyr BP) Age (cal. kyr BP) –20 –10 0 10 20 30 40 Δ (sea level) (m kyr–1) 0.0 0.05 0.1 0.15 0.2 0.25 C release (PgC yr–1) C release: Gradual 3 peaks a 0.0 0.5 1.0 1.5 2.0 MAR (μg cm–2 yr–1) MAR: HMW n-alkanes 10 × brGDGTs 180 190 200 210 220 230 240 250 260 270 280 b 0 10 20 30 40 50 60 70 80 90 100 CO2 data Simulated Δ(CO2): Simulated Δ(Δ 14C): Permafrost thaw, gradual Permafrost thaw, 3 peaks –300 –250 –200 –150 –100 –50 0 50 Δ14C IntCal13 Unexplained residual Permafrost thaw, gradual Permafrost thaw, 3 peaks c 100 150 200 250 300 350 400 450 atm. CO2 (ppm) atm. Δ(CO2) (ppm) atm. Δ14C (‰) atm. Δ(Δ14C) (‰) 12 14 16 18 10 12 14 16 18 10 12 14 16 18 Fig. 4 Simulated impacts of sea level triggered coastal erosion and related permafrost thawing on atmospheric carbon reservoirs using the global carbon cycle model BICYCLE. a Assumed carbon release from permafros thaw of 85 PgC from 18 to 10.8 kyrs BP, either gradual (orange) or in 3 short periods of 200 yr duration connected with rapid sea level rise (red) For comparison our new MAR data and a reconstruction of sea level change14 are also shown. b Simulated anomalies in atmospheric CO2 levels for the two carbon release scenarios and reconstructed CO2 data (mean ± 1σ) from ice cores12, 13 for comparison (data as in ref. 69). c Simulated anomalies in atmospheric Δ14C. The unexplained residual (mean (blue) ± 1σ uncertainty (grey)) shows Δ(Δ14C) that is not explained by changes in 14C d A l 14C d b h b l \| / leading to a true atmospheric CO2 peak of more than 20 ppm, that might have been recorded as a CO2 rise of 12 ppm in about two centuries in the EPICA Dome C ice core. However, this earlier interpretation3 was based on a CO2 record with lower resolution, while newer CO2 data from the WAIS Divide ice core12 provide more constraints on the amplitude and allow, due to a reﬁned understanding of gas enclosure processes in the WAIS Divide ice core33, only little overshoot of the true atmo- spheric signal when compared to the ice core-based CO2 rise. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w Based on the published chronologies and our assumption of a synchronicity of the onset of terrestrial carbon releases and abrupt sea level rises supported by the U/Th dated 14C available for the 14.6 ka event, it seems unlikely that at 16.5 kyrs BP the rapid CO2 rise is related to sea-level induced per- mafrost erosion. However, future improvements of age model uncertainties might support different conclusions. Age (cal. kyr BP) Simulated Δ(Δ 14C): –300 –250 –200 –150 –100 –50 0 50 Δ14C IntCal13 Unexplained residual Permafrost thaw, gradual Permafrost thaw, 3 peaks c 100 150 200 250 300 350 400 450 atm. Δ14C (‰) atm. Δ(Δ14C) (‰) 10 12 14 16 18 c c Our model-based extrapolations of carbon release from per- mafrost thawing and degradation through coastal erosion on the global carbon cycle are a rough ﬁrst estimate, which needs further support from independent data. Admittedly, some uncertainties exist in our assessment, e.g. the simulated atmospheric carbon anomalies are model-dependent, and our model has a rather small airborne fraction when compared to others (Supplementary Fig. 1a), implying that carbon released into the atmosphere is quickly taken up by the ocean. Models with higher airborne fractions would therefore simulate larger CO2 amplitudes based on the amounts of released carbon we estimated in our study. The extrapolated amount of carbon on the Arctic shelf also contains uncertainties on the order of 50% in the estimate of the Yedoma organic carbon content. Furthermore, some of the permafrost- derived carbon is found in our sediment cores in the Okhotsk Sea, underlining the uncertainties associated with the estimated remineralization rate of 66%, which is potentially too high. The current range of estimates for carbon loss upon thaw varies from 2 to ~80% of the initial carbon stock35,36. Fig. 4 Simulated impacts of sea level triggered coastal erosion and related permafrost thawing on atmospheric carbon reservoirs using the global carbon cycle model BICYCLE. a Assumed carbon release from permafrost thaw of 85 PgC from 18 to 10.8 kyrs BP, either gradual (orange) or in 3 short periods of 200 yr duration connected with rapid sea level rise (red). For comparison our new MAR data and a reconstruction of sea level change14 are also shown. b Simulated anomalies in atmospheric CO2 levels for the two carbon release scenarios and reconstructed CO2 data (mean ± 1σ) from ice cores12, 13 for comparison (data as in ref. 69). Discussion O d Our records indicate a contribution of degrading permafrost to atmospheric CO2 and Δ14C levels, the magnitude of which we attempt to estimate based on an extrapolation of our results using a carbon cycle model. On a global scale, the shelf areas of the Okhotsk Sea are relatively small, implying that their ﬂooding alone could not explain a signiﬁcant contribution to the rise in atmospheric CO2 concentration. The Bering, Chukchi, and East Siberian Shelves (together with the shelf areas of the Okhotsk Sea summarized as Arctic shelves from here on) were likely ﬂooded at around the same time, resulting in substantial emission of 14C- Our results show that the sea-level rise-induced rapid mobili- zation of old permafrost-derived carbon likely coincided with the abrupt rises in the atmospheric CO2 record12 at 14.6 and 11.5 kyrs BP, but not at 16.5 kyrs BP (Fig. 5a, c, d). At 14.6 kyrs BP we simulate a CO2 peak of 6 ppm (Fig. 5a) together with a drop in Δ14C of 6 or 8 ‰ (Fig. 5b) depending on whether a pre- depositional carbon age of either 5 or 10 kyrs was assumed. For this event, permafrost thawing has already been suggested as a possible cause3, but assuming greater carbon release of 125 PgC NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w c Simulated anomalies in atmospheric Δ14C. The unexplained residual (mean (blue) ± 1σ uncertainty (grey)) shows Δ(Δ14C) that is not explained by changes in 14C production rate. Anomalies in Δ14C caused by the two carbon release scenarios with difference in the pre-depositional ages of the released carbon (5 kyrs: broken lines; 10 kyrs: solid lines). IntCal1311 atmospheric Δ14C for comparison Constraints on the underlying processes responsible for the abrupt rises in atmospheric CO2 have been provided by δ13C analyses of CO22,37. It has been concluded that terrestrial carbon release alone cannot fully account for the atmospheric CO2 increase at 14.6 and 11.5 kyrs BP, which would have led to a drop in atmospheric δ13C-CO2. In agreement with the results from our 7 7 NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w 220 230 240 250 atm. CO2 (ppm) atm. Δ14C (‰) atm. CO2 (ppm) atm. CO2 (ppm) 14.0 Age (cal. kyr BP) a –10 0 10 20 30 atm. Δ(CO2) (ppm) atm. Δ(CO2) (ppm) atm. Δ(CO2) (ppm) atm. Δ(Δ14C) (‰) 14.6 kyr-event 200 210 220 230 16.0 Age (cal. kyr BP) –10 0 10 20 30 16.5 kyr-event 250 260 270 280 11.0 Age (cal. kyr BP) 0 10 20 30 11.5 kyr-event 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 Age (cal. kyr BP) b c d –90 –80 –70 –60 –50 –40 –30 –20 –10 0 10 20 30 40 50 60 14.6 kyr-event 14.5 15.0 14.0 14.5 15.0 11.5 12.0 16.5 Zoom-in on proposed 3 events of coastal erosion-based carbon cycle changes. CO2 changes for a 14.6 kyr-event, c 11.5 kyr-event, d 16.5 kyr-event. are CO2 data from ice cores (refs. 12, 13, data as in ref. 69). b Radiocarbon impacts during 14.6 kyr-event. IntCal13 (black with grey uncertainty band). esolution U/Th-dated Δ14C from Tahiti corals (magenta)31. Linear change in the Tahiti-based data 14C calculated with the Breakﬁt software (cyan ne)70 and mean of Tahiti Δ14C data before and after break (bold black circles). Simulated Δ(Δ14C) based on 5 (red broken) and 10 (red thin) kyrs positional aged carbon and for a scenario with prescribed Δ(Δ14C) of −1250‰ (red thick) potentially possible from radiocarbon free CO2 (pre- tional age > 30 kyrs). Alternatively, a background trend in Δ(Δ14C) of −0.1‰ per year was added to the scenarios (blue lines). NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w All uncertainties σ 220 230 240 250 atm. CO2 (ppm) 14.0 Age (cal. kyr BP) a –10 0 10 20 30 atm. Δ(CO2) (ppm) 14.6 kyr-event 14.5 15.0 atm. CO2 (ppm) atm. Δ(CO2) (ppm) 250 260 270 280 11.0 Age (cal. kyr BP) 0 10 20 30 11.5 kyr-event c 11.5 12.0 a c atm. Δ14C (‰) atm. CO2 (ppm) atm. Δ(CO2) (ppm) atm. Δ(Δ14C) (‰) 200 210 220 230 16.0 Age (cal. kyr BP) –10 0 10 20 30 16.5 kyr-event 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 Age (cal. kyr BP) b d –90 –80 –70 –60 –50 –40 –30 –20 –10 0 10 20 30 40 50 60 14.6 kyr-event 14.0 14.5 15.0 16.5 atm. CO2 (ppm) atm. Δ(CO2) (ppm) ( ) ( ) 200 210 220 230 16.0 Age (cal. kyr BP) –10 0 10 20 30 16.5 kyr-event d 16.5 atm. Δ14C (‰) 14 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 Age (cal. kyr BP) b –90 –80 –70 –60 –50 –40 –30 –20 –10 0 10 20 30 40 50 60 14.6 kyr-event 14.0 14.5 15.0 b d Fig. 5 Zoom-in on proposed 3 events of coastal erosion-based carbon cycle changes. CO2 changes for a 14.6 kyr-event, c 11.5 kyr-event, d 16.5 kyr-event. Circles are CO2 data from ice cores (refs. 12, 13, data as in ref. 69). b Radiocarbon impacts during 14.6 kyr-event. IntCal13 (black with grey uncertainty band). High-resolution U/Th-dated Δ14C from Tahiti corals (magenta)31. Linear change in the Tahiti-based data 14C calculated with the Breakﬁt software (cyan bold line)70 and mean of Tahiti Δ14C data before and after break (bold black circles). Simulated Δ(Δ14C) based on 5 (red broken) and 10 (red thin) kyrs pre-depositional aged carbon and for a scenario with prescribed Δ(Δ14C) of −1250‰ (red thick) potentially possible from radiocarbon free CO2 (pre- depositional age > 30 kyrs). Alternatively, a background trend in Δ(Δ14C) of −0.1‰ per year was added to the scenarios (blue lines). All uncertainties are ± 1σ modelling exercise, this indicates that these CO2 peaks are probably difﬁcult to explain with permafrost degradation alone, but rather suggest a combination of terrestrial and marine pro- cesses occurring simultaneously. Methods St d Study area and core locations. The circulation in the Okhotsk Sea is dominated by the Okhotsk Gyre and includes the southward-ﬂowing East Sakhalin Current (ESC), which transports surface and deep waters from the northern shelves to the Kuril Basin41. During the sea-ice season in fall and winter, Dense Shelf Water (DSW) is formed and ﬂows south along the Sakhalin margin, transporting high concentrations of organic matter, lithogenic particles and suspended matter that are entrained by vigorous tidal mixing on the northwestern shallow continental shelf into a highly turbid water layer10,42. Combined with discharge from the Amur River these materials rapidly accumulate along the East Sakhalin margin10,42, making this location the primary depositional site for terrigenous sediments sup- plied to the Okhotsk Sea. The two cores used in this study were retrieved from the northeast Sakhalin margin within the framework of the German-Russian KOMEX I and KOMEX- SONNE projects in 1998 and 2004, respectively. Gravity core LV28-4-4 (51°08.475′N, 145°18.582′E, 9.3 m recovery) was collected from 674 m water depth during expedition LV28 with R/V Akademik Lavrentiev43 and piston core SO178-13-6 (52°43.881′N, 144°42.647′E, 23.7 m recovery) was collected from 713 m water depth during the expedition SO178 with R/V Sonne44. The two cores feature similar lithofacies, consisting mainly of silty clays with sand and occasional larger dropstones derived from sea-ice transported terrigenous matter. Samples for CSRA were extracted from freeze-dried and homogenized sediment for 48 h using a Soxhlet with a mixture of dichloromethane:methanol 9:1 (v:v). The total lipid extract was hydrolysed as described above and the retrieved FAs were derivatized to fatty acid methyl esters (FAMEs) by adding HCl and methanol of known Δ14C reacting in a nitrogen atmosphere at 80 °C overnight. FAMEs were extracted from the methylated solution with n-hexane and subsequently separated from polar compounds with silica gel chromatography. In preparation for puriﬁcation of individual FAMES, branched and unsaturated FAMEs were removed from the FAME-fraction extracted from core LV28-4-4 using urea adduction and a column of silica gel coated with silver nitrate, respectively. For samples of core SO178-13-6 it was sufﬁcient to clean samples with silica gel chromatography as urea adduction was not necessary. For CSRA the n-C26:0, and n-C28:0 alkanoic acids were puriﬁed using preparative capillary gas chromatography (PC-GC)51 performed on an Agilent HP6890N GC with a Gerstel Cooled Injection System (CIS) connected to a Gerstel preparative fraction collector52. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w the Pre-Boreal. This substantial activation of pre-aged carbon (5 to 10 kyrs at the time of deposition) supports modelling studies published in recent years, which considered this process as a possible cause for abrupt CO2 releases. High accumulation rates of pre-aged terrestrial biomarkers at times of rapid sea level rise (melt water pulse 1A and 1B) suggest that shelf erosion was the dominant process for carbon mobilization. However, ﬂuvial export of old carbon from degrading permafrost in the Amur hinterland represents another important process for mobilization, particularly during times of high river discharge (~10 kyrs BP). covers approximately the last 16 kyrs BP. Age control was obtained through 12 AMS 14C dates on G. bulloides and N. pachyderma sinistral, supplemented by eight AMS 14C dates on mollusks/gastropods. All AMS 14C ages were calibrated with a regional reservoir correction of R 500 ± 100 yr and the MARINE09 calibration curve47. The routine CLAMS48 written in R was used to ﬁnd a best ﬁt through the 2σ ranges of all age control points, resulting in a smooth spline ﬁt (0.3 smoothing factor) with a ﬁnal run with 10,000 iterations in the CLAMS routine46. The age models revealed particularly high sediment accumulation rates during the deglacial period (8–18 kyrs BP) for core SO178-13-6, whereas maxima in sediment accu- mulation are reached in the Holocene in core LV28-4-4. The cores were sampled with varying resolution between 5 and 40 cm for down core analyses. Large samples of 50–100 g (dry weight) sediment for compound-speciﬁc radiocarbon analysis (CSRA) were taken from 4 (core LV28-4-4) and 6 (core SO178-13-6) depth hor- izons. Note that the lowermost sample of LV28-4-4 at depth 926–928 cm is below the last AMS 14C date used for the age model. The depositional age was linearly interpolated from there. p y g g g y The extrapolated carbon cycle changes led to simulated CO2 changes which are about a quarter (16.5 kyrs BP) or a half (14.6 and 11.5 kyrs BP) of the size of the three individual peaks found in the WAIS Divide ice core, but on the long-term to less than 5% of the deglacial rise in atmospheric CO2 of ~90 ppm12. ARTICLE Moreover, only little (<10 ‰) to the residual in the atmospheric Δ14C decline across Termination I that is unexplained by changes in the 14C production rate are according to our results related to this permafrost carbon release. Altogether, this implies that deglacial changes in atmospheric CO2 and Δ14C, while largely controlled by oceanic processes, were additionally impacted by degrading permafrost, potentially partly accounting for the abrupt CO2 rises at 14.6 and 11.5 kyrs BP that so far have remained difﬁcult to explain3,12. Further investigations of permafrost-carbon mobili- zation from locations bordering the permafrost domain that have undergone signiﬁcant deglacial changes are needed to improve the quantiﬁcation and constrain the possible age ranges of the mobilized carbon as well as their potential climate feedback. Chlorophycean freshwater algae counts were carried out on 32 pollen samples of core LV28-4-4 (Pediastrum spp.). Sample preparation and counts were reported in detail in previous studies46. Analytical methods. We present accumulation rates and concentrations (Sup- plementary Fig. 2) of two groups of terrigenous biomarkers, i.e., long-chain n- alkanes and branched glycerol dialkyl glycerol tetraethers (brGDGTs). Long-chain n-alkanes like long-chain n-alkanoic acids are primarily derived from leaf-waxes of higher plants, but their concentrations can more reliably be determined by gas chromatography, as the analytical procedures are more robust. To assure that their concentration records are not affected by petrogenic input and reliably reﬂect degrading permaforst, we compared them with concentration records of brGDGTs derived from soil bacteria. For down-core biomarker analysis total lipids were extracted from freeze-dried, homogenized sediment (2–5 g) using a three-step ultrasonic extraction with (i) dichloromethane, (ii) dichloromethane:methanol 1:1 (v:v) and (iii) methanol. Total lipid extracts were hydrolysed with 0.1 M potassium hydroxide (KOH) in methanol:water 9:1 (v:v) at 80 °C for 2 h in order to separate n-alkanoic acids (from here on called fatty acids (FAs)) from neutral lipids (NLs). Neutral compounds were extracted with n-hexane. Subsequently, the pH was adjusted to 1 by adding 37% HCl and FAs were extracted with dichloromethane. NLs were further split into three subfractions (apolar compounds, aldehydes and ketones, polar compounds) by silica gel chromatography. The n-alkane con- centrations of core LV-28-4 were determined using a HP 5890 GC and of core SO178-13-6 using an Agilent 7890A GC. Both GCs were equipped with an Agilent J&W DB-5ms column and a ﬂame ionization detector (FID). ARTICLE Each compound was identiﬁed based on retention time and comparison with an n-alkane standard. Quantiﬁcation was achieved using an internal standard (squalane) added prior to extraction. brGDGTs were analysed with minor modiﬁcations according to ref. 49. Brieﬂy, the polar fraction was ﬁltered through 0.45 µm PTFE syringe ﬁlters and dissolved in hexane:isopropanol 99:1 (v:v). Samples were analysed using an Agilent 1200 series HPLC coupled to an Agilent 6120 single quadrupole MS via an atmospheric pressure chemical ionization interface (APCI). Chromatographic separation was achieved by normal-phase chromatography using a Prevail Cyano column (Grace, 3 µm, 150 mm × 2.1 mm) maintained at 30 °C. brGDGTs were identiﬁed using selective ion monitoring50 and quantiﬁcation was achieved using an internal standard (C46-GDGT) added prior to extraction. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w Furthermore, both events took place at the same time as an abrupt warming in the Northern Hemisphere indicating a change in the bipolar seesaw38. Conse- quently, some ocean circulation changes are also expected to have occurred, potentially obscuring terrestrial-based carbon release39. Since permafrost thawing and degradation including thermo- karst lake and wetland development likely result in outgassing of both CO2 and CH4, these processes would potentially have con- tributed to the concurrent rise in CH4 at 11.5 kyrs BP12. However, radiocarbon analyses of CH4 from air extracted from the hor- izontal ice core in Taylor Glacier, Antarctica, covering the Younger Dryas-Pre-Boreal transition constrain that the modelling exercise, this indicates that these CO2 peaks are probably difﬁcult to explain with permafrost degradation alone, but rather suggest a combination of terrestrial and marine pro- cesses occurring simultaneously. Furthermore, both events took place at the same time as an abrupt warming in the Northern Hemisphere indicating a change in the bipolar seesaw38. Conse- quently, some ocean circulation changes are also expected to have occurred, potentially obscuring terrestrial-based carbon release39. contribution from old, 14C-free, carbon—which includes methane hydrates, permafrost and methane trapped under ice—to the rapid CH4 rise at 11.5 kyrs BP was <19%40. This further supports the idea that the abrupt rises in both greenhouse gases (CO2 and CH4) at the end of the last deglaciation might not have been caused by permafrost thawing and degradation alone. None- theless, our results of the ﬂooded shelf and hinterland permafrost thawing inherently imply the rapid subsequent initiation of wetland formation in the large Amur and other Siberian catch- ments, which established signiﬁcant boreal ‘young carbon’ CH4 sources in lockstep with our observed activation of previously frozen, inert old carbon from terrestrial reservoirs. Since permafrost thawing and degradation including thermo- karst lake and wetland development likely result in outgassing of both CO2 and CH4, these processes would potentially have con- tributed to the concurrent rise in CH4 at 11.5 kyrs BP12. However, radiocarbon analyses of CH4 from air extracted from the hor- izontal ice core in Taylor Glacier, Antarctica, covering the Younger Dryas-Pre-Boreal transition constrain that the Our ﬁndings provide the ﬁrst direct evidence for rapid mobi- lization of old permafrost-derived carbon in boreal to subarctic East Asia during the last glacial termination, prior to the onset of NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w T3527-100MG, LOT 018K3760, fossil F14Ctrue; In order to assess the procedure blank, we processed n-hexadecanoic acid (n- C16:0) from apple peel collected in 2013 (modern F14Ctrue) and n-triacontanoic acid (n-C30:0; Sigma, Prod. No. T3527-100MG, LOT 018K3760, fossil F14Ctrue; Supplementary Table 2). F14Ctrue of n-C30:0 has been reported58. The F14Ctrue of the apple peel was obtained from AMS-analyses of bulk apple peel (3 samples, graphitized and analysed at ETH Zürich) assuming that the F14C of n C Previous simulations with the same model60,61 show a 14C production-corrected residual of atmospheric Δ14C, which needs to be explained by carbon cycle changes. This residual in atmospheric Δ14C, using the 14C production rate estimate based on the geomagnetic ﬁeld reconstruction62 GLOPIS-75, has a large uncertainty band of about ± 80‰ around a mean decline of 100‰ across the last deglaciation (18–11 kyrs BP) (Fig. 4b). Note that in a more recent study using a similar carbon cycle box model63 this 14C production corrected Δ14C residual is by about a factor of two larger than these results obtained with the BICYCLE model. Supplementary Table 2). F14Ctrue of n-C30:0 has been reported58. The F14Ctrue of the apple peel was obtained from AMS-analyses of bulk apple peel (3 samples, equals the F14C of bulk apple peel. The F14Ctrue and F14Csample of apple peel and n- C30:0 as well as the respective sample sizes are given in the Supplementary Table 2. a1 and a2 (Eq. 3) were assessed from linear regression (Supplementary Fig. 3). For the blank assessment, it has to be acknowledged that the processed n-C16:0 and n- C30:0 were methylated for GC analysis. This means that F14Csample is affected by the F14C of the added methyl-group (F14Cmethyl) while the F14Ctrue (unprocessed g To put our carbon cycle simulation results into context with other models64,65, we compare their model response to an external perturbation. In detail, the fraction of carbon injected into the atmosphere that stays there, the so-called airborne fraction, is compared for a pulse of 100 PgC injected to the atmosphere within 1 year for pre-industrial (PI) climate conditions (Supplementary Fig. 1a). This quantity is a function of time and evaluates our simulated anomalies in atmospheric CO2. The results of a model intercomparison project (MIP)64 are restricted to 1 kyr. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w We are here mainly interested in the overall effect, therefore prescribed CO2 release rates from permafrost thawing were either kept constant over the last deglaciation, or restricted to three peaks potentially connected with rapid sea level rise. In the latter scenario carbon is released to the atmosphere in three 200-year time-windows starting at 16.5, 14.6 and 11.5 kyrs BP. The amount of released carbon in the three peaks is 20%, 40% and 40% of the total released carbon of 85 PgC, respectively, to approximately account for the amplitude differences in the maxima in our MAR record. These carbon releases are assigned with a 14C signature that has been constructed from a pre-depositional age of either 5 or 10 kyrs derived from the CSRA analyses of the two sediment cores discussed in this study (Supplementary Fig. 1b). Alternatively, we added another scenario in which 14C-free carbon is entering the atmosphere as proposed before3, which would imply a pre-depositional aging of the carbon of up to 35 kyrs resulting in approximately a doubling of the simulated Δ14C peak (e.g. −12 ‰ for the 14.6 kyrs BP event, Fig. 5b). However, the Tahiti-based Δ14C anomaly in focus in ref. 3 is even not entirely met, when the background trend of −0.1‰ per year, potentially caused by marine processes and changes in the 14C production rate, is added to our simulation results (Fig. 5b). For a strict process separation 14C production rate was kept constant here, at 25% higher than modern times in order to obtain a simulated atmospheric Δ14C level of 400‰, similar to what is found in the IntCAL13 14C stack around 18 kyrs BP11. Estimated impact on atmospheric CO2 and Δ14C. Using the well-known carbon cycle box model BICYCLE59, we estimated how much the coastal erosion throughout the Arctic Ocean might have impacted both atmospheric CO2 and Δ14C. For this aim, we perturbed simulation results covering the last glacial cycle. In our control run (using the atmosphere-ocean subsystem including carbonate compensation to simulated ocean-sediment interactions) the carbon content of the terrestrial biosphere was kept constant. In our additional simulations it was pre- scribed to release CO2 from thawing permafrost across the last deglaciation (18–10.8 kyrs BP). Data availability Data obtained in this study are deposited in Pangaea (https://doi.pangaea.de/10.1594/ PANGAEA.890865) ð5Þ σm ¼ mblank ´ 1 R2 Received: 25 October 2017 Accepted: 13 August 2018 Received: 25 October 2017 Accepted: 13 August 2018 Received: 25 October 2017 Accepted: 13 August 2018 The R2 of the n-C16:0 regression was considered only, as the n-C30:0 regression was based on n = 2 (Supplementary Fig. 3). Blank correction (of the CSRA results from the biomarker samples) and error propagation was performed after56. The obtained F14C values were corrected for F14Cmethyl by isotopic mass balance. Calculation of terrigenous biomarker ages at deposition. We use the measured Δ14C signature of our terrigenous biomarkers to calculate the age at deposition as follows: any carbon of terrestrial plants has its origin in atmospheric CO2 and therefore will incorporate carbon with the isotopic 14C signature of the atmosphere at the time of photosynthetic production. Using the atmospheric Δ14C record of IntCal13, we can therefore calculate the radioactive decay of 14C (using the decay constant λ = 8267−1 yr −1) of any sample as function of age and can derive the Δ14C signature this sample should have during the time of the measurement nowadays, in our case in the year 2013 and 2014 for the cores LV28-4-4 and SO178-13-6, respectively. By using this IntCAL13- based age calculation as look-up table, we are able to determine from our measured NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w In order to evaluate our results for the whole period of the last deglaciation, we extrapolate this MIP-based airborne fraction with exponential equations obtained from regression analysis of Earth system model simulation results65. While this approach provides only an approximation of the evolution of the mean value, we keep the relative width of the 2σ uncertainty band, based on MIP for 1 kyr, to show the likely uncertainty range or model spread for longer time periods. y g p y p equivalents) is not. Hence, when determining the mblank and F14Cblank as discussed above and shown in Supplementary Fig. 3, the methyl group of the processed n- C16:0 and n-C30:0 FAMEs affects the slope of the regression lines. As a result, this would count towards the unknown blank. In order to remove the contributions of the methyl group from the blank assessment, the F14Ctrue values the unprocessed n- C16:0 and n-C30:0 FAs would have if they were methylated has to be calculated. This was achieved by combining the F14Ctrue of the bulk apple peel and the n-C30:0 FA with the F14Cmethyl through isotopic mass balance. y g p The uncertainties of F14Cblank and mblank (σF14C and σm) can be inferred from the regression coefﬁcient (R2)57 as R2 indicates the certainty F14C and m are predicted with by the regression line. Under this assumption σF14C and σm can be obtained from: predicted with by the regression line. Under this assumption σF14C and σm can be obtained from: σF14C ¼ F14Cblank ´ 1 R2 ð4Þ σm ¼ mblank ´ 1 R2 ð5Þ ð4Þ NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w using Oxalic Acid II (NIST SRM 4990C) and radiocarbon free CO2 gas. AMS results are reported as fraction modern carbon (F14C; Supplementary Table 1), conventional radiocarbon ages (14C ages given in 14C yr BP), and Δ14C55. Δ14C in which year the biomarkers have been photosynthetically ﬁxed. The age at deposition can then be derived from the difference between the time of photosynthetic production and the deposition age (Table 1). The uncertainty of the age at deposition is estimated from the uncertainty in the Δ14C measurement, to which an additional 1σ- uncertainty in age of 10 years is added, which accounts for the uncertainty of Δ14C in the IntCAL13 record. This uncertainty offset fully accounts for any potential error propagation for the range of our Δ14C data (from −358‰ to −932‰). 14C blank assessment and corrections. In order to report accurate radiocarbon dates of terrigenous biomarker compounds, a correction for extraneous carbon of unknown composition that is introduced to the sample during processing is required (procedure blank). Possible contamination during sample preparation prior to AMS analysis include carbon added through column bleed and carry-over as well as atmospheric carbon during vacuum line handling and combustion due to leakages. Therefore, every measured F14C of a processed biomarker sample (F14Csample) is a composite of the true F14C of the biomarker (F14Ctrue) and the F14C of the blank (F14Cblank). Blank correction of AMS measured F14Csample requires the determination of F14Cblank and the size of the blank (mblank)56. Assuming constant F14Cblank and a constant mass of the blank (mblank), there is an inverse linear relationship between the F14Csample, and the sample size57 (msample; Eq. 1): Estimated impact on atmospheric CO2 and Δ14C. Using the well-known carbon cycle box model BICYCLE59, we estimated how much the coastal erosion throughout the Arctic Ocean might have impacted both atmospheric CO2 and Δ14C. For this aim, we perturbed simulation results covering the last glacial cycle. In our control run (using the atmosphere-ocean subsystem including carbonate compensation to simulated ocean-sediment interactions) the carbon content of the terrestrial biosphere was kept constant. In our additional simulations it was pre- scribed to release CO2 from thawing permafrost across the last deglaciation (18–10.8 kyrs BP). Methods St d The GC was equipped with a Restek Rtx-XLB fused silica capillary column (30 m, 0.53 mm diameter, 0.5 µm ﬁlm thickness). All samples were injected stepwise with 5 µL per injection. Puriﬁed compounds were transferred to pre-combusted quartz glass tubes and 150 µg pre-combusted copper (II)-oxide was added as oxidizing agent. Quartz tubes were evacuated (10−5 mbar) on a vacuum line and ﬂame-sealed with a hydrogen/oxygen torch. The sealed tubes were combusted at 950 °C for 4 h. The resulting CO2 was stripped from water and quantiﬁed. p g As both cores were retrieved from a relatively dynamic sedimentary setting, we took care to select sites that were both (1) representative of the depositional environment targeted (river discharge and DSW transport), and (2) undisturbed by secondary processes, such as sediment redistribution, mass wasting, chaotic facies, etc., as evidenced through prior extensive seismic survey works. Both cores were retrieved from areas surveyed with a purpose-built high-resolution sub-bottom proﬁling system (SES 2000 DS), with decimetre-resolution to sediment penetration depths of about 30 m below sediment surface. No disturbances, but ﬂat, continuous, undisturbed sedimentary facies across several hundred metres or even kilometres were recorded on both core locations. For the SO178-13-6 location, in addition the shipboard PARASOUND sub-bottom proﬁler system was extensively used to conﬁrm the absence of slumping, turbidites, mass wasting or other processes that might obfuscate or invalidate our assumptions about our sedimentary recording system of southward transport processes along the Sakhalin margin. In summary, both locations together represent an optimal approach for recording the depositional history of terrigenous transport from the Amur and Siberian hinterland. Compound-speciﬁc radiocarbon analysis. The isotopic ratio (14C/12C) of the CO2 samples derived from the individual n-alkanoic acids was determined by Accelerator Mass Spectrometry (AMS). The measurements were carried out on the MICADAS-system equipped with a gas-ion source53,54 at the Laboratory of Ion Beam Physics, ETH Zürich. Samples were normalized and background subtracted Sediment chronology. Core SO178-13-6 covers the last 17 kyrs. The chronology was established through an AMS 14C-anchored stratigraphic framework based on XRF scanning and core-to-core correlations for the Okhotsk Sea during the last deglaciation45. The chronostratigraphic age models for both cores used here have been published in detail previously and are used here without changes46. LV28-4-4 9 NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w We are here mainly interested in the overall effect, therefore prescribed CO2 release rates from permafrost thawing were either kept constant over the last deglaciation, or restricted to three peaks potentially connected with rapid sea level rise. In the latter scenario carbon is released to the atmosphere in three 200-year time-windows starting at 16.5, 14.6 and 11.5 kyrs BP. The amount of released carbon in the three peaks is 20%, 40% and 40% of the total released carbon of 85 PgC, respectively, to approximately account for the amplitude differences in the maxima in our MAR record. These carbon releases are assigned with a 14C signature that has been constructed from a pre-depositional age of either 5 or 10 kyrs derived from the CSRA analyses of the two sediment cores discussed in this study (Supplementary Fig. 1b). Alternatively, we added another scenario in which 14C-free carbon is entering the atmosphere as proposed before3, which would imply a pre-depositional aging of the carbon of up to 35 kyrs resulting in approximately a doubling of the simulated Δ14C peak (e.g. −12 ‰ for the 14.6 kyrs BP event, Fig. 5b). However, the Tahiti-based Δ14C anomaly in focus in ref. 3 is even not entirely met, when the background trend of −0.1‰ per year, potentially caused by marine processes and changes in the 14C production rate, is added to our simulation results (Fig. 5b). For a strict process separation 14C production rate was kept constant here, at 25% higher than modern times in order to obtain a simulated atmospheric Δ14C level of 400‰, similar to what is found in the IntCAL13 14C stack around 18 kyrs BP11. F14Csample ¼ a ´ 1 msample ! þF14Ctrue ð1Þ ð1Þ where the slope (a) is deﬁned as: ð2Þ a ¼ mblank F14Cblank F14Ctrue Given these relationships, mblank and F14Cblank can be derived from the intersections of two linear regression functions obtained from processing several, ideally different sized, samples of at least two different materials (1,2) with known (different) F14Ctrue (Supplementary Fig. 3) as: mblank ¼ a1 a2 F14Ctrue1 F14Ctrue2 ð Þ ð3Þ ð3Þ In order to assess the procedure blank, we processed n-hexadecanoic acid (n- C16:0) from apple peel collected in 2013 (modern F14Ctrue) and n-triacontanoic acid (n-C30:0; Sigma, Prod. No. NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications ARTICLE Biogenic and lithogenic particle ﬂuxes in the western region of the Sea of Okhotsk: implications for lateral material transport and biological productivity. J. Geophys. Res. Oceans 109, C09S13 (2004). 0-50,000 years cal BP. Radiocarbon 55, 1869–1887 (2013). 12. Marcott, S. A. et al. Centennial-scale changes in the global carbon cycle during the last deglaciation. Nature 514, 616–619 (2014). g p y p y 43. Biebow, N. & Hütten, E. Cruise Reports: KOMEX I and II. GEOMAR Report no. 82 (GEOMAR Research Centre for Marine Sciences, Kiel, 1999). 13. Monnin, E. et al. Atmospheric CO2 concentrations over the last glacial termination. Science 291, 112–114 (2001). 44. Dullo, W. C., Biebow, N. & Georgeleit, K. SO178-KOMEX Cruise Report: Mass Exchange Processes and Balances in the Okhotsk Sea. GEOMAR Report (GEOMAR Research Centre for Marine Geosciences, Kiel, 2004). 14. Lambeck, K., Rouby, H., Purcell, A., Sun, Y. & Sambridge, M. Sea level and global ice volumes from the Last Glacial Maximum to the Holocene. Proc. Natl Acad. Sci. USA 111, 15296–15303 (2014). (GEOMAR Research Centre for Marine Geosciences, Kiel, 2004 45. Max, L. et al. Pulses of enhanced North Paciﬁc Intermediate Water ventilation from the Okhotsk Sea and Bering Sea during the last deglaciation. Clim. Past. 10, 591–605 (2014). 15. Lantuit, H. et al. The Arctic Coastal Dynamics Database: a new classiﬁcation scheme and statistics on Arctic Permafrost Coastlines. Estuaries Coasts 35, 383–400 (2012). 46. Lembke-Jene, L. et al. Deglacial variability in Okhotsk Sea Intermediate Water ventilation and biogeochemistry: Implications for North Paciﬁc nutrient supply and productivity. Quat. Sci. Rev. 160, 116–137 (2017). 16. Hoque, M. A. & Pollard, W. H. Stability of permafrost dominated coastal cliffs in the Arctic. Polar Sci. 10, 79–88 (2016). 17. Vonk, J. E. et al. Preferential burial of permafrost-derived organic carbon in Siberian-Arctic shelf waters. J. Geophys. Res. Oceans 119, 8410–8421 (2014). 47. Reimer, P. J. et al. IntCal09 and Marine09 radiocarbon age calibration curves, 0–50,000 years cal BP. Radiocarbon 51, 1111–1150 (2009). y 48. Blaauw, M. Methods and code for ‘classical’ age-mode sequences. Quat. Geochronol. 5, 512–518 (2010). 18. Walter, K., Edwards, M., Grosse, G., Zimov, S. & Chapin, F. Thermokarst lakes as a source of atmospheric CH4 during the last deglaciation. Science 318, 633–636 (2007). 48. Blaauw, M. Methods and code for ‘classical’ age-modelling of radiocarbon sequences. Quat. Geochronol. 5, 512–518 (2010). 49. Hopmans, E., Schouten, S., Pancost, R., van der Meer, M. ARTICLE et al. Assessment for paleoclimatic utility of terrestrial biomarker records in the Okhotsk Sea sediments. Deep-Sea Res Pt Ii 61-64, 85–92 (2012). 55. Stuiver, M. & Polach, H. Discussion: reporting of 14C data. Radiocarbon 19, 355–363 (1977). 27. Seki, O., Meyers, P. A., Kawamura, K., Zheng, Y. & Zhou, W. Hydrogen isotopic ratios of plant wax n-alkanes in a peat bog deposited in northeast China during the last 16kyr. Org. Geochem. 40, 671–677 (2009). 56. Wacker, L. & Christl, M. Data Reduction for Small Samples. Error Propagation using the Model of Constant Contamination. Annual Report of Ion Beam Physics, ETH Zürich 36 (2012). 28. Mokhova, L., Tarasov, P., Bazarova, V. & Klimin, M. Quantitative biome reconstruction using modern and late Quaternary pollen data from the southern part of the Russian Far East. Quat. Sci. Rev. 28, 2913–2926 (2009). 57. Shah, S. & Pearson, A. Ultra-microscale (5–25 μg C) analysis of individual lipids by 14C AMS: assessment and correction for sample processing blanks. Radiocarbon 49, 69–82 (2007). 29. MacDonald, G. M. et al. Rapid early development of circumarctic peatlands and atmospheric CH4 and CO2 variations. Science 314, 285–288 (2006). 58. Rethemeyer, J. et al. Status report on sample preparation facilities for 14C analysis at the new CologneAMS center. Nucl. Instrum. Methods Phys. Res. B 294, 168–172 (2013). 30. Brosius, L. S. et al. Using the deuterium isotope composition of permafrost meltwater to constrain thermokarst lake contributions to atmospheric CH4 during the last deglaciation. J. Geophys. Res. 117, G01022 (2012). 31. Durand, N. et al. Comparison of 14C and U-Th ages in Corals from IODP #310 Cores Offshore Tahiti. Radiocarbon 55, 1947–1974 (2013). 59. Köhler, P., Fischer, H. & Schmitt, J. Atmospheric δ13CO2 and its relation to pCO2 and deep ocean δ13C during the late Pleistocene. Paleoceanography 25, PA1213 (2010). 32. Tanski, G. et al. Transformation of terrestrial organic matter along thermokarst-affected permafrost coasts in the Arctic. Sci. Total Environ. 581- 582, 434–447 (2017). 60. Köhler, P., Muscheler, R. & Fischer, H. A model-based interpretation of low- frequency changes in the carbon cycle during the last 120,000 years and its implications for the reconstruction of atmospheric Δ14C. Geochem. Geophys. Geosyst. 7, Q11N06 (2006). 33. WAIS Divide Project Members. Precise interpolar phasing of abrupt climate change during the last ice age. Nature 520, 661–665 (2015). 61. Skinner, L. C., Fallon, S., Waelbroeck, C., Michel, E. & Barker, S. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w 35. Knoblauch, C., Beer, C., Sosnin, A., Wagner, D. & Pfeiffer, E. M. Predicting long-term carbon mineralization and trace gas production from thawing permafrost of Northeast Siberia. Glob. Change Biol. 19, 1160–1172 (2013). 4. Crichton, K. A., Bouttes, N., Roche, D. M., Chappellaz, J. & Krinner, G. Permafrost carbon as a missing link to explain CO2 changes during the last deglaciation. Nat. Geosci. 9, 683–686 (2016). g 5. Vonk, J. E. et al. Activation of old carbon by erosion of coastal and subsea permafrost in Arctic Siberia. Nature 489, 137–140 (2012). g 36. Schädel, C. et al. Circumpolar assessment of permafrost C quality and its vulnerability over time using long-term incubation data. Glob. Change Biol. 20, 641–652 (2013). 6. Schneider von Deimling, T. et al. Observation-based modelling of permafrost carbon ﬂuxes with accounting for deep carbon deposits and thermokarst activity. Biogeosciences 12, 3469–3488 (2015). 37. Bauska, T. K. et al. Carbon isotopes characterize rapid changes in atmospheric carbon dioxide during the last deglaciation. Proc. Natl Acad. Sci. USA 113, 3465–3470 (2016). y g 7. Schuur, E. A. G. et al. Climate change and the permafrost carbon feedback. Nature 520, 171–179 (2015). ( ) 38. Barker, S. et al. Interhemispheric Atlantic seesaw response during the last deglaciation. Nature 457, 1097–1102 (2009). 8. Vandenberghe, J. et al. The Last Permafrost Maximum (LPM) map of the Northern Hemisphere: permafrost extent and mean annual air temperatures, 25-17 ka BP. Boreas 43, 652–666 (2014). 39. Schmittner, A. & Galbraith, E. D. Glacial greenhouse-gas ﬂuctuations controlled by ocean circulation changes. Nature 456, 373–376 (2008). 9. Vaks, A. et al. Speleothems Reveal 500,000-year history of Siberian Permafrost. Science 340, 183–186 (2013). 40. Petrenko, V. V. et al. Minimal geological methane emissions during the Younger Dryas–Preboreal abrupt warming event. Nature 548, 443–446 (2017). 10. Nakatsuka, T., Toda, M., Kawamura, K. & Wakatsuchi, M. Dissolved and particulate organic carbon in the Sea of Okhotsk: transport from continental shelf to ocean interior. J. Geophys. Res. Oceans 109, C09S14 (2004). 41. Ohshima, K. I. et al. Sverdrup balance and the cyclonic gyre in the Sea of Okhotsk. J. Phys. Oceanogr. 34, 513–525 (2004). p y 11. Reimer, P. J. et al. IntCal13 and Marine13 radiocarbon age calibration curves 0-50,000 years cal BP. Radiocarbon 55, 1869–1887 (2013). y g 42. Nakatsuka, T. et al. ARTICLE & Sinninghe Damsté, J. Analysis of intact tetraether lipids in archaeal cell material and sediments by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Rapid Commun. Mass Spectrom. 14, 585–589 (2000). 19. Walter Anthony, K. et al. Methane emissions proportional to permafrost carbon thawed in Arctic lakes since the 1950s. Nat. Geosci. 9, 679–682 (2016). 19. Walter Anthony, K. et al. Methane emissions proportional to permafrost carbon thawed in Arctic lakes since the 1950s. Nat. Geosci. 9, 679–682 (2016). 20. Tesi, T. et al. Massive remobilization of permafrost carbon during post-glacial warming. Nat. Commun. 7, 13653 (2016). carbon thawed in Arctic lakes since the 1950s. Nat. Geosci. 9, 679 682 (2016). 20. Tesi, T. et al. Massive remobilization of permafrost carbon during post-glacial warming. Nat. Commun. 7, 13653 (2016). g 21. Mann, P. J. et al. Utilization of ancient permafrost carbon in headwaters of Arctic ﬂuvial networks. Nat. Commun. 6, 7856 (2015). 50. Sinninghe Damsté, J., Hopmans, E., Pancost, R., Schouten, S. & Geenevasen, J. Newly discovered non-isoprenoid glycerol dialkyl glycerol tetraether lipids in sediments. Chem. Commun. 1683–1684. https://doi.org/10.1039/b004517i (2000). 22. Tachibana, Y., Oshima, K. & Ogi, M. Seasonal and interannual variations of Amur River discharge and their relationships to large-scale atmospheric patterns and moisture ﬂuxes. J. Geophys. Res. 113, D16102 (2008). 51. Eglinton, T., Aluwihare, L., Bauer, J., Druffel, E. & McNichol, A. Gas chromatographic isolation of individual compounds from complex matrices for radiocarbon dating. Anal. Chem. 68, 904–912 (1996). 23. Dykoski, C. et al. A high-resolution, absolute-dated Holocene and deglacial Asian monsoon record from Dongge Cave, China. Earth Planeti Sci. Lett. 233, 71–86 (2005). 52. Kusch, S., Rethemeyer, J., Schefuß, E. & Mollenhauer, G. Controls on the age of vascular plant biomarkers in Black Sea sediments. Geochim Cosmochim. Acta 74, 7031–7047 (2010). 24. Strauss, J. et al. Deep Yedoma permafrost: a synthesis of depositional characteristics and carbon vulnerability. Earth-Sci. Rev. 172, 75–86 (2017). 53. Synal, H.-A., Stocker, M. & Suter, M. MICADAS: a new compact radiocarbon AMS system. Nucl. Instrum. Methods Phys. Res. A 259, 7–13 (2007). 25. Ficken, K. J., Li, B., Swain, D. L. & Eglinton, G. An n-alkane proxy for the sedimentary input of submerged/ﬂoating freshwater aquatic macrophytes. Org. Geochem. 31, 745–749 (2000). 54. Wacker, L. et al. A versatile gas interface for routine radiocarbon analysis with a gas ion source. Nucl. Instrum. Methods Phys. Res. B 294, 315–319 (2013). g 26. Seki, O. References 1. Ciais, P. et al. Large inert carbon pool in the terrestrial biosphere during the Last Glacial Maximum. Nat. Geosci. 5, 74–79 (2012). 2. Hugelius, G. et al. Estimated stocks of circumpolar permafrost carbon with quantiﬁed uncertainty ranges and identiﬁed data gaps. Biogeosciences 11, 6573–6593 (2014). 2. Hugelius, G. et al. Estimated stocks of circumpolar permafrost carbon with quantiﬁed uncertainty ranges and identiﬁed data gaps. Biogeosciences 11, 6573–6593 (2014). 3. Köhler, P., Knorr, G. & Bard, E. Permafrost thawing as a possible source of abrupt carbon release at the onset of the Bolling/Allerod. Nat. Commun. 5, 5520 (2014). 10 Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 68. Rasmussen, S. O. et al. Synchronization of the NGRIP, GRIP, and GISP2 ice cores across MIS 2 and palaeoclimatic implications. Quat. Sci. Rev. 27, 18–28 (2008). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 69. Köhler, P., Nehrbass-Ahles, C., Schmitt, J., Stocker, T. F. & Fischer, H. A 156 kyr smoothed history of the atmospheric greenhouse gases CO2, CH4, and N2O and their radiative forcing. Earth Syst. Sci. Data 9, 363–387 (2017). 70. Mudelsee, M. Break function regression. Eur. Phys. J. Spec. Top. 174, 49–63 (2009). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Author contributions 62. Laj, C., Kissel, C. & Beer, J. High Resolution Global Paleointensity Stack since 75 kyrs (GLOPIS-75) calibrated to absolute values, in Timescales of the Geomagnetic Field, eds J. E. T. Channell, D. V. K. Kent, W. Lowrie, and J. G. Meert (AGU Monogr. Ser. vol. 145) (Washington, DC: American Geophysical Union), 255–265. https://doi.org/10.1029/145gm19 (2004). G.M. designed the study. R.T. and L.L.-J. performed ﬁeld work and sampling. M.W., W.D. and J.H. carried out biomarker analyses, puriﬁcation of long-chain n-alkanoic acids, and preparation for compound-speciﬁc radiocarbon analysis. C.M. and L.W. performed AMS 14C analysis on the MICADAS system and helped with 14C data pro- cessing. V.M. was responsible for 14C blank correction. P.K. performed the carbon cycle modelling and calculated the pre-depositional ages. U.K. provided freshwater algae counts. M.W. and G.M. wrote the manuscript with input from L.L.-J., V.M., W.D., P.K. and L.W. All authors discussed the results and commented on the manuscript at different stages. 63. Hain, M. P., Sigman, D. M. & Haug, G. H. Distinct roles of the Southern Ocean and North Atlantic in the deglacial atmospheric radiocarbon decline. Earth Planet. Sci. Lett. 394, 198–208 (2014). 64. Joos, F. et al. Carbon dioxide and climate impulse response functions for the computation of greenhouse gas metrics: a multi-model analysis. Atmos. Chem. Phys. 13, 2793–2825 (2013). y 65. Lord, N. S., Ridgwell, A., Thorne, M. C. & Lunt, D. J. An impulse response function for the ‘long tail’ of excess atmospheric CO2 in an Earth system model. Glob. Biogeochem. Cycle 30, 2–17 (2016). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06080-w Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06080-w. 66. Brown, J., Ferrians, O. J., Jr, Heginbottom, J. A. & Melnikov, E. S. Circum- Arctic map of permafrost and ground-ice conditions. National Snow and Ice Data Center (1998). Competing interests: The authors declare no competing interests. ARTICLE ARTICLE ARTICLE ARTICLE Ventilation of the Deep Southern Ocean and Deglacial CO2 Rise. Science 328, 1147–1151 (2010). 34. Rostek, F. & Bard, E. Hydrological changes in eastern Europe during the last 40,000yr inferred from biomarkers in Black Sea sediments. Quatern Res 80, 502–509 (2013). 11 NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06080-w. © The Author(s) 2018 Competing interests: The authors declare no competing interests. 67. Lehner, B. & Döll, P. Development and validation of a global database of lakes, reservoirs and wetlands. J. Hydrol. 296, 1–22 (2004). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Acknowledgements We acknowledge the professional support of masters and crew of R/V Akademik M.A. Lavrentiev and R/V Sonne on expeditions LV28 and SO178. We thank Ralph Kreutz and Hendrik Grotheer for laboratory support. We thank Jens Strauss for valuable comments on the manuscript. G.M. acknowledges funding from the Helmholtz Association (grant no. VH-NG-202 also supporting M.W., and funds in the W2/W3 program supporting V. M. and J.H.). L.L.-J. and R.T. acknowledge support from the Helmholtz REKLIM Initiative and BMBF grant no. 03F0704A ‘Sino-German Paciﬁc-Arctic Experiment (SIGEPAX)’. U.K. acknowledges support from the VILLUM Foundation (grant no. 10100). This work contributes to PALMOD, the German Paleomodeling Research Project funded by BMBF. We acknowledge the professional support of masters and crew of R/V Akademik M.A. Lavrentiev and R/V Sonne on expeditions LV28 and SO178. We thank Ralph Kreutz and Hendrik Grotheer for laboratory support. We thank Jens Strauss for valuable comments on the manuscript. G.M. acknowledges funding from the Helmholtz Association (grant no. VH-NG-202 also supporting M.W., and funds in the W2/W3 program supporting V. M. and J.H.). L.L.-J. and R.T. acknowledge support from the Helmholtz REKLIM Initiative and BMBF grant no. 03F0704A ‘Sino-German Paciﬁc-Arctic Experiment (SIGEPAX)’. U.K. acknowledges support from the VILLUM Foundation (grant no. 10100). This work contributes to PALMOD, the German Paleomodeling Research Project funded by BMBF. 12 NATURE COMMUNICATIONS \| (2018) 9:3666 \| DOI: 10.1038/s41467-018-06080-w \| www.nature.com/naturecommunications
https://openalex.org/W3043300301	https://revistaseletronicas.pucrs.br/index.php/revistafamecos/article/download/33837/26090	Portuguese	null	Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas	Revista FAMECOS	2,020	cc-by	7,177	Recebido em: 2 abr. 2019. Aprovado em: 21 ago. 2019. Publicado em: 16 jul. 2020. OPEN ACCESS OPEN ACCESS https://dx.doi.org/10.15448/1980-3729.2020.1.33837 REVISTA FAMECOS mídia, cultura e tecnologia Revista FAMECOS, Porto Al gre, v. 27, p. 1-11, jan.-dez. 2020 e-ISSN: 1980-3729 \| ISSN-L: 1415-0549 Tramas del tiempo en el teleperiodismo local: temporalidades sociales en el programa Terra de Minas Tramas del tiempo en el teleperiodismo local: temporalidades sociales en el programa Terra de Minas RESUMO: O presente artigo tem como objetivo analisar a forma como o programa Terra de Minas (TV Globo Minas) aborda diferentes temporalidades como forma de estabelecer uma imagem da cidade de Belo Horizonte. A partir das contribuições dos Estudos Culturais, este artigo assume o passado como algo não finalizado, mas é convocado no presente podendo assumir lugares de disputa e oposição às formas dominantes. Do mesmo modo, o futuro pode ser vivido no presente, não como algo projetado, mas como aquilo que reconfigura elementos do do- minante ou que instaura algo novo. A análise textual do programa, embasada em seus elementos visuais, sonoros e verbais, sinaliza para pelo menos três convocações das temporalidades sociais: o passado como patrimônio, o futuro como aprimoramento do passado e o passado como construção dos sujeitos. 1 Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil. Silva1 id orcid.org/0000-0003-3661-6475 fernandamauricio@gmail.com Recebido em: 2 abr. 2019. Aprovado em: 21 ago. 2019. Publicado em: 16 jul. 2020. Recebido em: 2 abr. 2019. Aprovado em: 21 ago. 2019. Publicado em: 16 jul. 2020. Palavras-chave: Telejornalismo local. Temporalidades sociais. Terra de Minas. ABSTRACT: This article analyzes how the program called Terra de Minas (TV Globo Minas) addresses different temporalities as a way to establish an image of the city of Belo Horizonte. Based on the contributions of Cultural Studies, this article assumes that the past is not something that is finalized, but it is sum- moned in the present and may assume places of dispute and opposition to the dominant forms. In the same way, the future can be lived in the present, not as something projected, but as that which reconfigures elements of the dominant or that establishes something new. The textual analysis of the program, based on its visual, sound and verbal elements, signals for at least three calls of social temporalities: the past as patrimony, the future as an improvement of the past and the past as a construction of oneself. Keywords: Local telejournalism. Social temporality. Terra de Minas. RESUMEN: El presente artículo tiene como objetivo analizar la forma en que el programa Tierra de Minas (TV Globo Minas) aborda diferentes temporalidades como forma de establecer una imagen de la ciudad de Belo Horizonte. A partir de las contribuciones de los Estudios Culturales, este artículo asume que el pa- sado no es algo finalizado, pero es convocado en el presente pudiendo asumir lugares de disputa y oposición a las formas dominantes. De la misma manera, el futuro puede ser vivido en el presente, no como algo proyectado, sino como aquello que reconfigura elementos del dominante o que instaura algo nuevo. El análisis textual del programa, basado en sus elementos visuales, sonoros y verbales, señala para al menos tres convocatorias de las temporalidades sociales: el pasado como patrimonio, el futuro como perfeccionamiento del pasado y el pasado como construcción de los sujetos. Palabras clave: Teleperiodismo local. Temporalidades sociales. Terra de Minas. Artigo está licenciado sob forma de uma licença Creative Commons Atribuição 4.0 Internacional. 1 Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil. 2/11 Revista FAMECOS, Porto Al gre, v. 27, p. 1-11, jan.-dez. 2020 \| e-33837 Revista FAMECOS, Porto Al gre, v. 27, p. 1-11, jan.-dez. 2 Nos anos de 2009 e 2010, a TV Globo Minas fez uma campanha publicitária que tinha como eixo elementos que a emissora considera relevantes na formação identitária do estado. Os spots iam ao ar ao longo da programação e consistiam em uma trilha musical de artistas locais acompanhados por imagens de paisagens do estado. 3 TERRA DE MINAS. O programa. Minas, Rede Globo, 31 jan. 2017. Disponível em: https://redeglobo.globo.com/globominas/terrademi- nas/noticia/o-programa.ghtml. Acesso em: 18 out. 2017. Silva1 id 2020 \| e-33837 disputam a construção de uma imagem que põe em contradição a ideia de “cidade do futuro” e a necessidade de preservação do passado são algumas das questões de fundo deste artigo. Introdução Terra de Minas é um programa de grandes reportagens que estreou em 2001 na TV Globo Minas. Com edições semanais, o jornalístico tem como parte de sua pauta mostrar diferentes localidades do estado e diferentes modos de viver, especialmente em cidades do interior. A capital mineira, Belo Horizonte (BH), se constrói no programa como símbolo da modernidade, o que se ratifica nas imagens da igreja de São Francisco, do Edifício Niemeyer e na paisagem urbana. Assim, BH representa um lugar de contradição para a afirmação do discurso hegemônico sobre estado defendido no Terra de Minas: ao mesmo tempo que traz “o progresso” e insere o estado em um cenário de modernidade, essa mesma modernidade impede a manutenção dos valores tradicionais que definem, segundo a emissora, o mineiro: simpatia, tranquilidade, simplicidade2. A análise do programa apresenta oito reportagens exibidas entre os anos de 2015 e 2017, que demonstram os deslocamentos das temporalidades acionados enquanto forma midiática. Antes, porém, faremos uma breve contextualização do programa Terra de Minas no cenário do telejornalismo local, buscando compreender suas matrizes culturais e sua inserção no gênero televisivo. Em seguida, trataremos a questão da temporalidade nos estudos comunicacionais trazendo as contribuições, principalmente, dos Estudos Culturais. Deste modo, este artigo assume, como lugar teórico- metodológico, um olhar para o tempo enquanto categoria que se mantém dinâmica na vida social e é acionada pelos produtos midiáticos em uma relação próxima à vida cotidiana. Muito do programa Terra de Minas consiste em dar visibilidade aos locais onde se pode encontrar tais valores tradicionais. As práticas e diferentes modos de viver dizem de outra temporalidade, de um passado que o programa procura resgatar enquanto resíduo, enquanto aspecto ainda ativo na sociedade. Mas o que dizer da cidade de Belo Horizonte retratada no programa? De que forma tradição e modernidade dialogam no mesmo espaço televisivo? Que tensionamentos são encontrados entre passado, presente e futuro? O objetivo deste artigo é fomentar uma discussão sobre as distintas temporalidades acionadas pelo programa Terra de Minas para retratar a cidade de Belo Horizonte. O programa projeta ao telespectador uma experiência temporal, uma forma de estar no mundo que articula dimensões identitárias a uma relação específica com o espaço-tempo. De que forma ele o faz, como passado, presente e futuro aparecem articulados nas formas do programa, e ainda, tomando Belo Horizonte como cenário e problema, de que forma as temporalidades O Terra de Minas e o telejornalismo local Terra de Minas é um programa produzido e exibido pela TV Globo Minas, uma versão regional da Rede Globo de Televisão. Indo ao ar nas tardes de sábado desde outubro de 2001, o programa possui a seguinte descrição: Juliana Perdigão [apresentadora] leva ao público as histórias, as tradições e os personagens fascinantes de Minas Gerais aos sábados. Ela apresenta o Terra de Minas, programa que viaja pelo estado para mostrar o patrimônio histórico e cultural mineiro3 (TERRA DE MINAS, [2017], grifo nosso). Apesar do forte sentido de pertencimento construído pelo programa, ele também é transmitido para mais de 130 países pela Globo Internacional. Assim, a emissora aposta no local como elemento de exportação, como marca das características do estado que, por vezes, se confundem com marcas do próprio Brasil. Por conta Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 3/11 Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 3/11 que engloba produção e recepção. Os laços de pertencimento de que nos falam Coutinho e Mata (2010) são ratificados por um sentido de “estar no mundo” compartilhado no momento da transmissão do programa. Por esta razão, tem sido cada vez mais comum, nos programas jornalísticos, os telespectadores enviarem fotos e vídeos em sua residência, em espaços públicos das cidades, em momentos de lazer, ou seja, pessoas comuns em sua vida cotidiana exibem-se na programação local em uma relação contínua entre o tempo midiático e o tempo da experiência do dia a dia4. disso, o Terra de Minas possui um forte tom turístico, como se se endereçasse ao turista de passagem. Terra de Minas é um programa de grandes reportagens que assume um modelo próximo aos programas de turismo vistos principalmente na TV fechada, mas guarda as convenções dominantes do telejornalismo presentes em qualquer manual de redação. O programa perscruta vilarejos, valoriza monumentos, mostra curiosidades, ensina receitas ancestrais, tudo isso sob o enfoque de uma tradição que constitui a identidade local. A pesquisa em comunicação possui uma ampla literatura a respeito das relações entre televisão local e formação/representação da identidade. Em muitas dessas abordagens, o objeto de análise são os telejornais diários exibidos pelas emissoras regionais dispersas pelo País (MATA, 2011; COUTINHO; MATA, 2010; LISBOA FILHO; ENNINGER, 2013). 4 O Terra de Minas, por exemplo, utiliza imagens de pessoas comuns nos cenários explorados pelo programa: cidades históricas, cachoeiras etc. Tudo isso reforça o papel do telejornalismo em possibilitar a partilha do mesmo tempo-espaço (aqui-agora) em uma experiência contínua. O presente é vivo, portanto, porque é histórico, porque permite a reconfigura- ção constante do passado e do futuro. Todo narrar, todo esforço de configurar a experiência temporal – midiático in- clusive – resulta, então, desse agir, se constitui como uma operação de pro- dução de sentido, de configuração de mundos, a partir da proposição de uma experiência do tempo, ao configurar presente, passado e futuro. (RIBEIRO; LEAL; GOMES, 2017, p. 39). O Terra de Minas e o telejornalismo local Lisboa Filho e Enninger (2013) explicam que a delimitação de um território, a partilha de certos valores e práticas e a disseminação de um mito fundador são recursos empregados para a construção de um sentido de unicidade. Coutinho e Mata (2010) defendem que a programação televisiva atua na formação de um laço de pertencimento que oculta os dissensos e procura estabelecer um consenso a respeito do que significa “pertencer”. Em um estudo sobre a construção identidade Buscando caracterizar a experiência estética a partir do prazer gerado pela socialização dos indivíduos, Mead (1926) chama atenção para o fato de que as experiências, quando vividas em conjunto, são apreendidas a partir de uma dimensão de deleite, que envolve o simples fato de colocar as pessoas em contato em direção a uma finalidade comum. Para Mead, a experiência parte da necessidade de interpretar as complexidades da vida social em termos dos objetivos e dos esforços dos indivíduos em alcançá-los. Com base nisso, o autor reconhece no cinema e no jornalismo formas de obter prazer por meio da recepção coletiva do produto. A simples leitura dos jornais (diariamente, pela manhã, por exemplo) e o contato com assuntos comuns provocam uma recepção prazerosa, uma vez que ultrapassa o nível do pessoal e une os indivíduos em torno de questões vividas socialmente. Sendo assim, o caráter generalizante dos assuntos pautados pelo jornalismo permite uma experiência comunitária e a identificação do grupo social ao qual o indivíduo faz parte. Em um estudo sobre a construção identidade juiz-forana em telejornais da TV Alterosa, TVE e Globo Minas, Mata afirma que A TV local, por seu pioneirismo e papel de integração, não deixou de unir os laços sociais da modernidade, inspi- rando-se nas suas tradições regionais para elaborar seus programas, mesmo com toda influência das grandes redes (MATA, 2011, p. 42). O telejornalismo local, por buscar maior proximidade com sua audiência (COUTINHO; MATA, 2010), constitui-se como um espaço profícuo para dar sentido às experiências comuns e transmiti-las a outros. São esses significados que, segundo Mead (1926), permitem a percepção do “mundo real” e a socialização dos indivíduos a partir de um sentimento de apreciação e prazer Não é a apenas a televisão local que gera sentidos de pertencimento por uma vinculação mais evidente com elementos reconhecíveis pelos públicos que deles partilham – os bairros, os eventos, as celebrações etc. O Terra de Minas e o telejornalismo local – mas é papel do próprio jornalismo conferir uma experiência 4/11 Revista FAMECOS, Porto Al gre, v. 27, p. 1-11, jan.-dez. 2020 \| e-33837 É por partilhar essa forma de entender as temporalidades que efetuamos um percurso de aproximação aos Estudos Culturais, corrente de investigação que teve, desde sua origem, uma preocupação central com o tempo e suas tramas (SILVA; GUTMANN, 2017). Embasados em Martín- Barbero, Ribeiro, Leal e Gomes destacam que a “multiplicidade de temporalidades, multiplicidade de histórias, com seus próprios ritmos e com suas próprias lógicas” (MARTÍN-BARBERO, 2006, p. 43) atravessam os processos culturais e midiáticos, estabelecendo uma nova perspectiva de observação dos fenômenos. Ainda de acordo com Ribeiro, Leal e Gomes (2017, p. 53), Martín-Barbero encontra apoio nas formulações de Raymond Williams para afirmar que cada sociedade vive uma heterogeneidade de temporalidades, ou seja, em um mesmo momento histórico vive- se aspectos arcaicos, residuais e emergentes. Assim, retomaremos as formulações de Williams para entendermos de que forma as distintas temporalidades convivem simultaneamente na sociedade e configuram o processo cultural. pelo simples fato de ser compartilhado com outros. Assim, se o telejornalismo local permite uma experiência no e do espaço em busca da construção de laços sociais, faz-se relevante entender também como a categoria do tempo pode ratificar esses valores. temporalidades midiáticas A questão das temporalidades encontra-se em crescimento nas pesquisas em comunicação, não tanto no sentido de meramente atribuir-lhe um caráter histórico, mas pelo reconhecimento de que a cultura contemporânea é atravessada por tempos distintos. Se a linearidade histórica já ocupou o ponto de partida para a consideração dos fenômenos comunicacionais, na atualidade percebe-se que os tempos possuem movimentos dinâmicos, implicando diversos ires-e-vires em sentidos múltiplos. A experiência com o tempo, portanto, está se modificando. Elementos do passado emergem no presente e reconfiguram-se em novas práticas. Para Musse, Vargas e Nicolau, Williams (1979) considera que a cultura é marcada por um jogo de disputas entre valores distintos assumidos como dominantes em um certo período histórico. Assim, formas hegemônicas a todo momento concorrem com formas alternativas e oposicionais (WILLIAMS, 2011b) para se estabelecer como dominante. Essas disputas podem provocar transformações (leves ou severas) nos modos de viver de uma sociedade em um dado momento histórico, assim, como transforma sua arte, sua literatura, sua linguagem, suas práticas cotidianas. Para Williams, a cultura é um processo sempre dinâmico e em transformação, que se evidencia em convenções dominantes, mas que não descarta formas já vividas no passado. Para dar conta dessas diferentes categorias de tempo, o autor formulou as noções de arcaico, residual e emergente, que trataremos a seguir. [...] cada mídia e cada processo co- municacional engendra um ou mais regimes de temporalidade. Cada um deles traduz o tempo e sua percepção de maneira peculiar conforme o uso social e simbólico que a sociedade faz dessa mediatização (MUSSE; VARGAS; NICOLAU, 2017, p. 8). Essa trama requer, primeiramente, considerar o presente como algo não cristalizado em processos solidificados, mas de forma fluida e em permanente transformação. Concordamos com Ribeiro, Leal e Gomes quando afirmam que O residual diz respeito aos aspectos vividos no passado que permanecem ativos no processo cultural por meio de uma reconfiguração no presente, podendo apresentar-se como alternativo Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 5/11 Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 5/11 Fernanda Mauricio da Silva Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas procuramos demonstrar de que forma passado, presente e futuro estão concatenados provocando distintas formas de ver o tempo. ou opositor. temporalidades midiáticas A relação entre o dominante e o residual convoca uma tradição seletiva, ou seja, “a forma pela qual, a partir de toda uma área possível do passado e do presente, certos significados e práticas são escolhidos e enfatizados, enquanto outros significados e práticas são negligenciados e excluídos” (WILLIAMS, 2011b, p. 54). A noção de residual segundo o autor não pode confundir-se com a de arcaico, ou seja, das formas que tiveram uma existência no passado, mas deixaram de existir completamente. 5 Segundo Gomes (2011), as noções de dominante, residual e emergente estão presentes na hipótese cultural da “estrutura de sentimen- to”, termo que designa os aspectos vividos que se encontram em processo, sempre em uma posição cambiante. Para uma consideração mais detalhada e uma análise de um produto comunicacional a partir da noção de estrutura de sentimento, ver Silva e Gutmann (2017). Continuidades e rupturas temporais no Terra de Minas Propõe-se, então, uma análise do programa Terra de Minas com base em seus elementos sonoros, visuais e verbais. Para tanto, selecionamos, aleatoriamente, seis edições do programa que tiveram ao menos uma reportagem centrada em elementos da capital: uma em 2015 (7 mar. 2015), três em 2016 (7 maio 2016a; 14 maio 2016b; 25 jun. 2016c) e duas em 2017 (18 nov. 2017a; 9 dez. 2017a). Foi com base nelas que localizamos distintas formas de aparição e tratamento das temporalidades que revelam uma presença tensionada entre elas. Discutiremos a seguir as principais delas. Já o emergente refere-se àquilo que está presente na sociedade de forma ainda virtual. São os “novos valores, novas práticas, novas relações e tipos de relação [que] estão sendo continuamente criados” (WILLIAMS, 1979, p. 126) e que resistem à cultura dominante. O conceito de emergente não pode se confundir com o de “inovação” que, segundo o autor, representa a reprodução da ordem dominante a fim de ratificá-la e dar-lhe força. A inovação acontece dentro da cultura dominante, enquanto o emergente teria o papel de negá-la, atuando como uma forma cultural de oposição. Deste modo, para Williams (1979), em um mesmo momento estão incorporadas diversas temporalidades, que, postas em relação, possibilitam olhar as articulações entre presente, passado e futuro5. o analista precisa considerar as diversas temporalidades sociais em qualquer aná- lise da cultura e estar atento a um certo senso de movimento, de processo históri- co, de conexões com o futuro e o passado, de articulações complexas entre esses elementos dominantes e os residuais e emergentes (GOMES, 2011, p. 44). O passado como patrimônio Ela mostra que às vezes inter- romper a pressa com um cafezinho coado na hora faz um bem danado pra gente. Por ser absorvida pela modernidade, Belo Horizonte torna-se cenário de suas consequências positivas e negativas: ao mesmo tempo que representa o “progresso”, representa a fuga dos valores ancestrais do estado. Diferentemente das demais localidades que visita, quando fala de BH, o Terra de Minas costuma mostrar a ação negativa do tempo para então revelar algo positivo. Mantém-se o tom afirmativo do programa a respeito do estado e do “ser mineiro”, buscando-se valorizar ações que resgatem a cidade dos problemas vividos. Segundo Musse, Viviane Possato: ela faz isso aqui no centro da cidade mesmo. Imagine você andando apressado no meio desta mul- tidão. De repente surge o convite: tomar um cafezinho logo cedo (TERRA DE MINAS, 2015). A atuação das jornalistas enfatiza os sentidos pretendidos pela reportagem: Viviane Possato instaura um aqui (rua) - agora (horário de movimento) reconhecido pelo telespectador residente em grandes centros urbanos. A partilha da experiência do tempo apressado do presente torna-se uma maneira do programa vincular-se à audiência, por isso, estratégia semelhante se repete em diversas edições do programa: a repórter Iana Coimbra coloca-se em um movimentado cruzamento de veículos no centro da cidade e pergunta: “qual a imagem que você tem do centro de Belo Horizonte? Trânsito? Barulho? Ruas cheias?” (7 maio 2016a); o repórter Vladmir Vilhaça usa a expressão “ritmo frenético” para definir as ruas do centro da capital enquanto a imagem mostra um cruzamento movimentado (14 maio 2016b). O Terra de Minas destaca a pressa como um efeito negativo da contemporaneidade, assim como a fragilidade das relações interpessoais, o que também é validado nas falas de pessoas comuns: [...] o cidadão típico da Modernidade Tardia, pelo menos aquele que vive nos grandes centros urbanos, tende a estar quase sempre em trânsito e conectado a algum suporte eletrônico, que lhe permita estabelecer contatos a longa distância, mas, certamente, o desassocia do ambiente do seu entorno. Tais aspectos, provenientes de uma rápida observação, estabelecem as bases de uma nova rede de sociabili- dade, pautada menos no face a face e mais nas relações mediadas por uma série de gadgets tecnológicos. [...] Da mesma forma, as relações afetivas com o patrimônio, com a paisagem urbana, parecem sofrer um desgaste natural (MUSSE, 2013, p. 229-230). O passado como patrimônio O Terra de Minas usa referências ao passado para legitimar os modos de vida do estado através das histórias que certos personagens têm para contar e que revelam algo do passado. O programa refere-se ao passado como um tempo onde se viviam as autênticas (e puras) relações sociais, ao contrário do presente, que é definido pela correria. Terra de Minas alonga o passado e busca preservá-lo como um patrimônio no presente. A dicotomia “passado tranquilo” x “presente acelerado” é o modo mais evidente de posicionamento das distintas temporalidades sociais vividas em um mesmo momento histórico. Isto se mostra tanto nas falas dos agentes do programa, quanto dos sujeitos entrevistados. Na abertura da reportagem que inicia o programa no dia 7 de março 2015, enquanto a apresentadora Juliana Perdigão encontra-se em um café no centro da cidade de Belo Horizonte, a repórter Viviane Possato está ao lado de fora, na rua, reforçando o sentido de correria: É a partir dessas noções que Gomes afirma que Isto se mostra tanto nas falas dos agentes do programa, quanto dos sujeitos entrevistados. Na abertura da reportagem que inicia o programa no dia 7 de março 2015, enquanto a apresentadora Juliana Perdigão encontra-se em um café no centro da cidade de Belo Horizonte, a repórter Viviane Possato está ao lado de fora, na rua, reforçando o sentido de correria: Tendo em vista as noções de residual, emergente e dominante, mas também de arcaico e de tradição seletiva, procedemos à análise do programa Terra de Minas em um esforço inicial de desvendar, ao menos em parte, essas complexas articulações das múltiplas temporalidades que atravessam o programa. Nesta análise, 11 Revista FAMECOS, Porto Al gre, v. 27, p. 1-11, jan.-dez. 2020 \| e-33837 6/11 6/11 Revista FAMECOS, Porto Al gre, v. 27, p. 1-11, jan.-dez. 2020 \| e-33837 Minas (autêntica, verdadeira) não se encontra na capital. No entanto, nos últimos anos, o programa tem se situado mais em Belo Horizonte. Juliana Perdigão: Para começar a gente vai falar da arte de fazer café, ou melhor, de uma artista que decidiu fazer arte com o café. Ela mostra que às vezes inter- romper a pressa com um cafezinho coado na hora faz um bem danado pra gente. Juliana Perdigão: Para começar a gente vai falar da arte de fazer café, ou melhor, de uma artista que decidiu fazer arte com o café. O futuro como aprimoramento do passado dialoga com aquela tradição seletiva engendrada pelo programa e sua emissora. A legitimação social do Terra de Minas e sua longevidade perpassam o seu papel enquanto defensor do passado, enquanto espaço de preservação desse patrimônio. É recorrente o uso de expressões que indicam essa revelação: “Mas a nossa equipe encontrou moradores que conseguem manter essa amizade durante décadas” (TERRA DE MINAS, 14 maio 2016b); “No meio de tantos prédios, ruas e avenidas nem sempre a gente para observar as novidades que surgem por aí” (TERRA DE MINAS, 7 maio 2016a). Um estudo da socióloga Mônica Abdala sobre os hábitos de vida em cidades do interior de Minas Gerais e Goiás aponta que certas práticas comuns no passado já entraram em desuso. No entanto, afirma a autora que a Cabe, aqui, uma breve consideração sobre o que o Terra de Minas entende por “modernidade”, que também se traduz por expressões como “progresso” ao longo do programa. Ao mesmo tempo que o programa esclarece a que tipo de tradição ele se refere – os hábitos de vida da sociedade rural, a simplicidade das pequenas casas, os afetos da comunidade – ele também dá ampla visibilidade aos edifícios com traços modernistas, aos artistas, aos avanços técnicos. Por este motivo, na ocasião do aniversário de 120 anos da capital mineira, o programa colocou em seu primeiro bloco trechos de reportagens antigas em que se podia ver os espaços públicos do centro da cidade do modo como se vivia “nas primeiras décadas” do século XX. Com isso, o programa convoca o telespectador a uma posição de deslocamento, de estranhamento com os espaços que ele frequenta ainda hoje: a Praça da Liberdade, o Parque Municipal, onde se podia passear de barco, aspectos que convocam alguma nostalgia, mas que naturalizam as formas contemporâneas de viver. [...] experiência coletiva, permeada por formas de cooperação, solidariedade e vizinhança, em meios rurais e pequenas cidades que se tornaram centros rurais urbanizados [...] também podem ser apreendidos por meio da valorização e ressignificação das tradições em meio à vida urbana (ABDALA, 2011, p. 127). Ou seja, marcas da vida comunitária do passado ainda encontram espaço para preservação de diversos agentes e, em certa medida, o Terra de Minas procura validar esse outro modo de viver como uma alternativa à correria e fugacidade. O programa almeja constituir o residual como dominante. O passado como patrimônio É contra esses efeitos da modernidade que o Terra de Minas se constrói. Enquanto o contemporâneo é marcado pela aceleração e correria, o passado tem um outro ritmo, mais lento, em que o afeto e as relações interpessoais podem ser vividos de forma autêntica e profunda. A modernidade trouxe facilidades para o dia a dia, mas “roubou” sabor e afeto. O programa legitima, por meio das vozes dos sujeitos entrevistados, um ritmo lento da vida cotidiana. Maria das Graças (sonora, pessoa co- mum): hoje em dia, as pessoas estão muito olhando ele, ele, ele. [voltadas para si mesmas]. Esse trabalho filan- trópico, essa reciprocidade, essa forma de agradar está muito difícil nos nossos tempos (TERRA DE MINAS, 7 mar. 2015). Este tensionamento entre presente e passado oculta outro: o Interior x capital. Não apenas o modo de viver da simplicidade está em outro tempo, como também em outro espaço: no campo, longe do “progresso” e da “modernidade”. Por este motivo, o Terra de Minas privilegia as cidades do interior como cenário para suas reportagens. É como se quisesse construir uma dimensão de que Para que ainda se encontre algum traço dos valores que configuram o ser mineiro projetado pelo programa, é preciso que ele o revele aos telespectadores. O jornalismo do Terra de Minas assume um papel de desvendamento e revelação (FRANCISCATO, 2005), de tirar do segredo os lugares que escondem um modo de vida que ainda Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 7/11 Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 7/11 Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 7/11 O futuro como aprimoramento do passado Rocha (2017) explica que a América Latina entrou no que se considera modernidade de um modo próprio estabelecendo valores típicos regionais. Segundo a autora, entender a modernidade latino-americana é “entender que a televisão [...] cumpre papel decisivo na articulação entre o mundo simbólico do rural, do popular e a racionalidade técnico-instrumental do urbano” (ROCHA, 2017, p. 116). Por este motivo, falar em modernidade implica considerar suas contradições e mestiçagens. É nesse sentido que Silva (2017) afirma que Minas entrou na modernidade a partir de formas próprias. Por causa disso, Belo Horizonte do Terra de Minas quase que se restringe ao centro da cidade, espaço que mais sofreu os efeitos das políticas públicas em nome da modernização, e que, hoje, sofre com o desgaste da ausência de manutenção dos espaços públicos. Deste modo, o programa valoriza a cidade, pensada como “cidade do futuro”, mas pouco problematiza as consequências desse futuro projetado, quase como se fossem provocadas naturalmente pelo tempo. Assim, uma outra forma de tratar o tempo no Terra de Minas é não apenas preservar os hábitos do passado, mas acenar para um futuro que aprimora as experiências ancestrais. No Terra de Minas, apesar da forte presença de um passado que o programa busca preservar, há o reconhecimento de que a cidade vivida no presente resulta da projeção de um futuro que prometia o bem comum. Se o tempo incidiu sobre a cidade trazendo consequências negativas, os avanços técnicos provenientes do “progresso” permitem um aprimoramento da experiência do passado no Revista FAMECOS, Porto Al gre, v. 27, p. 1-11, jan.-dez. 2020 \| e-33837 8/11 vez que seus projetos de futuro são fundados no sonho do progresso e do sucesso na vida urbana. (ABDALA, 2011, p. 137). Os efeitos perversos pouco aparecem problematizados no programa, que se por um lado quer oferecer resistência aos avanços do capitalismo, eles aparecem como um traço que o tempo traz por si só. O Terra de Minas sinaliza, no presente, para um futuro mais positivo e otimista que pode romper o caos do cotidiano. Esse futuro não oferece uma ruptura com o passado, uma vez que o utiliza como referência e como marco para uma continuidade. presente. Assim, se não é possível viver no sítio para ter alimentos frescos, que se façam hortas urbanas. Este foi o tema de uma das reportagens exibidas no programa no dia 18 de novembro de 2017. O futuro como aprimoramento do passado Na cobertura de um edifício localizado no centro da capital, um grupo interdisciplinar de pesquisadores (biólogos, nutricionistas, engenheiros etc.) criaram uma horta que produz alimentos sem agrotóxico e conservantes. O repórter Vladmir Vilhaça ressalta o estranhamento da produção: “horta em terraço, assim, no centro da cidade, dá certo? As plantas sobrevivem assim?” (TERRA DE MINAS, 18 nov. 2017a). A modernidade possibilita a reconfiguração de práticas do presente que se articulam ao passado, mas as transformam. A tecnologia funciona, então, como instrumento de resgate e aprimoramento do que havia de positivo no passado. O mesmo sentido se nota no tratamento do processo de restauração de obras de arte, monumentos e edifícios da cidade, tema recorrente no programa, como a reportagem sobre a restauração dos cinemas de rua em Belo Horizonte. Após entrevistar moradores da região que guardam na memória experiências naquelas salas, a reportagem exibe o presidente da Fundação Municipal de Cultura, que explica o processo de restauração do prédio do Cine Santa Tereza, edifício inaugurado em 1948 que, após sofrer o desgaste do tempo, foi restaurado e reativado, mantendo os traços arquitetônicos de sua origem. Assim, o programa pretende instaurar um sentido de que a experiência do passado pode ser vivida hoje, mas com mais conforto. O passado como construção de si No entanto, de que passado o Terra de Minas está falando? A que momento histórico o programa se refere quando aciona um tempo que já não é mais em sua plenitude, mas que almeja seu retorno? Se o passado é um patrimônio que precisa ser preservado, isso decorre do fato de que, no programa, o passado se materializa nos sujeitos. Ao ativar constantemente a memória dos entrevistados, Terra de Minas deixa ver permanentemente um sentimento de nostalgia ou saudade. Assim, o programa endossa “o mito do passado mais feliz” (WILLIAMS, 2011a). Mais feliz porque quem efetua o exercício de rememorar o faz a partir do afeto, da juventude, da infância, de um tempo em que, supostamente, havia maior pureza e simplicidade. Sendo assim, o passado não possui um tempo definido, ele é o tempo de quem o viveu. São poucos os momentos em que Terra de Minas dá precisão nas datas e períodos. O passado datado entra no programa enquanto dimensão legitimadora dos patrimônios – das igrejas, dos edifícios etc. Mas a dimensão preferencial do passado no programa é da história de vida, o passado do sujeito. Deste modo, o programa não faz distinção entre a infância que se deu nos anos 1950 e a dos anos 1980, a conjuntura que conforma as práticas vividas em um e outro tempo pouco se expressa para dar significado temporal. Por esse motivo, o programa utiliza expressões genéricas como “antigamente” para falar do tempo histórico. Ao que parece, há uma vertente da modernidade que é aceita: o programa orgulha-se dos museus, da literatura, dos monumentos, mas eles ganham maior relevo quando associados a certa historicidade que, certamente, encontra respaldo nas políticas públicas de promoção de cultura. 6 Hoje, aquela casa abriga uma ONG que cuida de menores em situação de risco por meio da arte. O passado como construção de si Embora lamente os danos da modernidade, o programa pouco problematiza a conjuntura desse mesmo passado que procura preservar: a chegada das monoculturas destinadas ao mercado global; a criação intensiva do gado; o desinteresse dos jovens em continuar os projetos dos pais, pequenos agricultores familiares, uma Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 9/11 Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 9/11 Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 9/11 teve como gancho a pesquisa realizada por duas jovens publicitárias em seu trabalho de conclusão de curso, cujo objetivo era mapear todas as salas de cinema de bairro em BH, contar suas histórias e aviltar o testemunho de pessoas que tenham frequentado aqueles espaços. Sendo assim, ao lado da experiência de quem viveu o passado, há o desejo de saber das novas gerações. A infância/juventude é um tempo frequentemente recorrido pelo programa, como na fala da artista plástica Maria Thereza que organiza diariamente um café nas ruas do centro de Belo Horizonte. Após mostrar a casa onde a artista viveu em sua infância6 e o relato do cotidiano de seu avô, ela conclui: “é como se eu estivesse relembrando esse passado que eu vivi. Esse cheiro de café constante na casa, com bolo. E eu estivesse reeditando essa história aqui na rua” (TERRA DE MINAS, 7 mar. 2015). O tempo da infância, o tempo vivido legitima-se no Terra de Minas e põe em contato a experiência de passado e presente na voz dos sujeitos. Em geral, a tentativa é de fazer uma crítica ao presente buscando restaurá-lo, é tomar o passado como horizonte para a construção de um modo de viver mais afetivo. Terra de Minas procura evidenciar situações em que encontra espontaneamente pessoas passando pela rua no momento da gravação do programa para contar suas histórias. Para falar sobre os murais urbanos, o programa interpela duas entrevistadas por meio de expressões como “a senhora lembra”? (18 nov. 2017a). Na reportagem que abordou a renovação dos cinemas de rua em BH, a sonora de Ieda Ferreira, que estava passando no momento de gravação, ratificou a mesma posição ao lembrar sua juventude: Horizontes para uma agenda de investigação A partir do Estudos Culturais, Rocha (2017) afirma que a mestiçagem é uma condição da modernidade latino-americana. Isso se comprova na forma como a imagem da cidade de Belo Horizonte se constrói no Terra de Minas. BH é, ao mesmo tempo, a materialização da modernidade e do progresso do estado, e as tradições do interior. As duas dimensões coexistem no programa, que pouco problematiza suas contradições. Ieda Ferreira (advogada): tinha a roupa de vir ao cinema. Eu fiz uma vez um vestido lilás para vir ao Paté [...] e até hoje eu tenho um pouco da nostalgia do lilás. Era muito bom! Pena que você [repórter] não tenha vivido essa época (TERRA DE MINAS, 25 jun. 2016c). Ao fazer essa articulação, Terra de Minas constrói para si um lugar de distinção no telejornalismo local: enquanto os telejornais diários ratificam os efeitos negativos da modernidade – o trânsito intenso, a violência etc. – o Terra de Minas atua como fomentador de uma versão afetiva que a cidade carrega como continuidade do passado. Ao tratar Minas Gerais como um local onde o passado permanece, como se o tempo fosse linear, o programa, justamente, ratifica a temporalidade não linear e comprova que as distintas temporalidades vividas estão em articulação no mesmo momento histórico. Tanto quanto o parecer técnico de um especialista, o testemunho de quem viu e viveu possui forte peso na construção informativa e afetiva do programa. Como consequência, certos personagens são preferenciais na legitimação do discurso. Especialistas e fontes oficias têm lugares assegurados, mas também idosos, anciãos que tenham vivido aquele passado projetado no programa para assegurar uma experiência autêntica de outra temporalidade no presente. Com isso, Terra de Minas parece assumir também o papel de transmitir as experiências de geração em geração, fortalecendo um vínculo de pertencimento e criando um sentido de comunidade. Ainda sobre a reportagem sobre a restauração dos cinemas de rua, a produção Há, também, um certo senso de arcaico no programa. A preservação, nesse caso, opera como uma tentativa de manter estática no tempo a experiência de um tempo que não se vive mais. 6 Hoje, aquela casa abriga uma ONG que cuida de menores em situação de risco por meio da arte. /11 Revista FAMECOS, Porto Al gre, v. 27, p. 1-11, jan.-dez. Horizontes para uma agenda de investigação Gêneros midiáticos e identidade. Belo Horizonte: PPGCOM UFMG, 2017, p. 115-132. Horizontes para uma agenda de investigação 2020 \| e-33837 Diferentemente do passado como patrimônio, em que há uma tentativa de evidenciar os valores e práticas que permanecem ativos como resíduo, o arcaico diz de algo que ficou no passado e torna- se peça de museu, como no caso dos enxovais do Museu da Renda (9 dez. 2017b). Diferentemente do passado como patrimônio, em que há uma tentativa de evidenciar os valores e práticas que permanecem ativos como resíduo, o arcaico diz de algo que ficou no passado e torna- se peça de museu, como no caso dos enxovais do Museu da Renda (9 dez. 2017b). MATA, Jhonatan. Um telejornal para chamar de seu: identidade, representação e inserção popular no telejor- nalismo local. Dissertação (Mestrado em Comunicação) – Faculdade de Comunicação, UFJF, Juiz de Fora, 2011. MEAD, George Herbert. The nature of aesthetics ex- perience. International Journal of Ethics, [s. l.], v. 36, n. 4, p. 382-393, 1926. https://doi.org/10.1086/inteje- thi.36.4.2377635. A análise das temporalidades em programas televisivos mostra-se como um fértil campo de investigação para pesquisadores brasileiros. As temporalidades dizem da experiência do tempo nas sociedades contemporâneas que são atravessadas por políticas públicas de ocupação do espaço, tecnologias que transformam as formas de sociabilidade entre outros fatores que modificam a percepção sobre estar no tempo, o que também se traduz nos produtos midiáticos. Este artigo procurou levantar algumas dessas possibilidades, ainda de modo inicial, mas com a finalidade de apontar pistas para análises futuras. MUSSE, Christina. Cultura, televisão e imaginário ur- bano. Matrizes, [s. l.], v. 7, n. 1, p. 223-234, jan./jun. 2013. https://doi.org/10.11606/issn.1982-8160.v7i1p223-234. MUSSE, Christina; VARGAS, Herom; NICOLAU, Marcos. Apresentação – Temporalidades: dos conceitos às apli- cações midiáticas. In: MUSSE, C.; VARGAS, H.; NICOLAU, ( ) C i ã ídi lid d MUSSE, Christina; VARGAS, Herom; NICOLAU, Marcos. Apresentação – Temporalidades: dos conceitos às apli- cações midiáticas. In: MUSSE, C.; VARGAS, H.; NICOLAU, M. (org.). Comunicação, mídias e temporalidades. Salvador: Edufba, 2017. p. 7-16. M. (org.). Comunicação, mídias e temporalidades. Salvador: Edufba, 2017. p. 7-16. RIBEIRO, Ana Paula; LEAL, Bruno; GOMES, Itania. A historicidade dos processos comunicacionais: elemen- tos para uma abordagem. In: MUSSE, C.; VARGAS, H.; NICOLAU, M. (org.). Comunicação, mídias e tempora- lidades. Salvador: Edufba, 2017, p. 37-58. ROCHA, Simone. “Ele é o atraso e você a moder- nidade”: experiências anacrônicas da modernidade latino-americana na televisualidade brasileira, o caso da telenovela Duas Caras. In: FRANÇA, V.; COHEN, E.; GOMES, I. (org.). MARTÍN-BARBERO, Jesús. Dos meios às mediações: comunicação, cultura e hegemonia. Rio de Janeiro: Editora UFRJ, 2006, p. 11-21. Referências Fernanda Mauricio da Silva Tramas do tempo no telejornalismo local: temporalidades sociais no programa Terra de Minas 11/11 11/11 Fernanda Mauricio da Silva Doutora e Pós-doutora em Comunicação e Cultura Contemporâneas pela Universidade Federal da Bahia (UFBA). Professora do Programa de Pós-Graduação em Comunicação e do Departamento de Comunica- ção Social da Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil. Referências SILVA, Fernanda Mauricio da; GUTMANN, Juliana. O feminino dá o tom: resultados de uma análise histórica e cultural do talk show no Brasil. Texto apresentado no XXVI Encontro Anual da Compós, São Paulo, jun. 2017. ABDALA, Mônica Chaves. Saberes e sabores: tradições culturais populares do interior de Minas e de Goiás. História: Questões & Debates, Curitiba, n. 54, p. 125-158, jan./jun. 2011. Editora UFPR. https://doi.org/10.5380/ his.v54i1.25743. SILVA, Marcos Vinícius. O estilo televisivo e a figuração da mineiridade em programas de caráter regional. 2017. Dissertação (Mestrado em Comunicação Social) – Faculdade de Filosofia e Ciências Humanas, UFMG, Belo Horizonte, 2017. COUTINHO, Iluska; MATA, Jhonatan. Telejornalismo a serviço do público: a voz do povo em cena. Revista Famecos, Porto Alegre, v. 17, n. 1, p. 65-73. jan./abr. 2010. https://doi.org/10.15448/1980-3729.2010.1.6881. TERRA DE MINAS. Programa TV Globo Minas. 7 mar. 2015. FRANCISCATO, Carlos Eduardo. A fabricação do pre- sente: como o jornalismo reformulou a experiência do tempo nas sociedades ocidentais. São Cristóvão: Editora UFS; Aracaju: Fundação Oviêdo Teixeira, 2005. 274 p. TERRA DE MINAS. Programa TV Globo Minas. 7 maio 2016a. TERRA DE MINAS. Programa TV Globo Minas. 14 maio 2016b. GOMES, Itania. Raymond Williams e a hipótese cultural da estrutura de sentimento. In: GOMES, Itania; JANOTTI, Jeder (org.). Comunicação e estudos culturais. Salva- dor: EDUFBA, 2011. p. 29-48. GOMES, Itania. Raymond Williams e a hipótese cultural da estrutura de sentimento. In: GOMES, Itania; JANOTTI, Jeder (org.). Comunicação e estudos culturais. Salva- dor: EDUFBA, 2011. p. 29-48. TERRA DE MINAS. Programa TV Globo Minas. 25 jun. 2016c. TERRA DE MINAS. Programa TV Globo Minas. 18 nov. 2017a. LISBOA FILHO, Flavi Ferreira; ENNINGER, Rossana Zott. Identidade e televisão regional: conceitos e aproxi- mações. Intercom – Sociedade Brasileira de Estudos Interdisciplinares da Comunicação, XXXVI Congresso Brasileiro de Ciências da Comunicação – Manaus/ AM – 4 a 7 set. 2013. Disponível em http://w3.ufsm.br/ estudosculturais/arquivos/trabalhos-eventos/IDEN- TIDADE%20E%20TELEVIS%C3%83O%20REGIONAL%20 CONCEITOS%20E%20APROXIMA%C3%87%C3%95ES. pdf. Acesso em: 20 mar. 2018. TERRA DE MINAS. Programa TV Globo Minas. 9 dez. 2017b. WILLIAMS, Raymond. O campo e a cidade: na história e na literatura. São Paulo: Companhia das Letras, 2011a. WILLIAMS, Raymond. Cultura. Rio de Janeiro: Paz e Terra, 2011b. WILLIAMS, Raymond. Marxismo e Literatura. Rio de Janeiro: Zahar Editores, 1979. p. 179-184. MARTÍN-BARBERO, Jesús. Dos meios às mediações: comunicação, cultura e hegemonia. Rio de Janeiro: Editora UFRJ, 2006, p. 11-21. Endereço para correspondência Fernanda Mauricio da Silva Universidade Federal de Minas Gerais Av. Antônio Carlos, 6.627, Sala 4.238, 4º Andar Pampulha, 31.270-901 Belo Horizonte, MG, Brasil Fernanda Mauricio da Silva Universidade Federal de Minas Gerais Av. Antônio Carlos, 6.627, Sala 4.238, 4º Andar Pampulha, 31.270-901 Belo Horizonte, MG, Brasil Fernanda Mauricio da Silva Universidade Federal de Minas Gerais Av. Antônio Carlos, 6.627, Sala 4.238, 4º Andar Pampulha, 31.270-901 Belo Horizonte, MG, Brasil Belo Horizonte, MG, Brasil
https://openalex.org/W3012335389	https://europepmc.org/articles/pmc7143966?pdf=render	English	null	Synthesis of 4′-Substituted-2′-Deoxy-2′-α-Fluoro Nucleoside Analogs as Potential Antiviral Agents	Molecules/Molecules online/Molecules annual	2,020	cc-by	9,969	Received: 3 February 2020; Accepted: 28 February 2020; Published: 11 March 2020 Abstract: Nucleoside analogs are widely used for the treatment of viral diseases (Hepatitis B/C, herpes and human immunodeﬁciency virus, HIV) and various malignancies. ALS-8176, a prodrug of the 4′-chloromethyl-2′-deoxy-2′-ﬂuoro nucleoside ALS-8112, was evaluated in hospitalized infants for the treatment of respiratory syncytial virus (RSV), but was abandoned for unclear reasons. Based on the structure of ALS-8112, a series of novel 4′-modiﬁed-2′-deoxy-2′-ﬂuoro nucleosides were synthesized. Newly prepared compounds were evaluated against RSV, but also against a panel of RNA viruses, including Dengue, West Nile, Chikungunya, and Zika viruses. Unfortunately, none of the compounds showed marked antiviral activity against these viruses. Keywords: nucleoside; virus; polymerase inhibitors; respiratory syncytial virus; Zika; Dengue; West Nile; Chikungunya Molecules 2020, 25, 1258; doi:10.3390/molecules25061258 molecules Through this work we focused on small groups r been introduced on the 4′-position of a nucleoside analog. These modiﬁcations included www.mdpi.com/journal/molecules Synthesis of 4′-Substituted-2′-Deoxy-2′-α-Fluoro Nucleoside Analogs as Potential Antiviral Agents Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, 1760 Haygood Drive, Atlanta, GA 30322, USA; mahesh.kasthuri@emory.edu (M.K.); cli35@emory.edu (C.L.); kverma2@emory.edu (K.V.); olivia.ollinger@emory.edu (O.O.R.); lyndsey.dickson@emory.edu (L.D.); alouisemccormick@gmail.com (L.M. leda.bassit@emory.edu (L.B.); famblar@emory.edu (F.A.) * Correspondence: rschina@emory.edu molecules molecules molecules sis of 4′-Substituted-2′-Deoxy-2′-α-Fluoro side Analogs as Potential Antiviral Agents huri, Chengwei Li, Kiran Verma, Olivia Ollinger Russell, Lyndsey Dickson, rmick, Leda Bassit, Franck Amblard and Raymond F. Schinazi * DS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory hool of Medicine, 1760 Haygood Drive, Atlanta, GA 30322, USA; uri@emory.edu (M.K.); cli35@emory.edu (C.L.); kverma2@emory.edu (K.V.); @emory.edu (O.O.R.); lyndsey.dickson@emory.edu (L.D.); alouisemccormick@gmail.com (L.M.); mory.edu (L.B.); famblar@emory.edu (F.A.) dence: rschina@emory.edu ebruary 2020; Accepted: 28 February 2020; Published: 11 March 2020 ucleoside analogs are widely used for the treatment of viral diseases (Hepatitis B/C, human immunodeﬁciency virus, HIV) and various malignancies. ALS-8176, a prodrug of omethyl-2′-deoxy-2′-ﬂuoro nucleoside ALS-8112, was evaluated in hospitalized infants ment of respiratory syncytial virus (RSV), but was abandoned for unclear reasons. Based ture of ALS-8112, a series of novel 4′-modiﬁed-2′-deoxy-2′-ﬂuoro nucleosides were Newly prepared compounds were evaluated against RSV, but also against a panel of s, including Dengue, West Nile, Chikungunya, and Zika viruses. Unfortunately, none of nds showed marked antiviral activity against these viruses. nucleoside; virus; polymerase inhibitors; respiratory syncytial virus; Zika; Dengue; hikungunya on d nucleoside and nucleotide analogs are now the cornerstone of antiviral and anticancer ies [1,2] and among them, 4′-substituted nucleosides have attracted a great deal of gure 1). Balapiravir (1), the prodrug of 4′-azidocytidine, was one of the early hits a potent and selective inhibitor of hepatitis C virus (HCV) RNA polymerase [3]. Further, ﬂuoro-2′-deoxyadenosine (2) (EFdA/MK-8591/islatravir), in its triphosphate form, is a t nucleoside reverse transcriptase translocation inhibitor (NRTTI) which is right now the treatment and pre-exposure prophylaxis of HIV-1 infection via subdermal implant [4]. -C-cyano-2-amino-2′-deoxyadenosine (CAdA) (3) [5] was also reported as a highly potent oth HBV and HIV-1 replication while E-CFCP (4), another 4′-C-cyano nucleoside analog, to be a subnanomolar inhibitor of HBV replication [6]. ALS-8176/lumicitabine (6), a LS-8112, a 4′-chloromethyl-2′-deoxy-2′-ﬂuorocytidine analog, was evaluated in a phase 2 or the treatment of respiratory syncytial virus (RSV) infections which was terminated asons [7]. We recently reported that ALS-8112 also displayed potent anti-Nipah virus ro while also displaying in vitro toxicity [8]. Based on the potential of ALS-8112, we wish in, the synthesis and the antiviral evaluation of new 4′-substituted-2′-deoxy-2′-ﬂuoro oside analogs. Although numerous 4′-substitutions have already been introduced on the ﬀold, these modiﬁcations remained basic and included mostly simple groups such as N3, ethynyl, cycloalkyl, ethers and thioethers. 2 1 Chemistry 2.1. Chemistry 2.1. Chemistry Scheme 1. Synthesis of key intermediates 8 and 9 from commercially available 2′-deoxy-2′-α- fluorocytidine 7. Targeted 4′-substituted-2′-deoxy-2′-α-fluoro nucleoside derivatives 11, 14, 17, 20, 25, and 26 were prepared from key intermediates 8 and/or 9 obtained from commercially available 2′-deoxy-2′-α- fluorocytidine 7 following the chemistry described by Wang et al. [10] (Scheme 1). The synthesis of 4′-difluoromethoxy analog 11 was achieved by the reaction of 9 with a reactive Cu-difluorocarbene complex obtained by the reaction of CuI with FSO2CF2CO2H [11], followed by removal of the trityl groups in 80% aqueous acetic acid (Scheme 2). We first thought to prepare the desired azetidine analog 14 by reacting an activated 5′-methyltriflate intermediate with azetidine in presence of an organic base (Et3N or pyridine). However, under these conditions, we were unable to observe formation of the desired compound. We hypothesized that the relatively bulky azetidine ring could not reach the sterically hindered 5′- position due to the presence of the nearby large 5′- and 3′- Targeted 4′-substituted-2′-deoxy-2′-α-ﬂuoro nucleoside derivatives 11, 14, 17, 20, 25, and 26 were prepared from key intermediates 8 and/or 9 obtained from commercially available 2′-deoxy-2′-α-ﬂuorocytidine 7 following the chemistry described by Wang et al. [10] (Scheme 1). The synthesis of 4′-diﬂuoromethoxy analog 11 was achieved by the reaction of 9 with a reactive Cu-diﬂuorocarbene complex obtained by the reaction of CuI with FSO2CF2CO2H [11], followed by removal of the trityl groups in 80% aqueous acetic acid (Scheme 2). We ﬁrst thought to prepare the desired azetidine analog 14 by reacting an activated 5′-methyltriﬂate intermediate with azetidine in presence of an organic base (Et3N or pyridine). However, under these conditions, we were unable to observe formation of the desired compound. We hypothesized that the relatively bulky azetidine ring could not reach the sterically hindered 5′- position due to the presence of the nearby large 5′- and 3′- monomethoxytrityl groups. Therefore, we subsequently evaluated an intramolecular reductive cyclization via the use of a primary halogeno alkylamine. The oxidation of 9 to the corresponding aldehyde with Dess Martin periodinane followed by reaction with 3-bromopropylamine in the presence of MgSO4 led to the formation of imine intermediate 12 which was subsequently reduced with NaBH4. Finally, the newly formed amine displaced the terminal bromine to form the desired azetidine derivative 13 [12,13]. Treatment of 13 under acidic conditions gave the targeted compound 14 (Scheme 3). 1. Introduction A selection of 4′-substituted nucleoside analogs displaying antiviral activity and structures of targeted 4′- and 5′-substituted-2′-deoxy-2′-ﬂuoro cytidine analogs (A). Figure 1. A selection of 4′-substituted nucleoside analogs displaying antiviral activity and structures of targeted 4′- and 5′-substituted-2′-deoxy-2′-fluoro cytidine analogs (A). Figure 1. A selection of 4′-substituted nucleoside analogs displaying antiviral activity and structures of targeted 4′- and 5′-substituted-2′-deoxy-2′-ﬂuoro cytidine analogs (A). Figure 1. A selection of 4′-substituted nucleoside analogs displaying antiviral activity and structures of targeted 4′- and 5′-substituted-2′-deoxy-2′-fluoro cytidine analogs (A). Figure 1. A selection of 4′-substituted nucleoside analogs displaying antiviral activity and structures of targeted 4′- and 5′-substituted-2′-deoxy-2′-ﬂuoro cytidine analogs (A). 1. Introduction Modiﬁed nucleoside and nucleotide analogs are now the cornerstone of antiviral and anticancer chemotherapies [1,2] and among them, 4′-substituted nucleosides have attracted a great deal of attention (Figure 1). Balapiravir (1), the prodrug of 4′-azidocytidine, was one of the early hits identiﬁed as a potent and selective inhibitor of hepatitis C virus (HCV) RNA polymerase [3]. Further, 4′-ethynyl-2-ﬂuoro-2′-deoxyadenosine (2) (EFdA/MK-8591/islatravir), in its triphosphate form, is a highly potent nucleoside reverse transcriptase translocation inhibitor (NRTTI) which is right now evaluated for the treatment and pre-exposure prophylaxis of HIV-1 infection via subdermal implant [4]. In addition, 4′-C-cyano-2-amino-2′-deoxyadenosine (CAdA) (3) [5] was also reported as a highly potent inhibitor of both HBV and HIV-1 replication while E-CFCP (4), another 4′-C-cyano nucleoside analog, was reported to be a subnanomolar inhibitor of HBV replication [6]. ALS-8176/lumicitabine (6), a prodrug of ALS-8112, a 4′-chloromethyl-2′-deoxy-2′-ﬂuorocytidine analog, was evaluated in a phase 2 clinical trial for the treatment of respiratory syncytial virus (RSV) infections which was terminated for unclear reasons [7]. We recently reported that ALS-8112 also displayed potent anti-Nipah virus activity in vitro while also displaying in vitro toxicity [8]. Based on the potential of ALS-8112, we wish to report herein, the synthesis and the antiviral evaluation of new 4′-substituted-2′-deoxy-2′-ﬂuoro cytidine nucleoside analogs. Although numerous 4′-substitutions have already been introduced on the ALS-8112 scaﬀold, these modiﬁcations remained basic and included mostly simple groups such as N3, alkyls, vinyl, ethynyl, cycloalkyl, ethers and thioethers. Through this work we focused on small groups that had never been introduced on the 4′-position of a nucleoside analog. These modiﬁcations included Molecules 2020, 25, 1258; doi:10.3390/molecules25061258 www.mdpi.com/journal/molecules 2 of 13 2 of 13 Molecules 2020, 25, 1258 small heterocyclic rings (azetidine, oxetane and isoxazole), but also a unique diﬂuoromethyl ether group. In parallel, we also evaluated the eﬀect, in terms of antiviral potency, of a methyl group on the 5′-methylene portion of ALS-8112, a modiﬁcation known to be tolerated by other viral polymerases [9]. unique difluoromethyl ether group. In parallel, we also evaluated the effect, in terms of antiviral potency, of a methyl group on the 5′-methylene portion of ALS-8112, a modification known to be tolerated by other viral polymerases [9]. Figure 1. A selection of 4′-substituted nucleoside analogs displaying antiviral activity and structures of targeted 4′- and 5′-substituted-2′-deoxy-2′-fluoro cytidine analogs (A). Figure 1. 2 1 Chemistry 2.1. Chemistry The 4′-oxetane analog 17 was obtained from 9 by, ﬁrst, oxidation to the corresponding aldehyde followed by a Johnson-Corey-Chaykovsky epoxidation and consecutive ring-expansion. Thus, compound 9 was oxidized by treatment with Dess-Martin periodinane to the corresponding aldehyde which was treated with 10 equivalents of trimethyloxosulfonium iodide in presence of tBuOK for 4 days to provide oxetane derivative 16 as a single isomer. Final deprotection under acidic conditions aﬀorded the desired 4′-oxetane analog 17 in 48% yield over 3 steps (Scheme 3). Stereochemistry of the oxetane 3 of 13 Molecules 2020, 25, 1258 ring in compound 17 could not be assessed with certitude by NMR analysis, therefore, crystals were grown from methanol by slow evaporation. Results from X-ray structure determination of 17 led us to ascertain the S-conﬁguration of the 5′-carbon (Figure 2). Synthesis of 4′-isoxazole analog 20 was achieved from intermediate 9 by ﬁrst oxidation to the 5-aldehyde intermediate followed by a Van Leusen cyclization reaction using tosylmethyl isocyanide (TosMIC) in the presence of K2CO3 [14] and ﬁnal deprotection with acetic acid. Molecules 2020, 25, x FOR PEER REVIEW 5 of 13 Figure 2. The ORTEP drawing of nucleoside 17 from X-ray crystal analysis. Figure 2. The ORTEP drawing of nucleoside 17 from X-ray crystal analysis. Figure 1. A selection of 4′-substituted nucleoside analogs displaying antiviral activity and structures of targeted 4′- and 5′-substituted-2′-deoxy-2′-fluoro cytidine analogs (A). sults Chemistry ules 2020, 25, x FOR PEER REVIEW 3 of zation via the use of a primary halogeno alkylamine. The oxidation of 9 to the correspondin hyde with Dess Martin periodinane followed by reaction with 3-bromopropylamine in th ence of MgSO4 led to the formation of imine intermediate 12 which was subsequently reduce NaBH4. Finally, the newly formed amine displaced the terminal bromine to form the desire dine derivative 13 [12,13]. Treatment of 13 under acidic conditions gave the targeted compoun cheme 3). The 4′-oxetane analog 17 was obtained from 9 by, first, oxidation to the correspondin Figure 2. The ORTEP drawing of nucleoside 17 from X-ray crystal analysis. Figure 2. The ORTEP drawing of nucleoside 17 from X-ray crystal analysis. ative 13 [12,13]. Treatment of 13 under acidic conditions gave the targe The 4′-oxetane analog 17 was obtained from 9 by first oxidation to the Targeted 5′-methyl derivatives 25 and 26 were prepared by following the chemistry described in Scheme 4. 2 1 Chemistry 2.1. Chemistry Protected intermediate 8 was oxidized under Pfitzner–Moffatt conditions and then reacted with MeMgCl to give the desired methylated intermediate as a 1/1 mixture. This compound was then oxidized to the corresponding ketone 21 under Pfitzner–Moffatt conditions and the tert butyldimethylsilyl (TBS) group was removed using tetra-n-butylammonium fluoride (TBAF). 22 wa then reacted with Tf2O in pyridine to form a triflate intermediate which was directly treated with LiCl or LiBr in DMF to give the corresponding halogeno derivatives 23 and 24, respectively. Finally reduction with NaBH4 and removal of the monomethoxytrityl groups under acidic condition afforded the desired compounds 25 and 26 as 1/1 mixtures of isomers at the 5′- position. Scheme 1. Synthesis of key intermediates 8 and 9 from commercially available 2′-deoxy-2′-α- Scheme 1. Synthesis of key intermediates 8 and 9 from commercially available 2′-deoxy-2′-α- aldehyde followed by a Johnson-Corey-Chaykovsky epoxidation and consecutive ring-expansion Thus, compound 9 was oxidized by treatment with Dess-Martin periodinane to the corresponding aldehyde which was treated with 10 equivalents of trimethyloxosulfonium iodide in presence o BuOK for 4 days to provide oxetane derivative 16 as a single isomer. Final deprotection under acidic conditions afforded the desired 4′-oxetane analog 17 in 48% yield over 3 steps (Scheme 3) Stereochemistry of the oxetane ring in compound 17 could not be assessed with certitude by NMR analysis, therefore, crystals were grown from methanol by slow evaporation. Results from X-ray tructure determination of 17 led us to ascertain the S-configuration of the 5′-carbon (Figure 2) Synthesis of 4′-isoxazole analog 20 was achieved from intermediate 9 by first oxidation to the 5 aldehyde intermediate followed by a Van Leusen cyclization reaction using tosylmethyl isocyanide mo /1 m ved cidi ion. dati mon 1/1 m ved p / p Scheme 1. Synthesis of key intermediates 8 and 9 from commercially available 2′-deoxy-2′-α- fluorocytidine 7. Scheme 1. Synthesis of key intermediates 8 and 9 from commercially available 2′-deoxy-2′-α- ﬂuorocytidine 7. hyde intermediate followed by a Van Leusen cyclization reaction using tosylmethyl isocyan MIC) in the presence of K2CO3 [14] and final deprotection with acetic acid. p / p Scheme 1. Synthesis of key intermediates 8 and 9 from commercially available 2′-deoxy-2′-α- fluorocytidine 7. Scheme 1. Synthesis of key intermediates 8 and 9 from commercially available 2′-deoxy-2′-α- ﬂuorocytidine 7. 2 1 Chemistry 2.1. Chemistry 4 of 13 f Molecules 2020, 25, 1258 Molecules 2020, 25, 1258 4 of 1 Molecules 2020, 25, x FOR PEER REVIEW 4 of 13 Molecules 2020, 25, x FOR PEER REVIEW 4 of 13 Scheme 3. Synthesis of compounds 14, 17 and 20. Reagents and conditions: a) i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; ii) 3-bromopropylamine, MgSO4, DCM, rt, 6 h, Quant. b) NaBH4, 40 °C, 2 h, 72% over 3 steps; c) i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; ii) trimethyloxosulfonium iodide, KOtBu, tBuOH, 65% over 2 steps. d) i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; ii) tosylmethyl isocyanide, K2CO3, MeOH, reflux, 2 h, 74% over 2 steps. e) 80% aq. AcOH, rt, 16 h, 14 (68%), 17 (73%) and 20 (76%). Scheme 3. Synthesis of compounds 14, 17 and 20. Reagents and conditions: (a) (i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; (ii) 3-bromopropylamine, MgSO4, DCM, rt, 6 h, Quant. (b) NaBH4, 40 ◦C, 2 h, 72% over 3 steps; (c) (i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; (ii) trimethyloxosulfonium iodide, KOtBu, tBuOH, 65% over 2 steps. (d) (i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; (ii) tosylmethyl isocyanide, K2CO3, MeOH, reﬂux, 2 h, 74% over 2 steps. (e) 80% aq. AcOH, rt, 16 h, 14 (68%), 17 (73%) and 20 (76%). Scheme 3. Synthesis of compounds 14, 17 and 20. Reagents and conditions: a) i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; ii) 3-bromopropylamine, MgSO4, DCM, rt, 6 h, Quant. b) NaBH4, 40 °C, 2 h, 72% over 3 steps; c) i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; ii) trimethyloxosulfonium iodide, KOtBu, tBuOH, 65% over 2 steps. d) i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; ii) tosylmethyl isocyanide, K2CO3, MeOH, reflux, 2 h, 74% over 2 steps. e) 80% aq. AcOH, rt, 16 h, 14 (68%), 17 (73%) and 20 (76%). Scheme 3. Synthesis of compounds 14, 17 and 20. Reagents and conditions: (a) (i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; (ii) 3-bromopropylamine, MgSO4, DCM, rt, 6 h, Quant. (b) NaBH4, 40 ◦C, 2 h, 72% over 3 steps; (c) (i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; (ii) trimethyloxosulfonium iodide, KOtBu, tBuOH, 65% over 2 steps. (d) (i) Dess-Martin periodinane, pyridine, DCM, 3 h, rt; (ii) tosylmethyl isocyanide, K2CO3, MeOH, reﬂux, 2 h, 74% over 2 steps. (e) 80% aq. 2 1 Chemistry 2.1. Chemistry hyde intermediate followed by a Van Leusen cyclization reaction using tosylmethyl isocyani MIC) in the presence of K2CO3 [14] and final deprotection with acetic acid. 2′-deoxy-2′-α-fluoro nucleoside deri diates 8 and/or 9 obtained from co he chemistry described by Wang et 1 was achieved by the reaction of 9 action of CuI with FSO2CF2CO2H [1 tic acid (Scheme 2). We first thoug tivated 5′-methyltriflate intermedia dine). However, under these cond d W h h i d h h Targeted 4′-substituted-2′-deoxy-2′-α-fluoro nucleoside derivatives 11, 14, 17, 20, 25, and 26 were prepared from key intermediates 8 and/or 9 obtained from commercially available 2′-deoxy-2′-α- fluorocytidine 7 following the chemistry described by Wang et al. [10] (Scheme 1). The synthesis of 4′-difluoromethoxy analog 11 was achieved by the reaction of 9 with a reactive Cu-difluorocarbene complex obtained by the reaction of CuI with FSO2CF2CO2H [11], followed by removal of the trityl groups in 80% aqueous acetic acid (Scheme 2). We first thought to prepare the desired azetidine analog 14 by reacting an activated 5′-methyltriflate intermediate with azetidine in presence of an organic base (Et3N or pyridine). However, under these conditions, we were unable to observe formation of the desired compound. We hypothesized that the relatively bulky azetidine ring could not reach the sterically hindered 5′- position due to the presence of the nearby large 5′- and 3′- monomethoxytrityl groups Therefore we subsequently evaluated an intramolecular reductive Scheme 2. Synthesis of compound 11. Reagents and conditions: a) CuI, FSO2CF2CO2H, CH3CN, 60 °C, 2 h, 22%. b) 80% aq AcOH, rt, 16 h, 68%. Scheme 2. Synthesis of compound 11. Reagents and conditions: (a) CuI, FSO2CF2CO2H, CH3CN, 60 ◦C, 2 h, 22%. (b) 80% aq AcOH, rt, 16 h, 68%. y (Et under ble to mation of the desired compound. We hypothesized that the relatively bulky azetidine ring cou reach the sterically hindered 5′- position due to the presence of the nearby large 5′- and nomethoxytrityl groups Therefore we subsequently evaluated an intramolecular reducti Scheme 2. Synthesis of compound 11. Reagents and conditions: a) CuI, FSO2CF2CO2H, CH3CN, 60 °C, 2 h, 22%. b) 80% aq AcOH, rt, 16 h, 68%. Scheme 2. Synthesis of compound 11. Reagents and conditions: (a) CuI, FSO2CF2CO2H, CH3CN, 60 ◦C, 2 h, 22%. (b) 80% aq AcOH, rt, 16 h, 68%. 2 1 Chemistry 2.1. Chemistry AcOH, rt, 16 h, 14 (68%), 17 (73%) and 20 (76%). Targeted 5′-methyl derivatives 25 and 26 were prepared by following the chemistry described in Scheme 4. Protected intermediate 8 was oxidized under Pﬁtzner–Moﬀatt conditions and then reacted with MeMgCl to give the desired methylated intermediate as a 1/1 mixture. This compound was then oxidized to the corresponding ketone 21 under Pﬁtzner–Moﬀatt conditions and the tert-butyldimethylsilyl (TBS) group was removed using tetra-n-butylammonium ﬂuoride (TBAF). 22 was then reacted with Tf2O in pyridine to form a triﬂate intermediate which was directly treated with LiCl or LiBr in DMF to give the corresponding halogeno derivatives 23 and 24, respectively. Finally, 5 of 13 22 was d with 5 of 13 22 was d with Molecules 2020, 25, 1258 butyldimethylsilyl ( th t d ith T reduction with NaBH4 and removal of the monomethoxytrityl groups under acidic conditions aﬀorded the desired compounds 25 and 26 as 1/1 mixtures of isomers at the 5′-position. LiCl or LiBr in DMF to give the corresponding halogeno derivatives 23 and 24, respectively. Finally, reduction with NaBH4 and removal of the monomethoxytrityl groups under acidic conditions afforded the desired compounds 25 and 26 as 1/1 mixtures of isomers at the 5′- position. Scheme 4. Synthesis of compounds 25 and 26. Reagents and conditions: a) i) DCC, pyridine DMSO, TFA, rt, overnight; ii) MeMgCl, THF, 86% over 2 steps. b) DCC, pyridine DMSO, TFA, 87%. c) TBAF, 95%; d) i) Tf2O, pyridine; ii) LiX, DMF. e) (i) NaBH4, MeOH; X = Cl 89%, X = Br 93%; f) 80% aq. AcOH, 60 °C, 16 h, X = Cl 81%, X = Br 48%. Scheme 4. Synthesis of compounds 25 and 26. Reagents and conditions: (a) (i) DCC, pyridine DMSO, TFA, rt, overnight; (ii) MeMgCl, THF, 86% over 2 steps. (b) DCC, pyridine DMSO, TFA, 87%. (c) TBAF, 95%; (d) (i) Tf2O, pyridine; (ii) LiX, DMF. (e) (i) NaBH4, MeOH; X = Cl 89%, X = Br 93%; (f) 80% aq. AcOH, 60 ◦C, 16 h, X = Cl 81%, X = Br 48%. Scheme 4. Synthesis of compounds 25 and 26. Reagents and conditions: a) i) DCC, pyridine DMSO, TFA, rt, overnight; ii) MeMgCl, THF, 86% over 2 steps. b) DCC, pyridine DMSO, TFA, 87%. c) TBAF, 95%; d) i) Tf2O, pyridine; ii) LiX, DMF. 2 2 Antiviral and Toxicity Evaluation 2.2. Antiviral and Toxicity Evaluation y Based on their structural similarity with ALS-8112, compounds 11, 14, 17, 20, 25 and 26 were tested against RSV replicon-containing adenocarcinomic human alveolar basal epithelial A549 cells (kindly provided by Apath, L.L.C, New York, NY, USA), but unfortunately none of them display antiviral activity in this system when evaluated up to 10 µM. It is worth noting that they did not show Based on their structural similarity with ALS-8112, compounds 11, 14, 17, 20, 25 and 26 were tested against RSV replicon-containing adenocarcinomic human alveolar basal epithelial A549 cells (kindly provided by Apath, L.L.C, New York, NY, USA), but unfortunately none of them display antiviral activity in this system when evaluated up to 10 µM. It is worth noting that they did not show toxicity either, up to 100 µM, in a panel of cell lines, including primary human peripheral blood mononuclear (PBM) cells, human lymphoblastoid cells (CEM), African Green monkey (Vero) cells and human liver hepatocarcinoma (HepG2) cells. The excellent safety proﬁle of these compounds led us to further evaluate them against a panel of RNA viruses (Dengue (DENV), West Nile (WNV), Chikungunya (CHIKV), and Zika viruses (ZIKV)) but, once again, none of them displayed antiviral activity when tested up to 20 µM for ZIKV or 30 µM for DENV, WNV, or CHIKV. 2 1 Chemistry 2.1. Chemistry e) (i) NaBH4, MeOH; X = Cl 89%, X = Br 93%; f) 80% aq. AcOH, 60 °C, 16 h, X = Cl 81%, X = Br 48%. Scheme 4. Synthesis of compounds 25 and 26. Reagents and conditions: (a) (i) DCC, pyridine DMSO, TFA, rt, overnight; (ii) MeMgCl, THF, 86% over 2 steps. (b) DCC, pyridine DMSO, TFA, 87%. (c) TBAF, 95%; (d) (i) Tf2O, pyridine; (ii) LiX, DMF. (e) (i) NaBH4, MeOH; X = Cl 89%, X = Br 93%; (f) 80% aq. AcOH, 60 ◦C, 16 h, X = Cl 81%, X = Br 48%. 3.1. Synthesis 1H NMR (400 MHz, CDCl3): δ 7.5–6.64 (m, 38H), 6.22 (t, J = 74.8, Hz, 1H), 6.14 (d, J = 7.6 Hz, 1H), 6.09 (d, J = 19.7 Hz, 1H), 4.54 (dd, J = 24.5 Hz, J = 5.0 Hz, 1H), 4.32 (q, J = 12.0 Hz, 2H), 4.24 (d, J = 7.6 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.73 (s, 3H), 3.61 (dd, J = 70.6 Hz, J = 10.0 Hz, 1H), 3.22 (dd, J = 52.0 Hz, J = 5.0 Hz, 1H). 19F NMR (376 MHz, CDCl3): δ –84.11 (d, J = 74.8 Hz), −185.8 (m). 13C NMR (101 MHz, CDCl3): δ 165.3, 159.1, 158.7, 158.6, 154.4, 144.3, 144.0, 143.8, 143.67, 143.6, 143.3, 141.3, 135.8, 134.5, 134.3, 131.0, 130.8, 130.7, 129.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.6, 127.5, 127.4, 127.1, 127.0, 118.7, 116.1, 113.5, 113.4, 113.3, 113.2, 112.9, 94.8, 94.0, 89.4, 89.1, 88.2, 88.1, 87.4, 86.7, 72.2, 72.0, 703, 64.3, 62.3, 55.2. HRMS for C71H62F3N3O8 (M + H]. Calcd: m/z 1142.4489, found: m/z 1142.4549. 4-Amino-1-((2R,3R,4R,5S)-5-((difluoromethoxy)methyl)-3-fluoro-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran- 2-yl)pyrimidin-2(1H)-one 11: Compound 10 (150 mg, 0.13 mmol) was treated with 3 mL of 80% acetic acid in water (v/v) at 50–60 ◦C for 12 h. Volatiles were evaporated under reduced pressure and the residue co-evaporated with toluene (3 × 5 mL). The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 90/10) to give compound 11 (29 mg, 68%). 1H NMR (400 MHz, MeOD-d4): δ 8.09 (d, J = 7.6 Hz, 1H), 6.43 (t, J = 75.6, Hz, 1H), 6.17 (dd, J = 15.4 Hz, J = 3.6 Hz, 1H), 5.93 (d, J = 7.5 Hz, 1H), 5.17 (dq, J = 53.5 Hz, J = 3.6 Hz, 1H), 4.56 (dd, J = 15.5 Hz, J = 5 Hz, 1H), 4.09 (q, J = 12.0 Hz, 2H), 4.09 (dd, J = 24.0 Hz, J = 11.2 Hz, 2H). 19F NMR (376 MHz, MeOD-d4): δ −85.6 (d, J = 73.0 Hz), −186.68 (m). 13C NMR (101 MHz, MeOD-d4): δ165.2, 156.8, 142.2, 120.2, 117.7, 115.2, 94.9, 94.5, 92.7, 87.4, 87.1, 86.5, 69.3, 69.2, 65.2, 61.5. HRMS for C11H14F3N3O5 (M + H]. Calcd: m/z 326.0885, found: m/z 326.0950. 1-((2R,3R,4R,5R)-5-(Azetidin-1-ylmethyl)-3-fluoro-4-((4-methoxyphenyl)diphenylmethoxy)-5-(((4-methoxyphenyl) diphenylmethoxy)methyl)tetrahydrofuran-2-yl)-4-(((4-methoxyphenyl) diphenylmethyl)amino)pyrimidin-2(1H)-one 13: To a solution of 9 (200 mg, 0.18 mmol) in dichloromethane (7 mL) was added pyridine (0.14 mL, 1.83 mmol) and Dess-Martin periodinane (173 mg, 0.41 mmol) at 0 ◦C. 3.1. Synthesis Anhydrous solvents were purchased from Aldrich Chemical Company, Inc. (Milwaukee, Wisconsin, USA). Reagents were purchased from commercial sources. Unless noted otherwise, the materials used in the examples were obtained from readily available commercial suppliers or synthesized by standard methods known to one skilled in the art of chemical synthesis. 1H, 13C, and 19F spectra were taken on a Bruker AscendTM 400 spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA) at rt and reported in ppm downﬁeld from internal tetramethylsilane (for 1H-NMR). NMR processing was performed with MestReNova version 10.0.2-15465. Deuterium exchange and decoupling experiments were utilized to conﬁrm proton assignments. Signal multiplicities are represented by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet), br (broad), bs (broad singlet), m (multiplet). All J-values are in Hz and calculated by Mnova or MestReNova 6 of 13 Molecules 2020, 25, 1258 programs (V 14.1.1). Mass spectra were determined on a Waters Acquity UPLC using electrospray ionization (Waters Corporation, Milford, MA, USA). Analytic TLC was performed on Analtech GHLF silica gel plates (Analtech, Newark, DE, USA), and preparative TLC on Analtech GF silica gel plates (Analtech, Newark, DE, USA). Column chromatography was performed on Combiﬂash Rf200 or via reverse-phase high performance liquid chromatography. 1H, 13C and 19F-NMR spectra for compounds 11, 14, 17, 20, 25 and 26 are available online in Supplementary Materials at (Figures S1–S18). 1-((2R,3R,4R,5S)-5-((Diﬂuoromethoxy)methyl)-3-ﬂuoro-4-((4-methoxyphenyl)diphenylmethoxy)-5-(((4- methoxyphenyl)diphenylmethoxy)methyl)tetrahydrofuran-2-yl)-4-(((4-methoxyphenyl) diphenylmethyl)amino) pyrimidin-2(1H)-one 10: To a solution of 9 (650 mg, 0.59 mmol) in acetonitrile (10 mL) was added CuI (22.2 mg, 0.12 mol). The resulting reaction mixture was heated to 60 ◦C and a solution of 2,2-diﬂuoro-2-(ﬂuorosulfonyl) acetic acid (89.7 µL, 0.89 mmol) in acetonitrile (2 mL) was added slowly over 10 min. After 2 h at 60 ◦C, the reaction was cooled down to room temperature, quenched with a saturated solution of NaHCO3 (50 mL) and stirred for 30 min at this temperature. The solution was then extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with brine (20 mL), dried over Na2SO4, ﬁltered and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give compound 10 (150 mg, 22%). 3.1. Synthesis The reaction mixture was then stirred for 2 h at 40 C before being quenched at room temperature with a saturated solution of ammonium chloride (7 mL). The mixture was then diluted with ethyl acetate (35 mL). The organic layer was separated, washed with brine (7 mL), dried over Na2SO4, ﬁltered and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give compound 13 (149 mg, 86 %). 1H NMR (400 MHz, CDCl3): δ 7.44–6.60 (m, 38H), 6.20 (d, J = 18.0 Hz, 1H), 4.45 (dd, J = 27.0 Hz, J = 3.8 Hz, 1H), 4.08 (d, J = 18.0 Hz, 1H), 3.9 (d, J = 10.0 Hz, 1H), 3.77 (s, 6H), 3.73 (s, 3H), 3.59 (d, J = 10.0 Hz, 1H), 3.12–2.96 (m, 6H), 2.86 (d, J = 13.2 Hz, 1H), 1.91 (m, 2H). 19F NMR (376 MHz, CDCl3): δ −185.36 (m). 13C NMR (101 MHz, CDCl3): δ 165.1, 159.1,158.6, 158.5, 154.7, 144.4, 144.3, 144.0, 143.9, 143.7, 141.1,135.7, 134.8, 134.3, 131.0, 130.9, 129.9, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 126.9, 126.8, 113.3, 113.1, 112.8, 94.7, 94.6, 92.7, 88.7, 87.8, 87.5, 71.5, 71.4, 70.1, 62.9, 60.6, 55.2, 18.8. HRMS for C73H67FN4O7 (M + H]. Calcd: m/z 1131.4994, found: m/z 1131.5055. 4-Amino-1-((2R,3R,4R,5R)-5-(azetidin-1-ylmethyl)-3-fluoro-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl) pyrimidin-2(1H)-one 14: Compound 13 (230 mg, 0.2 mmol) was treated with 4 mL of 80% acetic acid in water (v/v) at 50–60 ◦C for 12 h. Volatiles were evaporated under reduced pressure and co-evaporated with toluene (3 × 5 mL). The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 90/10) to aﬀord 14 (43 mg, 68%). 1H NMR (400 MHz, MeOD-d4): δ 8.02 (d, J = 7.5 Hz, 1H), 6.18 (dd, J = 15.2 Hz, J = 3.6 Hz, 1H), 5.9 (d, J = 7.5 Hz, 1H), 5.1 (dq, J = 53.6 Hz, J = 3.7 Hz, 1H), 4.45 (dd, J = 15.7 Hz, J = 5.2 Hz, 1H), 3.71 (dd, J = 81.0 Hz, J = 11.8 Hz, 2H), 3.38 (m, 4H), 2.88 (dd, J = 14.5 Hz, J = 13.6 Hz, 2H), 2.14 (m, 2H). 19F NMR (376 MHz, MeOD-d4): δ −204.24 (dt, J = 53.6 Hz, J = 15.4 Hz). 13C NMR (101 MHz, MeOD-d4): δ166.4, 156.8, 141.8, 94.8, 94.7, 92.9, 88.5, 88.1, 86.8, 71.0, 70.9, 63.7, 60.7, 60.6, 56.5, 17.8. 3.1. Synthesis The resulting reaction mixture was stirred at room temperature for 3 h, then quenched with 10 mL of Na2S2O3 and Na2CO3 (1:1 mixture). The solution was extracted with dichloromethane (3 × 20 mL) and the combined organic layers were washed with brine (20 mL), dried over Na2SO4, ﬁltered and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give the desired aldehyde (168 mg, 84%). To a solution of the freshly prepared aldehyde (168 mg, 0.15 mmol) in dichloromethane (5 mL) was added MgSO4 (170 mg). After 5 min, 3-bromopropylamine hydrogen chloride (37 mg, 0.17 mmol) and pyridine (19 µL, 0.23 mmol) were added and the resulting reaction mixture was stirred at room temperature for 16 h. After completion, the reaction was ﬁltered through celite and concentrated under reduced pressure. The resulting crude 7 of 13 Molecules 2020, 25, 1258 product was dissolved in methanol (5 mL) before addition of sodium borohydride (6 mg, 0.15 mmol). The reaction mixture was then stirred for 2 h at 40 ◦C before being quenched at room temperature with a saturated solution of ammonium chloride (7 mL). The mixture was then diluted with ethyl acetate (35 mL). The organic layer was separated, washed with brine (7 mL), dried over Na2SO4, ﬁltered and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give compound 13 (149 mg, 86 %). 1H NMR (400 MHz, CDCl3): δ 7.44–6.60 (m, 38H), 6.20 (d, J = 18.0 Hz, 1H), 4.45 (dd, J = 27.0 Hz, J = 3.8 Hz, 1H), 4.08 (d, J = 18.0 Hz, 1H), 3.9 (d, J = 10.0 Hz, 1H), 3.77 (s, 6H), 3.73 (s, 3H), 3.59 (d, J = 10.0 Hz, 1H), 3.12–2.96 (m, 6H), 2.86 (d, J = 13.2 Hz, 1H), 1.91 (m, 2H). 19F NMR (376 MHz, CDCl3): δ −185.36 (m). 13C NMR (101 MHz, CDCl3): δ 165.1, 159.1,158.6, 158.5, 154.7, 144.4, 144.3, 144.0, 143.9, 143.7, 141.1,135.7, 134.8, 134.3, 131.0, 130.9, 129.9, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 126.9, 126.8, 113.3, 113.1, 112.8, 94.7, 94.6, 92.7, 88.7, 87.8, 87.5, 71.5, 71.4, 70.1, 62.9, 60.6, 55.2, 18.8. HRMS for C73H67FN4O7 (M + H]. Calcd: m/z 1131.4994, found: m/z 1131.5055. 3.1. Synthesis Compound 16 (610 mg, 0.546 mmol) was treated with 10 mL of 80% acetic acid in water (v/v) at 50–60 ◦C for 12 h. The volatiles were then evaporated under reduced pressure and the residue was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 85/15) to give compound 17 (119 mg, 73%). 1H NMR (400 MHz, DMSO-d6): δ 7.89 (d, J = 7.4 Hz, 1H), 7.32 (NH2, 2H), 6.35 (dd, J = 11.4, 7.0 Hz, 1H), 5.80 (d, J = 7.4 Hz, 1H), 5.56 (br s, 1H), 5.25 (br s, 1H), 5.12 (ddd, J = 53.2, 6.8, 1.6 Hz, 1H), 4.96 (t, J = 7.4 Hz, 1H), 3.54 (m, 1H), 3.45 (m, 1H), 2.74 (m, 1H), 2.54 (m, 1H). 19F NMR (376 MHz, DMSO-d6): δ −210.35, (ddd, J = 53.2, 12, 3.6 Hz). 13C NMR (101 MHz, DMSO-d6): δ 166.1, 155.8, 141.8, 95.4, 93.1, 91.2, 89.29, 89.26, 86.3, 86.0, 82.6, 70.6, 70.4, 69.0, 61.8, 24.9. HRMS for C12H17FN3O5 (M + H). Calcd: m/z 302.1152, found: m/z 302.1142. 1-((2R,3R,4R,5R)-3-Fluoro-4-((4-methoxyphenyl)diphenylmethoxy)-5-(((4-methoxyphenyl) diphenylmethoxy) methyl)-5-(oxazol-5-yl)tetrahydrofuran-2-yl)-4-(((4-methoxyphenyl) diphenylmethyl)amino)pyrimidin-2(1H)-one 19: To a solution of 9 (200 mg, 0.18 mmol) in dichloromethane (7 mL) was added pyridine (0.14 mL, 1.83 mmol) and Dess-Martin periodinane (173 mg, 0.41 mmol) at 0 ◦C. The resulting reaction mixture was stirred at room temperature for 3 h, then quenched with 10 mL of Na2S2O3 and Na2CO3 (1:1 mixture). The solution was extracted with dichloromethane (3 × 20 mL) and the combined organic layers were washed with brine (20 mL), dried over Na2SO4, ﬁltered and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give the desired aldehyde intermediate (168 mg, 84%). To a solution of this aldehyde (168 mg, 0.15 mmol) in methanol (2 mL) was added sequentially p-toluenesulfonylmethyl isocyanide (TosMIC) (30 mg, 0.15 mmol) and K2CO3 (63 mg, 0.45 mmol). After 2 h at 65 ◦C, the volatiles were evaporated under reduced pressure, water (5 mL) was added and the solution stirred for 5 min. The organic content was extracted with ethyl acetate (3 × 5 mL), combined organic layer were washed with brine (5 mL), dried over Na2SO4, ﬁltered and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give 19 (147 mg, 88%). 3.1. Synthesis 1H NMR (400 MHz, CDCl3): δ 7.93 (s, 1H), 7.73–6.9 (m, 39H), 6.77 (d, J = 12 Hz, 2H), 6.70 (d, J = 12 Hz, 4H), 6.05 (d, J = 20.4 Hz, 1H), 4.66 (dd, J = 21.6 Hz, J = 4.4 Hz, 1H), 4.47 (d, J = 7.6 Hz, 1H), 3.77 (s, 3H), 3.76 (s, 3H), 3.74 (s, 3H), 3.68 (q, J = 10.0 Hz, 2H), 3.41 (dd, J = 51.6 Hz, J = 24.8 Hz, 1H). 19F NMR (376 MHz, CDCl3): δ −185.53 (m). 13C NMR (101 MHz, CDCl3): δ 165.5, 159.0, 158.7, 158.6, 154.3, 150.7, 150.2, 144.2, 144.1, 143.9, 143.8, 143.7, 143.2, 141.9, 135.7, 134.6, 134.4, 130.9, 130.6, 129.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.6, 127.5, 127.3, 127.1, 127.0, 125.3, 113.5, 113.1, 112.9, 94.9, 93.4, 91.7, 91.5, 91.4, 88.1, 87.1, 85.5, 73.6, 73.5, 70.4, 64.6, 55.2, 55.1. HRMS for C72H61FN4O8 (M + H]. Calcd: m/z 1129.45, found: m/z 1129.4543. 4-Amino-1-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-5-(oxazol-5-yl)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 20: Compound 19 (400 mg, 0.35 mmol) was treated with 8 mL of 80% acetic acid in water (v/v) at 50–60 ◦C for 12 h. The volatiles were then evaporated under reduced pressure and the residue co-evaporated with toluene (3 × 10 mL). The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 90/10) to give compound 20 (83.6 mg, 76%). 1H NMR (400 MHz, MeOD-d4): δ 8.21 (s, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.22 (s, 1H), 6.23 (dd, J = 18.0 Hz, J = 2.0 Hz, 1H), 5.93 (d, J = 7.5 Hz, 1H), 5.17 (dq, J = 53.5 Hz, J = 2.0 Hz, 1H), 4.79 (dd, J = 20.0 Hz, J = 5 Hz, 1H), 3.98 (q, J = 12.0 Hz, 2H). 19F NMR (376 MHz, MeOD-d4): δ −198.45 (dt, J = 53.0 Hz, J = 19.6 Hz). 13C NMR (101 MHz, MeOD-d4): δ 166.5, 156.5, 151.8, 150.0, 142.3, 124.1, 94.9, 94.6, 92.7, 90.8, 90.5, 85.9, 70.4, 70.3, 63. 3. HRMS for C12H13FN4O5 (M + H]. Calcd: m/z 313.09, found: m/z 313.0935. 1-((2R,3R,4R,5S)-5-Acetyl-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoro-4-((4-methoxyphenyl) diphenylmethoxy) tetrahydrofuran-2-yl)-4-(((4-methoxyphenyl)diphenylmethyl)amino) pyrimidin-2(1H)-one 21: To a solution of pyridine (0.21 mL, 2.51 mmol) in DMSO (4 mL) was added TFA (0.16 mL, 2.12 mmol) at 0 ◦C. The mixture was then stirred at room temperature for 10 min before being added dropwise to a solution of 8 (1.8 g, 1.93 mmol) and DCC (1.473 g, 7.14 mmol) in DMSO (10 mL). 3.1. Synthesis HRMS for C13H19FN4O4 (M + H]. Calcd: m/z 315.1390, found: m/z 315.1456. (2R,3R,4R,5S)-3-Fluoro-4-((4-methoxyphenyl)diphenylmethoxy)-5-(((4-methoxyphenyl) diphenylmethoxy)methyl) -5-((S)-oxetan-2-yl)tetrahydrofuran-2-yl)-4-(((4-methoxyphenyl) diphenylmethyl)amino)pyrimidin-2(1H)-one 16: To a solution of 9 (200 mg, 0.18 mmol) in dichloromethane (7 mL) was added pyridine (0.14 mL, 1.83 mmol) and Dess-Martin periodinane (173 mg, 0.41 mmol) at 0 ◦C. The resulting reaction mixture was stirred at room temperature for 3 h, then quenched with 10 mL of Na2S2O3 and Na2CO3 (1:1 mixture). The solution was extracted with dichloromethane (3 × 20 mL) and the combined organic layers were washed with brine (20 mL), dried over Na2SO4, ﬁltered and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give the desired aldehyde intermediate (168 mg, 84%). A solution of trimethyl oxosulfonium iodide (0.425 g, 1.91 mmol) and potassium tert-butoxide (0.43 g, 3.83 mmol) in 3 mL of tert-butanol was stirred at 30 ◦C for 30 min before addition of the freshly prepare aldehyde (168 mg, 0.19 mmol) in tert-butanol (3 mL). The resulting mixture was stirred at 50 ◦C for 4 days. After being cooled down to room temperature, the mixture was poured into a saturated solution of ammonium chloride (10 mL) and then extracted with dichloromethane (2 × 10 mL). The combined organic layers were dried over sodium sulfate, ﬁltered, and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 97/3) to give 16 (133 mg, 78%). 1H NMR (400 MHz, CDCl3): δ 6.4–7.5 (m, 44H), 5.4 (t, J = 7.6 Hz, 1H), 4.62 (m, 2H), 4.40 (m, 1H), 3.95 (m, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 3.74 (s, 3H), 3.63 (d, J = 10.6 Hz, 1H), 2.84 (m, 1H), 2.68 (m, 1H). 19F NMR (376 MHz, CDCl3): δ −186.83 (dt, J = 53.4, 25.6 Hz). 13C NMR(101 MHz, CDCl3): δ 165.0, 159.1, 158.7, 158.5, 154.7, 144.3, 144.2, 143.8, 143.6, 143.3, 143.2, 141.1, 135.6, 134.7, 134.2, 130.9, 130.8, 129.9, 129.2, 129.0, 128.8, 128.7, 128.4, 128.3, 128.2, 128.2, 127.9, 127.9, 127.7, 127.6, 127.4, 127.3, 127.2, 127.15, 113.4, 113.2, 112.7, 95.0, 94.2, 92.3, 89.6, 88.0, 87.8, 87.6, 87.4, 82.7, 77.2, 71.3, 71.2, 70.1, 69.1, 62.0, 55.2, 42.7, 42.7, 24.5. HRMS for C72H65FN3O8 (M + H). Calcd: m/z 1118.4756, found: m/z 1118.4758. 8 of 13 Molecules 2020, 25, 1258 4-Amino-1-((2R,3R,4R,5R)-3-ﬂuoro-4-hydroxy-5-(hydroxymethyl)-5-((S)-oxetan-2-yl) tetrahydrofuran-2-yl) pyrimidin-2(1H)-one 17. 3.1. Synthesis The reaction mixture 9 of 13 Molecules 2020, 25, 1258 was stirred overnight, quenched by adding water (20 mL) and ethyl acetate (20 mL). The precipitate was removed by ﬁltration and washed with ethyl acetate (30 mL). The ﬁltrate was extracted with dichloromethane (3 × 100 mL) and the combined organic layers were washed with a saturated solution of NaHCO3 (50 mL), dried with Na2SO4. ﬁltered and concentrated under reduced pressure. The resulting residue was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give the desired crude aldehyde. To a solution of this aldehyde (1.565 g, 1.68 mmol) in THF (10 mL) at −78 ◦C was slowly added MeMgCl (3.0 M solution in THF, 5.6 mL, 16.8 mmol). The mixture was stirred for 30 min at room temperature and then quenched at −78 ◦C with methanol (5 mL). The volatiles were then evaporated under reduced pressure and the residue was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 98/2) to give the desired hydroxy intermediate as a 1:1 mixture of isomers (1.44 g, 91%). To a solution of pyridine (0.282 mL, 3.5 mmol) in DMSO (3 mL) was added TFA (0.227 mL, 2.96 mmol) at 0 ◦C. The mixture was then stirred at room temperature for 10 min before being added dropwise to a solution of the freshly prepared hydroxy intermediate (1.44 g, 1.52 mmol) and DCC (3.12 g, 5.62 mmol) in DMSO (10 mL). The reaction mixture was stirred overnight and quenched by adding water (15 mL) and ethyl acetate (15 mL). The precipitate was removed by ﬁltration and washed with ethyl acetate (15 mL). The ﬁltrate was extracted with dichloromethane (3 × 50 mL) and the combined organic layers were washed with a saturated solution of NaHCO3 (30 mL), dried with Na2SO4. ﬁltered and concentrated under reduced pressure. The resulting residue was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 95/5) to give 21 (1.25 g, 87%). 1H NMR (400 MHz, CDCl3) δ: 6.6–7.2 (m, 29H), 5.22 (d, J = 21.88 Hz, 1H), 4.87 (d, J = 7.52 Hz, 1H), 4.52 (dd, J = 22.32, 5.0 Hz, 1H), 4.37 (d, J = 11.4 Hz, 1H), 4.09 (d, J = 11.4 Hz, 1H), 4.05 (dd, J = 53.64, 5.0 Hz, 1H), 3.69 (s, 1H), 3.08 (s, 3H), 1.89 (s, 3H), 0.78 (s, 9H), −0.0001 (s, 3H), -0.0222 (s, 3H). 3.1. Synthesis The resulting crude 10 of 13 10 of 13 Molecules 2020, 25, 1258 product was puriﬁed by ﬂash column chromatography (Hexanes/ Ethyl acetate, 100/0 to 50/50) to give compound 23 (113 mg, 87%). 1H NMR (400 MHz, CDCl3): δ 6.8–7.3 (m, 29H), 5.14 (dd, J = 20.8, 5.0 Hz, 1H), 5.07 (d, J = 24.2 Hz, 1H), 5.01 (1H, d, J = 7.5 Hz, 1H), 4.65 (d, J = 12.1 Hz, 1H), 4.27 (dd, J = 53.9, 5.0 Hz, 1H), 3.98 (d, J = 12.1 Hz, 1H), 3.80 (s, 6H), 3.48 (s, 1H), 1.99 (s, 3H), 19F NMR (376 MHz, CDCl3): δ 179.8 (ddd, J = 57.8, 26, 22.9 Hz). 13C NMR (101 MHz CDCl3): δ 205.0, 166.1, 159.0, 158.9, 153.9, 145.2, 143.9, 143.8, 143.4, 135.6, 134.4, 131.3, 129.9, 128.8, 128.51, 128.47, 127.9, 127.8, 127.7, 127.41, 127.35, 113.7, 113.2, 97.1, 96.8, 94.9, 92.5, 91.3, 90.6, 88.6, 74.5, 74.4, 70.7, 55.29, 55.27, 50.9, 43.3, 43.2, 24.9. HRMS for C51H46ClFN3O6 (M + H). Calcd: m/z 850.3059, found: m/z 850.3052, 852.3044. 1-((2R,3R,4R,5S)-5-Acetyl-5-(bromomethyl)-3-ﬂuoro-4-((4-methoxyphenyl)diphenylmethoxy) tetrahydrofuran- 2-yl)-4-(((4-methoxyphenyl)diphenylmethyl)amino)pyrimidin-2(1H)-one 24: Title compound 24 was obtained from 22 using the same procedure as for compound 23 and replacing LiCl by LiBr. Yield: 88%. 1H NMR (400 MHz, CDCl3): δ 6.8–7.3 (m, 29H), 5.15 (dd, J = 20.9, 5.0 Hz, 1H), 5.06 (d, J = 25.5, 1H), 5.01 (d, J = 7.6 Hz, 1H), 4.49 (d, J = 11.3 Hz, 1H), 4.29 (dd, J = 55.6, 11.3 Hz, 1H), 3.85 (d, J = 11.3 Hz, 1H), 3.80 (s, 6H), 1.97 (s, 3H). 19F NMR (376 MHz, CDCl3): δ 205.0 13C NMR (101 MHz CDCl3): δ 205.0, 166.1, 159.0, 158.9, 145.2, 143.9, 143.8, 143.4, 135.5, 134.4, 131.3, 129.9, 129.0, 128.8, 128.5, 128.47, 127.9, 127.8, 127.7, 127.4, 127.35, 113.7, 113.2, 97.0, 96.6, 94.9, 92.5, 90.74, 90.68, 88.6, 74.5, 74.3, 70.7, 55.3, 32.1, 24.8. HRMS for C51H46BrFN3O6 (M + H). Calcd: m/z 894.2554, found: m/z 894.2541, 896.2532. 4-Amino-1-((2R,3R,4R,5R)-5-(chloromethyl)-3-ﬂuoro-4-hydroxy-5-(1-hydroxyethyl)tetrahydrofuran-2-yl) pyrimidin-2(1H)-one 25: To a solution of 23 (150 mg, 0.177 mmol) in methanol (3 mL) was added sodium borohydride (35 mg, 0.9 mmol) portion wise. The mixture was stirred for 30 min at room temperature, quenched with water (20 mL) and extracted with dichloromethane (3 × 20 mL). The combined organic layers were dried over sodium sulfate, ﬁltered and concentrated under reduced pressure. 3.1. Synthesis The resulting crude product was puriﬁed by ﬂash column chromatography (Hexanes/Ethyl acetate, 100/0 to 33/67) to give the desired hydroxy intermediate (134 mg, 89%) as a 1:1 mixture of isomers. A solution of the mixture (134 mg, 0.159 mmol) in 80% acetic acid (10 mL) was heated at 60–65 ◦C overnight. The volatiles were then evaporated under reduced pressure and the residue was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 90/10) to give 25 (42 mg, 88%) as a 1:1 mixture of isomers. 1H NMR (400 MHz, DMSO-d6): δ 7.88 (d, J = 7.4 Hz, 0.5H), 7.87 (d, J = 7.4 Hz, 0.5H), 7.39 (bs, 0.5H), 7.35 (bs, 0.5H), 7.31(bs, 0.5H), 7.29 (bs, 0.5H), 6.19 (dd, J = 13.1, 6.6 Hz, 0.5H), 6.07 (dd, J = 16.2, 4.2 Hz, 0.5H), 5.95 (bs, 0.5H), 5.88 (bs, 0.5H), 5.81 (d, J = 7.4 Hz, 0.5H), 5.76 (d, J = 7.4 Hz, 0.5H), 5.50 (bs, 0.5H), 5.11 (m, 1H), 4.51 (dd, J = 13.8, 5.3 Hz, 0.5H), 4.37 (bs, 0.5H), 4.05 (m, 1H), 3.91 (d, J = 11.0 Hz, 0.5H), 3.85 (d, J = 12.8 Hz, 0.5H), 3.78 (d, J = 12.7 Hz, 0.5H), 1.15 (d, J = 6.6 Hz, 1.5H), 1.08 (d, J = 6.4 Hz, 1.5H). 19F NMR (376 MHz, DMSO-d6): δ −199.2 (dt, J = 57.5, 15.3 Hz), −206.2 (dd, J = 57.5, 13.7 Hz). 13C NMR (101 MHz, DMSO-d6): δ 166.2, 166.1, 155.6, 142.4, 142.0, 95.6, 95.0, 94.7, 93.5, 92.9, 91.6, 88.6, 88.5, 87.7, 87.3, 86.1, 85.8, 70.2, 70.1, 86.4, 68.3, 67.2, 66.6, 47.5, 41.1, 18.5, 17.4. HRMS for C11H16ClFN3O4 (M + H). Calcd: m/z 308.0813, found: m/z 308.0804, 310.0773. 4-Amino-1-((2R,3R,4R,5R)-5-(bromomethyl)-3-fluoro-4-hydroxy-5-(1-hydroxyethyl) tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 26: Title compound 26 was obtained as a 1:1 mixture of isomers from 24 using the same procedure as for compound 25. Yield: 48% over two steps. 3.1. Synthesis 19F NMR (376 MHz, CDCl3): δ −182.4 (dt, J = 53.6, 22.3Hz). 13C NMR (101 MHz CDCl3): δ 207.3, 165.9, 158.9, 158.8, 154.1, 144.2, 144.09, 144.05, 143.99, 143.6, 135.7, 134.8, 131.1, 130.0, 129.1, 128.9, 128.6, 128.4, 127.8, 127.7, 127.6, 127.4, 127.4, 127.3, 113.7, 113.1, 95.8, 95.4, 94.6, 92.7, 92.4, 90.5, 88.4, 73.5, 73.3, 70.6, 63.12, 63.09, 55.3, 49.2, 34.0, 26.2, 26.0, 25.6, 25.0, 18.4, −5.3, −5.4. HRMS for C57H61FN3O7Si (M + H). Calcd: m/z 946.4263, found: m/z 946.4250. 1-((2R,3R,4R,5S)-5-Acetyl-3-fluoro-5-(hydroxymethyl)-4-((4-methoxyphenyl)diphenylmethoxy) tetrahydrofuran- 2-yl)-4-(((4-methoxyphenyl)diphenylmethyl)amino)pyrimidin-2(1H)-one 22: To a solution of 21 (1.0 g, 1.06 mmol) in THF (5 mL) was added TBAF (1M in THF, 2 mL, 2.0 mmol) at 0 ◦C. The mixture was stirred at room temperature for 7 h, water (30 mL) was added and extracted with dichloromethane (3 × 30 mL). The combined organic layers were dried over sodium sulfate, ﬁltered and concentrated under reduced pressure. The resulting crude product was puriﬁed by ﬂash column chromatography (dichloromethane/methanol, 100/0 to 98/2) to give compound 22 (837 mg, 95%). 1H NMR (400 MHz, CDCl3): δ 6.8–7.26 (m, 29H), 5.24 (d, J = 23.8 Hz, 1H), 5.04 (dd, J = 20.0, 5.5 Hz, 1H), 5.0 (d, J = 7.5 Hz, 1H), 4.1-4.4 (m, 3H), 3.79 (s, 6H), 2.03 (s, 3H), 19F NMR (376 MHz, CDCl3): δ −180.5, (dt, J = 56.9, 26.4 Hz), 13C NMR (101 MHz CDCl3): δ 208.5, 166.1, 159.0, 158.9, 154.0, 145.0, 144.00, 143.96, 143.93, 143.50, 135.6, 134.6, 131.1, 130.0, 128.8, 128.7, 128.5, 128.4, 128.0, 127.9, 127.7, 127.5, 127.4, 113.7, 113.2. 97.3, 97.0, 94.8, 92.4, 90.5, 88.5, 73.8, 73.6, 70.7, 62.4, 55.30, 55.28, 25.1. HRMS for C51H47FN3O7 (M + H). Calcd: m/z 832.3398, found: m/z 832.3388. 1-((2R,3R,4R,5S)-5-Acetyl-5-(chloromethyl)-3-ﬂuoro-4-((4-methoxyphenyl)diphenylmethoxy) tetrahydrofuran- 2-yl)-4-(((4-methoxyphenyl)diphenylmethyl)amino)pyrimidin-2(1H)-one 23: 22 (128 mg, 0.154 mmol) was co-evaporated with toluene twice then dissolved in dichloromethane (3 mL). Pyridine (0.14 mL, 1.54 mmol) was added to the solution and the mixture was cooled to −78 ◦C. Triﬂic anhydride (52 µL, 0.3 mmol) was then added and the mixture was stirred at 0 ◦C for 40 min. The volatiles were then evaporated under reduced pressure and the residue was dissolved in DMF (3 mL) before addition of LiCl (33 mg, 0.77 mmol). The mixture was stirred overnight, quenched with a saturated solution of NaHCO3 (20 mL) and extracted with dichloromethane (3 × 20 mL). The combined organic layers were dried over sodium sulfate, ﬁltered and concentrated under reduced pressure. 3.3. Toxicity Assays Cytotoxicity assays. In vitro cytotoxicity was determined using the CellTiter 96 non-radioactive cell proliferation colorimetric assay (MTT assay, Promega, Madison, WI, USA) in primary human peripheral blood mononuclear (PBM) cells, human T lymphoblast (CEM) and human hepatocellular carcinoma (HepG2 or Huh7) cell lines. Toxicity levels were measured as the concentration of test compound that inhibited cell proliferation by 50% (CC50). 3.2.2. Zika Virus (ZIKV) Human hepatoma (Huh7) cells were exposed to the newly synthesized drugs or 7-deaza-7- ﬂuoro-2′-C-methyl adenosine (positive control) at concentrations up to 20 µM immediately following infection with ZIKV (multiplicity of infection, MOI = 0.5) Puerto Rican strain (PRVABC59) to assess antiviral activity. Cell cytopathic eﬀect (CPE) MTS assay (Promega, Madison, WI, USA) was measured ﬁve days after compound addition to determine the levels of replication inhibition [16,17]. 3.2.3. Dengue Virus serotype 2 (DENV-2), West Nile Virus (WNV) or Chikungunya (CHIKV) DENV2 or WNV replicon-harboring baby hamster kidney (BHK) cells and CHIKV replicon-harboring Huh7 cells were exposed to the newly synthesized drugs or 7-deaza-7-ﬂuoro- 2′-C-methyl adenosine or β-D-N4-hydroxycytidine (positive controls) at concentrations up to 30 µM to assess antiviral activity. Renilla luciferase levels (Promega, Madison, WI, USA) were quantiﬁed 48 h after test compounds addition to determine the levels of replication inhibition (EC50, µM) [18]. 3.1. Synthesis 1H NMR (400 MHz, DMSO-d6) δ 7.876 (d, J = 7.4 Hz, 0.5H), 7.872 (d, J = 7.4 Hz, 0.5H), 7.32 (m, 2H), 6.2 (dd, J = 12.9, 6.7 Hz, 0.5H), 6.05 (dd, J = 16.5, 4.0 Hz, 0.5H), 5.94 (d, J = 5.4 Hz, 0.5H), 5.88 (d, J = 6.2 Hz, 0.5H), 5.80 (d, J = 7.4 Hz, 0.5H), 5.75 (d, J = 7.4 Hz, 0.5H), 5.49 (d, J = 5.4 Hz, 0.5H), 5.30 (d, J = 4.3 Hz, 0.5H), 5.10 (m, 1H), 4.54 (m, 0.5H), 4.36 (m, 0.5H), 4.129 (m, 0.5H), 4.04 (m, 0.5H), 3.75 (d, J = 10.1 Hz, 0.5H), 3.74 (d, J = 12.0 Hz, 0.5H), 3.65 (d, J = 12.0 Hz, 0.5H), 1.14 (d, J = 6.6 Hz, 1.5H), 1.07 (d, J = 6.4 Hz, 1.5H). 19F NMR (376 MHz, DMSO-d6): δ −198.1 (dt, J = 58.1, 16.2 Hz), -206.2 (dd, J = 57.5, 13.4 Hz). 13C NMR (101 MHz, DMSO-d6): δ 166.2, 166.1, 155.60, 155.58, 142.4, 142.0, 95.6, 95.0, 93.6, 93.1, 91.7, 88.00, 88.99, 87.8, 87.7, 87.3, 86.0, 85.7, 70.3, 70.2, 68.4, 68.29, 68.27, 67.2, 37.4, 32.2, 18.3, 17.3. HRMS for C11H16BrFN3O4 (M + H). Calcd: m/z 352.0308, found: m/z 352.0301, 354.0276. 11 of 13 Molecules 2020, 25, 1258 3.4. Crystallography Single colorless plate crystals of compound 17 were recrystallised from methanol by slow evaporation. A suitable crystal with dimensions 0.41 × 0.30 × 0.15 mm3 was selected and mounted on a loop with paratone oil on a XtaLAB Synergy-S diﬀractometer (Rigaku Oxford Diﬀraction, Wroclaw, Poland). The crystal was kept at a steady T = 99.9(4) K during data collection. The structure was solved with the ShelXT [19] solution program using dual-space recycling methods and by using Olex2 (V1.3-alpha) [20] as the graphical interface. The model was reﬁned with ShelXL 2018/3 [21] using full matrix least squares minimization on F2. Results from X-ray structure determination of 17 are the following. Crystal data for C12H16FN3O5, Mr = 301.28, orthorhombic, P212121 (No. 19), a = 7.3409(5) Å, b = 8.2950(5) Å, c = 19.9788(15) Å, α = β = γ = 90◦, V = 1216.56(14) Å3, T = 99.9(4) K, Z = 4, Z′ = 1, µ(Cu Kα) = 1.192, 11534 reﬂections measured, 2161 unique (Rint = 0.0568) which were used in all calculations. The ﬁnal wR2 was 0.1111 (all data) and R1 was 0.0417 (I > 2σ(I)). (More details available in Supplementary Materials.) 3.2.1. RSV Replicon Assay RSV replicon cell lines were obtained from Apath, LLC (Brooklyn, NY, NY, USA) and were cultured as previously described [15]. Ribavirin and ALS-8112, synthesized by following reported procedures [11] were used as positive controls. Compounds were dissolved in dimethyl sulfoxide (DMSO) to a 40 mM concentration and serially diluted to the desired compound concentrations. Anti-viral activity was measured after 72 h by using Renilla-Glo reagent kit (Promega, Madison, WI, USA), according to manufacturer’s instruction. References 1. Seley-Radtke, K.L.; Yates, M.K. The evolution of nucleoside analogue antivirals: A review for chemists and non-chemists. Part 1: Early structural modiﬁcations to the nucleoside scaﬀold. Antiviral Res. 2018, 154, 66–86. [CrossRef] [PubMed] Shelton, J.; Lu, X.; Hollenbaugh, J.; Cho, J.-H.; Amblard, F.; Schinazi, R.F. Metabolism, biochemical actions and chemical synthesis of anticancer nucleosides. Chem. Rev. 2016, 119, 14379–14455. [CrossRef] [PubMed 3. Coats, S.J.; Garnier-Amblard, E.C.; Amblard, F.; Ehteshami, M.; Zhang, H.W.; Zhou, L.; Bondada, L.; Chavre, S.; Amiralaei, S.; Boucle, S.; et al. Chutes and ladders in hepatitis C nucleoside drug development. Antivir. Res. 2014, 102, 119–147. [CrossRef] [PubMed] 3. Coats, S.J.; Garnier-Amblard, E.C.; Amblard, F.; Ehteshami, M.; Zhang, H.W.; Zhou, L.; Bondada, L.; Chavre, S.; Amiralaei, S.; Boucle, S.; et al. Chutes and ladders in hepatitis C nucleoside drug development. Antivir. Res. 2014, 102, 119–147. [CrossRef] [PubMed] 4. Markowitz, M.; Saraﬁanos, S.G. 4’-Ethynyl-2-ﬂuoro-2’-deoxyadenosine, MK-8591: A novel HIV-1 reverse transcriptase translocation inhibitor. Curr. Opin. HIV AIDS 2018, 13, 294–299. [CrossRef] [PubMed] 4. Markowitz, M.; Saraﬁanos, S.G. 4’-Ethynyl-2-ﬂuoro-2’-deoxyadenosine, MK-8591: A novel HIV-1 reverse transcriptase translocation inhibitor. Curr. Opin. HIV AIDS 2018, 13, 294–299. [CrossRef] [PubMed] 5. Takamatsu, Y.; Tanaka, Y.; Kohgo, S.; Murakami, S.; Singh, K.; Das, D.; Venzon, D.J.; Amano, M.; Higashi-Kuwata, N.; Aoki, M.; et al. 4’-modiﬁed nucleoside analogs: Potent inhibitors active against entecavir-resistant hepatitis B virus. Hepatology 2015, 62, 1024–1036. [CrossRef] [PubMed] 5. Takamatsu, Y.; Tanaka, Y.; Kohgo, S.; Murakami, S.; Singh, K.; Das, D.; Venzon, D.J.; Amano, M.; Higashi-Kuwata, N.; Aoki, M.; et al. 4’-modiﬁed nucleoside analogs: Potent inhibitors active against entecavir-resistant hepatitis B virus. Hepatology 2015, 62, 1024–1036. [CrossRef] [PubMed] 6. Mitsuya, H. A novel long-acting anti-HBV nucleoside, E-CFCP, potently blocks the infectivity and replication of wild-type and drug-resistant HBVs in human-liver-chimeric mice with potental QW oral dosing schedule capabilities. Presented at the HEP DART, Kauai, HI, USA, 8–12 December 2019. 6. Mitsuya, H. A novel long-acting anti-HBV nucleoside, E-CFCP, potently blocks the infectivity and replication of wild-type and drug-resistant HBVs in human-liver-chimeric mice with potental QW oral dosing schedule capabilities. Presented at the HEP DART, Kauai, HI, USA, 8–12 December 2019. 7. Patel, K.; Kirkpatrick, C.M.; Nieforth, K.A.; Chanda, S.; Zhang, Q.; McClure, M.; Fry, J.; Symons, J.A.; Blatt, L.M.; Beigelman, L.; et al. Respiratory syncytial virus-A dynamics and the eﬀects of lumicitabine, a nucleoside viral replication inhibitor, in experimentally infected humans. J. Antimicrob. Chemother. 2019, 74, 442–452. [CrossRef] [PubMed] 8. 4. Conclusions Based on the structure of anti-RSV agent ALS-8112, a series of 4′- and 5′- substituted-2′-deoxy-2′- ﬂuoro cytidine nucleoside analogs were synthesized in 10 to 13 steps from commercially available 2′-deoxy-2′-α-ﬂuorocytidine. Nucleosides analogs with an azetidine, an oxetane, and an isoxazole 12 of 13 12 of 13 Molecules 2020, 25, 1258 ring, as well as a diﬂuoromethyl ether group, four groups never previously introduced at the 4′-position of a nucleoside, were successfully prepared. Interestingly, the formation of the 4′-oxetane ring via a Johnson–Corey–Chaykovsky epoxidation and consecutive ring-expansion was completely stereoselective, as determined by single crystal X-ray diﬀraction. We hypothesized that this selectivity could be attributed to the monomethoxytrityl groups hindering one face of the molecule. Final 4′- and 5′-substituted nucleosides (11, 14, 17, 20, 25, and 26) were evaluated for antiviral activity but unfortunately, none of them showed marked activity when tested against RSV, ZIKV, DENV-2, WNV, or CHIKV. Supplementary Materials: The following are available online. Figures S1–S18: 1H, 13C and 19F-NMR spectra for compounds 11, 14, 17, 20, 25 and 26, Table S1: Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2 × 103) for 14. Ueq is deﬁned as 1/3 of the trace of the orthogonalised Uij; Table S2: Anisotropic Displacement Parameters (×104) for 14. The anisotropic displacement factor exponent takes the form: −2π2[h2a2 × U11+ ... +2hka × b* × U12]; TableS3: Bond Lengths in Å for 14; Table S4: Bond Angles in ◦for 14; Table S5: Torsion Angles in ◦for 14; Table S6: Hydrogen Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2 × 103) for 14. Ueq is deﬁned as 1/3 of the trace of the orthogonalised Uij. Author Contributions: M.K. and C.L.: Synthesis of the tested compounds and writing of the original draft; L.B., L.M. and K.V.: In vitro antiviral assays; O.O.R. and L.D.: In vitro toxicity assays; F.A. and R.F.S.: Project conception and supervision; writing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by NIH Grant 1-R01-AI-132833, and 5P30-AI-50409 (CFAR). Acknowledgments: This paper is dedicated to our friend and colleague Dr. Piet Herdewijn who is retiring in 2020. We thank Dr. John Bacsa, Emory X-ray Crystallography Facility for the X-ray structural analysis. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Lo, M.K.; Amblard, F.; Flint, M.; Chatterjee, P.; Kasthuri, M.; Li, C.; Russell, O.; Verma, K.; Bassit, L.; Schinazi, R.F.; et al. Potent in vitro activity of β-D-4′-chloromethyl-2′-deoxy-2′-ﬂuorocytidine against Nipah virus. Antiviral Res. 2019, accepted. [CrossRef] [PubMed] 9. Beigelman, L.; Blatt, L.; Wang, G.; Rajwanshi, V.K.; Dyatkina, N.; Smith, D.B. Substituted Nucleotide Analogs. U.S. Patent 20120070411A1, 22 March 2012. 13 of 13 Molecules 2020, 25, 1258 10. Wang, G.; Deval, J.; Hong, J.; Dyatkina, N.; Prhavc, M.; Taylor, J.; Fung, A.; Jin, Z.; Stevens, S.K.; Serebryany, V.; et al. Discovery of 4’-chloromethyl-2’-deoxy-3’,5’-di-O-isobutyryl-2’-ﬂuorocytidine (ALS-8176), a ﬁrst-in-class RSV polymerase inhibitor for treatment of human respiratory syncytial virus infection. J. Med. Chem. 2015, 58, 1862–1878. [CrossRef] [PubMed] 11. Levchenko, K.; Datsenko, O.P.; Serhiichuk, O.; Tolmachev, A.; Iaroshenko, V.O.; Mykhailiuk, P.K. Copper-Catalyzed O-Diﬂuoromethylation of Functionalized Aliphatic Alcohols: Access to Complex Organic Molecules with an OCF2H Group. J. Org. Chem. 2016, 81, 5803–5813. [CrossRef] [PubMed] 12. De Kimpe, N.; De Smaele, D. Synthesis of aziridines and azetidines from N-(ω-haloalkyl) imines. Tetrahedron Lett. 1994, 35, 8023–8026. 13. Lia, G. A facile and eﬃcient synthesis of N-benzylazetidine. Synth. Commun. 2001, 31, 565–568. 14. Van leusen, A.M.; Hoogenboom, B.E.; Siderius, H. A novel synthesis and eﬃcient synthesis of oxazoles from tosylmethylisocyanide and carbonyl compounds. Tetrahedron Lett. 1972, 23, 2369–2372. [CrossRef] 15. Malykhina, O.; Yednak, M.A.; Collins, P.L.; Olivo, P.D.; Peeples, M.E. A respiratory syncytial virus replicon that is noncytotoxic and capable of long-term foreign gene expression. J. Virol. 2011, 85, 4792–4801. [CrossRef] [PubMed] 16. Zmurko, J.; Marques, R.E.; Schols, D.; Verbeken, E.; Kaptein, S.J.F.; Neyts, J. The viral polymerase inhibitor 7-deaza-2′-C-methyladenosine is a potent inhibitor of in vitro Zika virus replication and delays disease progression in a robust mouse infection model. PLoS Negl. Trop. Dis. 2016, 10, e0004695. [CrossRef] [PubMed] 17. Gavegnano, C.; Bassit, L.C.; Cox, B.C.; Hsiao, H.-M.; Johnson, E.L.; Suthar, M.; Chakraborty, R.; Schinazi, R.F. Jak inhibitors modulate production of replication competent Zika virus in human Hofbauer, trophoblasts and neuroblastoma cells. Pathogens Immunity. 2017, 2, 199–218. [CrossRef] [PubMed] 18. Ehteshami, M.; Tao, S.; Zandi, K.; Hsiao, H.M.; Jiang, Y.; Hammond, E.; Amblard, F.; Russell, O.; Merits, A.; Schinazi, R.F. Characterization of β-D-N(4)-hydroxycytidine as a novel inhibitor of Chikungunya virus. Antimicrob. Agents Chemother. 2017, 61, e02395-16. [CrossRef] [PubMed] 19. Sheldrick, G.M. Crystal structure reﬁnement with ShelXL. Acta Cryst. 2015, C71, 3–8. 20. Dolomanov, O.V.; Bourhis, L.J.; Gildea, R.J.; Howard, J.A.K.; Puschmann, H. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Olex2: A complete structure solution, reﬁnement and analysis program. J. Appl. Cryst. 2009, 42, 339–341. [CrossRef] 21. Sheldrick, G.M. ShelXT-Integrated space-group and crystal-structure determination. Acta Cryst. 2015, A71, 3–8. [CrossRef] [PubMed] Sample Availability: Samples of the compounds are not available from the authors. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2293774405	https://europepmc.org/articles/pmc4784881?pdf=render	English	null	Glucose Augments Killing Efficiency of Daptomycin Challenged Staphylococcus aureus Persisters	PloS one	2,016	cc-by	9,855	Marcel Prax1,2, Lukas Mechler1, Christopher Weidenmaier3, Ralph Bertram1,4* 1 Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Lehrbereich Mikrobielle Genetik, Auf der Morgenstelle 28, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany, 2 Paul-Ehrlich-Institut, Mikrobiologische Sicherheit, Paul-Ehrlich-Str. 51–59, 63225 Langen, Germany, 3 Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Medizinische Mikrobiologie und Hygiene, Elfriede-Aulhorn-Str. 6, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany, 4 Klinikum Nürnberg Medical School GmbH, Research Department, Paracelsus Medical University, Nuremberg, Germany a1111 * ralph.bertram@pmu.ac.at RESEARCH ARTICLE OPEN ACCESS Editor: Dirk-Jan Scheffers, University of Groningen, Groningen Institute for Biomolecular Sciences and Biotechnology, NETHERLANDS Received: September 1, 2014 Accepted: February 21, 2016 Published: March 9, 2016 Copyright: © 2016 Prax et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2016 Prax et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Glucose Augments Killing Efficiency of Daptomycin Challenged Staphylococcus aureus Persisters Marcel Prax1,2, Lukas Mechler1, Christopher Weidenmaier3, Ralph Bertram1,4* Introduction Eradication of harmful bacteria in the human body is often cumbersome due to drug resistance and drug tolerance particularly in biofilm embedded cells [1–7]. Biofilms accommodate a high percentage of persister cells which are in a non-dividing and metabolically less active state [8]. Persisters are regarded as genetically identical variants among a population of unicellular organisms that tolerate and survive high concentrations of antibiotics over extended periods of time [9–12]. This kind of phenotypic heterogeneity is a successful bet-hedging strategy to endure hostile conditions, such as antibiotic treatment or immune response and provides a rationale for recurrent or chronic bacterial infections [9,13,14]. The level of persister cells among a clonal bacterial culture is influenced by nutrient limitation, growth phase, various stresses, quorum sensing and other factors [15–17]. Compared to the identification of numer- ous persister-genes, information available on metabolic aspects of persisters is more limited Data Availability Statement: All relevant data are within the paper and its Supporting Information files. OPEN ACCESS Treatment of Staphylococcus aureus in stationary growth phase with high doses of the anti- biotic daptomycin (DAP) eradicates the vast majority of the culture and leaves persister cells behind. Despite resting in a drug-tolerant and dormant state, persister cells exhibit metabolic activity which might be exploited for their elimination. We here report that the addition of glucose to S. aureus persisters treated with DAP increased killing by up to five- fold within one hour. This glucose-DAP effect also occurred with strains less sensitive to the drug. The underlying mechanism is independent of the proton motive force and was not observed with non-metabolizable 2-deoxy-glucose. Our results are consistent with two hypotheses on the glucose-DAP interplay. The first is based upon glucose-induced carbo- hydrate transport proteins that may influence DAP and the second suggests that glucose may trigger the release or activity of cell-lytic proteins to augment DAP’s mode of action. Citation: Prax M, Mechler L, Weidenmaier C, Bertram R (2016) Glucose Augments Killing Efficiency of Daptomycin Challenged Staphylococcus aureus Persisters. PLoS ONE 11(3): e0150907. doi:10.1371/journal.pone.0150907 Editor: Dirk-Jan Scheffers, University of Groningen, Groningen Institute for Biomolecular Sciences and Biotechnology, NETHERLANDS Received: September 1, 2014 Accepted: February 21, 2016 Published: March 9, 2016 Copyright: © 2016 Prax et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Dirk-Jan Scheffers, University of Groningen, Groningen Institute for Biomolecular Sciences and Biotechnology, NETHERLANDS Received: September 1, 2014 Accepted: February 21, 2016 Published: March 9, 2016 Editor: Dirk-Jan Scheffers, University of Groningen, Groningen Institute for Biomolecular Sciences and Biotechnology, NETHERLANDS Received: September 1, 2014 Accepted: February 21, 2016 Published: March 9, 2016 Editor: Dirk-Jan Scheffers, University of Groningen, Groningen Institute for Biomolecular Sciences and Biotechnology, NETHERLANDS Editor: Dirk-Jan Scheffers, University of Groningen, Groningen Institute for Biomolecular Sciences and Biotechnology, NETHERLANDS Received: September 1, 2014 Accepted: February 21, 2016 Published: March 9, 2016 Copyright: © 2016 Prax et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. * ralph.bertram@pmu.ac.at Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grant BE4038/ 2 within the priority programme 1316 “host adapted metabolism of bacterial pathogens” of the Deutsche Forschungsgemeinschaft (www.dfg.de). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 1 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters [18]. A change in carbon source utilization upon glucose limitation stimulates persister forma- tion in E. coli [19] and accordingly, E. coli persisters maintain glycerol and glucose metabolism [20–22]. De novo synthesis of amino acids was observed with persister cells of the notorious pathogen Staphylococcus aureus [23], which is causative of skin infections, osteomyelitis, endo- carditis, bacteremia and further illnesses [24–27]. Multiple antibiotic resistant S. aureus strains continue to pose a formidable challenge in hospitals and in the community [28]. The bacteri- cidal lipopeptide daptomycin (DAP) is one of few antibiotics that is generally effective against many S. aureus strains [29], as well as other Gram positive bacteria [30–32]. The amphiphilic character of DAP in combination with calcium cations facilitates the incorporation into the bacterial membrane [33]. According to the current model, oligomerization of DAP leads to pore formation and increased permeability for ions resulting in perturbation of the proton motive force (PMF) and cell death [34]. DAP is highly efficient also against S. aureus cells in stationary phase, which are tolerant towards a broad range of other antibiotics [35]. As shown previously, the eradication efficiency of S. aureus by DAP is enhanced upon combination with other antibiotics [36,37] or D-cycloserine [38]. First cases of DAP non-susceptible strains were documented in hospitals briefly after introduction of the drug [39]. Such strains frequently exhibit changes in the cell envelope [40–42]. To prevent resistance formation and selection for non-susceptible strains due to prolonged drug-treatments [7], it is necessary to develop new efficient therapeutic strategies, with a special focus on targeting persister cells [43]. A new means for persister eradication in biofilms was achieved by a combination therapy with rifampicin and the acyldepsipeptide antibiotic ADEP4, leading to the permanent activa- tion of protease ClpP [44]. Furthermore, the administration of carbohydrates increases per- sister killing by aminoglycosides due to their dependency on the proton motive force (PMF) [45,46]. We here show that supplementing cultures of DAP challenged S. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. aureus cells with spe- cific carbohydrates in vitro leads to accelerated killing, which intriguingly also pertains to strains less susceptible to this drug. According to our data, the underlying mechanism is not- dependent on the PMF but may be dependent on metabolization of glucose. Unraveling the molecular basis and exploiting this phenomenon provides perspectives for a powerful anti-per- sister therapy. PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters Table 1. List of strains used in this study. S. aureus strains Description Reference SA113 (ATCC 35556) NCTC8325 derivative, agr-,rsbU-. agr: accessory gene regulator quorum-sensing system; rsbU: positive regulator of σB [50] MSSA 616, 621 clinical isolates, DAP MIC, 0.5 mg/L [42] MSSA 701, 703 clinical isolates, DAP MIC, 2.0 mg/L [42] HG003 NCTC8325 derivative, rsbU and tcaR repaired [51] HG003 D6 Derivative of HG003 carrying SNP in SAOUHSC_00670 (pitA) and SAOUHSC_02622 (gltS). [48] USA300 NE39 ptsG- (phosphotransferase system, glucose-speciﬁc IIABC component) [47] USA300 NE172 ptsG- (phosphotransferase system, glucose-speciﬁc IIABC component) [47] USA300 NE427 fumC- (fumarate hydratase) [47] USA300 NE476 fbaA- (fructose bisphosphate aldolase) [47] USA300 NE491 icd- (isocitrate dehydrogenase) [47] USA300NE1003 mqo- (malate:quinone oxidoreductase) [47] USA300 NE1046 fdh- (formate dehydrogenase accessory protein) [47] USA300 NE1407 pyk- (pyruvate kinase) [47] doi:10.1371/journal.pone.0150907.t001 Table 1. List of strains used in this study. S. aureus strains Description Reference SA113 (ATCC 35556) NCTC8325 derivative, agr-,rsbU-. agr: accessory gene regulator quorum-sensing system; rsbU: positive regulator of σB [50] MSSA 616, 621 clinical isolates, DAP MIC, 0.5 mg/L [42] MSSA 701, 703 clinical isolates, DAP MIC, 2.0 mg/L [42] HG003 NCTC8325 derivative, rsbU and tcaR repaired [51] HG003 D6 Derivative of HG003 carrying SNP in SAOUHSC_00670 (pitA) and SAOUHSC_02622 (gltS). [48] USA300 NE39 ptsG- (phosphotransferase system, glucose-speciﬁc IIABC component) [47] USA300 NE172 ptsG- (phosphotransferase system, glucose-speciﬁc IIABC component) [47] USA300 NE427 fumC- (fumarate hydratase) [47] USA300 NE476 fbaA- (fructose bisphosphate aldolase) [47] USA300 NE491 icd- (isocitrate dehydrogenase) [47] USA300NE1003 mqo- (malate:quinone oxidoreductase) [47] USA300 NE1046 fdh- (formate dehydrogenase accessory protein) [47] USA300 NE1407 pyk- (pyruvate kinase) [47] doi:10.1371/journal.pone.0150907.t001 Table 1. List of strains used in this study. MIC determination The MIC of all strains used in this study towards DAP was determined as described before [35] in triplicates with at least two independent cultures grown in TSB medium. The MIC was defined as the lowest antibiotic concentration that inhibited growth after 24 hours incubation at 37°C without shaking. Glucose and acetate determination Glucose and acetate concentrations in culture supernatants were both determined using enzy- matic kit systems (R-Biopharm, Art. No. 10716251035, Art. No. 10148261035) according to the manufacturer’s instructions but with the following modifications: first, the supernatant of each sample was diluted 1:10 with water prior to the reaction. For the glucose amount determi- nation, the assay was scaled down to one third of the final volume recommended in the man- ual. The extinction was measured photometrically at λ = 340 nm using a Hitachi spectrometer U-2900. The acetate measurement was performed using the plate reader SpectraMax 340 (Molecular Devices) and 96 well plates (Greiner) loaded with a final volume of 200 μl of the assay mix. Glucose (Roth, Karlsruhe, Germany) added to cultures was sterilized by filter of 0.2 μm pore size. Bacteria, growth conditions, and working solutions Bacteria, growth conditions, and working solutions Bacterial strains used in this study are listed in Table 1. Unless stated otherwise, cultures were incubated at 37°C with aeration and shaking (150 rpm) in tryptic soy broth (TSB). To provide stationary growth phase cultures, incubations were performed overnight. In the course of this study, two different types of TSB were used, either composed of casein peptone (pancreatic) (17 g/L), soy peptone (A3SC) (3 g/L), NaCl (5 g/L), K2HPO4 (2.5 g/L) and glucose (2.5 g/L, sterile filtered and added after autoclaving, or a ready-to-use powder purchased from Sigma, both at an approximate 1:10 culture-to-flask volume ratio. Daptomycin (DAP, analytic grade powder; designated ‘Cubicin’, Novartis Pharma, Nuremberg, Germany) was prepared freshly prior to each application, filter sterilized (0.2 μM pore size, Whatman, Dassel, Germany) and used to challenge stationary-phase S. aureus cells. 100-fold the MIC of DAP solution had been determined as 400 mg/L for S. aureus SA113 before [35] and 150 mg/L corresponding to 250-fold the MIC was determined and used for NARSA strains [47]. Strain HG003 D6 [48] was treated with 400 mg/L corresponding to 100-fold the MIC and the clinical strains with 400 mg/L corresponding to 200-fold and 800-fold the MIC for strains 616/621 and 701/703 respec- tively. Ca2+ cations, required for DAP activity [49], were provided as CaCl2 (Merck, Darmstadt, Germany) at a final concentration of 50 μg/mL. 2 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Addition of glucose accelerates DAP-dependent killing of stationary growth phase S. aureus in vitro Three SA113 cultures were grown identically in TSB medium to stationary growth phase at which the medium is depleted for glucose [23]. 100-fold the MIC of DAP was added at time point t = 0h to each of the cultures and at t = 3h, 5h or 7h, respectively, one culture each was supplemented with glucose at a final concentration of 5 g/L The viable counts indicated signifi- cantly enhanced killing when glucose was added at t = 3h compared to a culture challenged with DAP only (Fig 1). We now refer to the observation of enhanced DAP killing upon addi- tion of glucose as the “Glc-DAP effect”. Between t = 0h and t = 1h, the number of CFU decreased to approximately 0.05% of the initial amount. Within the next two hours, the eradi- cation slowed down, reflecting a typical biphasic killing behavior [9]. Of note, the addition of glucose three or five hours after DAP challenge resulted in an eradication of persister cells in the culture within the next five hours. In comparison, an SA113 culture exposed to 100-fold the MIC of DAP but no glucose still contained more than 1.6x103 CFU/mL after ten hours. Cells residing in a DAP-tolerant state for a longer period (up to 7 hours after addition of the drug) also were susceptible to the Glc-DAP effect, albeit less pronounced. Notably, a compara- ble behavior was observed when stationary growth phase cells were washed and resuspended in PBS buffer, ruling out that other components in the spent medium are causative for the Glc- DAP effect (S1 Fig). Upon supplementing the DAP-containing medium with lower concentrations of glucose (50 or 100 mg/L), killing of strain SA113 was only slightly affected, whereas higher glucose con- centration markedly enhanced and killing efficiency (S2 Fig). The maximal effect was observed with 1 g/L glucose, whereas higher concentrations (up to 5 g/L) did not accelerate killing fur- ther. To account for glucose consumption we performed all further experiments using 5 g/L glucose. Results Cells of stationary growth phase S. aureus SA113 cultures maintain an active amino acid anab- olism during high-level DAP treatment for several hours [23]. We assume that persister cells recalcitrant to eradication by this drug contribute significantly to this metabolic activity. Nota- bly, a closer inspection of killing curves of our previous study suggested an increased killing efficiency by DAP after the addition of glucose. We here scrutinized this effect in more detail. Statistical analysis Data are expressed as mean ± SD. Statistical analysis is described for each experiment in the corresponding figure legend. For all comparisons, a P value of 0.05 indicated statistical signif- icance. All statistical analyses were performed with GraphPad Prism version 4 and 5. CFU determination Overnight cultures of S. aureus SA113 were treated with 100-fold the MIC of DAP. Glucose was added at indicated time-points to final concentrations of 5 g/L (25 mM) unless stated oth- erwise. For the experiments with other compounds, glucose, fructose, ribose, xylose, glycerol, pyruvate, succinate, arginine or 2-deoxy-glucose were added at indicated time-points to final concentrations of 5 mM each. This concentration was chosen due to poorer aqueous solubility of some of the compounds compared to glucose. Viable counts were analyzed by CFU analysis on non-selective TSB agar plates. Cultures with viable counts below a threshold of 100 CFU/ mL were judged as sterilized. All viable count experiments were conducted using at least three biological replicates. 3 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters Glucose-enhanced killing of stationary growth phase S. aureus is DAP- specific and independent from cell division Delta CFU/mL (n = 3) of glucose added after 3h (from 4h to 6h), 5h (6h to 8h) and 7h (8h to 10 h) and delta CFU/mL for the same time points of the control without Glc, were compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test. *p<0.001. n.s.: not significant. doi:10.1371/journal.pone.0150907.g001 doi:10.1371/journal.pone.0150907.g001 doi:10.1371/journal.pone.0150907.g001 doi:10.1371/journal.pone.0150907.g001 did not induce killing by these antibiotics, which would be expected if the reversion to a repli- cating mode was responsible for the reinstated drug susceptibility. We therefore conclude that nutrient-dependent triggering of cell growth is not a reason for the Glc-DAP effect. Enhanced killing by DAP is specific to selected carbohydrates Are other metabolites also capable of accelerating DAP-dependent killing of stationary growth phase S. aureus? We chose fructose, ribose, glycerol, pyruvate, succinate and arginine, all of which are substrates or intermediates of catabolic pathways or anaplerotic reactions of S. aureus (Fig 2A) to test this hypothesis. The compounds were added to final concentrations of 5 mM each three hours after the onset of DAP treatment. Xylose and 2-deoxy-glucose, which are both transported into the cytoplasm of S. aureus but are not further metabolized [52,53], served as controls. Killing was enhanced with fructose, glycerol, succinate and arginine, but only glu- cose showed a significant effect compared to the DAP-only control. Eradication kinetics were unaffected with xylose or 2-deoxy-glucose (Fig 2B). As a further control, cultures were grown overnight in TSB-like medium in which glucose had been replaced by fructose. Also these were eradicated more efficiently by the addition of glucose, ruling out that an adaptation of metabolism to glucose during the cell cycle was mainly causative for the Glc-DAP effect (data not shown). Glucose-enhanced killing of stationary growth phase S. aureus is DAP- specific and independent from cell division At this point, it was conceivable that glucose induced killing of DAP challenged cells was the result of nutrient-dependent induction of cell division, rendering the cells generally more vul- nerable to antibiotics. We have previously shown that stationary growth phase S. aureus cul- tures are extremely tolerant to a number of antibiotics in vitro, even at elevated drug- concentrations [35]. This was consistent with observations in the present study, in which drugs targeting the cell envelope (penicillin, 100-fold the MIC, 2 mg/L, or vancomycin, 100-fold the MIC, 400 mg/L) did not discernibly decrease the life count (S3 Fig). The addition of glucose concentrations [35]. This was consistent with observations in the present study, in which drugs targeting the cell envelope (penicillin, 100-fold the MIC, 2 mg/L, or vancomycin, 100-fold the MIC, 400 mg/L) did not discernibly decrease the life count (S3 Fig). The addition of glucose 4 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters Fig 1. Time dependent killing of SA113. SA113 cells were treated with 100-fold the MIC of DAP starting at t = 0h. Glucose was added to cultures to final concentrations of 5 g/L each at time points 3h (triangles), 5h (circles), or 7h (squares), respectively, as indicated by arrows. CFU concentrations of a culture treated with DAP only (diamonds) were measured as a control. For statistical analysis area the delta of the CFU/mL was calculated 1 h and 4 h after glucose addition. Delta CFU/mL (n = 3) of glucose added after 3h (from 4h to 6h), 5h (6h to 8h) and 7h (8h to 10 h) and delta CFU/mL for the same time points of the control without Glc, were compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test. p<0.001. n.s.: not significant. doi:10.1371/journal.pone.0150907.g001 Fig 1. Time dependent killing of SA113. SA113 cells were treated with 100-fold the MIC of DAP starting at t = 0h. Glucose was added to cultures to final concentrations of 5 g/L each at time points 3h (triangles), 5h (circles), or 7h (squares), respectively, as indicated by arrows. CFU concentrations of a culture treated with DAP only (diamonds) were measured as a control. For statistical analysis area the delta of the CFU/mL was calculated 1 h and 4 h after glucose addition. The Glc-DAP effect is independent of the proton motive force The proton motive force (PMF) is generated by the electron transport chain and reduction equivalents originating from glycolysis and the TCA cycle. In order to examine a possible involvement of the PMF in the Glc-DAP effect, the uncoupler carbonyl cyanide m-chlorophe- nyl hydrazone (CCCP) was added to DAP challenged cells one hour before the addition of 5 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters Fig 2. Influence of selected carbohydrates on DAP challenged S. aureus cultures. A) Schematic overview of tested metabolites and their entrance into the metabolism of S. aureus and genes responsible for selected metabolic reactions; fructose (fru), glyceraldehyde-3-phosphate (GAP), Dihydroxyacetone phosphate (DHAP), phosphoenolpyruvate (PEP). B) Selected compounds were added to final concentrations of 5 mM at t = 3h and the CFU values were determined after 24 hours. Groups were compared to the untreated control group by 1-way ANOVA with Dunnett's Multiple Comparison Test. p<0.05. doi:10.1371/journal.pone.0150907.g002 Fig 2. Influence of selected carbohydrates on DAP challenged S. aureus cultures. A) Schematic overview of tested metabolites and their entrance into the metabolism of S. aureus and genes responsible for selected metabolic reactions; fructose (fru), glyceraldehyde-3-phosphate (GAP), Dihydroxyacetone phosphate (DHAP), phosphoenolpyruvate (PEP). B) Selected compounds were added to final concentrations of 5 mM at t = 3h and the CFU values were determined after 24 hours. Groups were compared to the untreated control group by 1-way ANOVA with Dunnett's Multiple Comparison Test. p<0.05. doi:10.1371/journal.pone.0150907.g002 Fig 2. Influence of selected carbohydrates on DAP challenged S. aureus cultures. A) Schematic glucose. CCCP impedes energizing of membranes by scavenging protons, rapidly leading to a lack of ATP in the cell [54]. As shown previously, the activity of DAP against exponential growth phase S. aureus is unaffected by CCCP [32]. In our experiments, CCCP treatment of cultures during exponential growth phase ceased growth independent of DAP (S4 Fig). Treat- ment of cells with CCCP prior to the addition of DAP resulted in approximately 10-fold 6 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters elevated persister levels compared to the untreated control (Fig 3), in agreement to results with CCCP-treated exponential phase E. coli cultures [55]. The Glc-DAP effect, however, was still observed in the presence of CCCP, suggesting a PMF independent mechanism. Analysis of S. aureus strains defective in glucose transport and catabolism We next inspected selected steps of glucose transport and catabolism by exploiting specific mutant strains of the S. aureus USA300 JE2 based NARSA Transposon Mutant Library [47]. The first two chosen strains exhibited disrupted phosphotransferase (PTS) systems, namely the glucose-PTS specific IIABC component, SAUSA300_2476 (NE39) and the glucose-PTS spe- cific IIBC component domain protein, SAUSA300_0191 (NE172). Compared to our PTS-posi- tive control strain NE1046 (fdhD, formate dehydrogenase, SAUSA300_2231), both PTS mutants’ killing curves showed a less pronounced incongruity between challenge with Glc- DAP and DAP only (Fig 4). The reduced but still discernible Glc-DAP effect may be rational- ized by redundant PTS systems or residual glucose transport due to secondary uptake systems of S. aureus [52]. The results hitherto suggest the involvement of one or more metabolic path- way(s) in the Glc-DAP phenomenon. In order to take a deeper look into central carbon metab- olism, further transposon mutants affected at certain branch points of glycolysis, or TCA cycle, respectively, were examined (Fig 2A). Results obtained with these mutants were inconclusive as DAP susceptibility among the strains varied considerably even without glucose. However, a trend towards an accelerated killing of all the tested strains upon the addition of glucose was observed (S5 Fig). The Glc-DAP effect is not affected by physiologic changes in the pH The interplay between oxygen supply and an excess of glucose can lead to an overload of meta- bolic pathways [56]. In S. aureus this results in accumulation of acetate and lactate stemming from pyruvate and concomitant acidification of the medium [57]. We determined both the pH value and the amount of acetate in DAP-containing cultures before and after the addition of glucose. The medium became slightly alkalized during the first three hours of DAP treatment (S6A Fig). After the addition of glucose the pH value rapidly decreased from 7.8 to about 7.4 and then leveled off, arguably due to glucose metabolism. The concentration of acetate was sta- ble for the first three hours and rose upon glucose addition (S6B Fig). An artificial adjustment of the pH value in the medium, to resemble the glucose-dependent changes, did not influence the killing behavior by DAP only (data not shown). Thus, it is unlikely that acidification may be causative of the Glc-DAP effect. The Glc-DAP effect is independent of the proton motive force elevated persister levels compared to the untreated control (Fig 3), in agreement to results with CCCP-treated exponential phase E. coli cultures [55]. The Glc-DAP effect, however, was still observed in the presence of CCCP, suggesting a PMF independent mechanism. Strains less susceptible to DAP are also subject to the Glc-DAP effect At t = 3h (arrows), glucose was added to cultures (filled symbols). Cultures without CCCP pretreatment (squares) were handled identically. For statistical analysis endpoint ODs after eight hours were compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test. p<0.05. doi:10.1371/journal.pone.0150907.g003 doi:10.1371/journal.pone.0150907.g003 glucose after 24 hours of incubation (Fig 5B). Similar results were obtained for the two less tol- erant strains 616 and 621 (Fig 5C). While DAP is regarded as non-lytic against S. aureus [59], the experiments with strains 616, 621, 701 and 703 revealed a drastic decrease in OD578 values after a 24 hour period of Glc-DAP treatment (Fig 5D). The OD578 of the samples treated with glucose after 24 hours of incubation (Fig 5B). Similar results were obtained for the two less tol- erant strains 616 and 621 (Fig 5C). While DAP is regarded as non-lytic against S. aureus [59], the experiments with strains 616, 621, 701 and 703 revealed a drastic decrease in OD578 values after a 24 hour period of Glc-DAP treatment (Fig 5D). The OD578 of the samples treated with glucose after 24 hours of incubation (Fig 5B). Similar results were obtained for the two less tol- erant strains 616 and 621 (Fig 5C). While DAP is regarded as non-lytic against S. aureus [59], the experiments with strains 616, 621, 701 and 703 revealed a drastic decrease in OD578 values after a 24 hour period of Glc-DAP treatment (Fig 5D). The OD578 of the samples treated with Fig 4. Time-dependent killing of PTS-mutants. Transposon mutants were treated with 250-fold the MIC of DAP. Glucose was added to the culture at t = 3h (arrow). NE39 (glucose-PTS specific IIABC component, circle), NE172 (glucose-PTS specific IIBC component domain protein, square). Strain NE1046 (formate dehydrogenase, triangle) served as control. For statistical analysis, the area under curve (AUC) was calculated from the time point of glucose (Glc) addition (3h). AUCs (n = 3) of all groups where compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test, according to which the differences were not significant. doi:10.1371/journal.pone.0150907.g004 Fig 4. Time-dependent killing of PTS-mutants. Transposon mutants were treated with 250-fold the MIC of DAP. Glucose was added to the culture at t = 3h (arrow). NE39 (glucose-PTS specific IIABC component, circle), NE172 (glucose-PTS specific IIBC component domain protein, square). Strain NE1046 (formate dehydrogenase, triangle) served as control. Strains less susceptible to DAP are also subject to the Glc-DAP effect We next investigated, whether the addition of glucose also increases DAP-dependent killing of strains with decreased susceptibility to this drug. Strain HG003 D6 [48] had previously been isolated as a highly DAP-tolerant mutant generated by cyclic treatment with high doses of the drug. The addition of glucose and DAP led to a tremendous killing also of this strain (Fig 5A) whereas the culture treated with DAP only still contained more than 1x 108 CFU/mL seven hours after drug-addition. The parent strain HG003 was highly susceptible for the Glc-DAP effect reflecting a more pronounced killing behavior than SA113 (Fig 1). In addition, two DAP sensitive strains (616, 621; MIC = 0.5 mg/L) and two less DAP-susceptible strains (701, 703; MIC = 2 mg/L), all isolated from a patient with relapsing endocarditis during DAP therapy [42,58] were tested for the Glc-DAP effect. Intriguingly, the killing efficiency was increased about 100-fold (strain 703) and 600-fold (strain 701) compared to the treatment without PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 7 / 15 Glucose/Daptomycin Killing of S. aureus Persisters Fig 3. Influence of the proton motive force. CCCP pretreatment of DAP challenged SA113 cultures. Stationary phase SA113 cells were treated with 50 μM CCCP (triangles) at t = -1h, corresponding to one hour before the addition of 100-fold the MIC of DAP. At t = 3h (arrows), glucose was added to cultures (filled symbols). Cultures without CCCP pretreatment (squares) were handled identically. For statistical analysis endpoint ODs after eight hours were compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test. p<0.05. d i 10 1371/j l 0150907 003 Fig 3. Influence of the proton motive force. CCCP pretreatment of DAP challenged SA113 cultures. Stationary phase SA113 cells were treated with 50 μM CCCP (triangles) at t = -1h, corresponding to one hour before the addition of 100-fold the MIC of DAP. At t = 3h (arrows), glucose was added to cultures (filled symbols). Cultures without CCCP pretreatment (squares) were handled identically. For statistical analysis endpoint ODs after eight hours were compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test. p<0.05. Fig 3. Influence of the proton motive force. CCCP pretreatment of DAP challenged SA113 cultures. Stationary phase SA113 cells were treated with 50 μM CCCP (triangles) at t = -1h, corresponding to one hour before the addition of 100-fold the MIC of DAP. Strains less susceptible to DAP are also subject to the Glc-DAP effect This apparent cell lysis was observed with all four strains, irrespective of their sensitivity towards the drug. Glc-DAP decreased by more than 75% compared to the cultures treated with DAP only. This apparent cell lysis was observed with all four strains, irrespective of their sensitivity towards the drug. Strains less susceptible to DAP are also subject to the Glc-DAP effect For statistical analysis, the area under curve (AUC) was calculated from the time point of glucose (Glc) addition (3h). AUCs (n = 3) of all groups where compared by 1-way ANOVA with Bonferroni's Multiple Comparison Test, according to which the differences were not significant. 8 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters Fig 5. Time-dependent killing of strains with different susceptibilities towards DAP. Stationary phase cultures of HG003, HG003 D6 and the clinical S. aureus strains 616, 621, 701 and 703 were treated with 100-fold, or 250-fold the MIC of DAP, respectively. At t = 3h, glucose was added (arrow) to the cultures (filled symbols) and CFU values were determined over time. For statistical analysis endpoint ODs after 24 hours for -Glc and +Glc for each strain were compared with unpaired t-test (with Welch’s correction for unequal variances if appropriate) *p<0.001. A) Growth-phase-dependent DAP-tolerant strain HG003 D6 (square) and parent wild type strain HG003 (triangle). B) DAP-tolerant strains 701 (square) and 703 (triangle) (MIC = 2 mg/L). C) Sensitive strains 616 (square) and 621 (triangle) (MIC = 0.5 mg/L). D) Optical densities after 24h of incubation. Strains were challenged with 100-fold the MIC of DAP from t = 0h. Identically treated cultures were supplemented with glucose from t = 3h. doi:10 1371/journal pone 0150907 g005 Fig 5. Time-dependent killing of strains with different susceptibilities towards DAP. Stationary phase cultures of HG003, HG003 D6 and the clinical S. aureus strains 616, 621, 701 and 703 were treated with 100-fold, or 250-fold the MIC of DAP, respectively. At t = 3h, glucose was added (arrow) to the cultures (filled symbols) and CFU values were determined over time. For statistical analysis endpoint ODs after 24 hours for -Glc and +Glc for each strain were compared with unpaired t-test (with Welch’s correction for unequal variances if appropriate) *p<0.001. A) Growth-phase-dependent DAP-tolerant strain HG003 D6 (square) and parent wild type strain HG003 (triangle). B) DAP-tolerant strains 701 (square) and 703 (triangle) (MIC = 2 mg/L). C) Sensitive strains 616 (square) and 621 (triangle) (MIC = 0.5 mg/L). D) Optical densities after 24h of incubation. Strains were challenged with 100-fold the MIC of DAP from t = 0h. Identically treated cultures were supplemented with glucose from t = 3h. doi:10.1371/journal.pone.0150907.g005 Glc-DAP decreased by more than 75% compared to the cultures treated with DAP only. Discussion Metabolite induced killing of bacterial persisters has been associated with PMF generation and concomitant uptake of aminoglycoside compounds [20,45,46]. We here show that the lipopep- tide antibiotic DAP also exhibits enhanced killing efficiency of S. aureus in the presence of glu- cose. Of note, this effect was also observed with a number of strains with low DAP- susceptibility. Our experiments with the uncoupler CCCP furthermore indicate that the Glc- DAP effect is not merely a consequence of PMF generation. Instead, the metabolism of glucose t b i l f th Gl DAP ff t hi h ith i t ith l t Metabolite induced killing of bacterial persisters has been associated with PMF generation and concomitant uptake of aminoglycoside compounds [20,45,46]. We here show that the lipopep- tide antibiotic DAP also exhibits enhanced killing efficiency of S. aureus in the presence of glu- cose. Of note, this effect was also observed with a number of strains with low DAP- Metabolite induced killing of bacterial persisters has been associated with PMF generation and concomitant uptake of aminoglycoside compounds [20,45,46]. We here show that the lipopep- tide antibiotic DAP also exhibits enhanced killing efficiency of S. aureus in the presence of glu- cose. Of note, this effect was also observed with a number of strains with low DAP- susceptibility. Our experiments with the uncoupler CCCP furthermore indicate that the Glc- DAP effect is not merely a consequence of PMF generation. Instead, the metabolism of glucose appears to be crucial for the Glc-DAP effect, which was neither prominent with low concentra- tions of glucose, nor with the non-metabolizable 2-deoxy-glucose. The observations of our study are consistent with two hypotheses for the mechanistic basis of the Glc-DAP effect. The first one suggests an influence of Glc-transport proteins on DAP’s mode of action, while the susceptibility. Our experiments with the uncoupler CCCP furthermore indicate that the Glc- DAP effect is not merely a consequence of PMF generation. Instead, the metabolism of glucose appears to be crucial for the Glc-DAP effect, which was neither prominent with low concentra- tions of glucose, nor with the non-metabolizable 2-deoxy-glucose. The observations of our study are consistent with two hypotheses for the mechanistic basis of the Glc-DAP effect. The first one suggests an influence of Glc-transport proteins on DAP’s mode of action, while the PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 9 / 15 Glucose/Daptomycin Killing of S. Discussion g g p y Notably, our experiments were based upon in vitro stationary growth phase cultures that were challenged with DAP concentrations that exceed serum concentrations in patients treated with this drug by more than tenfold [74,75]. Certainly, the systemic application of glucose to enhance DAP-dependent killing of S. aureus persisters in a patient is limited, as the blood sugar level in the human body is normally subject to homeostatic regulation. Of note, glycemia of non-diabetic humans is in a comparable range as the glucose concentrations that we deter- mined to enhance DAP’s function. The importance of glucose as an adjuvant for DAP may thus have gone unnoticed in patients so far. It may be an option to improve DAP-efficiency in the treatment of non-invasive acute bacterial skin and skin-structure infections by increasing local concentrations of glucose. Hopefully, recent achievements regarding the eradication of persister cells will also aid in reducing the formation of drug resistant cells that pose an ever- growing issue in public and clinical health [4,39,76–78]. Discussion aureus Persisters second is based upon Glc induced and DAP-specific lysis of cells. Based upon our observations with a number of S. aureus transposon-mutants, it is conceivable that components of the Glc PTS transport system may serve as receptors or targets of DAP. Accordingly, Glc-dependent induction of specific PTS transporters [60,61] will increase susceptibility to DAP. A similar effect was observed in Lactococcus lactis with bacteriocins that share biochemical features with DAP [62]. Moreover, mutations in PTS proteins confer a high degree of DAP-non-susceptibil- ity in Enterococcus faecium [63]. Regarding the integrity of S. aureus cells upon DAP treatment, contradictory results have been reported. Electron microscopic images have shown tremen- dous morphological changes of S. aureus cells during DAP challenge, but not lysis [64], in agreement with another study also suggesting a lysis-independent mechanism of this drug [59]. However, autolysis after DAP addition was observed, at least partially, in some S. aureus strains during exponential phase [65]. Cell lysis may be augmented by intrinsic murein hydro- lases. A potentiated lysis of exponential phase Staphylococcus cohnii upon addition of glucose was described with Pep 5, a cationic bactericidal peptide produced by Staphylococcus. epidermi- dis 5 [66]. It is conceivable that carbohydrates in combination with specific drugs induce a sui- cidal mechanism in persister cells comparable to programmed cell death [67]. Of note, carbohydrate metabolism influences murein hydrolase activity in S. aureus [68,69]. For exam- ple, the pleiotropic regulator CcpA activates transcription of the hydrolase activator cidA in the presence of glucose [70]. cidA is part of the cidABC operon which together with lrgAB is involved in the regulation of murein hydrolase activity and autolysis [69,71,72]. According to our previous data, an upregulation of the TCA cycle activity may lead to an overflow metabo- lism [23] and acetate derived from pyruvate activates cidABC and lrgAB transcription. Further studies are required to verify these speculations in regard to the Glc-DAP effect. q y p g Recently, a resuscitation promoting factor of S. aureus was postulated that is involved in shifting dormant cells back to a dividing state [73]. This factor can be ruled out as responsible for the Glc-DAP effect which we also observed in buffered solution, devoid of components found in culture supernatants. Discussion Although the regulatory network of hydrolase activity is still not well understood, it should be considered as a potential target for the development of new anti- persister therapies of S. aureus. Artificial activation of peptidoglycan hydrolases could thereby lead to a random lysis process with fatal consequences for the cells independent of both the sus- ceptibility towards antibiotics and their physiological state. It would be interesting to investi- gate the significance of the Glc-DAP effect in the treatment of staphylococcal infection. Notably, our experiments were based upon in vitro stationary growth phase cultures that were challenged with DAP concentrations that exceed serum concentrations in patients treated with this drug by more than tenfold [74,75]. Certainly, the systemic application of glucose to enhance DAP-dependent killing of S. aureus persisters in a patient is limited, as the blood sugar level in the human body is normally subject to homeostatic regulation. Of note, glycemia of non-diabetic humans is in a comparable range as the glucose concentrations that we deter- mined to enhance DAP’s function. The importance of glucose as an adjuvant for DAP may thus have gone unnoticed in patients so far. It may be an option to improve DAP-efficiency in the treatment of non-invasive acute bacterial skin and skin-structure infections by increasing local concentrations of glucose. Hopefully, recent achievements regarding the eradication of persister cells will also aid in reducing the formation of drug resistant cells that pose an ever- growing issue in public and clinical health [4,39,76–78]. Recently, a resuscitation promoting factor of S. aureus was postulated that is involved in shifting dormant cells back to a dividing state [73]. This factor can be ruled out as responsible for the Glc-DAP effect which we also observed in buffered solution, devoid of components found in culture supernatants. Although the regulatory network of hydrolase activity is still not well understood, it should be considered as a potential target for the development of new anti- persister therapies of S. aureus. Artificial activation of peptidoglycan hydrolases could thereby lead to a random lysis process with fatal consequences for the cells independent of both the sus- ceptibility towards antibiotics and their physiological state. It would be interesting to investi- gate the significance of the Glc-DAP effect in the treatment of staphylococcal infection. Supporting Information NE427 (fumC-, fuma- rate hydratase, diamonds), NE476 (fba-, fructose bisphosphate aldolase, squares), NE491 (icd-, isocitrate dehydrogenase, triangles), NE1003 (mqo-, malate-quinone oxidoreductase, x-mark), NE1046 (fdh-, formate dehydrogenase, circles), NE1407 (pyk-, pyruvate kinase, asterisks). For statistical analysis area under curve (AUC) was calculated from the time point of glucose (Glc) addition (3h). AUCs (n = 3) of all groups where compared to NE1046 fdh by 1-way ANOVA with Dunnett's Multiple Comparison Test. (TIF) S6 Fig. pH and acetate/glucose measurement. Cultures were treated with 100-fold the MIC of DAP at t = 0h. A) Glucose was added (filled squares) at t = 3h (arrow) and pH values were determined over time. B) Acetate (triangle) and glucose (square) measurement of a culture with glucose added at t = 3h. (TIF) Author Contributions Conceived and designed the experiments: MP LM RB. Performed the experiments: MP LM. Analyzed the data: MP LM CW RB. Wrote the paper: MP RB. Conceived and designed the experiments: MP LM RB. Performed the experiments: MP LM. Analyzed the data: MP LM CW RB. Wrote the paper: MP RB. Acknowledgments We thank Raphaela Künzel for technical assistance and Fritz Götz for support. Supporting Information S1 Fig. Time-dependent killing of SA113 cells in PBS. Stationary phase SA113 cells grown in TSB were harvested and resuspended in PBS. At t = 0h, 100-fold the MIC of DAP was added to 10 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters the cell suspensions. At t = 3h, one cell suspension was supplemented with glucose (filled square), the other was left unaffected (open square) and CFU concentrations were determined over time. (TIF) the cell suspensions. At t = 3h, one cell suspension was supplemented with glucose (filled square), the other was left unaffected (open square) and CFU concentrations were determined over time. (TIF) S2 Fig. Influence of the glucose concentration on the efficiency of the Glc-DAP effect. Sta- tionary phase SA113 cells were treated with 100-fold the MIC of DAP at t = 0h. At t = 3h, dif- ferent amounts of glucose were added and CFU values were determined after another four hours. Pearson’s r coefficient: r = -0,704. (TIF) S3 Fig. Penicillin and vancomycin treatment of SA113 ± glucose. Stationary phase SA113 cells were treated with 100-fold the MIC of penicillin (square) or 100-fold the MIC of vanco- mycin (triangle) at t = 0h. Glucose was added at t = 3h (arrow and filled symbols). (TIF) S4 Fig. Effect of CCCP on growth of SA113. SA113 was grown in TSB supplemented with glucose (t = 0h, squares), 100 μM CCCP (t = 0h, diamonds), glucose and CCCP (t = 0h, trian- gles), or glucose (t = 0h) and CCCP (t = 3h) (circles), respectively. (TIF) S5 Fig. Investigation of enzymatic branch points in glycolysis and TCA cycle. Time depen- dent killing of stationary phase cultures with 250-fold the MIC of DAP. NE427 (fumC-, fuma- rate hydratase, diamonds), NE476 (fba-, fructose bisphosphate aldolase, squares), NE491 (icd-, isocitrate dehydrogenase, triangles), NE1003 (mqo-, malate-quinone oxidoreductase, x-mark), NE1046 (fdh-, formate dehydrogenase, circles), NE1407 (pyk-, pyruvate kinase, asterisks). For statistical analysis area under curve (AUC) was calculated from the time point of glucose (Glc) addition (3h). AUCs (n = 3) of all groups where compared to NE1046 fdh by 1-way ANOVA with Dunnett's Multiple Comparison Test. (TIF) S5 Fig. Investigation of enzymatic branch points in glycolysis and TCA cycle. Time depen- dent killing of stationary phase cultures with 250-fold the MIC of DAP. 1. Moy JA, Caldwell-Brown D, Lin AN, Pappa KA, Carter DM (1990) Mupirocin-resistant Staphylococcus aureus after long-term treatment of patients with epidermolysis bullosa. J Am Acad Dermatol 22: 893– 895. PMID: 2112168 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 References 1. Moy JA, Caldwell-Brown D, Lin AN, Pappa KA, Carter DM (1990) Mupirocin-resistant Staphylococcus aureus after long-term treatment of patients with epidermolysis bullosa. J Am Acad Dermatol 22: 893– 895. PMID: 2112168 1. Moy JA, Caldwell-Brown D, Lin AN, Pappa KA, Carter DM (1990) Mupirocin-resistant Staphylococcus aureus after long-term treatment of patients with epidermolysis bullosa. J Am Acad Dermatol 22: 893– 895. PMID: 2112168 2. Endimiani A, Blackford M, Dasenbrook EC, Reed MD, Bajaksouszian S, Hujer AM, et al. (2011) Emer- gence of linezolid-resistant Staphylococcus aureus after prolonged treatment of cystic fibrosis patients 2. Endimiani A, Blackford M, Dasenbrook EC, Reed MD, Bajaksouszian S, Hujer AM, et al. (2011) Emer- gence of linezolid-resistant Staphylococcus aureus after prolonged treatment of cystic fibrosis patients PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 11 / 15 Glucose/Daptomycin Killing of S. aureus Persisters in Cleveland, Ohio. Antimicrob Agents Chemother 55: 1684–1692. doi: 10.1128/AAC.01308-10 PMID: 21263048 3. Mariani PG, Sader HS, Jones RN (2006) Development of decreased susceptibility to daptomycin and vancomycin in a Staphylococcus aureus strain during prolonged therapy. J Antimicrob Chemother 58: 481–483. PMID: 16847029 4. Skiest DJ (2006) Treatment failure resulting from resistance of Staphylococcus aureus to daptomycin. J Clin Microbiol 44: 655–656. PMID: 16455939 5. Babra C, Tiwari J, Costantino P, Sunagar R, Isloor S, Hedge N, et al. (2013) Human methicillin-sensi- tive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens. J Basic Microbiol. 6. Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15: 167–193. PMID: 11932229 7. Dortet L, Anguel N, Fortineau N, Richard C, Nordmann P (2013) In vivo acquired daptomycin resistance during treatment of methicillin-resistant Staphylococcus aureus endocarditis. Int J Infect Dis 17: e1076–1077. doi: 10.1016/j.ijid.2013.02.019 PMID: 23578850 8. Wood TK, Knabel SJ, Kwan BW (2013) Bacterial persister cell formation and dormancy. Appl Environ Microbiol 79: 7116–7121. doi: 10.1128/AEM.02636-13 PMID: 24038684 9. Lewis K (2010) Persister cells. Annu Rev Microbiol 64: 357–372. doi: 10.1146/annurev.micro.112408. 134306 PMID: 20528688 10. Balaban NQ, Gerdes K, Lewis K, McKinney JD (2013) A problem of persistence: still more questions than answers? Nat Rev Microbiol 11: 587–591. PMID: 24020075 11. Casadesús J, Low DA (2013) Programmed heterogeneity: epigenetic mechanisms in bacteria. J Biol Chem 288: 13929–13935. doi: 10.1074/jbc.R113.472274 PMID: 23592777 12. Helaine S, Kugelberg E (2014) Bacterial persisters: formation, eradication, and experimental systems. Trends Microbiol 22: 417–424. doi: 10.1016/j.tim.2014.03.008 PMID: 24768561 13. PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 References Brady RA, Leid JG, Calhoun JH, Costerton JW, Shirtliff ME (2008) Osteomyelitis and the role of biofilms in chronic infection. FEMS Immunol Med Microbiol 52: 13–22. PMID: 18081847 26. Moore CL, Osaki-Kiyan P, Haque NZ, Perri MB, Donabedian S, Zervos MJ (2012) Daptomycin versus vancomycin for bloodstream infections due to methicillin-resistant Staphylococcus aureus with a high vancomycin minimum inhibitory concentration: a case-control study. Clin Infect Dis 54: 51–58. doi: 10. 1093/cid/cir764 PMID: 22109947 27. Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339: 520–532. PMID: 970904 28. DeLeo FR, Otto M, Kreiswirth BN, Chambers HF (2010) Community-associated meticillin-resistant Staphylococcus aureus. Lancet 375: 1557–1568. doi: 10.1016/S0140-6736(09)61999-1 PMID: 20206987 29. French GL (2006) Bactericidal agents in the treatment of MRSA infections—the potential role of dapto- mycin. J Antimicrob Chemother 58: 1107–1117. PMID: 17040922 30. Fuchs PC, Barry AL, Brown SD (2002) In vitro bactericidal activity of daptomycin against staphylococci. J Antimicrob Chemother 49: 467–470. PMID: 11864946 31. Steenbergen JN, Alder J, Thorne GM, Tally FP (2005) Daptomycin: a lipopeptide antibiotic for the treat- ment of serious Gram-positive infections. J Antimicrob Chemother 55: 283–288. PMID: 15705644 32. Mascio CT, Alder JD, Silverman JA (2007) Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells. Antimicrob Agents Chemother 51: 4255–4260. PMID: 17923487 33. Humphries RM, Pollett S, Sakoulas G (2013) A Current Perspective on Daptomycin for the Clinical Microbiologist. Clin Microbiol Rev 26: 759–780. doi: 10.1128/CMR.00030-13 PMID: 24092854 34. Muraih JK, Pearson A, Silverman J, Palmer M (2011) Oligomerization of daptomycin on membranes. Biochim Biophys Acta 1808: 1154–1160. doi: 10.1016/j.bbamem.2011.01.001 PMID: 21223947 35. Lechner S, Lewis K, Bertram R (2012) Staphylococcus aureus Persisters Tolerant to Bactericidal Anti- biotics. J Mol Microbiol Biotechnol 22: 235–244. doi: 10.1159/000342449 PMID: 22986269 36. Berti AD, Wergin JE, Girdaukas GG, Hetzel SJ, Sakoulas G, Rose WE (2012) Altering the proclivity towards daptomycin resistance in methicillin-resistant Staphylococcus aureus using combinations with other antibiotics. Antimicrob Agents Chemother 56: 5046–5053. doi: 10.1128/AAC.00502-12 PMID: 22802248 37. Werth BJ, Sakoulas G, Rose WE, Pogliano J, Tewhey R, Rybak MJ (2013) Ceftaroline increases mem- brane binding and enhances the activity of daptomycin against daptomycin-nonsusceptible vancomy- cin-intermediate Staphylococcus aureus in a pharmacokinetic/pharmacodynamic model. Antimicrob Agents Chemother 57: 66–73. doi: 10.1128/AAC.01586-12 PMID: 23070161 38. References Fauvart M, De Groote VN, Michiels J (2011) Role of persister cells in chronic infections: clinical rele- vance and perspectives on anti-persister therapies. J Med Microbiol 60: 699–709. doi: 10.1099/jmm.0. 030932-0 PMID: 21459912 14. Tuchscherr L, Medina E, Hussain M, Völker W, Heitmann V, Niemann S, et al. (2011) Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection. EMBO Mol Med 3: 129–141. doi: 10.1002/emmm.201000115 PMID: 21268281 15. Que YA, Hazan R, Strobel B, Maura D, He J, Kesarwani M, et al. (2013) A quorum sensing small vola- tile molecule promotes antibiotic tolerance in bacteria. PLoS One 8: e80140. doi: 10.1371/journal. pone.0080140 PMID: 24367477 16. Leung V, Levesque CM (2012) A stress-inducible quorum-sensing peptide mediates the formation of persister cells with noninherited multidrug tolerance. J Bacteriol 194: 2265–2274. doi: 10.1128/JB. 06707-11 PMID: 22366415 17. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K (2004) Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230: 13–18. PMID: 14734160 18. Amato SM, Fazen CH, Henry TC, Mok WW, Orman MA, Sandvik EL, et al. (2014) The role of metabo- lism in bacterial persistence. Front Microbiol 5: 70. doi: 10.3389/fmicb.2014.00070 PMID: 24624123 19. Amato SM, Orman MA, Brynildsen MP (2013) Metabolic control of persister formation in Escherichia coli. Mol Cell 50: 475–487. doi: 10.1016/j.molcel.2013.04.002 PMID: 23665232 20. Orman MA, Brynildsen MP (2013) Establishment of a Method To Rapidly Assay Bacterial Persister Metabolism. Antimicrobial Agents and Chemotherapy 57: 4398–4409. doi: 10.1128/AAC.00372-13 PMID: 23817376 21. Orman MA, Brynildsen MP (2013) Dormancy is not necessary or sufficient for bacterial persistence. Antimicrob Agents Chemother 57: 3230–3239. doi: 10.1128/AAC.00243-13 PMID: 23629720 22. Bokinsky G, Baidoo EE, Akella S, Burd H, Weaver D, Alonso-Gutierrez J, et al. (2013) HipA-Triggered Growth Arrest and beta-Lactam Tolerance in Escherichia coli Are Mediated by RelA-Dependent ppGpp Synthesis. J Bacteriol 195: 3173–3182. doi: 10.1128/JB.02210-12 PMID: 23667235 23. Lechner S, Prax M, Lange B, Huber C, Eisenreich W, Herbig A, et al. (2014) Metabolic and transcrip- tional activities of Staphylococcus aureus challenged with high-doses of daptomycin. Int J Med Micro- biol 304: 931–940. doi: 10.1016/j.ijmm.2014.05.008 PMID: 24980509 24. Gould IM (2009) Antibiotics, skin and soft tissue infection and meticillin-resistant Staphylococcus aureus: cause and effect. Int J Antimicrob Agents 34 Suppl 1: S8–11. doi: 10.1016/S0924-8579(09) 70542-4 PMID: 19560675 12 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters 25. PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 References Gasch O, Pillai S, Dakos J, Miyakis S, Moellering R Jr., Eliopoulos G (2013) Daptomycin in vitro activity against methicillin-resistant Staphylococcus aureus is enhanced by D-cycloserine in a mechanism associated with a decrease in cell surface charge. Antimicrob Agents Chemother. 39. Hayden MK, Rezai K, Hayes RA, Lolans K, Quinn JP, Weinstein RA (2005) Development of Daptomy- cin resistance in vivo in methicillin-resistant Staphylococcus aureus. J Clin Microbiol 43: 5285–5287. PMID: 16207998 40. Bayer AS, Schneider T, Sahl HG (2013) Mechanisms of daptomycin resistance in Staphylococcus aureus: role of the cell membrane and cell wall. Ann N Y Acad Sci 1277: 139–158. doi: 10.1111/j.1749- 6632.2012.06819.x PMID: 23215859 41. Song Y, Rubio A, Jayaswal RK, Silverman JA, Wilkinson BJ (2013) Additional routes to Staphylococ- cus aureus daptomycin resistance as revealed by comparative genome sequencing, transcriptional profiling, and phenotypic studies. PLoS One 8: e58469. doi: 10.1371/journal.pone.0058469 PMID: 23554895 42. Bertsche U, Weidenmaier C, Kuehner D, Yang SJ, Baur S, Wanner S, et al. (2011) Correlation of dapto- mycin resistance in a clinical Staphylococcus aureus strain with increased cell wall teichoic acid pro- duction and D-alanylation. Antimicrob Agents Chemother 55: 3922–3928. doi: 10.1128/AAC.01226-10 PMID: 21606222 43. Cohen NR, Lobritz MA, Collins JJ (2013) Microbial persistence and the road to drug resistance. Cell Host Microbe 13: 632–642. doi: 10.1016/j.chom.2013.05.009 PMID: 23768488 44. Conlon BP, Nakayasu ES, Fleck LE, LaFleur MD, Isabella VM, Coleman K, et al. (2013) Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503: 365–370. doi: 10.1038/ nature12790 PMID: 24226776 45. Allison KR, Brynildsen MP, Collins JJ (2011) Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473: 216–220. doi: 10.1038/nature10069 PMID: 21562562 13 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 Glucose/Daptomycin Killing of S. aureus Persisters 46. Barraud N, Buson A, Jarolimek W, Rice SA (2013) Mannitol Enhances Antibiotic Sensitivity of Persister Bacteria in Pseudomonas aeruginosa Biofilms. PLoS One 8: e84220. doi: 10.1371/journal.pone. 0084220 PMID: 24349568 47. Fey PD, Endres JL, Yajjala VK, Widhelm TJ, Boissy RJ, Bose JL, et al. (2013) A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio 4: e00537–00512. doi: 10.1128/mBio.00537-12 PMID: 23404398 48. Mechler L, Herbig A, Paprotka K, Fraunholz M, Nieselt K, Bertram R (2015) A novel point mutation pro- motes growth phase-dependent daptomycin tolerance in Staphylococcus aureus. Antimicrob Agents Chemother 59: 5366–5376. doi: 10.1128/AAC.00643-15 PMID: 26100694 49. References Hanberger H, Nilsson LE, Maller R, Isaksson B (1991) Pharmacodynamics of daptomycin and vanco- mycin on Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca2+ and albumin on these drugs. Antimicrob Agents Che- mother 35: 1710–1716. PMID: 1659305 50. Iordanescu S, Surdeanu M (1976) Two restriction and modification systems in Staphylococcus aureus NCTC8325. J Gen Microbiol 96: 277–281. 51. Herbert S, Ziebandt AK, Ohlsen K, Schäfer T, Hecker M, Albrecht D, et al. (2010) Repair of global regu- lators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates. Infect Immun 78: 2877–2889. doi: 10.1128/IAI.00088-10 PMID: 20212089 52. Götz F, Bannerman T., Schleifer KH. (2006) The Genera Staphylococcus and Macrococcus. The Pro- karyotes 4: 5–75. 53. Wick AN, Drury DR, Nakada HI, Wolfe JB (1957) Localization of the primary metabolic block produced by 2-deoxyglucose. J Biol Chem 224: 963–969. PMID: 13405925 54. Cavari BZ, Avi-Dor Y (1967) Effect of carbonyl cyanide m-chlorophenylhydrazone on respiration and respiration-dependent phosphorylation in Escherichia coli. Biochem J 103: 601–608. PMID: 4962086 55. Kwan BW, Valenta JA, Benedik MJ, Wood TK (2013) Arrested protein synthesis increases persister- like cell formation. Antimicrob Agents Chemother 57: 1468–1473. doi: 10.1128/AAC.02135-12 PMID: 23295927 56. Paczia N, Nilgen A, Lehmann T, Gatgens J, Wiechert W, Noack S (2012) Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms. Microb Cell Fact 11: 122. doi: 10.1186/1475-2859-11-122 PMID: 22963408 57. Somerville GA, Said-Salim B, Wickman JM, Raffel SJ, Kreiswirth BN, Musser JM (2003) Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for host-pathogen interac- tions. Infect Immun 71: 4724–4732. PMID: 12874354 58. Fischer A, Yang SJ, Bayer AS, Vaezzadeh AR, Herzig S, Stenz L, et al. (2011) Daptomycin resistance mechanisms in clinically derived Staphylococcus aureus strains assessed by a combined transcrip- tomics and proteomics approach. J Antimicrob Chemother 66: 1696–1711. doi: 10.1093/jac/dkr195 PMID: 21622973 59. Cotroneo N, Harris R, Perlmutter N, Beveridge T, Silverman JA (2008) Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus. Antimicrob Agents Chemother 52: 2223–2225. doi: 10. 1128/AAC.01410-07 PMID: 18378708 60. Jahreis K, Pimentel-Schmitt EF, Bruckner R, Titgemeyer F (2008) Ins and outs of glucose transport sys- tems in eubacteria. FEMS Microbiol Rev 32: 891–907. doi: 10.1111/j.1574-6976.2008.00125.x PMID: 18647176 61. PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 References Stülke J, Martin-Verstraete I, Zagorec M, Rose M, Klier A, Rapoport G (1997) Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT. Mol Microbiol 25: 65– 78. PMID: 11902727 62. Diep DB, Skaugen M, Salehian Z, Holo H, Nes IF (2007) Common mechanisms of target cell recogni- tion and immunity for class II bacteriocins. Proc Natl Acad Sci U S A 104: 2384–2389. PMID: 17284603 63. Humphries RM, Kelesidis T, Tewhey R, Rose WE, Schork N, Nizet V, et al. (2012) Genotypic and phe- notypic evaluation of the evolution of high-level daptomycin nonsusceptibility in vancomycin-resistant Enterococcus faecium. Antimicrob Agents Chemother 56: 6051–6053. doi: 10.1128/AAC.01318-12 PMID: 22948885 64. Wale LJ, Shelton AP, Greenwood D (1989) Scanning electronmicroscopy of Staphylococcus aureus and Enterococcus faecalis exposed to daptomycin. J Med Microbiol 30: 45–49. PMID: 2550648 65. Gustafson JE, Berger-Bachi B, Strassle A, Wilkinson BJ (1992) Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrob Agents Chemother 36: 566–572. PMID: 1320363 PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 14 / 15 Glucose/Daptomycin Killing of S. aureus Persisters 66. Bierbaum G, Sahl HG (1985) Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes. Arch Microbiol 141: 249–254. PMID: 4004448 67. Bayles KW (2014) Bacterial programmed cell death: making sense of a paradox. Nat Rev Microbiol 12: 63–69. doi: 10.1038/nrmicro3136 PMID: 24336185 68. Rice KC, Nelson JB, Patton TG, Yang SJ, Bayles KW (2005) Acetic acid induces expression of the Staphylococcus aureus cidABC and lrgAB murein hydrolase regulator operons. Journal of Bacteriology 187: 813–821. PMID: 15659658 69. Yang SJ, Rice KC, Brown RJ, Patton TG, Liou LE, Park YH, et al. (2005) A LysR-type regulator, CidR, is required for induction of the Staphylococcus aureus cidABC operon. Journal of Bacteriology 187: 5893–5900. PMID: 16109930 70. Seidl K, Muller S, Francois P, Kriebitzsch C, Schrenzel J, Engelmann S, et al. (2009) Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus. BMC Microbiol 9: 95. doi: 10.1186/1471- 2180-9-95 PMID: 19450265 71. Rice KC, Bayles KW (2003) Death's toolbox: examining the molecular components of bacterial pro- grammed cell death. Molecular Microbiology 50: 729–738. PMID: 14617136 72. Groicher KH, Firek BA, Fujimoto DF, Bayles KW (2000) The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance. Journal of Bacteriology 182: 1794–1801. PMID: 10714982 73. PLOS ONE \| DOI:10.1371/journal.pone.0150907 March 9, 2016 References Pascoe B, Dams L, Wilkinson TS, Harris LG, Bodger O, Mack D, et al. (2014) Dormant Cells of Staphy- lococcus aureus Are Resuscitated by Spent Culture Supernatant. PLoS One 9: e85998. doi: 10.1371/ journal.pone.0085998 PMID: 24523858 74. Van der Auwera P (1989) Ex vivo study of serum bactericidal titers and killing rates of daptomycin (LY146032) combined or not combined with amikacin compared with those of vancomycin. Antimicrob Agents Chemother 33: 1783–1790. PMID: 2556079 75. Kullar R, Chin JN, Edwards DJ, Parker D, Coplin WM, Rybak MJ (2011) Pharmacokinetics of single- dose daptomycin in patients with suspected or confirmed neurological infections. Antimicrob Agents Chemother 55: 3505–3509. doi: 10.1128/AAC.01741-10 PMID: 21502620 76. Friedman L, Alder JD, Silverman JA (2006) Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob Agents Chemother 50: 2137–2145. PMID: 16723576 77. Peleg AY, Miyakis S, Ward DV, Earl AM, Rubio A, Cameron DR, et al. (2012) Whole genome character- ization of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staph- ylococcus aureus. PLoS One 7: e28316. doi: 10.1371/journal.pone.0028316 PMID: 22238576 78. Gasch O, Camoez M, Dominguez MA, Padilla B, Pintado V, Almirante B, et al. (2013) Emergence of resistance to daptomycin in a cohort of patients with methicillin-resistant Staphylococcus aureus persis- tent bacteraemia treated with daptomycin. J Antimicrob Chemother. 15 / 15
https://openalex.org/W3194366761	https://flore.unifi.it/bitstream/2158/1244708/1/2021%20DeSantis%2cMaltagliati%2cPetrucci%20SOCI%20Cultural%20Distance%20of%20Foreigners.pdf	English	null	Retirement? Other Ways Out of the Labour Market Are Far More Worrying for Health: Results from a Matching Approach Study	Journal of aging and health	2,021	cc-by	14,347	* Gustavo De Santis gustavo.desantis@unifi.it Abstract The presence of foreigners in a host country is a contentious issue: opponents claim, among other things, that the cultural distance between them and natives is too large to permit inte‑ gration. However, it is impossible to ascertain whether this is true in the absence of a clear, standardised system for measuring cultural distance (whether it be by nationality, length of stay, educational level, etc.). In this paper, we argue that a recently proposed method, called DBS or Distance Between Strata, fits this scope. We present the methodology under a new light, investigate several of its properties, and apply it to two Italian surveys of 2011–13. Results indicate, first, that no group is homogeneous: Italians, for instance, display a clear North to South gradient. Second, foreigners are not all equally culturally distant from Ital‑ ian natives: the ranking of their cultural distances largely conforms to expectation. Finally, Italians with a foreign origin are, as expected, close to Italians tout court, which suggests that cultural convergence is taking place. Keywords Clusters · Cultural distance · Multidimensional scaling · Foreigners in Italy Keywords Clusters · Cultural distance · Multidimensional scaling · Foreigners in Italy So Close, So Far. The Cultural Distance of Foreigners in Italy Accepted: 25 March 2021 © The Author(s) 2021 Social Indicators Research Social Indicators Research Social Indicators Research https://doi.org/10.1007/s11205-021-02676-w https://doi.org/10.1007/s11205-021-02676-w ORIGINAL RESEARCH 1 DiSIA ‑ Dept. of Statistics, Computer Science, Applications, University of Florence, Florence, Italy * Gustavo De Santis gustavo.desantis@unifi.it 1 DiSIA ‑ Dept. of Statistics, Computer Science, Applications, University of Florence, Florence, Italy 1 Introduction Cultural distance is frequently cited as a potential source of problems: it complicates the management of multinational enterprises, reduces commercial exchanges, arouses sus‑ picion and sometimes hostility, and slows down the integration of foreigners in the host country, etc. There are also potential benefits to it: for instance, it attracts curious tourists, helps to address complex problems from different angles, and contrasts the tendency of the world towards standardisation, under the pressure of globalisation. But whilst these ben‑ efits sometimes occur, they seem to materialize only rarely.i i This issue is of great cultural and social significance, but research into it is still yet to progress because the abstract concepts of culture and cultural distance are difficult to define, impossible to measure directly (they are latent constructs), and hard to measure even indirectly, because the corresponding manifest variables are typically questionable and partial. 123456789) 1 3 56789) 3 G. De Santis et al. In the first part of this paper, we briefly discuss the notion of cultural distance, high‑ lighting two separate issues. The first regards the definition of culture and its empirical indicators. Despite the relevance of the topic, we do not devote much space to it because our data do not permit us any choice: we must stick to the few variables that we have (Sect. 3). The second issue relates to the type of distance to be measured, distinguishing between what we will call here the collective and the individual approach. The former, which is a far more widely accepted concept, relies on the idea of a ‘representative’ (e.g., average, or modal) value, such as national culture. Groups have their ‘characteris‑ tic’ traits, and distances are built. The latter approach, the one that we will adopt here, relates instead to the statistical notion of variability, and can be broadly described as an attempt to estimate how likely it is for individuals of group A to find someone ‘like them’ in group B. In Sect. 3 we present our data. By merging two similar but independent Italian sur‑ veys of 2011 and 2013, we try to assess the cultural distance between Italians and for‑ eigners in Italy. Unfortunately, overlap between the two surveys is rather limited; this forced us to use just a few survey questions, focused on what may be called ‘culturally- driven use of time (with a focus on selected items)’. 1 Introduction For brevity’s sake, we will some‑ times refer to this as (revealed, or actual manifestations of) culture. While we admit that this may be a misnomer, and that in all cases the variables that we have may at best reveal only part of the general picture (cultural distance between groups), we believe that this may nonetheless prove of interest for three main reasons.i The first is that we do not know fully what people’s internal beliefs are, but we may observe external social behaviour, which serves as a reflection of culture (and other var‑ iables, to be sure). This is precisely what our variables measure, albeit only partially and imperfectly. The second reason is that the issue (potential problems caused by the presence of ‘aliens’ in the country) is deeply felt in Italy and is a cause of considerable political con‑ troversy. Despite this, to the best of our knowledge, no scientific evidence has ever been produced on the subject. Is the cultural distance between natives and immigrants large or small? Is it increasing or decreasing over time? Is it the same for all groups of foreigners? The third reason lies in the DBS (Distance Between Strata) method, introduced just a few years ago (De Santis, Maltagliati, and Salvini, 2016). This presentation improves on the preceding ones (see also Mucciardi and De Santis, 2017; De Santis and Mucciardi, 2017) in a few respects (1) we illustrate its overall philosophy more clearly, (2) we show that treating ordinal scales as interval scales does not worsen, and arguably improves, the performance of the method (Sect. 4), and, perhaps most importantly, (2) we show that treating ordinal scales as interval scales does not worsen, and arguably improves, the performance of the method (Sect. 4), and, perhaps most importantly, (3) we argue that, within limits, two of the critical choices of this method (the clustering criterion and the number of clusters) are less problematic than it may seem: in a large majority of the applications that we subjected to our sensitivity analysis (Sect. 7), results do not essentially change. (3) we argue that, within limits, two of the critical choices of this method (the clustering criterion and the number of clusters) are less problematic than it may seem: in a large majority of the applications that we subjected to our sensitivity analysis (Sect. 7), results do not essentially change. 1 3 1 Introduction (#1.a) foreigners coming from EU countries should be culturally closer to Italians than foreigners coming from other parts of the world; Hy. (#1.a) foreigners coming from EU countries should be culturally closer to Italians than foreigners coming from other parts of the world; Hy. (#2) the cultural distance should be smaller for people with some common back‑ ground, be it in geographical or socio-economic development terms; Hy. (#3) based on a recent survey (Istat, 2020, p. 22), the Chinese, and less so also the Filipinos and the Peruvians living in Italy, should be the national groups most culturally distant from Italians, given their linguistic difficulties (in the first two cases) and their ten‑ dency to form homogeneous but also relatively close communities (all of them). Hy. (#3) based on a recent survey (Istat, 2020, p. 22), the Chinese, and less so also the Filipinos and the Peruvians living in Italy, should be the national groups most culturally distant from Italians, given their linguistic difficulties (in the first two cases) and their ten‑ dency to form homogeneous but also relatively close communities (all of them). Surprisingly, though based on clustering, the proposed DBS method does not require that clusters be understood, interpreted, or labelled. However, it is instructive to do so and to understand why certain national groups are close to or far from others; this is what Sect. 6 does. Section 7 reports the main results of our sensitivity analysis. We attempted several alter‑ native paths (e.g., different clustering procedures and different numbers of clusters) and we compared the outcomes. As we show, in the large majority of cases the results are highly consistent, which reinforces our claim that they are robust and not model-induced.i Substantive and methodological conclusions are drawn in the eighth and final Section 1 Introduction We produced a number of different results (Sects. 5 and 6). The first, and possibly the most significant, is that heterogeneity characterises all groups, including Italians (by macro-region of residence), who show a clear North–South gradient in their cul‑ turally-related use of time. The presence of heterogeneity suggests, on the one hand, 1 3 1 3 So Close, So Far. The Cultural Distance of Foreigners in Italy that stereotypes have scant scientific basis and, on the other, that measuring cultural distances on ‘average’ (or ‘representative’) profiles is questionable. that stereotypes have scant scientific basis and, on the other, that measuring cultural distances on ‘average’ (or ‘representative’) profiles is questionable. i Among people of foreign origin living in Italy, we can distinguish those who are no longer foreigners, because they acquired Italian nationality at some point, and those who remain foreigners by country (or world region) of origin. This is, we believe, of the greatest interest, both in itself (which foreigners are closer to Italians? Can we discern a pattern in the cultural distance between groups of foreigners?) and as a check of the method: we can‑ not prove that the method works, but we can show that it leads to very reasonable results. Indeed, assessing the validity of a (relatively) new measure is not an easy task. One way to do it is to see how closely its results conform to assumptions, or at least very strong expectations. We designed two checks based on our assumptions: the method needs to pass these tests to prove worthy of further consideration. These are:i Check (A) Italians (by macro-region of residence, of which we have five) should be culturally close to each other and more or less in geographical order, from North to South; Check (B) people of foreign origin but now with an Italian citizenship should be cultur‑ ally closer to Italians (with Italian origin – simply Italians from now on) than foreigners are; As our method passes both checks (Section 5) we deem it reliable, and we use it to test the following hypotheses: Hy. (#1) foreigners should form relatively close cultural clusters when they come from the same world region (e.g., Latin America or Africa), which implies that Hy. (#1) foreigners should form relatively close cultural clusters when they come from the same world region (e.g., Latin America or Africa), which implies that Hy. 2 The Notion and Use of Cultural Distance Culture may be defined in several ways, whether it be according to norms, values, or beliefs. In all cases, two broad approaches can be identified, which we will label ‘col‑ lective’ and ‘individual’, respectively. The former is more popular and intuitive. In a famous interview, for instance, the social psychologist Hofstede defines culture as the ‘collective programming of the mind that distinguishes one group or category of peo‑ ple from another’ (http://www.geerthofstede.nl/). Shortly after, in the same interview, 3 3 G. De Santis et al. Hofstede simplifies his own definition to ‘the unwritten rules of the social game’. This definition implies group homogeneity, which leads to the definition of cultural differ‑ ence as ‘a difference in human values that are rooted in national culture, which affect individuals’ attitude and behaviour’ (Poh Chuin, 2019). These two sources are not chosen at random: they derive from the business and administration world, where the main objective is to translate the overarching idea of culture into something measurable and useful, such as for managing multinational enter‑ prises (Hofstede, 1980) and understanding what guides international commerce (Poh Chuin, 2019), tourism (Ng et al., 2007; Petit & Seetaram, 2019), cultural consumption (Schwartz, 2013), including the success of TV programmes (Berg, 2017; Ksiazek & Webster, 2008), the tendency of customers to complain (Luria et al., 2016), the entre‑ preneurial success of women (Naidu & Chand, 2017), and human development (Gam‑ lath, 2017). These ideas are so deeply rooted in this context that Guiso et al. (2006) assert that ‘the ultimate validity of the notion of culture resides in its ability to enhance our understanding of economic behaviour’. However, cases when culture is evoked are also frequent outside the economic sphere. 2 The Notion and Use of Cultural Distance For instance: • terrorism in OECD countries tends to increase with immigration, but not among immigrants from culturally close countries (Böhmelt & Bove, 2020),fi • the necessity and difficulty of integrating (sometimes just assimilating) foreigners, whose cultural difference is perceived as a menace, has inspired and continues to inspire the immigration policy of several countries, such as Switzerland (Piñeiro & Haller, 2012),f • potential migrants are culturally different from the rest of the population in their origin countries, they select their destination country on the basis of cultural traits (Docquier et al., 2020), and they contribute to the cultural modification not only of the destination but also of the origin country thanks to the new ideas that they come into contact with and bring back home (Fargues, 2011), • democratic stability or breakdown may depend on the depth of internal cultural divi‑ sions (McDoom & Gisselquist, 2020), • national cultures that attribute greater importance to economic success tend to breed feelings of inferiority in the poor; this, while boosting economic activity, may prove socially disruptive (Steckermeier & Delhey, 2019). • national cultures that attribute greater importance to economic success tend to breed feelings of inferiority in the poor; this, while boosting economic activity, may prove socially disruptive (Steckermeier & Delhey, 2019). In all cases, the attempt to define and measure a ‘typical’ cultural profile (for instance, of a country) and to use this profile to ‘explain’ a dependent variable is rarely successful (Beugelsdijk et al., 2015; Shenkar, 2001), and, when it is, suspicions arise regarding the choice of the manifest variables used to define the latent (non-observable) dimension of culture. If these manifest variables are chosen with the dependent variable already in mind, as seems inevitable, results may be biased. While several indicators may have a weak theoretical basis (Ortega-Villa & Ley-Garcia, 2018), and some, includ‑ ing our own, are clearly driven more by data availability than by a-priori reasoning (e.g., Vieira et al., 2020), it is also reasonable to suggest that finding suitable empirical data is a particularly serious obstacle in this field. i Part of the problem may lie in the often implicit assumption that ‘there are larger cultural differences between countries than within countries’ (Ng et al., 2007). 2 The Notion and Use of Cultural Distance How‑ ever, this assumption may be questioned, which leads to the second line of research on culture, the ‘individual’ approach, which is the style of approach that we follow here. 1 3 So Close, So Far. The Cultural Distance of Foreigners in Italy We follow Beugelsdijk et al.’s (2015) suggestion to build variance-based measures of cultural distance, based on an idea that can be traced back to Byrne and Nelson’s (1965) observation that individuals are attracted by those who resemble them. We follow Beugelsdijk et al.’s (2015) suggestion to build variance-based measures of cultural distance, based on an idea that can be traced back to Byrne and Nelson’s (1965) observation that individuals are attracted by those who resemble them. The point can be better illustrated with an example. Imagine three countries ‒ A, B (reference), and C ‒ and imagine that, on some reliable scale of ‘national culture’, these three countries score, respectively, 90, 100, and 120. Imagine further that a multinational enterprise, based in B, wants to open a branch abroad, in country A or C, based on cultural distance. At first sight, country A is a better choice, because the average cultural distance is smaller, 10 instead of 20. However, imagine that the citizens of country A (potential employees and customers of our enterprise) are extremely homogeneous, so that each of them scores approximately 90, whereas those of country C are highly heterogeneous, scor‑ ing, say, between 80 and 160. In this case, there will be a reasonable share of them scoring approximately 100, thus proving culturally close to our enterprise, coming from B. The example could be made more realistic, by allowing for some variability within our (B) enterprise, because not all of its managers and staff will score exactly 100. However, the point is that, in the search for somebody who resembles us, it may be unwise to rely only on group (country) means; it is better to consider also (or even primarily) internal vari‑ ability. Beugelsdijk et al. (2015) do so using variance. However, when individual data are available, it seems preferable to measure cultural distance directly at the individual level, as we do here (Sect. 4).ii To summarise, two separate issues are worth considering. The first refers to the specific cultural aspects to include in the analysis. 1 There were, and still are, almost 200 different national groups of foreigners in Italy, from very large (e.g., more than 800 thousand Rumanians) to very small (two – units – coming from small and distant countries such as Oman, Bahrein, Palau, and Brunei). Data elaborated with the SAS software (version 9.4). 2 The Notion and Use of Cultural Distance Scholars who are unable to design their own sur‑ vey must work with the empirical data they have at hand, frequently less than optimal. Our case is no exception, as discussed in Sect. 3. The second issue is whether it is preferable to use the average-based (‘collective’) approach or the variability-based (‘individual’) approach. With the former, researchers look for the typical, or representative, individual of a group. With the latter, which is our preference, researchers look at the composition (or ‘mix’) of individuals within groups, and it is these internal distributions (not their averages, or any other representative value) that make two groups close, or far away, from each other. 3 Data: Foreigners, Former Foreigners, and Italians in Italy (2011–13) Our data come from two Istat surveys (Istat is the National Institute of Statistics in Italy). The first is the multipurpose survey on the Condition and Social Integration of Foreign Citizens in Italy – CSIFCI (‘Condizione e Integrazione Sociale dei Cittadini Stranieri’, 2011–12). At that time, foreigners in Italy accounted for approximately 4 million (6.8%) of all residents, and comparatively little was known about them. They were in principle included in all surveys, but they always ended up by being too few to be analysed sepa‑ rately, especially by origin.1 Adapting to their case the rationale (and the questionnaire) of the multipurpose surveys that Istat routinely implemented in those years, 25,326 of them were interviewed on this occasion. 1 3 3 G. De Santis et al. To compare foreigners in Italy with Italians, we used the ‘standard’ 2013 multipur‑ pose survey (ADL-Aspects of Daily Life, or ‘Aspetti della vita quotidiana’) with 20,275 respondents. Unfortunately, to merge the two surveys we were forced to use only the questions that were (virtually) identical in the two cases, which turned out to be relatively few (Table 1). Based on the 11 manifest variables that we could retain for the analysis, our (implicit) defi‑ nition of ‘culture’ focuses on the way respondents spend part of their (free) time, alone or with others, and on related activities: surfing the internet, or reading books, newspapers, and magazines; attending cultural events (cinema, theatre, music); talking about politics. Admittedly, it is not only their cultural background that matters here; the outcome depends also on several other variables that we cannot keep under control, such as their resources, and the ‘cultural supply’ of their environment. All of these factors are also linked to the respondents’ age, which is known. As our method is not well suited to include covariates (see Sect. 4), we needed to limit the hetero‑ geneity of the group retaining only respondents in their adult (18–64) years. As we wanted to deal with sufficiently large national groups, we further limited our sample to nations (or groups of supposedly homogenous nationalities) with at least 100 members, and we dis‑ carded the rest.2 These restrictions explain why, of the 20,275 respondents to the ADL-2013 Survey (Aspects of Daily Living), only 11,481 appear in Table 1: these we will call ‘Italians’ in the rest of this paper. 3 Data: Foreigners, Former Foreigners, and Italians in Italy (2011–13) Conversely, of the original 25,326 respondents of the 2011–12 ‘Foreign‑ ers’ Survey, only 15,007 survive in our analyses. Among these, there are 448 persons of foreign origin but with (later acquired) Italian nationality, and 14,559 foreigners. Globally, we have 26,488 observations (Table 2). 2 We also dropped the 4,251 respondents who were Italians since birth, but were nonetheless interviewed in the CSIFCI Survey, because they co-resided with foreigners. 4 The DBS Method (Distance Between Strata) As the DBS method that we will apply here has been extensively illustrated elsewhere (De Santis, Maltagliati, and Salvini, 2016; De Santis and Mucciardi, 2017; Mucciardi and De f Santis, 2017), what we are offering in these pages is an alternative, graphical illustration. Imagine that we observe 13 units (e.g., individuals) belonging to three different groups (from now on, ‘strata’): Triangles, Squares, and Circles. These strata can be all types of collective units: nations (e.g., Thailand, South Korea, and China–so that initials match), ethnic groups, soccer teams, etc. Imagine further that we classify these individuals on two manifest variables and that, given the outcome (Fig. 1), we form three clusters. Notice that our methodological contribution is not in the clustering method, but in what comes next: assuming that these clusters are meaningful, we use them to characterise the strata to which the clustered units belong. We do this by looking at how the strata-specific units distribute (proportionally) among clusters. The easiest way to conceptualise this is to imagine the strata as ‘planets’ in an N-dimen‑ sional space, where N + 1 is the number of clusters. The coordinates of these ‘planets’ (strata) are the observed proportions. For instance, ‘Circle’ has a third of its members in 1 1 3 1 3 So Close, So Far. 4 The DBS Method (Distance Between Strata) The Cultural Distance of Foreigners in Italy Table 1 Questions used for clustering taken from two Istat surveys, codes, and distribution of respondents (Italy, 2011–13) Bold indicates totals, of rows and columns CSIFCI Condition and Social Integration of Foreign Citizens in Italy; ADL Aspects of Daily Life Sources: Istat (2016a,b) Variable Respondents by survey Answers Code CSIFCI ADL All Use of PC Daily 1 5674 5158 10,832 Often 2 2560 1820 4380 Rarely 3 2054 1183 3237 Never 4 4719 3320 8039 Use of Internet Daily 1 5510 4859 10,369 Often 2 2597 2101 4698 Rarely 3 1981 1194 3175 Never 4 4919 3327 8246 Attendance to theatre (past 12 months) Never 1 13,715 9268 22,983 1–3 times 2 1046 1781 2827 4 + times 3 246 432 678 Attendance to cinema (12 months) Never 1 10,061 5652 15,713 1–3 times 2 2735 3431 6166 4 + times 3 2211 2398 4609 Attendance to sport events (12 months) Never 1 12,507 8138 20,645 1–3 times 2 1653 2017 3670 4 + times 3 847 1326 2173 Attendance to live music (12 months) Never 1 12,196 8372 20,568 1–3 times 2 2067 1906 3973 4 + times 3 744 1203 1947 Go dancing (all types) (12 months) Never 1 11,846 8568 20,414 1–3 times 2 1592 1458 3050 4 + times 3 1569 1455 3024 Read newspapers (usual week) No 1 8358 4782 13,140 1–2 days 2 3669 3333 7002 3 + days 3 2980 3366 6346 Read magazines Yes 1 4125 6233 10,358 No 2 10,882 5248 16,130 Read books (12 months) Yes 1 4606 5351 9957 No 2 10,401 6130 16,531 Talk about politics Daily 1 995 1875 2870 A few days/week 2 1985 3568 5553 Once a week 3 809 677 1486 A few days/month 4 1388 1585 2973 A few days/year 5 809 1142 1951 Never 6 9021 2634 11,655 Total 15,007 11,481 26,488 e 1 Questions used for clustering taken from two Istat surveys, codes, and distribution of respondents y, 2011–13) 1 3 G. De Santis et al. 4 The DBS Method (Distance Between Strata) Table 2 Respondents in our sample, by gender and national group, Italy 2011–13 Bold indicates (grand) total Country/Region Label Males Females All Country/Region Label Males Females All Italy—North West I_NW 1018 1058 2076 Albania alb 1016 872 1888 Italy—North East I_NE 1348 1396 2744 Bangladesh bgd 163 78 241 Italy—Centre I_CE 911 938 1849 Bulgaria bgr 92 187 279 Italy—South I_SO 1840 1834 3674 Brazil bra 31 125 156 Italy—Islands I_IS 564 574 1138 China chn 265 256 521 Germany deu 30 73 103 Algeria dza 75 32 107 Ecuador ecu 79 143 222 Egypt egy 89 40 129 France fra 37 71 108 Italians of foreign origin IT2 233 215 448 Ghana gha 66 53 119 India ind 167 104 271 Centre Nord Europe * NEU 77 114 191 Kosovo kos 60 58 118 South Europe * SEU 38 71 109 Sri Lanka lka 184 149 333 Former Yugosla‑ via * FYU 76 72 148 Morocco mor 724 588 1312 East Europe * EEU 24 136 160 Moldova mda 109 251 360 Middle East * MEA 50 70 120 North Macedonia mkd 137 111 248 Central America * CAM 57 156 213 Nigeria nga 59 68 127 South America * SAM 53 102 155 Pakistan pak 95 48 143 Africa AFR 170 172 342 Peru per 70 134 204 Philippines phl 172 235 407 Poland pol 121 417 538 Romania rou 1410 2085 3495 Russian Fed rus 15 101 116 Senegal sen 182 51 233 Serbia srb 53 61 114 Tunisia tun 278 154 432 All 12,368 14,120 26,488 Ukraine ukr 130 667 797 Table 2 Respondents in our sample, by gender and national group, Italy 2011–13 All countries listed in alphabetical order of their label. Groups of countries are in capital letters (cf. Figure 3) countries listed in alphabetical order of their label. Groups of countries are in capital letters (cf. Figure 3) Sources: Istat (2016a) for the Italians and Istat (2016b) for the rest Respondents aged 18 64 years and countries listed in alphabetical order of their label. Groups of countries are in capital letters (cf. Figure 3) Sources: Istat (2016a) for the Italians and Istat (2016b) for the rest. Respondents aged 18–64 years, and with no missing values in their answers to the questions listed in Table 1 Sources: Istat (2016a) for the Italians and Istat (2016b) for the rest. CAM: Barbados, Costa Rica, Cuba, Dominica, Dominican Republic, El Salvador, Guatemala, Honduras, Mexico, Panama 4 The DBS Method (Distance Between Strata) Respondents aged 18–64 years, a with no missing values in their answers to the questions listed in Table 1 NEU: Austria, Belgium, Denmark, Finland, Ireland, Luxembourg, Netherlands, Norway, Sweden, Switzer‑ land, UK SEU: Cyprus, Greece, Portugal, Spain SEU: Cyprus, Greece, Portugal, Spain FYU: Bosnia and Herzegovina, Montenegro, Croatia, Slovenia FYU: Bosnia and Herzegovina, Montenegro, Croatia, Slovenia EEU: Belarus, Czech Republic, Estonia, Hungary, Latvia, Lithuania, Slovakia MEA: Armenia, Azerbaijan, Georgia, Iraq, Israel, Jordan, Lebanon, Palestine, Syria, Turkey CAM: Barbados, Costa Rica, Cuba, Dominica, Dominican Republic, El Salvador, Guatemala, Honduras, Mexico, Panama CAM: Barbados, Costa Rica, Cuba, Dominica, Dominican Republic, El Salvador, Guatemala, Honduras, Mexico, Panama SAM: Argentina, Bolivia, Chile, Colombia, Paraguay, Uruguay, Venezuela SAM: Argentina, Bolivia, Chile, Colombia, Paraguay, Uruguay, Venezuela AFR: Angola, Benin, Burkina Faso, Burundi, Cameroon, Cape Verde, Congo, Côte d’Ivoire, Democratic Republic of the Congo, Djibouti, Eritrea, Ethiopia, Gambia, Guinea, Guinea-Bissau, Kenya, Liberia, Libya, Madagascar, Mali, Mauritania, Mauritius, Seychelles, Sierra Leone, Somalia, South Africa, Sudan, Tanza‑ nia, Togo AFR: Angola, Benin, Burkina Faso, Burundi, Cameroon, Cape Verde, Congo, Côte d’Ivoire, Democratic Republic of the Congo, Djibouti, Eritrea, Ethiopia, Gambia, Guinea, Guinea-Bissau, Kenya, Liberia, Libya, Madagascar, Mali, Mauritania, Mauritius, Seychelles, Sierra Leone, Somalia, South Africa, Sudan, Tanza‑ nia, Togo AFR: Angola, Benin, Burkina Faso, Burundi, Cameroon, Cape Verde, Congo, Côte d’Ivoire, Democratic Republic of the Congo, Djibouti, Eritrea, Ethiopia, Gambia, Guinea, Guinea-Bissau, Kenya, Liberia, Libya, Madagascar, Mali, Mauritania, Mauritius, Seychelles, Sierra Leone, Somalia, South Africa, Sudan, Tanza‑ nia, Togo 1 3 So Close, So Far. The Cultural Distance of Foreigners in Italy each cluster, while ‘Triangle’ has 50% of its members in cluster A, 25% in cluster B, and another 25% in cluster C. This leads to Fig. 2. In this case, a bi-dimensional space suffices to represent our strata despite the appar‑ ent tri-dimensionality of their coordinates (N + 1 = 3), because we work with proportions, the sum of which is one, which reduces the degrees of freedom (to two, in this example). When clusters are more than three, an exact bi-dimensional representation of the strata Fig. 1 Imaginary individu‑ als belonging to three strata (Triangles, Squares, and Circles), classified on the basis of two manifest variables and grouped in three clusters Source: illus‑ trative example. Strata could be, for instance, nations (e.g., T = Thailand; S = South Korea; C = China) Fig. 4 The DBS Method (Distance Between Strata) 2 Imaginary strata (Trian‑ gle, Square, and Circle) classified on the basis of the proportional distribution of their members among the previously-formed clusters Source: see Fig. 1 Fig. 1 Imaginary individu‑ als belonging to three strata (Triangles, Squares, and Circles), classified on the basis of two manifest variables and grouped in three clusters Source: illus‑ trative example. Strata could be, for instance, nations (e.g., T = Thailand; S = South Korea; C = China) Fig. 2 Imaginary strata (Trian‑ gle, Square, and Circle) classified on the basis of the proportional distribution of their members among the previously-formed clusters Source: see Fig. 1 each cluster, while ‘Triangle’ has 50% of its members in cluster A, 25% in cluster B, and another 25% in cluster C. This leads to Fig. 2.fi each cluster, while ‘Triangle’ has 50% of its members in cluster A, 25% in cluster B, and another 25% in cluster C. This leads to Fig. 2.fi In this case, a bi-dimensional space suffices to represent our strata despite the appar‑ ent tri-dimensionality of their coordinates (N + 1 = 3), because we work with proportions, the sum of which is one, which reduces the degrees of freedom (to two, in this example). When clusters are more than three, an exact bi-dimensional representation of the strata (planets in a hyper-space with N + 1 dimensions) becomes impossible. However, accept‑ able approximations are generally offered by ad-hoc dimension-reducing statistical tech‑ niques, such as factor analysis or, as in our case, MDS – multidimensional scaling. This bi-dimensional plot, incidentally, is not a necessary ingredient of the DBS method, but it helps to understand its results.i The final step consists of calculating how far these ‘planets’ (our strata) are from each other (in Fig. 2), which can be done in the simplest possible way, i.e. calculating Euclidean distances, the extension of Pythagoras’s formula to an N-dimensional space.f Note how different this approach is from using strata’s average values. In Fig. 1, for instance, one could also easily calculate the average of each stratum on both dimensions 1 3 G. De Santis et al. (manifest variables) and use these three barycentres as strata-representative values. In so doing, however, internal variability gets lost: one would obtain the same results if the units that belong to the same stratum had different values with the same average. As mentioned, Beugelsdijk et al. 4.1 Cautions Caution is needed in interpreting results. First, the maximum possible distance between strata is √2, regardless of the number of clusters (N + 1), and the maximum empirical dis‑ tance that has ever been found until now is even smaller, approximately 1.2 (Mucciardi and De Santis, 2017, Table 3). Even more importantly, results are relative, not only to the manifest variables, but also to the terms of comparison. In Figs. 1 and 2, for instance, if we added a new stratum (Diamonds), and if its members were culturally distant from all those previously observed, either the number of clusters would change, or, with the same number of clusters, a different distribution of respondents among clusters would be observed. In all cases, the coordinates of the previous ‘planets’ (or strata: Triangles, Squares, and Circles) would change and so would the distances between them. In this example, remembering that the maximum possible distance is √2, these distances would shrink, to ‘accommodate’ the heterogeneous newcomer in the new strata space. In other words, Triangles, Squares, and Circles would now appear closer to one another, and all of them far from Diamonds. This happens because the method builds a strata space (of the type shown in Fig. 2) that adapts automatically to what is analysed. For instance, Poland may appear an outlier in a map focused on the distance between France and the UK, but very close to France and the UK in a map that also includes China, and all four of them very close to one another if the chart also includes the Moon.i A few more observations are in order. The first is that manifest variables can be consid‑ ered all together (as we will do here) or subdivided into assumedly homogenous categories, ideally referring to the same cultural sphere, e.g., ethics, family, and religion. In the latter case, cultural distances will be evaluated by ‘area’, or ‘sphere’.fi Secondly, the members of the strata must be sufficiently numerous for their distribution among clusters to be robust to random variations. Unfortunately, as the number of clusters is a priori unknown (see below), it is not easy to determine the minimum number of mem‑ bers of each stratum that is required for the method to work properly. 4 The DBS Method (Distance Between Strata) (2015) try to correct for this loss of information by allowing for internal variance, thus creating a sort of ‘acceptance zone’ around these barycentres: units that lie in that zone are reasonably similar to the nationally representative values. We submit that the proposed DBS (Distance Between Strata) method, while moving along the same lines, performs better, because it preserves individual values, and lets the strata space (of Fig. 2) reflect the exact distribution of respondents among clusters. 1 3 4.1 Cautions In this paper, we set this minimum to 100 and we worked (primarily) with five clusters; this ensures we remain in the safety zone and, besides, our sensitivity tests (when we progressively increased the number of clusters up to 70–Sect. 7) confirm that our results are extremely robust. i The third observation is that, beyond being members of a stratum, respondents have other characteristics (e.g., sex, age, and education) that will affect their answers (manifest variables), interfering with the connection of interest (stratum-culture). In ‘normal’ mod‑ els, one would introduce these characteristics as covariates, to keep them ‘under control’. However, this case is different, because individuals characterise their strata only indirectly, by forming clusters, and the inclusion of individual covariates becomes impossible at this 1 3 So Close, So Far. The Cultural Distance of Foreigners in Italy Table 3 Respondents by national group (stratum) and five clusters (Cl, A to E), proportions and total. CL Cluster. The last column displays the distances from Italy (average). With 42 national groups, the to number of distances is (42·41/2 =) 861. Sources and labels: see Table 2 Italics denote proportions 4.1 Cautions Cu tural distance of each stratum from Italy (2011–13) Area/Group Label Cl_A Cl_B Cl_C Cl_D Cl_E No Distance from Italy Italy, North-West I_NW 0.395 0.168 0.091 0.125 0.221 2076 0.053 Italy, North-East I_NE 0.439 0.168 0.078 0.109 0.207 2744 0.082 Italy, Centre I_CE 0.427 0.172 0.107 0.117 0.177 1849 0.057 Italy, South I_SO 0.309 0.227 0.164 0.132 0.168 3674 0.091 Italy, Islands I_IS 0.336 0.224 0.153 0.113 0.174 1138 0.064 ITALY ITA 0.377 0.193 0.120 0.121 0.189 11,481 Africa AFR 0.158 0.295 0.205 0.047 0.295 342 0.287 Albania alb 0.150 0.247 0.281 0.091 0.231 1888 0.288 Bangladesh bgd 0.050 0.373 0.340 0.037 0.199 241 0.442 Bulgaria bgr 0.161 0.258 0.262 0.047 0.272 279 0.289 Brazil bra 0.308 0.167 0.096 0.026 0.404 156 0.248 Central America CAM 0.249 0.239 0.141 0.014 0.357 213 0.242 China chn 0.044 0.284 0.207 0.012 0.453 521 0.456 Germany deu 0.437 0.107 0.029 0.068 0.359 103 0.226 Algeria dza 0.206 0.336 0.215 0.056 0.187 107 0.251 Ecuador ecu 0.149 0.293 0.198 0.045 0.315 222 0.300 East Europe EEU 0.206 0.200 0.119 0.069 0.406 160 0.281 Egypt egy 0.217 0.248 0.171 0.062 0.302 129 0.218 France fra 0.472 0.139 0.037 0.352 108 0.245 Former Yugoslavia FYU 0.203 0.284 0.155 0.095 0.264 148 0.215 Ghana gha 0.059 0.345 0.286 0.050 0.261 119 0.402 India ind 0.114 0.303 0.343 0.030 0.210 271 0.374 Italians (foreign origin) it2 0.286 0.203 0.132 0.078 0.301 448 0.152 Kosovo kos 0.153 0.220 0.229 0.034 0.364 118 0.318 Sri Lanka lka 0.075 0.306 0.216 0.027 0.375 333 0.396 Moldova mda 0.258 0.244 0.108 0.069 0.319 360 0.191 Middle East MEA 0.200 0.225 0.150 0.017 0.408 120 0.304 North Macedonia mkd 0.117 0.214 0.407 0.056 0.206 248 0.394 Morocco mor 0.098 0.302 0.376 0.063 0.161 1312 0.399 North-Central Europe NEU 0.450 0.073 0.042 0.016 0.419 191 0.299 Nigeria nga 0.134 0.339 0.283 0.071 0.173 127 0.331 Pakistan pak 0.154 0.252 0.301 0.042 0.252 143 0.310 Peru per 0.162 0.324 0.142 0.049 0.324 204 0.295 Philippines phl 0.098 0.224 0.253 0.034 0.391 407 0.380 Poland pol 0.199 0.260 0.173 0.045 0.323 538 0.251 Romania rou 0.142 0.273 0.244 0.054 0.287 3495 0.302 Russia (Fed) rus 0.216 0.190 0.112 0.026 0.457 116 0.327 South America SAM 0.284 0.213 0.103 0.071 0.329 155 0.177 Senegal sen 0.159 0.335 0.193 0.069 0.245 233 0.281 South Europe SEU 0.339 0.128 0.064 0.046 0.422 109 0.262 Serbia srb 0.167 0.219 0.254 0.035 0.325 114 0.298 Tunisia tun 0.104 0.329 0.285 0.100 0.183 432 0.347 Ukraine ukr 0.141 0.301 0.226 0.065 0.267 797 0.297 Tot Tot 26,488 Italics denote proportions CL Cl Th l l di l h di f I l ( ) Wi h 42 i l h le 3 Respondents by national group (stratum) and five clusters (Cl, A to E), proportions and total. 4.1 Cautions Cul‑ l distance of each stratum from Italy (2011–13) CL Cluster. The last column displays the distances from Italy (average). With 42 national groups, the total number of distances is (42·41/2 =) 861. Sources and labels: see Table 2 1 3 G. De Santis et al. stage, while the introduction of a summary measure (e.g., the mean age of the members of the cluster) would contradict the individual nature of the approach (and lead to insignifi‑ cant results – not shown here). stage, while the introduction of a summary measure (e.g., the mean age of the members of the cluster) would contradict the individual nature of the approach (and lead to insignifi‑ cant results – not shown here). Combining covariates (e.g., nationality and sex) and creating more homogeneous strata is a possibility, but their number increases multiplicatively, and soon becomes incom‑ patible with the requirement mentioned above, that a reasonable balance be maintained between the number of observations per (homogeneous) stratum and the number of clus‑ ters. Another possibility is to selects units (respondents, in our case) that are similar in structural terms, as we did here (respondents aged 18–64 years). Fourthly, in this paper, we treated ordinal, Likert-type scales as interval scales, using the values indicated in Table 1. We did this for several reasons. Firstly, we did it because, as discussed by De Santis, Maltagliati, and Salvini (2016), to treat these answers as nominal requires long and painstaking transformations (Jaccard’s index of similarity), which exceed the computing capacity of most calculators and require ad-hoc solutions. Secondly, we did it because this choice is likely to be close to the average respondent’s perceptions, i.e. non- distortive, and is in all cases frequently adopted in cases like this (e.g., Carifio & Perla, 2007; Spitzer, Greulich, & Hammer, 2018; Wu & Leung, 2017). Finally, and perhaps most impor‑ tantly, because none of these variables is analysed directly. In combination with others, they are used to form clusters and (almost) any transformation that preserves the ranking of the answers leads to similar results (Harwell & Gatti, 2001; Hennig et al., 2015; Walesiak & Dudek, 2010). The fifth qualification is that clustering involves other arbitrary choices, among which are the questions of which clustering method to adopt and how many clusters to form. 4.1 Cautions Cer‑ tain methods, among them EM (De Santis and Mucciardi, 2017 and Mucciardi and De San‑ tis, 2017), have the advantage of an incorporated stopping rule (which depends itself on an arbitrary parameter predefined by the researcher), but they are not available in all statistical packages. Here, after considering several options, we eventually opted for the Ward method (minimal internal variance within clusters), because it tends to create clusters of compara‑ ble dimensions, a non-trivial advantage for the DBS method, which looks precisely at how the members of a certain stratum distribute among clusters. As the ultimate purpose of the procedure is to construct a matrix of distances between strata (in this case, between national groups, of Italians and foreigners living in Italy) we checked that different clustering meth‑ ods (and different numbers of clusters within each of them) resulted in comparable distances (Sect. 7). Table 4 Average cultural distances within and between Italian macro-regions (2011–13) 5 On the Heterogeneity of Foreigners (and Italians) in Italy Table 5 Cultural distance between Italy, North-Western and Southern Italy, and selected world regions (2011–13) The countries included in NEU and SEU are listed at the end of Table 2 Source: Istat (2016a,2016b) Area Label Distance from Italy All NW South Germany deu 0.226 0.178 0.299 France fra 0.245 0.206 0.319 North-Central Europe NEU 0.299 0.256 0.367 South Europe SEU 0.262 0.228 0.304 Other foreign origin 0.309 0.329 0.260 Table 5 Cultural distance between Italy, North-Western and Southern Italy, and selected world regions (2011–13) preserving most of the initial information (the correlation between the original and the transformed distances is 0.992).i preserving most of the initial information (the correlation between the original and the transformed distances is 0.992).i Let us first check whether the method passes the two tests that we had set from the beginning: A. Are Italians culturally closer to each other than to all other nationalities? Are the five macro-regions of residence more or less in geographical order, from North to South? A. Are Italians culturally closer to each other than to all other nationalities? Are the five macro-regions of residence more or less in geographical order, from North to South? The answer to both questions is yes. Italian macro-regions form a separated and rel‑ atively homogeneous subgroup (top left of Figure 3). Within that, the regions of the North-Centre form a sub-cluster, with strong internal homogeneity, clearly separated from the other, composed of South and Islands (Table 4). However, despite this marked cultural divide, regardless of the region of residence, Italians are closer to other Italians than they are to any other national group. B. Are people of foreign origin, but now with an Italian citizenship, culturally closer to Italians (with Italian origin) than foreigners are? B. Are people of foreign origin, but now with an Italian citizenship, culturally closer to Italians (with Italian origin) than foreigners are? Yes. The distance between Italians and these ‘late’ Italians is 0.152 (Table 3). This number means nothing in itself. However, compared to the others of Table 3, it tells us that this distance is larger than, although comparable to, that between Italians (Table 4), but smaller than that between Italians and any other foreign group.i We can move on to determining whether our expectations are satisfied. 1. Do foreigners form relatively close cultural clusters when they come from the same world region (e.g., Latin America or Africa)? Table 5 Cultural distance between Italy, North-Western and Southern Italy, and selected world regions (2011–13) 5 On the Heterogeneity of Foreigners (and Italians) in Italy Several results emerge from our analysis. For the sake of brevity, in this section we will present only those that we obtained from what we believe is the best clusterisation method: Ward, with five clusters. Robustness checks are discussed in Sect. 7. 3 1 So Close, So Far. The Cultural Distance of Foreigners in Italy Fig. 3 Bi-dimensional representation (with MDS – multidimensional scaling) of the 861 distances between the national groups listed in Table 3. Italy 2011–13. Note: with 42 national groups, there are (42·41/2 =) 861 distances. Sources and labels: see Table 2 Fig. 3 Bi-dimensional representation (with MDS – multidimensional scaling) of the 861 distances between the national groups listed in Table 3. Italy 2011–13. Note: with 42 national groups, there are (42·41/2 =) 861 distances. Sources and labels: see Table 2 Fig. 3 Bi-dimensional representation (with MDS – multidimensional scaling) of the 861 distances between the national groups listed in Table 3. Italy 2011–13. Note: with 42 national groups, there are (42·41/2 =) 861 distances. Sources and labels: see Table 2 Table 4 Average cultural distances within and between Italian macro-regions (2011–13) Source: Istat (2016a, b) Area Average distance Within North and Centre 0.051 Within South and Islands 0.035 Between these two areas 0.137 Between the five macro regions 0.101 Table 4 Average cultural distances within and between Italian macro-regions (2011–13) Table 4 Average cultural distances within and between Italian macro-regions (2011–13) The single most important result is displayed in Table 3, where we present the dis‑ tribution of our observations among the five clusters that we formed (A–E), and where strata (national groups) appear in alphabetical order of their label. While Table 3 con‑ tains all the information, Fig. 3 is arguably easier to interpret: using MDS (multidi‑ mensional scaling), we projected the distances of Table 3 on a bi-dimensional plan, 3 1 G. De Santis et al. 1 3 5 On the Heterogeneity of Foreigners (and Italians) in Italy Are immigrants from EU countries cultur‑ ally closer to Italians than immigrants coming from elsewhere? 1. Do foreigners form relatively close cultural clusters when they come from the same world region (e.g., Latin America or Africa)? Are immigrants from EU countries cultur‑ ally closer to Italians than immigrants coming from elsewhere? The answer is yes in both cases. Let us start with the second question. The cultural distances between Italy and the (few) available European nationalities range between .226 (Germany) and .299 (other northern European countries), but the distance between Italy and other (non-European) nationalities is larger, .309 (Table 5). Closer inspection 1 3 So Close, So Far. The Cultural Distance of Foreigners in Italy Table 6 Regression of cultural distance on geographical and development distance, selected countries (and nationalities of foreigners in Italy 2011–13) Source: for cultural distances, Istat (2016a,b). For the HDI index, http://hdr.undp.org/en/content/2019- human-development-index-ranking. Geographical distance computed by the authors, using the geographical coordinates of capital cities All distances Italy vs other countries Coeff Std error t statistics Coeff Std error t statistics Intercept 0.1767 0.0124 14.20 0.2963 0.0280 10.59 Geographical distance 0.0002 0.0014 0.15 0.0065 0.0042 1.55 HDI Distance 0.4124 0.0640 6.44 0.2545 0.1511 1.68 Adj. R-squared: 0.089 0.158 Observations 406 (29 countries) 28 (28 countries) le 6 Regression of cultural distance on geographical and development distance, selected countries (and onalities of foreigners in Italy 2011–13) rce: for cultural distances, Istat (2016a,b). For the HDI index, http://hdr.undp.org/en/content/2019- man-development-index-ranking. Geographical distance computed by the authors, using the geographical rdinates of capital cities of Figure 3 and Table 5 reveals that the Centre-North of Italy is closer to the rest of Europe than Southern Italy, which is instead closer to immigrants from other origins (i.e., from less developed countries). This, too, is consistent with the available informa‑ tion on the degree of proximity of the central and northern part of Italy with the rest of Western Europe, not only in geographical but also in economic and commercial terms. As for the other countries, a graphical answer to the question of their cultural dis‑ tance can be found in Figure 3 where we encircled nationalities with a common ori‑ gin. Of course, there are exceptions and overlaps: for instance, Egypt is far from other Northern African countries, while Eastern Europe (in particular Romania and Bulgaria) appears to be very close to Africa. 5 On the Heterogeneity of Foreigners (and Italians) in Italy Overall, however, expectations are satisfied: geo‑ graphically homogeneous countries tend to be characterised by smaller cultural dis‑ tances (see also Table 6). 2. Is the cultural distance that we obtain positively correlated with the geographical and ‘development’ distance (the latter measured with, for instance, the HDI – Human Devel‑ opment Index)? 2. Is the cultural distance that we obtain positively correlated with the geographical and ‘development’ distance (the latter measured with, for instance, the HDI – Human Devel‑ opment Index)? 2. Is the cultural distance that we obtain positively correlated with the geographical and ‘development’ distance (the latter measured with, for instance, the HDI – Human Devel‑ opment Index)? We expected a similar background (in terms of geographic proximity, or socio-eco‑ nomic development of the country of origin, or both) to translate into a similar cultur‑ ally-driven use of time (as measured by our empirical indicators). Table 6 shows that this expectation is only partly satisfied. For the 29 single countries listed in Table 2, we measured the corresponding distances in terms of ‘culture’ (our dependent variable), geography (distance between capital cities, in kilometres), and development (absolute value of the difference of the HDIs for each pair of countries). We did this twice: both for the entire set of countries, with all the possible (29·28/2=406) distances, and for Italy only (28 distances between Italy and the other countries on the list). In both cases, 1 3 G. De Santis et al. Fig. 4 Cultural distance from Italians (2011–12) and proportion of students (2nd generation immigrants) declaring that they feel Italian (2014–15)Note: the nationalities displayed in this figure are those reported in Istat (2020), Table 2.1, p. 23 Source: As for the cultural distance, Istat (2016a,b); as for the proportion of students (of secondary schools, aged 11–18 years) declaring they feel Italian (out of a representative sample of over 42 thousand respondents), Istat (2020, p. 23) Fig. 4 Cultural distance from Italians (2011–12) and proportion of students (2nd generation immigrants) declaring that they feel Italian (2014–15)Note: the nationalities displayed in this figure are those reported in Istat (2020), Table 2.1, p. 23 Source: As for the cultural distance, Istat (2016a,b); as for the proportion of students (of secondary schools, aged 11–18 years) declaring they feel Italian (out of a representative sample of over 42 thousand respondents), Istat (2020, p. 23) the results are scarcely conclusive: the independent variables have the expected sign, but they are generally not significant (exception: ‘development distance’ for the whole set of countries), and the goodness of fit is very low. 3. Are the Chinese, and less so also the Filipinos and the Peruvians, particularly far from the Italians, as another Istat survey suggests (Istat, 2020)? 3. Are the Chinese, and less so also the Filipinos and the Peruvians, particularly far from the Italians, as another Istat survey suggests (Istat, 2020)? 3 Or (not shown here) give similar indications of cultural integration, such as ‘My Italian is good’, ‘I think in Italian’, ‘I have Italian friends’, and ‘I go to parties and gatherings organised by Italians’. 4 These immigrants (from Algeria, Egypt, Kosovo, Middle East, Morocco, Pakistan, Senegal, and Tunisia) are called ‘Muslims’ in Fig. 5, for brevity’s sake. Note, however, that we ignore their individual religious affiliation. 2. Is the cultural distance that we obtain positively correlated with the geographical and ‘development’ distance (the latter measured with, for instance, the HDI – Human Devel‑ opment Index)? Note, first, that the 2014–15 Istat survey (published in 2020) that we are using here as a term of comparison is focused on a different target of respondents: the second genera‑ tion of foreigners (born in Italy by foreign parents, or who immigrated at young ages), attending junior and senior secondary school (11–18 years). The connection between this group of selected youngsters and the group that we analyse in the rest of the paper (foreigners aged 18–64 years) is rather loose, and relies on two assumptions: that cer‑ tain cultural traits are preserved and passed on to the next generation and that they are reflected in the 11 empirical variables that we use. With these cautions in mind, our results seem to be in line with expectations (Figure 4): the greater the cultural distance of the group, the smaller the proportion of young respondents from that group who declare that ‘they feel Italian’.3 1 3 1 3 So Close, So Far. The Cultural Distance of Foreigners in Italy Fig. 5 Unit distribution among five clusters of four selected strata: Italy-South and Italy-Islands, China, and immigrants from Muslims countries (‘Muslims’). Ward method. Note: CL = Cluster. ‘Muslims’ are immi‑ grants from Algeria, Egypt, Kosovo, Middle East, Morocco, Pakistan, Senegal, and Tunisia Fig. 5 Unit distribution among five clusters of four selected strata: Italy-South and Italy-Islands, China, and immigrants from Muslims countries (‘Muslims’). Ward method. Note: CL = Cluster. ‘Muslims’ are immi‑ grants from Algeria, Egypt, Kosovo, Middle East, Morocco, Pakistan, Senegal, and Tunisia Fig. 5 Unit distribution among five clusters of four selected strata: Italy-South and Italy-Islands, China, and immigrants from Muslims countries (‘Muslims’). Ward method. Note: CL = Cluster. ‘Muslims’ are immi‑ grants from Algeria, Egypt, Kosovo, Middle East, Morocco, Pakistan, Senegal, and Tunisia 6 Interpreting Clusters Note: the darker the circle, the more that activity is practised by the members of that cluster PC 1.7 0.1 0.0 0.0 2.2 Internet 1.7 0.1 0.0 0.0 2.3 Theathre 2.3 0.2 0.2 0.7 1.1 Cinema 1.7 0.5 0.3 0.5 1.5 Sports events 1.9 0.4 0.3 0.9 1.2 Live music 1.9 0.4 0.2 0.8 1.3 Dancing 1.4 0.6 0.2 0.5 1.7 Newspapers 1.7 0.7 0.5 1.0 0.9 Magazines 1.5 0.8 0.6 1.1 0.9 Books 1.6 0.7 0.4 0.9 1.2 Politics 3.1 0.0 0.0 2.6 0.0 Clusters CL_A CL_B CL_C CL_D CL_E Fig. 6 Illustrative characterisation of the main characteristics of the five clusters (A to E). Ward method. Note: the darker the circle, the more that activity is practised by the members of that cluster Fig. 6 Illustrative characterisation of the main characteristics of the five clusters (A to E). Ward meth Note: the darker the circle, the more that activity is practised by the members of that cluster Fig. 6 Illustrative characterisation of the main characteristics of the five clusters (A to E). Ward method. Note: the darker the circle, the more that activity is practised by the members of that cluster respondents are in cluster D, which hosts only 1% of the Chinese. Conversely, 45% of the Chinese are in cluster E, which includes only about 17% of the Italians. Immigrants from Muslim countries lie somewhere in between: if we considered only the members of this group belonging to cluster A (12%), we should conclude that they are closer to the Chinese. Instead, if we focused only on the proportion belonging to cluster E (21%), we should come to the opposite conclusion. In fact, their correct allocation is better assessed by looking at the entire distribution, and it transpires that immigrants from Mus‑ lim countries are approximately halfway between southern Italians and the Chinese. What characterises the members of these clusters is illustrated in Fig. 6 and Table 7. Cluster A, for instance, is composed of respondents who frequently use personal com‑ puters, access the internet, go out (theatre, cinema, music, and sport events), read books and newspapers, and like to talk about politics. More than 30% of Italians are like this. Cluster C is just the opposite: the members of this group do very little of any of these activ‑ ities–and this describes more than 30% of immigrants from Muslim countries. 6 Interpreting Clusters Cluster E groups respondents who, while active with computers and on the internet (even more than those of cluster A, actually), limit their outings to dancing, and rarely, if ever, talk about politics. 45% of the Chinese are more or less like this. The other clusters can be character‑ ised in a similar way, by looking both at Fig. 6 and Table 7. 6 Interpreting Clusters The DBS method does not require scholars to interpret and label the resulting clusters; indeed, we reached our conclusions on the relative cultural distance of the various groups of foreigners from one another, and from Italians, without commenting on our clusters.fi However, once a sufficiently reliable result has been reached, it may be worthwhile to stop and consider what the members of a given cluster have in common and why certain national groups differ from, or are instead close to, others. f Let us start with Fig. 5, where we compare four groups, two of which are very similar (Italians, and on top of that, both from the southern part of the country), while the others are immigrants, from China and, separately, from predominantly Muslims countries.4 The two Italian subgroups are very similar: not because they are internally homogeneous, but because the five typologies that we identify (i.e. people belonging to clusters A–E) have very similar proportions in the two areas. Conversely, the Chinese appear to be culturally distant from this standard because the distribution of their members among the five clusters is markedly different. For instance, more than 30% of (southern) Italians are in cluster A, where only less than 5% of the Chinese can be found. Approximately 12% of the Italian 1 3 G. De Santis et al. PC 1.7 0.1 0.0 0.0 2.2 Internet 1.7 0.1 0.0 0.0 2.3 Theathre 2.3 0.2 0.2 0.7 1.1 Cinema 1.7 0.5 0.3 0.5 1.5 Sports events 1.9 0.4 0.3 0.9 1.2 Live music 1.9 0.4 0.2 0.8 1.3 Dancing 1.4 0.6 0.2 0.5 1.7 Newspapers 1.7 0.7 0.5 1.0 0.9 Magazines 1.5 0.8 0.6 1.1 0.9 Books 1.6 0.7 0.4 0.9 1.2 Politics 3.1 0.0 0.0 2.6 0.0 Clusters CL_A CL_B CL_C CL_D CL_E Fig. 6 Illustrative characterisation of the main characteristics of the five clusters (A to E). Ward method. 1 3 7 Sensitivity Analysis This section is devoted to a sensitivity analysis, which shows that several plausible alterna‑ tive choices would not have meaningfully affected our results.i f The first question that we will explore is on the best number of clusters. For each clustering method (e.g., Ward), we know the proportion of the total variance explained 1 3 So Close, So Far. 7 Sensitivity Analysis The Cultural Distance of Foreigners in Italy Table 7 Clustering variables, distribution of units by five (Ward) clusters, and relative proportions Variable Answers Values Absolute frequencies by cluster % Relative proportions within clusters A B C D E Tot A B C D E Use of PC Daily 1 4770 256 3 5803 10,832 41 1.742 0.101 0.000 0.003 2.214 Often 2 1466 2307 9 42 556 4380 17 1.324 2.248 0.011 0.113 0.525 Rarely 3 430 2088 174 501 44 3237 12 0.525 2.753 0.288 1.831 0.056 Never 4 30 1555 4756 1693 5 8039 30 0.015 0.826 3.173 2.491 0.003 Use of Internet Daily 1 4537 142 3 5687 10,369 39 1.731 0.058 0.000 0.003 2.267 Often 2 1652 2313 3 52 678 4698 18 1.391 2.101 0.003 0.131 0.597 Rarely 3 462 2058 165 448 42 3175 12 0.576 2.767 0.279 1.669 0.055 Never 4 45 1693 4771 1736 1 8246 31 0.022 0.876 3.103 2.491 0.001 Attendance to theatre (past 12 months) Never 1 4907 5820 4782 2019 5455 22,983 87 0.845 1.081 1.116 1.039 0.981 1–3 times 2 1392 351 136 180 768 2827 11 1.948 0.530 0.258 0.753 1.123 4 + times 3 397 35 21 40 185 678 3 2.316 0.220 0.166 0.698 1.128 Attendance to cinema (12 months) Never 1 2541 4425 4170 1646 2931 15,713 59 0.640 1.202 1.423 1.239 0.771 1–3 times 2 2153 1232 550 414 1817 6166 23 1.381 0.853 0.478 0.794 1.218 4 + times 3 2002 549 219 179 1660 4609 17 1.718 0.508 0.255 0.459 1.489 Attendance to sport events (12 months) Never 1 4263 5437 4533 1795 4617 20,645 78 0.817 1.124 1.178 1.029 0.924 1–3 times 2 1412 563 268 284 1143 3670 14 1.522 0.655 0.392 0.915 1.287 4 + times 3 1021 206 138 160 648 2173 8 1.859 0.405 0.341 0.871 1.233 Attendance to live music (12 months) Never 1 4272 5368 4613 1808 4507 20,568 78 0.822 1.114 1.203 1.040 0.906 1–3 times 2 1480 639 267 292 1295 3973 15 1.474 0.686 0.360 0.869 1.347 4 + times 3 944 199 59 139 606 1947 7 1.918 0.436 0.163 0.845 1.287 Go dancing (all types) (12 months) Never 1 4572 5182 4580 1959 4121 20,414 77 0.886 1.083 1.203 1.135 0.834 1–3 times 2 1063 574 232 152 1029 3050 12 1.379 0.803 0.408 0.590 1.395 4 + times 3 1061 450 127 128 1258 3024 11 1.388 0.635 0.225 0.501 1.720 G. 7 Sensitivity Analysis De Santis et al. Relative proportions are the ratio between observed and expected frequency, e.g., ‘Daily use of PC’ (first line) is observed in 10,832 cases out of 26,488 (41%). In cluster A, however, this ratio is 4,770 out of 6,696 (71% – not reported in the table). The ratio between the two figures (.71/.41) gives 1.742. Values greater than 1.5 are in bold (and smaller than 0.5 underlined) because they characterise clusters A to E (to be read by column) Source: Istat (2016a b) ( ) Variable Answers Values Absolute frequencies by cluster % Relative proportions within clusters A B C D E Tot A B C D E Read newspapers (usual week) No 1 1540 3689 3685 1000 3226 13,140 50 0.464 1.198 1.504 0.900 1.015 1–2 days 2 2353 1489 721 698 1741 7002 26 1.329 0.908 0.552 1.179 1.028 3 + days 3 2803 1028 533 541 1441 6346 24 1.747 0.691 0.450 1.009 0.939 Read magazines Yes 1 3874 2036 1070 1004 2374 10,358 39 1.480 0.839 0.554 1.147 0.947 No 2 2822 4170 3869 1235 4034 16,130 61 0.692 1.103 1.286 0.906 1.034 Read books (12 months) Yes 1 3972 1721 730 754 2780 9957 38 1.578 0.738 0.393 0.896 1.154 No 2 2724 4485 4209 1485 3628 16,531 62 0.652 1.158 1.365 1.063 0.907 Talk about politics Daily 1 2245 625 2870 11 3.094 0.000 0.000 2.576 0.000 A few days/week 2 4060 1484 9 5553 21 2.892 0.000 0.000 3.162 0.007 Once a week 3 387 444 123 532 1486 6 1.030 1.275 0.000 0.979 1.480 A few days/month 4 4 1563 9 7 1390 2973 11 0.005 2.244 0.016 0.028 1.933 A few days/year 5 1030 105 816 1951 7 0.000 2.253 0.289 0.000 1.729 Never 6 3169 4825 3661 11,655 44 0.000 1.161 2.220 0.000 1.298 Total 6696 6206 4939 2239 6408 26,488 So Close, So Far. The Cultural Distance of Foreigners in Italy Fig. 7 Share of variance explained, and increases in the explained share of variance, by number of clus‑ ters (Ward method) How to read the figure. With the Ward method, our preferred choice, if one uses three clusters, approximately 74% of the original variance is explained (thick line, left scale)). As this share is approximately 67% with two clusters, the improvement (or marginal progress) is of approximately 7% (dot‑ ted line, right scale). 7 Sensitivity Analysis Source: Istat (2016a, 2016b) Fig. 7 Share of variance explained, and increases in the explained share of variance, by number of clus‑ ters (Ward method) How to read the figure. With the Ward method, our preferred choice, if one uses three clusters, approximately 74% of the original variance is explained (thick line, left scale)). As this share is approximately 67% with two clusters, the improvement (or marginal progress) is of approximately 7% (dot‑ ted line, right scale). Source: Istat (2016a, 2016b) Fig. 7 Share of variance explained, and increases in the explained share of variance, by number of clus‑ ters (Ward method) How to read the figure. With the Ward method, our preferred choice, if one uses three clusters, approximately 74% of the original variance is explained (thick line, left scale)). As this share is approximately 67% with two clusters, the improvement (or marginal progress) is of approximately 7% (dot‑ ted line, right scale). Source: Istat (2016a, 2016b) Fig. 8 Correlation of results (distances between nationalities) between the case with five clusters and the case with N clusters. Ward (left) and other methods (right). How to read the figure. With the Ward method (left panel), if one uses 6 clusters instead of 5 (standard of reference), the resulting 861 cultural distances are almost the same (or, more precisely, are an almost perfect linear transformation of the baseline case – i.e., produce the same substantial result): the correlation is 0.996. As the number of clusters increases, the correlation of the 861 cultural distances with the reference case (5 clusters) declines, but only very slightly: with 50 clusters, for instance, it is still .882. The same happens in the right panel, where we show the results of the same test for different clustering criteria (Average linkage, Centroid, …). The only exception is the average linkage, where a discontinuity emerges in the passage from 35 to 40 clusters. Source: Istat (2016a, 2016b) Fig. 8 Correlation of results (distances between nationalities) between the case with five clusters and the case with N clusters. Ward (left) and other methods (right). How to read the figure. With the Ward method (left panel), if one uses 6 clusters instead of 5 (standard of reference), the resulting 861 cultural distances are almost the same (or, more precisely, are an almost perfect linear transformation of the baseline case – i.e., produce the same substantial result): the correlation is 0.996. 7 Sensitivity Analysis As the number of clusters increases, the correlation of the 861 cultural distances with the reference case (5 clusters) declines, but only very slightly: with 50 clusters, for instance, it is still .882. The same happens in the right panel, where we show the results of the same test for different clustering criteria (Average linkage, Centroid, …). The only exception is the average linkage, where a discontinuity emerges in the passage from 35 to 40 clusters. Source: Istat (2016a, 2016b) with N-1 clusters, and its improvement in passing from N-1 to N clusters. Figure 7 shows that this improvement declines very rapidly: it is slightly higher than 3% in the passage from four to five clusters (when about 81% of the total variance is explained), but it drops to less than 1% after that. This justifies our choice of stopping at five clusters. ii Besides, the results do not change in any relevant way as the number of clusters var‑ ies. With 42 national groups (five are Italians, one is Italians with foreign origin, and 36 are foreigners) we have (42·41/2 =861) cultural distances. These distances change 1 3 3 G. De Santis et al. Fig. 9 Correlation of results (861 between nationalities) between the standard (Ward, 5 clusters) and other clustering methods (with 5 clusters, except for the Average method, also used with 50 clusters) Source: Istat (2016a, 2016b) Fig. 9 Correlation of results (861 between nationalities) between the standard (Ward, 5 clusters) and other clustering methods (with 5 clusters, except for the Average method, also used with 50 clusters) Source: Istat (2016a, 2016b) almost linearly with the number of clusters and are therefore very strictly correlated to those that one finds working with just five clusters (Fig. 8, left). ii This consistency (virtually the same results as the number of clusters vary) is not lim‑ ited to the Ward method; it also emerges with almost all the other clustering methods that we tried: Centroid, Complete linkage, EML, Median linkage, and Single linkage (Fig. 8, right). In short, the number of clusters does not affect results in any significant way.ii fi This permits us to compare methods on a predefined number of clusters (five, except for the Average linkage method, for which we selected both five‒representative of what hap‑ pens up to 35 clusters ‒ and 50 ‒ representative of what happens for 40 or more clusters; Fig. 5 This method is based on the minimum distance between pairs of observations, and tends to generate elon‑ gated and irregular clusters. 8 Conclusions A few notes of caution are in order. The first is that emigrants are a selected subgroup: their cultural orientation may not be representative of that of their home country (see Doc‑ quier et al., 2020) or, depending on the length of their stay abroad, it may no longer be. If anything, however, if emigrants were positively selected towards emigration and towards a given destination at the start, or if they have somewhat adapted to the cultural habits of the host country, or both, they should be closer to natives than their fellow citizens are (that is, those who have remained in their country of origin). In short, our results are likely an underestimate of the cultural distances that would emerge comparing residents. The main note of caution, however, comes from the ‘cultural’ variables that we used for our analysis. We could not choose them because we were data-constrained; we considered only the questions that were asked, with the same wording, in the two Istat surveys that we merged (Istat, 2016a, 2016b), one of which focused on immigrants in Italy. Our empirical variables are therefore few, only 11, and they refer primarily to what respondents do in their (free) time. This use, while surely culturally driven, is also influenced by several other variables that we could not keep under control, such as personal resources, availability of free time, and the endowment of the areas where respondents live. In other words, we are also measuring the socio-economic standing of our respondents, together with their cul‑ tural orientation and their constraints, and we cannot determine how relevant each of these factors is.i However, first, this is what always happens, in various degrees, with empirical indica‑ tors. Secondly, it was impossible to do any better at this stage: future surveys will hopefully cover an increasing number of appropriate cultural dimensions. Thirdly, this weakness can also be used to defend our approach: even with the few and perfectible manifest variables that we had at our disposal, the method produced an output that passed all our preliminary checks, proved robust to alternative specifications (e.g., clustering method and number of clusters), and ‘makes sense’. 7 Sensitivity Analysis 9).fi The correlation with our reference (Ward) method is sufficiently high overall (above 60%), except for the Single linkage method.5 In three cases, the correlation with Ward is extremely high (above 90%): Complete linkage, EML, and Average linkage (this one, how‑ ever, only with a large number of clusters). In short, the conclusion of this section is that, as it always happens with clustering, sev‑ eral alternatives are possible, not all leading to the same results. However, Ward’s cluster‑ ing method seems preferable in this type of application, because it tends to generate clus‑ ters of comparable dimensions, i.e., with approximately the same number of units. As the DBS (Distance Between Strata) method is based on the distribution of units (from different strata) among clusters, it is better to avoid an excessive concentration of units in the same cluster, if this is at all possible. In this case in particular, it transpires that similar results emerge with other robust clustering methods, which reinforces our claim that Ward’s clus‑ tering criterion should be the preferred choice when using the DBS method. 1 3 So Close, So Far. The Cultural Distance of Foreigners in Italy 8 Conclusions We began with an expectation: that the closer they were ‘at the start’ (better: the closer was their country of origin, in terms of geographical distance and in terms of development, measured through the Human Devel‑ opment Index), the closer they would turn out to be in cultural terms. This expectation found only moderate empirical support, and surely needs further investigation, with better data and alternative methods.fi The nationalities that a subsequent Istat survey indicates as the most difficult to inte‑ grate in Italian society (e.g., the Chinese and the Filipinos) appear to be the farthest (or at least among the farthest) in our data, despite the use of different target populations, differ‑ ent indicators, and a very different methodology. Based on this, and on the other pieces of evidence presented above, we submit that our ranking (in Table 3) may be used as a pos‑ sible starting point to obtain at least an indication of the cultural distance of the various national subgroups of immigrants in Italy.i We suggest three main lines of future research. The first is to check whether these results are robust to alternative approaches. For instance, one could go into much greater depth by separating the two databases that we merged here, ADL for Italians and CSIFCI for foreigners. The comparison between Italians and immigrants becomes impossible, but one could verify, on a much wider and more appropriate set of questions, if the results for the two subgroups (separately: Italians by macro region of origin; foreigners by national‑ ity) remain at least roughly the same. The second step is to apply this methodology to more recent databases, as soon as they become available, and to determine how cultural distances evolve over time, both in gen‑ eral, and for specific national subgroups. Is there convergence towards the Italian ‘stand‑ ard’ (which, incidentally, may not remain constant)? If yes, how quick is it? Can this con‑ vergence be related to covariates such as length of stay, marital status, and labour market participation? This leads us to the third step: how to use our results. Even without going as far as Guiso et al. (2006), who view culture exclusively as a way of predicting economic behaviour, it seems reasonable to wonder how these findings can help us understand Italian society, and the socio-economic (and demographic) behaviours of her increasingly diverse actors. 8 Conclusions The cultural distances that we find are in fact consistent with expectations, and with what alternative sources suggest (e.g., a later Istat survey, conducted in 2014–15; Istat, 2020).i Among the expectations that were fulfilled, there is the distinction between the vari‑ ous parts of Italy, which also emerges in our data, with the well-known geographical gra‑ dient, North to South. Further, we found that people with Italian nationality but foreign origin are relatively close to Italians tout court, definitely closer than all foreigners living in Italy, including other Europeans, such as Germans and French. Their distance from the Italian ‘standard’ (assuming its existence) is 0.155 (on a 0‒√2 scale), comparable with the distance that separates ‘the two Italies’ (North to South, equalling 0.137), which can be conveniently used as a standard of reference in this peculiar context, where the metric is conventional and otherwise impossible to appreciate. Unfortunately, we do not have panel data (not even time series) for any of the meas‑ ures presented here and we cannot be sure about the correct interpretation. We offer two, not necessarily alternative, explanations: convergence and selection. The former implies that people at different stages of their assimilation into Italian society (first foreigners, then Italians with foreign origin) are also characterised by varying degrees of proximity to the Italian ‘culture’. In this interpretation, cultural orientations (influencing the use of time, which is what we measure here) change over time. The latter, instead (i.e., selection), is a mechanism that induces people with greater affinity to the Italian culture to come, stay, and eventually acquire the Italian nationality. In this interpretation, cultural orientations do not 3 G. De Santis et al. need to change; they simply differ between individuals. The truth likely lies somewhere in between, but, as mentioned, the lack of longitudinal data prevents us from exploring the matter in greater depth. need to change; they simply differ between individuals. The truth likely lies somewhere in between, but, as mentioned, the lack of longitudinal data prevents us from exploring the matter in greater depth. As for foreigners, their average cultural distance from Italians is approximately 0.300, but they are not homogeneous ‒ far from it, in fact. Acknowledgements Anonymous referees greatly helped us to improve this paper, which also benefitted from two sources of economic support: (1) JPI MYBL/CREW Project (Joint Programme Initiative: More Years Better Life, 2016 Call. CREW: Care, retirement and wellbeing of older people across different welfare regimes». MIUR Decree: n. 3266/2018; Official Bulletin no. 32 7. Feb 2019), and (2) MIUR-PRIN 2017 Grant (Italian Ministry of University and Research, Prot. N. 2017W5B55Y). Author contributions AP suggested that the method be applied to this specific topic and took care of the lit‑ erature review. MM collected and elaborated the data. GDS wrote the paper. All authors extensively debated the paper in all of its stages and approved its final version. Declarations Conflict of interest The authors declare that they have no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com‑ mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 8 Conclusions An additional difficulty is that cultural proximity can be both a cause (for instance of greater or lesser economic success) and an effect (for instance, of intermarriage). Disentangling the causal chain appears to be particularly problematic, in this case. Much remains to be done, but no progress is possible until a reliable measure of cultural distances becomes available. The one that we have proposed in these pages hopefully will pave the way for advancements in this field. Acknowledgements Anonymous referees greatly helped us to improve this paper, which also benefitted from two sources of economic support: (1) JPI MYBL/CREW Project (Joint Programme Initiative: More Years Better Life, 2016 Call. CREW: Care, retirement and wellbeing of older people across different welfare regimes». MIUR Decree: n. 3266/2018; Official Bulletin no. 32 7. Feb 2019), and (2) MIUR-PRIN 2017 Grant (Italian Ministry of University and Research, Prot. N. 2017W5B55Y). Author contributions AP suggested that the method be applied to this specific topic and took care of the lit‑ erature review. MM collected and elaborated the data. GDS wrote the paper. All authors extensively debated the paper in all of its stages and approved its final version. 1 3 1 So Close, So Far. The Cultural Distance of Foreigners in Italy Funding Open access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agree‑ ment.. Financial support from the Italian MIUR is gratefully acknowledged, twice. In part through the JPI MYBL/CREW Project (Joint Programme Initiative: More Years Better Life, 2016 Call. CREW: Care, retire‑ ment and wellbeing of older people across different welfare regimes». MIUR Decree: n. 3266/2018; Official Bulletin no. 32 7. Feb 2019), and in part thanks to the 2017 MiUR-PRIN Grant (Italian Ministry of Univer‑ sity and Research, Prot. N. 2017W5B55Y. Code Availability (software application or custom code): SAS programs available on request. Data Availability (data transparency): Istat surveys. Individual data available on request. Data Availability (data transparency): Istat surveys. Individual data available on request. References Cultural proximity and audience behavior: The role of language in patterns of polarization and multicultural fluency. Journal of Broadcasting & Electronic Media, 52(3), 485–503. https://doi.org/10.1080/08838150802205876. p g Luria, G., Levanon, A., Yagil, D., & Gal, I. (2016). Status, national culture and customers’ propensity to complain. Social Indicators Research, 126, 309–330. https://doi.org/10.1007/s11205-015-0884-y. McDoom, O. S., & Gisselquist, R. M. (2020). The measurement of ethnic and religious divisions: spatial, temporal, and categorical dimensions with evidence from Mindanao, the Philippines. Social Indicators Research, 129, 863–891. https://doi.org/10.1007/s11205-015-1145-9. , p g Mucciardi, M., & De Santis, G. (2017). Cultural versus objective distances: the DBS-EM approach. Social Indicators Research, 30(3), 867–882. Naidu, S., & Chand, A. (2017). National culture, gender inequality and women’s success in micro, small and medium enterprises. Social Indicators Research, 130, 647–664. https://doi.org/10.1007/ s11205-015-1203-3. Ng, S. I., Lee, J. A., & Soutar, G. N. (2007). Tourists’ intention to visit a country: The impact of cultural distance. Tourism Management, 28(6), 1497–1506. Ortega-Villa, L. M., & Ley-Garcia, J. (2018). Analysis of cultural indicators: A comparison of their con‑ ceptual basis and dimensions. Social Indicators Research, 137, 413–439. https://doi.org/10.1007/ s11205-017-1588-2.f Petit, S., & Seetaram, N. (2019). Measuring the effect of revealed cultural preferences on tourism e Journal of Travel Research, 58(8), 1262–1273. https://doi.org/10.1177/0047287518807582. Piñeiro, E., & Haller, J. (2012). Learning to live together - Towards a new integration society, in Z. Bek‑ erman, & T. Geisen, International handbook of migration, minorities and education, pp. 85–100. Springer. Poh Chuin, T. (2019). Belt and road initiative: The case of Malaysia. In The Belt and Road strategy in international business and administration, W. Liu, Z. Zhang, J.-X. Chen, & S.-B. Tsai, IGI Global, pp. 176–199, doi: https://doi.org/10.4018/978-1-5225-8440-7.ch011.f Schwartz S. H. (2013). National culture as value orientations: Consequences of value differences and cul‑ tural distance, in Handbook of the Economics of Art and Culture, Eds: V. Ginsburgh & D. Throsby, Elsevier/North Holland. Vol 2: 547–586. doi: https://doi.org/10.1016/B978-0-444-53776-8.00020-9. Shenkar, O. (2001). Cultural distance revisited: Towards a more rigorous conceptualization and measure‑ ment of cultural differences. Journal of International Business Studies, 32(4), 519–535. f Spitzer, S., Greulich, A. & Hammer, B. (2018). The subjective cost of young children: A European compari‑ son, VID Working Paper 12. Steckermeier, L. C., & Delhey, J. (2019). Better for everyone? Egalitarian culture and social wellbeing in Europe. Social Indicators Research, 143, 1075–1108. https://doi.org/10.1007/s11205-018-2007-z. Vieira, C. C., Ribeiro, F. N., Vaz de Melo, P. O. References Berg, M. (2017). The importance of cultural proximity in the success of Turkish dramas in Qatar. Interna- tional Journal of Communication, 11(2017), 3415–3430. Beugelsdijk, S., Maseland, R., Onrust, M., van Hoorn, A., & Slangen, A. (2015). Cultural distance in inter‑ national business and management: From mean-based to variance-based measures. The International Journal of Human Resource Management, 26(2), 165–191. https://doi.org/10.1080/09585192.2014. 922355. Byrne, D., & Nelson, D. (1965). Attraction as a linear function of proportion of positive reinforcements. Journal of Personality and Social Psychology, 1(6), 659–663. https://doi.org/10.1037/h0022073.f Böhmelt, T., & Bove, V. (2020). Does cultural proximity contain terrorism diffusion? Journal of Peace Research, 57(2), 251–264. https://doi.org/10.1177/0022343319864425.i Carifio, J., & Perla, R. J. (2007). Ten common misunderstandings, misconceptions, persistent myths and urban legends about Likert scales and Likert response formats and their antidotes. Journal of Social Sciences, 3(3), 106–116. ( ) De Santis, G., Maltagliati, M., & Salvini, S. (2016). A measure of the cultural distance between countries. Social Indicators Research, 126(3), 1065–1087. De Santis, G., & Mucciardi, M. (2017). From Euclidean distances to APC models. Quality and Quantity, 51(2), 829–846. Docquier, F., Tansel, A., & Turati, R. (2020). Do emigrants self-select along cultural traits? Evidence from the MENA countries. International Migration Review, 54(2), 388–422. https://doi.org/10.1177/01979 18319849011. Fargues, Ph. (2011). International migration and the demographic transition: a two-way interaction. Interna- tional Migration Review, 45(3), 588–614. https://doi.org/10.1111/j.1747-7379.2011.00859.x. Gamlath, S. (2017). Human development and national culture: A multivariate exploration. Social Indicators Research, 133, 907–930. https://doi.org/10.1007/s11205-016-1396-0.f Guiso, L., Sapienza, P., & Zingales, L. (2006). Does culture affect economic outcomes? Journal of Eco- nomic Perspectives, 20(2), 23–48. Harwell, M., & Gatti, G. (2001). Rescaling ordinal data to interval data in educational research. Review Educational Research, 71(1), 105–131. , ( ), Hennig, C., Meila, M., Murtagh, F., & Rocci, R. (Eds.). (2015). Handbook of cluster analysis (1st ed.). Chapman and Hall/CRC. https://doi.org/10.1201/b19706f Hennig, C., Meila, M., Murtagh, F., & Rocci, R. (Eds.). (2015). Handbook of cluster analysis (1st e Chapman and Hall/CRC. https://doi.org/10.1201/b19706f p p g Hofstede, G. (1980). Culture’s consequences: international differences in work-related values. . Sage. 1 3 3 3 G. De Santis et al. ISTAT (2016b) Aspetti della vita quotidiana: file per la ricerca, https://www.istat.it/it/archivio/129916 ISTAT (2020) Identità e percorsi di integrazione delle seconde generazioni in Italia [Cultural identity and integration of second generations in Italy], Rome. https://www.istat.it/it/files//2020/04/Identit%C3% A0-e-percorsi.pdf. p p Ksiazek, T. B., & Webster, J. G. (2008). Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References S., Benevenuto, F., & Zagheni. E. (2020). Using Facebook data to measure cultural distance between countries: The case of Brazilian cuisine. In Proceedings of TheWeb Conference 2020 (WWW’20), April 20–24, Taipei, Taiwan. ACM, New York, NY, USA, 7 pages. doi: https://doi.org/10.1145/3366423.3380082. p g p g Walesiak M., & Dudek A. (2010). Finding groups in ordinal data: An examination of some clustering proce‑ dures. In: Locarek-Junge H., Weihs C. (eds) Classification as a tool for research. Studies in classifica‑ tion, data analysis, and knowledge organization. Springer. https://doi.org/10.1007/978-3-642-10745-0_ 19 Walesiak M., & Dudek A. (2010). Finding groups in ordinal data: An examination of some clustering proce‑ dures. In: Locarek-Junge H., Weihs C. (eds) Classification as a tool for research. Studies in classifica‑ tion, data analysis, and knowledge organization. Springer. https://doi.org/10.1007/978-3-642-10745-0_ 19 Wu, H., & Leung, S. O. (2017). Can Likert scales be treated as interval scales? A simulation study. Journal of Social Service Research, 43(4), 527–532. https://doi.org/10.1080/01488376.2017.1329775. Wu, H., & Leung, S. O. (2017). Can Likert scales be treated as interval scales? A simulation study. Journal of Social Service Research, 43(4), 527–532. https://doi.org/10.1080/01488376.2017.1329775. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3 1
https://openalex.org/W2026376385	https://ccsenet.org/journal/index.php/ass/article/download/23526/15021	English	null	The Development of a Conceptual Model Promoting Learners’ Ownership in an Online Learning Environment	Asian social science	2,012	cc-by	3,975	The Development of a Conceptual Model Promoting Learners’ Ownership in an Online Learning Environment Divina C. Casim1 & Yong-Chil Yang2 1 Pampanga Agricultural College, Philippines 2 Andong National University, Andong, Korea Correspondence: Yong-Chil Yang, Andong National University, Andong, Kyungbuk 750-749, Korea. Tel: 82-54-820-5585. E-mail: ycyang@andong.ac.kr Received: October 15, 2012 Accepted: October 25, 2012 Online Published: December 31, 2012 doi:10.5539/ass.v9n1p9 URL: http://dx.doi.org/10.5539/ass.v9n1p9 Received: October 15, 2012 Accepted: October 25, 2012 Online Published: December 31, 2012 doi:10.5539/ass.v9n1p9 URL: http://dx.doi.org/10.5539/ass.v9n1p9 Keywords: ownership, conceptual model, online learning Keywords: ownership, conceptual model, online learning Asian Social Science; Vol. 9, No. 1; 2013 ISSN 1911-2017 E-ISSN 1911-2025 Published by Canadian Center of Science and Education Asian Social Science; Vol. 9, No. 1; 2013 ISSN 1911-2017 E-ISSN 1911-2025 Published by Canadian Center of Science and Education Abstract The purpose of this study was to explore the development of a conceptual model that promotes ownership in an online learning environment. It proceeds toward building a comprehensive online learning model that illustrates the interrelationship among learning motivation, cognition and meta-cognition. Those components can be employed into strategies to promote the learner ownership that is assumed to be an essential factor influencing learners’ achievement. The main methods of the study were literature review about ownership and development research on building of a conceptual model. A conceptual model of learners’ ownership in an online learning environment is proposed into integration of three factors of learning motivation, cognition, and meta-cognition and Milner-Bolotin’s three categories of ownership (2001), which are composed of personal value, control and responsibility. Since the model is a hypothetical and conceptual, further research for testing its theoretical validity is needed. 1. Introduction The fast growing information technology extensively increases online learning environments (OEL) such as e-learning, mobile learning, or smart learning. Although OEL is characterized by being taught anytime, anywhere, anyhow and any content, it poses dangers to learners’ experiences like early withdrawal or drop-out from online learning due to poor learning skills or sense of isolation. Thus, it is required that online learners have effective learning skills to proceed responsibly online learning, and then to enable them to become self-regulated learners. In order to help learners effectively in online settings, Milner-Bolotin (2001) suggested learners have ownership to online learning settings, and that there are three categories of ownership to online learning. According to the suggestion, the categories of ownerships are composed of three components. First, finding personal value is about understanding how the knowledge might be useful and can be connected to the acquired knowledge to his/her prior knowledge. Second, feeling in control pertains to learners’ involvement in making decisions and being proactive rather than reactive learner. And third, taking responsibility means being accountable for the process of learning as well as the results. Actually, ownership has been a common concept in many areas like psychology, economics, political science, philosophy, sociology, but it is comparatively a new concept in the field of education and training. With the emergence of constructivism as another epistemology in learning, ownership has also started gaining attention from educational researchers and theorists. Although it has already been initially explored for its potential benefit, further research is still required to conduct its affordances in the aspect of human learning. On the other hand, Pierce et al. (2003) in their conceptual examination of psychological ownership proposed three human motives that serve as roots of this psychological state: efficacy and effectiveness, self-identity, and having a place to dwell. First, an efficacy and effectiveness can influence desired outcomes in our environment and control object in any way (Furby, 1978). In learning setting, an efficacy and/or effectiveness is also considered to be a very strong determinant to learners’ motivation as illustrated in the work of Bandura (1986). It shows that self-efficacy influences on how individuals feel, think, motivate themselves and behave. Thus, it influences learners’ choice and the amount of effort that he puts forth and his persistence at a given task. It is 9 Vol. 9, No. 2.1 Finding Personal Value Learners need rich motivation at the very outset of any instruction. During the process of learning, poor motivation can also cause learners to quit the course in online learning easily since this type of setting leaving the course is as easy as clicking the mouse button. Recently, online designers have been focusing on identifying ways of attracting and retaining learners’ attention such as use of graphics, color, sound and animation (Ritchie & Hoffman, 1997). However, these techniques are more of external stimuli which are just useful for perceptual arousal or gaining learners’ initial attention which is considered as a short-lived motivation. In online learning, a more internal motive is highly demanded to sustain learners’ motivation throughout the course. Establishing the usefulness or value of the material in relation to the learners’ traits is one way to enhance motivation among learners in online learning. Learning tasks must be linked with learners’ needs, goals, values and interests, personal beliefs and even volition to ensure higher motivation or involvement of the learners. Tasks that are useful or valuable to the learner would elicit not only his attention, but his/her interest to pursue and persist during learning at the same time. Personal values and interest are also important factors influencing learners’ motivation to learn. Every person comes into learning with a distinct set of stable or general beliefs about the desirability of a certain object or activity and with individual interests to be satisfied. Thus, instruction should try to meet these values and interest for the learners to get highly motivated. In the study of Milner-Bolotin (2001) on the effect of topic choice on learners’ interest, ownership, and motivation in an offline learning, significant correlation between the learner interest and ownership had been observed. Therefore, matching instruction to learners’ interest can promote ownership and motivation as well. 1. Introduction 1; 2013 Asian Social Science www.ccsenet.org/ass assumed that learners with higher self-efficacy would consider a difficult task as a challenge to master rather than treats to be avoided letting them approach learning with higher level of motivation and quickly recover their sense of efficacy after failures. assumed that learners with higher self-efficacy would consider a difficult task as a challenge to master rather than treats to be avoided letting them approach learning with higher level of motivation and quickly recover their sense of efficacy after failures. The second root of psychological ownership is self-identity. Pierce et al. (2003) proposes that ownership helps people come to define themselves, express their self-identity to others, and maintain the continuity of the self across time. Identity is the interface between the individual and society. An individual develops a sense of self-identity as a result of viewing oneself from the perspective of how others view him/her. As pleasure and comfort are found in interactions with objects, the socially shared meaning ascribed to those objects becomes part of the individuals’ self-identity (McCracken, 1986). In addition to affording power over others, they communicate the individuals’ identity to others. Lastly, having a place to dwell is the third human motive that serves as another root of psychological ownership. According to Weil (1952), to have a place is an important “need of the human soul” (p.41). This can be formed to the idea being emphasized by Maslow (1954) on hierarchy of human needs wherein safety or security is on the second level of the hierarchy. Humans have an innate need of protecting themselves from all types of harm. So, having a place to dwell is a very important necessity for them. The purpose of this study is to explore the development of a conceptual model that promotes ownership in an online learning environment. To conduct the study, Milner-Bolotin’s three categories of ownership are employed. A conceptual model of learners’ ownership in an online learning environment is to propose the integration of three factors of learning motivation, cognition, and meta-cognition, and Milner-Bolotin’s three categories of ownership (2001), which are composed of finding personal value, feeling in control and taking responsibility. 2. Three Categories of Ownership 2.1 Finding Personal Value 2. Three Categories of Ownership 2.2 Feeling in Control In online learning, being an open system confronts learners with greater control than in other delivery media. The most common and important things are controls given to learners in terms of navigation and presentation. One example is the freedom given to the learner in sequencing of information. Strict, linear, and designer-driven sequence is now being replaced by user-driven sequencing decision (Jones & Farquhar, 1997). Although it can be noted that greater control does not benefit all types of learners, learners are motivated by having control of their learning rather than the program totally controlling the learning process (Allesi & Trollip, 2001). When learners are required to do something to get a reward that a teacher control, resentment may occur because the teacher has taken over part of the learner’s area of control over his/her life (Keller, 1987). One of the most important aspects of learner control is the design of navigational tools that allow learners to control the sequence of the learning process in online learning (Yang, 1993). It has been suggested that navigational freedom has an impact to the learners’ control of the program. Learners feel that they have the control of the program when they 10 Vol. 9, No. 1; 2013 Asian Social Science www.ccsenet.org/ass are provided with navigational aids that offer many options than with few ones. Trollip and Allesi (2001) mentioned that because we cannot be certain what purposes learners have for a program, the incorporation of different navigation methods facilitates different purposes and customized to the individuals need. In addition, they suggest the following strategies in designing learner control in online learning: (a) allowing the learner temporary termination of the program and return to it later with the use of bookmarking and do not use timed pauses, (b) allowing optional pacing which means learners should decide how fast the learning processes occur, (c) providing multiple orientation device that the learner can use at any time whenever the need arise such as directions, help, complaints, and glossaries, (d) providing appropriate controls for the learners’ needs. These strategies clearly reflect that learners are given enough instructional options both for navigation and presentation process so they can customize the instruction based on their own preferences. 2.3 Taking Responsibility Strategies such as self-goal setting and planning, supporting learners’ cognitive strategies providing enough opportunities for learners’ use of knowledge, and providing opportunities for self-evaluation can be included. Planning and goal setting influence not only learners’ motivation but also their meta-cognition because they provide learners with criteria in evaluating their own performance. A good deal of research indicates that goal setting is related to different types of performance as well as learners belief related to achievement such as personal efficacy for self-regulated learning (Garavalia, & Gredler, 2002). Learners with learning goals focus their attention on process of learning (Scott, 1996). However researchers found that allowing learners to set their goals enhances self-efficacy and learning (Schunk, & Ertmer, 2000). Self-goal setting let learners experience control of their learning and be responsible for achieving the desired goals. Another way of allowing learners to take responsibility of their learning is supporting their cognitive strategies. Instruction has to provide more opportunities for learners in exercising their cognitive strategies. Learners have to be more active in their cognitive process and they have to be provided with good activities for exercising rehearsal, elaboration and organization strategies to aid their cognitive process during online learning (Kim, 2008). Rehearsal is the repetition of the information to aid encoding into the memory. It can be in an oral manner by way of reciting a word or any piece of information aloud or in a written form by taking notes or highlighting words in a rather passive manner. Elaboration involves paraphrasing or summarizing the material learned and creating a generative note taking (Pintrich, 1999). Learners organize and connect ideas actively in their notes rather than taking notes passively and linearly. They also create analogies to fully understand materials to be learned, and explain ideas related with learning materials to someone else. Organization strategies include selecting the main idea from the text, making an outline of the material and using variety of specific techniques for selecting and organizing the ideas in the material by utilizing methods. 3.1 Ownership and Motivation Based on Schunk & Ertmer’s Model of Motivated Learning (2000), there are various variables affecting learner’s motivation from the pre-task phase until the post-task that indicate the changing role of motivation from one phase to another. Considering these variables, it seems to be assumed that linking ownership strategies into some of these variables can be a very significant way to enhance motivation for the entire learning process. In the model proposed in this study, personal value is the first category of ownership strategies that can be linked into these motivational variables, especially in terms of goals, values, and needs to ensure well-motivated learners. Though it can be noticed that only goal, personal desires, and need variables are being emphasized, this study deemed it necessary to add values, personal beliefs, and volition as additional determinants of learners’ motivation especially in the pre-task and post-task phase. Volition can be considered for its potential influence on learners’ motivation for the whole learning process. Goal reflects one’s purpose and refers to quantity, quality, or rate of performance (Schunk, 1990). Goals motivate people to exert effort necessary to meet task demands and to persist on the task over time. Goals can be set by the individual himself or can be established by others. However, allowing the learners to set their own learning goals as a way of taking ownership in their learning has been assumed to be more effective. Researchers have found that allowing learners to set their goals enhances self-efficacy and learning, perhaps because self-set goals produce high goal commitment (Schunk, & Ertmer, 2000). It is likely that before one can totally proceed with instruction in an optimal way, learners believe that it is related to their personal goals and will meet their specific needs (Driscoll, 2005). 11 Vol. 9, No. 1; 2013 Asian Social Science www.ccsenet.org/ass 3.2 Ownership and Cognition Cognition is the mental process of knowing, including aspects such as awareness, perception, reasoning, and judgment. It involves thinking, perceiving, abstracting, synthesizing, organizing, or any other process that allows the individual to conceptualize the nature of the external world (Franken, 1998). In the pursuit of trying to explain human behaviors especially in articulating how learning occurs, there are three theoretical perspectives: behaviorism, cognitivism and constructivism. Among them, it is known that cognitivism and constructivism emphasized the human factor more than the environment in the formation and modification of one’s behavior. Behaviorists assume for a direct influence of the environment to behavior. In contrast, cognitive psychology places emphasis on unobservable constructs, such as the mind, memory, attitudes, motivation, thinking, reflection and other internal processes. It attempts to describe how information around the world enters through our senses is retained or forgotten. Ownership in learning can be explored for its potential implications to cognitive theory as it emphasized the significance of the role of attention and perception, memory, and active learning. 3.3 Ownership and Meta-cognition Meta-cognition is the process of cognition about cognition. Being composed by self-awareness, reflection, and self-assessment, it can be related to the responsibility component of ownership in learning. Self-awareness is the learners’ awareness of his own knowledge and ability levels. Reflection is the stopping and thinking about what one has been doing and where one is going while self-assessment refers to giving oneself tests either actual or mental to evaluate cognition process. The development of meta-cognitive ability is greatly influenced by a variety of variables. Understanding which skills and strategies help us process and remember information is necessary but not sufficient to enhance our achievement (Schunk, & Ertmer, 2000). There are times that learners are fully aware of what helps them learn better and yet do not use them. One of the main reasons is the lack of understanding as to why, how, when, and where to use them in a particular situation. Hence, helping learners to gain a full understanding about the use of meta-cognitive strategies can boost their self-efficacy for performing well in the assigned task. Table 1 shows the potential ownership strategies classified into three components of learning and three categories of ownership in online learning. Table 1. Potential ownership strategies between three components of learning & three categories of ownership Components of Learning Categories of Ownership Potential Ownership Strategies Motivation Personal Value Relating the tasks to learners’ needs and goals. Relating the tasks to learners’ values and interest. Relating the task to learners’ ability, and successful achievements in the past. Control Providing multiple navigational aids and higher navigational freedom. Providing ill-defined learning space. Responsibility Allowing learners to set their own learning goals. Supporting learners in sustaining their motivation throughout the whole process of learning. Cognition Personal Value Relating the task to learners’ prior knowledge and experience. Allowing learners to use their own learning styles. Control Providing enough instructional options. Making instruction highly interactive. Responsibility Supporting learners in the use of their cognitive strategies. Providing more opportunities for learners to use the newly gained knowledge. Meta-cognition Personal Value Guiding the learners to attribute the result of learning to their own effort rather than luck or ease of task when appropriate. Control Allowing learners to monitor their own learning. Allowing learners to manage their own learning resources. Responsibility Providing learners enough opportunities for self-evaluation Support learners in redefining goals and planning. Table 1. 3.3 Ownership and Meta-cognition Potential ownership strategies between three components of learning & three categorie ownership strategies between three components of learning & three categories of ownership 12 Vol. 9, No. 1; 2013 Asian Social Science www.ccsenet.org/ass 4. A Proposed Ownership Strategy Model A model of ownership strategy has been proposed with a spiral figure located at the center that depicts the process of learning with the active interplay of its three components. They are all indispensable variables of learning and they should work synergistically to achieve the desired learning outcomes. The following Figure 1 shows the whole illustration of the final version of the ownership strategy model. The spiral figure starts at its inner most part and continuously expands following a series of loops. This implies the close and active interplay of the three components of learning in such a way that each component depends on each other throughout the learning process to arrive at the desired outcomes. It also depicts the ever dynamic nature of learning that is continuously undergoing change, making education a lifetime process. The outermost part of the figure is considered to be the area of learners’ highest degree of ownership. The spiral figure is three boxes representing the categories of ownership with specific strategies under each category. Two-way directional arrows connect these three categories that illustrate their interrelatedness. Embedding the strategies into the components of learning is illustrated by three separate arrows from each category to link strategies directly to the individual component of learning. It is assumed that it is through the active interplay of all the variables in the model that learner ownership can be fostered. Figure 1. Proposed ownership strategies model in online learning Figure 1. Proposed ownership strategies model in online learning Figure 1. Proposed ownership strategies model in online learning Figure 1. Proposed ownership strategies model in online learning References Allesi, S. M., & Trollip, S. R. (2001). Multimedia for learning: Methods and development. Needham Heights, MA: Allyn & Bacon. Bandura, A. (1986). Social foundations of thought and action: A social-cognitive theory. Englewood Cliffs, NJ: Prentice-Hall. Dittmar, H. (1992). The social psychology of material possessions: To have is to be. New York: St. Ma Driscoll, M. P. (2005). Psychology of learning for instruction (3rd ed.). Needham Heights, MA: Allyn & Bacon. Eccles, J. S. (1983). Expectancies, values, and academic behaviors. In J. T. Spence (Ed.), Achievement and Achievement Motivation (pp.75-146). San Francisco, CA: Freeman. Furby, L. (1978). Possessions’ in humans: An exploratory study of its meaning and motivation. Social Behavior and Personality, 6(1), 49-65. http://dx.doi.org/10.2224/sbp.1978.6.1.49 Garavalia, L. S., & Gredler, M. E. (2002). An exploratory study of academia goal setting, achievement calibration and self-regulated learning. Journal of Instructional Psychology, 29(4), 221-230. Jones, M. G., & Farquhar, J. D. (1997). User interface design for web-based instruction. In B. H. Khan (Ed.), Web-based instruction. Englewood Cliffs, NJ: Educational Technology Publications. Keller, J. M. (1987). Development and use of the ARCS model of instructional design. Journal of Instructional Development, 10(3), 2-10. http://dx.doi.org/10.1007/BF02905780 Kim, C-S. (2008). Development and utilization of self-regulatory tools to support e-Learning. Unpublished doctoral dissertation. Andong National University, Andong, Korea. Maslow, A. H. (1954). Motivation and personality. New York: Harper. Maslow, A. H. (1954). Motivation and personality. New York: Harper. McCracken, G. (1986). Culture and Consumption: A theoretical account of the structure and movement of the cultural meaning of consumer goods. Journal of Consumer Research, 17, 398-411. Milner-Bolotin, M. (2001). The effects of topic choice in project-based instruction on undergraduate physical science students’ interest, ownership, and motivation. Unpublished doctoral dissertation. University of Texas at Austin, Texas, USA. Pierce, J. L., Kostova, T., & Dirks, K. T. (2003). The state of psychological ownership: integrating and extending a century of research. Review of General Psychology, 7(1), 84-107. http://dx.doi.org/10.1037/1089-2680.7.1.84 Pintrich, P. R. (1999). The Role of motivation in promoting and sustaining self-regulated learning. Journal of Educational Research, 31, 459-470. Ritchie, D. C., & Hoffman, B. (1997). Incorporating instructional design principles with the World Wide Web. In B. H. Khan (Ed.), Web-Based Instruction. Englewood Cliffs, New Jersey: Educational Technology Publications. Schunk, D. H. (1990). Goal setting and self-efficacy during self-regulated learning. Educational Psychologist, 25, 71-86. http://dx.doi.org/10.1207/s15326985ep2501_6 Schunk, D. H., & Ertmer, P. A. (2000). Self-regulation and academic learning: Self-efficacy enhancing interventions. In M. Boekaerts, P. 5. Concluding Remarks With the belief that ownership greatly influences learners’ achievement especially in online learning, this study was conducted to conceptualize ownership strategies model. Since online learning is desirable to be related with learners’ personal value, it should allow more learner control and require learners to take responsibility of their own learning. Specific strategies that have potential in promoting ownership were investigated and integrated the three categories of personal value, control, responsibility about learner ownership into three components of motivation, cognition and meta-cognition. It is indicated that for learners to develop ownership over their learning the task should be related to their personal characteristics. Relating the task to learners’ needs, goals, values, interest and abilities are only some of the strategies under the first category which is about finding personal value. Also, learners have to feel in control of the learning process by involving them in making 13 Asian Social Science www.ccsenet.org/ass Vol. 9, No. 1; 2013 decision as to how and what to learn. This can be done by designing good navigational aids that offer higher navigational freedom, providing ill-defined learning space, monitor and manage their learning resource and many others. Also, learners should feel responsible and accountable both for the process and outcomes of learning. This can be done by letting them set their own learning goals, supporting them in the use of their cognitive strategies and providing more opportunities to use the newly-gained knowledge. These potential ownership strategies will be embedded into the motivational, cognitive, and meta-cognitive design of instruction to promote learner ownership in online learning. References R. Pintrich, & M. Zeidner (Eds.), Handbook of self-regulation (pp. 631-649). San Diego, CA: Academic Press. http://dx.doi.org/10.1016/B978-012109890-2/50048-2 Scott, J. E. (1996). Self-efficacy: A key to literacy learning. Reading Horizon, 36, 195-213. Weil, S. (1952). The Need for Roots: Prelude to a declaration of duties towards mankind. London: Routledge. Yang, Y-C. (1995). The effects of self-regulatory skills and type of instructional control on learning from computer-based instruction. International Journal of Instructional Media, 20(3), 225-241. 14
https://openalex.org/W4386953660	https://www.biorxiv.org/content/biorxiv/early/2023/09/22/2023.09.18.558373.full.pdf	English	null	Effect of ethanolic extract of Cyperus rotundus reduces the surgical induced secondary lymphedema and oxidative stress in adult mice tail	bioRxiv (Cold Spring Harbor Laboratory)	2,023	cc-by	13,467	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 Effect of ethanolic extract of Cyperus rotundus reduces the surgical induced secondary 2 lymphedema and oxidative stress in adult mice tail . 3 4 Nikhil Pandey1, Priyanka Mishra1, 5 1Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University 6 7 Corresponding Author 8 Dr Nikhil Pandey, Senior Researcher 9 Department of Medicinal Chemistry 10 IMS-BHU 11 Varanasi 12 Email: nikhil.pandey1@bhu.ac.in 13 14 Abstract 15 Background: Lymphedema is clinically manifested as swelling in the extremities due to abnormal accumulation of protein 16 rich in the extravascular interstitial space resulting in irreversible structural changes to the limb (s). The aim of this 17 explorative work was to evaluate the effect of Cyperus rotundus root (CRR) ethanolic extract in a mouse tail model of 18 secondary lymphedema. Method: Mice were temporally anaesthetised under sterile condition and the skin from the tail was 19 removed from distal part of the trunk after leaving 1cm of distance. The animals were divided into Experimental control 20 (EC) and Cyperus rotundus root (CRR) 80 mg/kg b.w. /day) treated groups. Change in tail volume and circumference were 21 monitored for 20 days. TNFα, SOD and catalase were estimated from blood obtained through retro-orbital at day 0, 5, 10 22 and 15. Further TS of upper part of the tail skin was stained with H&E stain to document histological changes mRNA level 23 of COX-2 was estimated from the skin. RESULTS: EC group showed gradual rise in tail oedema post-surgery (PS), 24 elevated inflammation, oxidative stress and histopathological alterations. However in CRR (80 mg/kg b.w./day) treated 25 group shown the reduced tail oedema after post-surgery. TNFα, SOD and catalase levels were significantly less in CRR 26 then EC group supporting anti-inflammatory potential. CRR protected the tail from structural damage. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 3 4 Nikhil Pandey1, Priyanka Mishra1, 5 1Department of Medicinal Chemistry, Institute of Medical Sc 6 7 Corresponding Author 8 Dr Nikhil Pandey, Senior Researcher 9 Department of Medicinal Chemistry 10 IMS-BHU 11 Varanasi 12 Email: nikhil.pandey1@bhu.ac.in 13 14 Abstract 15 Background: Lymphedema is clinically manifested as swelling in the ex 16 rich in the extravascular interstitial space resulting in irreversible stru 17 explorative work was to evaluate the effect of Cyperus rotundus root ( 18 secondary lymphedema. Method: Mice were temporally anaesthetised un 19 removed from distal part of the trunk after leaving 1cm of distance. Th 20 (EC) and Cyperus rotundus root (CRR) 80 mg/kg b.w. /day) treated grou 21 monitored for 20 days. TNFα, SOD and catalase were estimated from b 22 and 15. Further TS of upper part of the tail skin was stained with H&E st 23 of COX-2 was estimated from the skin. RESULTS: EC group showe 24 elevated inflammation, oxidative stress and histopathological alteration 25 group shown the reduced tail oedema after post-surgery. TNFα, SOD 26 then EC group supporting anti-inflammatory potential. CRR protecte 27 regulated the expression of COX-2 in comparison to EC group. Conclusio 28 secondary lymphedema indicating it potential for therapeutic use. 29 30 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint It also down 27 regulated the expression of COX-2 in comparison to EC group. Conclusions: CRR ethanolic extract significantly attenuated 28 secondary lymphedema indicating it potential for therapeutic use. 29 1 Effect of ethanolic extract of Cyperus rotundus reduces the surgical induced secondary 2 lymphedema and oxidative stress in adult mice tail . 3 4 Nikhil Pandey1, Priyanka Mishra1, 5 1Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University 6 7 Corresponding Author 8 Dr Nikhil Pandey, Senior Researcher 9 Department of Medicinal Chemistry 10 IMS-BHU 11 Varanasi 12 Email: nikhil.pandey1@bhu.ac.in 13 14 Abstract 15 Background: Lymphedema is clinically manifested as swelling in the extremities due to abnormal accumulation of protein 16 rich in the extravascular interstitial space resulting in irreversible structural changes to the limb (s). The aim of this 17 explorative work was to evaluate the effect of Cyperus rotundus root (CRR) ethanolic extract in a mouse tail model of 18 secondary lymphedema. Method: Mice were temporally anaesthetised under sterile condition and the skin from the tail was 19 removed from distal part of the trunk after leaving 1cm of distance. The animals were divided into Experimental control 20 (EC) and Cyperus rotundus root (CRR) 80 mg/kg b.w. /day) treated groups. Change in tail volume and circumference were 21 monitored for 20 days. TNFα, SOD and catalase were estimated from blood obtained through retro-orbital at day 0, 5, 10 22 and 15. Further TS of upper part of the tail skin was stained with H&E stain to document histological changes mRNA level 23 of COX-2 was estimated from the skin. RESULTS: EC group showed gradual rise in tail oedema post-surgery (PS), 24 elevated inflammation, oxidative stress and histopathological alterations. However in CRR (80 mg/kg b.w./day) treated 25 group shown the reduced tail oedema after post-surgery. TNFα, SOD and catalase levels were significantly less in CRR 26 then EC group supporting anti-inflammatory potential. CRR protected the tail from structural damage. It also down 27 regulated the expression of COX-2 in comparison to EC group. Conclusions: CRR ethanolic extract significantly attenuated 28 secondary lymphedema indicating it potential for therapeutic use. 29 30 31 1 Effect of ethanolic extract of Cyperus rotundus red 2 lymphedema and oxidative stress in adult mice tail . 1 Graphical abstract 2 Key words: Secondary Lymphedema, Cyperus rotundus, mice tail, histology, anti-inflammatory, Natural compounds. 1. Introduction Key words: Secondary Lymphedema, Cyperus rotundus, mice tail, histology, anti-inflammatory, Natural compounds. 1. Introduction 1 Effect of ethanolic extract of Cyperus rotundus reduces the surgical induced secondary 2 lymphedema and oxidative stress in adult mice tail . 14 Abstract 15 Background: Lymphedema is clinically manifested as swelling in the extremities due to abnormal accumulation of protein 16 rich in the extravascular interstitial space resulting in irreversible structural changes to the limb (s). The aim of this 17 explorative work was to evaluate the effect of Cyperus rotundus root (CRR) ethanolic extract in a mouse tail model of 18 secondary lymphedema. Method: Mice were temporally anaesthetised under sterile condition and the skin from the tail was 19 removed from distal part of the trunk after leaving 1cm of distance. The animals were divided into Experimental control 20 (EC) and Cyperus rotundus root (CRR) 80 mg/kg b.w. /day) treated groups. Change in tail volume and circumference were 21 monitored for 20 days. TNFα, SOD and catalase were estimated from blood obtained through retro-orbital at day 0, 5, 10 22 and 15. Further TS of upper part of the tail skin was stained with H&E stain to document histological changes mRNA level 23 of COX-2 was estimated from the skin. RESULTS: EC group showed gradual rise in tail oedema post-surgery (PS), 24 elevated inflammation, oxidative stress and histopathological alterations. However in CRR (80 mg/kg b.w./day) treated 25 group shown the reduced tail oedema after post-surgery. TNFα, SOD and catalase levels were significantly less in CRR 26 then EC group supporting anti-inflammatory potential. CRR protected the tail from structural damage. It also down 27 regulated the expression of COX-2 in comparison to EC group. Conclusions: CRR ethanolic extract significantly attenuated 28 secondary lymphedema indicating it potential for therapeutic use. 1 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is ma The copyright holder for this prep this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1. Introduction Such prepared 29 inoculums were used to spread onto agar plates using sterile cotton to make a lawn of bacteria. Preparation of samples in 30 different chemical.10 grams of sample was mixed with 40 ml of different chemical such as chloroform, water and ethanol. 31 Sample mixed chemical was boiled in a boiling water bath for 30 minutes, cooled at room temperature and filtered 32 through whatsmann No .1 filter paper and collected in a sterile container for further use. Extracts was kept at 4°C to 33 preserve the antibacterial property before they were used for well diffusion assay with antibiotic disc. Antibacterial assay 34 A loopful of each bacterial isolates were prepared in agar plates using a sterile cotton swab from the inoculums. The plates 1 Therefore the finding new therapeutic option is absolutely necessary. In the present work, we chose Cyperus rotundus (CR) 2 which is known for its TNFα inhibitory activity (7) and is employed for its preventive action of the reduction of obesity and 3 the reducing of the blockage in vascular system. (8).CR has been earlier reported to inhibit inflammatory cascade.(9) 4 Therefore ,we hypothesised that CR could be used in the management of SL by assessing its activity on inflammatory 5 cytokines, oxidative stress and on histological changes observed in mice. Here based on the earlier work of Olszewski’s 6 lymphedema model we chose the moue tail model to induce SL (10)(11). 7 2 Material and Methods: 1 Therefore the finding new therapeutic option is absolutely necessary. In the present work, we chose Cyperus rotundus (CR) 2 which is known for its TNFα inhibitory activity (7) and is employed for its preventive action of the reduction of obesity and 3 the reducing of the blockage in vascular system. (8).CR has been earlier reported to inhibit inflammatory cascade.(9) 4 Therefore ,we hypothesised that CR could be used in the management of SL by assessing its activity on inflammatory 5 cytokines, oxidative stress and on histological changes observed in mice. Here based on the earlier work of Olszewski’s 6 lymphedema model we chose the moue tail model to induce SL (10)(11). 7 2 M i l d M h d 1. Introduction 6 More than 130 million people are affected with secondary lymphedema (SL), a chronic condition characterised by the 7 accumulation of fluid, proteins, and adipocytes in the interstitium, resulting in the swelling of the affected limb.(1)(2) .The 8 vascular network in the lymphatic system is vital to maintain the homeostasis of fluid in the tissue in order to maintain the 9 tissue fluid homeostasis and to conciliate the local inflammation and the immune response. As the anatomical and the 10 functional integrity of the vasculature in the lymphatic system is impaired, leads to the loss of fluid transport potential 11 resulting to the condition of lymphedema which is the easily recognizable characteristics (3) .It is characterised by 12 progressive swelling due to disrupt local lymph transport, leading to the permanent and disoriented changes in the tissue 13 architecture. Due to surgical intervention or by infection, people develop the secondary lymphedema which gets worse in 14 gradual manner mostly in developed countries. As SL is common and leads to loss of limb function due to vascular 15 insufficiency, still lacks pharmacological treatment. (4).Hence at present the use of the pharmacotherapy agents in clinical 16 and experimental studies are focusing on inhibiting inflammation. Earlier, approaches were made to reduce the chronic 17 lymphedema through inhibiting the NfkB for example sodium selenite shown to reduce the lymphedema volume by directly 18 inhibiting the expression of adhesion molecules and NfkB, But its further trial are needed to confirm these results. (6). 2 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: oRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 Therefore the finding new therapeutic option is absolutely necessary. In the present work, we chose Cyperus rotundus (CR) 2 which is known for its TNFα inhibitory activity (7) and is employed for its preventive action of the reduction of obesity and 3 the reducing of the blockage in vascular system. 1. Introduction (8).CR has been earlier reported to inhibit inflammatory cascade.(9) 4 Therefore ,we hypothesised that CR could be used in the management of SL by assessing its activity on inflammatory 5 cytokines, oxidative stress and on histological changes observed in mice. Here based on the earlier work of Olszewski’s 6 lymphedema model we chose the moue tail model to induce SL (10)(11). 7 2. Material and Methods: 8 2.1. Material: Characterization of extract : Anti-bacterial analysis : Cytotoxicity Study: 9 10 For surgical procedure -Carbon steel surgical blade No.23, Stainless steel split ring tweezer Ethanol (70%), sterile cotton 11 ,digital vernacular calliper(Mavotank Pvt ltd),For biochemical procedure- Mouse TNF alpha ELISA kit was purchased from 12 Elabscience (E-EL-M3063), TRIzol (Ambion),TURBO DNA-free Kit (Invitrogen),RevertAid First Strand cDNA Synthesis 13 kit (Thermo scientific). 14 15 2.2. Preparation and standardization of the extract: Cyperus rotundus roots (CRR) were procured from the botanical 16 garden and its authenticity was verified by Centre of Advanced study in Botany, Institute of Science, BHU (voucher no. 17 Cypera.2021/4). The 200g roots were powdered and then exhaustively extracted with ethanol in continuous Soxhlet 18 extractor for 36 hrs. The ethanol filtrate was evaporated to a dryness using a Buchi RE11 rotavapor and Buchi461 water 19 bath. The yield was 17.5 percent i.e 17.5 gm of crude Cyperus rotundus roots extract (weight of dried extract/weight of the 20 powdered root). Fresh solution of the crude CRR extract was prepared by dissolving a given quantity of the ethanol extract 21 in a small volume of tween 20 and made up to appropriate volume with the sterile water The Ethanol CRR extract was given 22 in the dose of 80 mg.kg/BW/day orally to the mice of the CRR group up to twenty days. The experimental control group 23 was given 1ml of drug vehicle (i.e 20% of Tween 80 in the appropriate volume with the sterile water). 24 2.3. Characterization of extract: 25 2.4. Anti-bacterial analysis: The bacterial isolates Escherichia coli and Staphylococcus aureus were isolated from the 26 lab. Bacterial cultures were maintained on nutrient agar (NA) plates and slants. They were sub cultured every 2 weeks and 27 subsequently stored at 4°C. Inoculum Preparation 28 Nutrient agar broth was inoculated with freshly sub cultured bacteria and incubated at 37°C for few hours. 2.1. Material: Characterization of extract : Anti-bacterial analysis : Cytotoxicity Study: During the given time scale the photography of the tail 27 change in inflammation in lower and upper part of the cut. The measurement of tail was don 28 2.5. Parameters studied 29 2.5. A. Measurement of morphological changes and tail oedema after post surgery 30 Mice were analyzed visually for assessing morphological condition on day 0, 5, 10 and 20 31 were measured in terms of volume and circumference measurement followed from previous 32 33 Determination of Volume measurement at inflamed area –At post - surgery every mice w 34 of tail diameter of by digital vernacular calliper of upper and lower ends of the swelling. Alon microlitre of extracts of samples on each well. All plates were incubating at 37°C for 18-24 hours and the zones of inhibition (diameter in mm) were measured on the agar plate. microlitre of extracts of samples on each well. All plates were incubating at 37°C for 18-24 hours and the zones of inhibition (diameter in mm) were measured on the agar plate. 3 4 2.5. Cytotoxicity Study: Cell lines: 5 a) MCF7-Human Breast adenocarcinoma cell line (From NCCS, Pune) ,Cell culture medium: DMEM- High Glucose - 6 (#AL111, Himedia) ,Adjustable multichannel pipettes and a pipettor (Benchtop, USA) ,Fetal Bovine Serum (#RM10432, 7 Himedia) ,MTT Reagent (5 mg/ml) (# 4060 Himedia) ,DMSO (#PHR1309, Sigma) ,Camptothecin (#C9911, Sigma) ,D- 8 PBS (#TL1006, Himedia) ,96-well plate for culturing the cells (From Corning,USA) ,T25 flask (# 12556009, Biolite - 9 Thermo) ,50 ml centrifuge tubes (# 546043 TORSON) ,1.5 ml centrifuge tubes (TORSON) ,10 ml serological pipettes 10 (TORSON) ,Centrifuge (Remi: R-8oC). Inverted Biological Light microscope (Biolinkz) ,37°C incubator with humidified 11 atmosphere of 5% CO2 (Healforce, China) 12 13 2.3. Animal and experimental design: The experimental protocols were approved by the Animal Ethical Committee of 14 IMS-BHU (letter No. Dean/2021/IAEC/2567). Inbred male mice Swiss albino strain (15-20gm) was purchased from the 15 Central Animal facility. They were acclimatized for 7 days in ambient conditions of temperature, and a day-light cycle of 16 12 hrs. normal room temperature with 45%-55% of the humidity. The normal diet was provided during the course of the 17 whole experiment. Animals were divided in the following group as shown in (Table 1) S.No. 2.5. Cytotoxicity Study: Cell lines: 5 a) MCF7-Human Breast adenocarcinoma cell line (From NCCS, Pune) ,Cell culture medium: DMEM- High Glucose - 6 (#AL111, Himedia) ,Adjustable multichannel pipettes and a pipettor (Benchtop, USA) ,Fetal Bovine Serum (#RM10432, 7 Himedia) ,MTT Reagent (5 mg/ml) (# 4060 Himedia) ,DMSO (#PHR1309, Sigma) ,Camptothecin (#C9911, Sigma) ,D- 8 PBS (#TL1006, Himedia) ,96-well plate for culturing the cells (From Corning,USA) ,T25 flask (# 12556009, Biolite - 9 Thermo) ,50 ml centrifuge tubes (# 546043 TORSON) ,1.5 ml centrifuge tubes (TORSON) ,10 ml serological pipettes 10 (TORSON) ,Centrifuge (Remi: R-8oC). Inverted Biological Light microscope (Biolinkz) ,37°C incubator with humidified 11 atmosphere of 5% CO2 (Healforce, China) 3 2.3. Animal and experimental design: The experimental protocols were approved by the Animal Ethical Committee of 4 IMS-BHU (letter No. Dean/2021/IAEC/2567). Inbred male mice Swiss albino strain (15-20gm) was purchased from the 5 Central Animal facility. They were acclimatized for 7 days in ambient conditions of temperature, and a day-light cycle of 6 12 hrs. normal room temperature with 45%-55% of the humidity. The normal diet was provided during the course of the 7 whole experiment. Animals were divided in the following group as shown in (Table 1) p g g p ( ) S.No. Group Intervention Route 1 I- Experimental control (n=30) 20% of Tween 80/day Per oral 2 II- Treatment (n=30) CRR (80 mg/kg b.w/day) Per oral Table 1- Experimental design of the study Table 1- Experimental design of the study 2.1. Material: Characterization of extract : Anti-bacterial analysis : Cytotoxicity Study: Group Intervention Route 1 I- Experimental control (n=30) 20% of Tween 80/day Per oral 2 II- Treatment (n=30) CRR (80 mg/kg b.w/day) Per oral 18 Table 1- Experimental design of the study 19 2.4. Model to induce SL: We carried the study for secondary lymphedema in the tails of Swiss albino mice (20-25gm). 20 The tail skin was removed after leaving 1cm of distance from the base of the trunk. Cut was introduced in sterile condition 21 and no vessels were damaged. The administration of ether was applied for the temporary anesthesia and skin and 22 subcutaneous tissue between from the base of 5-10 mm to the distal region of the tail was removed (fig1). The mice were 23 kept in separate cages to avoid the damage being caused by the other mice. We measured the volume of the tails every day 24 for 20 days, thickness and histological studies were also done to assess the lymphatic adequacy. The tail was ethically 25 amputated in aseptic condition on due time period for the examination of tail for mRNA expression and histological studies 26 for its gross examination of the tail. During the given time scale the photography of the tail was carried out to examine the 27 change in inflammation in lower and upper part of the cut. The measurement of tail was done with digital caliper. 28 2.5. Parameters studied 29 2.5. A. Measurement of morphological changes and tail oedema after post surgery 30 Mice were analyzed visually for assessing morphological condition on day 0, 5, 10 and 20 for both groups. Tail Odema 31 were measured in terms of volume and circumference measurement followed from previous study (12). 32 33 Determination of Volume measurement at inflamed area –At post - surgery every mice were taken for the measurement 34 of tail diameter of by digital vernacular calliper of upper and lower ends of the swelling. Along with the a ruler for measuring 2.1. Material: Characterization of extract : Anti-bacterial analysis : Cytotoxicity Study: CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is ma The copyright holder for this prepr this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 microlitre of extracts of samples on each well. All plates were incubating at 37°C for 18-24 h 2 (diameter in mm) were measured on the agar plate. 3 4 2.5. Cytotoxicity Study: Cell lines: 5 a) MCF7-Human Breast adenocarcinoma cell line (From NCCS, Pune) ,Cell culture med 6 (#AL111, Himedia) ,Adjustable multichannel pipettes and a pipettor (Benchtop, USA) ,Fet 7 Himedia) ,MTT Reagent (5 mg/ml) (# 4060 Himedia) ,DMSO (#PHR1309, Sigma) ,Camp 8 PBS (#TL1006, Himedia) ,96-well plate for culturing the cells (From Corning,USA) ,T2 9 Thermo) ,50 ml centrifuge tubes (# 546043 TORSON) ,1.5 ml centrifuge tubes (TORSO 10 (TORSON) ,Centrifuge (Remi: R-8oC). Inverted Biological Light microscope (Biolinkz) ,3 11 atmosphere of 5% CO2 (Healforce, China) 12 13 2.3. Animal and experimental design: The experimental protocols were approved by the 14 IMS-BHU (letter No. Dean/2021/IAEC/2567). Inbred male mice Swiss albino strain (15-2 15 Central Animal facility. They were acclimatized for 7 days in ambient conditions of temper 16 12 hrs. normal room temperature with 45%-55% of the humidity. The normal diet was pro 17 whole experiment. Animals were divided in the following group as shown in (Table 1) S.No. Group Intervention Rou 1 I- Experimental control (n=30) 20% of Tween 80/day Per 2 II- Treatment (n=30) CRR (80 mg/kg b.w/day) Per 18 Table 1- Experimental design of the study 19 2.4. Model to induce SL: We carried the study for secondary lymphedema in the tails of 20 The tail skin was removed after leaving 1cm of distance from the base of the trunk. Cut was 21 and no vessels were damaged. The administration of ether was applied for the tempo 22 subcutaneous tissue between from the base of 5-10 mm to the distal region of the tail was r 23 kept in separate cages to avoid the damage being caused by the other mice. We measured th 24 for 20 days, thickness and histological studies were also done to assess the lymphatic ad 25 amputated in aseptic condition on due time period for the examination of tail for mRNA exp 26 for its gross examination of the tail. 2.1. Material: Characterization of extract : Anti-bacterial analysis : Cytotoxicity Study: For surgical procedure -Carbon steel surgical blade No.23, Stainless steel split ring tweezer Ethanol (70%), sterile cotton ,digital vernacular calliper(Mavotank Pvt ltd),For biochemical procedure- Mouse TNF alpha ELISA kit was purchased from Elabscience (E-EL-M3063), TRIzol (Ambion),TURBO DNA-free Kit (Invitrogen),RevertAid First Strand cDNA Synthesis kit (Thermo scientific). 15 2.2. Preparation and standardization of the extract: Cyperus rotundus roots (CRR) were procured from the botanical 16 garden and its authenticity was verified by Centre of Advanced study in Botany, Institute of Science, BHU (voucher no. 17 Cypera.2021/4). The 200g roots were powdered and then exhaustively extracted with ethanol in continuous Soxhlet 18 extractor for 36 hrs. The ethanol filtrate was evaporated to a dryness using a Buchi RE11 rotavapor and Buchi461 water 19 bath. The yield was 17.5 percent i.e 17.5 gm of crude Cyperus rotundus roots extract (weight of dried extract/weight of the 20 powdered root). Fresh solution of the crude CRR extract was prepared by dissolving a given quantity of the ethanol extract 21 in a small volume of tween 20 and made up to appropriate volume with the sterile water The Ethanol CRR extract was given 22 in the dose of 80 mg.kg/BW/day orally to the mice of the CRR group up to twenty days. The experimental control group 23 was given 1ml of drug vehicle (i.e 20% of Tween 80 in the appropriate volume with the sterile water). 24 2.3. Characterization of extract: 25 2.4. Anti-bacterial analysis: The bacterial isolates Escherichia coli and Staphylococcus aureus were isolated from the 26 lab. Bacterial cultures were maintained on nutrient agar (NA) plates and slants. They were sub cultured every 2 weeks and 27 subsequently stored at 4°C. Inoculum Preparation 5 2.4. Anti-bacterial analysis: The bacterial isolates Escherichia coli and Staphylococcus aureus were isolated from the 6 lab. Bacterial cultures were maintained on nutrient agar (NA) plates and slants. They were sub cultured every 2 weeks and 7 subsequently stored at 4°C. Inoculum Preparation 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . 29 2.5. A. Measurement of morphological changes and tail oedema after post surgery 30 Mice were analyzed visually for assessing morphological condition on day 0, 5, 10 and 20 for both groups. Tail Odema 31 were measured in terms of volume and circumference measurement followed from previous study (12). 32 33 Determination of Volume measurement at inflamed area –At post - surgery every mice were taken for the measurement 34 of tail diameter of by digital vernacular calliper of upper and lower ends of the swelling. Along with the a ruler for measuring 4 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 the length ,digital vernacular calliper was used to find the thickness increase and decrease in the murine tail. Photos were 2 taken to measure the diameter of each region .Besides after converting the diameter into circumference by using 3 mathematical equation : 1 the length ,digital vernacular calliper was used to find the thickness increase and decrease in the murine tail. Photos we 2 taken to measure the diameter of each region .Besides after converting the diameter into circumference by usin 3 mathematical equation : 4 Finally the volume was measured by using the following mathematical equation where tail was assumed to be a sliced 5 cone. (13). 6 7 8 9 10 2.5. B. Measurement of biochemical changes 11 Determination of TNF-α level: Wounded part of the mice tail were surgically removed at day 0,5,10 and 15 wa 12 homogenised in cold PBS, centrifuged and supernatant was kept for TNF-α estimation through a procedure stated b 13 Elabscience mice ELISA kit (E-EL-M3063). 14 Determination of oxidative stress: Part of the inflamed area from the upper part of the wound was taken day 0,5,10 an 15 15 and placed in 1ml of PBS and used for further estimation. 0 2.5. B. Measurement of biochemical changes 11 Determination of TNF-α level: Wounded part of the mice tail were surgically removed at day 0,5,10 and 15 was 12 homogenised in cold PBS, centrifuged and supernatant was kept for TNF-α estimation through a procedure stated by 13 Elabscience mice ELISA kit (E-EL-M3063). 14 Determination of oxidative stress: Part of the inflamed area from the upper part of the wound was taken day 0,5,10 and 15 15 and placed in 1ml of PBS and used for further estimation. Superoxide dismutase (SOD) activity was quantified by 16 Beauchamp method where the rate of inhibition of nitro blue tetrazolium (NBT) reduction in the presence of riboflavin as 17 described (14).The catalase enzyme activity was measured by the Aebi’s method by observing the H2O2 breakdown at the 18 240nm (15). 19 2.5. C. Measurement of histological changes: The collected tail samples at day 5 and 11 were removed surgically from 20 both groups. It was fixed in formaldehyde and was dehydrated ,embedded in paraffin ,sectioned into slices (5 μm ) and 21 stained with haematoxylin and eosin (H&E).The Samples were observed at 10X magnification. 22 19 2.5. C. Measurement of histological changes: The collected tail samples at day 5 and 11 were removed surgically from 20 both groups. It was fixed in formaldehyde and was dehydrated ,embedded in paraffin ,sectioned into slices (5 μm ) and 21 stained with haematoxylin and eosin (H&E).The Samples were observed at 10X magnification. 22 29 2.5. A. Measurement of morphological changes and tail oedema after post surgery 6 7 8 9 C=2 π r V=h (C12+ C1C2+C22) /12 π was measured by using the following mathematical equation where tail was assumed to be a sliced 29 2.5. A. Measurement of morphological changes and tail oedema after post surgery Superoxide dismutase (SOD) activity was quantified b 16 Beauchamp method where the rate of inhibition of nitro blue tetrazolium (NBT) reduction in the presence of riboflavin a 17 described (14).The catalase enzyme activity was measured by the Aebi’s method by observing the H2O2 breakdown at th 18 240nm (15). 19 2.5. C. Measurement of histological changes: The collected tail samples at day 5 and 11 were removed surgically fro 20 both groups. It was fixed in formaldehyde and was dehydrated ,embedded in paraffin ,sectioned into slices (5 μm ) an 21 stained with haematoxylin and eosin (H&E).The Samples were observed at 10X magnification. 22 23 2.5. D. Measurement of mRNA expression of COX-2 24 Inflamed upper part of the tail was taken for the total RNA extraction on day 5, 10 and 15 from both the EC group and C 25 group. As per the user’s manual, dissolved RNA (DEPC-water) was quantified through spectrophotometer. 1mg of tissu 26 was subjected to TRIzol homogenation and RNA was extracted. It was further subjected to DNAse treatment as per th 27 protocol mentioned by TURBO DNA-free Kit (Invitrogen). Then for cDNA preparation,DNAse treated RNA samples we 28 reverse transcribed by using Superscript II RNase H-reverse transcriptase (RT) through random hexamers as per th 29 manufacturer’s instruction (Fermentas, Thermo Fisher Scientific,Waltham,MA,USA) . The synthesised cDNA was store 30 at -20˚ C to be directly used for reverse transcriptase polymerase chain reaction (RT-PCR) .Specific oligonucleotides we 31 used for the analysis of COX-2 (FP – 5' AAAGGCCTCCATTGACCAGA -3’, RP 5’- GTGCTCGGCTTCCAGTATTG 32 3’), product size 373 bp and GAPDH (glyceraldehyde 3- phosphate dehydrogenase) (FP 5 33 AGTGAGGAGCAGGTTGAGGA-3’ and (RP) 5’ -GAGGAGGGGAGATGATGTGA-3’ (Reverse primer), product siz 34 244 bp were synthesized from Euro film, India. The PCR reactions were performed in 25ml reaction mixtures containin 35 2ml cDNA, 2.0 mM of dNTP, and 2.5ml of 10 x standard Taq reaction buffer, 1.0 unit of Taq DNA polymerase (Ne 36 England BioLabs, Inc.) and 5μmol of each primer. The reactions were carried out in the Thermo Fischer MiniAmp therm C=2 π r V=h (C12+ C1C2+C22) /12 π 4 Finally the volume was measured by using the following mathematical equation where tail was assumed to be a sliced 5 cone. (13). 23 2.5. D. Measurement of mRNA expression of COX-2 The steps for GAPDH: initial denaturation at 94˚C for 3 3 min, followed by 35 cycles of 94˚C for 30 s, 55˚C for 30 s, 72˚C for 1min and final extension at 72˚C for 8 min. The 4 amplified products were separated on 1.5 %agarose gel electrophoresis containing ethidium bromide. The intensity of COX- 5 2 was determined using image quant Omega Flour TM (GEL Company, USA) using Omega Fluor Acquisition software. 6 The expression of COX-2 was expressed as per cent band intensity relative to that of GAPDH. All RT-PCR experiments 7 were performed in triplicate with the same results and the best result provided in the result section. 8 2.6. Statistical analysis: Results are expressed as Means ± SD with the p value<0.001 in volume, upper and lower 9 circumference of both the groups. Statistical significance was analyzed with unpaired student’s T-test for comparison 10 between the 2 groups day wise. The comparisons among the groups were performed through Graph pad (8.0.2) using one- 11 way ANOVA (Tukey’s multiple comparison tests) to compare the mean values of each group with each other. ,p < 0.05, 12 , p < 0.001, and *, p < 0.0001 is significantly different from experimental control group 13 3. Results: 1 cycler. PCR steps for COX-2: initial denaturation at 94 ˚C for 5 min- 1 cycle, followed by 35 cycles of 94 C for 45s, 55 C 2 for 45 s, 72 C for 1 min and final extension at 72˚ C for 10 min. The steps for GAPDH: initial denaturation at 94˚C for 3 3 min, followed by 35 cycles of 94˚C for 30 s, 55˚C for 30 s, 72˚C for 1min and final extension at 72˚C for 8 min. The 4 amplified products were separated on 1.5 %agarose gel electrophoresis containing ethidium bromide. The intensity of COX- 5 2 was determined using image quant Omega Flour TM (GEL Company, USA) using Omega Fluor Acquisition software. 6 The expression of COX-2 was expressed as per cent band intensity relative to that of GAPDH. All RT-PCR experiments 7 were performed in triplicate with the same results and the best result provided in the result section. 8 2.6. Statistical analysis: Results are expressed as Means ± SD with the p value<0.001 in volume, upper and lower 9 circumference of both the groups. 23 2.5. D. Measurement of mRNA expression of COX-2 p 24 Inflamed upper part of the tail was taken for the total RNA extraction on day 5, 10 and 15 from both the EC group and CR 25 group. As per the user’s manual, dissolved RNA (DEPC-water) was quantified through spectrophotometer. 1mg of tissue 26 was subjected to TRIzol homogenation and RNA was extracted. It was further subjected to DNAse treatment as per the 27 protocol mentioned by TURBO DNA-free Kit (Invitrogen). Then for cDNA preparation,DNAse treated RNA samples were 28 reverse transcribed by using Superscript II RNase H-reverse transcriptase (RT) through random hexamers as per the 29 manufacturer’s instruction (Fermentas, Thermo Fisher Scientific,Waltham,MA,USA) . The synthesised cDNA was stored 30 at -20˚ C to be directly used for reverse transcriptase polymerase chain reaction (RT-PCR) .Specific oligonucleotides were 31 used for the analysis of COX-2 (FP – 5' AAAGGCCTCCATTGACCAGA -3’, RP 5’- GTGCTCGGCTTCCAGTATTG- 32 3’), product size 373 bp and GAPDH (glyceraldehyde 3- phosphate dehydrogenase) (FP 5’- 33 AGTGAGGAGCAGGTTGAGGA-3’ and (RP) 5’ -GAGGAGGGGAGATGATGTGA-3’ (Reverse primer), product size 34 244 bp were synthesized from Euro film, India. The PCR reactions were performed in 25ml reaction mixtures containing 35 2ml cDNA, 2.0 mM of dNTP, and 2.5ml of 10 x standard Taq reaction buffer, 1.0 unit of Taq DNA polymerase (New 36 England BioLabs, Inc.) and 5μmol of each primer. The reactions were carried out in the Thermo Fischer MiniAmp thermal 5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: ioRxiv preprint 1 cycler. PCR steps for COX-2: initial denaturation at 94 ˚C for 5 min- 1 cycle, followed by 35 cycles of 94 C for 45s, 55 C 2 for 45 s, 72 C for 1 min and final extension at 72˚ C for 10 min. 23 2.5. D. Measurement of mRNA expression of COX-2 Statistical significance was analyzed with unpaired student’s T-test for comparison 10 between the 2 groups day wise. The comparisons among the groups were performed through Graph pad (8.0.2) using one- 11 way ANOVA (Tukey’s multiple comparison tests) to compare the mean values of each group with each other. ,p < 0.05, 12 *, p < 0.001, and , p < 0.0001 is significantly different from experimental control group 13 3. Results: . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 14 3. A. Effect of CRR on tail morphology and kinetics 15 1: Morphological changes 16 We carried out an experimental model of the post-acute -surgical based lymphedema. We observed that following the 17 incision, the murine shown the acquired lymphatic swelling in both groups however the EC group (fig1) showed more 18 swelling. The hair follicles on the inflamed area were erected and the shiny texture was started to appear as the marked area 19 started to swell. Inflammation area of the tail near the edema was hairless and this condition was continuous in both the 20 upper and lower area of the edema area. These changes were less prominent in CRR group (fig 2) 6 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint Figu Morphological assessment of mice tail at day 0,5,10 and 20 in EC group. 1 Figure 1: Morphological assessment of mice tail at day 0,5,10 and 20 in EC group. Figure 1: Figure 1: Figure 1: Morphological assessment of mice tail at day 0,5,10 and 20 in EC group. 7 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: oRxiv preprint 2: Tail oedema post surgery changes 5 1) On volume- The gradual increase of the tail’s total volume at post-surgical was observed using the formula mentioned 6 in methodology. After the assessment of volume of the mice’s tail in both groups, the CRR group shown the significant 7 decrease in volume compared to volume of the EC group. Significant difference (p<0.001) was observed from day 6 till end 8 in comparison to EC group. and this difference was continued till the end point of the experiment. When observing the 9 volume change, the most distinct increase was observed in day 11 where highest difference was observed. Though CRR 10 group shown to attain the highest swelling area on the day 8 and day 9 but it was still low as compared to the EC which 11 shown the highest swelling on the day 11th . Continuous decrease in volume was presented after day 9 for CRR and day 11 12 for EC. Furthermore, according to the assessment of the diameter the higher swelling was observed on the upper (Proximal) 13 compared to lower (Distal) area in all the mice of the two groups. (Figure 3 and 4) Experimental Control Experimental Control 14 15 Fig3 Graphical representation of the change in the volume of both groups. The Statistical analysis was done with 16 Student’s t-test. The results were considered significant at p<0.001. 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 1 2 3 1.1356 1.1992 1.2899 1.3536 1.4818 1.5912 1.6640 1.7230 1.6624 1.6106 1.5700 1.5121 1.4690 1.4364 1.4105 1.3946 1.3544 1.3348 1.2750 1.2540 1.3234 1.3523 1.4701 1.5401 1.5811 1.9348 1.6812 1.8342 1.9709 2.0778 2.1574 2.0665 2.0668 2.0335 1.9429 1.8637 1.7630 1.6773 1.6410 1.6203 Days VOULUME Experimental Control Cyperus Rotundus ,p<0.001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 1 2 3 1.1356 1.1992 1.2899 1.3536 1.4818 1.5912 1.6640 1.7230 1.6624 1.6106 1.5700 1.5121 1.4690 1.4364 1.4105 1.3946 1.3544 1.3348 1.2750 1.2540 1.3234 1.3523 1.4701 1.5401 1.5811 1.9348 1.6812 1.8342 1.9709 2.0778 2.1574 2.0665 2.0668 2.0335 1.9429 1.8637 1.7630 1.6773 1.6410 1.6203 Days VOULUME yp ,p<0.001 14 15 Fig3 Graphical representation of the change in the volume of both groups. The Statistical analysis was done with 16 Student’s t-test. The results were considered significant at p<0.001. 2: Tail oedema post surgery changes 3 B Eff f CRR bi h i l Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 0.0 0.2 0.4 0.6 0.360 0.370 0.380 0.400 0.420 0.440 0.450 0.460 0.460 0.460 0.460 0.440 0.440 0.430 0.420 0.420 0.420 0.410 0.410 0.400 0.420 0.450 0.470 0.470 0.490 0.510 0.520 0.520 0.513 0.510 0.500 0.500 0.473 0.472 0.451 0.451 0.423 Days UC Experimental Control Cyperus Rotundus Upper Circumference , p<0.001 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 0.0 0.2 0.4 0.6 0.370 0.380 0.390 0.420 0.420 0.430 0.450 0.460 0.450 0.440 0.440 0.430 0.430 0.420 0.420 0.420 0.410 0.410 0.390 0.390 0.389 0.408 0.421 0.434 0.439 0.449 0.464 0.475 0.492 0.502 0.507 0.504 0.513 0.511 0.498 0.490 0.480 0.472 0.464 0.459 Days LC Experimental Control Cyperus Rotundus Lower Circumference ,p<0.001 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 0.0 0.2 0.4 0.6 0.360 0.370 0.380 0.400 0.420 0.440 0.450 0.460 0.460 0.460 0.460 0.440 0.440 0.430 0.420 0.420 0.420 0.410 0.410 0.400 0.420 0.450 0.470 0.470 0.490 0.510 0.520 0.520 0.513 0.510 0.500 0.500 0.473 0.472 0.451 0.451 0.423 Days UC Experimental Contr Cyperus Rotundus Upper Circumference , p<0.001 l Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 0.0 0.2 0.4 0.6 0.370 0.380 0.390 0.420 0.420 0.430 0.450 0.460 0.450 0.440 0.440 0.430 0.430 0.420 0.420 0.420 0.410 0.410 0.390 0.390 0.389 0.408 0.421 0.434 0.439 0.449 0.464 0.475 0.492 0.502 0.507 0.504 0.513 0.511 0.498 0.490 0.480 0.472 0.464 0.459 Days LC Experimental Control Cyperus Rotundus Lower Circumference *,p<0.001 Days Days 4 Fig 4 Graphical representation of the change in the circumference of the tail in both groups. 2: Tail oedema post surgery changes 17 18 19 Days 15 Fig3 Graphical representation of the change in the volume of both groups. The Statistical analysis was done with 16 Student’s t-test. The results were considered significant at p<0.001. 20 8 8 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 2 Fig 4 Graphical representation of the change in the circumference of the tail in both groups. Upper (a),lower(b) The Statistical analysis was done with Student’s t-test. The results were considered significant at p<0.001. 2: Tail oedema post surgery changes Upper (a),lower(b) The 5 Statistical analysis was done with Student’s t-test. The results were considered significant at p<0.001. 6 3 ff f C i i 7 1) On TNFα level: As the disruption to the lymphatic vasculature start the edema and inflammation. Hence to assess the 8 inflammation in both the experimental control and Cyperus rotundus administered mice, inflammation was measured .The 9 experimental group have shown the significant increment in the TNF-alpha ,but in the case of CRR it was significantly 10 lower in a mouse model of acquired lymphedema.(p<0.001) (Fig 5). Highest inflammation in the EC and CR were 11 170.73pg/ml and 148.221pg/ml respectively. 7 1) On TNFα level: As the disruption to the lymphatic vasculature start the edema and inflammation. Hence to assess the 8 inflammation in both the experimental control and Cyperus rotundus administered mice, inflammation was measured .The 9 experimental group have shown the significant increment in the TNF-alpha ,but in the case of CRR it was significantly 10 lower in a mouse model of acquired lymphedema.(p<0.001) (Fig 5). Highest inflammation in the EC and CR were 11 170.73pg/ml and 148.221pg/ml respectively. 9 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 0 5 10 15 20 0 50 100 150 200 D TNF alpha (pg/ml) Experimental Contro Cyprus Rotundus * ** 1 2 Fig5 Level of TNFα in experimental control vs Cyperus rotundus at day 0, 5, 10, 15 and 20. Values represented as mean 3 ± SD (n=5). The comparisons among the groups were performed using one-way ANOVA (Tukey’s multiple comparison 4 tests) to compare the mean values of EC with CR. (p < 0.05, p < 0.001, and *p < 0.0001 is significantly different from 5 the EC group in day wise.) 6 2. On antioxidant enzyme status: The increased inflammation in the wounded area and its oxidative stress was determined 7 by measuring antioxidant enzymes (SOD and CAT). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 2: Tail oedema post surgery changes It was observed that CRR group shown decreased SOD and CAT 8 levels as compared to EC group .CRR treatment significantly elevated the SOD and CAT levels (p < 0.01) as equated to the 0 5 10 15 20 0 50 100 150 200 Day TNF alpha (pg/ml) Experimental Contro Cyprus Rotundus * ** 2 Fig5 Level of TNFα in experimental control vs Cyperus rotundus at day 0, 5, 10, 15 and 20. Values represented as mean 3 ± SD (n=5). The comparisons among the groups were performed using one-way ANOVA (Tukey’s multiple comparison 4 tests) to compare the mean values of EC with CR. (p < 0.05, p < 0.001, and *p < 0.0001 is significantly different from 5 the EC group in day wise.) 2 Fig5 Level of TNFα in experimental control vs Cyperus rotundus at day 0, 5, 10, 15 and 20. Values represented as mean 3 ± SD (n=5). The comparisons among the groups were performed using one-way ANOVA (Tukey’s multiple comparison 4 tests) to compare the mean values of EC with CR. (p < 0.05, *p < 0.001, and p < 0.0001 is significantly different from 5 the EC group in day wise.) 6 2. On antioxidant enzyme status: The increased inflammation in the wounded area and its oxidative stress was determined 7 by measuring antioxidant enzymes (SOD and CAT). It was observed that CRR group shown decreased SOD and CAT 8 levels as compared to EC group .CRR treatment significantly elevated the SOD and CAT levels (p < 0.01) as equated to the 9 EC group as evident from (Figure 6 (a) (b)). 10 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 0 5 10 15 0.0 0.5 1.0 1.5 SOD U/mg Exp Cyp ** 1 2 Fig 6 (a) SOD level in experimental control vs cyperus rotundus at day 0, 5, 10 and 15. Values represented as mean ± 3 SD (n=5). The comparisons among the groups were performed using one-way ANOVA (Tukey’s multiple comparison tests) 4 to compare the mean values of EC with CR. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 2 3 4 5 6 7 8 9 10 comparison tests) to compare the mean values of EC with CR. (p < 0.05, p < 0.001, and *p < 0.0001 is significantly different from the EC group day wise) 2: Tail oedema post surgery changes (p < 0.05, *p < 0.001, and p < 0.0001 is significantly different from the EC 5 group day wise) 0 5 10 15 0.0 0.5 1.0 Day SOD U/mg Experimental Contro Cyprus Rotundus ** Day 2 Fig 6 (a) SOD level in experimental control vs cyperus rotundus at day 0, 5, 10 and 2 Fig 6 (a) SOD level in experimental control vs cyperus rotundus at day 0, 5, 10 and 15. Values represented as mean ± 3 SD (n=5). The comparisons among the groups were performed using one-way ANOVA (Tukey’s multiple comparison tests) 4 to compare the mean values of EC with CR. (p < 0.05, p < 0.001, and p < 0.0001 is significantly different from the EC 5 group day wise) 6 7 8 Fig 6 (b) Catalase level in experimental control vs Cyperus rotundus at day 0, 5, 10 and 15. Values represented as 9 mean ± SD (n=5). The comparisons among the groups were performed using one-way ANOVA (Tukey’s multiple 0 5 10 15 0.0 0.5 1.0 1.5 Day Catalase (U/mg) Experimental Control Cyprus Rotundus 0 5 10 15 0.0 0.5 1.0 1.5 Day Catalase (U/mg) Experimental Control Cyprus Rotundus ** 8 Fig 6 (b) Catalase level in experimental control vs Cyperus rotundus at day 0, 5, 10 and 15. Values represented as 9 mean ± SD (n=5). The comparisons among the groups were performed using one-way ANOVA (Tukey’s multiple 11 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is mad The copyright holder for this prepr this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 3. C. Effect of CRR on histological changes in mice tail H&E staining of a cross section of the mouse tail shown to have different thickness in the EC tail in comparison to the CRR treated tail. While there are many infiltrated cells at the site of lymphedema in EC groups a lesser number of the inflammatory cells was observed in the CRR group (Fig.7(B)(D)).As the higher thickening in the dermal ,perivascular and peri- lymphatic site can be seen EC group as compared to CRR administered group. Likewise, in EC group there was the marked inflammatory response, infiltration of leukocytes and higher collagen deposition in comparison to CRR group. 10 10 11 12 Fig.7 Histological observation (10X) in experimental control on day 5 (A) and day 11 (C) showed the thicker dermal, 13 hypodermic perivascular and peri lymphatic layer . However in CRR treated group on day 5 (B) and day 11(D) there is 14 reduced infiltration of lymphatic fluid and cells as compared to EC group. 15 12 Fig.7 Histological observation (10X) in experimental control on day 5 (A) and day 11 (C) showed the thicker dermal, 13 hypodermic perivascular and peri lymphatic layer . However in CRR treated group on day 5 (B) and day 11(D) there is 14 reduced infiltration of lymphatic fluid and cells as compared to EC group. 12 Fig.7 Histological observation (10X) in experimental control on day 5 (A) and day 11 (C) showed the thicker dermal, 13 hypodermic perivascular and peri lymphatic layer . However in CRR treated group on day 5 (B) and day 11(D) there is 14 reduced infiltration of lymphatic fluid and cells as compared to EC group. 15 12 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: oRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 2 Fig8. Immunostaining observation (10X) of CD 4 in the experimental control on day 5 (a) was observed high as compared 3 to day 5 (b) of the CRR. 3. C. Effect of CRR on histological changes in mice tail In the day 11 (c) the pre-fasical lymphatics are dilated (arrow head) as compared to day 11 of 4 CRR (d) and CD4 expression is higher in (c) as compared to (d). 1 2 Fig8. Immunostaining observation (10X) of CD 4 in the experimental control on day 5 (a) was observed high as compared 3 to day 5 (b) of the CRR. In the day 11 (c) the pre-fasical lymphatics are dilated (arrow head) as compared to day 11 of 4 CRR (d) and CD4 expression is higher in (c) as compared to (d). 5 6 7 3. D Effect of CRR on mRNA level of COX-2- 8 In the RT-PCR, the expression of the COX-2 was observed in both the EC and CR group, the findings indicate the up 9 regulation of cox-2 in experimental group in EC Day 5, 10 and 15, in comparison to the Cyperus rotundus treated mice. In 10 the day 15 we observed more down regulation of the expression in COX2 in both the EC and CR, but in comparison with 11 day wise manner, CR group shown lower expression of cox-2 .In highest marked swelled area (DAY 11) of EC, the cox2 12 was highest but on the same day the CR group shown the lower expression of COX2 as well as in other parameters. 13 2 Fig8. Immunostaining observation (10X) of CD 4 in the experimental control on day 5 (a) was observed high as compared 3 to day 5 (b) of the CRR. In the day 11 (c) the pre-fasical lymphatics are dilated (arrow head) as compared to day 11 of 4 CRR (d) and CD4 expression is higher in (c) as compared to (d). 3. D Effect of CRR on mRNA level of COX-2- 8 In the RT-PCR, the expression of the COX-2 was observed in both the EC and CR group, the findings indicate the up 9 regulation of cox-2 in experimental group in EC Day 5, 10 and 15, in comparison to the Cyperus rotundus treated mice. 3. C. Effect of CRR on histological changes in mice tail In 10 the day 15 we observed more down regulation of the expression in COX2 in both the EC and CR, but in comparison with 11 day wise manner, CR group shown lower expression of cox-2 .In highest marked swelled area (DAY 11) of EC, the cox2 12 was highest but on the same day the CR group shown the lower expression of COX2 as well as in other parameters. 8 In the RT-PCR, the expression of the COX-2 was observed in both the EC and CR group, the findings indicate the up 9 regulation of cox-2 in experimental group in EC Day 5, 10 and 15, in comparison to the Cyperus rotundus treated mice. In 10 the day 15 we observed more down regulation of the expression in COX2 in both the EC and CR, but in comparison with 11 day wise manner, CR group shown lower expression of cox-2 .In highest marked swelled area (DAY 11) of EC, the cox2 12 was highest but on the same day the CR group shown the lower expression of COX2 as well as in other parameters. 13 13 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: oRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 2 Fig 8 (a) (b) mRNA level of COX-2 in experimental control vs Cyperus rotundus at day 5, 10 and 15. Values 3 represented as mean ± SD (n=3). The comparisons among the groups were performed using one-way ANOVA 4 (Tukey’s multiple comparison tests) to compare the mean values of EC with CR. (p < 0.05, p < 0.001, and 5 p < 0.0001 is significantly different from the EC group day wise 6 4. 3. C. Effect of CRR on histological changes in mice tail DISCUSSION: EC DAY 5 EC DAY 10 EC DAY 15 CR DAY 5 CR DAY 10 CR DAY 15 0.0 0.2 0.4 0.6 0.8 1.0 COX-2/GAPDH EC DAY 5 EC DAY 10 EC DAY 15 CR DAY 5 CR DAY 10 CR DAY 15 EC DAY 5 EC DAY 10 EC DAY 15 CR DAY 5 CR DAY 10 CR DAY 15 0.0 0.2 0.4 0.6 0.8 1.0 COX-2/GAPDH EC DAY 15 CR DAY 5 CR DAY 10 CR DAY 15 ** 2 Fig 8 (a) (b) mRNA level of COX-2 in experimental control vs Cyperus rotundus at day 5, 10 and 15. Values 3 represented as mean ± SD (n=3). The comparisons among the groups were performed using one-way ANOVA 4 (Tukey’s multiple comparison tests) to compare the mean values of EC with CR. (p < 0.05, p < 0.001, and 5 **p < 0.0001 is significantly different from the EC group day wise 6 4. DISCUSSION: 4. DISCUSSION: 7 Till date many research had been conducted on lymphedema but the molecular mechanism and targeted therapy remains 8 debatable. The most common non-pharmacological treatment is decongestive physiotherapy which is used as supportive 9 care for post management (16). Since the core question lies in the pathogenesis of lymphedema and its progression. It is an 10 important domain to identify and utilise new therapeutic targets as currently there is no drug available in common person 11 domain . The uncovering of the new compounds which can act on the various different pathways during the lymphedema 12 is utmost important task for patients. As the secondary lymphedema induction in animals is utilised by researcher to 13 understand the pathophysiology(17). How the therapeutic agent could be used to observe the effects of it on the site of the 14 oedema is another important point for the study of SL .Towards this objective here for the first time we have validated the 15 therapeutic response of Cyperus rotundus root extract (CRR) in mouse model of secondary lymphedema. In this work we 16 evaluated the effect of ethanolic extract of CRR in terms of change in the tail oedema post-surgery, biochemical, 17 morphological and molecular changes in comparison to experimental control. 18 18 14 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: ioRxiv preprint 1 As roots of CR claims to have good efficiency as anti-oxidant and anti-inflammatory agent, owing to presence of high 2 flavonoids and polyphenols in polar and non-polar extract of CR(18). 4. DISCUSSION: In silico studies indicated the effect of CRR as an 3 inhibitor of 5-lypoxygenease and leukotriene A4 hydrolase (LTA4H) reported by Fenanir et al (19) .Thus it could be that 4 the reduced swelling after post-surgery in CRR group could be due to the effect of cyperus the provides a insight attenuating 5 swelling in mice tail can be supported by earlier reports from the in-silico studies indicating the role of CRR as Apart from 6 this, another feature of lymphedema which is the deposition of adipose tissue which may triggered by subtle dysfunction or 7 the injury in the lymphatic system . Cyperus rotundus root extract effect in the decrescent of swelling is strongly supporting 8 its earlier described role as downregulating of pro-inflammatory cytokines genes. Molecule’s present in CR has been 9 reported to inhibit the pro-inflammation via the inhibition of the NF-κB and STAT3 pathways and ROS. (20)(21).As in the 10 condition of SL, some of the studies have shown the expression of the pro-inflammatory gene which are been upregulated 11 in animal models and patients with lymphedema. Hence the expression of pro-inflammatory gene such as the TNF-alpha 12 are seen to be at increased state(22). 13 1 As roots of CR claims to have good efficiency as anti-oxidant and anti-inflammatory agent, owing to presence of high 2 flavonoids and polyphenols in polar and non-polar extract of CR(18). In silico studies indicated the effect of CRR as an 3 inhibitor of 5-lypoxygenease and leukotriene A4 hydrolase (LTA4H) reported by Fenanir et al (19) .Thus it could be that 4 the reduced swelling after post-surgery in CRR group could be due to the effect of cyperus the provides a insight attenuating 5 swelling in mice tail can be supported by earlier reports from the in-silico studies indicating the role of CRR as Apart from 6 this, another feature of lymphedema which is the deposition of adipose tissue which may triggered by subtle dysfunction or 7 the injury in the lymphatic system . Cyperus rotundus root extract effect in the decrescent of swelling is strongly supporting 8 its earlier described role as downregulating of pro-inflammatory cytokines genes. Molecule’s present in CR has been 9 reported to inhibit the pro-inflammation via the inhibition of the NF-κB and STAT3 pathways and ROS. 4. DISCUSSION: (20)(21).As in the 10 condition of SL, some of the studies have shown the expression of the pro-inflammatory gene which are been upregulated 11 in animal models and patients with lymphedema. Hence the expression of pro-inflammatory gene such as the TNF-alpha 12 are seen to be at increased state(22). 13 14 Another factor is the increased ROS level which causes higher oxidative stress in the higher lymphedema fluid . These 15 implications have been addressed in literatures on how the ROS are able to disturb the lymphatic contractions and how 16 the robust anti-oxidant defence could play vital role in the secondary lymphedema . In the lymphedema ,COX pathway 17 sits in centre for the production of ROS in the surgically induced lymphedema site and due to increased ROS it also 18 enhances lipid peroxidation .CRR have the potency to decrease the lipid peroxidation (23). 19 20 Therefore, due to high inflammation and increased ROS at the inflamed area brings the subcutaneous fat deposition and 21 increased in fat thickness in the case of the mice tail skin which can be observed in histological analysis (24) (25) (26). 22 Considering all these cascading effects arising after the induction of surgical based lymphedema ,we propose that CRR 23 can effectively downregulate the ROS level via inhibiting the COX -2 expression and also lowers the TNF alpha level. 24 We utilised the mouse tail edema model to research this hypothesis .As shown in figures above . It was supported by the 25 volume change in mice tail (Fig 3.) and change in circumferential area of both upper and lower area of surgical area also 26 established this(Fig 4 (a)(b)). 27 28 Hence ,from this study our results shows that CRR ethanolic extract shown its potency as anti-inflammatory and 29 antioxidant property in the mice tail of lymphedema. Though more refined and single compounds based studies in future 30 could aid in the management of secondary lymphedema and this could shed more light on the Cyperus rotundus’s derived 31 compounds in the initiation of therapeutic use of it in future. 32 33 Conclusions – Our work showed roots of Cyperus rotundus can be used to target secondary lymphedema by decreasing 34 the level of inflammatory cytokines and reactive oxygen species. This was morphologically supported by change in the tail 35 volume and circumference. 4. DISCUSSION: It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 1 CRR significantly reduced it after the post-surgery . However further studies are required for Cyperus r 2 potential plant as an additional treatment option in future. 3 4 Acknowledgement: NP and PM is thankful to ICMR and UGC respectively for providing the financial as 5 Conflict of Interest: Authors have no conflict of interest . 6 7 8 9 References: 10 11 1. Rockson, Stanley G. "Lymphedema." The American journal of medicine 110, no. 4 (2001 12 2. Rockson, Stanley G., and Kahealani K. Rivera. "Estimating the population burden of 13 lymphedema." Annals of the New York Academy of Sciences 1131, no. 1 (2008): 147-15 14 3. Rockson, Stanley G. "Diagnosis and management of lymphatic vascular disease." Journa 15 American College of Cardiology 52, no. 10 (2008): 799-806. 16 4. Borman, Pınar. "Lymphedema diagnosis, treatment, and follow-up from the view point of 17 medicine and rehabilitation specialists." Turkish journal of physical medicine and rehabilit 18 3 (2018): 179. 19 5. Birmingham, Brian, and Asokumar Buvanendran. "Nonsteroidal anti-inflammatory drugs, 20 acetaminophen, and COX-2 inhibitors." In Practical Management of Pain, pp. 553-568. M 21 6. Pfister, Christina, Horst Dawzcynski, and Franz-Josef Schingale. "Sodium selenite and ca 22 lymphedema: Biological and pharmacological effects." Journal of Trace Elements in Med 23 Biology 37 (2016): 111-116. 24 7. Shin, Ji-Sun, Yujin Hong, Hwi-Ho Lee, Byeol Ryu, Young-Wuk Cho, Nam-Jung Kim, Dae 25 and Kyung-Tae Lee. "Fulgidic acid isolated from the rhizomes of Cyperus rotundus suppr 26 induced iNOS, COX-2, TNF-α, and IL-6 expression by AP-1 inactivation in RAW264. 7 27 macrophages." Biological and Pharmaceutical Bulletin 38, no. 7 (2015): 1081-1086. 28 8. Venkatasubramanian, Padma, Subrahmanya K. Kumar, and Venugopalan SN Nair. "Cyp 29 rotundus, a substitute for Aconitum heterophyllum: Studies on the Ayurvedic concept of A 30 Pratinidhi Dravya (drug substitution)." Journal of Ayurveda and integrative medicine 1, no 31 33. 32 9. Jung, Seung-Hyun, Su Jung Kim, Bo-Gyu Jun, Kyung-Tae Lee, Seon-Pyo Hong, Myung 33 Dae Sik Jang, and Jung-Hye Choi. "α-Cyperone, isolated from the rhizomes of Cyperus r 34 inhibits LPS-induced COX-2 expression and PGE2 production through the negative regu 35 signalling in RAW 264.7 cells." Journal of ethnopharmacology 147, no. 1 (2013): 208-214 1 CRR significantly reduced it after the post-surgery . 27 19 26 4. DISCUSSION: However further studies are required for Cyperus rotundus to be a 2 potential plant as an additional treatment option in future. 3 4 Acknowledgement: NP and PM is thankful to ICMR and UGC respectively for providing the financial assistance.. 5 Conflict of Interest: Authors have no conflict of interest . 6 4 Acknowledgement: NP and PM is thankful to ICMR and UGC respectively for providing the financial assistance.. 5 Conflict of Interest: Authors have no conflict of interest . 6 25 18 22 28 4. DISCUSSION: We have a Swiss albino mice tail model for inducing secondary lymphedema, which can be 36 used to stimulate a trait of human secondary lymphedema and provides the insights of functional and structural changes of 37 lymphedema. Our work provides insight on the importance of COX-2 in development of secondary lymphedema and how 14 Another factor is the increased ROS level which causes higher oxidative stress in the higher lymphedema fluid . These 15 implications have been addressed in literatures on how the ROS are able to disturb the lymphatic contractions and how 16 the robust anti-oxidant defence could play vital role in the secondary lymphedema . In the lymphedema ,COX pathway 17 sits in centre for the production of ROS in the surgically induced lymphedema site and due to increased ROS it also 18 enhances lipid peroxidation .CRR have the potency to decrease the lipid peroxidation (23). 19 Hence ,from this study our results shows that CRR ethanolic extract shown its potency as anti-inflammatory and antioxidant property in the mice tail of lymphedema. Though more refined and single compounds based studies in future could aid in the management of secondary lymphedema and this could shed more light on the Cyperus rotundus’s derived compounds in the initiation of therapeutic use of it in future. 33 Conclusions – Our work showed roots of Cyperus rotundus can be used to target secondary lymphedema by decreasing 34 the level of inflammatory cytokines and reactive oxygen species. This was morphologically supported by change in the tail 35 volume and circumference. We have a Swiss albino mice tail model for inducing secondary lymphedema, which can be 36 used to stimulate a trait of human secondary lymphedema and provides the insights of functional and structural changes of 37 lymphedema. Our work provides insight on the importance of COX-2 in development of secondary lymphedema and how 15 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 24 20 29 21 17 16 23 References: 1. Rockson, Stanley G. "Lymphedema." The American journal of medicine 110, no. 4 (2001): 288-295. 2. Rockson, Stanley G., and Kahealani K. Rivera. "Estimating the population burden of lymphedema." Annals of the New York Academy of Sciences 1131, no. 1 (2008): 147-154. 3. Rockson, Stanley G. "Diagnosis and management of lymphatic vascular disease." Journal of the American College of Cardiology 52, no. 10 (2008): 799-806. 4. Borman, Pınar. "Lymphedema diagnosis, treatment, and follow-up from the view point of physical medicine and rehabilitation specialists." Turkish journal of physical medicine and rehabilitation 64, no. 3 (2018): 179. 5. Birmingham, Brian, and Asokumar Buvanendran. "Nonsteroidal anti-inflammatory drugs, acetaminophen, and COX-2 inhibitors." In Practical Management of Pain, pp. 553-568. Mosby, 2014. 6. Pfister, Christina, Horst Dawzcynski, and Franz-Josef Schingale. "Sodium selenite and cancer related lymphedema: Biological and pharmacological effects." Journal of Trace Elements in Medicine and Biology 37 (2016): 111-116. 7. Shin, Ji-Sun, Yujin Hong, Hwi-Ho Lee, Byeol Ryu, Young-Wuk Cho, Nam-Jung Kim, Dae Sik Jang, and Kyung-Tae Lee. "Fulgidic acid isolated from the rhizomes of Cyperus rotundus suppresses LPS- induced iNOS, COX-2, TNF-α, and IL-6 expression by AP-1 inactivation in RAW264. 7 macrophages." Biological and Pharmaceutical Bulletin 38, no. 7 (2015): 1081-1086. 8. Venkatasubramanian, Padma, Subrahmanya K. Kumar, and Venugopalan SN Nair. "Cyperus rotundus, a substitute for Aconitum heterophyllum: Studies on the Ayurvedic concept of Abhava Pratinidhi Dravya (drug substitution)." Journal of Ayurveda and integrative medicine 1, no. 1 (2010): 33. 9. Jung, Seung-Hyun, Su Jung Kim, Bo-Gyu Jun, Kyung-Tae Lee, Seon-Pyo Hong, Myung Sook Oh, Dae Sik Jang, and Jung-Hye Choi. "α-Cyperone, isolated from the rhizomes of Cyperus rotundus, inhibits LPS-induced COX-2 expression and PGE2 production through the negative regulation of NFκB signalling in RAW 264.7 cells." Journal of ethnopharmacology 147, no. 1 (2013): 208-214. 16 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 10. Olszewski, W., Z. Machowski, J. 27 17 16 28 26 15 18 24 25 19 References: Sokolowski, and J. Nielubowicz. "Experimental lymphedema in dogs." J. Cardiovasc. Surg 9, no. 2 (1968): 178-183. 11. Rutkowski, Joseph M., Monica Moya, Jimmy Johannes, Jeremy Goldman, and Melody A. Swartz. "Secondary lymphedema in the mouse tail: Lymphatic hyperplasia, VEGF-C upregulation, and the protective role of MMP-9." Microvascular research 72, no. 3 (2006): 161-171. 12. Taylor, Richard, Upali W. Jayasinghe, Louise Koelmeyer, Owen Ung, and John Boyages. "Reliability and validity of arm volume measurements for assessment of lymphedema." Physical therapy 86, no. 2 (2006): 205-214. 13. Taylor, Richard, Upali W. Jayasinghe, Louise Koelmeyer, Owen Ung, and John Boyages. "Reliability and validity of arm volume measurements for assessment of lymphedema." Physical therapy 86, no. 2 (2006): 205-214. 14. Beauchamp, Charles, and Irwin Fridovich. "Superoxide dismutase: improved assays and an assay applicable to acrylamide gels." Analytical biochemistry 44, no. 1 (1971): 276-287. 15. Aebi, Hugo. "[13] Catalase in vitro." Methods in enzymology 105 (1984): 121-126. 16. Borman, P., 2018. Lymphedema diagnosis, treatment, and follow-up from the view point of physical medicine and rehabilitation specialists. Turkish journal of physical medicine and rehabilitation, 64(3), p.179. 17. Pandey, Nikhil, Priyanka Mishra, and Yamini Bhusan Tripathi. "CYPERUS ROTUNDUS IN THE MANAGEMENT OF METABOLIC SYNDROME–BENEFIT IN THE TREATMENT OF METABOLIC SYNDROME." (2021). 18. Fenanir, Fares, Abderrahmane Semmeq, Yacine Benguerba, Michael Badawi, Marie-Antoinette Dziurla, Smain Amira, and Hocine Laouer. "In silico investigations of some Cyperus rotundus compounds as potential anti-inflammatory inhibitors of 5-LO and LTA4H enzymes." Journal of Biomolecular Structure and Dynamics (2021): 1-16. 19. Seo, Yun-Ji, Miran Jeong, Kyung-Tae Lee, Dae Sik Jang, and Jung-Hye Choi. "Isocyperol, isolated from the rhizomes of Cyperus rotundus, inhibits LPS-induced inflammatory responses via suppression of the NF-κB and STAT3 pathways and ROS stress in LPS-stimulated RAW 264.7 cells." International immunopharmacology 38 (2016): 61-69. 19. Seo, Yun-Ji, Miran Jeong, Kyung-Tae Lee, Dae Sik Jang, and Jung-Hye Choi. "Isocyperol, isolated from the rhizomes of Cyperus rotundus, inhibits LPS-induced inflammatory responses via suppression of the NF-κB and STAT3 pathways and ROS stress in LPS-stimulated RAW 264.7 cells." International immunopharmacology 38 (2016): 61-69. 20. Ramadhani, Abu Hanifah, Wirdatun Nafisah, Hary Isnanto, Tri Kurniawati Sholeha, Yoga Dwi Jatmiko, Hideo Tsuboi, and Muhaimin Rifa'i. "Immunomodulatory Effects of Cyperus rotundus Extract on 7, 12 Dimethylbenz [a] anthracene (DMBA) Exposed BALB/c Mice." Pharmaceutical Sciences 27, no. 1 (2020): 46-55. 20. Ramadhani, Abu Hanifah, Wirdatun Nafisah, Hary Isnanto, Tri Kurniawati Sholeha, Yoga Dwi Jatmiko, Hideo Tsuboi, and Muhaimin Rifa'i. 14 13 29 12 23 22 20 21 6 10 8 33 9 6 7 8 References: "Immunomodulatory Effects of Cyperus rotundus Extract on 7, 12 Dimethylbenz [a] anthracene (DMBA) Exposed BALB/c Mice." Pharmaceutical Sciences 27, no. 1 (2020): 46-55. 21. Ly, Catherine L., Raghu P. Kataru, and Babak J. Mehrara. "Inflammatory manifestations of lymphedema." International journal of molecular sciences 18, no. 1 (2017): 171. 21. Ly, Catherine L., Raghu P. Kataru, and Babak J. Mehrara. "Inflammatory manifestations of lymphedema." International journal of molecular sciences 18, no. 1 (2017): 171. 21. Ly, Catherine L., Raghu P. Kataru, and Babak J. Mehrara. "Inflammatory manifestations of lymphedema." International journal of molecular sciences 18, no. 1 (2017): 171. 17 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 22, 2023. ; https://doi.org/10.1101/2023.09.18.558373 doi: bioRxiv preprint 22. Chang, Ting-Chen, Yih-Huei Uen, Cheng-Hung Chou, Joen-Rong Sheu, and Duen-Suey Chou. "The role of cyclooxygenase-derived oxidative stress in surgically induced lymphedema in a mouse tail model." Pharmaceutical biology 51, no. 5 (2013): 573-580. 23. Aschen, Seth, Jamie C. Zampell, Sonia Elhadad, Evan Weitman, Marina De Brot Andrade, and Babak J. Mehrara. "Regulation of adipogenesis by lymphatic fluid stasis part II: expression of adipose differentiation genes." Plastic and reconstructive surgery 129, no. 4 (2012): 838. 24. Benoit, JOSEPH N., and DAVID C. Zawieja. "Effects of f-Met-Leu-Phe-induced inflammation on intestinal lymph flow and lymphatic pump behavior." American Journal of Physiology-Gastrointestinal and Liver Physiology 262, no. 2 (1992): G199-G202. 25. Zawieja, D. C., and K. L. Davis. "Inhibition of the active lymph pump in rat mesenteric lymphatics by hydrogen peroxide." Lymphology 26, no. 3 (1993): 135-142. 26. Pandey, Nikhil, Priyanka Mishra, and Yamini Bhusan Tripathi. "Cyperus rotundus root extract inhibits progress of lymphedema in mouse tail model." bioRxiv (2021). 18
https://openalex.org/W4390056643	https://www.qeios.com/read/9J86JS/pdf	English	null	Review of: "Targeting Alzheimer's disease hallmarks with the Nrf2 activator Isoeugenol"	null	2,023	cc-by	431	Qeios, CC-BY 4.0 · Review, December 21, 2023 Potential competing interests: No potential competing interests to declare. The authors have worked on isoeugenol, as an activator of Nrf2 and having potential to treat AD. Different in vivo and in vitro tests are conducted targeting the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) as a master controller of metabolism, neuroinflammationa and proteostais in AD. This work was conducted in vitro (in mice microglia cells exposed to LPS and neuronal cells overexpressing the human APP with Swedish mutation, N2a-APPswe) and in vivo (in the AD double transgenic mice, APP/PS1, intranasally administered with Isoeugenol), at an early (6-month-old animals) and late (11-month-old animals) AD stage. Overall, the results showed that Isoeugenol exhibit a good pharmacokinetic and pharmacodynamic profile. Isoeugenol activates Nrf2 and displays antioxidant and anti-inflammatory effects and reduced the levels of Aβ peptides in in vitro and in vivo models of AD. In addition, its positive effect on metabolism was also demonstrated in vivo, as it reduced the triglyceride and LDL cholesterol levels in treated AD mice. Importantly, Isoeugenol improved the memory deficits observed in APP/PS1 mice, which was more evident in older animals (11-month-old), reinforcing its potential in ameliorating AD hallmarks, even at a late stage. The article is having multiple studies placed on one table, causing a lot of confusion and showing an asymmetric data. The authors might reconsider the title in order to address all the studies provided, or the authors may only include the data related to Nrf2 activation. Review of: "Targeting Alzheimer's disease hallmarks with the Nrf2 activator Isoeugenol" Dr. Shehla Akbar1 1 Cecos Univeristy of Information Technology Dr. Shehla Akbar1 1 Cecos Univeristy of Information Technology Potential competing interests: No potential competing interests to declare. Introduction a. The information about the AD is too long, the introduction must be precise and to the point. a. The information about the AD is too long, the introduction must be precise and to the point. b. The results must not be added in the introduction part. Qeios ID: 9J86JS · https://doi.org/10.32388/9J86JS Qeios, CC-BY 4.0 · Review, December 21, 2023 Methods Phramcokinetic Studies Phramcokinetic Studies a. Why was the brain samples homogenized in NaCl? Usually the brain samples are homogenized in the solvents of mobile phase. a. Why was the brain samples homogenized in NaCl? Usually the brain samples are homogenized in the solvents of mobile phase. b. The detail of HPLC method development and validation is not provided. Qeios ID: 9J86JS · https://doi.org/10.32388/9J86JS 1/2 Qeios, CC-BY 4.0 · Review, December 21, 2023 Qeios ID: 9J86JS · https://doi.org/10.32388/9J86JS 2/2
https://openalex.org/W2997858965	https://europepmc.org/articles/pmc6941818?pdf=render	English	null	Assertive, trainable and older dogs are perceived as more dominant in multi-dog households	PloS one	2,020	cc-by	10,067	RESEARCH ARTICLE Assertive, trainable and older dogs are perceived as more dominant in multi-dog households RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Lisa J. WallisID, Ivaylo B. Iotchev, Enikő Kubinyi Department of Ethology, Eo¨tvo¨s Lora´nd University, Budapest, Hungary Lisa J. WallisID, Ivaylo B. Iotchev, Enikő Kubinyi Department of Ethology, Eo¨tvo¨s Lora´nd University, Budapest, Hungary a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * lisa.wallis@live.co.uk * lisa.wallis@live.co.uk a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Social dominance is an important and widely used concept, however, different interpreta- tions have led to ambiguity in the scientific literature and in popular science. Even though in ethology dominance is an attribute of dyadic encounters, and not a characteristic of the indi- vidual, ‘dominance’ has often been referred to as a personality trait in animals. Since few studies have specifically examined the link between personality traits and dominance status, we investigated this in dogs living in multi-dog households using a questionnaire, which required owners to specify whether the dog had a dominant or submissive status, and com- prised items of both the features of the individual (i.e. personality traits) and previous social experience (interactions with group members and strangers). Four distinct personality factors emerged from 23 behavioural items by principal component analysis, labelled as assertiveness, trainability, intraspecific aggression and independence. Binomial logistic regression was used to examine how the demographic information of the dogs and the per- sonality factors predicted the owner’s estimate of the dog’ status as dominant or submissive. The personality factor assertiveness accounted for 34% of the variance in dominance sta- tus, trainability 5% and dog age contributed 4%. Dogs perceived as dominant scored more highly on the factors assertiveness and trainability, which can help explain why ‘dominance’ has often been suggested to be a personality trait, rather than a dyad-specific social status according to different traditions in behavioural research. Similar to the ‘social dominance’ trait in humans, owner ascribed dominance showed a quadratic trajectory in cross-sectional mean change across the lifespan, increasing during adulthood and then maintaining high levels until old age. Overall, our study proposes a multifactorial background of dominance relationships in pet dogs, suggesting that not only previous experience of social interactions between individuals but also age and personality traits influence owner perceived domi- nance status in multi-dog households. OPEN ACCESS Citation: Wallis LJ, Iotchev IB, Kubinyi E (2020) Assertive, trainable and older dogs are perceived as more dominant in multi-dog households. PLoS ONE 15(1): e0227253. https://doi.org/10.1371/ journal.pone.0227253 Introduction 1158?m=178), all to EK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1158?m=178), all to EK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dominance is used to describe social relationships among group-living animals. It is an impor- tant and widely used concept, however, there is little agreement regarding its meaning, and dif- ferent interpretations have often led to ambiguity in the scientific literature and in popular science. In ethology, dominance is a relative measure, an attribute of dyadic encounters and refers to a consistent outcome in favour of the same dyad member and a default yielding response of its opponent rather than escalation [1]. Dominance status is typically determined by examining the outcomes of aggressive interactions within dyads (agonistic dominance), rit- ualized and/or greeting signals that are independent of context (formal dominance) and moti- vation to obtain valued resources (competitive ability, measured through pairwise competition tests). When dominance is operationalized as competitive ability, the consistent winner is dominant, and the loser—subordinate. Competing interests: The authors have declared that no competing interests exist. Historically, in human psychology, social dominance is considered as an aspect or trait of personality [2]. Personality describes a specific pattern of behaviour, thoughts, and feelings that persist through time and across situations [3]. Personality traits refer to measurable aspects of personality that vary between individuals, but remain relatively consistent within individuals across time and context [4]. Factor analysis (or principle component analysis) can be used to identify personality factors/traits that are robust across investigations, samples and time. Confusingly, in some studies in animals, if a behavioural factor identified through factor analysis associated with dominance status or rank, it was often labelled as ‘dominance’ [5], which contributed to the ambiguity around the term, leading to the assumption that domi- nance is a personality trait. For example, meta-analyses of research on temperament and per- sonality (the two terms are often used interchangeably [6]) traits in dogs have suggested that across studies, ‘social dominance’ (a terminology adopted by e.g. [7–9]), is one of six [10], or seven recurring personality factors investigated in the dog [11]. Jones and Gosling [11] found that dominance was characterised by behaviours such as refusing to move out of the way, bul- lying other dogs, guarding food, and eating first. Demographic variables and personality traits as predictors of perceived dominance status in dogs Editor: Nicolas Chaline, Universidade de São paulo, BRAZIL Editor: Nicolas Chaline, Universidade de São paulo, BRAZIL Received: November 1, 2018 Accepted: December 16, 2019 Published: January 3, 2020 Copyright: © 2020 Wallis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Grant Agreement No. 680040; https://erc.europa.eu/), from the Ja´nos Bolyai Research Scholarship of the Hungarian Academy of Sciences, and from the U´NKP-Bolyai+ Scholarship (https://ttk.elte.hu/content/uj-nemezti- kivalosag-program-unkp-2018-2019-es-tanev.t. 1 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Introduction One suitable species to examine the link between personality traits and dominance status is the domestic dog. Many households contain more than one dog, which allows owners to observe the formation of dominance relationships. Even in single dog households, the emer- gence of dog parks has facilitated socialisation between dogs, which has enabled owners to view interactions between familiar dogs on a regular basis. Several studies have already utilised owner questionnaires to determine the dominance status of dogs in multi-dog households [26,27,41,42]. When dominance is considered as an attribute of dyadic encounters, and not a property of individuals, the perception of each dogs’ status can be based on consistent patterns in the outcome of interactions within dyads [23,43]. For example, in multi-dog households, dogs perceived as dominant by the owner have priority access to certain resources (for exam- ple, resting places and food rewards), undertake specific tasks (defend the group during per- ceived threats and lead other dog/s during walks), display dominance (win fights and over mark), have characteristic personality traits (measured using single item statements), and are usually older than subordinates [26]. The fact that owner estimates of dominance status corre- spond to previously established behavioural markers of dominance displays in dogs, suggests that dominance relationships are robust and well-perceivable components of companion dog behaviour. It furthermore shows that owner-derived reports about dominance status have external validity. Additionally, there is evidence that owners scoring of their dogs’ behavioural traits via questionnaire is also valid, as it corresponds to observational measurements of behav- iour. For example, extraverted dogs spent more time in dyads in off-leash parks, highly amica- ble dogs spent more time in play, and neurotic dogs displayed a higher frequency of lowered or hunched postures [44]. In free ranging and/or pet dogs, dominance status has been found to be associated with the personality factors motivation, trainability, sociability, impulsivity, and aggression. Impor- tantly, the relationships tend to be less strong in larger dog packs and to be more specifically related to the formal dominance style [26,27,45]. Age, sex, leadership and reproductive success have also been found to be related to dominance status in free ranging and/or pet dogs [24,26,46–49]. Theoretical models predict that intra-specific dominance, especially when tied to consistent leadership, is only useful in small groups characterized by asymmetric distribu- tion of experience and familiarity with the environment [50,51]. Introduction Therefore, dominance is often referred to as a personality trait of dogs, both in the literature [2] and by laymen [12,13]. This misuse of the term has contributed to the rise of dominance related aversive training techniques, such as hit- ting, shaking, growling, staring, and using other physical force, such as the ‘alpha roll’, and the ‘dominance down’; all of which in most cases provoke fearful or defensively aggressive behav- iour in the dog [14]. Ethologists have argued against the existence of dominance as a personality trait. Individu- als living in a group can be dominant or submissive with different partners, and thus domi- nance status within dyads is flexible, which does not fit to the definition of personality traits. In some relationships, the context of the behavioural interaction proves important. For exam- ple, individual competitive ability and differences in motivation to obtain valued resources can interact to produce different outcomes in different contexts. Relationships between dyads also involves affiliative behaviours in addition to, or even in the absence of dominance behaviours. Some dyads avoid each other and thus do not interact, so it is not possible to easily determine their relationship [15]. The fact that animals can form complex dynamic relationships that dif- fer between dyads argues against the concept of dominance (and submissiveness) as a person- ality trait. The relationship between dominance rank (position in a hierarchy) and related behaviours has been investigated in many species (for example; elephants (Loxodonta africana and Elephas maximus) [16], bottlenose dolphins (Tursiops truncatus) [17], chimpanzees (Pan troglodytes) [18–21], dogs (Canis familiaris) [15,22–27], hyena (Crocuta crocuta) [28], gorilla (Gorilla gorilla) [29–31], female zebra finches (Taeniopygia guttata) [32], great tits (Parus major) [33], PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 2 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs mountain chickadees (Poecile gambeli) [34], starlings (Sturnus vulgaris) [35], barnacle geese (Branta leucopsis) [36], male rainbowfish (Melanotaenia duboulayi) [37], and brown trout (Salmo trutta) [38]). However, far fewer studies have attempted to determine whether natural variation in personality can predict social status [27,37,39]. This is particularly surprising given the theoretical links between evolutionary game theory and the maintenance of animal personality [40]. Such studies would help clarify the correct terminology and ensure that per- sonality traits/factors and dominance status/rank are not treated equivalently. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Subjects The questionnaire was filled in online by 396 owners of more than one dog (90.1% of which were women), for a total of 550 dogs, in Hungarian. The questionnaire was advertised in a social media Dog Ethology group, between 14th June 2014 and 6th February 2015, and specifi- cally targeted owners with more than one dog in their household. Since we were interested in the personal knowledge and experience of the owner when examining owners’ perception of the dominance status of their dogs, we provided no training, explanation, or definition of dominance. Dogs were on average 5.0 years old (± 3.13 SD), weighted 22.7 kg (± 13.54 SD), 54% of the total sample were female, and 54% of the total sample were neutered. Regarding breed, 4.4% were of unknown/mixed breed, and the most frequently present breeds in the sample were the German shepherd dog (8%), Border collie (6.0%), Vizsla (5.3%), Spaniel (including Cocker and Springer 4.7%), Dachshund (4.6%), Golden retriever (4.0%), Labrador retriever (3.6%), and Belgian shepherd (3.3%). All other breeds were represented by less than 3% of the sample. Please refer to the supplementary materials for tables with a breakdown of the breeds and breed groups (S1 and S2 Tables). According to dogs housing conditions, 26.6% of the dogs were kept in the house, 16.4% in the garden, and 57.0% both in the garden and in the house. We allocated the dogs to three groups according to their training level; 43.3% did not received any formal training, 28.1% received basic and 28.6% participated in specialised training (e.g. agility, hunting). Procedure The questionnaire consisted of demographic and keeping conditions questions about individ- ual dogs (age in years, weight in kg, sex, neuter status, where the dog is kept, and training level), followed by one question about the perceived dominance status of the dog in the group (dominant N = 252, submissive, N = 116, both (or “I do not know”) N = 149, NA (missing data) N = 33 (the owner provided no answer to this question). The third part consisted of 23 questions about the dogs’ behaviour and personality (Table 1). For the questions about dog behaviour and personality we used a 1–5 scale system (Likert scale: 1 = no/not/never, 5 = very/ very much/frequently). Introduction The more knowledgeable individual can convey a true advantage to other group members. Dominance follows almost automatically from this asymmetrical arrangement [23,52]. The likelihood of formal domi- nance and/or leadership can be expected to increase with age for this reason, since aged ani- mals have more experience if the environment is stable across generations. In the current study, we investigated how the owner perceived dominance rank of dogs liv- ing in multi-dog households is related to their personality traits, derived from a behavioural questionnaire by factor analysis, and dog demographic information using a pilot sample. Instead of using existing questionnaires, we developed a new one to include both the owners’ estimation of their dogs’ personality traits, and their dogs’ previous experience with other dogs in the household, and during meetings with other individuals. The questionnaire items were pre-selected to address behaviours frequently studied in dog personality research that might be related to dominance status [53]. We chose the terminology that owners most often use PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 3 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs (e.g. ‘smart’ instead of ‘trainable’). In contrast to previous studies we used age in years to inves- tigation nonlinear relationship with dominance rank. We hypothesized that dominant dogs as perceived by the owners will be older, and possess specific personality traits, such as high asser- tiveness, confidence or boldness, high physical aggression, and high trainability, in contrast to subordinate dogs. Statistical analysis Analyses were performed in SPSS 22.0 (factor analysis) and R (binomial logistic regression and graphs) [54]. In order to reduce the number of items, and to determine the underlying structure of the data, a principle component analysis (PCA) with Varimax rotation [55] and a default of maximum 25 iterations was used with the Maximum Likelihood method on the 23 questions concerning dog behaviour and personality traits on the full sample (N = 500, NA = 50). For details please refer to supplementary materials S1 File. The solution that explained >50% of the variance and with factor eigenvalues > 1 was accepted. In addition, items that did not load on any of the factors (below 0.5) were removed from the analysis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 4 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs Table 1. Questionnaire items related to dog characteristics, behaviours, and previous social experiences. Item number Short name of item Characteristics/behaviours/social experiences 1 Fit How fit is your dog? 2 Smart How smart is your dog? 3 Calm How easily does your dog calm down if it is nervous? 4 Leading type Is your dog the leading type? 5 Cunning How cunning is your dog? 6 Read people well How well can your dog “read human thoughts”? 7 Best rest How often does your dog acquire the best resting place? 8 Temper How often does your dog display his/her temper? 9 Break rules How often does your dog cunningly try to break the rules? 10 Interfere How much does your dog interfere in other dogs’ fights? 11 Fast learner Is your dog a fast learner? 12 Win play fights How often does your dog win play-fights with other dogs? 13 Stubborn Is your dog “devious” (does he/she often get his/her own way)? 14 Slow Is your dog a slow (lazy) type? 15 Pack defence Does your dog remain in the front if the pack faces real or apparent threat? 16 Look down Does your dog appear to look down on other dogs? 17 Socialized How well is your dog socialized? 18 Mount others How often does your dog try to mount other dogs outside the breeding season? 19 Adaptive How well does your dog adapt to your other dogs? 20 Challenge others How often does your dog initiate rough interactions with other dogs? Statistical analysis 21 Novelty seeking Does your dog quickly respond to novel or distressing stimuli? 22 Fighting How often does your dog fight with other dogs, including strangers? 23 Obedience Is your dog obedient? https://doi.org/10.1371/journal.pone.0227253.t001 Subsequently, the items of each final factor were tested for internal consistency with Cron- bach’s alpha. The resulting factor structure was then used as a template to calculate the factor scores for each individual dog, in order to allow missing values, with the provision of a mini- mum of two values per factor, to maximise the sample size (N = 542). We calculated the trait scores by taking the mean of the items loading with at least 0.5 on a given factor (items that loaded negatively on a factor were inverted (e.g. calm and socialized)). Subsequently, the items of each final factor were tested for internal consistency with Cron- bach’s alpha. The resulting factor structure was then used as a template to calculate the factor scores for each individual dog, in order to allow missing values, with the provision of a mini- mum of two values per factor, to maximise the sample size (N = 542). We calculated the trait scores by taking the mean of the items loading with at least 0.5 on a given factor (items that loaded negatively on a factor were inverted (e.g. calm and socialized)). We then used binomial logistic regression to test how the demographic information of the dogs (age in years, weight in kg, sex, neuter status, where the dog is kept, and training level), and the factors obtained with the personality trait factor analysis would predict the owner’s estimate of the dog as dominant or submissive (rank status). After removing dogs that were identified as “both” (sometimes dominant, sometimes submissive (N = 182)) and those with missing information ((NA) N = 28), the sample size using the full model was 332 individuals, and in the reduced model 343 individuals were included. Due to the small sample size, we only examined main effects, but included quadratic terms for the continuous personality factor scores and age in years. Non-significant terms (P > 0.05) were removed stepwise from the model. A pseudo R-squared value was calculated to determine how well the model explains the data. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Descriptive information of the canine personality factors The intraspecific aggression factor was positively skewed, with half the dogs scoring between 1.80 and 3.33. Trainability was the most negatively skewed of the factors, with half the dogs scoring between 3.75 to 4.75. At least one dog obtained the maximum score possible on each of the four factors, apart from for intraspecific aggression. The largest range of scores was obtained for the assertiveness and independence factors while the intraspecific aggression fac- tor had the smallest range. The median scores and percentiles for each of the personality trait factors are shown in Fig 1. Factor analysis of dog characteristics, behaviours and previous social experience questionnaire items Seventeen items contributing to four factors were found to explain 57.8% of the total variance (Table 2), while six items were excluded (listed in short form: fit, best rest, slow, mount others, adaptive, and novelty seeking). The factors were labelled as assertiveness (22.77% of variance explained, Cronbach alpha = 0.76), trainability (16.88% of variance explained, Cronbach alpha = 0.73), intraspecific aggression (10.48% of variance explained, Cronbach alpha = 0.73), and independence (7.71% of variance explained, Cronbach alpha = 0.73). The loadings of the items on the factors are shown in Table 2. Statistical analysis The R package rcompanion and the command nagelkerke were used to produce a pseudo R squared value for the fixed effects in comparison to the intercept only model. For details of the R code, and results from the models please refer to supplementary materials S2 File. Possi- ble dependence between owner responses of dogs living in the same household was addressed in the supplementary materials (S3 File). Graphs were produced in R using the ggplot package PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 5 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs and the geom_smooth function to plot the smoothed conditional mean and confidence inter- vals. Significant predictors were mean centred before plotting, by subtracting the sample mean from each observation, in order to make the intercept more meaningful. Ethics statement The data was collected using an online questionnaire designed to assess the dogs’ demo- graphic data, personality, and keeping conditions via owner report. According to the current Hungarian law (1998. e´vi XXVIII. To¨rve´ny—the Animal Protection Act, 3rd para- graph, 9th point), non-invasive observational data collection on dog demographics and behaviour are not considered as animal experiments and are therefore allowed to be con- ducted without any special permission from the University Institutional Animal Care and Use Committee (UIACUC). The filling out of the questionnaires was voluntary and anony- mous so the study did not violate respondents’ privacy. Informed consent was included in the introductory text of the questionnaires. Ethical approval or an ethical waiver from an Institutional Research Board or equivalent for collecting survey data from human partici- pants was not necessary, as we did not collect private, identifiable information about human third parties. Binomial logistic regression of dominance status, dog demographics and PCA factors The final model revealed significant associations with the personality trait factor scales of assertiveness and trainability, as well as dog age in years. We calculated the pseudo R-squared measure to indicate how well the model explains the data. McFadden pseudo R squared was 0.43 indicating an excellent model fit. A significant polynomial relationship between the factor score assertiveness and the proba- bility that dogs were allocated a ‘dominant’ or ‘subordinate’ status by the owner was found. Dogs that were described as more assertive were significantly more likely to be dominant, than PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 6 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs Table 2. Results of the factor analysis of the behaviour and personality trait items. Column 1: Item numbers that loaded > 0.5 on at least one factor. Column 2: The short form of the name of the item. Columns 3–6: Loadings of individual items across the four factors—assertiveness, trainability, intraspecific aggression, and indepen- dence. Loadings > 0.5 are shown in boldface. The percentage of variance explained, Cronbach’s alpha value and Eigenvalue for each factor are shown in the last rows of the table Table 2. Results of the factor analysis of the behaviour and personality trait items. Column 1: Item numbers that loaded > 0.5 on at least one factor. Column 2: The short form of the name of the item. Columns 3–6: Loadings of individual items across the four factors—assertiveness, trainability, intraspecific aggression, and indepen- dence. Loadings > 0.5 are shown in boldface. The percentage of variance explained, Cronbach’s alpha value and Eigenvalue for each factor are shown in the last rows of th t bl short form of the name of the item. Columns 3–6: Loadings of individual items across the four factors—assertiveness, trainability, intraspecific aggression, and indepen- dence. Loadings > 0.5 are shown in boldface. The percentage of variance explained, Cronbach’s alpha value and Eigenvalue for each factor are shown in the last rows of the table. Item Short form Assertiveness Trainability Intraspecific aggression Independence 12 Win play fights 0.76 0.09 -0.05 0.04 15 Pack defence 0.71 0.06 0.08 -0.05 4 Leading type 0.67 0.15 0.09 0.29 16 Look down 0.63 0.04 0.09 0.22 10 Interfere 0.61 -0.01 0.39 0.06 2 Smart 0.05 0.83 0.02 0.13 11 Fast learner 0.06 0.83 0.01 0.10 6 Read people well 0.19 0.68 -0.23 0.15 23 Obedience 0.03 0.62 -0.11 -0.37 3 Calm 0.14 0.10 -0.70 -0.13 20 Challenge others 0.39 0.05 0.70 -0.03 8 Temper 0.08 0.03 0.68 0.22 22 Fighting 0.47 -0.03 0.61 -0.05 17 Socialized -0.09 0.36 -0.61 0.05 9 Break rules 0.06 -0.10 0.13 0.80 5 Cunning 0.14 0.27 0.00 0.74 13 Stubborn 0.14 0.05 0.07 0.73 Explained Variance 22.77% 16.88% 10.48% 7.71% Cronbach Alpha 0.76 0.73 0.73 0.73 Eigenvalues 3.87 2.87 1.78 1.31 htt //d i /10 1371/j l 0227253 t002 dogs that scored lower in assertiveness (see Fig 2, Table 3). Dogs that had a higher than average assertiveness score (3.29) had a greater than 75% probability of being allocated a “dominant” status. On average, a one-unit change in assertiveness is associated with an exp(41.248−2×- 10.479) change in the odds of being dominant. dogs that scored lower in assertiveness (see Fig 2, Table 3). Dogs that had a higher than average assertiveness score (3.29) had a greater than 75% probability of being allocated a “dominant” status. On average, a one-unit change in assertiveness is associated with an exp(41.248−2×- 10.479) change in the odds of being dominant. dogs that scored lower in assertiveness (see Fig 2, Table 3). Dogs that had a higher than average assertiveness score (3.29) had a greater than 75% probability of being allocated a “dominant” status. On average, a one-unit change in assertiveness is associated with an exp(41.248−2×- 10.479) change in the odds of being dominant. Fig 1. Median and quartiles of the personality trait factor scores. https://doi.org/10.1371/journal.pone.0227253.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 7 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 7 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs Fig 2. The influence of the dog personality factor assertiveness on dog rank allocation. Fitted logistic regression curve (smoothed conditional mean) showing that the dogs’ probability of being classified as ‘dominant’ (1.0) or ‘submissive’ (0.0) by the owner (Y -axis), is dependent on the factor assertiveness (mean centred, M = 3.39). The dots show the individual data points, the blue line is the predicted probability that a dog is dominant, and the shaded areas show the confidence intervals. https://doi org/10 1371/journal pone 0227253 g002 Fig 2. The influence of the dog personality factor assertiveness on dog rank allocation. Fitted logistic regression curve (smoothed conditional mean) showing that the dogs’ probability of being classified as ‘dominant’ (1.0) or ‘submissive’ (0.0) by the owner (Y -axis), is dependent on the factor assertiveness (mean centred, M = 3.39). The dots show the individual data points, the blue line is the predicted probability that a dog is dominant, and the shaded areas show the confidence intervals. https://doi.org/10.1371/journal.pone.0227253.g002 https://doi.org/10.1371/journal.pone.0227253.g002 Additionally, we found a significant linear relationship between the factor scale trainability and the proportion of dogs that were allocated a ‘dominant’ status. Dogs that were described as more trainable were significantly more likely to be labelled dominant, than dogs that scored lower in trainability (see Fig 3, Table 3). Holding all other predictors at a fixed value, we see a 73% increase in the odds of being dominant for a one-unit increase in trainability score. Finally, we found a significant quadratic relationship between the demographic variable age in years and the probability that dogs were allocated a dominant status. Dogs that were older were significantly more likely to be dominant according to the owner, than dogs that were younger (see Fig 4, Table 3). Dogs of one year of age had around a 50% probability of being dominant, which rose to 80% at the age of eight. By age 10, the probability of being dominant began to plateau, and the youngest and oldest dogs showed the greatest variability. On average, a one-year change in age is associated with an exp(12.408−2×-8.167) change in the odds of being dominant. Table 3. Results and parameter estimates (±SE) from the binomial generalised linear model investigating which factors affect whether the dog was allocated a “dominant” status. Predictor p-value Estimate SE 95% Wald confidence interval (lower and upper) Odds Ratio Assertiveness: linear 0.000 41.248 4.788 31.863 50.633 8.20e+17 Assertiveness: quadratic 0.038 -10.479 5.055 -20.387 -0.571 2.81e-05 Age in years: linear 0.001 12.408 3.812 4.936 19.881 2.45e+05 Age in years: quadratic 0.019 -8.167 3.493 -15.012 -1.322 2.84e-04 Trainability 0.029 0.027 0.013 0.003 0.052 1.73 https://doi.org/10.1371/journal.pone.0227253.t003 from the binomial generalised linear model investigating which factors affect whether the dog was allocated a sults and parameter estimates (±SE) from the binomial generalised linear model investigating which factors affect wheth status Table 3. Results and parameter estimates (±SE) from the binomial generalised linear model investigating which factors “dominant” status. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 8 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs Fig 3. The influence of the dog personality factor trainability on dog rank allocation. Fitted logistic regression curves (smoothed conditional means) showing that the dogs’ probability of being classified as ‘dominant’ (1.0) or ‘submissive’ (0.0) by the owner (Y-axis), is dependent on the factor trainability (mean centred, M = 4.25). The dots show the individual data points, the blue line is the predicted probability that a dog is dominant, and the shaded areas show the confidence intervals. https://doi org/10 1371/journal pone 0227253 g003 Fig 3. The influence of the dog personality factor trainability on dog rank allocation. Fitted logistic regression curves (smoothed conditional means) showing that the dogs’ probability of being classified as ‘dominant’ (1.0) or ‘submissive’ (0.0) by the owner (Y-axis), is dependent on the factor trainability (mean centred, M = 4.25). The dots show the individual data points, the blue line is the predicted probability that a dog is dominant, and the shaded areas show the confidence intervals. Fig 3. The influence of the dog personality factor trainability on dog rank allocation. Fitted logistic regression curves (smoothed conditional means) showing that the dogs’ probability of being classified as ‘dominant’ (1.0) or ‘submissive’ (0.0) by the owner (Y-axis), is dependent on the factor trainability (mean centred, M = 4.25). The dots show the individual data points, the blue line is the predicted probability that a dog is dominant, and the shaded areas show the confidence intervals. https://doi.org/10.1371/journal.pone.0227253.g003 Weight, sex, neuter status, training level, keeping place, and the personality trait factors intraspecific aggression and independence had no significant effect on the estimated domi- nance rank. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Discussion The current study aimed to identify demographic and personality factors associated with dogs’ dominance status as perceived by the owner using a questionnaire. The main finding is that dogs assigned a dominant status displayed higher assertiveness and trainability and were older than subordinate dogs. The remaining two traits, intraspecific aggression and independence, weight, sex, neuter status, where the dog is kept, and training level had no association with owner perceived dominance status. The factors trainability and (intraspecific) aggression correspond to already established per- sonality factors in dogs (reviews: 8 studies aggression, 11 responsiveness to training [51], and 30 aggression and 34 responsiveness to training [52]). However, independence and assertive- ness are less widespread. Independence refers to the dogs’ tendency to make decisions inde- pendently of the owner. Two previous studies have identified a factor/items which they also labelled or described as “independence” [56,57]. Assertiveness’ associated items point to per- ceived confidence, initiative and persistence in social interactions. It has been described in other species, but so far has only been suggested to be linked to dominance status in dogs [58]. However, previous studies have defined analogous traits to assertiveness, such as ‘boldness’ [59–61] (which increases with age in dogs [62,63]), and ‘confidence’, ‘courage’, ‘self-confi- dence’, and ‘motivation’ [64–68]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 9 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs Fig 4. The influence of dog age in years on dog rank allocation. Fitted logistic regression curve (smoothed conditional mean) showing that the dogs’ probability of being classified as ‘dominant’ (1.0) or ‘submissive’ (0.0) by the owner (Y-axis), is dependent on dogs’ age in years (mean centred, M = 5.13). The dots show the individual data points, the blue line is the predicted probability that a dog is dominant, and the shaded areas show the confidence intervals. https://doi.org/10.1371/journal.pone.0227253.g004 Fig 4. The influence of dog age in years on dog rank allocation. Fitted logistic regression curve (smoothed conditional mean) showing that the dogs’ probability of being classified as ‘dominant’ (1.0) or ‘submissive’ (0.0) by the owner (Y-axis), is dependent on dogs’ age in years (mean centred, M = 5.13). The dots show the individual data points, the blue line is the predicted probability that a dog is dominant, and the shaded areas show the confidence intervals. https://doi.org/10.1371/journal.pone.0227253.g004 Fig 4. The influence of dog age in years on dog rank allocation. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Discussion Fitted logistic regression curve (smoothed conditional mean) showing that the dogs’ probability of being classified as ‘dominant’ (1.0) or ‘submissive’ (0.0) by the owner (Y-axis), is dependent on dogs’ age in years (mean centred, M = 5.13). The dots show the individual data points, the blue line is the predicted probability that a dog is dominant, and the shaded areas show the confidence intervals. https://doi.org/10.1371/journal.pone.0227253.g004 https://doi.org/10.1371/journal.pone.0227253.g004 https://doi.org/10.1371/journal.pone.0227253.g004 The fact that dogs perceived as dominant were more assertive is not surprising. Previous studies have shown that dominant individuals undertake specific tasks such as defending the group during perceived threats and leading other dogs during walks (which corresponds to the items ‘pack defence’ and ‘leading type’). They also display dominance through consistently winning fights (item ‘wins play fights’) [26]. Interestingly, in free ranging dogs, participation in intergroup conflicts increases with a decreasing ratio of the number of rivals [69], and the number of affiliative partners involved [70]. Given that most dogs living in multi-dog house- holds have relatively strong affiliative bonds, and most interactions with strangers occur in small groups, or singularly, this might help explain why some owners observed higher levels of pack defence and leading in the more dominant animals. In their review, Gosling and John [5] found that the personality factor ‘dominance’ emerged as a clear factor in 7 animal studies out of 19. Although this factor was interpreted as ‘confi- dence’ in rhesus macaques (Macaca mulatta) and ‘assertiveness’ in hyenas (Crocuta crocuta), all measures correlated substantially with dominance rank. This suggests that these personality factors (labelled dominance, assertiveness, confidence etc. in previous studies) describe typical dominance related behaviours that are displayed to familiar individuals that live in a group set- ting. For example, assertiveness also corresponds to the previously described trait of ‘motiva- tion’ in dogs [67,68], as suggested by Bradshaw et al. [71]. Highly trainable dogs also tended to be allocated a dominant status by owners. Dogs high in trainability were reported to be smart, fast learners, could “read people” well, and were obe- dient. Previously we found that dogs allocated a dominant status within dyads were rated as “smarter” than subordinates [26] and more controllable [27]. In addition, studies have demon- strated rank-related effects on cognitive ability. Discussion For example, in starlings (Sturnus vulgaris) PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 10 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs and pheasants (Phasianus colchicus), the fastest learners occupied the highest competitive ranks [35,72], and in rhesus macaques, dominant individuals showed superior learning capaci- ties when tested, and subordinates “played dumb” when learning in mixed social groups. The subordinates avoided socially difficult situations by inhibiting their behaviour, and missed out on desirable food items, in order to minimise potential retaliation from dominant individuals [73]. Therefore, it is conceivable that in the current study, subordinate dogs are not less smart but inhibit their behaviour in the home environment in order to avoid conflicts with the domi- nant animal. In contrast to our predictions, dogs labelled as dominant by owners did not show higher scores in the factor ‘intraspecific aggression’. Some dogs allocated a dominant status by the owner may have a formal dominance relationship (display ritualized and/or greeting signals that are independent of context) with the other dog in the household and adopt a non-con- frontational attitude with stranger dogs, and thus may rarely show aggression, or they may have a non-interactive relationship with other dogs, which would also result in low intraspe- cific aggression scores. Indeed, field studies in both dogs and macaques [49,74] suggest that agonistic dominance (associated with higher aggression) is less likely than formal dominance to predict leadership. Aggressive interactions are usually influenced by motivation and context (e.g. reproductive activity) [75], and occur more often in less well-established relationships, and therefore may not correspond to the underlying hierarchy [24,76]. Hence, our results imply that individuals rated as dominant by owners were more likely expressing formal dominance. Although part of this study’s aim was to additionally assess the influence of demographic and keeping conditions on dominance, none of the chosen factors (weight in kg, sex, neuter status, where the dog is kept, and training level) reached significance. The only exception was the age of the dog in years. Overall, we found a quadratic relationship between age in years and the probability that dogs were allocated a dominant status. In humans, the personality trait ‘social dominance’ shows a very similar quadratic trajectory in cross-sectional mean trait change across adulthood [77], as does ‘dominance’ in male chimpanzees [78]. Discussion Age also pre- dicted dominance rank in our previous study [26], where overall 66% of dominant individuals were the older animal in the dyad. Older dogs were more likely to be classified as dominant than younger dogs, in agreement with the literature for both wolves and dogs [22,23,46,47,79]. One-year old dogs had a 50% probability of being classified as dominant, which seems high given their age and amount of experience. However, since we do not know the age of their partner dog/s, perhaps they were the oldest in a group of young dogs, or they were all a similar age. Interestingly, in free-ranging dogs, subadults (who tend to be in the middle of the hierarchy) target more dominance inter- actions of all types at other subadult individuals, which may indicate instability in the hierar- chy [76]. Although adolescent dogs may be sexually mature, dogs do not tend to show fully adult behaviour until 2–3 years of age [80]. Most adolescent dogs go through a hormonal surge which affects their behaviour, decreasing their ability to pay attention and respond to previ- ously learned cues [81,82]. High activity levels and motivation, as well as deficiencies in execu- tive control during this period, might result in owners interpreting their dog’s behaviour as an expression of dominance, and lead them to conclude that the dog is dominant. An important limitation of the current study is that 90% of the owners surveyed were Hun- garian females, which prevented the examination of sex effects. Previous studies have found owner sex differences in the perception of dog behaviour; male owners perceive their dogs to be more disobedient [83], bold, and less sociable and trainable in comparison to female owners [63]. Therefore, there are likely to be differences in the way male and female owners perceive dominance rank and related behaviours. Future studies should aim to include more males, PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 11 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs people from other countries/cultures, and in addition, they should take into account the char- acteristics of the other canine members of the group (including sex, breed and age). This study focused on examining owner perceived dominance status, associated dominance related behaviours, and personality factors, and did not attempt to define all the different types of relationships found previously in dogs. Conclusion Overall, our study suggests a multifactorial background of dominance relationships in pet dogs. It is likely that dominance status is not only determined by previous experience of social interactions between group members as suggested by ethologists but also by personality factors as proposed by psychologists [85]. Since owners based their answers on their estimation of their dogs’ characteristics, and previous experience with their dog’s behaviour when socialising with other dogs in the household, and during meetings with other individuals, our study com- bined both the features of the individual and previous intraspecific social interactions. Our work adds to a growing line of evidence that some personality factors, namely assertiveness and trainability can increase the odds that an owner ascribes the dog a higher status within the household. The identified link between assertiveness as a personality trait and dominance sta- tus is probably the basis of the confusion regarding the term ‘dominance’ among lay people and also partially in science. Furthermore, while future studies need to more directly address the distinction between different dominance styles, the observation that assertiveness was a better predictor of dominance status than intraspecific aggression in our study, suggests that mainly formal dominance (i.e. dominance displayed without aggression) influences owners’ perception of dogs’ social rank in multi-dog households. Finally, the weight, sex, neuter status, and training level of the dog did not influence owner perceived dominance status. Importantly, our results suggest that labelling an animal personality factor as ‘dominance’ is an incorrect use of the term. Therefore, in order to facilitate the discrimination between per- sonality traits and dominance as a status in dyads, or as a rank in dog social groups, we suggest using ‘assertive’ when describing related personality traits, and ‘dominance’ when referring to status/ranks between individuals with an established relationship. Discussion For example, we were not able to determine whether dogs’ relationships were characterised by agonistic dominance, formal dominance, or compet- itive ability, which might differ between contexts. Although the observed pattern of low intra- specific aggression, high trainability and higher age of the dominant animals are found in the literature on free-ranging dogs and are particularly associated with the formal type, additional studies are necessary to clarify the relationship between dominance status, dominance related behaviours, and associated personality factors in dogs. Furthermore, how owner demographics (e.g. the number of people in the household, their amount of experience with dogs, their sex, age and/or personalities) influence how they perceive dominance and dominance related behaviours between dogs in their household, should also be established. To obtain a more complete picture of the factors influencing social relationships in dogs, questions pertaining to agonistic and formal dominance behaviours, previous experiences, social contexts, play, affilia- tion, passive interactions, sleeping proximities, and other tactile communication within multi- dog households should be addressed [84]. S2 Table. Count of the number of dogs in each breed group of the Fe´de´ration Cynologique Internationale (FCI), including the percentage of the overall sample. (DOCX) S2 File. R code. Full details of the binomial generalized linear model before and after reduc- tion carried out in R. (DOCX) S3 File. GEE models. Investigation of possible dependence between dogs living in the same household. (DOCX) Acknowledgments We would like to thank all the Hungarian owners who filled in the online survey. We thank Borba´la Turcsa´n for her contribution to the design of the questionnaires, and additionally for her helpful comments on the manuscript. Zso´fia Kőva´go´ for her statistical analysis during the development of the study, and Zolta´n Vajo´ for initiating the questionnaire and formulating some items. Project administration: Enikő Kubinyi. Supervision: Enikő Kubinyi. Writing – review & editing: Lisa J. Wallis, Ivaylo B. Iotchev, Enikő Kubinyi. Writing – review & editing: Lisa J. Wallis, Ivaylo B. Iotchev, Enikő Kubinyi. Writing – review & editing: Lisa J. Wallis, Ivaylo B. Iotchev, Enikő Kubinyi. Supporting information S1 Table. Break down of all the dog breeds present in the sample, including count of the dogs and the percentage of the overall sample. The percentages marked in bold are all breeds with a greater than three percentage in the sample population. (DOCX) 12 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Demographic variables and personality traits as predictors of perceived dominance status in dogs S2 Table. Count of the number of dogs in each breed group of the Fe´de´ration Cynologique Internationale (FCI), including the percentage of the overall sample. (DOCX) S1 File. Principle component analysis. A principle component analysis (PCA) with Varimax rotation and a default of maximum 25 iterations was used with the Maximum Likelihood method on the 23 questions concerning dog behaviour and personality traits on the full sample (N = 500, NA = 50). (DOCX) S2 Table. Count of the number of dogs in each breed group of the Fe´de´ration Cynologique Internationale (FCI), including the percentage of the overall sample. (DOCX) S1 File. Principle component analysis. A principle component analysis (PCA) with Varimax rotation and a default of maximum 25 iterations was used with the Maximum Likelihood method on the 23 questions concerning dog behaviour and personality traits on the full sample (N = 500, NA = 50). (DOCX) S2 File. R code. Full details of the binomial generalized linear model before and after reduc- tion carried out in R. (DOCX) S3 File. GEE models. Investigation of possible dependence between dogs living in the same household. (DOCX) Author Contributions Conceptualization: Enikő Kubinyi. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Conceptualization: Enikő Kubinyi. Data curation: Lisa J. Wallis. References 1. Drews C. The Concept and Definition of Dominance in Animal Behaviour. Intergovernmental Panel on Climate Change, editor. Behaviour. Cambridge: Cambridge University Press; 1993; 125: 283–313. https://doi.org/10.1163/156853993X00290 1. Drews C. The Concept and Definition of Dominance in Animal Behaviour. Intergovernmental Panel on Climate Change, editor. Behaviour. Cambridge: Cambridge University Press; 1993; 125: 283–313. https://doi.org/10.1163/156853993X00290 2. Roberts BW, Walton KE, Viechtbauer W. Patterns of mean-level change in personality traits across the life course: a meta-analysis of longitudinal studies. Psychol Bull. 2006; 132: 1–25. https://doi.org/10. 1037/0033-2909.132.1.1 PMID: 16435954 3. John OP, Robins RW, Pervin LA. Handbook of Personality: Theory and Research. Handb Personal Theory Res. 2008; 3: 881. 1. Drews C. The Concept and Definition of Dominance in Animal Behaviour. Intergovernmental Panel on Climate Change, editor. Behaviour. Cambridge: Cambridge University Press; 1993; 125: 283–313. https://doi.org/10.1163/156853993X00290 2. Roberts BW, Walton KE, Viechtbauer W. Patterns of mean-level change in personality traits across the life course: a meta-analysis of longitudinal studies. Psychol Bull. 2006; 132: 1–25. https://doi.org/10. 1037/0033-2909.132.1.1 PMID: 16435954 3. John OP, Robins RW, Pervin LA. Handbook of Personality: Theory and Research. Handb Personal Theory Res. 2008; 3: 881. 13 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Demographic variables and personality traits as predictors of perceived dominance status in dogs 4. Bell AM, Hankison SJ, Laskowski KL. The repeatability of behaviour: a meta-analysis. Anim Behav. Elsevier Ltd; 2009; 77: 771–783. https://doi.org/10.1016/j.anbehav.2008.12.022 PMID: 24707058 5. Gosling SD, John OP. Personality Dimensions in Nonhuman Animals. Curr Dir Psychol Sci. 1999; 8: 69–75. https://doi.org/10.1111/1467-8721.00017 6. McCrae RR, Costa PT, Ostendorf F, Angleitner a, Hrebı´ckova´ M, Avia MD, et al. Nature over nurture: temperament, personality, and life span development. J Pers Soc Psychol. 2000; 78: 173–86. Available: http://www.ncbi.nlm.nih.gov/pubmed/10653513 7. James WT. Social organization among dogs of different temperaments, terriers and beagles, reared together. J Comp Physiol Psychol. 1951; 44: 71–77. https://doi.org/10.1037/h0061218 PMID: 14814243 8. Beaudet R, Chalifoux A, Dallaire A. Predictive value of activity level and behavioral evaluation on future dominance in puppies. Appl Anim Behav Sci. 1994; 40: 273–284. https://doi.org/10.1016/0168-1591 (94)90068-X 9. Campbell WE. A behaviour test for puppy selection. Mod Vet Pract. [American Veterinary Publications]; 1972; 53: 29–33. Available: http://www.ncbi.nlm.nih.gov/pubmed/5080472 10. Fratkin JL, Sinn DL, Patall EA, Gosling SD. Personality Consistency in Dogs: A Meta-Analysis. Widdig A, editor. PLoS One. 2013; 8: e54907. https://doi.org/10.1371/journal.pone.0054907 PMID: 23372787 11. Jones AC, Gosling SD. References Temperament and personality in dogs (Canis familiaris): A review and evalua- tion of past research. Appl Anim Behav Sci. 2005; 95: 1–53. https://doi.org/10.1016/j.applanim.2005. 04.008 12. Dan D. Dominant Vs Submissive Dog Personality Traits [Internet]. 2018. https://theonlinedogtrainer. com/best-behaved-dog-breeds-does-breed-determine-a-dogs-personality/ 13. Familypet. What is the definition of a dominant dog [Internet]. https://familypet.com/what-is-the- definition-of-a-dominant-dog/ 14. Herron ME, Shofer FS, Reisner IR. Survey of the use and outcome of confrontational and non-confron- tational training methods in client-owned dogs showing undesired behaviors. Appl Anim Behav Sci. 2009; 117: 47–54. https://doi.org/10.1016/j.applanim.2008.12.011 15. Smuts B, Sandel AA, Trisko RK. Affiliation, dominance and friendship among companion dogs. Behav- iour. 2016; 153: 693–725. https://doi.org/10.1163/1568539X-00003352 16. Yasui S, Konno A, Tanaka M, Idani G, Ludwig A, Lieckfeldt D, et al. Personality Assessment and Its Association With Genetic Factors in Captive Asian and African Elephants. 2013; https://doi.org/10. 1002/zoo.21045 PMID: 22996044 17. Frick EE, Frick EE. Establishing a Link Between Personality and Social Rank in a Group of Bottlenose Dolphins (Tursiops truncatus) by. 2016; 18. Anestis SF. Behavioral style, dominance rank, and urinary cortisol in young chimpanzees (Pan troglo- dytes). 2005; 1245–1268. 19. Buirski P, Plutchik R, Kellerman H. Sex differences, dominance, and personality in the chimpanzee. Anim Behav. 1978; 26: 123–129. https://doi.org/10.1016/0003-3472(78)90011-8 PMID: 565175 20. Pederson AK, King JE, Landau VI. Chimpanzee (Pan troglodytes) personality predicts behavior. J Res Pers. 2005; 39: 534–549. https://doi.org/10.1016/j.jrp.2004.07.002 21. King JE, Figueredo AJ. The Five-Factor Model plus Dominance in Chimpanzee Personality. J Res Pers. 1997; 31: 257–271. https://doi.org/10.1006/jrpe.1997.2179 22. Trisko RK, Smuts BB. Dominance relationships in a group of domestic dogs (Canis lupus familiaris). Behaviour. 2015; 152: 677–704. https://doi.org/10.1163/1568539X-00003249 23. Bonanni R, Cafazzo S, Valsecchi P, Natoli E. Effect of affiliative and agonistic relationships on leader- ship behaviour in free-ranging dogs. Anim Behav. Elsevier Ltd; 2010; 79: 981–991. https://doi.org/10. 1016/j.anbehav.2010.02.021 24. Cafazzo S, Valsecchi P, Bonanni R, Natoli E. Dominance in relation to age, sex, and competitive con- texts in a group of free-ranging domestic dogs. Behav Ecol. 2010; 21: 443–455. https://doi.org/10.1093/ beheco/arq001 25. Bonanni R, Cafazzo S. The Social Organisation of a Population of Free-Ranging Dogs in a Suburban Area of Rome. The Social Dog. Elsevier; 2014. pp. 65–104. 26. Kubinyi E, Wallis LJ. Dominance in dogs as rated by owners corresponds to ethologically valid markers of dominance. PeerJ. Cold Spring Harbor Laboratory; 2019; 7: e6838. https://doi.org/10.7717/peerj. 6838 PMID: 31119074 27. A´ kos Z, Beck R, Nagy M, Vicsek T, Kubinyi E. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 References Leadership and Path Characteristics during Walks Are Linked to Dominance Order and Individual Traits in Dogs. Faisal AA, editor. PLoS Comput Biol. 2014; 10: e1003446. https://doi.org/10.1371/journal.pcbi.1003446 PMID: 24465200 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 14 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs 28. Gosling SD. Personality Dimensions in Spotted Hyenas (Crocuta crocuta). J Comp Psychol. 1998; 112: 107–118. https://doi.org/10.1037/0735-7036.112.2.107 PMID: 9642781 29. Gold KC, Maple TL. Personality Assessment in the Gorilla and Its Utility As a Management Tool. 1994; 522. 30. Eckardt W, Steklis HD, Steklis NG, Fletcher AW, Stoinski TS, Weiss A. Personality dimensions and their behavioral correlates in wild Virunga mountain gorillas (Gorilla beringei beringei). J Comp Psychol. 2015; 129: 26–41. https://doi.org/10.1037/a0038370 PMID: 25528652 31. Kuhar CW, Stoinski TS, Lukas KE, Maple TL. Gorilla Behavior Index revisited: Age, housing and behav- ior. Appl Anim Behav Sci. 2006; 96: 315–326. https://doi.org/10.1016/j.applanim.2005.06.004 32. David M, Auclair Y, Ce´zilly F. Personality predicts social dominance in female zebra finches, Taeniopy- gia guttata, in a feeding context. Anim Behav. Elsevier Ltd; 2011; 81: 219–224. https://doi.org/10.1016/ j.anbehav.2010.10.008 33. Dingemanse NJ, De Goede P. The relation between dominance and exploratory behavior is context- dependent in wild great tits. Behav Ecol. 2004; 15: 1023–1030. https://doi.org/10.1093/beheco/arh115 34. Fox RA, Ladage LD, Roth TC, Pravosudov VV. Behavioural profile predicts dominance status in moun- tain chickadees, Poecile gambeli. Anim Behav. 2009; 77: 1441–1448. https://doi.org/10.1016/j. anbehav.2009.02.022 PMID: 20161203 35. Boogert NJ, Reader SM, Laland KN. The relation between social rank, neophobia and individual learn- ing in starlings. Anim Behav. 2006; 72: 1229–1239. https://doi.org/10.1016/j.anbehav.2006.02.021 36. Kurvers RHJM, Eijkelenkamp B, van Oers K, van Lith B, van Wieren SE, Ydenberg RC, et al. Personal- ity differences explain leadership in barnacle geese. Anim Behav. 2009; 78: 447–453. https://doi.org/ 10.1016/j.anbehav.2009.06.002 37. Colle´ter M, Brown C. Personality traits predict hierarchy rank in male rainbowfish social groups. Anim Behav. 2011; 81: 1231–1237. https://doi.org/10.1016/j.anbehav.2011.03.011 38. Adriaenssens B, Johnsson JI. Shy trout grow faster: Exploring links between personality and fitness- related traits in the wild. Behav Ecol. 2011; 22: 135–143. https://doi.org/10.1093/beheco/arq185 39. David M, Auclair Y, Ce´zilly F. Personality predicts social dominance in female zebra finches, Taeniopy- gia guttata, in a feeding context. Anim Behav. Elsevier Ltd; 2011; 81: 219–224. https://doi.org/10.1016/ j.anbehav.2010.10.008 40. Briffa M, Sneddon LU, Wilson AJ. Animal personality as a cause and consequence of contest behav- iour. Biol Lett. 2015; 11. https://doi.org/10.1098/rsbl.2014.1007 PMID: 25808004 41. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 References Vienna, Austria: R Foundation for Statistical Computing; 2013. http://www.r-project.org 55. Gudjonsson G. An easy guide to factor analysis. Pers Individ Dif. 1994; 17: 302. https://doi.org/10.1016/ 0191-8869(94)90040-X 56. Cattell RB, Korth B. The isolation of temperament dimensions in dogs. Behav Biol. 1973; 9: 15–30. https://doi.org/10.1016/s0091-6773(73)80165-8 PMID: 4738708 57. Wahlgren K, Lester D. The big four: personality in dogs. Psychol Rep. Ammons Scientific; 2003; 92: 828. https://doi.org/10.2466/pr0.2003.92.3.828 PMID: 12841450 58. Ley JM, Bennett PC. Understanding Personality by Understanding Companion Dogs. Anthrozoos A Multidiscip J Interact People Anim. 2007; 20: 113–124. https://doi.org/10.2752/175303707X207909 59. Gosling SD, Bonnenburg A V. An integrative approach to personality research in anthrozoology: Rat- ings of six species of pets and their owners. Anthrozoos. 1998; 11: 148–155. https://doi.org/10.2752/ 089279398787000661 60. Svartberg K, Forkman B. Personality traits in the domestic dog (Canis familiaris). Appl Anim Behav Sci. 2002; 79: 133–155. https://doi.org/10.1016/S0168-1591(02)00121-1 61. Svartberg K. Shyness–boldness predicts performance in working dogs. Appl Anim Behav Sci. 2002; 79: 157–174. 62. Starling MJ, Branson N, Thomson PC, McGreevy PD. Age, sex and reproductive status affect boldness in dogs. Vet J. Elsevier Ltd; 2013; 197: 868–872. https://doi.org/10.1016/j.tvjl.2013.05.019 PMID: 23778256 63. Kubinyi E, Turcsa´n B, Miklo´si A. Dog and owner demographic characteristics and dog personality trait associations. Behav Processes. 2009; 81: 392–401. https://doi.org/10.1016/j.beproc.2009.04.004 PMID: 19520239 64. Humphrey ES. “Mental tests” for shepherd dogs: An attempted classification and evaluation of the vari- ous traits that go to make up “temperament” in the german shepherd dog. J Hered. Oxford University Press; 1934; 25: 129–136. https://doi.org/10.1093/oxfordjournals.jhered.a103899 65. Goddard ME, Beilharz RG. Individual variation in agonistic behaviour in dogs. Anim Behav. 1985; 33: 1338–1342. https://doi.org/10.1016/S0003-3472(85)80195-0 66. Ruefenacht S, Gebhardt-Henrich S, Miyake T, Gaillard C. A behaviour test on German Shepherd dogs: heritability of seven different traits. Appl Anim Behav Sci. 2002; 79: 113–132. 67. Ley JM, Bennett PC, Coleman GJ. A refinement and validation of the Monash Canine Personality Ques- tionnaire (MCPQ). Appl Anim Behav Sci. 2009; 116: 220–227. https://doi.org/10.1016/j.applanim.2008. 09.009 68. Ley J, Bennett P, Coleman G. Personality dimensions that emerge in companion canines. Appl Anim Behav Sci. 2008; 110: 305–317. https://doi.org/10.1016/j.applanim.2007.04.016 69. Bonanni R, Natoli E, Cafazzo S, Valsecchi P. Free-ranging dogs assess the quantity of opponents in intergroup conflicts. Anim Cogn. 2011; 14: 103–115. https://doi.org/10.1007/s10071-010-0348-3 PMID: 20845053 70. Bonanni R, Valsecchi P, Natoli E. Pattern of individual participation and cheating in conflicts between groups of free-ranging dogs. Anim Behav. Elsevier Ltd; 2010; 79: 957–968. https://doi.org/10.1016/j. anbehav.2010.01.016 71. References Pongra´cz P, Ba´nhegyi P, Miklo´si A´ . When rank counts—dominant dogs learn better from a human dem- onstrator in a two-action test. Behaviour. 2012; 149: 111–132. https://doi.org/10.1163/ 156853912X629148 42. Pongra´cz P, Vida V, Ba´nhegyi P, Miklo´si A. How does dominance rank status affect individual and social learning performance in the dog (Canis familiaris)? Anim Cogn. 2008; 11: 75–82. https://doi.org/ 10.1007/s10071-007-0090-7 PMID: 17492317 43. Schenkel R. Submission: Its features and function in the wolf and dog. Integr Comp Biol. 1967; 7: 319– 329. https://doi.org/10.1093/icb/7.2.319 44. Ottenheimer Carrier L, Cyr A, Anderson RE, Walsh CJ. Exploring the dog park: Relationships between social behaviours, personality and cortisol in companion dogs. Appl Anim Behav Sci. Elsevier B.V.; 2013; 146: 96–106. https://doi.org/10.1016/j.applanim.2013.04.002 45. Muner Kroll Dantas de Castro M. Assessing the social organization of multi-dog households : Dog behaviour, hormones, personality, and demographics. Memorial University of Newfoundland. 2017. 46. Cafazzo S, Lazzaroni M, Marshall-Pescini S. Dominance relationships in a family pack of captive arctic wolves (Canis lupus arctos): the influence of competition for food, age and sex. PeerJ. 2016; 4: e2707. https://doi.org/10.7717/peerj.2707 PMID: 27904806 47. Bonanni R, Cafazzo S, Abis A, Barillari E, Valsecchi P, Natoli E. Age-graded dominance hierarchies and social tolerance in packs of free-ranging dogs. Behav Ecol. 2017; 28: 1004–1020. https://doi.org/ 10.1093/beheco/arx059 48. Cafazzo S, Bonanni R, Valsecchi P, Natoli E. Social variables affecting mate preferences, copulation and reproductive outcome in a pack of free-ranging dogs. PLoS One. 2014; 9. https://doi.org/10.1371/ journal.pone.0098594 PMID: 24905360 49. Bonanni R, Cafazzo S, Valsecchi P, Natoli E. Effect of affiliative and agonistic relationships on leader- ship behaviour in free-ranging dogs. Anim Behav. Elsevier Ltd; 2010; 79: 981–991. https://doi.org/10. 1016/j.anbehav.2010.02.021 50. Conradt L, Roper TJ. Consensus decision making in animals. Trends Ecol Evol. 2005; 20: 449–456. https://doi.org/10.1016/j.tree.2005.05.008 PMID: 16701416 15 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 Demographic variables and personality traits as predictors of perceived dominance status in dogs 51. Conradt L, Roper TJ. Group decision-making in animals. Nature. 2003; 421: 155–158. https://doi.org/ 10.1038/nature01294 PMID: 12520299 52. de Waal FBM. Dominance “style” and primate social organization. Comparative Socioecology: The Behavioural Ecology of Humans and other Mammals. 1989. pp. 243–263. 53. Jones AC. Development and validation of a dog personality questionnaire [Internet]. The University of Texas at Austin, ProQuest Dissertations Publishing. University of Texas, Austin. 2008. http://ovidsp. ovid.com/ovidweb.cgi?T=JS&PAGE=reference&D=psyc6&NEWS=N&AN=2009-99140-320 54. R Core Team. R: A Language and Environment for Statistical Computing [Internet]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 References Bradshaw JWS, Blackwell E, Casey RA. Dominance in domestic dogs—A response to Schilder et al. (2014). J Vet Behav Clin Appl Res. Elsevier Inc; 2016; 11: 102–108. https://doi.org/10.1016/j.jveb.2015. 11.008 72. Langley EJG, van Horik JO, Whiteside MA, Madden JR. Group social rank is associated with perfor- mance on a spatial learning task. R Soc Open Sci. 2018; 5: 1–9. https://doi.org/10.1098/rsos.171475 PMID: 29515866 73. Drea CM, Wallen K. Low-status monkeys “play dumb” when learning in mixed social groups. Proc Natl Acad Sci U S A. 1999; 96: 12965–9. https://doi.org/10.1073/pnas.96.22.12965 PMID: 10536031 74. De Waal FB. Macaque social culture: development and perpetuation of affiliative networks. J Comp Psychol. 1996; 110: 147–154. https://doi.org/10.1037/0735-7036.110.2.147 PMID: 8681528 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 16 / 17 Demographic variables and personality traits as predictors of perceived dominance status in dogs 75. Sands J, Creel S, State M, Sands J, Creel S, State M, et al. Social dominance, aggression and faecal glucocorticoid levels in a wild population of wolves, Canis lupus. Anim Behav. 2004; 67: 387–396. https://doi.org/10.1016/j.anbehav.2003.03.019 76. Silk MJ, Cant MA, Cafazzo S, Natoli E, McDonald RA. Elevated aggression is associated with uncer- tainty in a network of dog dominance interactions. Proc R Soc B Biol Sci. 2019; 286: 20190536. https:// doi.org/10.1098/rspb.2019.0536 PMID: 31266423 77. Roberts BW, Mroczek D. Personality Trait Change in Adulthood. Curr Dir Psychol Sci. 2008; 17: 31–35. https://doi.org/10.1111/j.1467-8721.2008.00543.x PMID: 19756219 78. King JE, Weiss A, Sisco MM. Aping Humans: Age and Sex Effects in Chimpanzee (Pan troglodytes) and Human (Homo sapiens) Personality. J Comp Psychol. 2008; 122: 418–427. https://doi.org/10. 1037/a0013125 PMID: 19014265 79. Mech LD. Alpha status, dominance, and division of labor in wolf packs. Can J Zool. 1999; 77: 1196– 1203. https://doi.org/10.1139/z99-099 80. Miklo´si A´ . Dog behaviour, evolution, and cognition [Internet]. Oxford University Press.; 2008. https:// books.google.com/books?id=h9rsmyaywPUC&pgis=1 81. Lindsay S. Handbook of Applied Dog Behavior and Training, Volume Two [Internet]. Lindsay SR, editor. Assessment. Ames, Iowa, USA: Iowa State University Press; 2001. 82. Wallis LJ, Range F, Mu¨ller CA, Serisier S, Huber L, Zso´fi V. Lifespan development of attentiveness in domestic dogs: drawing parallels with humans. Front Psychol. 2014; 5: 71. https://doi.org/10.3389/ fpsyg.2014.00071 PMID: 24570668 83. Bennett PC, Rohlf VI. Owner-companion dog interactions: Relationships between demographic vari- ables, potentially problematic behaviours, training engagement and shared activities. Appl Anim Behav Sci. Elsevier B.V.; 2007; 102: 65–84. https://doi.org/10.1016/j.applanim.2006.03.009 84. Overall KL. PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 References Special issue: The “dominance” debate and improved behavioral measures—Articles from the 2014 CSF/FSF. J Vet Behav Clin Appl Res. Elsevier Ltd; 2016; 11: 1–6. https://doi.org/10.1016/j. jveb.2015.12.004 85. Chase ID, Tovey C, Spangler-Martin D, Manfredonia M. Individual differences versus social dynamics in the formation of animal dominance hierarchies. Proc Natl Acad Sci. 2002; 99: 5744–5749. https://doi. org/10.1073/pnas.082104199 PMID: 11960030 PLOS ONE \| https://doi.org/10.1371/journal.pone.0227253 January 3, 2020 17 / 17
https://openalex.org/W3198305805	https://link.springer.com/content/pdf/10.1007/JHEP07(2021)163.pdf	English	null	Critical behavior of the 2d scalar theory: resumming the N8LO perturbative mass gap	The Journal of high energy physics/The journal of high energy physics	2,021	cc-by	7,655	Published for SISSA by Springer Received: March 8, 2021 Accepted: July 9, 2021 Published: July 22, 2021 Received: March 8, 2021 Accepted: July 9, 2021 Published: July 22, 2021 Received: March 8, 2021 Accepted: July 9, 2021 Published: July 22, 2021 JHEP07(2021)163 Open Access, c⃝The Authors. Article funded by SCOAP3. Critical behavior of the 2d scalar theory: resumming the N8LO perturbative mass gap Gustavo O. Heymans and Marcus Benghi Pinto Departamento de Física, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil Gustavo O. Heymans and Marcus Benghi Pinto Departamento de Física, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil E-mail: gustavo.olegario@posgrad.ufsc.br, marcus.benghi@ufsc.br E-mail: gustavo.olegario@posgrad.ufsc.br, marcus.benghi@ufsc.br Abstract: We apply the optimized perturbation theory (OPT) to resum the perturbative series describing the mass gap of the bidimensional φ4 theory in the Z2 symmetric phase. Already at NLO (one loop) the method is capable of generating a quite reasonable non- perturbative result for the critical coupling. At order-g7 we obtain gc = 2.779(25) which compares very well with the state of the art N8LO result, gc = 2.807(34). As a novelty we investigate the supercritical region showing that it contains some useful complimentary information that can be used in extrapolations to arbitrarily high orders. Keywords: Discrete Symmetries, Nonperturbative Eﬀects Keywords: Discrete Symmetries, Nonperturbative Eﬀects ArXiv ePrint: 2103.00354 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP07(2021)163 Contents 1 Introduction 1 2 Reviewing the perturbative mass gap series 3 3 OPT resummation 5 4 Numerical results at N8LO 7 5 Exploring the supercritical region 9 6 Conclusions 11 1 3 5 7 9 11 JHEP07(2021)163 6 Conclusions 1 Introduction The bidimensional scalar φ4 model describes a simple non integrable super-renormalizable theory which displays rich phase transition patterns. When the original mass parameter, m2, is positive the model has a mass gap and remains invariant under the Z2 transfor- mation φ →−φ as far as one remains within the weak coupling regime. As the coupling (g) increases the mass gap decreases until the symmetry gets ultimately broken through a second order phase transition when a critical value, gc, is attained [1, 2]. The case m2 < 0 displays an even richer phase transition structure in which the Z2 symmetry that is broken for 0 < g < ˜gc gets restored at g = ˜gc through a second order transition [3]. As the coupling further increases the model returns to the broken phase at g = ˜g′ c. An interesting duality between the Z2 broken and unbroken phases, which allows to relate the three diﬀerent crit- ical couplings, was discovered by Chang [1]. Since its β function vanishes at all orders in perturbation theory the model represents a conformal ﬁeld theory at the critical coupling where it becomes gapless. It then lies within the same universality class as the bidimen- sional Ising model. These physically appealing characteristics combined with its simplicity suggest that the strongly coupled bidimensional φ4 model provides an excellent framework to test how accurately diﬀerent non-perturbative techniques describe critical parameters associated with the phase transitions. Indeed, a survey of the literature reveals that meth- ods such as lattice simulations [4–7], Hamiltonian truncations (HT) [8–17] as well as other resummation schemes [18] have been recently used to determine the numerical value of gc for the symmetric (m2 > 0) case. In some of the most recent investigations [19, 20] the self energy contributions to the physical mass have been perturbatively evaluated up to the N8LO before being Borel resummed to yield gc = 2.807(34). The availability of such a perturbative series provides us with an excellent opportunity to test alternative resummation techniques as those prescribed by variational methods such as the optimized perturbation theory (OPT) [21, 22] to be considered here. Let us point out that similar – 1 – approximations appear under acronyms such as LDE (linear δ expansion [23]), VPT (varia- tional perturbation theory [24]), and SPT (screened perturbation theory [25]). 1 Introduction The aim of most of these variational approximations is to resum an originally perturba- tive series by combining the easiness of perturbative evaluations with some optimization criterion in order to produce non-perturbative results. In this way the formal evaluations at each order generally involve only a handful of contributions which is certainly advanta- geous. A welcome feature is that the renormalization program can then be implemented by following the perturbative approach discussed in most standard textbooks before non- perturbative results be produced through optimization. One way to implement this kind of approximation is to deform the original theory by adding and subtracting a Gaussian term written in terms of an arbitrary variational (mass) parameter, η, which represents a Lagrange multiplier. For example, in the case of the φ4 scalar theory to be considered here one deforms the original theory by shifting the harmonic term m2φ2 →m2φ2 +(1−δ)η2φ2 while multiplying the original couplings by a dummy bookeeping parameter, δ. Then, as one can easily check, the deformed Lagrangian density interpolates between a free theory (δ = 0) and the original interacting case (δ = 1). Next, a physical quantity, Φ, is evaluated perturbatively in powers of δ up to a given order k producing Φ(k)(δ, η). One then sets δ = 1 (the original value) and ﬁxes the optimum η by requiring that it satisﬁes the so called principle of minimal sensitivity (PMS), (∂Φ)/(∂η)\|η = 0 [26, 27]. In most situations this variational criterion produces non-perturbative results since the optimal η turns out to de- pend on the couplings in a non trivial way (often, via self consistent relations). In general, as a result of implementing such a procedure, one can manage to turn a highly divergent series into a convergent expansion. The method is also known for exactly reproducing large-N results already at the ﬁrst non trivial order in many relevant situations [28]. Fi- nite N non-perturbative corrections are easily taken into account by considering few higher loop contributions. Another advantage concerns the case of an originally massless theory for in this case the variational mass also acts as an infra red regulator. A possible concern regards the OPT convergence properties since arbitrary manipulations of divergent series can lead to wrong results. 1 Introduction This important question has been addressed in the context of the anharmonic oscillator [29–31] where a rigorous convergence proof was established before being generalized to the case of renormalizable quantum ﬁeld theories [32]. JHEP07(2021)163 The OPT has been successfully employed in many diﬀerent physical situations in- volving symmetry breaking and phase transitions. In particular, applications related to condensed matter physics have shown that by including ﬁnite N corrections in a non- perturbative fashion the method was able to produce very accurate results regarding the critical dopant concentration in polyacetylene [33], the critical temperature for homoge- neous Bose gases [34], and the phase diagram of magnetized planar fermionic systems [35]. Applications to high energy physics include the evaluation of quark susceptibilities [36] and the phase diagram of eﬀective QCD models [37] among others. Regarding gauge theories at ﬁnite temperatures and non vanishing baryonic densities a variant of the method, with renormalization group properties, has been recently applied to determine the QCD equa- tion of state for dense hadronic matter at T = 0 [38] and T ̸= 0 [39, 40]. This variant, which has been originally dubbed renormalization group optimized perturbation theory – 2 – (RGOPT) [41, 42], has produced results which are in excellent agreement with the state of the art lattice QCD predictions [39, 40]. In the present work the OPT capabilities at strong couplings will be tested primarily to evaluate gc for the case m2 > 0. With this aim we shall resum the N8LO mass gap perturbative series which became recently available [20]. Our other goal is to investigate if some extra useful information can be acquired by exploring the supercritical region. The work is organized as follows. In the next section we review the N8LO mass gap perturbative series presented in ref. [20]. In section 3 the OPT method is illustrated with an application to the mass gap at the two ﬁrst non trivial orders. Numerical results for gc at N8LO are obtained in section 4. Then, in section 5, we explore the region where g > gc. Finally, our conclusions are presented in section 6. JHEP07(2021)163 2 Reviewing the perturbative mass gap series The well known φ4 model is described by the following Z2 invariant Lagrangian density The well known φ4 model is described by the following Z2 invariant Lagrangian density L = 1 2∂µφ∂µφ + m2 2 φ2 + λφ4 . (2.1) (2.1) In 2d, where the theory is super-renormalizable, λ has canonical dimensions [λ] = 2. In this particular case the coupling is ﬁnite so that βλ = 0 at all perturbative orders. Regarding the two point function the only primitive divergence stems from tadpole (direct) contributions which do not depend on the external momenta. Hence, no wave function renormalization is needed. Let us start by reviewing the perturbative evaluation of the physical mass squared. At the lowest perturbative order, an explicit evaluation using dimensional regularization in the MS scheme gives M2 PT = m2 + λ 3 π 1 ϵ + Lm + m2 ct , (2.2) (2.2) where we have deﬁned Lm ≡ln µ2 m2 , (2.3) (2.3) with µ representing an arbitrary energy scale. Within the MS renormalization scheme only the pole is eliminated by the counterterm, m2 ct, so that the renormalized physical mass squared reads 3 M2 PT = m2 + λ 3 πLm . (2.4) (2.4) Requiring this quantity to satisfy the Callan-Symanzik equation Requiring this quantity to satisfy the Callan-Symanzik equation (µ∂µ + βm2∂m2)M2 = 0 , (2.5) (2.5) one can ﬁx the mass anomalous dimension to [3] one can ﬁx the mass anomalous dimension to [3] βm2 = −λ 6 π . (2.6) βm2 = −λ 6 π . (2.6) – 3 – – 3 – Therefore, Therefore, m2(µ) = m2(µ0) −λ 3 π ln µ2 µ2 0 , (2.7) (2.7) where µ0 represents a reference scale. As already emphasized the only primitive divergence associated with the two point function is the one which appears in eq. (2.2). This means that by consistently considering the counterterm m2 ct = −λ3/(πϵ) the physical mass squared will remain ﬁnite at any perturbative order. Note also that βm2, as given by eq. (2.6), remains valid as higher orders are considered. 2 Reviewing the perturbative mass gap series In the case where tadpoles are present the ﬁnite perturbative physical mass squared at O(λ8) reads [20] JHEP07(2021)163 M2 PT = m2 + 3λ π Lm −9λ2 π2m2 Lm −3λ2 2m2 + λ3 (m2)2 ( 9 π + 63 2π3 ζ(3) + 27 π3 Lm + 9 2πLm + 27 2π3 L2 m ) − λ4 (m2)3 ( 14.655869(22) + 6 + 5π2 + 14ζ(3) Lm + 27 2π4 9 + π2 L2 m + 27 π4 L3 m ) + λ5 (m2)4 ( 65.97308(43) + 51.538171(63)Lm + 81 4π5 36 + 17π2 + 42ζ(3) L2 m + 81 2π5 11 + π2 L3 m + 243 4π5 L4 m ) + 81 2π6 105 + 37π2 + 84ζ(3) L3 m + 243 4π6 25 + 2π2 L4 m + 729 5π6 L5 m ) + λ7 (m2)6 ( 2077.703(36) + 1948.682(14)Lm + 828.4327(39)L2 m + 205.20516(19)L3 m + 243 8π7 675 + 197π2 + 420ζ(3) L4 m + 729 5π5 137 + 10π2 L5 m + 729 5π7 L6 m ) − λ8 (m2)7 ( 13771.04(54) + 13765.22(21)Lm + 6373.657(40)L2 m + 1778.1465(75)L3 m + 323.93839(27)L4 m + 2187 20π8 812 + 207π2 + 420ζ(3) L5 m ) + 323.93839(27)L4 m + 2187 20π8 812 + 207π2 + 420ζ(3) L5 m + 2187 20π8 147 + 10π2 L6 m + 6561 7π8 L7 m ) . + 323.93839(27)L4 m + 2187 20π8 812 + 207π2 + 420ζ(3) L5 m + 2187 20π8 147 + 10π2 L6 m + 6561 7π8 L7 m ) . (2.8) + 2187 20π8 147 + 10π2 L6 m + 6561 7π8 L7 m ) . (2.8) (2.8) Note that by setting µ = m all Lm dependent (tadpole) terms appearing in eq. (2.8) vanish and one retrieves the series considered in ref. [19]. However, as we shall explicitly see, these terms should not be discarded prior to implementing the OPT mass shift. The reason is that after expanding in powers of δ the Lm terms become ln[µ2/(m2+η2)] eventually giving non vanishing contributions even if one later chooses µ = m. – 4 – 3 OPT resummation Now, to implement the OPT procedure one considers eq. (2.8) with the following replace- ments [21] m2(µ) = m2(µ0) −3λ π ln µ2 µ2 0 −→m2(µ) + η2(1 −δ) = m2(µ0) + η2(1 −δ) −δ3λ π ln µ2 µ2 0 m2(µ) = m2(µ0) −3λ π ln µ2 µ2 0 −→m2(µ) + η2(1 −δ) = m2(µ0) + η2(1 −δ) −δ3λ π ln µ2 µ2 0 λ −→δλ , (3.1) (3.1) λ −→δλ , where µ0 is a reference scale. Next, by reexpanding to a given order δ(k) one obtains the OPT physical mass squared, M2. To understand how the method works it is convenient to extract the maximum of information in an analytical fashion. This can be achieved by considering the ﬁrst two non trivial lowest order contributions given by JHEP07(2021)163 M2(µ) = m2(µ0) −δ3λ π ln µ2 µ2 0 + η2(1 −δ) + δ3λ π ln µ2 [m(µ0)2 + η2] + δ2 3λη2 π[m(µ0)2 + η2] −δ2 9λ2 π2[m(µ0)2 + η2] ln µ2 [m(µ0)2 + η2] + δ2 9λ2 π2[m(µ0)2 + η2] ln µ2 µ2 0 −δ2 3λ2 2[m(µ0)2 + η2] + O(δ3) . (3.2) −δ2 9λ2 π2[m(µ0)2 + η2] ln µ2 [m(µ0)2 + η2] + δ2 9λ2 π2[m(µ0)2 + η2] ln µ2 µ2 0 −δ2 3λ2 2[m(µ0)2 + η2] + O(δ3) . (3.2) (3.2) −δ2 3λ2 2[m(µ0)2 + η2] + O(δ3) . (3.2) 2[m(µ0)2 + η2] + It is now a trivial matter to rearrange the logarithms to see that at any arbitrary scale, µ′, the OPT mass can be written as1 M2(µ′) = M2(µ0) where M2(µ0) = m2(µ0) + η2(1 −δ) + δ3λ π Lη + δ2 3λη2 π[m(µ0)2 + η2] −δ2 9λ2 π2[m(µ0)2 + η2]Lη −δ2 3λ2 2[m(µ0)2 + η2] + O(δ3) . (3.3) (3.3) The following deﬁnition has been used in the previous relation Lη ≡ln µ2 0 [m(µ0)2 + η2] . (3.4) (3.4) Having understood that the complete standard perturbative renormalization procedure is not spoiled by the OPT simple replacements we can turn to the optimization procedure. With this aim let us set µ0 = m(µ0) in eq. (3.3) so that our results can be directly compared to those of ref. [19] (or, equivalently, to those presented in ref. [20] for the particular case κ = Lm ≡0). Next, let us deﬁne the dimensionless coupling g ≡λ m2 . 3 OPT resummation (3.5) (3.5) Then, in units of m2 the physical mass squared becomes Then, in units of m2 the physical mass squared becomes M2 m2 = 1+ η2 m2 (1−δ)+δ3g π Lη+δ2 1 (1+η2/m2) " g 3η2 πm2 −g2 9 π2 Lη−g2 3 2 # +O(δ3). (3.6) (3.6) 1As in the purely perturbative case this feature remains valid as higher order contributions are considered. 1As in the purely perturbative case this feature remains valid as higher order contributions are considered. – 5 – 0.0 0.5 1.0 1.5 2.0 2.5 -0.5 0.0 0.5 1.0 g M²/m² 0.0 0.5 1.0 1.5 2.0 2.5 -0.5 0.0 0.5 1.0 g M²/m² Figure 1. The physical mass squared, in units of m2, as a function of g obtained with standard PT (dashed line) and OPT (continuous line) at the two loop level. The critical couplings occur at gc = p 2/3 = 0.82 (PT) and gc = 1.511 (OPT). Figure 1. The physical mass squared, in units of m2, as a function of g obtained with standard PT (dashed line) and OPT (continuous line) at the two loop level. The critical couplings occur at gc = p 2/3 = 0.82 (PT) and gc = 1.511 (OPT). JHEP07(2021)163 At order-δ the optimal solution is just η = 0 implying that the trivial perturbative solution M2/m2 = 1 is recovered. A non trivial result is obtained at order-δ2 where, after setting δ = 1 and applying the variational criterion [26, 27] At order-δ the optimal solution is just η = 0 implying that the trivial perturbative solution M2/m2 = 1 is recovered. A non trivial result is obtained at order-δ2 where, after setting δ = 1 and applying the variational criterion [26, 27] ∂M2 ∂η η = 0 , (3.7) (3.7) to eq. (3.6), one obtains as solutions the trivial η = 0 as well as the highly non-perturbative relation η2 = m2 " 3g π W π exp[1 + π/(3g) + π2/6] 3g ! −1 # , (3.8) (3.8) where W represents the Lambert-W function. Remark that exactly at g = 0 the non trivial solution, eq. (3.8), would give η2 = −m2 leading to divergences but obviously in this case one has a free theory and the optimal mass is just η = 0. 3 OPT resummation Figure 1 compares the OPT result for M2(g) with the standard PT prediction at order-g2 (two loop level) showing that a second order quantum transition takes place when M2(gc) = 0. Before pushing the evaluation of M2 to higher orders it is important to remark that the authors of ref. [20] noticed that by resumming the series for M, instead of the one for M2, much better results could be obtained due to the fact that M approaches the critical point smoothly. Therefore, for comparison purposes, we shall also resum this quantity to determine gc from the condition M(gc) = 0. After taking the square root of eq. (2.8), reexpanding to order λ8, and carrying out the OPT replacements represented by eqs. (3.1) one easily obtains the OPT series up to order-δ8. As we did in the M2 case let us start by considering M expanded to order-δ2. The explicit result reads M m = q 1 + η2/m2 −δ 1 2 p 1 + η2/m2 η2 m2 −g 3 πLη ! −δ2 η2 m2(1 + η2/m2)3/2 " η2 8m2 −g 1 2π 3 + 1 2Lη # −δ2g2 3 (1 + η2/m2)3/2 1 4 + 3 2π2 Lη 1 + 1 4Lη + O(δ3) . (3.9) (3.9) – 6 – – 6 – O(3) O(1) O(5) O(7) O(2) O(4) O(6) O(8) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 g η/m Figure 2. The optimized variational mass, in units of m, as a function of g obtained by resumming M (odd orders) and M 2 (even orders). O(3) O(1) O(5) O(7) O(2) O(4) O(6) O(8) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 g η/m Figure 2. The optimized variational mass, in units of m, as a function of g obtained by resumming M (odd orders) and M 2 (even orders). JHEP07(2021)163 After setting δ = 1 and applying the PMS condition one immediately notices that, contrary to what happens when resumming M2, optimizing M at order-δ yields η = 0 together with the non trivial result η2 = m2 3g π W π exp[2 + π/(3g)] 3g −1 , (3.10) (3.10) which is rather similar to the optimal mass obtained by resumming the squared mass at order-δ2. 3 OPT resummation However, in opposition to what happens in the M2 case, the optimization of M does not furnish a non trivial real result at order-δ2. Then, injecting η as given by eq. (3.10) back into eq. (3.9) at O(δ) yields gc = 3.760. Although numerically not so accurate (e.g., when compared to gc = 2.807(34) of ref. [19]), this is still a quite remarkable result given that it has been generated by resumming only a simple one loop contribution. This result also emphasizes the crucial role played by the OPT tadpole terms Lη which, in contrast to purely perturbative Lm, survive the scale choice µ = m. 4 Numerical results at N8LO Now, by following the steps which led to the order-δ2 mass gap result it is a simple matter to consider higher order corrections up to O(δ8) starting from the perturbative result, eq. (2.8). The whole procedure can be easily carried out in a numerical fashion. Exactly as it happened at NLO it turns out that real solutions for η can only be obtained at even(odd) orders when M2(M) is optimized. The complete set of results for the optimal η is displayed in ﬁgure 2. Using the results for η at even orders one can then investigate the physical mass squared to determine the critical coupling from M2(gc) = 0 as ﬁgure 3 illustrates. At the same time, at odd orders the critical coupling value should be determined from the condition M(gc) = 0. Figure 4 shows M(g) indicating that the value of gc decreases as more orders are considered in opposition to what happens when M2 is resummed. Table 1 compares the OPT predicted values for M(g), at some representative cou- pling values, with those furnished by Hamiltonian truncations [13, 14] and Borel resumma- tion [19]. The comparison indicates a particularly good agreement between our results and those from ref. [19]. As far as the critical coupling value is concerned ﬁgure 5 compares the OPT predictions, at diﬀerent δ orders, with the state of the art result obtained in ref. [19]. – 7 – 0.0 0.5 1.0 1.5 2.0 2.5 3.0 -0.5 0.0 0.5 1.0 g M²/m² O(2) O(4) O(6) O(8) 1.4 1.6 1.8 2.0 2.2 2.4 -0.2 -0.1 0.0 0.1 0.2 g M²/m² Figure 3. Left panel: the physical mass squared, in units of m2, as a function of g at even orders. Right panel: same as the left panel but zoomed. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 -0.5 0.0 0.5 1.0 g M²/m² O(2) O(4) O(6) O(8) 1.4 1.6 1.8 2.0 2.2 2.4 -0.2 -0.1 0.0 0.1 0.2 g M²/m² Figure 3. Left panel: the physical mass squared, in units of m2, as a function of g at even orders. Right panel: same as the left panel but zoomed. 4 Numerical results at N8LO JHEP07(2021)163 O(1) O(3) O(5) O(7) 2.6 2.8 3.0 3.2 3.4 3.6 3.8 -0.2 -0.1 0.0 0.1 0.2 g M/m 0 1 2 3 4 -0.5 0.0 0.5 1.0 g M/m O(1) O(3) O(5) O(7) 2.6 2.8 3.0 3.2 3.4 3.6 3.8 -0.2 -0.1 0.0 0.1 0.2 g M/m Figure 4. Left panel: the physical mass, in units of m, as a function of g at odd orders. Right panel: same as the left panel but zoomed. 0 1 2 3 4 -0.5 0.0 0.5 1.0 g M/m M/m Figure 4. Left panel: the physical mass, in units of m, as a function of g at odd orders. Right panel: same as the left panel but zoomed. g M(g) Ref. 0.979733(5) [13, 14] 0.02 0.9797313(4) [19] 0.9797315(1) this work 0.7494(2) [13, 14] 1 0.7507(5) [19] 0.750520(2) this work 0.345(2) [13, 14] 2 0.357(5) [19] 0.352838(14) this work Table 1. Comparing some OPT values for M(g), in units of m, with those predicted by Hamiltonian truncations [13, 14] and Borel resummation [19]. Table 1. Comparing some OPT values for M(g), in units of m, with those predicted by Hamiltonian truncations [13, 14] and Borel resummation [19]. The ﬁgure clearly indicates that also within the OPT resumming M produces a much faster convergence than resumming M2. The actual numerical values are presented in table 2. Not surprizingly, in comparison with gc = 2.807(34), our most accurate prediction happens at N7LO which represents the highest available odd order for which M can be optimized in the present work. – 8 – ⨯ ⨯ ⨯ ⨯ · · · · 1 2 3 4 5 6 7 8 1 2 3 4 Order gc Figure 5. Order by order predictions for the critical coupling. The dots correspond to values ob- tained by resumming M at odd orders while the crosses represent the values obtained by resumming M 2 at even orders. The straight line represents the prediction gc = 2.807(34) obtained in ref. [19] by resumming M to N8LO. ⨯ ⨯ ⨯ ⨯ · · · · 1 2 3 4 5 6 7 8 1 2 3 4 Order gc Order JHEP07(2021)163 Figure 5. Order by order predictions for the critical coupling. The dots correspond to values ob- tained by resumming M at odd orders while the crosses represent the values obtained by resumming M 2 at even orders. 4 Numerical results at N8LO The straight line represents the prediction gc = 2.807(34) obtained in ref. [19] by resumming M to N8LO. Resummed M Resummed M2 order gc order gc 1 3.76015 2 1.51147 3 2.89809 4 1.98859 5 2.80400 6 2.21970 7 2.77947 8 2.37301 Table 2. Critical couplings obtained from the OPT resummation of M(M 2) at odd(even) orders. Table 2. Critical couplings obtained from the OPT resummation of M(M 2) at odd(even) orders. Table 2. Critical couplings obtained from the OPT resummation of M(M 2) at odd(even) orders. 5 Exploring the supercritical region Left panel: the physical mass squared, in units of m2, as a function of g at even orders. For clarity the order-g2 result is not shown in this panel. Right panel: location of the minima from O(2) to O(8) (light dots). The dark dot locates the extrapolated minimum corresponding to an arbitrarily high order. Table 3. The ﬁrst and second critical points, g(w) c and g(s) c , from resumming M 2. ● ● ● ● O(4) O(6) O(8) 0 1 2 3 4 5 6 7 -0.5 0.0 0.5 1.0 g M²/m² ● ● ● ● ● 4 6 8 10 12 -5 -4 -3 -2 -1 0 g M²/m² Figure 6. Left panel: the physical mass squared, in units of m2, as a function of g at even orders. For clarity the order-g2 result is not shown in this panel. Right panel: location of the minima from O(2) to O(8) (light dots). The dark dot locates the extrapolated minimum corresponding to an arbitrarily high order. ● ● ● ● O(4) O(6) O(8) 0 1 2 3 4 5 6 7 -0.5 0.0 0.5 1.0 g M²/m² ● ● ● ● ● 4 6 8 10 12 -5 -4 -3 -2 -1 0 g M²/m² JHEP07(2021)163 Figure 6. Left panel: the physical mass squared, in units of m2, as a function of g at even orders. For clarity the order-g2 result is not shown in this panel. Right panel: location of the minima from O(2) to O(8) (light dots). The dark dot locates the extrapolated minimum corresponding to an arbitrarily high order. indeed the transition associated with g(s) c emerges as an artifact of our approximation the diﬀerence between g(s) c and g(w) c should further increase until g(s) c eventually disappears, as higher orders are considered. However, table 3 reveals that at increasing orders the diﬀerence between the two critical coupling values decreases reaching g(s) c ≃1.5g(w) c at N8LO. As a digression we note that, physically, the supercritical scenario observed in ﬁgure 6 reminds us of a reentrant phase which is a characteristic of many condensed matter systems. One of the best known examples is the symmetry pattern observed in potassium sodium tartrate tetrahydrate most commonly known as the Rochelle salt which, as the temperature increases, goes from a more symmetric orthorhombic crystalline structure to a less symmetric monoclinic structure at T ≃255 K [45]. 5 Exploring the supercritical region Having established the reliability of the OPT as an eﬃcient resummation tool let us now investigate what happens at coupling values greater than gc ≡g(w) c which represents a (supercritical) region that has been seldom explored. Our motivation stems from a very recent investigation [43] in the three dimensional case where the authors observed that a second transition, leading back to the symmetric phase, could in principle occur at some coupling value, g(s) c , greater than g(w) c . This intriguing possibility (which in that work occurs within a particular renormalization scheme) has not been fully explored in ref. [43] because g(s) c is reached after the theory has passed a phase transition. In this case, resummations of perturbative series are not guaranteed to work since g(w) c represents a non analytical point. Despite of that we shall see that by exploring this region one gets extra information that can be used to further improve convergence properties. To do that let us consider once again the physical mass squared, M2, since this quantity is often taken to represent the order parameter in studies related to phase transitions in scalar models [44] while M does not have a clear physical interpretation beyond gc. Our NNLO result suggests that after being broken at g(w) c the Z2 symmetry could get restored at a higher value, g(s) c . Since at this ﬁrst non trivial order g(s) c ≃14g(w) c one could dismiss the second transition on the grounds that it takes place at far too high couplings where the resummation is possibly less eﬀective as already emphasized. Within this point of view it would be reasonable to expect that if – 9 – Order g(w) c g(s) c 2 1.51 21.4 4 1.98 5.89 6 2.21 4.16 8 2.37 3.53 Table 3. The ﬁrst and second critical points, g(w) c and g(s) c , from resumming M 2. 4 1.98 5.89 6 2.21 4.16 8 2.37 3.53 Table 3. The ﬁrst and second critical points, g(w) c and g(s) c , from resumming M 2. ● ● ● ● O(4) O(6) O(8) 0 1 2 3 4 5 6 7 -0.5 0.0 0.5 1.0 g M²/m² ● ● ● ● ● 4 6 8 10 12 -5 -4 -3 -2 -1 0 g M²/m² Figure 6. 5 Exploring the supercritical region To see that let us ﬁrst recall that, as seen in the previous section, resumming M seems to be more eﬀective than resumming M2. Next, let us refer again to ﬁgure 6 to point out that each curve presents a minimum lying within the g(w) c −g(s) c interval. As one considers more perturbative contributions both, the interval and \|M2\|, decrease so it is plausible that at arbitrarily high orders the exact (and unique) critical value is the one which satisﬁes g(w) c = g(s) c ≡gc. Assuming that this is true one can use the low order results to estimate the critical coupling value at arbitrarily high orders through a simple extrapolation (see right panel of ﬁgure 6). By interpolating over the four available (even) orders we predict gc = 2.785 as the extrapolated value. This simple exercise shows that although M2 displays a much slower convergence than M one can nevertheless use the information contained within the supercritical region to substantially improve the resummation of the physical mass squared. Table 4 compares our best results with the most recent predictions for the critical coupling. Before closing this section it is important to emphasize that one should not interpret our extrapolated value, gc = 2.785, as representing the exact one given that it has been obtained through a very simple numerical extrapolation procedure. Rather, one should take this estimate as an indication that the physical mass squared can also be used to predict accurate critical coupling values provided that one uses the information stored within the supercritical region to accelerate convergence. 5 Exploring the supercritical region It then returns to be orthorhombic phase at T ≃297 K, till it melts at T ≃348 K therefore exhibiting intermediary inverse symmetry breaking through a reentrant phase (see ref. [46] for a detailed list of diﬀerent materials exhibiting this phenomenon). Naively, ﬁgure 6 could then lead us to think that a similar situation takes place in the present bidimensional case where, instead of the temperature, the coupling drives the (quantum) transition. However, although physically interesting, this exotic phase transition pattern is unlikely to emerge within the bidimensional φ4 model since our present investigation of M, at odd orders, indicates only one value at which M(gc) = 0. Moreover, the existence of a unique critical coupling is also supported by other robust investigations [13, 14, 19, 20]. Then, in the light of these results, we shall not assume that a reentrant transition really takes place within the simple theory being studied here – 10 – Method Year, ref. gc Lattice Monte Carlo 2009, [48] 2.70+0.025 −0.013 Uniform matrix product states 2013, [5] 2.766(5) Lattice Monte Carlo 2015, [4] 2.788(15)(8) Resummed perturbation theory 2015, [49] 2.75(1) LO renormalized HT 2015, [8] 2.97(14) raw HT 2016, [10] 2.78(6) NLO-HT 2017, [14] 2.76(3) Borel resummation 2018, [19] 2.807(34) OPT optimal M to N7LO 2021, This work 2.779(25) OPT optimal (extrapolated) M2 2021, This work 2.785 Table 4. Summary of recent predictions. JHEP07(2021)163 Table 4. Summary of recent predictions. (in opposition to what happens in some O(N) × O(N) scalar models [47]). Nevertheless, even when this pragmatic view is adopted the supercritical region may still oﬀer interesting possibilities regarding high order extrapolations. To see that let us ﬁrst recall that, as seen in the previous section, resumming M seems to be more eﬀective than resumming M2. Next, let us refer again to ﬁgure 6 to point out that each curve presents a minimum lying within the g(w) c −g(s) c interval. As one considers more perturbative contributions both, the interval and \|M2\|, decrease so it is plausible that at arbitrarily high orders the exact (and unique) critical value is the one which satisﬁes g(w) c = g(s) c ≡gc. (in opposition to what happens in some O(N) × O(N) scalar models [47]). Nevertheless, even when this pragmatic view is adopted the supercritical region may still oﬀer interesting possibilities regarding high order extrapolations. 6 Conclusions We have applied the OPT method to the bidimensional φ4 model in order to resum the N8LO perturbative series representing the mass gap within the MS renormalization scheme. Our main goal was to evaluate the numerical value of the critical coupling, gc, at which a second order phase transition signals the breaking of the Z2 symmetry. When the physical – 11 – mass squared is considered the OPT optimization criterion has real solutions only at even orders. In this case, the value gc = 1.511 found at NNLO increases as higher orders are considered reaching the value gc = 2.373 at N8LO. Following ref. [19] we have also resummed the series representing the mass, M, observing that for this quantity the OPT optimization criterion has real solutions only at odd orders. Our investigation conﬁrms that in this case a better convergence is achieved: as higher orders are considered, the value gc = 3.760 found at NLO decreases to gc = 2.779 at N7LO. It turns out that the latter value is in excellent agreement with the state of the art N8LO result, gc = 2.807(34), obtained in ref. [19] where M was (Borel) resummed. Our prediction also agrees very well with results obtained with methods such as Hamil- tonian truncation [8, 10, 14], lattice Monte Carlo [4, 48] and matrix product states [5] which predict gc to lie within the range 2.75–2.788. As emphasized in the text, the logarithmic terms of the form ln(µ2/m2) which are associated with tadpole contributions must be included in the perturbative evaluation (prior to the OPT resummation) even when one later decides to set µ = m. The reason is that within the OPT these terms become ln[µ2/(m2+η2)] so that even when µ = m the logarithms give important contributions as we have explicitly observed when resumming M at NLO. In this case, an order-g logarithmic term was crucial to generate a highly non-perturbative variational mass allowing us to show that a decent prediction for gc can readily be obtained even when only a simple tadpole contribution is considered. JHEP07(2021)163 Motivated by some recent observations [43] we have investigated the physical mass squared behavior in the supercritical domain. As a novelty, the present work shows that this (often neglected) region contains useful information which allows for high order extrapola- tions to be performed. 6 Conclusions Indeed, applying our extrapolation method to M2 has substantially improved the N8LO prediction generating values which are compatible with the OPT re- summation of M, at N7LO, as well as with other recent estimates. The present application demonstrates how a purely perturbative series can be eﬃciently resummed by the OPT procedure. Acknowledgments We thank the authors of ref. [19] for sending us some of their numerical data and for valuable comments. We are also grateful to Jean-Loïc Kneur, Rudnei Ramos and Paul Romatschke for related discussions. M.B.P. is partially supported by Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq-Brazil), Process No 303846/2017-8 and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior — (CAPES-Brazil) — Finance Code 001. G.O.H. thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior — (CAPES-Brazil) for a MSc scholarship. This work has also been ﬁnanced in part by Instituto Nacional de Ciência e Tecnologia de Física Nuclear e Aplicações (INCT- FNA), Process No. 464898/2014-5. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. – 12 – References [1] S.-J. Chang, Existence of a second-order phase transition in a two-dimensional ϕ4 ﬁeld theory, Phys. Rev. D 13 (1976) 2778 [Erratum ibid. 16 (1977) 1979] [INSPIRE]. [2] B. Simon and R.B. Griﬃths, The (φ4)2 ﬁeld theory as a classical Ising model, Commun. Math. Phys. 33 (1973) 145 [INSPIRE]. [3] M. Serone, G. Spada and G. Villadoro, λφ4 2 theory — Part II. the broken phase beyond NNNN(NNNN)LO, JHEP 05 (2019) 047 [arXiv:1901.05023] [INSPIRE]. [4] A. Milsted, J. Haegeman and T.J. Osborne, Matrix product states and variational methods applied to critical quantum ﬁeld theory, Phys. Rev. D 88 (2013) 085030 [arXiv:1302.5582] [INSPIRE]. JHEP07(2021)163 [5] P. Bosetti, B. De Palma and M. Guagnelli, Monte Carlo determination of the critical coupling in φ4 2 theory, Phys. Rev. D 92 (2015) 034509 [arXiv:1506.08587] [INSPIRE]. [6] S. Bronzin, B. De Palma and M. Guagnelli, New Monte Carlo determination of the critical coupling in φ4 2 theory, Phys. Rev. D 99 (2019) 034508 [arXiv:1807.03381] [INSPIRE]. [7] D. Kadoh, Y. Kuramashi, Y. Nakamura, R. Sakai, S. Takeda and Y. Yoshimura, Tensor network analysis of critical coupling in two dimensional φ4 theory, JHEP 05 (2019) 184 [arXiv:1811.12376] [INSPIRE]. [8] S. Rychkov and L.G. Vitale, Hamiltonian truncation study of the φ4 theory in two dimensions, Phys. Rev. D 91 (2015) 085011 [arXiv:1412.3460] [INSPIRE]. [9] S. Rychkov and L.G. Vitale, Hamiltonian truncation study of the φ4 theory in two dimensions. II. The Z2-broken phase and the Chang duality, Phys. Rev. D 93 (2016) 065014 [arXiv:1512.00493] [INSPIRE]. [10] Z. Bajnok and M. Lajer, Truncated Hilbert space approach to the 2d φ4 theory, JHEP 10 (2016) 050 [arXiv:1512.06901] [INSPIRE]. [11] M. Burkardt, S.S. Chabysheva and J.R. Hiller, Two-dimensional light-front φ4 theory in a symmetric polynomial basis, Phys. Rev. D 94 (2016) 065006 [arXiv:1607.00026] [INSPIRE]. [12] N. Anand, V.X. Genest, E. Katz, Z.U. Khandker and M.T. Walters, RG ﬂow from φ4 theory to the 2D Ising model, JHEP 08 (2017) 056 [arXiv:1704.04500] [INSPIRE]. [13] J. Elias-Miro, S. Rychkov and L.G. Vitale, High-Precision Calculations in Strongly Coupled Quantum Field Theory with Next-to-Leading-Order Renormalized Hamiltonian Truncation, JHEP 10 (2017) 213 [arXiv:1706.06121] [INSPIRE]. [14] J. Elias-Miro, S. Rychkov and L.G. Vitale, NLO Renormalization in the Hamiltonian Truncation, Phys. Rev. D 96 (2017) 065024 [arXiv:1706.09929] [INSPIRE]. [15] A.L. Fitzpatrick, J. Kaplan, E. Katz, L.G. Vitale and M.T. Walters, Lightcone eﬀective Hamiltonians and RG ﬂows, JHEP 08 (2018) 120 [arXiv:1803.10793] [INSPIRE]. [16] S.S. Chabysheva and J.R. References Hiller, Transitioning from equal-time to light-front quantization in φ4 2, Phys. Rev. D 102 (2020) 116010 [arXiv:1811.01685] [INSPIRE]. [16] S.S. Chabysheva and J.R. Hiller, Transitioning from equal-time to light-front quantization in φ4 2, Phys. Rev. D 102 (2020) 116010 [arXiv:1811.01685] [INSPIRE]. [17] A.L. Fitzpatrick, E. Katz and M.T. Walters, Nonperturbative Matching Between Equal-Time and Lightcone Quantization, JHEP 10 (2020) 092 [arXiv:1812.08177] [INSPIRE]. [17] A.L. Fitzpatrick, E. Katz and M.T. Walters, Nonperturbative Matching Between Equal-Time and Lightcone Quantization, JHEP 10 (2020) 092 [arXiv:1812.08177] [INSPIRE]. [18] P. Romatschke, Simple non-perturbative resummation schemes beyond mean-ﬁeld: case study for scalar φ4 theory in 1+1 dimensions, JHEP 03 (2019) 149 [arXiv:1901.05483] [INSPIRE]. [18] P. Romatschke, Simple non-perturbative resummation schemes beyond mean-ﬁeld: case study for scalar φ4 theory in 1+1 dimensions, JHEP 03 (2019) 149 [arXiv:1901.05483] [INSPIRE]. – 13 – [19] M. Serone, G. Spada and G. Villadoro, λφ4 Theory I: The Symmetric Phase Beyond NNNNNNNNLO, JHEP 08 (2018) 148 [arXiv:1805.05882] [INSPIRE]. [20] G. Sberveglieri, M. Serone and G. Spada, Renormalization scheme dependence, RG ﬂow, and Borel summability in φ4 Theories in d < 4, Phys. Rev. D 100 (2019) 045008 [arXiv:1905.02122] [INSPIRE]. [21] A. Okopińska, Nonstandard expansion techniques for the eﬀective potential in λϕ4 quantum ﬁeld theory, Phys. Rev. D 35 (1987) 1835 [INSPIRE]. [22] H. Yamada, Spontaneous symmetry breaking in QCD, Z. Phys. C 59 (1993) 67 [INSPIRE]. [22] H. Yamada, Spontaneous symmetry breaking in QCD, Z. Phys. C 59 (1993) 67 [INSPIRE]. [23] A Duncan and H F Jones Interpolating Lagrangians and U(1) Gauge Theory on the Lattice [22] H. Yamada, Spontaneous symmetry breaking in QCD, Z. Phys. C 59 (1993) 67 [INSPIRE]. [23] A. Duncan and H.F. Jones, Interpolating Lagrangians and U(1) Gauge Theory on the Lattice, Nucl. Phys. B 320 (1989) 189 [INSPIRE]. [23] A. Duncan and H.F. Jones, Interpolating Lagrangians and U(1) Gauge Theory on the Lattic Nucl. Phys. B 320 (1989) 189 [INSPIRE]. JHEP07(2021)163 [24] R.P. Feynman and H. Kleinert, Eﬀective Classical Partition Functions, Phys. Rev. A 34 (1986) 5080 [INSPIRE]. [25] F. Karsch, A. Patkos and P. Petreczky, Screened perturbation theory, Phys. Lett. B 401 (1997) 69 [hep-ph/9702376] [INSPIRE]. [26] P.M. Stevenson, Optimized Perturbation Theory, Phys. Rev. D 23 (1981) 2916 [INSPIRE]. [27] P. Stevenson, Sense and Nonsense in the Renormalization Scheme Dependence Problem, Nucl. Phys. B 203 (1982) 472 [INSPIRE]. [28] S.K. Gandhi, H.F. Jones and M.B. Pinto, The δ-expansion in the large-N limit, Nucl. Phys. References B 359 (1991) 429 [INSPIRE]. [29] R. Guida, K. Konishi and H. Suzuki, Improved convergence proof of the delta expansion and order dependent mappings, Annals Phys. 249 (1996) 109 [hep-th/9505084] [INSPIRE]. [30] B. Bellet, P. Garcia and A. Neveu, Convergent sequences of perturbative approximations for the anharmonic oscillator. 1. Harmonic approach, Int. J. Mod. Phys. A 11 (1996) 5587 [hep-th/9507155] [INSPIRE]. [31] B. Bellet, P. Garcia and A. Neveu, Convergent sequences of perturbative approximations for the anharmonic oscillator. 2. Compact time approach, Int. J. Mod. Phys. A 11 (1996) 5607 [hep-th/9507156] [INSPIRE]. [32] J.L. Kneur and D. Reynaud, (Borel) convergence of the variationally improved mass expansion and the O(N) Gross-Neveu model mass gap, Phys. Rev. D 66 (2002) 085020 [hep-th/0205133] [INSPIRE]. [33] H. Caldas, J.L. Kneur, M.B. Pinto and R.O. Ramos, Critical dopant concentration in polyacetylene and phase diagram from a continuous four-Fermi model, Phys. Rev. B 77 (2008) 205109 [arXiv:0804.2675] [INSPIRE]. [34] J.-L. Kneur, A. Neveu and M.B. Pinto, Improved optimization of perturbation theory: Applications to the oscillator energy levels and Bose-Einstein condensate critical temperature, Phys. Rev. A 69 (2004) 053624 [cond-mat/0401324] [INSPIRE]. [35] J.-L. Kneur, M.B. Pinto and R.O. Ramos, Phase diagram of the magnetized planar Gross-Neveu model beyond the large-N approximation, Phys. Rev. D 88 (2013) 045005 [arXiv:1306.2933] [INSPIRE]. [36] T.E. Restrepo, J.C. Macias, M.B. Pinto and G.N. Ferrari, Dynamical Generation of a Repulsive Vector Contribution to the Quark Pressure, Phys. Rev. D 91 (2015) 065017 [arXiv:1412.3074] [INSPIRE]. – 14 – [37] L. Ferroni, V. Koch and M.B. Pinto, Multiple Critical Points in Eﬀective Quark Models, Phys. Rev. C 82 (2010) 055205 [arXiv:1007.4721] [INSPIRE]. [38] J.-L. Kneur, M.B. Pinto and T.E. Restrepo, Renormalization group improved pressure for cold and dense QCD, Phys. Rev. D 100 (2019) 114006 [arXiv:1908.08363] [INSPIRE]. [39] J.-L. Kneur, M.B. Pinto and T.E. Restrepo, QCD pressure: renormalization group optimized perturbation theory confronts lattice, arXiv:2101.02124 [INSPIRE]. [40] J.-L. Kneur, M.B. Pinto and T.E. Restrepo, Renormalization group improved pressure for hot and dense quark matter, arXiv:2101.08240 [INSPIRE]. [41] J.L. Kneur and A. Neveu, Renormalization Group Improved Optimized Perturbation Theory: Revisiting the Mass Gap of the O(2N) Gross-Neveu Model, Phys. Rev. D 81 (2010) 125012 [arXiv:1004.4834] [INSPIRE]. JHEP07(2021)163 JHEP07(2021)163 [42] J.-L. Kneur and A. Neveu, αS from Fπ and Renormalization Group Optimized Perturbation Theory, Phys. Rev. D 88 (2013) 074025 [arXiv:1305.6910] [INSPIRE]. [43] G. Sberveglieri, M. Serone and G. References Spada, Self-Dualities and Renormalization Dependence of the Phase Diagram in 3d O(N) Vector Models, JHEP 02 (2021) 098 [arXiv:2010.09737] [INSPIRE]. [44] M.B. Pinto and R.O. Ramos, High temperature resummation in the linear δ expansion, Phys. Rev. D 60 (1999) 105005 [hep-ph/9903353] [INSPIRE]. [45] F. Jona and G. Shirane, Ferroeletric Crystals, Monographs in Solid State Physics, Pergamon Press, Oxford U.K. (1962). [46] N. Schupper and N.M. Shnerb, Inverse melting and inverse freezing: A spin model, Phys. Rev. E 72 (2005) 046107 [cond-mat/0502033]. [47] M.B. Pinto, R.O. Ramos and J.E. Parreira, Phase transition patterns in relativistic and nonrelativistic multi-scalar-ﬁeld models, Phys. Rev. D 71 (2005) 123519 [hep-th/0506131] [INSPIRE]. [48] D. Schaich and W. Loinaz, An Improved lattice measurement of the critical coupling in φ4 2 theory, Phys. Rev. D 79 (2009) 056008 [arXiv:0902.0045] [INSPIRE]. [49] A. Pelissetto and E. Vicari, Critical mass renormalization in renormalized φ4 theories in two and three dimensions, Phys. Lett. B 751 (2015) 532 [arXiv:1508.00989] [INSPIRE]. [49] A. Pelissetto and E. Vicari, Critical mass renormalization in renormalized φ4 theories in two and three dimensions, Phys. Lett. B 751 (2015) 532 [arXiv:1508.00989] [INSPIRE]. – 15 –
W2989671033.txt	http://ijasos.ocerintjournals.org/tr/download/article-file/885696	en		ANALYSIS OF THE METHODS FOR REGISTRATION OF IMAGES RECEIVED FROM THE INFORMATION SYSTEMS OF NON-MOTORIZED AIRCRAFTS	International e-journal of advances in social sciences	2,019	cc-by	4,541	IJASOS- International E-Journal of Advances in Social Sciences, Vol. V, Issue 15, December 2019 ANALYSIS OF THE METHODS FOR REGISTRATION OF IMAGES RECEIVED FROM THE INFORMATION SYSTEMS OF NON-MOTORIZED AIRCRAFTS Venelin Terziev1 and Teodora Petrova2 1 Full Member of the Russian Academy of Natural History, Professor, Eng., D.Sc. (National Security), D.Sc. (Economics), D.Sc. (Social Activities), Ph.D., Russian Academy of Natural History, Moscow, Russia, Vasil Levski National Military University, Veliko Tarnovo, Bulgaria University of Rousse, Rousse, Bulgaria, terziev@skmat.com 2 Assoc. Prof. Ph.D., Faculty of Aviation, Vasil Levski National Military University, Dolna Mitropoliya, District Pleven, Bulgaria, teodorapetrova33@abv.bg Abstract The non-motorized air systems for intelligence, monitoring and control of the earth surface have gained currency and are used for various tactic flight’s tasks and missions. The non-motorized aircrafts (NMA) and the air-monitoring systems that include board and land part are key elements of these systems. The world experience in using NMA for these uses shows that they are most suitable where the exploitation conditions are very extreme and there is an unacceptable risk for operations of piloted aviation. Such are intelligence and observation of strictly guarded sites, zones, where military operations are conducted as well as regions with large scale fires and floods. The use of people in these conditions is connected with actual threat for their lives and practically, NMA as a tool for collecting and processing of information is irreplaceable. Keywords: registration of images, methods, information systems, non-motorized aircrafts. 1. INTRODUCTION The non-motorized aircrafts find wider and wider application in the recent years for solving wide range of tasks in military science, ecology, environment monitoring, calamities and averages. Non-motorized aircraft systems for intelligence, monitoring and control of the earth surface have gained wide distribution and are used in various tactic flight’s tasks and missions. Key elements of these systems are the non-motorized aircrafts (NMA) and the systems for air monitoring that include board and land part. The world experience in using NMA for these uses shows that they are most suitable where the exploitation conditions are very extreme and there is an unacceptable risk for operations of piloted aviation. Such are intelligence and observation of strictly guarded sites, zones, where military operations are conducted as well as regions with large scale fires and floods. The use of people in these conditions is connected to actual threat for their lives and practically, NMA as a tool for collecting and processing of information is irreplaceable. 2. ANALYSIS OF THE METHODS FOR REGISTRATION OF THE IMAGES RECEIVED FROM THE INFORMATION SYSTEMS OF NON-MOTORIZED AIRCRAFTS Monitoring the air space, the earth and water surface, depending on the particular tasks that are to be solved, air-pictures, hydro-meteorological situation, atmosphere study, radio-metric control of calamity areas, http://ijasos.ocerintjournals.org 1448 IJASOS- International E-Journal of Advances in Social Sciences, Vol. V, Issue 15, December 2019 seismic control, control of the status of gas and oil pipelines, the electricity supply network, geological studies, under-surface earth sounding, study of the ice situation, see motion could be made. The scope of civil NMA application is: • Small objects finding – air, over water, land; • Air transport management – in hardly accessible areas, at vehement calamities and averages, of temporary airways in civil aviation; • Control of sea vessels movement – searching for and finding vessels, warning for average situations in the ports, control of sea borders, control of proper fishing; • Development of regional and cross-regional telecommunication networks – the connecting systems including the mobile ones, tele-radio-broadcasting, the navigation systems; • Taking air pictures and control of the earth surface – cartography, inspection and observation of contractual obligations, control of the hydrologic and meteorological situation; • Environmental situation control – radiation control, gas and chemical control, control of gas and oil pipelines, test of seismic sensors; • Ensuring agricultural work and geologic studies – defining soil characteristics, study for minerals, under surface (up to 100 m) earth sounding; • Ocean studies – study of the ice, study of the sea motion, searching for fish shoals. A range of useful load is developed for NMA that includes digital camera, different types of tele-cameras and sensors, system for laser mapping (LIDAR), radar for synthesized aperture, which allows satisfaction of wide scope of clients’ requirements. The data from the useful NMA load could be transferred in real time to a remote terminal placed in an automobile, plane or helicopter that execute target tasks (search for victims, for extinguishing fire, etc.). The remote terminal could be based, in particular, on the grounds of a portable computer and to include also receiving device with antenna. Solving such type of problems, various types of sensors and systems are used, in particular, monitoring visualizing systems working in the optic spectrum range – 0,2 – 12 . Despite their variety, each type of monitoring system in the optic range has limitations and specific possibilities when loaded on NMA. Using radar with synthesized aperture gives great possibilities because it is designed for work in any weather conditions. In normal mode of functioning, it ensures receiving radio-location images of a terrain with resolution 1 meter. For a twenty-four hours, an image of an area of 138 000 km2 at a distance of 200 km2 could be received. In points mode, a picture with size of the terrain of 2×2 km is received, and for 24 hours, more than 1900 images could be received with resolution of 0,3 m. In the third mode, the radar could accompany moving targets if its speed is over 7 km/h. A day digital-optic camera that ensures receiving images with high resolution. Sensor (1024×1,024 pixels), connected to a telephoto lens with focal length 1750 mm. Depending on the programme, there are two modes of functioning. The first is scanning in a strip 10 km wide. And the second one is detailed image of a terrain of 2×2 km. An infrared (IR) sensor (640×480 pixels) that uses the same telephoto lens is used for receiving night images. Number of channels for connection could be used for transferring the information to the users. Along the satellite channel, the speed of transferring information is 50 Mbit/sec. Information with speed 137 Mbit/sec. could be transmitted along a channel of UHF range. The interest toward NMA is provoked by their inexpensive exploitation and avoiding the risk for the crew. The limitation of the exploitation load is defined by the physiologic abilities of the person, the possibility for maintaining monitoring at multiple points for a very short period of time. NMA could bring benefit and economies not only for the military but in the civil field. Their possibilities depend on a parameter such as height of flight up to 30 km. At such a height, the non-motorized aircraft could compete with the satellites that are at a height of about 400 kilometers. They could track everything that happens on a territory with area of about a million of square kilometers, which turns them into kind of „aerodynamic satellite”. The non-motorized aircrafts could take the functions of satellite groupings and to execute them in real time mode within the limits of a whole region. 24 satellites are necessary to make photo- and movie pictures from space or to monitor any objects, but http://ijasos.ocerintjournals.org 1449 IJASOS- International E-Journal of Advances in Social Sciences, Vol. V, Issue 15, December 2019 even then the information from them would be submitted once in an hour. The point is that the satellite is over the monitored object for only 15-20 minutes and then gets out of its visibility zone and gets back to this very same spot after making a revolution in a circle around the Earth. In contracts to the satellite, the nonmotorized aircraft follows the point of monitoring constantly. It works at a height of 20 km more than 24 hours but returns to the base and another one takes its shift in the sky. One more appliance stays in reserve. This is great economy because the non-motorized aircrafts are considerably cheaper than the satellites. The non-motorized aircrafts could compete with the satellites in the field of establishing telecommunication networks and in the navigation systems. The non-motorized aircrafts could be assigned continuous circular twenty-four hours monitoring of the earth surface in a wide range. Using them, we could create an information field of the country that includes control and management of the air and water transport, because these appliances could have functions of land, air and satellite locators (the current information from them gives a full picture of what happens in the sky, in the water and on lands). The NMA advantages are in the fact that first, they are considerably cheaper than the piloted aircrafts. Also, pilots have to be trained and this costs a lot of money. As a result of the absence of a crew onboard, the expenses for executing a task reduce considerably. Second, the light-weighted (in comparison to the piloted aircrafts) non-motorized aircrafts consume less fuel. A more realistic perspective opens before them even at a possible transition to cryogenic fuel. Third, in contrast to the piloted aircrafts, the NMA do not need airports of special surface. NMA have gained wide popularity thanks to their possibility to receive various radio-technical information from terrain, where placing an operator (observer) is difficult. The progress of computing equipment and the digital technologies for signals processing (Bezryadin, 2003; Gruzman et al. 2000; Prett; 1982) allows considerable improvement of the quality, speed and volume of images procession and ensuring their transmission over big distances. Alongside, digital technologies have their disadvantages that influence their effectiveness when used in monitoring tools. The technical monitoring systems are realized as optic appliances systems, placed immediately in the NMA body and solidly attached to its construction or with the help of gyro-stabilized platforms that ensure turning of the systems’ optic axes to all sides with preset speed. As a rule, the appliance’s optic axis solid connection with the NMA construction is used with the viewing aviation cameras and optic appliances in the front or other hemispheres of the aircraft. The optic sensors and cameras differ considerably in dimensions and bulk and the realization of such specific requirements as distance to the object for photographing and the high terrain’s resolution lead inevitably to these systems bulk and value increase. Practically, all optic appliances mounted in the aircraft’s construction have the possibility for a certain angular shifting for compensation of the aircraft’s angular position towards Earth’s surface and of the aircraft’s angular fluctuation regarding the normal coordinate system’s axes (Petrov, 2010). The use of such appliances allows solving great amount of practical tasks but in order to get a quality result, the fulfillment of strict enough requirements towards the aircraft’s stabilization in the air at the moment of monitoring is necessary. Unfortunately, such systems’ functioning principle as a whole does not allow realizing modes of accompanying of targets, and when trying to realize such a mode, great amount of difficulties and limitations occur. The optic appliances placed on gyro-stabilized platforms appear to be more flexible. The world practice has accumulated considerable experience in using such appliances and the market of electronic-optic monitoring and intelligence systems offers sufficiently wide range of such appliances (Petrov, 2010a; Petrova, 2019; Petrov, 2015; Petrov, Miron, 2019а). The using of gyro-stabilized platforms in the optic monitoring systems ensures the solution of the problem with accompanying a selected target at NMA evolutions in flight or change of the target’s spatial position. The joint mounting of television and infrared camera allows considerable improvement of the received image’s information, and using additional laser range-finder – target indicator gives possibility for the targets coordinates precise defining. Considering the great variety of the offered optic systems for NMA, the question with selection of equipment depending on the tasks to be executed arises, and to estimate the effectiveness of using the selected appliances. The following main tasks were reviewed in the process of study: http://ijasos.ocerintjournals.org 1450 IJASOS- International E-Journal of Advances in Social Sciences, Vol. V, Issue 15, December 2019 To estimate the possibilities of the systems for ecologic monitoring, used in NMA Necessity, actuality and main directions for development of the board systems for ecologic monitoring with using NMA. Finding, localization and announcing of arisen forest and fires and floods, taking into consideration the significance, place and tasks it is going to fulfill for improving the prevention and protection of country’s population at these natural calamities, averages and crises arousal. To ground the composition, block scheme, interconnections and criteria of evaluation in the ecologic monitoring systems on the base of modern requirements and regulating documents. The analysis of the used modern appliances and systems for air and ecologic monitoring shows the digital methods for procession of images received from the board system has gained wide application. Registering the images in the non-motorized aircrafts Registering cameras are used for registering the images. In them, the various points of the objects’ space are represented by the camera’s optic system in the images’ space at different distance from the focal plain. However, if the distance between the camera and the monitored scene exceeds the optic system’s focal distance significantly, then it can be considered the image is built in its focal plain. In this case, projective model of camera can be used, where the image of the three-dimension object is received through projecting in the focal plane through one single point, which is called optic centre. The straight line, which is perpendicular to the plane of the image and crosses this point, is called camera’s optic axis, and the optic axis’ cross point with the image’s plane – main point. The gyro-stabilized platform’s optic systems have acquired an important place in the NMA target loading. Their complexity and meaningful functional variety (accompanying a pint in space, calculating relative coordinates of a pint in space, etc.) constantly increase as well as the value of these appliances. That’s why the task for evaluating the effectiveness of applying optic systems on gyro-stabilized platforms turns to be up to date. And the complexity of tasks that non-motorized equipment could be assigned is constantly increasing. The constructively optic systems on gyro-stabilized platforms are performed in a manner, where the lens (and the system of sensors) ensure relative turning in two axes connected in coordinate system OX and OY with angular speeds of x and y. The utmost value of lens’s angular turning is usually selected based on the NMA purpose, where the optic system would be mounted, but generally a circular view and scanning from an angle of place of about 1100-1200 is ensured. The positive value of the alfa angle ensures view and accompanying of an object that are higher that the aircraft’s axis. Figure1. Deciphering scheme for defining the effectiveness of the used optic system of a gyro-platform. Such wide range of the lens’s diversion angle ensures sustainable accompanying of the object at aircraft’s manoeuvres and evolution with optic system. The theoretic effectiveness at applying optic system that ensures circular view is defined conditionally by the probability the searched object to be in the reviewed area Freviewed (fig.1). Defining that indicator, the following conditions have to be taken into consideration: http://ijasos.ocerintjournals.org 1451 IJASOS- International E-Journal of Advances in Social Sciences, Vol. V, Issue 15, December 2019  The aircraft to have speed of flight Vflight > 0,   2 2 x y  The angular speed at lens’s optic axis’ turning to have limitation. With imitation modeled system „optic appliance – operator” it appears that the angular speed of optic axis’ turning could not be bigger than (0,06…0,07)/L (L-the inclined distance to the centre of the reviewed area in meters).  The angular speed of lens’s turning depends on the flight’s speed, because of which the Vflight and the value of the aircraft’s angular speed of turning towards the normal coordinate system at perfect evolution should be taken into consideration. Otherwise, blurred image would be seen on the screen, where nothing could be possible to define.  According to theoretic calculations, the foreseeable area has the shape of a circle and it is supposed that the earth surface is smooth and close to the surface of a sphere with radius equal to the Earth’s radius. In reality, earth’s surface differs considerably from the ideal one by all indicators received for ideal surface.  The searched object is usually situated where nobody is looking for it. The conditional probability of finding an object with given parameters could be defined by the expression: Pобн  Pно .Pво .Pпоо , P Where но is the probability the object to be within the limits of the foreseeable area at a certain moment of time. This tactic parameter does not depend on the optic system’s features and characteristics. Because of F P that it is accepted it is equal to 1,0; поо - the probability the searched object to be in the area оц . It is not seen for this parameter to be dependable on the strategy for searching (i.e. on the law of lens’s optic axis space position change in time) for aircrafts (A) having Vflight > 0. This dependence, as it would be later seen, has no any sense. That’s why the value of Pпоо  K зон . Pпоо could be defined by the following expression: Fобз Fобз . In this case, Vflight > 0 should be considered when the foreseeable area in the time has shape different from circle. Then: Pпоо  2 Rобз Fобз  2Vпол Rобз . PВО is the probability for instantaneous object’s separation on the background of the spreading surface. It depends on the volume of the information coming for the searched object. If the volume of information is sufficient for accepting the hypothesis that this is the object we search for, then PВО =1,0. For simplicity and clearness, the following condition is accepted: if the object’s projection occupies 1 pixel and more of the operator’s screen, then PВО =1,0. The limited angular speed of lens’s optic axis turning considerably limits the potential possibilities that are preset in the gyro-stabilized platform’s optic-electronic device. The analysis shows that at flight speed of 30 km/h, such device becomes useless: it cannot realize its possibilities and the probability to get on the searched object in the area hardly reaches value of 0,05. The view radius’s increase sharply limits the probability of getting on the searched object in the area. In this regard, the optic system as main appliance which is solidly attached to the aircraft’s construction (aviation cameras, ТV cameras looking downwards, etc.) has significant advantage: with them can be made on the grounds of the data: Pпоо reaches values of 0,95-0,98 of range. Number of conclusions http://ijasos.ocerintjournals.org 1452 IJASOS- International E-Journal of Advances in Social Sciences, Vol. V, Issue 15, December 2019  Choosing optic system for NMA, what we expect as final result of the non-motorized aviation system should be very carefully considered,  The availability of the most perfect from technical point of view optic system on the board of NMA does not yet guarantee successful applying in practice,  The bigger the size of searching area of the earth surface is, the more difficult the use of small NMA and micro NMA is for solving practical problems: in this case their effectiveness would not bare any critics. Necessity, actuality and main directions for developing board systems for ecologic monitoring using NMA. Finding, localization and announcing of arisen forest and fires and floods, taking into consideration the significance, place and tasks it is going to fulfill for improving the prevention and protection of country’s population at these natural calamities, averages and crises arousal. The digital procession of images appears to be that part of signals digital procession (SDP), where the signal serves as image. The word „image” means „reproduction or representation of the shape of a human or object”. The physical mechanism that creates the mentioned “reproduction or representation” has an essential significance. In daily life, getting an image is associated with the eyesight, i.e. with stimulating eyes’ retina, and in this case, the image forming subdues to the laws of optics. However, technology has found many other ways for getting an image without the participation of retina. Recently, images are gotten often with the help of system of sensors that register such types of energy, to which the organ of eye sight does not react (here, synthesized radio-locators are included for example, the acoustic holography and the systems using penetrating radiation). Fortunately, having the whole variety of cases, where images are created and registered, they could be described with the help of the general mathematical apparatus. Presence of distortion in the received images is one of the characteristics of the used optic systems. They could find expression in contrast reduction, geometric distortions, blurring, diluted image as result of camera’s movement, etc. These impose the study of various algorithms for the received images restoration and improvement (Fisenko, Fisenko, 2008; Antonov, 2017; Antonov, 2018). The images from radars with synthesized aperture have characteristically specific distortions different from those of the optic ones. The fight with them is held most effectively at the very creation of the image, not through procession of already received image as far as the trajectory and the parameters of NMA movement is necessary to be considered. The described mathematical models of the optic and radar images creating systems allow for elaborating, analyzing and studying of different algorithms for distorted images’ restoration as well as for their quality improvement. Review and analysis of different types of algorithms for distorted images’ restoration is necessary to be made on the basis of the forming systems’ models. The algorithms should be optimized for various types of distortions – blurring, camera moving, geometric distortions, etc (Enimanev, 2001; Novakova, Enimanev, Andonov, 2007; Enimanev, 2007a-b; Enimanev, 2016; Enimanev, 2019b). 3. CONCLUSION The various theoretically grounded and studied practically applied methods for procession of air-photo and radiolocation images offer algorithms for consecutive use of various methods aiming ensuring better possibilities for analysis of the information from the images. The great quantity of results of the application of various methods illustrates the peculiarities at applying the various algorithms and the effectiveness with their usage. Studies show that analyzing the objects in the images, the consecutive using of several algorithms for processing is necessary until reaching the desired result. Part of the results included in the monograph is published in the authors’ articles, and greater part is received in the process of working on the monograph itself. The received results show that the various algorithms and methods’ effectiveness is not the same, which requires the operators, who analyze the visual information to be well acquainted to their possibilities and peculiarities. http://ijasos.ocerintjournals.org 1453 IJASOS- International E-Journal of Advances in Social Sciences, Vol. V, Issue 15, December 2019 REFERENCE LIST Bezryadin S.N. (2003). Osnovnoy nedostatok sensorov sovremennayh tsifrovayh kamer, 2003 (Безрядин С.Н. 2003. Основной недостаток сенсоров современных цифровых камер, 2003). Gruzman I.S., Kirichuk V.S., Kosayh V.P., Peretyagin G.I., Spektor A.A. (2000). Tsifrovaya obrabotka izobrazheniy v informatsionnayh sistemah: Uchebnoe posobie.- Novosibirsk: Izd-vo NGTU, 2000. – 168 (Грузман И.С., Киричук В.С., Косых В.П., Перетягин Г.И., Спектор А.А. 2000. Цифровая обработка изображений в информационных системах: Учебное пособие.- Новосибирск: Изд-во НГТУ, 2000. – 168). Prett U. (1982). Tsifrovaya obrabotka izobrazheniy. Kn.1. - M.: Mir, 1982 (Прэтт У. 1982. Цифровая обработка изображений. Кн.1. - М.: Мир, 1982). Petrov, Zhivo. (2010). Algoritam za savmestna obrabotka na signali ot GPS I INS s izpolzvane na kalmanov filtar, Nauchni trudove na rusenskiya universitet - 2010, tom 49, seriya 3.2 str.18-22, ISSN 1311-3321 (Петров, Живо. 2010. Алгоритъм за съвместна обработка на сигнали от GPS И INS с използване на калманов филтър, Научни трудове на русенския университет - 2010, том 49, серия 3.2 стр.1822, ISSN 1311-3321). Petrov, Zhivo. (2010a). Algoritam za savmestna obrabotka na signali ot GPS I INS s izpolzvane na filtar Monte Karlo, Nauchni trudove na rusenskiya universitet - 2010, tom 49, seriya 3.2, str.23-27, ISSN 1311-3321 (Петров, Живо. Алгоритъм за съвместна обработка на сигнали от GPS И INS с използване на филтър Монте Карло, Научни трудове на русенския университет - 2010, том 49, серия 3.2, стр.23-27, ISSN 1311-3321). Petrova, Teodora. (2019). Izsledvane i sintez na algoritmi za obrabotka na radiolokatsionni i optichni izobrazheniya s povisheno kachestvo, Dolna Mitropoliya, 2019, ISBN 978-954-713-117-0 (Петрова, Теодора. Изследване и синтез на алгоритми за обработка на радиолокационни и оптични изображения с повишено качество, Долна Митрополия, 2019, ISBN 978-954-713-117-0). Petrov, Zhivo. (2015). Izsledvane na mikroelektromehanichni inertsialni senzori, Pleven 2015 g, ISBN 978954-713-107-1 (Петров, Живо. 2015. Изследване на микроелектромеханични инерциални сензори, Плевен 2015 г, ISBN 978-954-713-107-1). Petrov, Zh., Miron, L. (2019а). Opportunities Of a Genetic Algorithm For Static Calibration Of MEMS Accelerometers, International Scientific Conference Aviation Faculty, National Military University, 2019, p. 246, ISBN 978-954-713-123-1. Fisenko V. T., Fisenko T. Yu. (2008). Kompyyuternaya obrabotka i razpoznavanie izobrazheniy, SankPeterburg, ITMO, 2008 (Фисенко В. Т., Фисенко Т. Ю. 2008. Компьютерная обработка и разпознавание изображений, Санк-Петербург, ИТМО, 2008). Antonov S. I. (2017). Comparative analysis of the armament and equipment support modules in the field of command and control information systems of NATO armies, International Scientific Journal "Security & Future“, Year I, Issue 4, p.p. 163-167, 2017, WEB ISSN 2535-082X; PRINT ISSN 2535-0668. Antonov S. I. (2018). Researching the capabilities of information technologies for education in design, 3D modeling and visualization of the working principles of specific armament elements, International Scientific Journal „Mathematical modeling 2018“, Year 2, Issue 4, 2018, p.p. 156-159, ISSN (PRINT) 2535-0986, ISSN (WEB) 2603-2929. Novakova, A., Enimanev, K., Andonov, K. (2007). Izsledvane na netnite ikonomii na energiya ot atmosferniya vazduh pri sahranenie na produktsiya. // Nauchno spisanie za selskostopanska i gorska tehnika, Ekologiya i badeshte, Sofiya, 6, 2007, 3, s. 3-13, ISSN 1312-0751 (Новакова, А., Ениманев, К., Андонов, К. Изследване на нетните икономии на енергия от атмосферния въздух при съхранение на продукция. // Научно списание за селскостопанска и горска техника, Екология и бъдеще, София, 6, 2007, 3, с. 3-13, ISSN 1312-0751). Enimanev, Krasimir. (2007a). Algoritam i programa za optimalno orazmeryavane izolatsiyata na energoikonomichni stopanski sgradi. // Selskostopanska tehnika, Sofiya, XLIV, 3/ 2007, s. 29-36, ISSN 0037-1718 (Ениманев, Красимир. Алгоритъм и програма за оптимално оразмеряване изолацията на енергоикономични стопански сгради. // Селскостопанска техника, София, XLIV, 3/ 2007, с. 29-36, ISSN 0037-1718). Enimanev, Krasimir. (2007b). Mehanizatsiya i avtomatizatsiya v zhivotnovadstvoto. // Selskostopanska tehnika, Sofiya, XLIV, 3/ 2007, s. 29-36, ISSN 0037-1718 (Ениманев, Красимир. Механизация и http://ijasos.ocerintjournals.org 1454 IJASOS- International E-Journal of Advances in Social Sciences, Vol. V, Issue 15, December 2019 автоматизация в животновъдството. // Селскостопанска техника, София, XLIV, 3/ 2007, с. 29-36, ISSN 0037-1718). Enimanev, Krasimir. (2016). Kompleksna prostranstvena zavisimost na infrastrukturnite elementi pri planirano izgrazhdane na teritorialnite sistemi. // Mezhdunarodna nauchna konferentsiya, Ikonomichesko blagosastoyanie chrez spodelyane na znaniya, 09-10 noemvri 2016, Svishtov, 1, 2016, s. 331-335, ISBN 978-954-23-1185-0 (Ениманев, Красимир. Комплексна пространствена зависимост на инфраструктурните елементи при планирано изграждане на териториалните системи. // Международна научна конференция, Икономическо благосъстояние чрез споделяне на знания, 09-10 ноември 2016, Свищов, 1, 2016, с. 331-335, ISBN 978-954-23-1185-0). Enimanev, Krasimir. (2019b). Energoefektivno osiguryavane na mikroklimata v zatvoreni agrarni sgradi chrez unifitsirani modulni elementi. Fakultet „Aviatsionen“, Natsionalen Voenen Universitet „Vasil Levski“, Dolna Mitropoliya, 2019, 224 str., ISBN 978-954-713-130-9 (Ениманев, Красимир. Енергоефективно осигуряване на микроклимата в затворени аграрни сгради чрез унифицирани модулни елементи. Факултет „Авиационен“, Национален Военен Университет „Васил Левски“, Долна Митрополия, 2019, 224 стр., ISBN 978-954-713-130-9). http://ijasos.ocerintjournals.org 1455
https://openalex.org/W2346328150	https://molecularcytogenetics.biomedcentral.com/track/pdf/10.1186/s13039-016-0247-7	English	null	de novo interstitial deletions at the 11q23.3-q24.2 region	Molecular cytogenetics	2,016	cc-by	3,675	© Su et al. 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Su et al. Molecular Cytogenetics (2016) 9:39 DOI 10.1186/s13039-016-0247-7 Su et al. Molecular Cytogenetics (2016) 9:39 DOI 10.1186/s13039-016-0247-7 Open Access Abstract Background: Jacobsen syndrome (JBS) is a contiguous gene deletion syndrome involving 11q terminal deletion. Interstitial deletions at distal 11q are rare and their contributions to the clinical phenotype of JBS are unknown. Case presentation: We presented the chromosome microarray (CMA) data and the clinical features of two individuals carrying a non-overlapping de novo deletion each at the 11q23.3-q24.2 region in an effort to analyze the correlation between region of deletion at 11q and phenotype. Both deletions are likely pathogenic for patient’s condition. The deletion at 11q23.3q24.1 is associated with short stature, relative microcephaly, failure to thrive, hypotonia and sleeping disorder. The deletion at 11q24.2 involves HEPACAM and our patient’s clinical presentation (relative macrocephaly, abnormal MRI, mild developmental delay and seizure) is not inconsistent with Megalencephalic leukoencephalopathy with subcortical cysts 2B. Conclusions: Our finds support the notion that more than one critical region at 11q23.3-qter are responsible for the variable clinical presentation of JBS, thus JBS is a true contiguous gene deletion syndrome where multiple loci contributed to the clinical characteristics of JBS. Small interstitial deletions at 11q23.3-q24.2 and their associated unique features also suggest emerging novel genomic disorders. Keywords: Jacobsen syndrome, Contiguous gene deletion syndrome, Chromosomal microarray, In cobsen syndrome, Contiguous gene deletion syndrome, Chromosomal microarray, Interstitial deletion the genotype–phenotype correlation. Since the first re- port of an 11q24.1q24.3 interstitial deletion character- ized by CMA [10], eight cases with 11q23-qter interstitial deletion detected by CMA had been docu- mented in literature (see Additional file 1: Table S1). The breakpoints of the 11q23-qter interstitial deletions are variable and deletions range from 2.89 to 12.8 Mb in size. Clinical presentations of affected individuals are also very heterogeneous, thus complicating the geno- type–phenotype correlation analysis [4, 5, 10–14]. Pa- tient with interstitial deletion at 11q23-qter region could share some of the JBS features such as hypotonia [9], macrocephaly [11, 14], microcephaly [5], trigonocephaly [4, 7, 9], some of dysmorphic facial features [4, 5, 7–14], limbs anomalies [7, 11, 13], congenital heart defect (CHD) [4, 5, 7, 9, 10, 12], but often did not exhibit the whole constellation of JBS [13, 14]. The most consistent feature among all patients with 11q23-qter interstitial deletion is DD/ID. Smallest overlapping regions helped to define some critical regions or candidate genes at the distal 11q region [4–11]. de novo interstitial deletions at the 11q23.3-q24.2 region Jiasun Su1, Rongyu Chen1, Jingsi Luo1, Xin Fan1, Chunyun Fu1, Jin Wang1, Sheng He1, Xuyun Hu1, ShuJie Zhang1, Shang Yi1, Shaoke Chen1* and Yiping Shen1,2* * Correspondence: chenshaoke123@163.com; yiping.shen@childrens.harvard.edu 1Department of Genetic and Metabolic Central Laboratory, Guangxi Maternal and Child Health Hospital, No59 Xiangzhu Road, Nanning, China Full list of author information is available at the end of the article Method Chromosomal microarray (CMA) analyses DNA sample was extracted from peripheral blood lym- phocytes using standard protocol, microarray was per- formed using Agilent 244 K array for patient 1 and the illumina HumanSNP cyto-12 array for patient 2. Discussion Interstitial deletions on 11q are rare and the genotype- phenotype study had been hindered by the small number of cases reported and a lack of sufficient details about patient’s clinical phenotype. So far there are only 11 interstitial deletion cases at the 11q23.3-q25 region have been reported (Additional file 1: Table S1). Chromosome microarray data were available for eight of them. Guerin et al. reported a patient with a 2.899 Mb interstitial dele- tion at 11q24.2-q24.3, presented with trigonocephaly, hypertelorism and deep-set eyes [4]; Taoyun Ji et al. re- ported a patient with a 4.11 Mb interstitial deletion at 11q25, presented with severe DD, microcephaly and some facial dysmorphism [5]; Tyson et al. described a patient with a 4.74 Mb deletion at 11q24.2q24.3, pre- sented with macrocephaly, ID and abnormal MRI [11]; SO J et al. reported a woman with a 3.162 Mb interstitial deletion at 11q24.2-q24.3, presented with periventricular nodular heterotopia and transverse limb reduction defect [13]; Yamamoto et al. identified a 20-month-old boy with a 5.3 Mb interstitial deletion at 11q23.3q24.2 and Results A 1.6 Mb deletion at the region of 11q23.3-q24.1 (chr11:120410050–122085906) (hg19) was detected in the genome of P1. A 0.76 Mb deletion at 11q24.2 (chr11:124635144–125390604) (hg19) was detected in the genome of P2 (Fig. 1). Parental CMA demonstrated that both deletions were de novo. She had a history of sleeping disorder. Notably, her parents reported that she would vocalize around bed- time, often it is associated with body rocking and head banging. She would wake up and bang her head at mid- night. She would sit Indian style on her bed and leaned forward until she banged her head on the mattress. This occurred in a fairly fast and rhythmic pattern. Often she would moan during such a movement and many times the moan escalated into very loud scream. She also had the rocking behavior during the daytime that she would move her back up against a hard surface and then rocked her lower back and buttocks on the floor. She was diagnosed as jactatio capitis nocturna and such be- havior had improved but not resolved entirely. Case presentation Patient 1 Patient 1 (P1) was a 5-year-old girl born to a 29-year-old woman at 41 weeks of gestational age. Pregnancy and delivery were uneventful. Her birth weight was 2.8 kg (~15thpercentile) and birth length around 48.2 cm (~15thpercentile). She had an apparently healthy twin brother and sister. At around 5–6 months of age she was noticed to have global delay including unable to roll over, delayed crawling, sitting and standing. She was at a 5-month developmental level at the age of one. She had a history of failure to thrive and hypotonia. She came to Genetics Clinic due to dysmorphic features and global developmental delay. At 5-years of age, her height was 101 cm (−2.11 SD), weight 14 kg (−2.58 SD) and head circumference 48.3 cm (7th percentile). She had overlap- ping toes, dysmorphic features including mild hyperte- lorism (inner canthal distance 27 mm, +1SD), prominent forehead, flat facial profile, broad nose, smooth philtrum with thin upper lip, upslanting palpebral fissures with epicanthal folds. Her brain MRI was normal. No other defect was noticed. Background h The 11q terminal deletion, also known as Jacobsen syn- drome (OMIM #147791), is a contiguous gene deletion syndrome involving the deletion of 11qter. Several hun- dred 11q terminal deletion patients had been described since it was first reported by Jacobsen in 1973 [1]. The deletions observed in JBS patients range from 7 to 20 Mb in size and are almost always associated with 11q terminal deletions and the furthest breakpoint is located at 11q23.3. Typical features of JBS include developmen- tal delay (DD)/intellectual disability (ID), dysmorphic fa- cial features, platelet disorder and multiple congenital defects [1–6]. Interstitial deletions from 11q23 to 11qter are rare and their clinical significance is currently un- known. Some of previously reported 11q23-qter intersti- tial deletions were characterized by karyotyping analysis [7–9]. CMA analysis made it possible to better define * Correspondence: chenshaoke123@163.com; yiping.shen@childrens.harvard.edu 1Department of Genetic and Metabolic Central Laboratory, Guangxi Maternal and Child Health Hospital, No59 Xiangzhu Road, Nanning, China Full list of author information is available at the end of the article Page 2 of 5 Page 2 of 5 Su et al. Molecular Cytogenetics (2016) 9:39 raised his head around 3–4 months of age, rolled over around 6–7 months of age, and pronounced first word at about 10 months of age. His developmental quotient (DQ) and the mental index (MI) suggested that he had mild de- velopment delay by the Denver Development Screening Test score (DQ = 51 and MI = 63). At the age of 10 months, his weight was 9.4 kg (30th percentile) and height 72.5 cm (25th percentile). He presented with a rela- tive macrocephaly (circumference was 47.5 cm (+1.3SD)), mild hypertelorism, low nasal bridge, thin upper lip and strabismus. The magnetic resonance imaging (MRI) re- vealed the widening of the frontotemporal lobe, full bilat- eral ventricles and deep parietal sulci. He had normal electroencephalogram and electromyography. He was di- agnosed with acute pharyngitis, secondary seizure, psy- chomotor retardation and mild development delay as well as cerebral hypogenesis. Here, we reported two individuals carrying non- overlapping interstitial deletions at 11q23.3-q24.2 region with different degrees of DD/ID and some dysmorphic features in an effort to further define genotype–pheno- type correlation. Patient 2 Patient 2 (P2) was a boy who came to hospital due to fever, mild developmental delay and seizure at the age of 10 months. He was a full term second child to a healthy parent who had a healthy 4-year-elder daughter. He was delivered via cesarean section without complication dur- ing pregnancy or delivery. His birth weight was 3.25 kg (25–50th percentile) and birth length was 50 cm (25– 50th percentile). He had no significant medical history prior to presentation. No family history of seizure, autism, mental retardation or other neurologic impairments. He Su et al. Molecular Cytogenetics (2016) 9:39 Page 3 of 5 Fig. 1 a The deletion (shaded in red) at 11q23.3-q24.1 in P1 detected by Agilent 4X180K array. b The facial features of P2. Large forehead, mild hypertelorism, low nasal bridge, thin upper lip and strabismus. The illumina array detected a small deletion (red rectangle) at 11q24.2 Fig. 1 a The deletion (shaded in red) at 11q23.3-q24.1 in P1 detected by Agilent 4X180K array. b The facial features of P2. Large forehead, mild hypertelorism, low nasal bridge, thin upper lip and strabismus. The illumina array detected a small deletion (red rectangle) at 11q24.2 haploinsufficiency of each region is likely associated with DD/ID. presented with white matter abnormalities, prenatal macrocephaly and mild developmental delay [14]. Two patients reported here added to this list. P1 presented with short stature, DD/ID, relative microcephaly, sleep- ing disorder, some dysmorphic features and P2 exhibited mild DD/ID, relative macrocephaly, mild hypertelorism. A summary of the clinical features of the eight previ- ously reported patients with 11q23-qter interstitial dele- tions and two present cases is listed in Table 1. The prevalence of phenotype among these interstitial dele- tion patients can not be quantified for the small number, but the summarization collectively support the notion that Jacobsen syndrome is a contiguous gene deletion syndrome where multiple genes (regions) involved within the deletion region play roles to the syndromic phenotypes. It is interesting to note that patient 1 exhibited the characteristic features of jactatio capitis nocturna, also known as rhythmic movement disorder (RMD). Most of RMD will spontaneously resolve by 4 years of age [15]. Our patient’s condition had improved but not resolved entirely at age 5. The underlying cause of RMD is cur- rently unknown, the deletion in P1 may provide a clue for investigating the molecular mechanism of RMD. Patient 2 13 RefSeq genes including 10 OMIM genes (GRIK4, LRRC35, TECTA, SC5DL, SORL1, MIR100HG, MIR125B1, BLID, MIRLET7A2 and MIR100) are involved in the deletion. But it is unclear which gene or genes are likely responsible for the sleeping disorder. GRIK4 (OMIM #600282) en- codes a protein that belongs to the glutamate-gated ionic channel family. Glutamate functions as the major excita- tory neurotransmitter in the central nervous system through activation of ligand-gated ion channels and G protein-coupled membrane receptors. Takenouchi T et al. and Pickard BS et al. suggested that the haploinsufficiency of GRIK4 was related to DD, mental retardation, schizo- phrenia and bipolar disorder [16, 17]. The developmental delay present in our patient may be explained by the GRIK4 deletion. DD/ID is a consistent feature of JBS. It is also a common feature of patients with 11q interstitial deletions (Table 1). Taoyun Ji et al. proposed a critical region for DD/ID near the telomere defined by a 4.1 Mb deletion (case 3 in Fig. 2) [5]. The deletion detected in our P2 is the smallest in size, it overlaps with deletions detected in case 1 [13] and 4 [11]. All three cases had the clinical presentation of DD/ ID, based on this observation, we propose a novel DD/ID locus at 11q24.2 (chr11:124635144–125390604). Similarly, the deletion detected in our P1 may define another novel DD/ID locus at 11q23.3-q24.1 (chr11: 120410050– 122085906). Thus there are multiple loci on 11q that the The deletion interval in P2 encompassed 6 OMIM genes (ROBO3, ROBO4, HEPACAM, HEPN1, PKNOX2 and FEZ1). Sequence variants in HEPACAM (OMIM Su et al. Molecular Cytogenetics (2016) 9:39 Page 4 of 5 Table 1 Summary of clinical features of the eight patients with 11q23-qter interstitial deletions and our present cases Clinical Findings Previously cases P1 P2 Number 8 1 1 Region 11q23-q25 11q23.3q24. Author details 1 1Department of Genetic and Metabolic Central Laboratory, Guangxi Maternal and Child Health Hospital, No59 Xiangzhu Road, Nanning, China. 2Department of Laboratory Medicine, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA. 2Department of Laboratory Medicine, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA. Received: 20 January 2016 Accepted: 17 April 2016 Consent Written informed consent was obtained from the par- ents of the proband for publication of this Case Report and any accompanying images. The consent form was approved by the ethical committee of Guangxi Maternal and Child Health Hospital, China. A copy of the written consent is available for review by the editor of this journal. 11. Tyson C, Qiao Y, Harvard C, Liu X, et al. Submicroscopic deletions of 11q24-25 in individuals without Jacobsen syndrome: re-examination of the critical region by high-resolution array-CGH. Mol Cytogenet. 2008;1:23. 12. Van Zutven LJ, van Bever Y, Van Nieuwland CC, et al. Interstitial 11q deletion derived from a maternal ins(4;11)(p14;q24.2q25): a patient report and review. Am J Med Genet A. 2009;149A(7):1468–75. 13. So J, Stockley T, Stavropoulos DJ. Periventricular nodular heterotopia and transverse limb reduction defect in a woman with interstitial 11q24 deletion in the Jacobsen syndrome region. Am J Med Genet A. 2014;164A(2):511–5. 14. Yamamoto T, Shimada S, Shimojima K, et al. Leukoencephalopathy associated with 11q24 deletion involving the gene encoding hepatic and glial cell adhesion molecule in two patients. Eur J Med Genet. 2015 Sep; 58(9):492–6. 14. Yamamoto T, Shimada S, Shimojima K, et al. Leukoencephalopathy associated with 11q24 deletion involving the gene encoding hepatic and glial cell adhesion molecule in two patients. Eur J Med Genet. 2015 Sep; 58(9):492–6. Patient 2 11q24.2 Deletion size (Mb) 2.89–12.8 1.6 0.76 Gender 3 m/5f f m Age Ranges from newborn to adult 4 years 10 months Birth weight Low-normal ~15thpercentile 25–50th percentile Hypotonia - + - Macrocephaly 2 - relative macrocephaly Microcephaly 2 relative microcephaly - Trigonocephaly 1 - - Prominent forehead 1 + - Hypertelorism 2 mild mild Palpebral fissure anomalies 4 + - Ear anomalies 4 - - Nasal anomalies 3 + + Mouth anomalies 3 + + Limb anomalies 2 + - Cardiovascular anomalies 4 - - Hematological anomalies 3 - - Developmental delay/intellectual disability 6 + + Social interaction difficulties 2 - NA Clinical findings: +, present; −, absent, f female, m male, NA not applicable Clinical findings: +, present; −, absent, f female, m male, NA not applicable interstitial deletion 11q24 and clinical features of MLC [14]. P2 was presented with relative macrocephaly, abnor- mal MRI mild developmental delay and seizure, which is not inconsistent with MLC2B. The much smaller deletion detected in P2 overlap with the interstitial deletion in pa- tient 2 of Yamamoto’s report. The smallest overlapping re- gion between this two cases can exclude the involvement of FEZ1 (OMIM #604825) gene which plays a role in axonal outgrowth and has been proposed as a candidate gene for abnormal MRI by Tyson et al. [11]. #611642) have been shown to cause Megalencephalic leukoencephalopathy with subcortical cysts 2A (an auto- somal recessive form MLC2A, OMIM #613925) and 2B (an autosomal dominant form MLC2B, OMIM #613926), both of which are characterized with macrocephaly, ab- normal MRI and variable degree of intellectual disability [18–20]. The clinical presentation of the autosomal form is milder, some features improve with age. Recently hap- loinsufficiency of HEPACAM was considered as a cause of two patients with heterozygous deletion at 11q23.3q24.2 Fig. 2 Schematic representation referred to interstitial deletion at 11q23-qter region cases reported (UCSC Genome Browser, hg19) Su et al. Molecular Cytogenetics (2016) 9:39 Page 5 of 5 Page 5 of 5 Page 5 of 5 Additional file Additional file 1: Clinical features from the literature of patients with 11q23-qter interstitial deletions. (DOCX 20 kb) 15. Khan A, Auger RR, Kushida CA, et al. Rhythmic movement disorder. Sleep Med. 2008 Mar;9(3):329–30. 15. Khan A, Auger RR, Kushida CA, et al. Rhythmic movement disorder. Sleep Med. 2008 Mar;9(3):329–30. 16. Takenouchi T, Hashida N, Torii C, et al. 1p34.3 deletion involving GRIK3: Further clinical implication of GRIK family glutamate receptors in thepathogenesis of developmental delay. Am J Med Genet A. 2014;164A(2): 456–60. 16. Takenouchi T, Hashida N, Torii C, et al. 1p34.3 deletion involving GRIK3: Further clinical implication of GRIK family glutamate receptors in thepathogenesis of developmental delay. Am J Med Genet A. 2014;164A(2): 456–60. Competing interests The authors declare that they have no competing interests. 17. Pickard BS, Malloy MP, Christoforou A, et al. Cytogenetic and genetic evidence supports a role for the kainate-type glutamate receptor gene, GRIK4, in schizophrenia and bipolar disorder. Mol Psychiatry. 2006;11(9):847–57 17. Pickard BS, Malloy MP, Christoforou A, et al. Cytogenetic and genetic evidence supports a role for the kainate-type glutamate receptor gene, GRIK4, in schizophrenia and bipolar disorder. Mol Psychiatry. 2006;11(9):847–57. Authors’ contributions Wrote the manuscript: SJS, YPS. Conceived and designed the experiments: SKC, YPS. Performed the experiments: SJS, RYC and YCF. XF, JSL, JW, SY were involved in SNP array analysis. SH and DHL helped to revise the manuscript. All authors read and approved the final manuscript. 18. Lopez-Hernandez T, Ridder MC, Montolio M, Capdevila-Nortes X, et al. Mutant glialCAM causes megalencephalic leukoencephalopathy with subcortical cysts, benign familial macrocephaly, and macrocephaly with retardation and autism. Am J Hum Genet. 2011;88:422–32. 18. Lopez-Hernandez T, Ridder MC, Montolio M, Capdevila-Nortes X, et al. Mutant glialCAM causes megalencephalic leukoencephalopathy with subcortical cysts, benign familial macrocephaly, and macrocephaly with retardation and autism. Am J Hum Genet. 2011;88:422–32. 19. Lopez-Hernandez T, Sirisi S, Capdevila-Nortes X, Montolio M, et al. Molecular mechanisms of MLC1 and GLIALCAM mutations in megalencephalic leukoencephalopathy with subcortical cysts. Hum Molec Genet. 2011;20: 3266–77. Acknowledgments We are grateful to the patients and families for participating in this study. We thank Dr. Sheng He for revised for this manuscript. This work was supported by grants from Guangxi Medical Plan Subject (Z2015238) and the project of science and technology of Guangxi Zhuang Autonomous Region (gui-ke-gong 14124004-1-8). 20. Arnedo T, Lopez-Hernandez T, Jeworutzki E, Capdevila-Nortes X, Sirisi S, Pusch M, Estevez R. Functional analyses of mutations in HEPACAM causing megalencephalic leukoencephalopathy. Hum Mutat. 2014;35(10):1175–8. 20. Arnedo T, Lopez-Hernandez T, Jeworutzki E, Capdevila-Nortes X, Sirisi S, Pusch M, Estevez R. Functional analyses of mutations in HEPACAM causing megalencephalic leukoencephalopathy. Hum Mutat. 2014;35(10):1175–8. Conclusions 5. Ji T, Wu Y, Wang H, Wang J, Jiang Y. Diagnosis and fine mapping of a deletion in distal 11q in two Chinese patients with developmental delay. J Hum Genet. 2010;55(8):486–9. 5. Ji T, Wu Y, Wang H, Wang J, Jiang Y. Diagnosis and fine mapping of a deletion in distal 11q in two Chinese patients with developmental delay. J Hum Genet. 2010;55(8):486–9. In summary, we described two rare interstitial de novo deletions in JBS region. ID/DD is a shared feature with JBS, supporting the notion that Jacobsen syndrome is a true contiguous gene deletion syndrome and critical re- gions of ID/DD exist in different regions of 11q ter- minal. Our study further defined a smallest critical region associate with DD/ID. Each interstitial deletion also presented with its unique features and suggested distinct novel genomic imbalance disorder. 6. Fryns JP, Kleczkowska A, Buttiens M, Marien P, van den Berghe H. Distal 11q monosomy. The typical 11q monosomy syndrome is due to deletion of subband 11q24.1. Clin Genet. 1986;30:255–60. 6. Fryns JP, Kleczkowska A, Buttiens M, Marien P, van den Berghe H. Distal 11q monosomy. The typical 11q monosomy syndrome is due to deletion of subband 11q24.1. Clin Genet. 1986;30:255–60. 10. Wenger SL, Grossfeld PD, Siu BL, et al. Molecular characterization of an 11q interstitial deletion in a patient with the clinical features of Jacobsen syndrome. Am J Med Genet A. 2006;140(7):704–8. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: References 1. Mattina T, Perrotta CS, Grossfeld P. Jacobsen syndrome. Orphanet J Rare Dis. 2009;4:9. 1. Mattina T, Perrotta CS, Grossfeld P. Jacobsen syndrome. Orphanet J Rare Dis. 2009;4:9. 2. Grossfeld PD, Mattina T, Lai Z, et al. The 11q terminal deletion disorder: a prospective study of 110 cases. Am J Med Genet A. 2004;129A(1):51–61. 3. Penny LA, Dell Aquila M, Jones MC, et al. Clinical and molecular characterization of patients with distal 11q deletions. Am J Hum Genet. 1995;56(3):676–83. 4. Guerin A, Stavropoulos DJ, Diab Y, et al. Interstitial deletion of 11q- implicating the KIRREL3 gene in the neurocognitive delay associated with Jacobsen syndrome. Am J Med Genet A. 2012;158A(10):2551–6. 4. Guerin A, Stavropoulos DJ, Diab Y, et al. Interstitial deletion of 11q- implicating the KIRREL3 gene in the neurocognitive delay associated with Jacobsen syndrome. Am J Med Genet A. 2012;158A(10):2551–6.
https://openalex.org/W3198371787	https://jgeb.springeropen.com/track/pdf/10.1186/s43141-021-00221-3	English	null	Disputed paternity presumption in Burkina Faso: determination of the biological fathers of children using ABO-rhesus/hemoglobin electrophoresis and STR assays	Journal of Genetic Engineering and Biotechnology /Journal of Genetic Engineering and Biotechnology	2,021	cc-by	6,822	© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract Background: In resource-limited countries, ABO, HLA, MNS, Kells, and hemoglobin electrophoresis are classic tests for the resolution of paternity disputes due to their affordable cost. The limitations of these tests in cases of disputed paternity require the use of Short Tandem Repeats (STR) for their certification. This study aimed to determine the biological fathers of children using ABO-rhesus/hemoglobin electrophoresis and STR assays in Burkina Faso, West Africa. Results: Of the fourteen trios studied, the ABO-rhesus/hemoglobin electrophoresis analysis revealed ten probable inclusion cases, three exclusion cases, and one undetermined paternity. DNA STR analysis found five inclusions of paternity out of the ten probable inclusions with ABO-rhesus/hemoglobin electrophoresis assay versus nine exclusions of paternity. Conclusion: This study showed that the implementation of the analysis of short tandem repeat is required to resolve increasing disputed filiation cases in Burkina Faso. Keywords: Paternity, ABO-rhesus, Hemoglobin electrophoresis of Short Tandem Repeat, Burkina Faso, West Africa is obtained by the examination of well-established blood group systems [3]. The issues in the use of these methods to include paternity of “alleged fathers” are re- lated to the random transmission of alleles in the general population. The late maturation of antigens is a barrier to the determination of filiation using the ABO system. Thus, the establishment of parentage, by the current tests such as ABO, HLA, MNS, and Kell, is not accurate, with low exclusion probabilities around 0.17 [4]. More- over, hemoglobin (Hb) electrophoresis can be used for paternity dispute cases based on the allelic variants between individuals [5, 6]. However, the technique is Background The biological determination of filiations is an old prob- lem. The analysis is based on the genetic polymorphisms of individuals and their Mendelian transmission [1, 2]. Referring to techniques based on blood group determin- ation, some authors showed that in affiliation cases, a combined exclusion chance for non-fathers of 99.995% Disputed paternity presumption in Burkina Faso: determination of the biological fathers of children using ABO-rhesus/ hemoglobin electrophoresis and STR assays Missa Millogo1,2, Serge Theophile Soubeiga2,3,4* , Bapio Valerie Jean Telesphore Bazie2,3,5, Theodora Mahoukede Zohoncon2,3,6, Abdoul Karim Ouattara2,3, Albert Theophane Yonli2,3 and Jacques Simpore2,3,6 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 https://doi.org/10.1186/s43141-021-00221-3 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 https://doi.org/10.1186/s43141-021-00221-3 Journal of Genetic Engineering and Biotechnology Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 https://doi.org/10.1186/s43141-021-00221-3 RESEARCH Open Access Open Access * Correspondence: theo.soubeiga@gmail.com 2Laboratory of Molecular Biology and Genetics (LABIOGENE) of University Joseph Ki-Zerbo, P.O. 03 BOX, 7021 Ouaga 03, Ouagadougou, Burkina Faso 3Biomolecular Research Centre Pietro Annigoni (CERBA)), P.O. 01 BOX 364, Ouagadougou Ouaga 01, Burkina Faso Full list of author information is available at the end of the article Page 2 of 9 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Page 2 of 9 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 of the hemoglobin type was performed using the HELENA electrophoresis chain (Helena Biosciences Europe, Queensway South, Gateshead Tyne, and Wea) according to the manufacturer’s instructions. Hemolysate was prepared by mixing 1 V of the whole blood with 3 V of Helena hemolysis reagent (0.005 M EDTA and 0.01% potassium cyanide). Electrophoresis was performed at 350 V for 25 min in a boric acid/Tris-EDTA buffer (pH 8.4, ionic strength = 0.035). limited when there is correspondence between the Hb genotype of the alleged father–mother and child. Taking these limitations into account, in the context of paternity research, it is necessary to combine several systems [ABO, rhesus, HLA, MNS, Kell, serum systems...] or to use other more efficient systems such as microsatellite genetic analysis, or “short tandem repeats” (STR) [7, 8]. Hemoglobin electrophoresis is complementary to ABO- rhesus because the first is for “exclusion” when the latter detected “inclusion”. limited when there is correspondence between the Hb genotype of the alleged father–mother and child. Taking these limitations into account, in the context of paternity research, it is necessary to combine several systems [ABO, rhesus, HLA, MNS, Kell, serum systems...] or to use other more efficient systems such as microsatellite genetic analysis, or “short tandem repeats” (STR) [7, 8]. Hemoglobin electrophoresis is complementary to ABO- rhesus because the first is for “exclusion” when the latter detected “inclusion”. STR is a polymorphic locus present in all eukaryotic genomes. They generally consisted of tandem matrices of short repeated sequences of 2 to 6 base pairs, and polymorphism occurs when the number of copies of the repeated sequence present at a given STR locus varies between individual chromosomes [9–11]. Hundreds of microsatellites have been studied and some are used as markers for the determination of genetic fingerprints to discriminate or genetically link individuals (families, im- migrants, etc.) [12]. They present a wide diversity and can be used in the identification of paternity testing cases [8, 13]. The application of STRs to the search for parentage in 877 paternity cases had in the past ruled out 35.2% of cases and found a probability of paternity of 99.9999% [14]. In Burkina Faso, the justice system is facing strong demands for paternity tests, causes of divorce, and family conflicts. Statistical analyses The paternity index (PI), which measures the weight of scientific evidence obtained from the paternity test, was calculated for each STR locus using the method de- scribed by Eisenberg, 2003. Then, the combined pater- nity index (CPI) was estimated by multiplying the individual paternity index with the others. The probabil- ity of paternity (POP), a conditional probability of know- ing whether an alleged father is the biological father of a child, was calculated using the following equation: CPI x Capillary electrophoresis The amplification fragments obtained were then ana- lyzed on the ABI 3130 Genetic Analyzer (Applied Bio- system, USA) on a 96-well plate containing 1 μL of PCR product, 8.7 μL of Hi-Di Formamide, and 0.3 μL of Gen- eScan 500 LIZ Size Standard followed by denaturation at 95°C for 3 min and immediate cooling on ice for 3 min. The electrophoresis was performed with Performance- Optimized Polymer 4 (POP4) with a capillary of 36 cm. After electrophoresis, GeneMapper® ID version v3.2.1 software was used to assemble the obtained sequences and compares them to the allele scale to determine the allele types present in each analyzed sample. Amplification by polymerase chain reaction (PCR) Amplification by polymerase chain reaction (PCR) PCR amplification was performed using 1.2 mm of bloodstained disc obtained by a punch on FTA paper previously soaked in blood and containing 5 to 20 ng of DNA. A multiplex PCR amplification of 16 loci of tan- dem repeat strap (polymorphic STR loci) was performed using the AmpFlSTR® identifiler® Direct kit (Applied Bio- systems, Foster City, CA, USA) according to the manu- facturer’s instructions. Among the 16 STRs, the Amelogenin marker was included to allow genetic iden- tification of the sex of each subject. The characteristics of the 16 STRs are shown in Table 1. The PCR was per- formed in 25 μL of reaction volume containing 5–20 ng DNA, 12.5 μL primers, and 12.5 μL Master Mix on the Gene Amp PCR System 9700 thermocycler (Applied Biosystems, USA) according to the following program: initial denaturation at 94°C for 11 min, 28 cycles of 9°C for 20 s, 59°C for 3 min, and 72°C for 1 min, and final extension at 60°C for 25 min. Sample collection Fourteen trios (mother-child-alleged father) were in- volved in the present study. They were referred to the Pietro Annigoni Biomolecular Research Centre for sam- ples (42) collection and paternity tests at the request of the Tribunal de Grande Instance de Ouagadougou. Written informed consent was obtained from partici- pants before blood sample collection on EDTA tube for blood grouping and Hb electrophoresis and on FTA paper (NucleiCard, Brescia, Italy). Despite the technical limitations with a high risk of misidentification of the biological father, ABO-rhesus/Hb electrophoresis has been used for a long time to resolve paternity disputed cases in Burkina Faso, because of their affordability and the absence of STR assays. For a deep identification, the present pioneer study aimed to determine the biological fathers of children using the old ABO-rhesus/Hb elec- trophoresis method and STR assays for the first time in Burkina Faso, West Africa. Carrying out blood grouping and hemoglobin electrophoresis The determination of the blood and rhesus groups was performed using the Beth-Vincent technique with Anti- A, Anti-B, Anti-AB, and Anti-D sera. The determination Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Page 3 of 9 Table 1 16 STR loci and alleles with their characteristics Locus Location on the chromosome Included alleles Fluorochrom D8S1179 8 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 6-FAM D21S11 21q11.2-q21 24, 24.2, 25, 26, 27, 28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36, 37, 38 D7S820 7q11.21-22 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 CSF1PO 5q33.3-34 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 D3S1358 3p 12, 13, 14, 15, 16, 17, 18, 19 VIC TH01 11p15.5 4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3 D13S317 13q22-31 8, 9, 10, 11, 12, 13, 14, 15 D16S539 16q24-qter 5, 8, 9, 10, 11, 12,13, 14, 15 D2S1338 2q35-37.1 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 D19S433 19q12-13.1 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2 NED vWA 12p12-pter 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 TPOX 2p23-2per 6, 7, 8, 9, 10, 11, 12, 13 D18S51 18q21.3 7, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 Amelogenin X: p22.1-22.3 Y: p11.2 X, Y PET D5S818 5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 FGA 4q28 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26.2, 27, 28, 29, 30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2 6-FAM 6-FAM VIC 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26.2, 27, 28, 29, 30, 30.2, 31.2, 32.2, 33.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2 0.5/[CPI x 0.5 + (1- 0.5)], the CPI is the combined pater- nity index and 0.5 is the prior probability [15]. 0.5/[CPI x 0.5 + (1- 0.5)], the CPI is the combined pater- nity index and 0.5 is the prior probability [15]. Inclusion and exclusion according to the STR analysis g y The analysis of the 16 STRs identified the DNA profile of each trio (mother-child-alleged father). These results revealed cases of inclusion and exclusion by comparing the child’s alleles with those of both parents and by cal- culating the PI and POP. Of the 14 trios, 5 alleged Evaluation of paternity inclusion and exclusion according to the test used Comparison of the results using the two methods re- vealed that 5 alleged fathers (35.71%) were included of paternity with the analysis of STR as opposed to 10 in- clusions (71.43%) of paternity found with the ABO- rhesus/Hb electrophoresis method. But all 5 inclusions reported by analysis of genetic polymorphism of DNA were also found by the ABO-rhesus/Hb electrophoresis method (Table 4). Table 5 compares the inclusion/exclu- sion results by methods used for each trio case. Eight out of nine exclusions found with STR assay were in- cluded using ABO genotyping. Moreover, 5 inclusions (trios 2, 11, 12, 14) found with Hb electrophoresis were excluded using STR assay. Inclusion and exclusion by the ABO/rhesus system and hemoglobin electrophoresis Of the 14 trios, the ABO/rhesus system showed only one case of exclusion while Hb electrophoresis reported two cases of exclusion. The trio affected by the exclusion revealed by the ABO/rhesus system is different from the other two found by Hb electrophoresis. Two cases were considered inconclusive because of fetal hemoglobin (Hb F) immaturity in children (Table 2). Carrying out blood grouping and hemoglobin electrophoresis fathers (trios 1, 3, 7, 8, and 13) were included in pater- nity while 9 (trios 2, 4, 5, 6, 9, 10, 11, 12, and 14) were excluded from paternity. The paternity index ranged from 0 to 37, 072, 170, and 900 and the highest POP was 99.99999999997% found in trio 3 (Table 3). Figures 1 and 2 show examples of inclusion and exclusion of paternity. Ethics approval and consent to participate This study was approved by the Institutional Ethics Committee of CERBA/LABIOGENE and The Tribunal de Grande Instance de Ouagadougou (Deliberation N°2019-19/III-015) and conducted according to the Declaration of Helsinki. Also, written informed consent was obtained before blood collection. Discussion So, the Hb electrophoresis technique had the benefit to identify exclusion cases not detected by the ABO-Rh Table 2 Inclusion and exclusion results according to the ABO-rhesus/hemoglobin electrophoresis Trio ABO/rhesus (hemoglobin electrophoresis) Inclusion and exclusion Conclusion Mother Child Alleged father ABO Rhesus Hemoglobin electrophoresis 1 B+ (AC) O+ (AA) A+ (AA) Inclusion Inclusion Inclusion Probable inclusion 2 A+ (AA) AB+ (AA) B+ (AA) Inclusion Inclusion Inclusion Probable inclusion 3 B+ (AC) B+ (AC) B- (AA) Inclusion Inclusion Inclusion Probable inclusion 4 O+ (AA) B+ (AF) A+ (AA) Exclusion Inclusion Inconclusive Exclusion 5 O+ (AA) B+ (AF) B+ (AS) Inclusion Inclusion Inconclusive Inconclusive 6 B+ (AC) B+ (AA) B+ (AS) Inclusion Inclusion Inclusion Probable inclusion 7 B+ (AC) B+ (AA) B+ (AA) Inclusion Inclusion Inclusion Probable inclusion 8 O+ (AA) B+ (AA) AB+ (AA) Inclusion Inclusion Inclusion Probable inclusion 9 O+ (AA) B+ (AC) AB+ (AA) Inclusion Inclusion Exclusion Exclusion 10 O+ (AA) A+ (AC) AB+ (AA) Inclusion Inclusion Exclusion Exclusion 11 B+ (AS) AB+ (AA) A+ (AS) Inclusion Inclusion Inclusion Probable inclusion 12 A+ (AA) O+ (AA) A+ (AS) Inclusion Inclusion Inclusion Probable inclusion 13 A+ (AA) A+ (AA) AB+ (AC) Inclusion Inclusion Inclusion Probable inclusion 14 A+ (AA) A+ (AA) A+ (AA) Inclusion Inclusion Inclusion Probable inclusion Table 2 Inclusion and exclusion results according to the ABO-rhesus/hemoglobin electrophoresis Trio ABO/rhesus (hemoglobin electrophoresis) Inclusion and exclusion Conclusion Mother Child Alleged father ABO Rhesus Hemoglobin electrophoresis 1 B+ (AC) O+ (AA) A+ (AA) Inclusion Inclusion Inclusion Probable inclusion 2 A+ (AA) AB+ (AA) B+ (AA) Inclusion Inclusion Inclusion Probable inclusion 3 B+ (AC) B+ (AC) B- (AA) Inclusion Inclusion Inclusion Probable inclusion 4 O+ (AA) B+ (AF) A+ (AA) Exclusion Inclusion Inconclusive Exclusion 5 O+ (AA) B+ (AF) B+ (AS) Inclusion Inclusion Inconclusive Inconclusive 6 B+ (AC) B+ (AA) B+ (AS) Inclusion Inclusion Inclusion Probable inclusion 7 B+ (AC) B+ (AA) B+ (AA) Inclusion Inclusion Inclusion Probable inclusion 8 O+ (AA) B+ (AA) AB+ (AA) Inclusion Inclusion Inclusion Probable inclusion 9 O+ (AA) B+ (AC) AB+ (AA) Inclusion Inclusion Exclusion Exclusion 10 O+ (AA) A+ (AC) AB+ (AA) Inclusion Inclusion Exclusion Exclusion 11 B+ (AS) AB+ (AA) A+ (AS) Inclusion Inclusion Inclusion Probable inclusion 12 A+ (AA) O+ (AA) A+ (AS) Inclusion Inclusion Inclusion Probable inclusion 13 A+ (AA) A+ (AA) AB+ (AC) Inclusion Inclusion Inclusion Probable inclusion 14 A+ (AA) A+ (AA) A+ (AA) Inclusion Inclusion Inclusion Probable inclusion because the Hb of the child (AC) was different from those of the mother (AA) and the presumed father (AA). Discussion To our knowledge, this study is considered the first study in Burkina Faso concerned with the disputed paternity presumption. Here, in addition to the ABO/ Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Page 4 of 9 rhesus hemoglobin electrophoresis method, we used STR assay for the identification of biological father in disputed paternity cases. The determination of paternity based on blood grouping, the rhesus factor combining Hb electrophoresis, had identified some limitations re- lated to the profile of Hb in young infants in trios 4 and 5. This could be explained by the fact that there is still a significant proportion of fetal hemoglobin (Hb F) in in- fants due to their very young age [16]. Considering the because the Hb of the child (AC) was different from those of the mother (AA) and the presumed father (AA). However, the presumed fathers of these trios were in- cluded by ABO/rhesus. A child inherits one copy of Hb from the mother and another from the biological father [17]. Based on this principle, the two presumed fathers (9 and 10) were automatically excluded from paternity. Legend: CPI combined paternity index, POP probability of paternity Discussion However, the presumed fathers of these trios were in- cluded by ABO/rhesus. A child inherits one copy of Hb from the mother and another from the biological father [17]. Based on this principle, the two presumed fathers (9 and 10) were automatically excluded from paternity. So, the Hb electrophoresis technique had the benefit to identify exclusion cases not detected by the ABO-Rh method. The other benefit of this technique is the possi- bility to diagnose hemoglobinopathies cases [18–20]. These match discrepancies would make it difficult for rhesus hemoglobin electrophoresis method, we used STR assay for the identification of biological father in disputed paternity cases. The determination of paternity based on blood grouping, the rhesus factor combining Hb electrophoresis, had identified some limitations re- lated to the profile of Hb in young infants in trios 4 and 5. This could be explained by the fact that there is still a significant proportion of fetal hemoglobin (Hb F) in in- fants due to their very young age [16]. Considering the blood grouping of parents and children, a match dis- crepancy was observed (trio4). Hemoglobin electrophor- esis also showed match discrepancies in trios 9 and 10 Table 3 Results of the combined paternity index (CPI) and the probability of paternity (POP) in trio cases N° Cases CPI POP Table 3 Results of the combined paternity index (CPI) and the probability of paternity (POP) in trio cases N° Cases CPI POP Conclusion of paternity 1 Trio 3, 263, 198, 110 0.99999999968 Inclusion 2 Trio 0 0.00 Exclusion 3 Trio 37, 072, 170, 900 0.99999999997 Inclusion 4 Trio 0 0.00 Exclusion 5 Trio 0 0.00 Exclusion 6 Trio 0 0.00 Exclusion 7 Trio 196, 349, 727 0.99999999490 Inclusion 8 Trio 12, 695, 452, 599 0.99999999992 Inclusion 9 Trio 0 0.00 Exclusion 10 Trio 0 0.00 Exclusion 11 Trio 0 0.00 Exclusion 12 Trio 0 0.00 Exclusion 13 Trio 2, 526, 793 0.99999996 Inclusion 14 Trio 0 0.00 Exclusion Legend: CPI combined paternity index POP probability of paternity chnology (2021) 19:130 Page 5 of 9 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Page 5 of 9 1 Inclusion of paternity for the trio 13: example of allele correspondence (allele 8) between alleged father and child for the locus CSF1PO Fig. Discussion This trend was consistent with the studies conducted by Souiden et al., 2007 [15]. The determination of STRs would cor- rect inclusion and exclusion errors induced by the ABO- rhesus technique associated with Hb electrophoresis. The ABO-rhesus system is easily suited to the search for the exclusion of paternity. For example, in trio 4, the mother had group O+, the child had group B+ and the alleged father had group A+. In this case, paternity was excluded with certainty and without recourse to other systems to confirm the result. Similarly, the rhesus system alone could reveal an exclusion, as is the case for example for an O- mother, O+ child, and O- father [16]. But in some cases, such as mother O+, child A1, father O+, and if the ABO system is the only exclusion system, paternity is excluded only if it can be shown that one of the parents does not have the Bombay phenotype [21]. On the other hand, the immaturity of antigens in all newborns should be considered; for example, in the fol- lowing example: mother O, child A2, father A1B, the child’s blood type should be checked a few months later before reporting an exclusion, as this could be a delay in Table 5 Comparison of Inclusion and exclusion results by method Trio ABO Rhesus Hemoglobin electrophoresis STR assay 1 Inclusion Inclusion Inclusion Inclusion 2 Inclusion Inclusion Inclusion Exclusion 3 Inclusion Inclusion Inclusion Inclusion 4 Exclusion Inclusion Inconclusive Exclusion 5 Inclusion Inclusion Inconclusive Exclusion 6 Inclusion Inclusion Inclusion Exclusion 7 Inclusion Inclusion Inclusion Inclusion 8 Inclusion Inclusion Inclusion Inclusion 9 Inclusion Inclusion Exclusion Exclusion 10 Inclusion Inclusion Exclusion Exclusion 11 Inclusion Inclusion Inclusion Exclusion 12 Inclusion Inclusion Inclusion Exclusion 13 Inclusion Inclusion Inclusion Inclusion 14 Inclusion Inclusion Inclusion Exclusion technique cannot affirm with certainty the biological father because there can be a match of the Hb when comparing the Hb of the trios (mother-child-father) without the presumed father being the biological father. Taking all these limitations into account, in the context of a paternity search, it is necessary to combine several systems (ABO, rhesus, HLA, MNS, Kell, serum sys- tems...) [4, 22] or to use other systems such as STR gen- etic analysis [7]. In this study, STRs solved all the cases studied, as it was based on DNA polymorphism analysis for the identification of an individual. Discussion 1 Inclusion of paternity for the trio 13: example of allele correspondence (allele 8) between alleged father and child for the locus CSF1PO Fig. 2 Exclusion of paternity for the trio 2: example of allele no correspondence between alleged father and child for the locus TPOX Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Page 6 of 9 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Page 6 of 9 the ABO-rhesus and Hb electrophoresis association t d t i t it I l th hi h f i Fig. 2 Exclusion of paternity for the trio 2: example of allele no correspondence between alleged father and child for the locus TPOX Fig. 2 Exclusion of paternity for the trio 2: example of allele no correspondence between alleged father and child for the locus TPOX the ABO-rhesus and Hb electrophoresis association to determine paternity. In general, the high frequencies of the ABO system alleles would make it difficult to include the presumed father in a paternity case, but could rather exclude him if, because of his blood type, he did not present the possibility of being the father. From the above, the ABO-rhesus technique associated with Hb electrophoresis used to determine paternity had limita- tions. The analysis of STRs consisted of determining the Table 4 Inclusion and exclusion with the ABO-rhesus/ hemoglobin electrophoresis and analysis of STR Paternity ABO-rhesus/Hb electrophoresis Analysis of STR Inclusion 10 (71.43%) 5 (35.71%) Exclusion 3 (21.43%) 9 (64.29%) Inconclusive 1 (7.14%) 0 (0.0%) Legend: STR Short Tandem Repeat, Hb hemoglobin Table 4 Inclusion and exclusion with the ABO-rhesus/ hemoglobin electrophoresis and analysis of STR Paternity ABO-rhesus/Hb electrophoresis Analysis of STR Inclusion 10 (71.43%) 5 (35.71%) Exclusion 3 (21.43%) 9 (64.29%) Inconclusive 1 (7.14%) 0 (0.0%) Legend: STR Short Tandem Repeat, Hb hemoglobin Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Page 7 of 9 Page 7 of 9 genetic markers in the DNA of each trio to compare the alleles of the alleged fathers with those of the children. The Identifiler Direct Kit made it possible to compare 15 alleles between the individuals in each trio. The gen- etic analysis of the fourteen paternity search trios com- prising mother, child, and alleged father revealed 64.29% of cases of exclusion compared to 21.43% with ABO- rhesus associated with Hb electrophoresis. Conclusion In Burkina Faso, ABO-rhesus/hemoglobin electrophor- esis tests have long been used for paternity exclusion. Because of the limitations of these conventional tests, STR analysis has become the reference technique not only for parentage testing but also for forensic analysis. 4. Salmon C, Cartron J, Rouger P (1991) Paternité: les groupes sanguins chez l'Homme. Masson ed, Paris 5. Szuberski J, Oliveira J, Hoyer JD (2012) A comprehensive analysis of hemoglobin variants by high-performance liquid chromatography (HPLC). Int J Lab Hematol 34(6):594–604. https://doi.org/10.1111/j.1751-553X.2012. 01440.x CPI: Combined paternity index; DNA: Deoxyribonucleic; HbF: Fetal hemoglobin; PCR: Polymerase chain reaction; PI: Paternity index; Discussion Some studies were done in African countries to determine alleles frequencies [33, 34]. Moreover, the STR analysis can be used in forensic investigations. Additionally, it would be more appropriate to carry out a study to determine the allelic frequencies of the 16 STRs specific to the popula- tion of Burkina Faso. References k 1. Wenk RE (2004) Testing for parentage and kinship. Curr Opin Hematol 11(5): 357–361. https://doi.org/10.1097/01.moh.0000137914.80855.8a 1. Wenk RE (2004) Testing for parentage and kinship. Curr Opin Hematol 11(5): 357–361. https://doi.org/10.1097/01.moh.0000137914.80855.8a 2. de Mazancourt P, Pfitzinger H (2005) DNA and paternity testing. Gynecol Obstet Fertil 33(7-8):461–463 3. Spielmann W, Kühnl P (1983) Blood group expert evaluation: relation between the extent of testing and the reliability of paternity determination. Reflections on revision and guidelines. J Legal Med 90(1):35–44. https://doi. org/10.1007/BF01886065 Discussion Additionally, it would be more appropriate to carry out a study to determine the allelic frequencies of the 16 STRs specific to the popula- tion of Burkina Faso. POP: Probability of paternity; POP4: Performance-optimized polymer 4; STR: Short tandem repeat POP: Probability of paternity; POP4: Performance-optimized polymer 4; STR: Short tandem repeat POP: Probability of paternity; POP4: Performance-optimized polymer 4; STR: Short tandem repeat paternity likelihood and power of exclusion of the ABO genotype was significantly higher than of the ABO phenotype. In 12 of 35 exclusion cases (34.3%) the ABO genotype also excluded the alleged father, whereas the ABO phenotype excluded the alleged father only in 7 cases (20%) [25]. As demonstrated in this study, the results of paternity tests were better when using STR analysis compared to serological tests. Every individual in the world can be identified at the molecular level based on an extremely greater level of polymorphism in the sequence of DNA which is inherited from biological parents and is identical in every cell of the body [26, 27]. In inclusion cases, the child shares the length of each STR loci with his parents because each biological parent shares 23 chromosomes for their child and in exclusion cases, the child’s length of STR loci differs between the father and mother [28]. In this study, genetic amplifica- tion of STRs excluded 9 presumed fathers and identified 5 others as the biological fathers of the children. STR markers provide sufficient discriminatory power to ex- clude or include an alleged father in contested paternity cases. Unlike ABO-rhesus/Hb electrophoresis techniques which are based on the agglutination of red blood cells and hemoglobin, the STR technique determines the gen- etic profile of the DNA which contains the genetic infor- mation that is unique to each individual [29]. Because of the limitations of ABO-Rhesus/Hb electrophoresis, the PCR technique became the standard process for DNA paternity testing because PCR technology allows ampli- fying a very small quantity of DNA to increase the amount of DNA up to billions of copies of the same DNA for testing and analysis [28, 30]. Furthermore, STR assays can be used to establish genetic affinity between populations [31]. In this study, the allele frequencies of the African American population provided in the kit’s user manual were used to calculate the paternity index because there are no great genetic variations between this population and West Africans [32]. Acknowledgements The authors would like to thank the Pietro Annigoni Biomolecular Research Centre (CERBA/LABIOGENE) for their material and financial support in carrying out this study. We also thank Dr. François GUIDET, Expert at the Court of Aix in Provence, France, and the International Criminal Court (The Hague) and Mr. Philippe SAMAIN at Forensic France for their contribution to the enrichment of this manuscript. Author details 1 1Direction of Scientific and Technical Police, Ouagadougou, Burkina Faso. 2Laboratory of Molecular Biology and Genetics (LABIOGENE) of University Joseph Ki-Zerbo, P.O. 03 BOX, 7021 Ouaga 03, Ouagadougou, Burkina Faso. 3Biomolecular Research Centre Pietro Annigoni (CERBA)), P.O. 01 BOX 364, Ouagadougou Ouaga 01, Burkina Faso. 4Research Institute of Health Sciences (IRSS)), P.O. 03. BOX 7192, Ouagadougou Ouaga 03, Burkina Faso. 5Research Institute of Applied and Technical Sciences (IRSAT)), P.O. 03 BOX 7047, Ouagadougou Ouaga 03, Burkina Faso. 6University of Saint Thomas d’Aquin (USTA)), P.O. 06 BOX 10212, Ouagadougou Ouaga 06, Burkina Faso. Received: 26 March 2021 Accepted: 30 July 2021 Authors’ contributions d l d h MM and STS analyzed the data and interpreted result. STS, BVJTB, and AKO drafted the manuscript. TMZ participated in the data collection and interpretation. MM, AKO, STS, BVJTB, and JS revised the manuscripts. JS designed the study. The authors read and approved the final manuscript Ethics approval and consent to participate This study was approved by the Institutional Ethics Committee of CERBA/ LABIOGENE and The Tribunal de Grande Instance de Ouagadougou (Deliberation N°2019-19/III-015) and conducted according to the Declaration of Helsinki. Also, written informed consent was obtained before blood collection. Discussion Based on the Bayesian probability law, we determined the PI and the CPI. These PIs maked it possible to determine the prob- ability of paternity of an alleged father “to be” or “not to be” the biological father of a child. The results of this study showed a CPI of more than 100 million, unlike a study conducted in Egypt which found a CPI of more than 1 million [23]. The CPI can be high depending on whether the PI calculated from the allele frequencies is high or low. In the ABO system, three alleles are pos- sible and therefore six possible genotypes are present in the human population. In contrast, STR multiplex markers produce a greater number of possible geno- types, as many alleles are present for each STR locus. Thus, although the ABO-rhesus/Hb electrophoresis is useful for excluding a person from paternity, this tech- nique cannot be used to declare a truth inclusion of pa- ternity. The conclusion of paternity from this technique is “Exclusion” or “probable inclusion.” The term “prob- able inclusion” means that there is a possibility of inclu- sion of paternity, but this situation needs reliable techniques to confirm. In paternity tests, the results of the probability of filiation would be either 0% to exclude someone in situations of paternity, siblings, etc., as the biological parent of a child or the same filiation or at least 99% to confirm someone as the biological parent. Legally, a 99% or greater probability of a biological rela- tionship is considered proof of paternity [23, 24]. The in- clusion of paternity comes from the fact that one of the child’s alleles is identical to one of the alleles of the al- leged father for all the markers studied. While the exclu- sion is explained by the fact that the child did not receive any allele from the alleged father for one or more STR markers. Our results with STR assays were similar to those conducted in Egypt where STR assay was used [23]. In Poland, analysis of results obtained between 1966 and 2014 from paternity testing revealed a percent- age of exclusions of 31% using serological tests (ABO, MNSs, Rh factors, Kell, Duffy, Kidd, white blood cells (HLA), serum proteins), 18% using RFLP tests and 20% using STR assay [24]. Discussion Another study in Germany using ABO genotyping by PCR-SSP and ABO grouping re- Page 8 of 9 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 Page 8 of 9 Page 8 of 9 Page 8 of 9 paternity likelihood and power of exclusion of the ABO genotype was significantly higher than of the ABO phenotype. In 12 of 35 exclusion cases (34.3%) the ABO genotype also excluded the alleged father, whereas the ABO phenotype excluded the alleged father only in 7 cases (20%) [25]. As demonstrated in this study, the results of paternity tests were better when using STR analysis compared to serological tests. Every individual in the world can be identified at the molecular level based on an extremely greater level of polymorphism in the sequence of DNA which is inherited from biological parents and is identical in every cell of the body [26, 27]. In inclusion cases, the child shares the length of each STR loci with his parents because each biological parent shares 23 chromosomes for their child and in exclusion cases, the child’s length of STR loci differs between the father and mother [28]. In this study, genetic amplifica- tion of STRs excluded 9 presumed fathers and identified 5 others as the biological fathers of the children. STR markers provide sufficient discriminatory power to ex- clude or include an alleged father in contested paternity cases. Unlike ABO-rhesus/Hb electrophoresis techniques which are based on the agglutination of red blood cells and hemoglobin, the STR technique determines the gen- etic profile of the DNA which contains the genetic infor- mation that is unique to each individual [29]. Because of the limitations of ABO-Rhesus/Hb electrophoresis, the PCR technique became the standard process for DNA paternity testing because PCR technology allows ampli- fying a very small quantity of DNA to increase the amount of DNA up to billions of copies of the same DNA for testing and analysis [28, 30]. Furthermore, STR assays can be used to establish genetic affinity between populations [31]. In this study, the allele frequencies of the African American population provided in the kit’s user manual were used to calculate the paternity index because there are no great genetic variations between this population and West Africans [32]. Some studies were done in African countries to determine alleles frequencies [33, 34]. Moreover, the STR analysis can be used in forensic investigations. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 6. Riou J, Szuberski J, Godart C, Wajcman H, Oliveira JL, Hoyer JD, Bardakdjian- Michau J (2018) Precision of CAPILLARYS 2 for the detection of hemoglobin Availability of data and materials N li bl Availability of data and materials Not applicable Competing interests p g The authors declare that they have no competing interests. Publisher’s Note 15. Eisenberg AJ. Parentage statistics strength of genetic evidence in parentage testing. 2003, https://www.promega.com/-/media/files/resources/ conference-proceedings/ishi-15/parentage-and-mixture-statistics workshop/ introductiontoparentagestatistics.pdf?la=en; (Accessed 25 May 2019). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 16. Souiden Y, Chaieb K, Romdhani M, Mahdouani K (2007) Contribution of the genetic fingerprintings compared to grouping ABO/Rhesus technique in the expertise of filiation. Ann Biol Clin (Paris) 65(6):663–670 17. Hasan MN, Fraiwan A, An R, Alapan Y, Ung R, Akkus A, Xu JZ, Rezac AJ, Kocmich NJ, Creary MS, Oginni T, Olanipekun GM, Hassan-Hanga F, Jibir BW, Gambo S, Verma AK, Bharti PK, Riolueang S, Ngimhung T, Suksangpleng T, Thota P, Werner G, Shanmugam R, Das A, Viprakasit V, Piccone CM, Little JA, Obaro SK, Gurkan UA (2020) Based microchip electrophoresis for point-of- care hemoglobin testing. Analyst 145(7):2525–2542. https://doi.org/10.1039/ C9AN02250C 18. Caboot JB, Allen JL (2014) Hypoxemia in sickle cell disease: significance and management. Paediatr Respir Rev 15(1):17–23. https://doi.org/10.1016/j. prrv.2013.12.004 19. Obaro SK, Iroh Tam PY (2016) Preventing Infections in sickle cell disease: the unfinished business. Pediatr Blood Cancer 63(5):781–785. https://doi.org/10.1 002/pbc.25911 19. Obaro SK, Iroh Tam PY (2016) Preventing Infections in sickle cell disease: the unfinished business. Pediatr Blood Cancer 63(5):781–785. https://doi.org/10.1 002/pbc.25911 20. Katamea T, Mjumbe CK, Omoy MN, Bafwafwa D, Ngombe M, Kasongo L et al (2020) Neonatal screening for lubumbashi drepacytosis: feasibility and suitable performance. Open Access Library Journal 7(3):1–12 20. Katamea T, Mjumbe CK, Omoy MN, Bafwafwa D, Ngombe M, Kasongo L et al (2020) Neonatal screening for lubumbashi drepacytosis: feasibility and suitable performance. Open Access Library Journal 7(3):1–12 21. Balgir RS (2005) Detection of a rare blood group “Bombay (Oh) Phenotype” among the Kutia Kondh Primitive Tribe of Orissa, India. Int J Hum Genet 5(3):193–198. https://doi.org/10.1080/09723757.2005.11885925 21. Balgir RS (2005) Detection of a rare blood group “Bombay (Oh) Phenotype” among the Kutia Kondh Primitive Tribe of Orissa, India. Int J Hum Genet 5(3):193–198. https://doi.org/10.1080/09723757.2005.11885925 22. Tongio MM, Dormoy A (1997) HLA genotyping: indications and limits. Transfus Clin Biol 5:6–12 23. El-Alfy SH, El-Hafez AFA (2012) Paternity testing and forensic DNA typing by multiplex STR analysis using ABI PRISM 310 Genetic Analyzer. J Genet Eng Biotechnol 10(1):101–112. https://doi.org/10.1016/j.jgeb.2012.05.001 24. Karpiewska A, Kowalczyk E, Dobosz T (2017) Paternity testing at the Department of Forensic Medicine of Wroclaw Medical University (Poland). Legal Med 26:18–24. Abbreviations Li L, Zhao SM, Zhang SH, Li CT, Liu Y, Lin Y, Liu JH (2012) Typing and polymorphism analysis of 16 STR loci on X chromosome. Fa Yi Xue Za Zhi 28(1):36–40 3 33. Rosa A, Ornelas C, Brehm A, Villems R (2006) Population data on 11 Y- chromosome STRs from Guiné-Bissau. Forensic Sci Int 157(2-3):210–217. https://doi.org/10.1016/j.forsciint.2005.04.005 12. Amankwaa AO (2019) Towards a reformed policy for immigrant DNA tests, a commentary. J Forensic Legal Med 66:117–119. https://doi.org/10.1016/j. jflm.2019.06.016 34. Semo AC, Carvalho MR, Bogas V, Serra A, Lopes V, Brito P, Sá FB, Porto MJ, Gonçalves IMT, Corte-Real F (2017) Allelic frequencies of 15 autosomal STRs from two main population groups (Makua and Changana) in Mozambique. Forensic Sci Int Genet Suppl Ser 6:e286–e2e8. https://doi.org/10.1016/j. fsigss.2017.09.139 13. Gao JS, Ye Y, Hou YP (2017) Application of SNP-STR Composed by D18S51 and Three SNPs of Its Flanking Region in Paternity Testing. Fa Yi Xue Za Zhi 33(7):607–610 14. Van Huffel V, Rouger P (1999) DNA polymorphism applied to paternity testing. Analysis of 877 cases. Transfus Clin Biol 6(4):236–244. https://doi. org/10.1016/s1246-7820(99)80034-2 Abbreviations 6. Riou J, Szuberski J, Godart C, Wajcman H, Oliveira JL, Hoyer JD, Bardakdjian- Michau J (2018) Precision of CAPILLARYS 2 for the detection of hemoglobin Page 9 of 9 Page 9 of 9 Page 9 of 9 Page 9 of 9 Millogo et al. Journal of Genetic Engineering and Biotechnology (2021) 19:130 (2021) 19:130 variants based on their migration positions. Am J Clin Pathol 149(2):172– 180. https://doi.org/10.1093/ajcp/aqx148 variants based on their migration positions. Am J Clin Pathol 149(2):172– 180. https://doi.org/10.1093/ajcp/aqx148 variants based on their migration positions. Am J Clin Pathol 149(2):172– 180. https://doi.org/10.1093/ajcp/aqx148 28. Essam K, Mona H, Diab AA (2014) Role of DNA in Paternity Testing. J Forensic Sci & Criminal Inves 14(2):555882 7. Schlenk J, Seidl S, Braunschweiger G, Betz P, Lederer T (2004) Development of a 13-locus PCR multiplex system for paternity testing. Int J Legal Med 118(1):55–61. https://doi.org/10.1007/s00414-003-0420-5 29. De Kock AA, Kloppers JJ (2021) The impact of motherless paternity testing in a South African population. Afr Health Sci 21(1):379–384. https://doi.org/1 0.4314/ahs.v21i1.48 30. Thomsen AR, Hallenberg C, Simonsen BT, Langkjær RB, Morling N (2009) A report of the 2002–2008 paternity testing workshops of the English-speaking working group of the International Society for Forensic Genetics. Forensic Sci Int Genet 3(4):214–221. https://doi.org/10.1016/j. fsigen.2009.01.016 8. Burguete-Argueta N, Martinez De la Cruz B, Camacho-Mejorado R, Santana C, Noris G, Lopez-Bayghen E et al (2016) Forensic-paternity effectiveness and genetics population analysis of six non-CODIS mini-STR loci (D1S1656, D2S441, D6S1043, D10S1248, D12S391, D22S1045) and SE33 in Mestizo and Amerindian populations from Mexico. Ann Hum Biol 43(6):563–571. https:// doi.org/10.3109/03014460.2015.1127424 31. Zeye MMJ, Li J, Ouedraogo SY, Zha L, Simpore J, Jifeng C (2021) Population data and genetic structure analysis based on 29 Y-STR loci among the ethnolinguistic groups in Burkina Faso. Int J Legal Med. https://doi.org/10.1 007/s00414-021-02544-9 9. Thomson JA, Pilotti V, Stevens P, Ayres KL, Debenham PG (1999) Validation of short tandem repeat analysis for the investigation of cases of disputed paternity. Forensic Sci Int 100(1-2):1–16. https://doi.org/10.1016/S0379-073 8(98)00199-6 32. Bryc K, Auton A, Nelson MR, Oksenberg JR, Hauser SL, Williams S, Froment A, Bodo JM, Wambebe C, Tishkoff SA, Bustamante CD (2010) Genome-wide patterns of population structure and admixture in West Africans and African Americans. Proc Natl Acad Sci U S A 107(2):786–791. https://doi.org/10.1 073/pnas.0909559107 10. Butler JM (2011) Forensic DNA testing. Cold Spring Harb Protoc 12:1438– 1450 11. Publisher’s Note https://doi.org/10.1016/j.legalmed.2017.02.002 25. Bugert P, Rink G, Kemp K, Klüter H (2012) Blood group ABO genotyping in paternity testing. Transfus Med Hemother 39(3):182–186. https://doi.org/1 0.1159/000339235 26. Mishra A, Sathyan S (2016) DNA Fingerprinting in disputed paternity. Med Phoenix 1(1):44–46 26. Mishra A, Sathyan S (2016) DNA Fingerprinting in disputed paternity. Med Phoenix 1(1):44–46 27. Asela PRM, Clarencia RD (2019) A paternity case based on short tandem repeat (str) using DNA finger printing technology. Int J Dev Res 9(12): 32230–32237
https://openalex.org/W4283022285	https://www.researchsquare.com/article/rs-1741339/latest.pdf	English	null	Anthropogenic signature of global agricultural soil phosphorus	Research Square (Research Square)	2,022	cc-by	17,357	Keywords: Posted Date: June 16th, 2022 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Anthropogenic signature of global agricultural soil phosphorus 1 Anthropogenic signature of global agricultural soil phosphorus 1 Abstract 2 The global phosphorus (P) cycle has been dramatically altered by human activities through the use of 3 mineral P fertilizers, often referred to anthropogenic P. The application of mineral P fertilizers on 4 agricultural soils has driven the planet beyond its safe operating space but the extent to which the 5 global P cycle relies on anthropogenic P has never been quantified. To fill this gap, we developed a 6 model that simulates, at the country scale, the evolution of agricultural soil available P by 7 distinguishing anthropogenic vs. natural P pools, and by accounting for farming practices, crop- 8 livestock recycling loop, and agricultural trade, over the 1950-2017 period. At the global scale we 9 found that the anthropogenic signature of soil available P was 45% ± 8% in 2017. The national 10 anthropogenic signatures varied according to the cumulative mineral P fertilizer use as well as to the 11 soil P availability in 1950. Despite having different historical trajectories, Western Europe, North 12 America and Asia displayed similar reliance on anthropogenic P, close to 60% in 2017. Conversely, 13 African soil anthropogenic signature remained around 30%. Trade did not modify the simulated 14 signatures. Overall, our results unravel the strong reliance of our soil fertility and food production 15 systems on anthropogenic P resources. 16 Abstract 2 The global phosphorus (P) cycle has been dramatically altered by human activities through the use of 3 mineral P fertilizers, often referred to anthropogenic P. The application of mineral P fertilizers on 4 agricultural soils has driven the planet beyond its safe operating space but the extent to which the 5 global P cycle relies on anthropogenic P has never been quantified. To fill this gap, we developed a 6 model that simulates, at the country scale, the evolution of agricultural soil available P by 7 distinguishing anthropogenic vs. natural P pools, and by accounting for farming practices, crop- 8 livestock recycling loop, and agricultural trade, over the 1950-2017 period. At the global scale we 9 found that the anthropogenic signature of soil available P was 45% ± 8% in 2017. The national 10 anthropogenic signatures varied according to the cumulative mineral P fertilizer use as well as to the 11 soil P availability in 1950. Anthropogenic signature of global agricultural soil phosphorus 1 Despite having different historical trajectories, Western Europe, North 12 America and Asia displayed similar reliance on anthropogenic P, close to 60% in 2017. Conversely, 13 African soil anthropogenic signature remained around 30%. Trade did not modify the simulated 14 signatures. Overall, our results unravel the strong reliance of our soil fertility and food production 15 systems on anthropogenic P resources. 16 Main text 17 The Anthropocene is characterised by the profound, anthropogenic disturbance of the global 18 biogeochemical cycles, which has led to drastic consequences for soil fertility, ocean and river 19 quality, greenhouse gas emissions and biodiversity losses (Steffen et al., 2015). In particular, the 20 global phosphorus (P) cycle has been altered in an unprecedented way, both in time and in space 21 (Elser and Bennett, 2011). At the core of these modifications stand phosphate rocks and their use in 22 agriculture, mainly as mineral P fertilizers but also, although at a lesser extent, as mineral feed for 23 livestock animals. Both mineral P fertilizers and mineral P feed are derived from industrial treatments 24 – to increase the P solubility of phosphate rocks – and are hereafter referred to as anthropogenic P. 25 The massive use of mineral P fertilizers combined with a sharp rise in livestock animals number – and 26 resulting manure production – have globally increased cropland soil available P, although with some 27 variations across world regions (Ringeval et al., 2017). The changes in croplands soil P fertility have 28 been triggered by both local and global modifications in the P cycle. At the local scale, P has been 29 added to agricultural soils through massive application of mineral P fertilizers –sometimes far above 30 crop P uptake – resulting in increasing soil P legacy, together with some P deficits in some regions 31 (MacDonald et al., 2011). Large amounts of P have also been transferred from grasslands to 32 1 croplands through the production, transport and soil application of animal manure (Sattari et al., 33 2016), a phenomenon much increased by the massive rise in global livestock population (Bouwman 34 et al., 2011). At the global scale, P has been displaced through the extraction and international trade 35 of phosphate rocks (Nesme et al., 2018). Anthropogenic signature of global agricultural soil phosphorus 1 Indirectly this traded P 39 has fertilized, at least partly, the agricultural soils of importing countries through the application of 40 animal and human manure - derived from traded feed and food - on cropland and grassland soils. 41 croplands through the production, transport and soil application of animal manure (Sattari et al., 33 2016), a phenomenon much increased by the massive rise in global livestock population (Bouwman 34 et al., 2011). At the global scale, P has been displaced through the extraction and international trade 35 of phosphate rocks (Nesme et al., 2018). Highly concentrated in a few places such as Morocco and 36 Western Sahara, phosphate rocks have been redistributed, yet unevenly, to the rest of the world, 37 thus reshaping the spatial distribution of soil P. More recently, the trade of agricultural products has 38 also contributed to displace large quantities of P worldwide (Lun et al., 2021). Indirectly this traded P 39 has fertilized, at least partly, the agricultural soils of importing countries through the application of 40 animal and human manure - derived from traded feed and food - on cropland and grassland soils. 41 croplands through the production, transport and soil application of animal manure (Sattari et al., 33 2016), a phenomenon much increased by the massive rise in global livestock population (Bouwman 34 et al., 2011). At the global scale, P has been displaced through the extraction and international trade 35 of phosphate rocks (Nesme et al., 2018). Highly concentrated in a few places such as Morocco and 36 Western Sahara, phosphate rocks have been redistributed, yet unevenly, to the rest of the world, 37 thus reshaping the spatial distribution of soil P. More recently, the trade of agricultural products has 38 also contributed to displace large quantities of P worldwide (Lun et al., 2021). Indirectly this traded P 39 has fertilized, at least partly, the agricultural soils of importing countries through the application of 40 animal and human manure - derived from traded feed and food - on cropland and grassland soils. 41 This alteration of the global P cycle has had several, positive and negative consequences. On one 42 hand, crop yields have increased as a result of an overall rise in soil P fertility, thereby helping to 43 achieve food security objectives (Mueller et al., 2012). Anthropogenic signature of global agricultural soil phosphorus 1 Highly concentrated in a few places such as Morocco and 36 Western Sahara, phosphate rocks have been redistributed, yet unevenly, to the rest of the world, 37 thus reshaping the spatial distribution of soil P. More recently, the trade of agricultural products has 38 also contributed to displace large quantities of P worldwide (Lun et al., 2021). Indirectly this traded P 39 has fertilized, at least partly, the agricultural soils of importing countries through the application of 40 animal and human manure - derived from traded feed and food - on cropland and grassland soils. 41 This alteration of the global P cycle has had several, positive and negative consequences. On one 42 hand, crop yields have increased as a result of an overall rise in soil P fertility, thereby helping to 43 achieve food security objectives (Mueller et al., 2012). On the other hand, our agriculture has 44 become dependent on phosphate rocks, an alarming situation due to the progressive exhaustion of 45 the easily accessible, remaining reserves of this fossil ressource (Cordell et al., 2009; Reijnders, 2014). 46 Although it will take centuries for phosphate rock reserves to be depleted, short-term challenges are 47 likely to arise in the next years to decades (Scholz and Wellmer, 2018). The prices of mineral P 48 fertilizers are likely to increase in most world regions and potential geopolitical conflicts might break 49 out, thus putting at risk the resilience of our global food systems (Barbieri et al., 2022; Cordell et al., 50 2009; Van Vuuren et al., 2010). 51 Those alterations of the global P cycle and of soil P fertility following anthropogenic P supply have 52 croplands through the production, transport and soil application of animal manure (Sattari et al., 33 2016), a phenomenon much increased by the massive rise in global livestock population (Bouwman 34 et al., 2011). At the global scale, P has been displaced through the extraction and international trade 35 of phosphate rocks (Nesme et al., 2018). Highly concentrated in a few places such as Morocco and 36 Western Sahara, phosphate rocks have been redistributed, yet unevenly, to the rest of the world, 37 thus reshaping the spatial distribution of soil P. More recently, the trade of agricultural products has 38 also contributed to displace large quantities of P worldwide (Lun et al., 2021). Anthropogenic signature of global agricultural soil phosphorus 1 84 In addition to soil P pools, we also modelled the anthropogenic signature of all P fluxes incoming to 85 or outgoing from agricultural soils (Figure 1). We assumed that P harvest had the same 86 anthropogenic signature of that of the soil labile P pool in which plant uptake occurred. Similarly, we 87 assumed that imported food and feed products had the same P signature as that of the soil labile P 88 pool of the countries where they came from. Because P losses from soils may apply to both labile and 89 stable P pools indifferently, we assumed that the signature of P losses was similar to that of the 90 overall soil P pools. The anthropogenic P signature of animal manure was assumed equal to that of 91 animal intake. By construction and because they are entirely processed from the chemical industry, 92 an anthropogenic signature of 1 was attributed to both mineral P fertilizers applied to soils and to 93 mineral feed supplements given to livestock (Figure 1). 94 In addition to soil P pools, we also modelled the anthropogenic signature of all P fluxes incoming to 85 or outgoing from agricultural soils (Figure 1). We assumed that P harvest had the same 86 anthropogenic signature of that of the soil labile P pool in which plant uptake occurred. Similarly, we 87 assumed that imported food and feed products had the same P signature as that of the soil labile P 88 pool of the countries where they came from. Because P losses from soils may apply to both labile and 89 stable P pools indifferently, we assumed that the signature of P losses was similar to that of the 90 overall soil P pools. The anthropogenic P signature of animal manure was assumed equal to that of 91 animal intake. By construction and because they are entirely processed from the chemical industry, 92 an anthropogenic signature of 1 was attributed to both mineral P fertilizers applied to soils and to 93 mineral feed supplements given to livestock (Figure 1). 94 We initialized the size of the P pools by assuming that, although some mineral P fertilizers were 95 sometimes applied to agricultural soils in the first half of the 20th century (Bouwman et al., 2011; 96 Grigg, 1987; Ringeval et al., 2014), anthropogenic soil P pools were negligible before 1950. Anthropogenic signature of global agricultural soil phosphorus 1 On the other hand, our agriculture has 44 become dependent on phosphate rocks, an alarming situation due to the progressive exhaustion of 45 the easily accessible, remaining reserves of this fossil ressource (Cordell et al., 2009; Reijnders, 2014). 46 Although it will take centuries for phosphate rock reserves to be depleted, short-term challenges are 47 likely to arise in the next years to decades (Scholz and Wellmer, 2018). The prices of mineral P 48 fertilizers are likely to increase in most world regions and potential geopolitical conflicts might break 49 fertilizers are likely to increase in most world regions and potential geopolitical conflicts might break 49 out, thus putting at risk the resilience of our global food systems (Barbieri et al., 2022; Cordell et al., 50 2009; Van Vuuren et al., 2010). 51 Those alterations of the global P cycle and of soil P fertility following anthropogenic P supply have 52 already been highlighted by recent synthesis studies (Chen and Graedel, 2016; Elser and Bennett, 53 2011; Lu and Tian, 2016). However, the specific and cumulated contribution of anthropogenic P 54 supply to P fertility of global agricultural soils has never been estimated. Providing such estimate is 55 key to assess both the anthropogenic disturbance of the global P cycle but also to estimate the past 56 and current reliance of agricultural soils and food production to the finite phosphate rock resources. 57 Here we address this knowledge gap by estimating the anthropogenic signature of available P in 58 agricultural soils at the global scale. We defined the anthropogenic signature of soil available P as the 59 ratio of anthropogenic over total soil available P, where total P refers to the sum of non- 60 anthropogenic P (considered as natural P) and anthropogenic P. 61 To achieve this objective, we developed a model that simulates for each country and with a yearly 62 time step, the evolution of agricultural soil P stocks (in both cropland and grassland soils) from 1950 63 to 2017, accounting for (i) direct P fertilizer inputs to soils but also (ii) P recycling from the crop- 64 livestock cycling loop and (iii) geographic displacement of P through the trade of agricultural 65 2 products (Figure 1). Anthropogenic signature of global agricultural soil phosphorus 1 By construction and because they are entirely processed from the chemical industry, 92 an anthropogenic signature of 1 was attributed to both mineral P fertilizers applied to soils and to 93 mineral feed supplements given to livestock (Figure 1). 94 We initialized the size of the P pools by assuming that, although some mineral P fertilizers were 95 sometimes applied to agricultural soils in the first half of the 20th century (Bouwman et al., 2011; 96 Grigg, 1987; Ringeval et al., 2014), anthropogenic soil P pools were negligible before 1950. The sizes 97 In our model, P harvested from cropland and grassland soils (𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑇𝑜𝑡) was computed as a 76 function of the size of the labile P pool (Equation 2). The two parameters involved in the relation 77 between P harvest and the size of the labile P pool were calibrated for each country. Similarly, and in 78 an attempt to take into account the soil P dynamic specificities of each country, we calibrated the P 79 exchange function between stable and labile soil P pools (Equation 3). Both calibrations were 80 conducted independently for each country and were performed so that the model outputs could 81 reproduce both P harvest time-series (from FAOSTAT) over the 1950-2017 period and the size of the 82 labile P pool at year 2005 (derived from the global modelling approach of (Ringeval et al., 2017) or 83 from observations for the few countries where data were available) (see Method). 84 In our model, P harvested from cropland and grassland soils (𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑇𝑜𝑡) was computed as a 76 function of the size of the labile P pool (Equation 2). The two parameters involved in the relation 77 between P harvest and the size of the labile P pool were calibrated for each country. Similarly, and in 78 an attempt to take into account the soil P dynamic specificities of each country, we calibrated the P 79 exchange function between stable and labile soil P pools (Equation 3). Both calibrations were 80 conducted independently for each country and were performed so that the model outputs could 81 reproduce both P harvest time-series (from FAOSTAT) over the 1950-2017 period and the size of the 82 labile P pool at year 2005 (derived from the global modelling approach of (Ringeval et al., 2017) or 83 from observations for the few countries where data were available) (see Method). Anthropogenic signature of global agricultural soil phosphorus 1 We modelled soil available P as the interaction between a labile P pool (LP) and a 66 stable P pool (SP), which is commonly done in the literature (Le Noë et al., 2020; Sattari et al., 2012; 67 Zhang et al., 2017). The labile P pool referred to soil P directly available for plant uptake while the 68 stable P pool acted as a slow-release buffer that could replenish the labile P pool following crop P 69 uptake. Each pool was further broken down into a natural (Nat) vs. an anthropogenic (Ant) 70 compartment. This allowed us to track the behaviour of P inputs according to their anthropic vs 71 natural origin in a similar way as isotopic labelling approaches (Sebilo et al., 2013). For each country, 72 the size of the four P pools (LPNat, LPAnt, SPNat, SPAnt) were simulated following the size of the previous 73 years, soil P inputs and outputs, and soil P exchanges between labile and stable P pools (see 74 Method). 75 products (Figure 1). We modelled soil available P as the interaction between a labile P pool (LP) and a 66 stable P pool (SP), which is commonly done in the literature (Le Noë et al., 2020; Sattari et al., 2012; 67 Zhang et al., 2017). The labile P pool referred to soil P directly available for plant uptake while the 68 stable P pool acted as a slow-release buffer that could replenish the labile P pool following crop P 69 uptake. Each pool was further broken down into a natural (Nat) vs. an anthropogenic (Ant) 70 compartment. This allowed us to track the behaviour of P inputs according to their anthropic vs 71 natural origin in a similar way as isotopic labelling approaches (Sebilo et al., 2013). For each country, 72 the size of the four P pools (LPNat, LPAnt, SPNat, SPAnt) were simulated following the size of the previous 73 years, soil P inputs and outputs, and soil P exchanges between labile and stable P pools (see 74 Method). 75 stable P pool (SP), which is commonly done in the literature (Le Noë et al., 2020; Sattari et al., 2012; 67 Zhang et al., 2017). The labile P pool referred to soil P directly available for plant uptake while the 68 stable P pool acted as a slow-release buffer that could replenish the labile P pool following crop P 69 uptake. Anthropogenic signature of global agricultural soil phosphorus 1 Each pool was further broken down into a natural (Nat) vs. an anthropogenic (Ant) 70 compartment. This allowed us to track the behaviour of P inputs according to their anthropic vs 71 natural origin in a similar way as isotopic labelling approaches (Sebilo et al., 2013). For each country, 72 the size of the four P pools (LPNat, LPAnt, SPNat, SPAnt) were simulated following the size of the previous 73 years, soil P inputs and outputs, and soil P exchanges between labile and stable P pools (see 74 Method). 75 In our model, P harvested from cropland and grassland soils (𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑇𝑜𝑡) was computed as a 76 function of the size of the labile P pool (Equation 2). The two parameters involved in the relation 77 between P harvest and the size of the labile P pool were calibrated for each country. Similarly, and in 78 an attempt to take into account the soil P dynamic specificities of each country, we calibrated the P 79 exchange function between stable and labile soil P pools (Equation 3). Both calibrations were 80 conducted independently for each country and were performed so that the model outputs could 81 reproduce both P harvest time-series (from FAOSTAT) over the 1950-2017 period and the size of the 82 labile P pool at year 2005 (derived from the global modelling approach of (Ringeval et al., 2017) or 83 from observations for the few countries where data were available) (see Method). 84 In addition to soil P pools, we also modelled the anthropogenic signature of all P fluxes incoming to 85 or outgoing from agricultural soils (Figure 1). We assumed that P harvest had the same 86 anthropogenic signature of that of the soil labile P pool in which plant uptake occurred. Similarly, we 87 assumed that imported food and feed products had the same P signature as that of the soil labile P 88 pool of the countries where they came from. Because P losses from soils may apply to both labile and 89 stable P pools indifferently, we assumed that the signature of P losses was similar to that of the 90 overall soil P pools. The anthropogenic P signature of animal manure was assumed equal to that of 91 animal intake. Anthropogenic signature of global agricultural soil phosphorus 1 The sizes 97 of the labile and stable natural P pools were estimated based on the observed crop and forage P 98 We initialized the size of the P pools by assuming that, although some mineral P fertilizers were 95 sometimes applied to agricultural soils in the first half of the 20th century (Bouwman et al., 2011; 96 Grigg, 1987; Ringeval et al., 2014), anthropogenic soil P pools were negligible before 1950. The sizes 97 of the labile and stable natural P pools were estimated based on the observed crop and forage P 98 3 3 harvest in 1950 and by assuming an equilibrium between the two soil P pools (see Methods). 99 harvest in 1950 and by assuming an equilibrium between the two soil P pools (see Methods). 99 harvest in 1950 and by assuming an equilibrium between the two soil P pools (see Methods). 99 harvest in 1950 and by assuming an equilibrium between the two soil P pools (see Methods). 99 Hereafter, we comment the signature of the soil labile P pool both in 2017 and throughout the 1950- 100 2017 period. 101 Hereafter, we comment the signature of the soil labile P pool both in 2017 and throughout the 1950- 100 2017 period. 101 102 103 Figure 1 - Structure of the model for a given country, with specific focus on soil P compartments. LP and SP refer to the labile 104 and stable soil P pools, respectively. The anthropogenic vs. natural soil P pools are represented in orange and green 105 respectively. The fluxes in solid lines were explicitly simulated. Conversely, those in dotted lines were not modelled because 106 they do not modify the anthropogenic signature of P pools. As illustrated by the pie charts, each flux was subdivided into 107 anthropogenic (orange) vs. natural (green) components. The blue box refers to a given country that interacts with the rest of 108 the world (other countries) through the trade of food and feed products. 109 103 Figure 1 - Structure of the model for a given country, with specific focus on soil P compartments. LP and SP refer to the labile 104 and stable soil P pools, respectively. The anthropogenic vs. natural soil P pools are represented in orange and green 105 respectively. The fluxes in solid lines were explicitly simulated. Anthropogenic signature of global agricultural soil phosphorus 1 Conversely, those in dotted lines were not modelled because 106 they do not modify the anthropogenic signature of P pools. As illustrated by the pie charts, each flux was subdivided into 107 anthropogenic (orange) vs. natural (green) components. The blue box refers to a given country that interacts with the rest of 108 the world (other countries) through the trade of food and feed products. 109 110 Despite large spatial variabilities, the current global anthropogenic signature of the soil P is high 111 Despite large spatial variabilities, the current global anthropogenic signature of the soil P is high 111 111 112 Figure 2 - Anthropogenic signature of the labile P pool of agricultural soils in 2017. Data are displayed per country. Countries 113 coloured in grey were excluded from the calculation either because of missing data (as for Democratic Republic of Congo, 114 Libya and Somalia among others) or because their calibration did not succeed (Cuba, Estonia, Jordan, Kazakhstan, Latvia, 115 Lithuania, Tajikistan, Turkmenistan, and Ukraine). 116 112 Figure 2 - Anthropogenic signature of the labile P pool of agricultural soils in 2017. Data are displayed per country. Countries 113 coloured in grey were excluded from the calculation either because of missing data (as for Democratic Republic of Congo, 114 Libya and Somalia among others) or because their calibration did not succeed (Cuba, Estonia, Jordan, Kazakhstan, Latvia, 115 Lithuania, Tajikistan, Turkmenistan, and Ukraine). 116 Our results showed that the use of anthropogenic P - derived from phosphate rocks - greatly 117 modified agricultural soil P fertility worldwide (Figure 2). In 2017, the global mean anthropogenic 118 signature of the soil labile P pool was 45 ±8 %, suggesting that almost half of the global agricultural 119 soil P fertility was derived from anthropic P supply. This result clearly mirrors the overall 120 intensification growth path of the world agriculture, which has relied on the massive use of 121 agricultural inputs and mineral fertilizers from the 1950s (Coomes et al., 2019). 122 Our findings also highlight large spatial variabilities of anthropogenic soil P signatures among world 123 regions (Figure 2). North America, Western Europe and Asia displayed the highest anthropogenic 124 signatures, with values of 69 ±8 %, 61 ± 8 % and 59 ± 6 %, respectively. Central and South America 125 had an intermediate anthropogenic signature of 41 ±9 %, close to that of Eastern Europe (39 ±10 %). 126 In Africa, the soil P anthropogenic signature remained moderate at 30 ±6 %. Finally, Oceania, which 127 includes Australia and New Zealand, had the lowest anthropogenic signature with value of 18 ±11 %. 128 We also found that the anthropogenic signatures of soil P were in fact explained by two main factors: 129 (i) the cumulative application of anthropogenic P - mostly as mineral P fertilizer - over the 1950-2017 130 period and (ii) the initial soil P status in 1950 – assimilated to natural P in our model (Section S8). Despite large spatial variabilities, the current global anthropogenic signature of the soil P is high 111 As a 131 result, the massive mineral P fertilization in North America, Western Europe and Asia since the mid- 132 20th century translated into high anthropogenic signatures in those regions. Besides, high initial soil 133 P pools in 1950 in Western Europe mechanistically ‘diluted’ the massive anthropogenic P supply to 134 agricultural soils experienced by this region, which has been three times higher than in North 135 Figure 2 - Anthropogenic signature of the labile P pool of agricultural soils in 2017. Data are displayed per country. Countries 113 coloured in grey were excluded from the calculation either because of missing data (as for Democratic Republic of Congo, 114 Libya and Somalia among others) or because their calibration did not succeed (Cuba, Estonia, Jordan, Kazakhstan, Latvia, 115 Lithuania, Tajikistan, Turkmenistan, and Ukraine). 116 Our results showed that the use of anthropogenic P - derived from phosphate rocks - greatly 117 modified agricultural soil P fertility worldwide (Figure 2). In 2017, the global mean anthropogenic 118 signature of the soil labile P pool was 45 ±8 %, suggesting that almost half of the global agricultural 119 soil P fertility was derived from anthropic P supply. This result clearly mirrors the overall 120 intensification growth path of the world agriculture, which has relied on the massive use of 121 agricultural inputs and mineral fertilizers from the 1950s (Coomes et al., 2019). 122 Our results showed that the use of anthropogenic P - derived from phosphate rocks - greatly 117 modified agricultural soil P fertility worldwide (Figure 2). In 2017, the global mean anthropogenic 118 signature of the soil labile P pool was 45 ±8 %, suggesting that almost half of the global agricultural 119 soil P fertility was derived from anthropic P supply. This result clearly mirrors the overall 120 intensification growth path of the world agriculture, which has relied on the massive use of 121 agricultural inputs and mineral fertilizers from the 1950s (Coomes et al., 2019). 122 Our findings also highlight large spatial variabilities of anthropogenic soil P signatures among world 123 regions (Figure 2). North America, Western Europe and Asia displayed the highest anthropogenic 124 signatures, with values of 69 ±8 %, 61 ± 8 % and 59 ± 6 %, respectively. Central and South America 125 had an intermediate anthropogenic signature of 41 ±9 %, close to that of Eastern Europe (39 ±10 %). Despite large spatial variabilities, the current global anthropogenic signature of the soil P is high 111 126 In Africa, the soil P anthropogenic signature remained moderate at 30 ±6 %. Finally, Oceania, which 127 includes Australia and New Zealand, had the lowest anthropogenic signature with value of 18 ±11 %. 128 5 5 America and Asia (Table S8). This explains why despite very different cumulated mineral P 136 fertilization (Figure S15), the three regions all displayed similar anthropogenic signatures in 2017. 137 Finally, and contrary to the stable P pool, the anthropogenic signature of the labile P pool was 138 dependant on a third and last driver: the P transfer between the soil P pools (Text S8), which 139 underlines the need to account for soil P dynamics when simulating soil P availability and its changes 140 over time. 141 Highly different trends in anthropogenic signature of soil P among countries over 1950-2017 142 143 Figure 3 – Temporal evolution of the anthropogenic signature of the soil labile P pool for eight contrasting countries (France, 144 the Netherlands, the United States of America, Brazil, India, China, Zimbabwe and Morocco) for the 1950-2017 period. 145 Mean (solid line) and standard deviation (blurred zone) data are shown. 146 Highly different trends in anthropogenic signature of soil P among countries over 1950-2017 142 143 Figure 3 – Temporal evolution of the anthropogenic signature of the soil labile P pool for eight contrasting countries (France, 144 the Netherlands, the United States of America, Brazil, India, China, Zimbabwe and Morocco) for the 1950-2017 period. 145 Mean (solid line) and standard deviation (blurred zone) data are shown. 146 Figure 3 Temporal evolution of the anthropogenic signature of the soil labile P pool for eight contrasting countries (France, 144 the Netherlands, the United States of America, Brazil, India, China, Zimbabwe and Morocco) for the 1950-2017 period. 145 Mean (solid line) and standard deviation (blurred zone) data are shown. 146 We found that the soil P anthropogenic signatures displayed highly variable historical trajectories 147 among countries (Figure 3), thereby reflecting contrasting and region-specific histories of soil P 148 fertilization (Figure S16-S17). More precisely, our results showed that the anthropogenic signature of 149 industrialised Western countries such as France, the Netherlands and the USA rose sharply from the 150 1950s to the 1970s (Figure 3), as a result of the very early and massive application of mineral P 151 fertilizers on agricultural soils in industrialised, Western countries. Despite large spatial variabilities, the current global anthropogenic signature of the soil P is high 111 This is 168 mainly due to small applications of mineral P fertilizers, resulting on strong limitation of crop yields 169 by soil P availability in those countries (Kvakíc et al., 2018). 170 Finally, the soil P anthropogenic signatures of most African countries e.g. Morocco and Zimbabwe 167 exhibited a late and slow take off, with values remaining low and close to 20-30% in 2017. This is 168 mainly due to small applications of mineral P fertilizers, resulting on strong limitation of crop yields 169 by soil P availability in those countries (Kvakíc et al., 2018). 170 Finally, the soil P anthropogenic signatures of most African countries e.g. Morocco and Zimbabwe 167 exhibited a late and slow take off, with values remaining low and close to 20-30% in 2017. This is 168 mainly due to small applications of mineral P fertilizers, resulting on strong limitation of crop yields 169 by soil P availability in those countries (Kvakíc et al., 2018). 170 Overall, these results illustrate the ‘Great Divergence’ (Pomeranz, 2000) that, from the 1950s, has 171 seen industrialised countries such as Europe and the USA standing out from the rest of the world in 172 term of growth and development, thanks among other things to the appropriation of overseas 173 resources, such as phosphate rocks (Le Noë et al., 2020). This development gap is being filled by most 174 Asian and South American countries, as illustrated in our estimated trends in anthropogenic 175 signature for the specific case of P. 176 Overall, these results illustrate the ‘Great Divergence’ (Pomeranz, 2000) that, from the 1950s, has 171 seen industrialised countries such as Europe and the USA standing out from the rest of the world in 172 term of growth and development, thanks among other things to the appropriation of overseas 173 resources, such as phosphate rocks (Le Noë et al., 2020). This development gap is being filled by most 174 Asian and South American countries, as illustrated in our estimated trends in anthropogenic 175 signature for the specific case of P. 176 Despite large spatial variabilities, the current global anthropogenic signature of the soil P is high 111 This development gap is being filled by most 174 Asian and South American countries, as illustrated in our estimated trends in anthropogenic 175 signature for the specific case of P. 176 Unexpectedly, the trade of feed and food products had almost no effects on soil P anthropogenic 177 signatures 178 Our results also showed that the soil P anthropogenic signatures of Brazil, India and China took off in 161 the 1970s and 1980s up to values of 60 ± 9%, 61 ± 8% and 73 ± 6% in 2017, respectively (Figure 3). 162 Those results reflect the Green Revolution and the sudden and sharp increase in mineral P fertilizer 163 use that transformed agriculture and soil fertility of most Asian and South American countries from 164 the 1970s (Figure S16). Interestingly, the signatures of those countries have become similar and even 165 higher compared to those of Western European countries in 2017. 166 Our results also showed that the soil P anthropogenic signatures of Brazil, India and China took off in 161 the 1970s and 1980s up to values of 60 ± 9%, 61 ± 8% and 73 ± 6% in 2017, respectively (Figure 3). 162 Those results reflect the Green Revolution and the sudden and sharp increase in mineral P fertilizer 163 use that transformed agriculture and soil fertility of most Asian and South American countries from 164 the 1970s (Figure S16). Interestingly, the signatures of those countries have become similar and even 165 higher compared to those of Western European countries in 2017. 166 Our results also showed that the soil P anthropogenic signatures of Brazil, India and China took off in 161 the 1970s and 1980s up to values of 60 ± 9%, 61 ± 8% and 73 ± 6% in 2017, respectively (Figure 3). 162 Those results reflect the Green Revolution and the sudden and sharp increase in mineral P fertilizer 163 use that transformed agriculture and soil fertility of most Asian and South American countries from 164 the 1970s (Figure S16). Interestingly, the signatures of those countries have become similar and even 165 higher compared to those of Western European countries in 2017. 166 Finally, the soil P anthropogenic signatures of most African countries e.g. Morocco and Zimbabwe 167 exhibited a late and slow take off, with values remaining low and close to 20-30% in 2017. Despite large spatial variabilities, the current global anthropogenic signature of the soil P is high 111 However, since the 1980s the 152 anthropogenic signatures of France and the Netherlands have stabilized around values of 70 ± 7% 153 and 47 ± 11% (in 2017), respectively. Conversely, the signature of the soil labile P pool in the USA was 154 still raising in 2017. The observed stabilization in France and in the Netherlands resulted from smaller 155 mineral P inputs over the last four decades, partly compensated by higher manure inputs with less 156 effects on the anthropogenic signatures (Figure 4). Indeed, when imported feed are small compared 157 to domestically produced feed – which is the general case –, manure simply recycles P from domestic 158 We found that the soil P anthropogenic signatures displayed highly variable historica 147 ound that the soil P anthropogenic signatures displayed highly variable historical trajectories 6 feed and fodder back to agricultural soils, without affecting the anthropogenic signature of 159 agricultural soils. 160 feed and fodder back to agricultural soils, without affecting the anthropogenic signature of 159 agricultural soils. 160 Our results also showed that the soil P anthropogenic signatures of Brazil, India and China took off in 161 the 1970s and 1980s up to values of 60 ± 9%, 61 ± 8% and 73 ± 6% in 2017, respectively (Figure 3). 162 Those results reflect the Green Revolution and the sudden and sharp increase in mineral P fertilizer 163 use that transformed agriculture and soil fertility of most Asian and South American countries from 164 the 1970s (Figure S16). Interestingly, the signatures of those countries have become similar and even 165 higher compared to those of Western European countries in 2017. 166 Finally, the soil P anthropogenic signatures of most African countries e.g. Morocco and Zimbabwe 167 exhibited a late and slow take off, with values remaining low and close to 20-30% in 2017. This is 168 mainly due to small applications of mineral P fertilizers, resulting on strong limitation of crop yields 169 by soil P availability in those countries (Kvakíc et al., 2018). 170 Overall, these results illustrate the ‘Great Divergence’ (Pomeranz, 2000) that, from the 1950s, has 171 seen industrialised countries such as Europe and the USA standing out from the rest of the world in 172 term of growth and development, thanks among other things to the appropriation of overseas 173 resources, such as phosphate rocks (Le Noë et al., 2020). Unexpectedly, the trade of feed and food products had almost no effects on soil P anthropogenic 177 signatures 178 Unexpectedly, the trade of feed and food products had almost no effects on soil P anthropogenic 177 signatures 178 7 179 Figure 4 – Temporal evolution of P inputs to agricultural soils over the 1950-2017 period, with distinction between anthropic 180 (in red) and natural (in green) origin of P fluxes. For comparison, P harvested from agricultural soils has also been displayed. 181 For clarity, we did not show the anthropic vs. natural origin of P embedded in sludge (in yellow), although they have been 182 considered in the model. Manure input was split in three categories: (i) manure that came from the consumption of 183 domestically produced feed and forage (ii) manure that came from the consumption of imported feed and forage, and (iii) 184 manure that came from the consumption of mineral P feed. Note that the Y-axis scale differs among countries. 185 Figure 4 – Temporal evolution of P inputs to agricultural soils over the 1950-2017 period, with distinction between anthropic 180 (in red) and natural (in green) origin of P fluxes. For comparison, P harvested from agricultural soils has also been displayed. 181 For clarity, we did not show the anthropic vs. natural origin of P embedded in sludge (in yellow), although they have been 182 considered in the model. Manure input was split in three categories: (i) manure that came from the consumption of 183 domestically produced feed and forage (ii) manure that came from the consumption of imported feed and forage, and (iii) 184 manure that came from the consumption of mineral P feed. Note that the Y-axis scale differs among countries. 185 Figure 4 – Temporal evolution of P inputs to agricultural soils over the 1950-2017 period, with distinction between anthropic 180 (in red) and natural (in green) origin of P fluxes. For comparison, P harvested from agricultural soils has also been displayed. 181 For clarity, we did not show the anthropic vs. natural origin of P embedded in sludge (in yellow), although they have been 182 considered in the model. Manure input was split in three categories: (i) manure that came from the consumption of 183 domestically produced feed and forage (ii) manure that came from the consumption of imported feed and forage, and (iii) 184 manure that came from the consumption of mineral P feed. Note that the Y-axis scale differs among countries. Implications for sustainable phosphate rock management 210 Thanks to our large scale and time-dependant modelling approach, our results helped to picture both 211 the geographical variations and temporal evolutions of the countries’ reliance on anthropogenic P. 212 Overall, we found that anthropogenic P has greatly contributed to build the current P fertility of most 213 agricultural soils, the average global anthropogenic signature of agricultural soil available P being 214 around 45%. We also found that the discrepancies in anthropogenic signatures among countries 215 were explained by both an uneven use of mineral P fertilizers since the Second World War and 216 inequalities in the inherited agricultural soil P stocks in 1950. In some cases, the use of mineral P 217 fertilizers has even exacerbated the existing inequalities in soil available P in 1950. For example, 218 Western Europe was the region that has fertilized the most with mineral P over the 1950-2017 219 period while its soil available P stocks in 1950, inherited from biogeochemical background and past 220 fertilization practices, were also among the highest in the world (Ringeval et al., 2017). Our findings 221 also illustrate that, on the other side of the gradient, many countries – especially in Africa – have very 222 little benefited from phosphate rocks for sustaining their soil P fertility, the anthropogenic signature 223 of their soils remaining below 30%. These results question the past distribution of phosphate rock 224 resources, which has been rather based on regional economic and financial ability to buy fertilizers 225 than on the search to maximise global food production (Langhans et al., 2022). 226 Thanks to our large scale and time-dependant modelling approach, our results helped to picture both 211 the geographical variations and temporal evolutions of the countries’ reliance on anthropogenic P. 212 Overall, we found that anthropogenic P has greatly contributed to build the current P fertility of most 213 agricultural soils, the average global anthropogenic signature of agricultural soil available P being 214 around 45%. We also found that the discrepancies in anthropogenic signatures among countries 215 were explained by both an uneven use of mineral P fertilizers since the Second World War and 216 inequalities in the inherited agricultural soil P stocks in 1950. In some cases, the use of mineral P 217 fertilizers has even exacerbated the existing inequalities in soil available P in 1950. Unexpectedly, the trade of feed and food products had almost no effects on soil P anthropogenic 177 signatures 178 199 imported feed and food products represented an important, indirect P input to their soils, the 196 anthropogenic signature of these imported products was generally close to that of their domestic 197 agricultural soils (Figure S28), thus resulting in neutral effect on soil P anthropogenic signature 198 (Figure S27). 199 We nevertheless found that, for some countries (such as the Netherlands, Saudi Arabia, Israel, 200 Belgium, Egypt, Lebanon, Portugal and Switzerland), imported feed and food products represented 201 an important input of anthropogenic P to agricultural soils. In these countries, P in animal manure 202 and sewage sludge derived from imported feed and food products accounted for 10% to 34% of their 203 total soil P inputs over the 2007-2017 period, with around half being from anthropic origin. More 204 generally, we found that, at the global scale, 53% of the P embedded in traded products was of 205 anthropic origin; a value amounting to 1.5 Mt P.yr-1 or 8% of the global use of mineral P fertilizers 206 (see Methods). Those results illustrate that food and feed trade displaces large amounts of 207 anthropogenic P that need to be accounted for when assessing the reliance of our global food system 208 to phosphate rock resources (Barbieri et al., 2022; Nanda et al., 2019). 209 We nevertheless found that, for some countries (such as the Netherlands, Saudi Arabia, Israel, 200 Belgium, Egypt, Lebanon, Portugal and Switzerland), imported feed and food products represented 201 an important input of anthropogenic P to agricultural soils. In these countries, P in animal manure 202 and sewage sludge derived from imported feed and food products accounted for 10% to 34% of their 203 total soil P inputs over the 2007-2017 period, with around half being from anthropic origin. More 204 generally, we found that, at the global scale, 53% of the P embedded in traded products was of 205 anthropic origin; a value amounting to 1.5 Mt P.yr-1 or 8% of the global use of mineral P fertilizers 206 (see Methods). Those results illustrate that food and feed trade displaces large amounts of 207 anthropogenic P that need to be accounted for when assessing the reliance of our global food system 208 to phosphate rock resources (Barbieri et al., 2022; Nanda et al., 2019). 209 Unexpectedly, the trade of feed and food products had almost no effects on soil P anthropogenic 177 signatures 178 185 Given that the trade of agricultural products displace annually 3Mt P.yr-1, equivalent to 27% of the P 186 traded through mineral P fertilizers (Nesme et al., 2018), we would have expected trade to 187 substantially modify the soil P anthropogenic signature of large importers such as China or the 188 Netherlands. Yet, we found that the trade of feed and food products had almost no impact on the 189 anthropogenic signatures of the soil P pools. The reasons for that were twofold. First, for 94% of the 190 countries studied, the soil P inputs derived from imported feed and food products (and applied to 191 soils as animal manure and sewage sludge) contributed to less than 5% of the cumulative P inputs 192 over the 1950-2017 period (Figure 4 for all countries except the Netherlands). As a result, for these 193 countries, imported products – whatever their anthropogenic P signature – had almost no effect on 194 soil P anthropogenic signatures. Second, for a handful of countries, despite the P embedded in 195 Given that the trade of agricultural products displace annually 3Mt P.yr-1, equivalent to 27% of the P 186 traded through mineral P fertilizers (Nesme et al., 2018), we would have expected trade to 187 substantially modify the soil P anthropogenic signature of large importers such as China or the 188 Netherlands. Yet, we found that the trade of feed and food products had almost no impact on the 189 anthropogenic signatures of the soil P pools. The reasons for that were twofold. First, for 94% of the 190 countries studied, the soil P inputs derived from imported feed and food products (and applied to 191 soils as animal manure and sewage sludge) contributed to less than 5% of the cumulative P inputs 192 over the 1950-2017 period (Figure 4 for all countries except the Netherlands). As a result, for these 193 countries, imported products – whatever their anthropogenic P signature – had almost no effect on 194 soil P anthropogenic signatures. Second, for a handful of countries, despite the P embedded in 195 8 imported feed and food products represented an important, indirect P input to their soils, the 196 anthropogenic signature of these imported products was generally close to that of their domestic 197 agricultural soils (Figure S28), thus resulting in neutral effect on soil P anthropogenic signature 198 (Figure S27). Implications for sustainable phosphate rock management 210 For example, 218 Western Europe was the region that has fertilized the most with mineral P over the 1950-2017 219 period while its soil available P stocks in 1950, inherited from biogeochemical background and past 220 fertilization practices, were also among the highest in the world (Ringeval et al., 2017). Our findings 221 also illustrate that, on the other side of the gradient, many countries – especially in Africa – have very 222 little benefited from phosphate rocks for sustaining their soil P fertility, the anthropogenic signature 223 of their soils remaining below 30%. These results question the past distribution of phosphate rock 224 resources, which has been rather based on regional economic and financial ability to buy fertilizers 225 than on the search to maximise global food production (Langhans et al., 2022). 226 The high reliance we found of the global soil P fertility to anthropogenic resources raises concerns 227 regarding the vulnerability of our agricultural systems to potential future P scarcity. Our results 228 The high reliance we found of the global soil P fertility to anthropogenic resources raises concerns 227 regarding the vulnerability of our agricultural systems to potential future P scarcity. Our results 228 9 9 indeed demonstrate that our agricultural systems have been built on the massive use of mineral P 229 fertilizers. Complemented by proxies on current P fertilizer use as well as on soil P stocks (Barbieri et 230 al., 2022), our result show that world regions are facing differentiated risks of P deficits. For instance, 231 Western Europe has high soil P stocks (Jones et al., 2020; Ringeval et al., 2017) but its high 232 anthropogenic P signature shows its strong reliance on mineral P fertilizers, suggesting some long- 233 term vulnerability (Le Noë et al., 2020). In contrast, countries such as India or Brazil are particularly at 234 risk as they use massive amounts of mineral P fertilizers to maintain high yields – as illustrated by the 235 recent increase in their soil P anthropogenic signatures – but, unlike European countries, their soil 236 available P stocks are small (Ringeval et al., 2017). It is worth noting that the anthropogenic signature 237 we simulated is rather conservative and will decrease only slightly even if mineral P fertilizer use is 238 drastically reduced. Implications for sustainable phosphate rock management 210 This is due to the fact that increasing P recycling on agricultural soils would affect 239 both anthropogenic and natural P fluxes in similar proportion to that of the soil P anthropogenic 240 signature. This makes our anthropogenic metric suitable to estimate the current reliance on 241 anthropogenic P but not appropriate to analyse how soil P fertility would be affected under future 242 farming scenarios. 243 Our work also offers a way to quantify the anthropogenic P content of agricultural food and feed 244 products by considering not only contemporary soil P inputs but also the inherited soil P legacy from 245 past anthropogenic P supply. Most studies estimating the P footprint of crop products have focused 246 on the annual P supply to soils under the considered crop (Barbieri et al., 2022; Lun et al., 2021; 247 Nesme et al., 2018). By labelling, the anthropic vs. natural origin of P in agricultural soils and in 248 agricultural products, our methodology helps to get more accurate estimates of P footprint that 249 account for past mineral P fertilization. Despite negligible effect of trade on soil P anthropogenic 250 signature, our results provide, for the first time, an estimate of the flux of anthropogenic P 251 exchanged between countries through agricultural trade. We found that this flux was equivalent to 252 around 8% of the global use of mineral P fertilizers. 253 To conclude, the quantification of the anthropogenic signature of agricultural soil P shed a new light 254 on our understanding of the Anthropocene, which we analysed through the spectrum of the global P 255 cycle. World regions have unequally benefited from the use of mineral P fertilizers, thus calling for 256 different desirable trajectories for the future. Where the signature is high there is a need to move 257 away from the massive use of anthropogenic P, thus helping to save phosphate rock resources for 258 regions that have little benefitted from these resources so far – and that are often facing high 259 undernourishment issues (Langhans et al., 2022). The distribution of the remaining phosphate rocks 260 reserves should thus be coordinated on a global scale in order to maximise world food production. Implications for sustainable phosphate rock management 210 280 Equation 1a 281 𝐿𝑃𝑁𝑎𝑡(𝑦+ 1) = 𝐿𝑃𝑁𝑎𝑡(𝑦) + 𝜃[𝑂𝐹𝑁𝑎𝑡(𝑦) + 𝑆𝐿𝑁𝑎𝑡(𝑦)] −𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑁𝑎𝑡(𝑦) −𝐿𝑂𝐿𝑃,𝑁𝑎𝑡(𝑦) + 𝑇𝑁𝑎𝑡(𝑦) 282 Equation 1b 283 𝐿𝑃𝐴𝑛𝑡(𝑦+ 1) = 𝐿𝑃𝐴𝑛𝑡(𝑦) + 𝐶𝐹(𝑦) + 𝜃[𝑂𝐹𝐴𝑛𝑡(𝑦) + 𝑆𝐿𝐴𝑛𝑡(𝑦)] −𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝐴𝑛𝑡(𝑦) −𝐿𝑂𝐿𝑃,𝐴𝑛𝑡(𝑦) + 𝑇𝐴𝑛𝑡(𝑦) 284 Equation 1c 285 Our model aims to simulate P fluxes within the agricultural system of each country, as well as P flows 265 between countries through the trade of agricultural products (Figure 1). As a result, not only did we 266 simulate soil P stocks and dynamics, but also the fluxes of P embedded in crop and livestock products 267 as well as P fluxes in animal manure and sewage sludge. 268 The P dynamics in agricultural soils (including both cropland and grassland) were simulated by using a 269 two-pool soil P model. Such models have been used in previous large scale studies and proved 270 successful to reproduce observed P harvest over long-term periods (Le Noë et al., 2020; Sattari et al., 271 2012; Zhang et al., 2017). For the specific purpose of our study, we distinguished the natural vs. 272 anthropic origin of each P flow and stock, following the methodology of a preliminary study 273 conducted at the French scale (Ringeval et al., 2014). Soil P pools were expressed in kgP.ha-1, while 274 fluxes were all expressed in kgP.ha-1.yr-1. We assumed that for each country the 0-30 cm soil horizon 275 was relevant to study the soil P dynamics following fertilizer inputs, plant uptake by roots and 276 erosion losses. 277 The P dynamics in agricultural soils (including both cropland and grassland) were simulated by using a 269 two-pool soil P model. Such models have been used in previous large scale studies and proved 270 successful to reproduce observed P harvest over long-term periods (Le Noë et al., 2020; Sattari et al., 271 2012; Zhang et al., 2017). For the specific purpose of our study, we distinguished the natural vs. 272 anthropic origin of each P flow and stock, following the methodology of a preliminary study 273 conducted at the French scale (Ringeval et al., 2014). Soil P pools were expressed in kgP.ha-1, while 274 fluxes were all expressed in kgP.ha-1.yr-1. We assumed that for each country the 0-30 cm soil horizon 275 was relevant to study the soil P dynamics following fertilizer inputs, plant uptake by roots and 276 erosion losses. Implications for sustainable phosphate rock management 210 261 To conclude, the quantification of the anthropogenic signature of agricultural soil P shed a new light 254 on our understanding of the Anthropocene, which we analysed through the spectrum of the global P 255 cycle. World regions have unequally benefited from the use of mineral P fertilizers, thus calling for 256 different desirable trajectories for the future. Where the signature is high there is a need to move 257 away from the massive use of anthropogenic P, thus helping to save phosphate rock resources for 258 regions that have little benefitted from these resources so far – and that are often facing high 259 undernourishment issues (Langhans et al., 2022). The distribution of the remaining phosphate rocks 260 reserves should thus be coordinated on a global scale in order to maximise world food production. 261 10 Methods 262 263 Modelling framework. 264 Our model aims to simulate P fluxes within the agricultural system of each country, as well as P flows 265 between countries through the trade of agricultural products (Figure 1). As a result, not only did we 266 simulate soil P stocks and dynamics, but also the fluxes of P embedded in crop and livestock products 267 as well as P fluxes in animal manure and sewage sludge. 268 The P dynamics in agricultural soils (including both cropland and grassland) were simulated by using a 269 two-pool soil P model. Such models have been used in previous large scale studies and proved 270 successful to reproduce observed P harvest over long-term periods (Le Noë et al., 2020; Sattari et al., 271 2012; Zhang et al., 2017). For the specific purpose of our study, we distinguished the natural vs. 272 anthropic origin of each P flow and stock, following the methodology of a preliminary study 273 conducted at the French scale (Ringeval et al., 2014). Soil P pools were expressed in kgP.ha-1, while 274 fluxes were all expressed in kgP.ha-1.yr-1. We assumed that for each country the 0-30 cm soil horizon 275 was relevant to study the soil P dynamics following fertilizer inputs, plant uptake by roots and 276 erosion losses. 277 For each country, we modelled the size of the soil P pools using the Equations below. We 278 distinguished between labile (LP) and stable (SP) P pools and between anthropic (subscript “Ant”) or 279 natural (subscript “Nat”) origin of P within each pool. Implications for sustainable phosphate rock management 210 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡 refers to the P in the plant biomass that is harvested 293 11 11 from both croplands and grasslands, both as grains for animal feed and human food consumption 294 (either domestically consumed or exported to other countries) and as forage. 295 from both croplands and grasslands, both as grains for animal feed and human food consumption 294 (either domestically consumed or exported to other countries) and as forage. 295 from both croplands and grasslands, both as grains for animal feed and human food consumption 294 (either domestically consumed or exported to other countries) and as forage. 295 PHarvest. For each country, 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑇𝑜𝑡 of crop and forage products was annually simulated based on 296 Equation 2. 297 PHarvest. For each country, 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑇𝑜𝑡 of crop and forage products was annually simulated based on 296 Equation 2. 297 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑇𝑜𝑡(𝑦) = 𝛽(1 −𝑒−𝛾.𝐿𝑃𝑇𝑜𝑡(𝑦)) Where 𝛽 (in kgP.ha-1.yr-1) refers to the maximum attainable yield without any P limitation, and γ (in 300 ha.kgP-1) depicts the ability of crops to extract soil P. 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑇𝑜𝑡 and 𝐿𝑃𝑇𝑜𝑡 refer respectively to P 301 exported from agricultural soils either for animal or human consumption (in kgP.ha-1.yr-1) and to the 302 size of the labile P pool (kgP.ha-1). The subscript “Tot” refers to the sum of both anthropogenic (Ant) 303 and natural (Nat) P. A calibration procedure (see below) was performed to determine the values of 𝛽 304 and 𝛾 for each country. 305 P transfers from the stable to the labile P pool. The net flux of P from the stable to the labile P pool 306 was also explicitly simulated, as described in Equation 3. 307 P transfers from the stable to the labile P pool. The net flux of P from the stable to the labile P pool 306 was also explicitly simulated, as described in Equation 3. 307 Where 𝜇𝑆𝑃𝑡𝑜𝐿𝑃 (in yr-1) represents the percentage of P in the stable P pool that transfers annually to 310 the labile P pool. The inverse of 𝜇𝑆𝑃𝑡𝑜𝐿𝑃 can also be interpreted as a mean residence time of P in the 311 stable P pool. For the labile P pool, a turnover rate of 0.2 yr-1 - corresponding to a mean residence 312 time of 5 years - was used as proposed by the literature (Sattari et al., 2012). Implications for sustainable phosphate rock management 210 277 For each country, we modelled the size of the soil P pools using the Equations below. We 278 distinguished between labile (LP) and stable (SP) P pools and between anthropic (subscript “Ant”) or 279 natural (subscript “Nat”) origin of P within each pool. 280 Equation 1a 281 𝐿𝑃𝑁𝑎𝑡(𝑦+ 1) = 𝐿𝑃𝑁𝑎𝑡(𝑦) + 𝜃[𝑂𝐹𝑁𝑎𝑡(𝑦) + 𝑆𝐿𝑁𝑎𝑡(𝑦)] −𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑁𝑎𝑡(𝑦) −𝐿𝑂𝐿𝑃,𝑁𝑎𝑡(𝑦) + 𝑇𝑁𝑎𝑡(𝑦) 282 Equation 1b 283 𝐿𝑃𝐴𝑛𝑡(𝑦+ 1) = 𝐿𝑃𝐴𝑛𝑡(𝑦) + 𝐶𝐹(𝑦) + 𝜃[𝑂𝐹𝐴𝑛𝑡(𝑦) + 𝑆𝐿𝐴𝑛𝑡(𝑦)] −𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝐴𝑛𝑡(𝑦) −𝐿𝑂𝐿𝑃,𝐴𝑛𝑡(𝑦) + 𝑇𝐴𝑛𝑡(𝑦) 284 Equation 1c 285 𝐿𝑃𝐴𝑛𝑡(𝑦+ 1) = 𝐿𝑃𝐴𝑛𝑡(𝑦) + 𝐶𝐹(𝑦) + 𝜃[𝑂𝐹𝐴𝑛𝑡(𝑦) + 𝑆𝐿𝐴𝑛𝑡(𝑦)] −𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝐴𝑛𝑡(𝑦) −𝐿𝑂𝐿𝑃,𝐴𝑛𝑡(𝑦) + 𝑇𝐴𝑛𝑡(𝑦) 284 Equation 1c 285 𝐿𝑃𝐴𝑛𝑡(𝑦+ 1) = 𝐿𝑃𝐴𝑛𝑡(𝑦) + 𝐶𝐹(𝑦) + 𝜃[𝑂𝐹𝐴𝑛𝑡(𝑦) + 𝑆𝐿𝐴𝑛𝑡(𝑦)] −𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝐴𝑛𝑡(𝑦) −𝐿𝑂𝐿𝑃,𝐴𝑛𝑡(𝑦) + 𝑇𝐴𝑛𝑡(𝑦) 284 Equation 1c 285 𝑆𝑃𝑋(𝑦+ 1) = 𝑆𝑃𝑋(𝑦) + (1 −𝜃)[𝑂𝐹𝑋(𝑦) + 𝑆𝐿𝑋(𝑦)] −𝐿𝑂𝑆𝑃,𝑋(𝑦) −𝑇𝑋(𝑦) 𝑊ℎ𝑒𝑟𝑒 𝑋∈{𝑁𝑎𝑡, 𝐴𝑛𝑡} 286 𝑆𝑃𝑋(𝑦+ 1) = 𝑆𝑃𝑋(𝑦) + (1 −𝜃)[𝑂𝐹𝑋(𝑦) + 𝑆𝐿𝑋(𝑦)] −𝐿𝑂𝑆𝑃,𝑋(𝑦) −𝑇𝑋(𝑦) 𝑊ℎ𝑒𝑟𝑒 𝑋∈{𝑁𝑎𝑡, 𝐴𝑛𝑡} here y refers to the year considered, X stands for the sub-scripts Nat and Ant, and SL, OF, CF an y y , p , , , LO correspond to soil P inputs as sewage sludge, animal manure, mineral P fertilizer and outputs as 288 losses via erosion to water bodies, respectively. Ө refers to the bioavailable fraction of manure and 289 sludge, which was assumed to equal 0.8 based on reported values from the literature (Ringeval et al., 290 2014; Sattari et al., 2012). T represents the net transfer of P from the stable to the labile P pool and 291 captures soil P dynamics such as adsorption/desorption, precipitation/solubilisation and 292 organisation/mineralisation processes. 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡 refers to the P in the plant biomass that is harvested 293 LO correspond to soil P inputs as sewage sludge, animal manure, mineral P fertilizer and outputs as 288 losses via erosion to water bodies, respectively. Ө refers to the bioavailable fraction of manure and 289 sludge, which was assumed to equal 0.8 based on reported values from the literature (Ringeval et al., 290 2014; Sattari et al., 2012). T represents the net transfer of P from the stable to the labile P pool and 291 captures soil P dynamics such as adsorption/desorption, precipitation/solubilisation and 292 organisation/mineralisation processes. Implications for sustainable phosphate rock management 210 Similarly to 𝛽 and 𝛾, 313 𝜇𝑆𝑃𝑡𝑜𝐿𝑃 was also calibrated for each country, thus counterbalancing the 0.2 fixed rate of transfer 314 from labile to stable P pool. 315 Input data. Input data were collected for each individual country. We detail hereafter how data were 316 collected or estimated. Mineral P fertilizer (CF) data were collected from FAOSTAT for the 1961-2017 317 period. Data for the 1950-1960 period were estimated from the spatially explicit dataset of (Lu and 318 Tian, 2016) by aggregating values per country. Because data were only available for the years 1950 319 and 1960, we performed a linear interpolation to estimate the missing years. Manure (OF) data were 320 estimated by considering 10 animal categories (cattle, poultry, asses, mules, goats, sheep, horses, 321 buffaloes, pigs and rabbits) and by multiplying the number of heads (FAOSTAT; Mitchell, 1998a, 322 1998b, 1993) by species-dependent P excretion rates (Sheldrick et al., 2003). All the manure 323 Input data. Input data were collected for each individual country. We detail hereafter how data were 316 collected or estimated. Mineral P fertilizer (CF) data were collected from FAOSTAT for the 1961-2017 317 period. Data for the 1950-1960 period were estimated from the spatially explicit dataset of (Lu and 318 Tian, 2016) by aggregating values per country. Because data were only available for the years 1950 319 and 1960, we performed a linear interpolation to estimate the missing years. Manure (OF) data were 320 estimated by considering 10 animal categories (cattle, poultry, asses, mules, goats, sheep, horses, 321 buffaloes, pigs and rabbits) and by multiplying the number of heads (FAOSTAT; Mitchell, 1998a, 322 1998b, 1993) by species-dependent P excretion rates (Sheldrick et al., 2003). All the manure 323 Input data. Input data were collected for each individual country. We detail hereafter how data were 316 collected or estimated. Mineral P fertilizer (CF) data were collected from FAOSTAT for the 1961-2017 317 period. Data for the 1950-1960 period were estimated from the spatially explicit dataset of (Lu and 318 Tian, 2016) by aggregating values per country. Because data were only available for the years 1950 319 and 1960, we performed a linear interpolation to estimate the missing years. Implications for sustainable phosphate rock management 210 The global use of 333 mineral P feed was then allocated to each country based on both their total number of livestock 334 heads and their consumption of mineral P fertilizer (see Section S2.5). Harvested P data were used to 335 calibrate our model. P harvest from grasslands was assumed to equal livestock forage P demand at 336 the country scale, assuming no trade of forage between countries. Livestock forage P demand was 337 estimated annually by multiplying livestock number of cattle, sheep and goats by their total energy 338 requirement (Barbieri et al., 2021), by the percentage of forage consumption in their diet (Herrero et 339 al., 2013), and by P concentration of forage. P harvest from croplands was estimated by considering 340 31 crop species (Table S5) and by collecting harvested data from FAOSTAT for the 1961-2017 period. 341 Crop yield data for the 1950-1960 period were estimated by assuming that crop-specific production 342 varied linearly with the country human population as in (Bouwman et al., 2011). Finally, crop P 343 harvest was estimated by multiplying crop yields by the species-dependent P concentration, derived 344 from (Comifer, 2009). The trade of agricultural products was considered by including 55 crop 345 products, representing more than 85% of the P traded through food and feed at the global scale 346 (Table S7). From 1986 to 2017, we extracted the data regarding the imported quantities of feed and 347 food products and the countries of origin from the FOATSAT Detailed Trade Matrix. From 1961 to 348 1985, the imported quantities were derived from the Food Balances matrix (FAOSTAT) and the 349 countries of origin were assumed to be roughly the same as those from the 1986-1991 period (see 350 Section S4). For feed, imported quantities and their origin for the 1950-1960 period were estimated 351 based on the information at year 1961 and scaled down based on the livestock density. For food, we 352 assumed that the traded quantities were negligible for the 1950-1960 period. Note that the 353 quantities and origin of imported food and feed products were only used to compute the 354 anthropogenic signatures of manure and sludge, respectively. 355 Model initialisation. To initiate the model, we had to determine the size of each soil P pool in 1950. 356 Model initialisation. Implications for sustainable phosphate rock management 210 Manure (OF) data were 320 estimated by considering 10 animal categories (cattle, poultry, asses, mules, goats, sheep, horses, 321 buffaloes, pigs and rabbits) and by multiplying the number of heads (FAOSTAT; Mitchell, 1998a, 322 1998b, 1993) by species-dependent P excretion rates (Sheldrick et al., 2003). All the manure 323 12 produced was assumed to reach agricultural soils, either on croplands or on grasslands. Losses (LO) of 324 P from soils were calculated by multiplying country-specific percentages of soil lost by erosion (Van 325 Oost et al., 2007) by the here-simulated P pool sizes, by assuming that the rate of losses affected 326 equally stable and labile soil P pools. Sludge P (SL) production was estimated by focussing on sewage 327 sludge coming from human food consumption only (thereby excluding P release from detergents). 328 We then estimated the fraction of P in sludge that would reach agricultural soils by multiplying each 329 national human P excretions by the fraction of sewage sludge that is treated and by the P removal 330 efficiency of treatments plants. The methodology and data used were derived from (Van Puijenbroek 331 et al., 2019). We assumed that Mineral P feed (MF) represented 5% of the global production of 332 phosphate rock (from FAOSTAT) annually (Cordell and White, 2014; Smil, 2000). The global use of 333 mineral P feed was then allocated to each country based on both their total number of livestock 334 heads and their consumption of mineral P fertilizer (see Section S2.5). Harvested P data were used to 335 calibrate our model. P harvest from grasslands was assumed to equal livestock forage P demand at 336 the country scale, assuming no trade of forage between countries. Livestock forage P demand was 337 estimated annually by multiplying livestock number of cattle, sheep and goats by their total energy 338 requirement (Barbieri et al., 2021), by the percentage of forage consumption in their diet (Herrero et 339 al., 2013), and by P concentration of forage. P harvest from croplands was estimated by considering 340 31 crop species (Table S5) and by collecting harvested data from FAOSTAT for the 1961-2017 period. 341 Crop yield data for the 1950-1960 period were estimated by assuming that crop-specific production 342 varied linearly with the country human population as in (Bouwman et al., 2011). Implications for sustainable phosphate rock management 210 Finally, crop P 343 harvest was estimated by multiplying crop yields by the species-dependent P concentration, derived 344 from (Comifer, 2009). The trade of agricultural products was considered by including 55 crop 345 products, representing more than 85% of the P traded through food and feed at the global scale 346 (Table S7). From 1986 to 2017, we extracted the data regarding the imported quantities of feed and 347 food products and the countries of origin from the FOATSAT Detailed Trade Matrix. From 1961 to 348 1985, the imported quantities were derived from the Food Balances matrix (FAOSTAT) and the 349 countries of origin were assumed to be roughly the same as those from the 1986-1991 period (see 350 Section S4). For feed, imported quantities and their origin for the 1950-1960 period were estimated 351 based on the information at year 1961 and scaled down based on the livestock density. For food, we 352 assumed that the traded quantities were negligible for the 1950-1960 period. Note that the 353 quantities and origin of imported food and feed products were only used to compute the 354 anthropogenic signatures of manure and sludge, respectively. 355 Model initialisation. To initiate the model, we had to determine the size of each soil P pool in 1950. 356 produced was assumed to reach agricultural soils, either on croplands or on grasslands. Losses (LO) of 324 P from soils were calculated by multiplying country-specific percentages of soil lost by erosion (Van 325 Oost et al., 2007) by the here-simulated P pool sizes, by assuming that the rate of losses affected 326 equally stable and labile soil P pools. Sludge P (SL) production was estimated by focussing on sewage 327 sludge coming from human food consumption only (thereby excluding P release from detergents). 328 We then estimated the fraction of P in sludge that would reach agricultural soils by multiplying each 329 national human P excretions by the fraction of sewage sludge that is treated and by the P removal 330 efficiency of treatments plants. The methodology and data used were derived from (Van Puijenbroek 331 et al., 2019). We assumed that Mineral P feed (MF) represented 5% of the global production of 332 phosphate rock (from FAOSTAT) annually (Cordell and White, 2014; Smil, 2000). Implications for sustainable phosphate rock management 210 To initiate the model, we had to determine the size of each soil P pool in 1950. 356 We hypothesised that before 1950 the application of mineral P fertilizer was negligible compared to 357 13 13 the use of other fertilizers, mainly animal manure. As a result, we considered that LPAnt (1949)= SPAnt 358 (1949)=0. In 1950, the LPAnt value was set equal to the inputs of mineral P fertilizer that year. The 359 SPAnt was null in 1950 because, by definition all mineral P fertilizers are applied to the labile P pool. 360 The size of the LPTot in 1950 was calculated based on the P harvest that year, using Equation 2, 361 possibly leading to significant effects of P harvest in 1950 on the initial soil P pool sizes. The size of 362 LPNat was then calculated as LPTot minus LPAnt. Finally, based on equilibrium between the labile and 363 stable P pools we determined SPNat (Equation 3 with T=0). 364 the use of other fertilizers, mainly animal manure. As a result, we considered that LPAnt (1949)= SPAnt 358 (1949)=0. In 1950, the LPAnt value was set equal to the inputs of mineral P fertilizer that year. The 359 SPAnt was null in 1950 because, by definition all mineral P fertilizers are applied to the labile P pool. 360 The size of the LPTot in 1950 was calculated based on the P harvest that year, using Equation 2, 361 possibly leading to significant effects of P harvest in 1950 on the initial soil P pool sizes. The size of 362 LPNat was then calculated as LPTot minus LPAnt. Finally, based on equilibrium between the labile and 363 stable P pools we determined SPNat (Equation 3 with T=0). 364 Calibration procedure. For each country, an independent calibration procedure was performed to 365 estimate the values of 𝛽, 𝛾 and 𝜇𝑆𝑃𝑡𝑜𝐿𝑃 parameters. The general idea was to test many triplets of 366 parameters, to select the ones that allow the simulations to match P harvest time-series available 367 data (see Section S2.2) and labile P pool size available data in 2005 (see Section S2.7). The adequacy 368 between the simulation outputs and the available data were quantified through calibration scores 369 (Equations 4 and 5). Depending on their calibration scores, some triplets were selected to run the 370 calculations and to compute an uncertainty related to the anthropogenic signatures. Implications for sustainable phosphate rock management 210 Values of 371 calibrated parameters are presented and discussed in Section S6.2. 372 Calibration started first by choosing the range of values to be tested for each parameter (see Section 373 S6.1). Note that for 𝛽, the ranges were country-dependent. Then, from all possible values we 374 produced triplets (𝛽𝑖, 𝛾𝑖, 𝜇𝑖) for which we ran the model and computed score 1 and score 2, as 375 described in Equations (4,5). From 1500 to 8000 triplets were tested for each country. 376 Calibration started first by choosing the range of values to be tested for each parameter (see Section 373 S6.1). Note that for 𝛽, the ranges were country-dependent. Then, from all possible values we 374 produced triplets (𝛽𝑖, 𝛾𝑖, 𝜇𝑖) for which we ran the model and computed score 1 and score 2, as 375 described in Equations (4,5). From 1500 to 8000 triplets were tested for each country. 376 Equation 4 – Score 1 377 𝑠𝑐𝑜𝑟𝑒1 = 1 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠 √1 𝑁 ∑(𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠(𝑖) −𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑠𝑖𝑚(𝑖))2 1950+𝑁 𝑖=1950 378 Equation 5 – Score 2 379 𝑠𝑐𝑜𝑟𝑒2 = \|𝐿𝑃𝑜𝑏𝑠(2005) −𝐿𝑃𝑠𝑖𝑚(2005) 𝐿𝑃𝑜𝑏𝑠(2005) \| 380 Equation 4 – Score 1 377 𝑠𝑐𝑜𝑟𝑒1 = 1 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠 √1 𝑁 ∑(𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠(𝑖) −𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑠𝑖𝑚(𝑖))2 1950+𝑁 𝑖=1950 378 Equation 5 – Score 2 379 𝑠𝑐𝑜𝑟𝑒2 = \|𝐿𝑃𝑜𝑏𝑠(2005) −𝐿𝑃𝑠𝑖𝑚(2005) 𝐿𝑃𝑜𝑏𝑠(2005) \| 380 Where N refers to the number of years studied (N=67), 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠 to the mean value of P harvest 381 available data for the time-period studied, 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠 and 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑠𝑖𝑚 to the P harvest time-series 382 available and simulated values, 𝐿𝑃𝑜𝑏𝑠 and 𝐿𝑃𝑠𝑖𝑚 to labile P pool size available and simulated value at 383 year 2005. 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠 was derived from international database (see Equation S3) and 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑠𝑖𝑚 384 was calculated with Equation 2. 385 Where N refers to the number of years studied (N=67), 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠 to the mean value of P harvest 381 available data for the time-period studied, 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠 and 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑠𝑖𝑚 to the P harvest time-series 382 available and simulated values, 𝐿𝑃𝑜𝑏𝑠 and 𝐿𝑃𝑠𝑖𝑚 to labile P pool size available and simulated value at 383 year 2005. 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑜𝑏𝑠 was derived from international database (see Equation S3) and 𝑃ℎ𝑎𝑟𝑣𝑒𝑠𝑡,𝑠𝑖𝑚 384 was calculated with Equation 2. 385 14 Score 1 calculates the normalized root mean square error (nRMSE) between the simulated total P 386 harvest and the P harvest available data over the 1950-2017 period. Implications for sustainable phosphate rock management 210 Score 2 computes the error 387 between the total labile P pool simulated at year 2005 and the total labile P pool available data 388 extracted either from (Ringeval et al., 2017) or from observations representative to country-scale 389 values for the few countries where observed soil P values were available (see Section S6.3). In the 390 main text, only results from the calibration performed with labile P pool available data from Ringeval 391 et al. 2017 were presented. 392 Based on these scores, triplets were selected for each country. When possible, all triplets that led to 393 score 1+ score 2 < 30% were selected and the corresponding countries were classified in category 1 394 (62% of countries). This category thus encompassed countries for which the model managed to 395 reproduce available data on both historical P harvest and the size of the labile P pool in 2005. For 396 some countries, no triplets were able to match the above condition. In this case and when possible 397 we selected the triplets for which score 1 < 30%. The corresponding countries were classified in 398 category 2 (29% of countries). For such countries, the parameters were thus only constrained by P 399 harvest. Finally, when no triplets led to score 1 < 30%, the corresponding countries were classified in 400 category 3 (7% of countries) and not further studied. 401 Based on these scores, triplets were selected for each country. When possible, all triplets that led to 393 score 1+ score 2 < 30% were selected and the corresponding countries were classified in category 1 394 (62% of countries). This category thus encompassed countries for which the model managed to 395 reproduce available data on both historical P harvest and the size of the labile P pool in 2005. For 396 some countries, no triplets were able to match the above condition. In this case and when possible 397 we selected the triplets for which score 1 < 30%. The corresponding countries were classified in 398 category 2 (29% of countries). For such countries, the parameters were thus only constrained by P 399 harvest. Finally, when no triplets led to score 1 < 30%, the corresponding countries were classified in 400 category 3 (7% of countries) and not further studied. Implications for sustainable phosphate rock management 210 Data on the trade of 442 agricultural products were used to determine the anthropogenic signature of animal manure and 443 sewage sludge, when animals and humans consumed imported products (see Section S3 and S4). For 444 several reasons, we underestimated the global P flows that occurred through the trade of 445 agricultural products. Indeed, we estimated that around 1MtP.yr-1 embedded in food and feed 446 products were traded at the global scale, while other studies reported values around 3MtP.yr-1 447 (Nesme et al., 2018). Our underestimation of these flows comes from (i) the fact that we excluded 448 several countries from our analysis (see above about the calibration procedure) and (ii) some 449 revisions of data from the Detailed Trade Matrix – because those data did not always match with 450 those from the Food Balances, we revised them downwards, notably about trade in soybean cakes. 451 However, the impact of these errors on the anthropogenic signatures of the different countries are 452 Regarding input data to the model, the uncertainties were also potentially large, although difficult to 438 quantify. In particular, we recognise large uncertainties in our estimates of P harvest from grasslands, 439 especially in some countries such as India (Fetzel et al., 2017). We explicitly chose to base our 440 estimates of P harvest from grasslands on forage demand and not on forage production given the 441 large uncertainty in Net Primary Productivity of grassland (Sun et al., 2021). Data on the trade of 442 agricultural products were used to determine the anthropogenic signature of animal manure and 443 sewage sludge, when animals and humans consumed imported products (see Section S3 and S4). For 444 several reasons, we underestimated the global P flows that occurred through the trade of 445 agricultural products. Indeed, we estimated that around 1MtP.yr-1 embedded in food and feed 446 products were traded at the global scale, while other studies reported values around 3MtP.yr-1 447 (Nesme et al., 2018). Our underestimation of these flows comes from (i) the fact that we excluded 448 several countries from our analysis (see above about the calibration procedure) and (ii) some 449 revisions of data from the Detailed Trade Matrix – because those data did not always match with 450 those from the Food Balances, we revised them downwards, notably about trade in soybean cakes. Implications for sustainable phosphate rock management 210 The uncertainties associated with our estimate of the anthropogenic 419 signatures of the soil available P are numerous and come from both the model itself (structure and 420 parameter values) and the input data used. Like all models, the one we used is imperfect and cannot 421 represent the dynamics and mechanisms at play in a fully accurate way. However, because of the 422 constraints inherent to global and time-dependant modelling, our model appeared as the most 423 adequate. We yet recognize limitations in using an only two-pool soil P model. Indeed, this model 424 cannot represent the full diversity of P forms and mechanisms at play in soils. More complex 425 mechanistic models exist but they remain hard to parametrize and are still under development 426 (Wang et al., 2022). Our yield response curve to soil P availability is also very simplistic as it considers 427 changes in yields only as a function of changes in soil available P while other factors such as water 428 supply or nitrogen availability should have been considered. Nevertheless, this type of P response 429 curves has been widely used throughout the literature and has repeatedly demonstrated its ability to 430 simulate the temporal evolutions of P harvest over time (Sattari et al., 2012; Zhang et al., 2017). In 431 addition, a special feature of our work has been to calibrate 𝛽, 𝛾 and 𝜇𝑆𝑃𝑡𝑜𝐿𝑃 for each country (see 432 Section S6.1), thereby helping to capture country-specific conditions. The calibration of β (referring 433 to the maximum attainable yield without any P limitation) allowed us to take into account other 434 limiting factors that would be country specific. The calibration of 𝜇𝑆𝑃𝑡𝑜𝐿𝑃 made it possible to take 435 into account the soil-type specificities (even if a calibration at the country level is questionable 436 because of the diversity of soil types within each country). 437 Regarding input data to the model, the uncertainties were also potentially large, although difficult to 438 quantify. In particular, we recognise large uncertainties in our estimates of P harvest from grasslands, 439 especially in some countries such as India (Fetzel et al., 2017). We explicitly chose to base our 440 estimates of P harvest from grasslands on forage demand and not on forage production given the 441 large uncertainty in Net Primary Productivity of grassland (Sun et al., 2021). Implications for sustainable phosphate rock management 210 401 In an effort to calibrate the model with observed data on soil available P pool instead of (simulated) 402 available values from (Ringeval et al., 2017), we collected data on soil available P measurements 403 (whatever the chemical extraction used) conducted on agricultural soils (Section S2.7). We found 404 enough data for 24 countries including European countries (Jones et al., 2020), the USA and Canada 405 (IPNI, 2015), Tanzania (Hengl et al., 2017), Botswana and Yemen (Batjes, 2010). For these countries, 406 we found that using the measured data to calibrate the model instead of the simulated ones did not 407 significantly change the anthropogenic signatures of the soil P pools (see Section S6.3). 408 In an effort to calibrate the model with observed data on soil available P pool instead of (simulated) 402 available values from (Ringeval et al., 2017), we collected data on soil available P measurements 403 (whatever the chemical extraction used) conducted on agricultural soils (Section S2.7). We found 404 enough data for 24 countries including European countries (Jones et al., 2020), the USA and Canada 405 (IPNI, 2015), Tanzania (Hengl et al., 2017), Botswana and Yemen (Batjes, 2010). For these countries, 406 we found that using the measured data to calibrate the model instead of the simulated ones did not 407 significantly change the anthropogenic signatures of the soil P pools (see Section S6.3). 408 Data analysis. The outputs of our model were the anthropogenic signatures of the fluxes and soil P 409 pools, as well as the size of the soil P pools. For each output, we computed a mean and a standard 410 deviation value, reflecting uncertainties in the parametrization of the three calibrated parameters. 411 The global and regional averages of the soil P anthropogenic signatures are mean values weighted by 412 the agricultural area of each country. We also chose to present detailed results for eight contrasted 413 countries (France, the Netherlands, the USA, Brazil, India, China, Zimbabwe and Morocco), to better 414 appreciate the temporal evolutions in soil P inputs and in soil P anthropogenic signatures. Following 415 the calibration procedure, these eight countries were all in category 1, which was a guarantee of 416 good quality results. We only displayed results for the anthropogenic signature of the labile P pool, 417 since that of the stable P pool were very similar. 418 15 Uncertainties and limitations. Implications for sustainable phosphate rock management 210 451 However, the impact of these errors on the anthropogenic signatures of the different countries are 452 16 16 likely to be small, given that the anthropogenic signatures are more sensitive to the signature of 453 imported products rather than on their quantities. Finally, we hypothesized that all the animal 454 manure produced was available for agricultural soil application, which is highly questionable. Other 455 studies have used global average but those were too uncertain to be included in our estimate (Chen 456 and Graedel, 2016). However, because manure application on agricultural soils is basically P recycling 457 that conserves anthropogenic signature, the possible overestimation of manure application in our 458 approach is likely to have small effects on the estimated anthropogenic soil P signatures. 459 For some countries, we recognise that we did not find any triplet of parameters that allowed the 460 model to replicate the historical available data on soil P harvest. As a result, these countries 461 (category 3) were excluded from our analyses. Conversely, the results we obtained for countries in 462 category 1 were the most reliable. The countries in category 2 were not able to match the constraints 463 on both P harvest and labile P pool size in 2005. For these countries, mainly located in Africa, we 464 recognise strong uncertainties in our estimates of the size of soil P pools and thus in the estimated 465 anthropogenic signatures. One of the reasons for the difficulties to match the two constraints comes 466 from the methodology we used to estimate the size of the soil labile P pool in 1950. In our study, the 467 latter was estimated from data on P harvest in 1950, while initial size of labile P pool in (Ringeval et 468 al., 2017) – used to constraint the size of the labile P pools in our model- were derived from a 469 dataset about soil biogeochemical background (Yang et al. 2013). However, although our 470 uncertainties on the size of the stable and labile soil P pools are large (see Section S9), our final 471 uncertainties on anthropogenic signatures remain reasonable (Figure S14). The difficulties in 472 providing accurate estimates of soil P content at large spatial scales is often raised in the literature 473 (Das et al. Implications for sustainable phosphate rock management 210 2019), even though more and more studies are tackling this issue (He et al., 2021; Hengl et 474 al., 2021; Ringeval et al., 2017). 475 Another limitation comes from neglecting mineral P fertilizer use before 1950. Although mineral P 476 fertilizer use was small for most countries in 1950 (Bouwman et al., 2011), several European 477 countries had already high fertilisation rates at that period. We nevertheless chose to start the 478 simulations in 1950 because of the critical lack of accurate, global data before that date. A sensitivity 479 test performed in Ringeval et al. (2014) – on which our methodology is based – has shown that our 480 estimates are likely to remain robust. Running the model for 30 years with soil inputs and outputs 481 related to the 1st year (~1950) before the transient run showed only a ~10% underestimation of the 482 anthropogenic signature of agricultural soils. 483 Another limitation comes from neglecting mineral P fertilizer use before 1950. Although mineral P 476 fertilizer use was small for most countries in 1950 (Bouwman et al., 2011), several European 477 countries had already high fertilisation rates at that period. We nevertheless chose to start the 478 simulations in 1950 because of the critical lack of accurate, global data before that date. A sensitivity 479 test performed in Ringeval et al. (2014) – on which our methodology is based – has shown that our 480 estimates are likely to remain robust. Running the model for 30 years with soil inputs and outputs 481 related to the 1st year (~1950) before the transient run showed only a ~10% underestimation of the 482 anthropogenic signature of agricultural soils. 483 Finally, our definition of anthropogenic P – which referred to the use of mineral P fertilizers and 484 mineral P feed derived from the extraction of phosphate rocks – is questionable. Indeed, other 485 Finally, our definition of anthropogenic P – which referred to the use of mineral P fertilizers and 484 mineral P feed derived from the extraction of phosphate rocks – is questionable. Indeed, other 485 17 anthropogenic practices such as animal manure application, deforestation and P fertility transfers 486 from grasslands to croplands have also modified the global P cycle. Implications for sustainable phosphate rock management 210 However, in a context of 487 increasing scarcity of phosphate rock reserves, we found it more relevant to quantify the 488 modification of the P cycle related solely to the use of this critical and highly processed resource. 489 Code and data availability 490 Data are available in the Supplementary Information file. The python code used for computing all 491 calculations is available at https://data.inrae.fr/privateurl.xhtml?token=4ddb8501-c41d-4ad6-8a09- 492 5c1ebb8289f9. 493 References 494 Barbieri, P., MacDonald, G.K., Bernard de Raymond, A., Nesme, T., 2022. Food system resilience to 495 phosphorus shortages on a telecoupled planet. Nat. Sustain. 5, 114–122. 496 https://doi.org/10.1038/s41893-021-00816-1 497 Barbieri, P., Pellerin, S., Seufert, V., Smith, L., Ramankutty, N., Nesme, T., 2021. The global option 498 space for organic agriculture under nitrogen limitations. Nat. Food 2, 363–372. 499 https://doi.org/https://dx.doi.org/10.1038/s43016-021-00276-y 500 Batjes, N.H., 2010. Inventory of P-Olsen data in the ISRIC-WISE soil database for use with QUEFTS: 501 (version 1.0)., Report - ISRIC World Soil Information. 502 Bouwman, L., Goldewijk, K.K., Van Der Hoek, K.W., Beusen, A.H.W., Van Vuuren, D.P., Willems, J., 503 Rufino, M.C., Stehfest, E., 2011. Exploring global changes in nitrogen and phosphorus cycles in 504 agriculture induced by livestock production over the 1900-2050 period. Proc. Natl. Acad. Sci. 505 110, 20882–20887. https://doi.org/10.1073/pnas.1206191109 506 Chen, M., Graedel, T.E., 2016. A half-century of global phosphorus flows, stocks, production, 507 consumption, recycling, and environmental impacts. Glob. Environ. Chang. 36, 139–152. 508 https://doi.org/10.1016/j.gloenvcha.2015.12.005 509 Comifer, 2009. Teneur en P, K et Mg des organes végétaux récoltés. COMIFER. 510 Coomes, O.T., Barham, B.L., MacDonald, G.K., Ramankutty, N., Chavas, J.-P., 2019. Leveraging total 511 factor productivity growth for sustainable and resilient farming. Nat. Sustain. 2, 22–28. 512 https://doi.org/https://doi.org/10.1038/s41893-018-0200-3 513 Cordell, D., Drangert, J.O., White, S., 2009. The story of phosphorus: Global food security and food fo 514 thought. Glob. Environ. Chang. 19, 292–305. https://doi.org/10.1016/j.gloenvcha.2008.10.009 515 Cordell, D., White, S., 2014. Life’s bottleneck: Sustaining the world’s phosphorus for a food secure 516 future. Annu. Rev. Environ. Resour. 39, 161–188. https://doi.org/10.1146/annurev-environ- 517 010213 113300 518 anthropogenic practices such as animal manure application, deforestation and P fertility transfers 486 from grasslands to croplands have also modified the global P cycle. However, in a context of 487 increasing scarcity of phosphate rock reserves, we found it more relevant to quantify the 488 modification of the P cycle related solely to the use of this critical and highly processed resource. Implications for sustainable phosphate rock management 210 Quantification of uncertainties in global 523 grazing systems assessment. Global Biogeochem. Cycles 31, 1089–1102. 524 https://doi.org/10.1002/2016GB005601 525 Van Bodegom, P.M., Wirsenius, S., Erb, K.H., 2017. Quantification of uncertainties in global grazing systems assessment. Global Biogeochem. Cycles 31, 1089–1102. https://doi.org/10.1002/2016GB005601 Grigg, D., 1987. The Industrial Revolution and land transformation., Land Transformation in 526 Agriculture. John Wiley & Sons Ltd, New York. 527 He, X., Augusto, L., Goll, D., Ringeval, B., Wang, Y., Helfenstein, J., Huang, Y., Yu, K., Wang, Z., Yang, Y., 528 Hou, E., 2021. Global patterns and drivers of soil total phosphorus concentration. Earth Syst. Sci. 529 Data Discuss. 14583375, 1–21. https://doi.org/10.5194/essd-2021-166 530 Hengl, T., Leenaars, J.G.B., Shepherd, K.D., Walsh, M.G., Heuvelink, G.B.M., Mamo, T., Tilahun, H., 531 Berkhout, E., Cooper, M., Fegraus, E., Wheeler, I., Kwabena, N.A., 2017. Soil nutrient maps of 532 Sub-Saharan Africa: assessment of soil nutrient content at 250 m spatial resolution using 533 machine learning. Nutr. Cycl. Agroecosystems 109, 77–102. https://doi.org/10.1007/s10705- 534 017-9870-x 535 Hengl, T., Miller, M.A.E., Križan, J., Shepherd, K.D., Sila, A., Kilibarda, M., Antonijević, O., Glušica, L., 536 Dobermann, A., Haefele, S.M., McGrath, S.P., Acquah, G.E., Collinson, J., Parente, L., 537 Sheykhmousa, M., Saito, K., Johnson, J.-M., Chamberlin, J., Silatsa, F.B.T., Yemefack, M., Wendt, 538 J., MacMillan, R.A., Wheeler, I., Crouch, J., 2021. African soil properties and nutrients mapped at 539 30 m spatial resolution using two-scale ensemble machine learning. Sci. Rep. 11, 1–18. 540 https://doi.org/10.1038/s41598-021-85639-y 541 Herrero, M., Havlík, P., Valin, H., Notenbaert, A., Rufino, M.C., Thornton, P.K., Blümmel, M., Weiss, F., 542 Grace, D., Obersteiner, M., 2013. Biomass use, production, feed efficiencies, and greenhouse 543 gas emissions from global livestock systems. Proc. Natl. Acad. Sci. U. S. A. 110, 20888–20893. 544 https://doi.org/10.1073/pnas.1308149110 545 IPNI, 2015. Soil Test Levels In North America. Summary Update: International Plant Nutrition Institute 546 (IPNI). 547 Jones, A., Fernandez-Ugalde, O., Scarpa, S., 2020. LUCAS 2015 Topsoil Survey. Presentation of dataset 548 and results. Luxembourg. https://doi.org/10.2760/616084 549 Kvakíc, M., Pellerin, S., Ciais, P., Achat, D.., Augusto, L., Denoroy, P., Gerber, J.., Goll, D., Mollier, A., 550 Mueller, N.., Wang, X., Ringeval, B., 2018. Quantifying the Limitation to World Cereal 551 Production Due To Soil Phosphorus Status. Global Biogeochem. Cycles 32, 143–157. 552 https://doi.org/10.1002/2017GB005754 553 Langhans, C., Beusen, A.H.W., Mogollón, J.M., Bouwman, A.F., 2022. Phosphorus for Sustainable 554 Development Goal target of doubling smallholder productivity. Nat. Sustain. 5, 57–63. Implications for sustainable phosphate rock management 210 489 anthropogenic practices such as animal manure application, deforestation and P fertility transfers 486 from grasslands to croplands have also modified the global P cycle. However, in a context of 487 increasing scarcity of phosphate rock reserves, we found it more relevant to quantify the 488 modification of the P cycle related solely to the use of this critical and highly processed resource. 489 Barbieri, P., Pellerin, S., Seufert, V., Smith, L., Ramankutty, N., Nesme, T., 2021. The global option 498 space for organic agriculture under nitrogen limitations. Nat. Food 2, 363–372. 499 https://doi.org/https://dx.doi.org/10.1038/s43016-021-00276-y 500 Batjes, N.H., 2010. Inventory of P-Olsen data in the ISRIC-WISE soil database for use with QUEFTS: 501 (version 1.0)., Report - ISRIC World Soil Information. 502 Bouwman, L., Goldewijk, K.K., Van Der Hoek, K.W., Beusen, A.H.W., Van Vuuren, D.P., Willems, J., 503 Rufino, M.C., Stehfest, E., 2011. Exploring global changes in nitrogen and phosphorus cycles in 504 agriculture induced by livestock production over the 1900-2050 period. Proc. Natl. Acad. Sci. 505 110, 20882–20887. https://doi.org/10.1073/pnas.1206191109 506 Chen, M., Graedel, T.E., 2016. A half-century of global phosphorus flows, stocks, production, 507 consumption, recycling, and environmental impacts. Glob. Environ. Chang. 36, 139–152. 508 https://doi.org/10.1016/j.gloenvcha.2015.12.005 509 Comifer, 2009. Teneur en P, K et Mg des organes végétaux récoltés. COMIFER. 510 Coomes, O.T., Barham, B.L., MacDonald, G.K., Ramankutty, N., Chavas, J.-P., 2019. Leveraging total 511 factor productivity growth for sustainable and resilient farming. Nat. Sustain. 2, 22–28. 512 https://doi.org/https://doi.org/10.1038/s41893-018-0200-3 513 Cordell, D., Drangert, J.O., White, S., 2009. The story of phosphorus: Global food security and food for 514 thought. Glob. Environ. Chang. 19, 292–305. https://doi.org/10.1016/j.gloenvcha.2008.10.009 515 Cordell, D., White, S., 2014. Life’s bottleneck: Sustaining the world’s phosphorus for a food secure 516 future. Annu. Rev. Environ. Resour. 39, 161–188. https://doi.org/10.1146/annurev-environ- 517 010213-113300 518 Elser, J., Bennett, E., 2011. A broken biogeochemical cycle. Nature 478, 29–31. 519 https://doi.org/https://doi.org/10.1038/478029a 520 FAO, 2022. FAOSTAT, Statistics Database. https://doi.org/https://www.fao.org/faostat/en/#data 521 Fetzel, T., Havlik, P., Herrero, M., Kaplan, J.O., Kastner, T., Kroisleitner, C., Rolinski, S., Searchinger, T., 522 FAO, 2022. FAOSTAT, Statistics Database. https://doi.org/https://www.fao.org/faostat/ 521 FAO, 2022. FAOSTAT, Statistics Database. https://doi.org/https://www.fao.org/faostat/en/#data 521 F t l T H lik P H M K l J O K t T K i l it C R li ki S S hi T 522 Fetzel, T., Havlik, P., Herrero, M., Kaplan, J.O., Kastner, T., Kroisleitner, C., Rolinski, S., Se 522 18 Van Bodegom, P.M., Wirsenius, S., Erb, K.H., 2017. Implications for sustainable phosphate rock management 210 555 https://doi.org/10.1038/s41893-021-00794-4 556 Le Noë, J., Roux, N., Gingrich, S., Erb, K.-H., Krausmann, F., Thieu, V., Silvestre, M., Garnier, J., 2020. 557 The phosphorus legacy offers opportunities for agro-ecological transition (France 1850–2075). 558 Environ. Res. Lett. 15. https://doi.org/10.1088/1748-9326/ab82cc 559 Lu, C., Tian, H., 2016. Global nitrogen and phosphorus fertilizer use for agriculture production in the 560 past half century: shifted hot spots and nutrient imbalance. Earth Syst. Sci. Data 9, 181–192. 561 https://doi.org/https://doi.org/10.5194/essd-9-181-2017 562 Lun, F., Sardans, J., Sun, D., Xiao, X., Liu, M., Li, Z., Wang, C., Hu, Q., Tang, J., Ciais, P., Janssens, I.A., 563 Obersteiner, M., Peñuelas, J., 2021. Influences of international agricultural trade on the global 564 phosphorus cycle and its associated issues. Glob. Environ. Chang. 69. 565 https://doi.org/10.1016/j.gloenvcha.2021.102282 566 19 MacDonald, G.K., Bennett, E.M., Potter, P.A., Ramankutty, N., 2011. Agronomic phosphorus 567 imbalances across the world’s croplands. Proc. Natl. Acad. Sci. U. S. A. 108, 3086–3091. 568 https://doi.org/10.1073/pnas.1010808108 569 Mitchell, B.R., 1998a. International Historical Statistics: Europe 1750 -1993. 570 Mitchell, B.R., 1998b. International Historical Statistics-Africa, Asia and Oceania 1750-1993. 571 Mitchell, B.R., 1993. International Historical Statistics - The Americas. 572 Mueller, N.D., Gerber, J.S., Johnston, M., Ray, D.K., Ramankutty, N., Foley, J.A., 2012. Closing yield 573 gaps through nutrient and water management. Nature 490, 254–257. 574 https://doi.org/10.1038/nature11420 575 Nanda, M., Cordell, D., Kansal, A., 2019. Assessing national vulnerability to phosphorus scarcity to 576 build food system resilience : The case of India ☆. J. Environ. Manage. 240, 511–517. 577 https://doi.org/10.1016/j.jenvman.2019.03.115 578 Nesme, T., Metson, G.S., Bennett, E.M., 2018. Global phosphorus flows through agricultural trade. 579 Glob. Environ. Chang. 50, 133–141. https://doi.org/10.1016/j.gloenvcha.2018.04.004 580 Pomeranz, K., 2000. The Great Divergence – China, Europe, and the Making of the Modern World 581 Economy. Princeton University Press. 582 Reijnders, L., 2014. Phosphorus resources, their depletion and conservation, a review. Resour. 583 Conserv. Recycl. 93, 32–49. https://doi.org/https://doi.org/10.1016/j.resconrec.2014.09.006 584 Ringeval, B., Augusto, L., Monod, H., Van Apeldoorn, D., Bouwman, L., Yang, X., Achat, D.L., Chini, 585 L.P., Van Oost, K., Guenet, B., Wang, R., Decharme, B., Nesme, T., Pellerin, S., 2017. Phosphorus 586 in agricultural soils: drivers of its distribution at the global scale. Glob. Chang. Biol. 23, 3418– 587 3432. https://doi.org/10.1111/gcb.13618 588 Ringeval, B., Nowak, B., Nesme, T., Delmas, M., Pellerin, S., 2014. Contribution of anthropogenic 589 phosphorus to agricultural soil fertility and food production. Global Biogeochem. Cycles 28, 590 743–756. Implications for sustainable phosphate rock management 210 https://doi.org/10.1002/ 2014GB004842 591 Sattari, S.Z., Bouwman, A.F., Giller, K.E., Van Ittersum, M.K., 2012. Residual soil phosphorus as the 592 missing piece in the global phosphorus crisis puzzle. Proc. Natl. Acad. Sci. U. S. A. 109, 6348– 593 6353. https://doi.org/10.1073/pnas.1113675109 594 Sattari, S.Z., Bouwman, A.F., Martinez Rodríguez, R., Beusen, A.H.W., Van Ittersum, M.K., 2016. 595 Negative global phosphorus budgets challenge sustainable intensification of grasslands. Nat. 596 Commun. 7. https://doi.org/10.1038/ncomms10696 597 Scholz, R.W., Wellmer, F.-H., 2018. Although there is no Physical Short-Term Scarcity of Phosphorus , 598 its Resource Efficiency Should be Improved. J. Ind. Ecol. 23, 313–318. 599 https://doi.org/10.1111/jiec.12750 600 Sebilo, M., Mayer, B., Nicolardot, B., Pinay, G., Mariotti, A., 2013. Long-term fate of nitrate fertilizer 601 in agricultural soils. Proc. Natl. Acad. Sci. U. S. A. 110, 18185–18189. 602 https://doi.org/10.1073/pnas.1305372110 603 Sebilo, M., Mayer, B., Nicolardot, B., Pinay, G., Mariotti, A., 2013. Long term fate of nitrate fertilizer 601 in agricultural soils. Proc. Natl. Acad. Sci. U. S. A. 110, 18185–18189. 602 https://doi.org/10.1073/pnas.1305372110 603 Sheldrick, W., Keith Syers, J., Lingard, J., 2003. Contribution of livestock excreta to nutrient balances. 604 Nutr. Cycl. Agroecosystems 66, 119–131. https://doi.org/10.1023/A:1023944131188 605 Smil, V., 2000. Phosphorus in the Environment: Natural Flows and Human Interferences. Annu. Rev. 606 Energy Environ. 25, 53–88. https://doi.org/https://doi.org/10.1146/annurev.energy.25.1.53 607 20 Steffen, W., Richardson, K., Rockström, J., Cornell, S.E., Fetzer, I., Bennett, E.M., Biggs, R., Carpenter, 608 S.R., De Vries, W., De Wit, C.A., Folke, C., Gerten, D., Heinke, J., Mace, G.M., Persson, L.M., 609 Ramanathan, V., Reyers, B., Sörlin, S., 2015. Planetary boundaries: Guiding human development 610 on a changing planet. Science (80-. ). 347. https://doi.org/10.1126/science.1259855 611 Sun, Y., Feng, Y., Wang, Y., Zhao, X., Yang, Y., Tang, Z., Wang, S., Su, H., Zhu, J., Chang, J., Fang, J., 612 2021. Field-Based Estimation of Net Primary Productivity and Its Above- and Belowground 613 Partitioning in Global Grasslands. J. Geophys. Res. Biogeosciences 126, 1–24. 614 https://doi.org/10.1029/2021JG006472 615 Van Oost, K., Quine, T.A., Govers, G., De Gryze, S., Six, J., Harden, J.W., Ritchie, J.C., McCarty, G.W., 616 Heckrath, G., Kosmas, C., Giraldez, J. V., Marques Da Silva, J.R., Merckx, R., 2007. The Impact of 617 Agricultural Soil Erosion on the Global Carbon Cycle. Science (80-. ). 318, 626–629. 618 https://doi.org/10.1126/science.1145724 619 Van Puijenbroek, P., Beusen, A.H.W., Bouwman, A.F., 2019. Global nitrogen and phosphorus in urban 620 waste water based on the Shared Socio-economic pathways. J. Environ. Manage. 231, 446–456. Implications for sustainable phosphate rock management 210 621 https://doi.org/10.1016/j.jenvman.2018.10.048 622 https://doi.org/10.1016/j.jenvman.2018.10.048 622 Van Vuuren, D.P., Bouwman, A.F., Beusen, A.H.W., 2010. Phosphorus demand for the 1970-2100 623 period: A scenario analysis of resource depletion. Glob. Environ. Chang. 20, 428–439. 624 https://doi.org/10.1016/j.gloenvcha.2010.04.004 625 Wang, Y., Huang, Y., Augusto, L., Goll, D.S., Helfenstein, J., Hou, E., 2022. Toward a Global Model for 626 Soil Inorganic Phosphorus Dynamics: Dependence of Exchange Kinetics and Soil Bioavailability 627 on Soil Physicochemical Properties. Global Biogeochem. Cycles 36, 1–20. 628 https://doi.org/10.1029/2021gb007061 629 Zhang, J., Beusen, A., van Apeldoorn, D., Mogollón, J., Yu, C., Bouwman, A., 2017. Spatiotemporal 630 dynamics of soil phosphorus and crop uptake in global cropland during the twentieth century. 631 Biogeosciences 14, 2055–2068. https://doi.org/10.5194/bg-2016-543 632 633 Competing interests 634 The authors declare no competing interests. 635 Van Vuuren, D.P., Bouwman, A.F., Beusen, A.H.W., 2010. Phosphorus demand for the 1970-2100 623 period: A scenario analysis of resource depletion. Glob. Environ. Chang. 20, 428–439. 624 https://doi.org/10.1016/j.gloenvcha.2010.04.004 625 Wang, Y., Huang, Y., Augusto, L., Goll, D.S., Helfenstein, J., Hou, E., 2022. Toward a Global Model for 626 Soil Inorganic Phosphorus Dynamics: Dependence of Exchange Kinetics and Soil Bioavailability 627 on Soil Physicochemical Properties. Global Biogeochem. Cycles 36, 1–20. 628 https://doi.org/10.1029/2021gb007061 629 Zhang, J., Beusen, A., van Apeldoorn, D., Mogollón, J., Yu, C., Bouwman, A., 2017. Spatiotemporal 630 dynamics of soil phosphorus and crop uptake in global cropland during the twentieth century. 631 Biogeosciences 14, 2055–2068. https://doi.org/10.5194/bg-2016-543 632 21 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SI.pdf
https://openalex.org/W2955633253	https://europepmc.org/articles/pmc6602202?pdf=render	English	null	Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm Bombyx mori	PloS one	2,019	cc-by	15,031	RESEARCH ARTICLE Sumihiro Matsumoto1, Natsumaro Kutsuna1¤, Ivana Daubnerova´ 2, Ladislav Roller2, Dusˇan Zˇitňan2, Hiromichi Nagasawa3, Shinji Nagata1* Sumihiro Matsumoto1, Natsumaro Kutsuna1¤, Ivana Daubnerova´ 2, Ladislav Roller2, Dusˇan Zˇitňan2, Hiromichi Nagasawa3, Shinji Nagata1* 1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan, 2 Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia, 3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: Research and Development Division, LPixel Inc., Tokyo, Japan * shinjin@edu.k.u-tokyo.ac.jp ¤ Current address: Research and Development Division, LPixel Inc., Tokyo, Japan * shinjin@edu.k.u-tokyo.ac.jp Abstract Our previous study demonstrated that predominant feeding inhibitory effects were found in the crude extracts of foregut and midgut of the silkworm Bombyx mori larvae. To address the entero-intestinal control crucial for the regulation of insect feeding behavior, the present study identified and functionally characterized feeding inhibitory peptides from the midgut of B. mori larvae. Purification and structural analyses revealed that the predominant inhibitory factors in the crude extracts were allatotropin (AT) and GSRYamide after its C-terminal sequence. In situ hybridization revealed that AT and GSRYamide were expressed in enter- oendocrine cells in the posterior and anterior midgut, respectively. Receptor screening using Ca2+-imaging technique showed that the B. mori neuropeptide G protein-coupled receptor (BNGR)-A19 and -A22 acted as GSRYamide receptors and BNGR-A5 acted as an additional AT receptor. Expression analyses of these receptors and the results of the peri- staltic motion assay indicated that these peptides participated in the regulation of intestinal contraction. Exposure of pharynx and ileum to AT and GSRYamide inhibited spontaneous contraction in ad libitum-fed larvae, while exposure of pharynx to GSRYamide did not inhibit contraction in non-fed larvae, indicating that the feeding state changed their sensitivity to inhibitory peptides. These different responses corresponded to different expression levels of their receptors in the pharynx. In addition, injection of AT and GSRYamide decreased esophageal contraction frequencies in the melamine-treated transparent larvae. These find- ings strongly suggest that these peptides exert feeding inhibitory effects by modulating intestinal contraction in response to their feeding state transition, eventually causing feeding termination. OPEN ACCESS Citation: Matsumoto S, Kutsuna N, Daubnerova´ I, Roller L, Zˇitňan D, Nagasawa H, et al. (2019) Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm Bombyx mori. PLoS ONE 14(7): e0219050. https://doi.org/10.1371/journal. pone.0219050 Editor: Man-yeon Choi, US Department of Agriculture, UNITED STATES Received: August 21, 2018 Accepted: June 16, 2019 Published: July 1, 2019 Copyright: © 2019 Matsumoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Man-yeon Choi, US Department of Agriculture, UNITED STATES Received: August 21, 2018 Accepted: June 16, 2019 Published: July 1, 2019 Copyright: © 2019 Matsumoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Development Fund (ERDF) (http://ec.europa.eu/ regional_policy/en/funding/erdf/) (ITMS: 26240220044) to DZ and LR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Most phytophagous insects live on or around their preferred host plants [1]. Observations of these insects have demonstrated the presence of a regularly occurring pattern switching from a feeding mode to a quiescent mode, indicating unidentified endogenous regulatory mechanisms in both upregulation and downregulation of feeding motivation. The regula- tory mechanisms in feeding motivation have been characterized by a number of physiologi- cal investigations, illuminating the importance of hemolymph factors corresponding to the nutritional status [2], eventually inducing in modifications in locomotor activities and neu- ronal signaling to maintain homeostasis [3, 4]. These biological homeostatic processes, including sequential feeding behavior and fluctuation in motivation, are known to be regu- lated by a number of neuropeptides in the central nervous system (CNS) and the peripheral organs [5, 6]. 26240220044) to DZ and LR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: NK is employed by LPixel Inc. There are no other competing interests to declare. This does not affect our adherence to PLOS ONE policies on data or materials sharing. including sequential feeding behavior and fluctuation in motivation, are known to be regu- lated by a number of neuropeptides in the central nervous system (CNS) and the peripheral organs [5, 6]. Among the feeding regulatory peptides in insects, several peptides are known as ‘brain-gut peptides’, which are expressed and produced in the brain and intestinal organs. Of the intesti- nal organs, the midgut possesses an important class of endocrine cells as well as the brain and CNS, where produce several neuropeptides, such as myosuppressin, neuropeptide F (NPF), tachykinin-related peptides (TRPs), and allatoregulatory peptides [7–10]. These peptides may regulate not only the physiological functions related to digestion [11] and intestinal lipid metabolisms [12] but also the intestinal contraction [13–15]. However, the current evidence is not adequate to evaluate the effects of “brain-gut peptides” on feeding regulation in insects. We have considered that the endogenous factors affecting the repetitive feeding state transition (feeding mode to quiescent mode) may be attributed to some of the feeding regulatory pep- tides [16]. Animals Eggs of the silkworm, Bombyx mori (a hybrid strain, Kinshu × Showa), were purchased from a silkworm egg-producing company, Ueda Sanshu Ltd. (Ueda, Japan). Larvae were reared in plastic containers at 26 ± 1˚C at 70 ± 10% relative humidity, and under 16 L:8 D lighting con- ditions. Larvae were fed a fresh artificial diet of Silkmate 2S purchased from Nippon Nosan Co. Ltd. (Yokohama, Japan). Enteroendocrine control of feeding behavior in B. mori Development Fund (ERDF) (http://ec.europa.eu/ regional_policy/en/funding/erdf/) (ITMS: 26240220044) to DZ and LR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Development Fund (ERDF) (http://ec.europa.eu/ regional_policy/en/funding/erdf/) (ITMS: 26240220044) to DZ and LR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This study was partially supported by the Japan Society for the Promotion of Science (JSPS) (https://www.jsps.go.jp/) KAKENHI (Grant Number 16K15066) to SN and grants from the Slovak Research and Development Agency (SRDA) (http:// www.apvv.sk/) (APVV-14-0556, APVV-16-0395 and VEGA 2/0080/18) and the European Regional 1 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Introduction In our previous study, significant feeding inhibitory factors that prolong the latency to the initiation of feeding (feeding-delaying activity) were observed in the extracts of the intes- tine (foregut, midgut) of B. mori larvae [17]. A previous biochemical study showed that B. mori RFamide has also similar feeding inhibitory activity [16]. In contrast, B. mori TRPs and short neuropeptide F (sNPF) -2 have feeding acceleratory effects that can shorten the latency to the initiation of feeding [16]. Although these reports demonstrate that endogenous endo- crine factors regulate feeding motivation, little is known about their mode of action or how feeding state transition occurs. In the present study, we purified and identified the predominant inhibitory factors from the midgut of B. mori larvae by assaying their behavioral activity on larval feeding. We further confirmed the inhibitory effects of these factors on intestinal contraction of the foregut and hindgut, illuminating a possible model for a feeding regulatory mechanism mediated by enter- oendocrine factors that modulate intestinal contraction. Chemicals and reagents The chemicals and reagents used in this study were purchased from Nacalai-tesque (Osaka, Japan). HPLC reagents (acetonitrile and trifluoroacetic acid [TFA]) were purchased from PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 2 / 24 Enteroendocrine control of feeding behavior in B. mori Kanto Kagaku (Tokyo, Japan). Fmoc derivatives of the amino acids were purchased from Watanabe Kagaku (Hiroshima, Japan). Purification of biologically feeding inhibitory peptides Midguts isolated from 500 2-day-old fifth-instar B. mori larvae fed ad libitum were homoge- nized in 90% methanol containing 1.0% acetic acid on ice. After centrifugation at 2,300 × g for 15 min, the supernatant was evaporated to dryness, redissolved in a 0.1% TFA aqueous solu- tion. The resulting solution was applied to a Sep-Pak C18 Cartridge (Waters, Milford, MA, USA) and the crude peptide fraction was collected as the fraction eluted with 60% acetonitrile containing 0.1% TFA after washing the un-adsorbed substances with a 0.1% TFA aqueous solution followed by 10% acetonitrile containing 0.1% TFA. The resulting eluent was subjected to reversed-phase high-performance liquid chromatography (RP-HPLC) on a UG 120Å ODS column (10 mm i.d. × 250 mm, Senshu Kagaku, Tokyo, Japan). The elution was performed with a linear gradient of 10–60% acetonitrile containing 0.1% TFA over 50 min at a flow rate of 2.0 mL/min. The elution was monitored by the absorbance at 280 nm. The biologically active fractions were evaporated to dryness, redissolved in a 0.1% TFA aqueous solution and then subjected to the next step of RP-HPLC using a PEGASIL ODS column (4.6 mm i.d. × 250 mm, Senshu Kagaku) in a linear gradient of 10–60% acetonitrile containing 0.1% TFA over 50 min at a flow rate of 1.0 mL/min. The elution was monitored by the absorbance at 280 nm. The purifications according to biological activities possibly until obtaining an isolated peptide were carried out by RP-HPLC using different of linear gradient program conditions moni- tored by absorbance at 225 nm. Mass spectra of the substances in the biologically active frac- tions were measured using a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer. Feeding behavioral assay A part of the eluents of the fractions from RP-HPLC as well as synthetic peptide solutions was evaporated to dryness and dissolved in 100 μL distilled water just prior to the bioassay. The feeding cycles of 2-day-old fifth-instar B. mori larvae were synchronized by diet deprivation for 16 h. After diet deprivation, the larvae were anesthetized by submerging in icy water for 15 min. Then, 100 μL sample was injected dorsolaterally into the hemolymph. After injection, the larvae were placed in front of artificial diet blocks, and then the latency to their first bites as the initiation of feeding (first bite) after sample-injection was measured as reported previously [16]. Statistical analysis was performed using a one-way analysis of variance (one-way ANOVA), then a post hoc Dunnett’s test. Reproducibility was confirmed at least twice by assays using different populations of larvae. MALDI-TOF mass spectrometry Molecular mass of peptides was measured by a MALDI-TOF mass spectrometer Voyager- DETM STR (Applied Biosystems, Foster City, CA, USA) in a positive ion mode. Samples were prepared by mixing with the supernatant of α-cyano-4-hydroxycinnamic acid saturated in 60% acetonitrile as a matrix at 1:1 ratio. Preparation of synthetic peptides Peptides were synthesized by the standard solid-phase methodology (Fmoc procedure) on an AAPPTEC APEX 396-SC synthesizer (AAPPTEC, Louisville, KY, USA). The resulting crude synthetic peptides after deprotection were purified using a Sep-Pak C18 Cartridge (Waters) and by RP-HPLC on a PEGASIL-300 ODS column (10 mm i.d. × 250 mm or 4.6 mm i.d. × 250 mm, Senshu Kagaku) using the identical linear gradient condition as that used for purifi- cation from midgut according to MALDI-TOF mass analyses. The synthetic peptides solutions were evaporated to dryness and dissolved in distilled water or B. mori Ringer’s solution (110 mM KCl, 4 mM NaCl, 15 mM MgCl2, 4 mM CaCl2, 5 mM KH2PO4, pH 6.5) for biological assay. The concentration of eluted synthetic peptide solutions was adjusted after measuring the peptide solution by Bradford protein assay [18]. The purities of the all used peptides in the experiments were confirmed to be higher than 97.0%. Amino acid sequence analysis and homology search The N-terminal amino acid sequence of the purified peptide was analyzed by a protein sequencer ProciseTM cLC (Applied Biosystems) in pulsed-liquid mode. A Basic Local Align- ment Search Tool (BLAST) search for the entire sequences of the obtained partial sequences PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 3 / 24 Enteroendocrine control of feeding behavior in B. mori was performed using the B. mori databases Silk Base (http://silkbase.ab.a.u-tokyo.ac.jp/) and KAIKO BLAST (http://kaikoblast.dna.affrc.go.jp/). A homology search of GSRYa-1 and -2 (S3 Fig) was performed by using GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and BLAST searches (blastn, tblastn, and Blastp). Reverse transcriptase polymerase chain reaction (RT-PCR) Total RNA was extracted from the isolated tissues of 2-day-old fifth-instar larvae fed ad libitum (brain, CNS, foregut, anterior, middle and posterior midgut, hindgut, Malpighian tubules, fat body, silk gland, testis, ovary, and hemocytes) using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The extracted total RNA was treated with DNase I (TaKaRa Bio, Shiga, Japan) at 37˚C for 1 h. cDNA was synthesized using total RNA with oligo (dT) and Superscript III Reverse Transcriptase (Invitrogen). RT-PCR was per- formed using the resulting cDNA as a template and the following primer sets: AT-Fw (5´-A GCAGGCAGTCTCACGAGTT-3´) and AT-Rv (5´-CGCAAACCGATTTTAACAGA-3´), GSRYamide-Fw (5´-GTGCGTGCGTGTACTACGAT-3´) and GSRYamide-Rv (5´-CATTGG CATGGCAAAGTCTA-3´), B. mori neuropeptide G protein-coupled receptor (BNGR)-A5- Fw (5´-TACGTGTTCCCTCAGCCCTA-3´) and BNGR-A5-Rv (5´-TTTGCACTTGAAGCA CCACG-3´), BNGR-A16-Fw (5´-TGGAGCTCCAACGGAATTCC-3´) and BNGR-A16-Rv (5´-GTGCTGGTATGTCCTGGCTT-3´), BNGR-A19-Fw (5´-AGAATGGGACCACGTGGA AC-3´) and BNGR-A19-Rv (5´-TGAAGCCGCGTCGATATCTC-3´), BNGR-A22-Fw (5´-AA GCCTCGACTTGGGAAAGG-3´) and BNGR-A22-Rv (5´-TGACTCGCGAACCAAACGTA- 3´). Ribosomal protein L3 (rpL3) was used for experimental control. Utilized primers were rpL3-Fw (5´-TGGCACACAAAGAAGCTACCC-3´) and rpL3-Rv (5´-TGACCAGCACGAG CTACAGTG-3´). Ex-Taq polymerase (TaKaRa Bio) was used for PCR. The PCR program was as follows. Amplification: 30 cycles of 15 s at 94˚C (3 min for the first cycle), 30 s at 55˚C, and 45 s at 72˚C. The PCR products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. The PCR products were subcloned into pGEM-T Easy vectors (Promega, Madison, WI, USA) and were confirmed by sequencing on an ABI PRIZM 310 Genetic Ana- lyzer (Applied Biosystems) using a Big Dye Terminator Ver.3.1 Cycle Sequencing Kit (Applied Biosystems). using the primer sets: AT-Fw (5´-AGCAGGCAGTCTCACGAGTT-3´) and AT-Rv (5´-CGG GTTGTTGAGAACCTCAT-3´), GSRYamide-Fw (5´-GTGCGTGCGTGTACTACGAT-3´) and GSRYamide-Rv (5´-CATTGGCATGGCAAAGTCTA-3´). The probe size of AT and GSRYamide were 360 nt and 521 nt, respectively. using the primer sets: AT-Fw (5´-AGCAGGCAGTCTCACGAGTT-3´) and AT-Rv (5´-CGG GTTGTTGAGAACCTCAT-3´), GSRYamide-Fw (5´-GTGCGTGCGTGTACTACGAT-3´) and GSRYamide-Rv (5´-CATTGGCATGGCAAAGTCTA-3´). The probe size of AT and GSRYamide were 360 nt and 521 nt, respectively. The midgut from fourth-instar larvae was isolated and fixed overnight at 4˚C in 4% parafor- maldehyde (PFA) in phosphate buffered saline (PBS), and washed with PBS containing 0.1% Tween 20 (PBST). The samples were digested with proteinase K (50 μg /mL PBST) for 10 min, washed with Glycine-PBST (2 mg Glycine/1 mL PBST), postfixed with 4% PFA for 1 h and washed with PBST. Prehybridization was performed in the hybridization solution (HS; 50% formamide, 5 × Saline sodium citrate, 100 mg/mL salmon testes DNA, 0.1% Tween 20, 50 mg/ mL heparin, RNase-free deionized water) at 48˚C for 1–2 h. The tissues were incubated in HS containing the DNA probe (1 volume of probe per 9 volumes of HS) specific for the AT and GSRYamide gene transcripts at 48˚C for 20–24 h. After hybridization, the tissues were washed with HS at 48˚C overnight and briefly with a mixture of HS-PBST (1:1) and PBST at room temperature. They were then blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich Co. LLC., St. Louis, MO, USA) in PBST for 10 min, incubated with an alkaline phosphatase (AP)-labelled anti-digoxigenin antibody (1:1000; Roche, Mannheim, Germany) overnight at 4˚C, and stained with a Nitroblue tetrazolium and 5-Bromo-4-chloro-3-indolyl phosphate solution (NBT-BCIP, Roche) diluted with 1:50 in AP buffer (100 mM Tris, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20, pH 9.5). After color development, they were washed with PBST, then mounted in glycerol. Negative controls were performed using a specific sense probe. Staining was detected using a LEICA M205FA stereomicroscope (Leica Microsystems, Wet- zlar, Germany) and a Nikon Eclipse 600 microscope with Nomarski differential interference contrast optics and photographed using an attached Nikon Coolpix 990 digital camera (Nikon, Tokyo, Japan). Images are presented as maximum projection of Z-stacks. Stacking was performed using CombineZP (shareware by Alan Hadley). PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 In situ hybridization Whole mount in situ hybridization was performed as described by Roller et al. [19] and Bedna´r et al. 2017 [20]. The digoxigenin-labeled single-stranded DNA probes were synthesized by asymmetric PCR. The sense and antisense probes of AT and GSRYamide were synthesized PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 4 / 24 Enteroendocrine control of feeding behavior in B. mori Ca2+-imaging assay of BNGR-A19, -A22, -A23, -A24, -A32, and -A33 Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37˚C in a humidified atmosphere containing 5% CO2. HEK293 cells seeded on a 35-mm glass-based dish (Iwaki, Tokyo, Japan) were co-transfected with pME18S plasmids carrying BNGR cDNAs and the promiscuous G protein alpha subunit (Gα15) using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. The Ca2+ indicator solution consisting of 2 μM Fluo-4 AM (Thermo Fisher Scientific Inc.) and 0.01% pluronic-F 127 (Sigma-Aldrich Co. LLC.) in Opti-MEM (Thermo Fisher Scientific Inc.) was added to the cells after 24 h transfection at 37˚C for 30 min in the dark. After incuba- tion, the cells were washed twice with Ringer’s solution (140 mM NaCl, 5.6 mM KCl, 2.0 mM CaCl2, 2.0 mM MgCl2, 9.4 mM D-glucose, 1.25 mM KH2PO4, and 5 mM HEPES, pH 7.4), and then the Ringer’s solution was added. The Ca2+-imaging assays were performed within 2 h after replacement with Ringer’s solution. Using a confocal laser-scanning microscope LSM5 Exciter (Zeiss, Oberkochen, Germany), the intensity of fluorescence in Fluo-4-loaded cells at 518 nm by excitation at 488 nm was recorded during measurement for 1400 s with the follow- ing supplements in the Ringer’s solution; synthetic peptides (10−10–10−5 M final concentra- tion) at 180, 360, 540, 720, 900, 1080, and 100 μM ATP (a ligand for an endogenous ATP receptor) at 1260 s as a positive experimental control for robust intracellular Ca2+ increase. Data were calculated by the responding fluorescent intensities of 20 cells which did not respond to the exposure to Ringer’s solution (at 0 s) and showed responses to exposure to the ATP solution, as the relative fluorescent intensity (0%, the minimum intensity; 100%, the max- imum intensity during monitoring). Phylogenetic analysis A phylogenetic tree was generated using CLUSTAL W by neighbor-joining method. Bootstrap of 1,000 replications was conducted to assess the relationships. The deduced amino acid sequences of 36 class A BNGRs; BNGR-A1 (NP_001127736), BNGR-A2 (NP_001127737), BNGR-A3 (NP_001127737), BNGR-A4 (NP_001127739), BNGR-A5 (NP_001127740), BNGR-A6-A (NP_001127741), BNGR-A6-B (NP_001165737), BNGR-A7 (NP_001127742), BNGR-A8 (NP_001127743), BNGR-A9 (NP_001127744), BNGR-A10 (NP_001127707), BNGR-A11 (NP_001127708), BNGR-A12 (NP_001127709), BNGR-A13 (NP_001127710), BNGR-A14 (NP_001127711), BNGR-A15 (NP_001127712), BNGR-A16 (NP_001127714), BNGR-A17 (NP_001127715), BNGR-A18 (NP_001127716), BNGR-A19 (NP_001127720), BNGR-A20 (NP_001127718), BNGR-A21 (NP_001127719), BNGR-A22 (NP_001127717), BNGR-A23 (NP_ 001127721), BNGR-A24 (NP_001127722), BNGR-A25 (NP_001127723), BNGR-A26 (NP_001127724), BNGR-A27 (NP_001127725), BNGR-A28 (NP_001127726), BNGR-A29 (NP_001127745), BNGR-A30 (NP_001127746), BNGR-A31 (NP_001127747), BNGR-A32 (NP_001127748), BNGR-A33 (NP_0011227749), BNGR-A34 (NP_001127750), BNGR-A35 (NP_001127751), B. mori diuretic hormone receptor variant X2 (DHR) (XM_02 1352745.1), Drosophila melanogaster myosuppressin receptor 1 isoform A (D-MsR1) (NP_647 713), D. melanogaster myosuppressin receptor 2 isoform A (D-MsR2) (NP_647711), D. mela- nogaster sNPF receptor isoform A (D-sNPFR) (NP_524176), D. melanogaster tachykinin-like receptor at 86C, isoform A (D-TkR86C) (NP_524304), D. melanogaster tachykinin-like recep- tor at 99D, isoform A (D-TkR99D) (NP_524556), Tribolium castaneum RYamide receptor PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 5 / 24 Enteroendocrine control of feeding behavior in B. mori (T-RYaR) (NP_001280539), and D. melanogaster RYamide receptor isoform A (D-RYaR) (NP_524525) were obtained from GenBank by BLAST searches (blastn, tblastn and Blastp). B. mori DHR was used as an outgroup. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Ca2+-imaging assay of BNGR-A5 and -A16 Activity of the BNGR-A5 and -16 receptors were analyzed using Chinese hamster ovary (CHO) cells transiently transfected with plasmids for expression of each BNGR, calcium-sensi- tive reporter aequorin and chimeric G protein that couple with receptor activation to an intra- cellular Ca2+-increase independent of the endogenous receptor signaling cascade. The entire open reading frames of BNGR-A5 and -16 were cloned into pcDNA3.1(+) vector (Invitrogen) with a Kozak translation initiation sequence incorporated at 5´end. Plasmids for codon opti- mized aequorin and chimeric G proteins were used as described previously [21–23]. CHO cells were maintained at 37˚C in a humidified atmosphere of 5% CO2 in DMEM Nutri- ent Mixture F-12 Ham (DMEM/F12) with L-glutamine, 15 mM HEPES, sodium bicarbonate and phenol red (Sigma-Aldrich Co. LLC.) enriched with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich Co. LLC.), 100 IU/mL penicillin and 100 μg/mL streptomycin (Life Tech- nologies, Carlsbad, CA, USA). Approximately 24–30 hours before transfection, we seeded appro- priate number of CHO cells in 10 mL of complete growth medium in 10 cm Petri dishes to achieve 60–80% confluency at the time of transfection. Each receptor assay was performed using transfected cells from two Petri dishes. For cell transfection, we prepared 20 μg DNA directly into 950 μL of serum- and antibiotic-free medium and incubated the mixture for 5 minutes at room temperature. Thereafter, we added 30 μL of warm FuGene HD (Promega) to the tube and incubated the transfection reagent to make DNA complex at room temperature for 25 min. We PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 6 / 24 Enteroendocrine control of feeding behavior in B. mori then removed the cell medium from Petri dishes, followed by covered the cells with 10 mL of fresh antibiotic-free culture medium and added the transfection mixture in a dropwise manner. Cells were allowed to grow for 24–48 hours (37˚C, 5% CO2) and used for the assay. The transfected cells were detached using PBS supplemented with 0.2% EDTA and rinsed of the culture plates with the assay medium—phenol red free DMEM/F12 with L-glutamine and 15 mM HEPES (PAN Biotech, Wimborne, Dorset, UK), 0.1% BSA (Sigma-Aldrich Co. LLC.) and 1% penicillin/streptomycin (Life Technologies). Then we centrifuged cells at 1000 rpm for 3 min, aspirated the medium and resuspended the cell pellet in the assay medium (10 mL). Ca2+-imaging assay of BNGR-A5 and -A16 We repeated centrifugation step (1000 rpm, 3 min), resuspended the pellet in the assay medium (5 mL) and coelenterazine H (Promega) was added at a final concentration of 5 μM. Cells were then main- tained in suspension with a stirrer at room temperature for 2.5–3 h in the dark to reconstitute the active holoenzyme aequorin. 30 min prior to the measurement cells were diluted threefold using the same BSA medium and 50 μL of cell suspension was injected into each well of a 96-well plate filled with 50 μL of tested compounds dissolved in the assay medium. Various peptide concentra- tions were loaded in triplicates and wells containing a negative control (assay medium without ligands) was included in each row to correct cell response of each well in the same row. Emitted luminescence signal corresponding to the ligand-induced intracellular Ca2+ release was recorded for 20 s using the GloMax-Multi+ Detection System (Promega). Wells containing ATP at a final concentration of 50 μM served as a positive control and 100 μM digitonin was used to measure the total receptor-independent cellular Ca2+-response. Each experiment was repeated three times for data analysis. Calculations and further analysis of the output data were done in Excel (Micro- soft, Redmond, WA, USA) and GraphPad Prism 6 software. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Observations of spontaneous contractions of the foregut and hindgut in dissected open larvae The opened head and abdominal portion of 2-day-old fifth-instar larvae (fed ad libitum and non-fed for 16 h) were washed twice with B. mori Ringer’s solution and incubated for 3 min at 27˚C to stabilize the movement. After incubation, the contractile movements of the larval foregut and hindgut [24], particularly the spontaneous contraction of the pharynx and ileum (predominantly contracting area in hindgut) were observed and recorded using a digital microscope USB2.0 DigiScope II v2TM (CHRONOS, Taipei, Taiwan). 100 μL of B. mori Ring- er’s solution was added to the examined gut at 60 s after starting recording. Furthermore, 100 μL of B. mori Ringer’s solution (vehicle control) and 100 μL peptide-containing solution (10−9–10−5 M of peptides dissolved in B. mori Ringer’s solution) were exposed over gut at 120 s after a recording start. The contractile movements were analyzed using ImageJ software (https://imagej.net/Fiji) with the Optic flow plug-in (Gaussian window MSE). The direction of contractile movement was represented by the color and arrows described in the ‘Direction’ map. The position of the lines used for the kymograph analyses (x-y, z-w) were determined by Optic flow analyses. The waveform of the kymographs, corresponding to the visible contrac- tions in the recorded movies, were quantified and converted to myograms. The number of contractions in 60 s was defined as the ‘contraction frequency’. Invisible contractions in the recorded movie and extremely small amplitudes in the myograms were considered as no sig- nificant contractions. The evaluation of visible contraction was defined by giving a threshold at the half length of the observed amplitude. The effect of synthetic peptides was represented by the ratio of the ‘contraction frequency’ at 60–120 s to 120–180 s (%) due to the extendedly various frequencies by individuality; the pharynx (30-40/60s) and hindgut (20-40/60s). This methodology by kymograph is substantially corresponding to the previously used assays for intestinal contraction [25–27]. Statistical analyses were performed compared with the vehicle PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 7 / 24 Enteroendocrine control of feeding behavior in B. mori control by one-way ANOVA, then a post hoc Dunnett’s test. Reproducibility was confirmed at least twice by assays using different populations of larvae. Quantitative polymerase chain reaction (Q-PCR) Total RNA was extracted from the isolated tissues (pharynx, esophagus, anterior and posterior midgut, pylorus-ileum, colon, and rectum) of 2-day-old fifth-instar larvae fed ad libitum and non-fed for 16 h. The first-strand cDNA was used as a template and each amplification was primed by a pair of primers: BNGR-A19-Fw (5´-ATGAAGAGTGGGCTGCATGG-3´) and BNGR-A19-Rv (5´-AATGTGACATCGCCAGCCA-3´). Ribosomal protein L3 (rpL3), Glycer- aldehyde-3-phosphate dehydrogenase (GAPDH) and α-tubulin were used for experimental control. Utilized primers were rpL3-Fw (5´-TCGTCCAAGTTCGGTCATGG-3´) and rpL3- Rv (5´-ACCCATGAATGCAGCCTTGT-3´), GAPDH-Fw (5´-TTCCTGCCTCTACTGGTGC T-3´) and GAPDH-Rv (5´-CCATTCCAGTCAGCTTGCCA-3´), α-tubulin-Fw (5´-CGCACT GGCACATACAGACA-3´) and α-tubulin-Rv (5´-TTGTTGGCCGCATCTTCCTT-3´). Q-PCR was performed using a Thermal Cycler Dice Real-Time System (TaKaRa Bio), and THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) and 0.4 μM of each primer. The thermal cycling conditions were as follows: initial denaturation at 95˚C for 1 min and then 45 cycles of 95˚C, 10 s; and 60˚C, 30 s. The single targeted Q-PCR product was confirmed by gen- erating a melt curve for all reactions. Data were analyzed using the Thermal Cycler Dice Real- Time System Single Software Version 5.11 (TaKaRa Bio). The results are represented as a ratio of BNGR-A19 and rpL3 by comparative CT method. Statistical analysis was performed to compare with the larvae fed ad libitum in each tissue by Student’s t-test. Observations of the esophageal contraction in intact larvae Transparent larvae were prepared by feeding an artificial diet containing 0.05% melamine (Wako Pure Chemical Industries) starting from 0-day-old fifth-instar larvae. Melamine-feed- ing enhanced the excretion of uric acid and rendered the larval integument transparent as pre- viously reported [28]. The visible effect took place in 2-day-old fifth-instar larvae compared to normally fed larvae. The feeding behavioral assay of transparent larva was performed in the same way as that of normal larvae. Each transparent larva was surrounded with a soft sponge and held with both sides by two slide glasses. Transmitted light enabled to illuminate the shape and movement of the shadowed esophagus portion. The contraction of foregut, particularly ‘forward contraction wave’ and ‘peristaltic squeeze’ at the esophagus [24] were observed using a digital microscope to measure the number of contractions per minute. The ‘peristaltic squeezes’ at 8 h and 16 h in non-fed larvae were unmeasurable because of the limit of detection in our observation using digital microscope. 100 μL of distilled water (vehicle control) and 100 μL peptide-containing solution (10−7–10−4 M of peptides dissolved in distilled water) were injected dorsolaterally into the hemolymph. After injection, the numbers of contractions per minute of the esophagus were counted from the data of 3 min observation. Statistical analysis was performed using a one-way ANOVA, then a post hoc Dunnett’s test. Reproducibility was confirmed at least twice by assay using different populations of larvae. Purification and identification of feeding inhibitory factors from the midgut of B. mori larvae To identify the feeding inhibitory factors in B. mori larvae, we purified these factors into two fractions at the sixth purification step of RP-HPLC according to their biological activities, PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 8 / 24 Enteroendocrine control of feeding behavior in B. mori which significantly prolonged the latency to the first bite after sample injection compared with those by vehicle injection (A1 and B3 in Fig 1A and 1B). The two purified biologically active fractions were then sequenced, consequently providing a single predominant N-terminal sequence in each fraction. BLAST search of the obtained N-terminal sequences in the B. mori databases and MALDI-TOF mass analyses (S1 Fig) revealed that the predominant peptides were allatotropin (AT) in fraction A1 and a peptide composed of 17 amino acids in fraction B3 (Fig 1C). The latter peptide was an ortholog of the recently discovered insect RY-amide [29, 30], and was accompanied by its paralogous peptide in the precursor peptide (S2 Fig); we then designated them as GSRYamides (GSRYa-1 and -2) after their common C-terminal amino acid sequences. The feeding inhibitory activities of these three peptides were confirmed by the use of synthetic peptides. Injection of AT and GSRYa-1 and -2 prolonged the latency to the first bite in a dose-dependent manner (ED50 = 1.0 × 10−6 M, 1.3 × 10−6 M, and 1.0 × 10−6 M, respectively) (Fig 1D). These data indicated that AT and GSRYa-1 and -2 are the predominant inhibitory peptides in the midgut of B. mori larvae. In addition, we further investigated the effects of these peptides on intestinal motility. Spatial expression analyses of AT, GSRYamide, and their cellular distribution of enteroendocrine cells (EEs) in the larval midgut We next analyzed the spatial expression pattern of the identified feeding inhibitory peptides (AT and GSRYamide) in the B. mori larvae. RT-PCR revealed that AT was expressed in the middle and posterior midgut, while GSRYamide was strongly expressed in the anterior and middle midgut as well as in the reproductive organs (testis and ovary) (Fig 2A and 2B). In addition, expression of AT and GSRYamide was observed in the brain. As previously reported [31], PCR products differing by approximately 100 bp due to alternatively spliced variants of AT and AT-like peptides have been observed in the brain and CNS. In the case of AT expres- sion in the midgut, we observed only the transcript for AT-0 encoding one of the AT sequence (Fig 2B). In situ hybridization revealed that AT-expressing EEs were located in the posterior midgut (Fig 2C), while GSRYamide-expressing EEs were present in the anterior midgut (Fig 2E). Most of the AT-expressing cells (30–35 μm) were pyramid-shaped EEs, whose broad ends might be in contact with the basal lamina (Fig 2D). In contrast, most GSRYamide-expressing cells (20– 25 μm) [19] were bottle-shaped EEs, having a broad basal region and cytoplasmic apical pro- cesses (Fig 2F). These data indicated that the AT and GSRYamide enteroendocrine peptides were expressed and produced in different regions and distinctive types of EEs in the midgut. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Expression of AT and GSRYa-1 and -2 receptors in the intestinal organs (A) and (B) RP-HPLC profiles of the final purification step of the midgut extracts (upper) and their feeding inhibitory activities (lower). Upper; The dashed lines represent the acetonitrile concentrations. The numbered areas and peaks indicate the fractions used for behavioral bioassays. The injected dose of each sample was 20 midgut equivalents of the fifth-instar larvae. Lower; Bars represent means of the latency to the first bite. An asterisk indicates a significant difference compared to vehicle-injected larvae by one-way ANOVA (P < 0.05). Data represent means + S.D. (n = 3). (C) Amino acid sequences of feeding inhibitory factors in the A1 and B3 fractions. N-terminal sequences identified by the protein sequencer are underlined. ‘a’ indicates C-terminal amidation. Masses are given in Dalton (Da) and represent monoisotopic uncharged mass (MS). (D) Dose-dependent effects of synthetic AT and GSRYa- 1 and -2 on the latency to the first bite. Asterisks () indicate significant differences compared with vehicle-injected larvae by one- way ANOVA (P < 0.01). Data represent means ± S.D. (n = 3). Purification and identification of feeding inhibitory factors from the midgut of B. mori larvae. (A) and (B) RP-HPLC f h f l f f h d ( ) d h f d h b (l ) Th Fig 1. Purification and identification of feeding inhibitory factors from the midgut of B. mori larvae. (A) and (B) RP-HPLC profiles of the final purification step of the midgut extracts (upper) and their feeding inhibitory activities (lower). Upper; The dashed lines represent the acetonitrile concentrations. The numbered areas and peaks indicate the fractions used for behavioral bioassays. The injected dose of each sample was 20 midgut equivalents of the fifth-instar larvae. Lower; Bars represent means of the latency to the first bite. An asterisk indicates a significant difference compared to vehicle-injected larvae by one-way ANOVA (P < 0.05). Data represent means + S.D. (n = 3). (C) Amino acid sequences of feeding inhibitory factors in the A1 and B3 fractions. N-terminal sequences identified by the protein sequencer are underlined. ‘a’ indicates C-terminal amidation. Masses are given in Dalton (Da) and represent monoisotopic uncharged mass (MS). (D) Dose-dependent effects of synthetic AT and GSRYa- 1 and -2 on the latency to the first bite. Asterisks () indicate significant differences compared with vehicle-injected larvae by one- way ANOVA (P < 0.01). Data represent means ± S.D. (n = 3). Expression of AT and GSRYa-1 and -2 receptors in the intestinal organs To validate the target organ of AT and GSRYa-1 and -2 in B. mori larvae, we analyzed the expression sites of receptors for AT and GSRYa-1 and -2. We, first, screened for the GSRYa-1 and -2 receptors by Ca2+-imaging technique from the members in the specific clade of BNGRs for the ligands with structural similarities at their C-terminal RF-amide sequences [32], which include receptors for the extended RF-amide peptides [33]; Bm-sNPFR, and Bm-TRPR (Fig 3). Of six BNGRs (BNGR-A19, -A22, -A23, -A24, -A32, and -A33), BNGR-A19 and -A22 expressed in HEK293 cells showed increased levels of intracellular Ca2+ after exposure to GSRYa-1 and -2 with different affinities (Fig 4A). BNGR-A19 responded to GSRYa-1 and -2 in a dose-response manner, with a higher affinity to GSRYa-1 than that to GSRYa-2 (Fig 4B, 4C and 4E). BNGR-A22 showed much weaker responses to GSRYa-1 and -2 than BNGR-A19 (Fig 4B, 4C and 4E). The different affinities of BNGR-A19 and -A22 to GSRYa-1 and -2 indi- cated that BNGR-A19 functions predominantly as the receptor for GSRYa-1 and -2 in B. mori. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 9 / 24 Enteroendocrine control of feeding behavior in B. mori Fig 1. Purification and identification of feeding inhibitory factors from the midgut of B. mori larvae. (A) and (B) RP-HPLC profiles of the final purification step of the midgut extracts (upper) and their feeding inhibitory activities (lower). Upper; The dashed lines represent the acetonitrile concentrations. The numbered areas and peaks indicate the fractions used for behavioral bioassays. The injected dose of each sample was 20 midgut equivalents of the fifth-instar larvae. Lower; Bars represent means of the latency to the first bite. An asterisk indicates a significant difference compared to vehicle-injected larvae by one-way ANOVA (P < 0.05). Data represent means + S.D. (n = 3). (C) Amino acid sequences of feeding inhibitory factors in the A1 and B3 fractions. N-terminal sequences identified by the protein sequencer are underlined. ‘a’ indicates C-terminal amidation. Masses are given in Dalton (Da) and represent monoisotopic uncharged mass (MS). (D) Dose-dependent effects of synthetic AT and GSRYa- 1 and -2 on the latency to the first bite. Asterisks () indicate significant differences compared with vehicle-injected larvae by one- way ANOVA (P < 0.01). Data represent means ± S.D. (n = 3). https://doi.org/10.1371/journal.pone.0219050.g001 Fig 1. Purification and identification of feeding inhibitory factors from the midgut of B. mori larvae. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Expression of AT and GSRYa-1 and -2 receptors in the intestinal organs https://doi.org/10.1371/journal.pone.0219050.g001 https://doi.org/10.1371/journal.pone.0219050.g001 Although the previous study reported that the AT receptor is assigned to be BNGR-A16 in Ca2 +-imaging technique using HEK293 cell [32] (Fig 3), we newly validated the response of BNGR-A5 (Fig 3 an arrow) as well as BNGR-A16 to AT by an aequorin-based bioluminescent calcium assay in CHO cells. BNGR-A5 showed much higher affinity to AT than BNGR-A16 (Fig 4D and 4E). This high response of BNGR-A5 to AT indicated that BNGR-A5 functions predominantly as the receptor for AT in B. mori. RT-PCR revealed that GSRYa-1 and -2 recep- tors (BNGR-A19 and -A22) were ubiquitously expressed in the larval body (Fig 4F). In con- trast, AT receptors (BNGR-A5 and -A16) were expressed in several tissues, including the brain, CNS, intestine (foregut, midgut, and hindgut), Malpighian tubules, fat body, silk gland and reproductive tissues (Fig 4F). These results implied that larval intestine (foregut, midgut, and hindgut) is one of the target organ of AT and GSRYa-1 and -2, which participates in intes- tinal contraction, as followingly described. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 10 / 24 Enteroendocrine control of feeding behavior in B. mori Fig 2. Expression analyses of AT and GSRYamide and the cellular distributions of EEs in the midgut. (A) A schematic diagram of B. mori larval intestine (three parts of the midgut). Dashed line indicates the outline of the larval body. (B) RT-PCR analyses of AT and GSRYamide (GSRYa). Ribosomal protein L3 (rpL3) was used as an experimental control. Brain (lane 1), CNS (lane 2), foregut (lane 3), anterior, middle and posterior midgut (lanes 4, 5 and 6), hindgut (lane 7), Malpighian tubules (lane 8), fat body (lane 9), silk gland (lane 10), testis (lane 11), ovary (lane 12), and hemocyte (lane 13). (C) In situ hybridization of AT. AT-expressing EEs were scattered in the posterior midgut of fourth-instar larvae. (D) A zoomed-in image of a pyramid-shaped EE expressing AT. (E) In situ hybridization of GSRYamide. GSRYamide-expressing EEs were scattered in the anterior midgut of fourth-instar larvae. (F) A zoomed-in image of bottle-shaped EE expressing GSRYamide. Scale bars = 100 μm in (C), (E) and 10 μm in (D), (F). Fig 2. Expression analyses of AT and GSRYamide and the cellular distributions of EEs in the midgut. (A) A schematic diagram of B. mori larval intestine (three parts of the midgut). Dashed line indicates the outline of the larval body. Expression of AT and GSRYa-1 and -2 receptors in the intestinal organs (B) RT-PCR analyses of AT and GSRYamide (GSRYa). Ribosomal protein L3 (rpL3) was used as an experimental control. Brain (lane 1), CNS (lane 2), foregut (lane 3), anterior, middle and posterior midgut (lanes 4, 5 and 6), hindgut (lane 7), Malpighian tubules (lane 8), fat body (lane 9), silk gland (lane 10), testis (lane 11), ovary (lane 12), and hemocyte (lane 13). (C) In situ hybridization of AT. AT-expressing EEs were scattered in the posterior midgut of fourth-instar larvae. (D) A zoomed-in image of a pyramid-shaped EE expressing AT. (E) In situ hybridization of GSRYamide. GSRYamide-expressing EEs were scattered in the anterior midgut of fourth-instar larvae. (F) A zoomed-in image of bottle-shaped EE expressing GSRYamide. Scale bars = 100 μm in (C), (E) and 10 μm in (D), (F). https://doi.org/10.1371/journal.pone.0219050.g002 https://doi.org/10.1371/journal.pone.0219050.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Effects of AT and GSRYa-1 and -2 on the spontaneous contraction of foregut and hindgut in B. mori larvae Because AT and GSRYamide were the predominant brain-gut peptides in the midgut that modulated feeding initiation, we next examined the effects of these peptides on intestinal motility, particularly on the spontaneous contraction of the foregut and the hindgut [24]. In the isolated foregut of B. mori larvae, “peristaltic squeeze” was remarkably observed at the epi- pharynx within the pharynx, whereas little contraction was observed in the esophagus (Fig 5A and 5B(a)–(c)). We, then, measured the movement of the x-y axis (Fig 5B(a)) by kymograph analysis followed by conversion to myogram (Fig 5C). In the hindgut, predominant intestinal contraction was observed at the first constricted part within the ileum (Fig 5A and 5B(d)–(f)). Therefore, the anteroposterior axis over the first constricted part was analyzed (z-w in Fig 5B (d) and 5D). Exposure of intestines isolated from the larvae fed ad libitum to AT and GSRYa-1 and -2 decreased relative frequency of contraction at the pharynx and ileum (Fig 5E) (S1 Movie and S2 Movie). Similarly, exposure of non-fed larvae to AT also decreased the frequency in the pharynx and ileum (Fig 5F). By contrast, exposure of the ileum in the intestines isolated from non-fed larvae to GSRYa-1 and -2 decreased contraction in a dose-response manner (Fig 5F right), whereas no changes were observed in the pharynx (Fig 5F left). PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 11 / 24 Enteroendocrine control of feeding behavior in B. mori Fig 3. Phylogenetic tree of neuropeptide G protein-coupled receptors in B. mori and other insects. Phylogenetic tree of BNGRs (bolded) and D. melanogaster and T. castaneum G protein-coupled receptors; D-MsR1, D-MsR2, D- sNPFR, D-TkR86C, D-TkR99D, D-RYaR, and T-RYaR. Deorphanized BNGRs are indicated in parentheses; Bm-ITPR (B. mori ion transport peptide receptor) [34], Bm-ETHR (B. mori ecdysis-triggering hormone receptor) [32], Bm- ATSCR (B. mori allatostatin C receptor) [32], Bm-ATR (B. mori allatotropin receptor) [32], Bm-CRZR (B. mori corazonin receptor) [35], Bm-AKHR (B. mori adipokinetic hormone receptor) [36], Bm-NPFR (B. mori neuropeptide F receptor) [37], Bm-sNPFR (B. mori short neuropeptide F receptor) [32, 38], Bm-NTLR (B. mori natalisin receptor) [39], Bm-TRPR (B. mori tachykinin-related peptide receptor) [40], Bm-ITPLR (B. mori ion transport peptide-like receptor) [34]. BNGR-A5 (an arrow) was newly validated as another AT receptor. B. mori DHR was used as an outgroup. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Effects of AT and GSRYa-1 and -2 on the spontaneous contraction of foregut and hindgut in B. mori larvae BNGR-A19, -A22, -A23, -A24, -A32, and -A33 in the clade (black bar) including T-RYaR and D-RYaR (gray-boxed) [29, 41] were screened as potential GSRYa-1 and -2 receptors. Fig 3. Phylogenetic tree of neuropeptide G protein-coupled receptors in B. mori and other insects. Phylogenetic tree of BNGRs (bolded) and D. melanogaster and T. castaneum G protein-coupled receptors; D-MsR1, D-MsR2, D- sNPFR, D-TkR86C, D-TkR99D, D-RYaR, and T-RYaR. Deorphanized BNGRs are indicated in parentheses; Bm-ITPR (B. mori ion transport peptide receptor) [34], Bm-ETHR (B. mori ecdysis-triggering hormone receptor) [32], Bm- ATSCR (B. mori allatostatin C receptor) [32], Bm-ATR (B. mori allatotropin receptor) [32], Bm-CRZR (B. mori corazonin receptor) [35], Bm-AKHR (B. mori adipokinetic hormone receptor) [36], Bm-NPFR (B. mori neuropeptide F receptor) [37], Bm-sNPFR (B. mori short neuropeptide F receptor) [32, 38], Bm-NTLR (B. mori natalisin receptor) [39], Bm-TRPR (B. mori tachykinin-related peptide receptor) [40], Bm-ITPLR (B. mori ion transport peptide-like receptor) [34]. BNGR-A5 (an arrow) was newly validated as another AT receptor. B. mori DHR was used as an outgroup. BNGR-A19, -A22, -A23, -A24, -A32, and -A33 in the clade (black bar) including T-RYaR and D-RYaR (gray-boxed) [29, 41] were screened as potential GSRYa-1 and -2 receptors. 12 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Enteroendocrine control of feeding behavior in B. mori Fig 4. Screening for AT and GSRYa-1 and -2 receptors and expression analyses of BNGRs. (A) Ca2+-imaging analyses of six BNGRs (BNGR-A19, -A22, -A23, -A24, -A32, and -A33) to GSRYa-1 and -2 using HEK293 cells. Circles of BNGR-A19 and -A22 indicate intracellular Ca2+ increase following exposure to GSRYa-1 and -2 in a dose-dependent manner. Dashes of BNGR-A23, -A24, -A32, and -A33 indicate no response in all doses. (B) and (C) Dose-response analyses of BNGR-A19 and -A22 to synthetic GSRYa-1 (closed and open circles) and GSRYa-2 (closed and open triangles). Data are shown as relative fluorescent intensities (mean ± S.E.). (D) The dose- response analyses of BNGR-A5 and -A16 to synthetic AT. The receptors were expressed in CHO cells and tested in an aequorin-based bioluminescent calcium assay. Data are shown as relative fluorescent intensities (mean ± S.E.). (E) RT-PCR analyses of BNGR-A5, -A16, -A19, and -A22, with rpL3 as an experimental control. Effects of AT and GSRYa-1 and -2 on the spontaneous contraction of foregut and hindgut in B. mori larvae Brain (lane 1), CNS (lane 2), foregut (lane 3), anterior, middle and posterior midgut (lanes 4, 5, and 6), hindgut (lane 7), Malpighian tubules (lane 8), fat body (lane 9), silk gland (lane 10), testis (lane 11), ovary (lane 12), and hemocyte (lane 13). https://doi org/10 1371/journal pone 0219050 g004 Fig 4. Screening for AT and GSRYa-1 and -2 receptors and expression analyses of BNGRs. (A) Ca2+-imaging analyses of six BNGRs (BNGR-A19, -A22, -A23, -A24, -A32, and -A33) to GSRYa-1 and -2 using HEK293 cells. Circles of BNGR-A19 and -A22 indicate intracellular Ca2+ increase following exposure to GSRYa-1 and -2 in a dose-dependent manner. Dashes of BNGR-A23, -A24, -A32, and -A33 indicate no response in all doses. (B) and (C) Dose-response analyses of BNGR-A19 and -A22 to synthetic GSRYa-1 (closed and open circles) and GSRYa-2 (closed and open triangles). Data are shown as relative fluorescent intensities (mean ± S.E.). (D) The dose- response analyses of BNGR-A5 and -A16 to synthetic AT. The receptors were expressed in CHO cells and tested in an aequorin-based bioluminescent calcium assay. Data are shown as relative fluorescent intensities (mean ± S.E.). (E) RT-PCR analyses of BNGR-A5, -A16, -A19, and -A22, with rpL3 as an experimental control. Brain (lane 1), CNS (lane 2), foregut (lane 3), anterior, middle and posterior midgut (lanes 4, 5, and 6), hindgut (lane 7), Malpighian tubules (lane 8), fat body (lane 9), silk gland (lane 10), testis (lane 11), ovary (lane 12), and hemocyte (lane 13). To confirm the change of sensitivity in the pharynx (including the slightly moving hypo- pharynx) (Fig 5B (b) and (c)), we measured the expression level of BNGR-A19 as a predomi- nant receptor of GSRYa-1 and -2 in order to address the different responses to GSRYa-1 and -2 between fed ad libitum and non-fed larvae. Quantitative-PCR revealed that BNGR-A19 expression in the hindgut (pylorus-ileum, colon, and rectum) did not fluctuate according to the feeding state (Fig 5G right), whereas a significant decrease in BNGR-A19 expression was observed in the foregut (pharynx and esophagus) (Fig 5G left). PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Effects of AT and GSRYa-1 and -2 on the esophageal contraction in B. mori larvae To observe intestinal contraction without opening the larval body, we prepared transparent larvae (Fig 6A(b)) [28] that exhibited similar inhibitory effects by injection of AT and GSRYa- 1 and -2 (Fig 6B) compared to those of normal larvae (Figs 1D and 6A (a)). In the cephalic and thoracic parts of transparent larvae, two different contractions were observed at the esophagus [24]; a continuous “forward contraction wave” (35–45 waves/min) from the junction between the esophagus and the midgut toward the pharynx (Fig 6C (a) and (b)), and a strong and inter- mittent “peristaltic squeeze” (5–6 pulses/min) at the esophagus junction (Fig 6C (a) and (c)) (S3 Movie). Both contractions were observed as constitutively continuous contractions in the PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 13 / 24 Enteroendocrine control of feeding behavior in B. mori Fig 5. Observation of the spontaneous contraction of the foregut and hindgut in the dissected larvae. (A) A schematic diagram of larval intestine (foregut and hindgut) of B. mori. The dashed line indicates the outline of the larval body. A double-headed arrow indicates a portion of ileum. (B) (a) and (d) Pharynx and Ileum. The white axis lines (x-y for the epipharynx and z-w for the first constricted part) indicate the segment used for kymograph analysis. (b), (c) and (e), (f) Optic flow images of peristaltic squeeze at the pharynx and contraction at the ileum, respectively. The colored arrows in the diagrams at the right indicate the substantial direction of contractile movement of the pharynx and ileum after the “Direction” map. (C) and (D) A representative kymograph (upper) and myogram (lower) of pharynx and ileum isolated from larvae fed ad libitum. Arrowheads indicate the timing of exposure to B. mori Ringer’s solution at 60 s, GSRYa-1 (10−7–6 M) and AT (107−6 M) solutions at 120 s. The waveform in the kymograph between 60 s and 180 s was converted to a myogram. The double- headed arrows indicate a single period of contraction. (E) and (F) The effects of synthetic AT and GSRYa-1 and -2 on spontaneous contraction as relative Enteroendocrine control of feeding behavior in B. mori S ONE \| https://doi org/10 1371/journal pone 0219050 July 1 2019 14 / 24 Fig 5. Observation of the spontaneous contraction of the foregut and hindgut in the dissected larvae. (A) A schematic diagram of larval intestine (foregut and hindgut) of B. mori. Effects of AT and GSRYa-1 and -2 on the esophageal contraction in B. mori larvae The dashed line indicates the outline of the larval body. A double-headed arrow indicates a portion of ileum. (B) (a) and (d) Pharynx and Ileum. The white axis lines (x-y for the epipharynx and z-w for the first constricted part) indicate the segment used for kymograph analysis. (b), (c) and (e), (f) Optic flow images of peristaltic squeeze at the pharynx and contraction at the ileum, respectively. The colored arrows in the diagrams at the right indicate the substantial direction of contractile movement of the pharynx and ileum after the “Direction” map. (C) and (D) A representative kymograph (upper) and myogram (lower) of pharynx and ileum isolated from larvae fed ad libitum. Arrowheads indicate the timing of exposure to B. mori Ringer’s solution at 60 s, GSRYa-1 (10−7–6 M) and AT (107−6 M) solutions at 120 s. The waveform in the kymograph between 60 s and 180 s was converted to a myogram. The double- headed arrows indicate a single period of contraction. (E) and (F) The effects of synthetic AT and GSRYa-1 and -2 on spontaneous contraction as relative PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 14 / 24 Enteroendocrine control of feeding behavior in B. mori frequencies of the pharynx and ileum in larvae fed ad libitum (E) and non-fed (F). B. mori Ringer’s solution was administered as a vehicle control (0 M) and 10−9–10−5 M of peptide solutions were examined (%). An asterisk indicates a significant difference compared to the effect of the vehicle control by one-way ANOVA (P < 0.05). Data represent means ± S.D. (n = 5). (G) Quantitative-PCR analysis of BNGR-A19 in pharynx, esophagus, anterior and posterior midgut, pylorus-ileum, colon, and rectum of larvae fed ad libitum and non-fed. Results are represented as the ratio of BNGR-A19 to rpL3 by Ct. An asterisk () indicates a significant difference compared with the larvae fed ad libitum in each tissue by Student’s t-test (P < 0.05). n.s. indicates no significant difference between two conditions. The data represent means + S.D. (n = 5). https://doi.org/10.1371/journal.pone.0219050.g005 larvae fed ad libitum through 24 h (Fig 6D; open squares). However, in the non-fed larvae, these contractions gradually decreased and increased after refeeding subsequently (Fig 6D; closed squares). Effects of AT and GSRYa-1 and -2 on the esophageal contraction in B. mori larvae Injection of AT and GSRYa-1 and -2 to these transparent larvae decreased numbers of forward contraction waves and peristaltic squeezes in a dose-response manner (Fig 6E) (S4 Movie), as observed in the non-fed larvae before peptide injection (Fig 6D; closed squares). Taken together, AT and GSRYa-1 and -2 function as feeding inhibitory peptides by modu- lating intestinal contraction accompanied by the feeding state transition, eventually influenc- ing feeding termination. In addition, the feeding state changed the sensitivity of the pharynx to GSRYa-1 and -2 by changing the expression of its receptor. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Identification of AT and GSRYa-1 and -2 as feeding inhibitory “brain-gut peptides” from B. mori larvae AT was originally isolated from extracts of heads of pharate adult tobacco hornworm, Man- duca sexta as an allatoregulatory peptide [42]. Immunohistochemistry has demonstrated the presence of AT-expressing cells in the brain, the CNS, and the midgut in B. mori [20, 43]. Although the characterization of intestinal function has not been elucidated in B. mori, the current study revealed the feeding regulatory function of AT by modulating intestinal contrac- tion, which is a function, distinctive from allatoregulatory roles [32, 44]. Consistently, it is pos- sible in other insect species that AT can modulate feeding motivation by its pleiotropic physiological and developmental functions [45–47] including myoregulatory activities on the foregut [25, 26, 48], midgut [27, 49] and hindgut [50, 51]. A database search showed that GSRYa-1 and -2 are orthologs of the recently discovered insect RYamide (S3 Fig). Peptides having C-terminal RYamide sequences constitute a neuro- peptide Y (NPY) family, which is conserved both in vertebrates and invertebrate. As NPY has been assigned to a neuropeptide contributing to feeding behaviors in mice [52], the NPY homolog, NPF is known to regulate larval feeding behavior in D. melanogaster [53, 54]. Insect RYamide was reported to suppress feeding motivation in proboscis extension response tests for measuring feeding sensitivity when RYamide was administered to the flies, D. melanogaster [29] and Phormia regina [55]. The results of the present study (Figs 1D and 6B) are consistent with the context in the previous reports that RYamide functions as a feeding inhibitory pep- tide. In addition, GSRYamide was expressed in the reproductive organs, especially in the testis (Fig 2B). Such expression pattern of any bioactive peptides in the reproductive tissues as that of GSRYamide expression may indicate the parental function of the bioactive peptides during reproduction and development. The myoinhibitory activities of AT and GSRYa-1 and -2 in the foregut (pharynx and esoph- agus) and hindgut (ileum) (Figs 5E, 5F and 6E) seem to be linked to the delay in feeding initia- tion in B. mori larvae (Figs 1D and 6B). The intestinal contraction, especially in the esophagus, was gradually weakened by the continuous non-feeding (quiescent) mode (Fig 6D), indicating that these peptides contribute to the shift of the larval feeding state from the feeding to non- PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 15 / 24 Enteroendocrine control of feeding behavior in B. mori Fig 6. Observation of esophageal contraction in intact larvae. Identification of AT and GSRYa-1 and -2 as feeding inhibitory “brain-gut peptides” from B. mori larvae The shadowed area matches roughly with the foregut and anterior midgut; (a) The positions of the foregut (pharynx and esophagus) and midgut (anterior) in the figure; (b) Forward contraction wave: continuous contraction waves (35–45 waves/min) from the junction of the esophagus and midgut toward the pharynx (arrows). The period of a single wave (between arrow (I) and arrow (II)) was approximately 1.3–1.7 seconds; (c) Peristaltic squeeze: the strong and intermittent contraction (5–6 pulses/min) at the junction of the esophagus (arrowheads). (D) Fluctuations of forward contraction wave and peristaltic squeeze per minute in the two different feeding states; fed ad libitum (open squares), non-fed for 16 h and subsequently refed for 8 h (closed squares). An arrow indicates the timing of refeeding. An asterisk () indicates a significant difference compared with the larvae fed ad libitum by one-way ANOVA (P < 0.05). Data represent means ± S.D. (n = 5). (E) Effects of synthetic AT (open circles), GSRYa-1 (closed circles), and GSRYa-2 (closed triangles) on forward contraction wave and peristaltic squeeze in the larvae fed ad libitum. Distilled water was used as a vehicle control (0 M) and 10−7–10−4 M of peptide solution were dorsolaterally injected. Asterisks () indicate significant differences compared with vehicle-injected larvae by one-way ANOVA (P < 0.05). Data represent means ± S.D. (n = 5). https://doi.org/10.1371/journal.pone.0219050.g006 feeding (quiescent) mode. In this study, AT showed inhibitory effect on intestinal contrac- tions, whereas AT in several other Lepidopteran species show a stimulatory effect as described above [25, 26, 48, 51]. To know the opposite effects of AT on contractive regulation among the lepidopteran species, detail of the target for AT should be addressed. In fact, similar opposite effect in feeding behavior by a neuropeptide between species has been previously observed. For example, sNPF stimulates food intake and body weight gain in D. melanogaster [56] and has feeding acceleratory effect in B. mori [16], whereas an opposite behavioral effect, an inhibi- tory effect on food intake, is observed in Schistocerca. gregaria, [57]. In the non-fed B. mori lar- vae, exposure of the pharynx to GSRYa-1 and -2 did not decrease its contractions (Fig 5F left), indicating that the feeding state transition by GSRYa-1 and -2 (Figs 1D and 6B) is likely to be induced only by the inhibition of hindgut (ileum) contraction. Identification of AT and GSRYa-1 and -2 as feeding inhibitory “brain-gut peptides” from B. mori larvae (A) Pictures of normal (a) and transparent larvae (b). (B) Effects of synthetic AT (open circles), GSRYa-1 (closed circles), and GSRYa-2 (closed triangles) on the latency to the first bite. Asterisks () indicate significant differences compared with vehicle-injected larvae by one-way ANOVA (P < 0.01). Data represent means ± S.D. (n = 3). (C) Schematic diagrams and pictures of larval thorax under transmitted light. The shadowed area matches roughly with the foregut and anterior midgut; (a) The positions of the foregut (pharynx and esophagus) and midgut (anterior) in the figure; (b) Forward contraction wave: continuous contraction waves (35–45 waves/min) from the junction of the esophagus and midgut toward the pharynx (arrows). The period of a single wave (between arrow (I) and arrow (II)) was approximately 1.3–1.7 seconds; (c) Peristaltic squeeze: the strong and intermittent contraction (5–6 pulses/min) at the junction of the esophagus (arrowheads). (D) Fluctuations of forward contraction wave and peristaltic squeeze per minute in the two different feeding states; fed ad libitum (open squares), non-fed for 16 h and subsequently refed for 8 h (closed squares). An arrow indicates the timing of refeeding. An asterisk () indicates a significant difference compared with the larvae fed ad libitum by one-way ANOVA (P < 0.05). Data represent means ± S.D. (n = 5). (E) Effects of synthetic AT (open circles), GSRYa-1 (closed circles), and GSRYa-2 (closed triangles) on forward contraction wave and peristaltic squeeze in the larvae fed ad libitum. Distilled water was used as a vehicle control (0 M) and 10−7–10−4 M of peptide solution were dorsolaterally injected. Asterisks () indicate significant differences compared with vehicle-injected larvae by one-way ANOVA (P < 0.05). Data represent means ± S.D. (n = 5). https://doi org/10 1371/journal pone 0219050 g006 Fig 6. Observation of esophageal contraction in intact larvae. (A) Pictures of normal (a) and transparent larvae (b). (B) Effects of synthetic AT (open circles), GSRYa-1 (closed circles), and GSRYa-2 (closed triangles) on the latency to the first bite. Asterisks () indicate significant differences compared with vehicle-injected larvae by one-way ANOVA (P < 0.01). Data represent means ± S.D. (n = 3). (C) Schematic diagrams and pictures of larval thorax under transmitted light. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Identification of AT and GSRYa-1 and -2 as feeding inhibitory “brain-gut peptides” from B. mori larvae During the states of hunger and subsequent lack of nutrition, extended feeding duration to ingest more food might occur in the pharynx and esophagus as a machinery to food entry the food by decreasing the sensitivities to feeding inhibitory GSRYa-1 and -2 following the reduced expression of its receptor (Fig 5G). level, this intriguing effect of AT and GSRYa-1 and -2 may be crucial in determining the unknown pleiotropic function of these peptides. In addition, the inhibition after exposure to AT and GSRYa-1, 2 did not resume even after washing with the vehicle without the ligands. AT and GSRYa 1, 2 did not resume even after washing with the vehicle without the ligands. In B. mori, 40 BNGRs are expressed in different tissues in distinct expression patterns [32]. Among the receptors, several receptors show ubiquitous expression patterns as observed in four BNGRs in this study (BNGR-A5, -A16, -A19, and -A22), indicating that GSRYa-1 and -2 may have pleiotropic functions as well as AT (Fig 4F). In this study, the expression patterns of those receptors in the peristaltic intestine (Fig 4F) covered the area where contraction was influenced by both AT and GSRYa-1 and -2 (Fig 7). Because different sensitivities of the phar- ynx to AT and GSRYa-1 and -2 were observed in the non-fed larvae (Fig 5F left), the feeding state can also be shifted or modified by altered expression levels of GSRYa-1 and -2 receptors. During the states of hunger and subsequent lack of nutrition, extended feeding duration to ingest more food might occur in the pharynx and esophagus as a machinery to food entry the food by decreasing the sensitivities to feeding inhibitory GSRYa-1 and -2 following the reduced expression of its receptor (Fig 5G). Identification of AT and GSRYa-1 and -2 as feeding inhibitory “brain-gut peptides” from B. mori larvae Although few studies have focused on these kinds of effects of different peptides in the species level or in the individual PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 16 / 24 Enteroendocrine control of feeding behavior in B. mori Fig 7. Proposed regulatory model of feeding behavior in B. mori larvae. AT and GSRYamide are secreted from the posterior and anterior midgut EEs. After circulating in the hemolymph, these peptides act directly on the foregut and hindgut to inhibit intestinal contraction. This myoinhibitory effect induces a larval feeding state transition from the feeding to the non-feeding (quiescent) mode, eventually influencing feeding termination. EEs: enteroendocrine cells, FG: foregut, MG: midgut, HG: hindgut. https://doi org/10 1371/journal pone 0219050 g007 Fig 7. Proposed regulatory model of feeding behavior in B. mori larvae. AT and GSRYamide are secreted from the posterior and anterior midgut EEs. After circulating in the hemolymph, these peptides act directly on the foregut and hindgut to inhibit intestinal contraction. This myoinhibitory effect induces a larval feeding state transition from the feeding to the non-feeding (quiescent) mode, eventually influencing feeding termination. EEs: enteroendocrine cells, FG: foregut, MG: midgut, HG: hindgut. https://doi.org/10.1371/journal.pone.0219050.g007 https://doi.org/10.1371/journal.pone.0219050.g007 level, this intriguing effect of AT and GSRYa-1 and -2 may be crucial in determining the unknown pleiotropic function of these peptides. In addition, the inhibition after exposure to AT and GSRYa-1, 2 did not resume even after washing with the vehicle without the ligands. In B. mori, 40 BNGRs are expressed in different tissues in distinct expression patterns [32]. Among the receptors, several receptors show ubiquitous expression patterns as observed in four BNGRs in this study (BNGR-A5, -A16, -A19, and -A22), indicating that GSRYa-1 and -2 may have pleiotropic functions as well as AT (Fig 4F). In this study, the expression patterns of those receptors in the peristaltic intestine (Fig 4F) covered the area where contraction was influenced by both AT and GSRYa-1 and -2 (Fig 7). Because different sensitivities of the phar- ynx to AT and GSRYa-1 and -2 were observed in the non-fed larvae (Fig 5F left), the feeding state can also be shifted or modified by altered expression levels of GSRYa-1 and -2 receptors. Model of feeding regulatory mechanism via control of intestinal contraction in B. mori larvae In other studies, AT and allatostatin (AST) regulate spontaneous contraction of the foregut of Helicoverpa armigera [25] and Lacanobia oleracea [26]. Furthermore, myosuppressin (leukomyosuppressin), a member of the structurally related RFamide peptide family [33], also regulates spontaneous contraction of the foregut of Lacano- bia oleracea [66] and the hindgut of Leucophaea maderae [13]. In fact, we have observed sev- eral fractions with weak inhibitory activities during the purification steps, which may correspond to the previous reports that those allatoregulatory peptides such as AST and FGLa- mide are present in the midgut and modulate the gut contraction. Taken together, midgut- derived AT and GSRYamide can contribute predominantly to the regulation of feeding behav- ior by modulating intestinal contraction as currently depicted in a model of “feeding-termina- tion loop” (Fig 7). First, AT and GSRYamide are secreted from the posterior and anterior midgut EEs, respectively. Once these secreted peptides circulate in the hemolymph, they act directly on the foregut and hindgut to inhibit intestinal contraction. These myoinhibitory effects induce the stagnation of the ingested food in the foregut and fecal passage through the hindgut, accompanied by a larval feeding state transition from the feeding to the non-feeding (quiescent) mode. Consequently, these peptides appear to induce feeding termination. A pre- vious study demonstrated that the arrest of pharynx movement by the stagnation of the ingested food induces the quiescent mode of feeding in B. mori [67]. In many insect species, foregut muscular cells are innervated from the brain and frontal gan- glion [68] within a short neural circuit involving the stomatogastric central pattern generator [5, 69]. Many neuropeptides, such as AST-A, RFamide, sNPF and TRPs including AT, have been observed in the stomatogastric nervous system (SNS) [5]. In particular, AT can contribute to the modulation of feeding via SNS by controlling spontaneous foregut contractions in many insect species, including D. melanogaster, and M. sexta [70–72]. The interaction of those neuropeptides, such as myoactive peptides that regulate intestinal contraction, may play an important role in the shift of the larval feeding state by harmonization of those neuropeptides. In B. mori larvae, pharynx movements for swallowing food are linked by a neuron projecting from brain and fron- tal ganglion [73]. In addition, AT-driving contractions of the foregut and hindgut may be pro- duced by neurons innervating from these parts of the intestine [20]. The expression of B. Model of feeding regulatory mechanism via control of intestinal contraction in B. mori larvae Previous ultrastructural studies have demonstrated that exocytosis occurs at both the basal and lateral surfaces of insect midgut EEs, depicting the possibilities of secretion of biologically active compounds [58, 59]. AT-producing EEs (Fig 2C and 2D) and GSRYamide-producing EEs (Fig 2E and 2F) also have the potential to deliver secretory granules containing peptidyl factors to the hemocoel side through a cellular membrane [60]. Those EEs have similar cellular morphology to EEs releasing other arthropod neuropeptide CCAP (Crustacean cardioactive peptide) observed in the American cockroach, Periplaneta americana [61]. CCAP has multiple roles, including gut contraction [61] as observed for AT and GSRYamide in the present study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 17 / 24 Enteroendocrine control of feeding behavior in B. mori Similarly, the presence of EEs secreting allatoregulatory peptides has also been reported in Dipteran and Hemipteran species [62–65]. In other studies, AT and allatostatin (AST) regulate spontaneous contraction of the foregut of Helicoverpa armigera [25] and Lacanobia oleracea [26]. Furthermore, myosuppressin (leukomyosuppressin), a member of the structurally related RFamide peptide family [33], also regulates spontaneous contraction of the foregut of Lacano- bia oleracea [66] and the hindgut of Leucophaea maderae [13]. In fact, we have observed sev- eral fractions with weak inhibitory activities during the purification steps, which may correspond to the previous reports that those allatoregulatory peptides such as AST and FGLa- mide are present in the midgut and modulate the gut contraction. Taken together, midgut- derived AT and GSRYamide can contribute predominantly to the regulation of feeding behav- ior by modulating intestinal contraction as currently depicted in a model of “feeding-termina- tion loop” (Fig 7). First, AT and GSRYamide are secreted from the posterior and anterior midgut EEs, respectively. Once these secreted peptides circulate in the hemolymph, they act directly on the foregut and hindgut to inhibit intestinal contraction. These myoinhibitory effects induce the stagnation of the ingested food in the foregut and fecal passage through the hindgut, accompanied by a larval feeding state transition from the feeding to the non-feeding (quiescent) mode. Consequently, these peptides appear to induce feeding termination. A pre- vious study demonstrated that the arrest of pharynx movement by the stagnation of the ingested food induces the quiescent mode of feeding in B. mori [67]. Similarly, the presence of EEs secreting allatoregulatory peptides has also been reported in Dipteran and Hemipteran species [62–65]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Model of feeding regulatory mechanism via control of intestinal contraction in B. mori larvae mori sNPF and TRPs, which have feeding acceleratory effects [16], and their receptor BNGRs (BNGR-A10, -24, and -A32) were confirmed in the brain and CNS [34, 40, 74]. Therefore, the expression of AT, GSRYamide and their receptor BNGRs (BNGR-A5, -16, -A19, and -A22) in the brain and CNS (Figs 2B and 4F) implies that AT and GSRYa-1 and -2 can function as neuro- transmitters and interact with sNPF or TRPs to control intestinal contractions in B. mori larvae. (BNGR-A10, -24, and -A32) were confirmed in the brain and CNS [34, 40, 74]. Therefore, the expression of AT, GSRYamide and their receptor BNGRs (BNGR-A5, -16, -A19, and -A22) in the brain and CNS (Figs 2B and 4F) implies that AT and GSRYa-1 and -2 can function as neuro- transmitters and interact with sNPF or TRPs to control intestinal contractions in B. mori larvae. Neuropeptides released from nerve terminal also function as endocrine factors in addition to as the neurotransmitters [75, 76]. AT produced from CNS travels through ventral nerve cord and gets released into the hemolymph to modulate the intestinal contraction. However, the number of AT-producing EEs in the whole midgut seems to be much higher than that in the CNS (Fig 2C) [20, 77]. Therefore, the main source of AT released into the hemolymph is probably midgut EEs. In addition, the previous report demonstrates that GSRYamide is pro- duced only in the interneurons within CNS in this species [19]. Therefore, midgut EEs are the only possible source of GSRYamide in the hemolymph. Our proposed regulatory model is more plausible for GSRYamide. Although it remains to be elucidated whether neurosecretory AT affects the intestinal con- tractions, our findings in this study suggest that hormonal AT and GSRYamide secreted from the midgut to the hemolymph can also contribute to the physiological functions, including regulation in intestinal contraction of B. mori. In addition, our “feeding-termination loop” PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 18 / 24 Enteroendocrine control of feeding behavior in B. mori model provides a response to acute EE-derived feeding regulatory signals without mediation via the brain or CNS, allowing for a smooth feeding state transition. Supporting information S1 Fig. MALDI-TOF mass analyses of the final step purified fractions A1 and B3. (A) MALDI-TOF mass spectrum of A1 fraction (Fig 1A). Monoisotopic ion peak ([M+H]+) at m/z 1486.9 consistent with the H+ adduct of uncharged AT is labeled. (B) MALDI-TOF mass spec- trum of B3 fraction (Fig 1B). Monoisotopic ion peak ([M+H]+) at m/z 1999.9 consistent with the H+ adduct of uncharged GSRYa-1 is labeled. (TIF) S2 Fig. cDNA and deduced amino acid sequences encoding GSRYa-1 and -2. The deduced mature GSRYa-1 and -2 are underlined by a bold line and a dashed line, respectively. Arrows (GSRYa-Fw and GSRYa-Rv) represent the forward and reverse primer sites for RT-PCR and in situ hybridization. The stop codon is indicated by an asterisk. (TIF) S3 Fig. Alignment of the amino acid sequences among B. mori GSRYamides and other insect RYamides. By BLAST (blastn, tblastn and Blastp) search in the GenBank database, simi- lar sequences to GSRYa-1 and -2 were obtained. B. mori GSRYa-1 and -2, RYa-1 and -2 of D. melanogaster [29], T. castaneum and Aedes aegypti [30] are aligned. Residues identical between peptides are shaded. (TIF) S1 Movie. The contraction of pharynx after exposure to B. mori Ringer’s solution and GSRYa-1. B. mori Ringer’s solution and GSRYa-1 (10−6 M) solution were exposed at 60 s and 120 s in Fig 5C (Arrowheads). After exposure to GSRYa-1, the contraction of pharynx was inhibited, while exposure of B. mori Ringer’s solution had little effect on the contraction. (MP4) S2 Movie. The contraction of ileum after exposure to B. mori Ringer’s solution and AT. B. mori Ringer’s solution and AT (10−6 M) solution were exposed at 60 s and 120 s in Fig 5D (Arrowheads). After exposure to AT, the contraction of ileum was inhibited, while exposure of B. mori Ringer’s solution had little effect on the contraction. (MP4) S3 Movie. The esophageal contraction. Forward contraction wave (Fig 6C (b)) and peristaltic squeeze (Fig 6C (c)) were visualized under the transmitted light in the transparent larvae. (MP4) S4 Movie. The esophageal contraction before and after injection of AT. AT (10−5 M) solu- tion was injected dorsolaterally into the hemolymph. After injection of AT, the forward con- traction wave and peristaltic squeeze were inhibited. (MP4) Acknowledgments We thank Dr. Takeshi Kawai of the Department of Applied Biological Chemistry, the Univer- sity of Tokyo, for technical support regarding chemical peptide synthesis. We thank Dr. Takeshi Kawai of the Department of Applied Biological Chemistry, the Univer- sity of Tokyo, for technical support regarding chemical peptide synthesis. Author Contributions References 1. Schoonhoven LM, van Loon JJA, Dicke M. Insect-Plant Biology. Oxford: Oxford University Press; 2005. 2. Simpson SJ, Raubenheimer D. The central role of the hemolymph in the regulation of nutrient intake in insects. Physiol Entomol. 1993; 18(4):395–403. WOS:A1993MP27600009. 3. Timmins WA, Reynolds SE. Physiological-mechanisms underlying the control of meal size in Manduca sexta larvae. Physiol Entomol. 1992; 17(1):81–9. https://doi.org/10.1111/j.1365-3032.1992.tb00993.x WOS:A1992HL35500011. 4. Abisgold JD, Simpson SJ. The effect of dietary-protein levels and hemolymph composition on the sensi- tivity of the maxillary palp chemoreceptors of locusts. J Exp Biol. 1988; 135:215–29. WOS: A1988M698500015. 5. Audsley N, Weaver RJ. Neuropeptides associated with the regulation of feeding in insects. Gen Comp Endocrinol. 2009; 162(1):93–104. https://doi.org/10.1016/j.ygcen.2008.08.003 WOS:000265906900012. PMID: 18775723 6. Nassel DR, Winther AME. Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol. 2010; 92(1):42–104. https://doi.org/10.1016/j.pneurobio.2010.04.010 WOS:000281187200004. PMID: 20447440 7. Lu D, Lee KY, Horodyski FM, Witten JL. Molecular characterization and cell-specific expression of a Manduca sexta FLRFamide gene. J Comp Neurol. 2002; 446(4):377–96. https://doi.org/10.1002/cne. 10205 WOS:000174966300006. PMID: 11954036 8. Roller L, Yamanaka N, Watanabe K, Daubnerova I, Zitnan D, Kataoka H, et al. The unique evolution of neuropeptide genes in the silkworm Bombyx mori. Insect Biochem Mol Biol. 2008; 38(12):1147–57. https://doi.org/10.1016/j.ibmb.2008.04.009 WOS:000264262200013. PMID: 19280707 9. Veenstra JA, Agricola H-J, Sellami A. Regulatory peptides in fruit fly midgut. Cell Tissue Res. 2008; 334 (3):499–516. https://doi.org/10.1007/s00441-008-0708-3 WOS:000262734300013. PMID: 18972134 10. Reichwald K, Unnithan GC, Davis NT, Agricola H, Feyereisen R. Expression of the allatostatin gene in endocrine-cells of the cockroach midgut. Proc Natl Acad Sci USA. 1994; 91(25):11894–8. https://doi. org/10.1073/pnas.91.25.11894 WOS:A1994PW70800027. PMID: 7991553 11. Spit J, Badisco L, Verlinden H, Van Wielendaele P, Zels S, Dillen S, et al. Peptidergic control of food intake and digestion in insects. Can J ZoolCan J Zool. 2012; 90(4):489–506. https://doi.org/10.1139/ z2012-014 WOS:000304677200006. 12. Song W, Veenstra JA, Perrimon N. Control of Lipid Metabolism by Tachykinin in Drosophila. Cell Rep. 2014; 9(1):40–7. https://doi.org/10.1016/j.celrep.2014.08.060 WOS:000344468100006. PMID: 25263556 13. Holman GM, Cook BJ, Nachman RJ. Isolation, primary structure and synthesis of leukomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1986; 85(2):329–33. https://doi.org/10.1016/0742-8413(86) 90202-1 WOS:A1986F166000012. 14. Schoofs L, Holman GM, Hayes TK, Nachman RJ, Deloof A. Locustatachykinin I and II, two novel insect neuropeptides with homology to peptides of the vertebrate tachykinin family. FEBS Lett. 1990; 261 (2):397–401. https://doi.org/10.1016/0014-5793(90)80601-e WOS:A1990CU75900044. PMID: 2311766 15. Author Contributions Funding acquisition: Ladislav Roller, Dusˇan Zˇitňan, Shinji Nagata. Funding acquisition: Ladislav Roller, Dusˇan Zˇitňan, Shinji Nagata. Funding acquisition: Ladislav Roller, Dusˇan Zˇitňan, Shinji Nagata. 19 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Enteroendocrine control of feeding behavior in B. mori Investigation: Sumihiro Matsumoto, Natsumaro Kutsuna, Ivana Daubnerova´, Ladislav Roller, Dusˇan Zˇitňan, Shinji Nagata. Investigation: Sumihiro Matsumoto, Natsumaro Kutsuna, Ivana Daubnerova´, Ladislav Roller, Dusˇan Zˇitňan, Shinji Nagata. Methodology: Natsumaro Kutsuna. Supervision: Hiromichi Nagasawa, Shinji Nagata. Writing – original draft: Sumihiro Matsumoto, Shinji Nagata. Writing – review & editing: Ladislav Roller, Dusˇan Zˇitňan. Investigation: Sumihiro Matsumoto, Natsumaro Kutsuna, Ivana Daubnerova´, Ladislav Roller, Dusˇan Zˇitňan, Shinji Nagata. Methodology: Natsumaro Kutsuna. Supervision: Hiromichi Nagasawa, Shinji Nagata. Supervision: Hiromichi Nagasawa, Shinji Nagata. Writing – original draft: Sumihiro Matsumoto, Shinji Nagata. Writing – review & editing: Ladislav Roller, Dusˇan Zˇitňan. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 References Schoofs L, Holman GM, Hayes TK, Kochansky JP, Nachman RJ, Deloof A. Locustatachykinin-III and locustatachykinin-IV—2 additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family. Regul Pept. 1990; 31(3):199–212. https://doi.org/10.1016/0167-0115(90)90006-i WOS:A1990EM84500006. PMID: 2132575 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 20 / 24 Enteroendocrine control of feeding behavior in B. mori 16. Nagata S, Morooka N, Matsumoto S, Kawai T, Nagasawa H. Effects of neuropeptides on feeding initia- tion in larvae of the silkworm, Bombyx mori. Gen Comp Endocrinol. 2011; 172(1):90–5. https://doi.org/ 10.1016/j.ygcen.2011.03.004 WOS:000290842200014. PMID: 21397600 17. Nagata S, Morooka N, Matsumoto S, Nagasawa H. Characterization of feeding-delaying factors from the silkworm Bombyx mori. Ann N Y Acad Sci. 2009; 1163:481–3. Epub 2009/05/22. https://doi.org/10. 1111/j.1749-6632.2009.04454.x PMID: 19456393. 18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utiliz- ing the principle of protein-dye binding. Anal Biochem. 1976; 72:248–54. Epub 1976/05/07. PMID: 942051. 19. Roller L, Cizmar D, Bednar B, Zitnan D. Expression of RYamide in the nervous and endocrine system of Bombyx mori. Peptides. 2016; 80:72–9. https://doi.org/10.1016/j.peptides.2016.02.003 WOS:000375936400010. PMID: 26896568 20. Bednar B, Roller L, Cizmar D, Mitrova D, Zitnan D. Developmental and sex-specific differences in expression of neuropeptides derived from allatotropin gene in the silkmoth Bombyx mori. Cell Tissue Res. 2017; 368(2):259–75. https://doi.org/10.1007/s00441-016-2556-x WOS:000399916800004. PMID: 28091775 21. Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR. Substitution of three amino acids switches recep- tor specificity of Gq alpha to that of Gi alpha. Nature. 1993; 363(6426):274–6. Epub 1993/05/20. https:// doi.org/10.1038/363274a0 PMID: 8387644. 22. Conklin BR, Herzmark P, Ishida S, Voyno-Yasenetskaya TA, Sun Y, Farfel Z, et al. Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation. Mol Pharmacol. 1996; 50 (4):885–90. Epub 1996/10/01. PMID: 8863834. 23. Vernon WI, Printen JA. Assay for intracellular calcium using a codon-optimized aequorin. Biotechni- ques. 2002; 33(4):730, 2, 4. Epub 2002/10/26. https://doi.org/10.2144/02334bm02 PMID: 12398176. 24. Hukuhara T, Satake S. Contractile movements of the larval fore- and hindguts of the silkworm, Bombyx mori. J Sericult Sci Japan. 1985; 54(1):82–6. 25. Duve H, East PD, Thorpe A. Regulation of lepidopteran foregut movement by allatostatins and allatotro- pin from the frontal ganglion. J Comp Neurol. 1999; 413(3):405–16. https://doi.org/10.1002/(sici)1096- 9861(19991025)413:3<405::aid-cne4>3.3.co;2-i WOS:000082661800004. PMID: 10502248 26. Matthews HJ, Audsley N, Weaver RJ. Interactions between allatostatins and allatotropin on spontane- ous contractions of the foregut of larval Lacanobia oleracea. J Insect Physiol. 2007; 53(1):75–83. https://doi.org/10.1016/j.jinsphys.2006.10.007 WOS:000244027300009. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 References Activation of Bombyx neuropeptide G protein- coupled receptor A4 via a Gα i-dependent signaling pathway by direct interaction with neuropeptide F from silkworm, Bombyx mori. Insect Biochem Mol Biol. 2014; 45:77–88. Epub 2014/01/01. https://doi. org/10.1016/j.ibmb.2013.12.007 PMID: 24374022. 38. Ma Q, Cao Z, Yu YN, Yan LL, Zhang WJ, Shi Y, et al. Bombyx neuropeptide G protein-coupled receptor A7 is the third cognate receptor for short neuropeptide F from silkworm. J Biol Chem. 2017; 292 (50):20599–612. https://doi.org/10.1074/jbc.M117.815191 WOS:000418267000021. PMID: 29084843 39. Jiang H, Lkhagva A, Daubnerova I, Chae HS, Simo L, Jung SH, et al. Natalisin, a tachykinin-like signal- ing system, regulates sexual activity and fecundity in insects. Proc Natl Acad Sci USA. 2013; 110(37): E3526–34. Epub 2013/08/28. https://doi.org/10.1073/pnas.1310676110 PMID: 23980168; PubMed Central PMCID: PMC3773796. 40. Nagai-Okatani C, Nagasawa H, Nagata S. Tachykinin-related peptides share a G protein-coupled receptor with ion transport peptide-like in the Silkworm Bombyx mori. PLoS One. 2016; 11(6): e0156501. https://doi.org/10.1371/journal.pone.0156501 MEDLINE:27248837. PMID: 27248837 41. Collin C, Hauser F, Krogh-Meyer P, Hansen KK, de Valdivia EG, Williamson M, et al. Identification of the Drosophila and Tribolium receptors for the recently discovered insect RYamide neuropeptides. Bio- chem Biophys Res Commun. 2011; 412(4):578–83. https://doi.org/10.1016/j.bbrc.2011.07.131 WOS:000295148500013. PMID: 21843505 42. Kataoka H, Toschi A, Li JP, Carney RL, Schooley DA, Kramer SJ. Identification of an allatotropin from adult Manduca sexta. Science. 1989; 243(4897):1481–3. https://doi.org/10.1126/science.243.4897. 1481 WOS:A1989T685700033. PMID: 17839751 43. Park C, Hwang JS, Kang SW, Lee BH. Molecular characterization of a cDNA from the silk moth Bombyx mori encoding Manduca sexta allatotropin peptide. Zoolog Sci. 2002; 19(3):287–92. https://doi.org/10. 2108/zsj.19.287 WOS:000176735000005. PMID: 12125926 44. Weaver RJ, Audsley N. Neuropeptide regulators of juvenile hormone synthesis: structures, functions, distribution, and unanswered questions. Ann N Y Acad Sci. 2009; 1163:316–29. https://doi.org/10. 1111/j.1749-6632.2009.04459.x WOS:000266493400030. PMID: 19456353 45. Koladich PM, Cusson M, Bendena WG, Tobe SS, McNeil JN. Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth, Pseudaletia unipuncta. Peptides. 2002; 23(4):645–51. https://doi.org/10.1016/s0196-9781(01)00658-1 WOS:000174578700007. PMID: 11897383 46. Lee KY, Horodyski FM, Chamberlin ME. Inhibition of midgut ion transport by allatotropin (Mas-AT) and Manduca FLRFamides in the tobacco hornworm Manduca sexta. J Exp Biol. 1998; 201(22):3067–74. WOS:000077543000006. 47. Oeh U, Dyker H, Losel P, Hoffmann KH. In vivo effects of Manduca sexta allatotropin and allatostatin on development and reproduction in the fall armyworm, Spodoptera frugiperda (Lepidoptera, Noctuidae). Invertebr Repr Dev. 2001; 39(3):239–47. https://doi.org/10.1080/07924259.2001.9652488 WOS:000171879700008. 48. Duve H, Audsley N, Weaver RJ, Thorpe A. References PMID: 17150228 27. Jose Villalobos-Sambucaro M, Nieves Lorenzo-Figueiras A, Luis Riccillo F, Anibal Diambra L, Gabriel Noriega F, Rafael Ronderos J. Allatotropin modulates myostimulatory and cardioacceleratory activities in Rhodnius prolixus (Stal). PLoS One. 2015; 10(4). https://doi.org/10.1371/journal.pone.0124131 WOS:000353212600050. PMID: 25897783 28. Tsujita M, Sakurai S. Melamine action in the phenocopy of pigment elimination from hypodermal cells of the silkworm larva. Proc Jap Acad. 1963; 39:513–8. 29. Ida T, Takahashi T, Tominaga H, Sato T, Kume K, Ozaki M, et al. Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila. Bio- chem Biophys Res Commun. 2011; 410(4):872–7. https://doi.org/10.1016/j.bbrc.2011.06.081 WOS:000293204600029. PMID: 21704020 30. Hauser F, Neupert S, Williamson M, Predel R, Tanaka Y, Grimmelikhuijzen CJP. Genomics and pepti- domics of neuropeptides and protein hormones present in the parasitic wasp Nasonia vitripennis. J Pro- teome Res. 2010; 9(10):5296–310. https://doi.org/10.1021/pr100570j WOS:000282257800039. PMID: 20695486 31. Nagata S, Matsumoto S, Mizoguchi A, Nagasawa H. Identification of cDNAs encoding allatotropin and allatotropin-like peptides from the silkworm, Bombyx mori. Peptides. 2012; 34(1):98–105. https://doi. org/10.1016/j.peptides.2012.01.002 WOS:000302851500014. PMID: 22265806 32. Yamanaka N, Yamamoto S, Zitnan D, Watanabe K, Kawada T, Satake H, et al. Neuropeptide receptor transcriptome reveals unidentified neuroendocrine pathways. PLoS One. 2008; 3(8):e3048. https://doi. org/10.1371/journal.pone.0003048 WOS:000264429000001. PMID: 18725956 33. Bechtold DA, Luckman SM. The role of RFamide peptides in feeding. J Endocrinol. 2007; 192(1):3–15. https://doi.org/10.1677/JOE-06-0069 WOS:000244958100002. PMID: 17210738 34. Nagai C, Mabashi-Asazuma H, Nagasawa H, Nagata S. Identification and characterization of receptors for ion transport peptide (ITP) and ITP-like (ITPL) in the Silkworm Bombyx mori. J Biol Chem. 2014; 289 (46):32166–77. https://doi.org/10.1074/jbc.M114.590646 WOS:000345314700044. PMID: 25278025 35. Yang J, Huang H, Yang H, He X, Jiang X, Shi Y, et al. Specific activation of the G protein-coupled recep- tor BNGR-A21 by the neuropeptide corazonin from the silkworm, Bombyx mori, dually couples to the G PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 21 / 24 Enteroendocrine control of feeding behavior in B. mori (q) and G(s) signaling cascades. J Biol Chem. 2013; 288(17):11662–75. Epub 2013/03/05. https://doi. org/10.1074/jbc.M112.441675 PMID: 23457297; PubMed Central PMCID: PMC3636857. 36. Shi Y, Huang H, Deng X, He X, Yang J, Yang H, et al. Identification and functional characterization of two orphan G-protein-coupled receptors for adipokinetic hormones from silkworm Bombyx mori. J Biol Chem. 2011; 286(49):42390–402. Epub 2011/10/20. https://doi.org/10.1074/jbc.M111.275602 PMID: 22009754; PubMed Central PMCID: PMC3234951. 37. Deng X, Yang H, He X, Liao Y, Zheng C, Zhou Q, et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 References Triple co-localisation of two types of allatostatin and an alla- totropin in the frontal ganglion of the lepidopteran Lacanobia oleracea (Noctuidae): innervation and action on the foregut. Cell Tissue Res. 2000; 300(1):153–63. https://doi.org/10.1007/s004410050056 WOS:000086880100015. PMID: 10805084 49. Lismont E, Vleugels R, Marchal E, Badisco L, Van Wielendaele P, Lenaerts C, et al. Molecular cloning and characterization of the allatotropin precursor and receptor in the desert locust, Schistocerca gre- garia. Front Neurosci. 2015; 9:84 https://doi.org/10.3389/fnins.2015.00084 WOS:000352968700001. PMID: 25814925 50. Santini MS, Ronderos JR. Allatotropin-like peptide released by Malpighian tubules induces hindgut activity associated with diuresis in the Chagas disease vector Triatoma infestans (Klug). J Exp Biol. 2007; 210(11):1986–91. https://doi.org/10.1242/jeb.004291 WOS:000246665600021. PMID: 17515423 51. Oeh U, Antonicek H, Nauen R. Myotropic effect of helicokinins, tachykinin-related peptides and Man- duca sexta allatotropin on the gut of Heliothis virescens (Lepidoptera: Noctuidae). J Insect Physiol. 2003; 49(4):323–37. Epub 2003/05/29. PMID: 12769986. 22 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Enteroendocrine control of feeding behavior in B. mori 52. Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y—a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature. 1982; 296(5858):659–60. https://doi.org/10.1038/ 296659a0 WOS:A1982NK38700048. PMID: 6896083 53. Brown MR, Crim JW, Arata RC, Cai HN, Chun C, Shen P. Identification of a Drosophila brain-gut pep- tide related to the neuropeptide Y family. Peptides. 1999; 20(9):1035–42. https://doi.org/10.1016/ s0196-9781(99)00097-2 WOS:000082606900003. PMID: 10499420 54. Wu Q, Wen TQ, Lee G, Park JH, Cai HN, Shen P. Developmental control of foraging and social behav- ior by the Drosophila neuropeptide Y-like system. Neuron. 2003; 39(1):147–61. https://doi.org/10.1016/ s0896-6273(03)00396-9 WOS:000184057400016. PMID: 12848939 55. Maeda T, Nakamura Y, Shiotani H, Hojo MK, Yoshii T, Ida T, et al. Suppressive effects of dRYamides on feeding behavior of the blowfly, Phormia regina. Zoological Lett. 2015; 1:35. Epub 2015/12/10. https://doi.org/10.1186/s40851-015-0034-z PMID: 26649188; PubMed Central PMCID: PMC4672552. 56. Lee KS, You KH, Choo JK, Han YM, Yu K. Drosophila short neuropeptide F regulates food intake and body size. J Biol Chem. 2004; 279(49):50781–9. https://doi.org/10.1074/jbc.M407842200 WOS:000225355800020. PMID: 15385546 57. Dillen S, Zels S, Verlinden H, Spit J, Van Wielendaele P, Vanden Broeck J. Functional characterization of the short neuropeptide F receptor in the desert locust, Schistocerca gregaria. PLoS One. 2013; 8(1): e53604. Epub 2013/01/12. https://doi.org/10.1371/journal.pone.0053604 PMID: 23308260; PubMed Central PMCID: PMC3537624. 58. Endo Y, Nishiitsutsujiuwo J. Exocytotic release of secretory granules from endocrine cells in the midgut of insects. Cell Tissue Res. 1982; 222(3):515–22. WOS:A1982NC86700004. References PMID: 7060100 59. Endo Y, Nishiitsutsujiuwo J. Gut endocrine cells in insects: the ultrastructure of the gut endocrine cells of the lepidopterous species. Biomed Research Tokyo. 1981; 2(3):270–80. WOS:A1981LU99100003. 60. Leite ACR, Evangelista LG. Ultrastructure of endocrine cells from the abdominal midgut epithelium of Lutzomyia longipalpis (Diptera: psychodidae). J Med Entomol. 2001; 38(5):749–52. https://doi.org/10. 1603/0022-2585-38.5.749 WOS:000171279100022. PMID: 11580051 61. Sakai T, Satake H, Minakata H, Takeda M. Characterization of crustacean cardioactive peptide as a novel insect midgut factor: Isolation, localization, and stimulation of alpha-amylase activity and gut con- traction. Endocrinology. 2004; 145(12):5671–8. https://doi.org/10.1210/en.2004-0722 WOS:000225109400033. PMID: 15358677 62. Hernandez-Martinez S, Li Y, Lanz-Mendoza H, Rodriguez MH, Noriega FG. Immunostaining for allato- tropin and allatostatin-A and -C in the mosquitoes Aedes aegypti and Anopheles albimanus. Cell Tissue Res. 2005; 321(1):105–13. Epub 2005/05/24. https://doi.org/10.1007/s00441-005-1133-5 PMID: 15909164; PubMed Central PMCID: PMC2647714. 63. Riccillo FL, Ronderos JR. Allatotropin expression during the development of the fourth instar larvae of the kissing-bug Triatoma infestans (Klug). Tissue Cell. 2010; 42(6):355–9. Epub 2010/09/08. https:// doi.org/10.1016/j.tice.2010.07.011 PMID: 20817237. 64. Sarkar NR, Tobe SS, Orchard I. The distribution and effects of Dippu-allatostatin-like peptides in the blood-feeding bug, Rhodnius prolixus. Peptides. 2003; 24(10):1553–62. Epub 2004/01/07. PMID: 14706534. 65. Sterkel M, Riccillo FL, Ronderos JR. Cardioacceleratory and myostimulatory activity of allatotropin in Triatoma infestans. Comp Biochem Physiol A Mol Integr Physiol. 2010; 155(3):371–7. https://doi.org/ 10.1016/j.cbpa.2009.12.002 WOS:000274752600012. PMID: 19995615 66. Matthews HJ, Audsley N, Weaver RJ. In vitro and in vivo effects of myo-active peptides on larvae of the tomato moth Lacanobia oleracea and the cotton leaf worm Spodoptera littoralis (Lepidoptera; Noctui- dae). Arch Insect Biochem Physiol. 2008; 69(2):60–9. https://doi.org/10.1002/arch.20265 WOS:000259276400002. PMID: 18780345 67. Kamioka S. Effect of stomodaeal nervous system on the movement of alimentary canal and dorsal ves- sel in the silkworm larva. J Sericult Sci Japan. 1953; 22(1):1–10. https://doi.org/10.11416/ kontyushigen1930.22.1 68. Willey RB. The morphology of the stomodeal nervous system in Periplaneta americana (L.) and other blattaria. J Morph. 1961; 108:219–61. https://doi.org/10.1002/jmor.1051080207 PMID: 13785379 69. Ayali A. The insect frontal ganglion and stomatogastric pattern generator networks. Neurosignals. 2004; 13(1–2):20–36. https://doi.org/10.1159/000076156 WOS:000220052700003. PMID: 15004423 70. Miles CI, Booker R. The role of the frontal ganglion in the feeding and eclosion behavior of the moth Manduca sexta. J Exp Biol. 1998; 201(11):1785–98. WOS:000074325400011. 71. Miles CI, Booker R. Octopod, a homeotic mutation of the moth Manduca sexta, affects development of both mesodermal and ectodermal structure. Dev Biol. 1993; 155(1):147–60. https://doi.org/10.1006/ dbio.1993.1014 WOS:A1993KF57100014. PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 References PMID: 8093235 23 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0219050 July 1, 2019 Enteroendocrine control of feeding behavior in B. mori 72. Hartenstein V, Tepass U, GruszynskideFeo E. Proneural and neurogenic genes control specification and morphogenesis of stomatogastric nerve cell precursors in Drosophila. Dev Biol. 1996; 173(1):213– 27. https://doi.org/10.1006/dbio.1996.0018 WOS:A1996TU03300018. PMID: 8575623 72. Hartenstein V, Tepass U, GruszynskideFeo E. Proneural and neurogenic genes control specification and morphogenesis of stomatogastric nerve cell precursors in Drosophila. Dev Biol. 1996; 173(1):213– 27. https://doi.org/10.1006/dbio.1996.0018 WOS:A1996TU03300018. PMID: 8575623 73. Sasaki K, Asaoka K. Swallowing motor pattern triggered and modified by sucrose stimulation in the lar- vae of the silkworm, Bombyx mori. J Insect Physiol. 2006; 52(5):528–37. https://doi.org/10.1016/j. jinsphys.2006.02.001 WOS:000237998900015. PMID: 16540116 74. Nagata S, Matsumoto S, Nakane T, Ohara A, Morooka N, Konuma T, et al. Effects of starvation on brain short neuropeptide F-1, -2, and -3 levels and short neuropeptide F receptor expression levels of the silkworm, Bombyx mori. Front Endocrinol (Lausanne). 2012; 3:3–. https://doi.org/10.3389/fendo. 2012.00003 MEDLINE:22649403. PMID: 22649403 75. Na¨ssel DR, Enell LE, Santos JG, Wegener C, Johard HAD. A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions. BMC Neurosci. 2008; 9. https://doi.org/10.1186/1471-2202-9-90 WOS:000260256900001. PMID: 18803813 76. Na¨ssel DR, Wegener C. A comparative review of short and long neuropeptide F signaling in inverte- brates: Any similarities to vertebrate neuropeptide Y signaling? Peptides. 2011; 32(6):1335–55. https:// doi.org/10.1016/j.peptides.2011.03.013 WOS:000292586200035. PMID: 21440021 77. Roller L, Beke G, Stano M, Daubnerova I, Mizoguchi A, Satake H, et al. BomBase—web atlas of the neuroendocine system in Bombyx mori. Online database resource. http://bombase.org/. 2017. 24 / 24
https://openalex.org/W2096026100	https://inserm.hal.science/inserm-00816439/document	English	null	Gender Differences in Transcriptional Signature of Developing Rat Testes and Ovaries following Embryonic Exposure to 2,3,7,8-TCDD	PloS one	2,012	cc-by	10,880	Gender differences in transcriptional signature of developing rat testes and ovaries following embryonic exposure to 2,3,7,8-TCDD. Solange Magre, Diane Rebourcet, Muhammad Ishaq, Richard Wargnier, Cyrille Debard, Emmanuelle Fouilloux-Meugnier, Hubert Vidal, Joëlle Cohen-Tannoudji, Brigitte Le Magueresse-Battistoni To cite this version: Solange Magre, Diane Rebourcet, Muhammad Ishaq, Richard Wargnier, Cyrille Debard, et al.. Gen- der differences in transcriptional signature of developing rat testes and ovaries following embryonic exposure to 2,3,7,8-TCDD.. PLoS ONE, 2012, 7 (7), pp.e40306. 10.1371/journal.pone.0040306. inserm-00816439 Abstract Dioxins are persistent organic pollutants interfering with endocrine systems and causing reproductive and developmental disorders. The objective of our project was to determine the impact of an in utero exposure to 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) on reproductive function of male and female offspring in the rat with a special emphasis on the immature period. We used a low dose of TCDD (unique exposure by oral gavage of 200 ng/kg at 15.5 days of gestation) in order to mirror a response to an environmental dose of TCDD not altering fertility of the progeny. We choose a global gene expression approach using Affymetrix microarray analysis, and testes of 5 days and ovaries of 14 days of age. Less than 1% of the expressed genes in gonads were altered following embryonic TCDD exposure; specifically, 113 genes in ovaries and 56 in testes with 7 genes common to both sex gonads. It included the repressor of the aryl hydrocarbon receptor (Ahrr), the chemokines Ccl5 and Cxcl4 previously shown to be regulated by dioxin in testis, Pgds2/Hpgds and 3 others uncharacterized. To validate and extend the microarray data we realized real-time PCR on gonads at various developmental periods of interest (from 3 to 25 days for ovaries, from 5 to the adult age for testes). Overall, our results evidenced that both sex gonads responded differently to TCDD exposure. For example, we observed induction of the canonic battery of TCDD- induced genes coding enzymes of the detoxifying machinery in ovaries aged of 3–14 days of age (except Cyp1a1 induced at 3–10 days) but not in testes of 5 days (except Ahrr). We also illustrated that inflammatory pathway is one pathway activated by TCDD in gonads. Finally, we identified several new genes targeted by TCDD including Fgf13 in testis and one gene, Ptgds2/Hpgds regulated in the two sex gonads. Editor: Dmitry I. Nurminsky, University of Maryland School of Medicine, United States of America Received December 19, 2011; Accepted June 7, 2012; Published July 9, 2012 Copyright: 2012 Magre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research project was supported by Agence Nationale pour la Recherche (ANR-06-PNRA-006-01, Dhyoxhyme project) and by Agence franc¸aise de se´curite´ sanitaire de l’environnement et du travail (AFSSET, EST-2006/1/33). HAL Id: inserm-00816439 https://inserm.hal.science/inserm-00816439v1 Submitted on 22 Apr 2013 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Gender Differences in Transcriptional Signature of Developing Rat Testes and Ovaries following Embryonic Exposure to 2,3,7,8-TCDD Solange Magre1, Diane Rebourcet2¤, Muhammad Ishaq1, Richard Wargnier1, Cyrille Debard2, Emmanuelle Meugnier2, Hubert Vidal2, Joe¨lle Cohen-Tannoudji1, Brigitte Le Magueresse-Battistoni2* 1 Universite´ Paris Diderot, Sorbonne Paris Cite´, Biologie Fonctionnelle et Adaptative, EAC CNRS 4413, Paris, France, 2 Universite´ Lyon 1, INSERM U1060, INRA U1235, CarMeN, Laboratoire Lyonnais de Recherche en Cardiovasculaire, Me´tabolisme, Diabe´tologie et Nutrition, Oullins, France Universite´ Paris Diderot, Sorbonne Paris Cite´, Biologie Fonctionnelle et Adaptative, EAC CNRS 4413, Paris, France, 2 Universite´ Lyon arMeN, Laboratoire Lyonnais de Recherche en Cardiovasculaire, Me´tabolisme, Diabe´tologie et Nutrition, Oullins, France Abstract DR is the recipient of subsides from ANR (ANR-06-PNRA-006-01) and Schering-Plough (FARO-grant-0119-10/Organon). RW is the recipient of subsides from ANR (ANR-CES-2008-011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare that DR received funding from Shering-Plough. There are no patents, products in products. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. The authors declare that DR received funding from Shering-Plough. There are no patents, products in development or marketed alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. RC Centre for Reproductive Health, University of Edinburgh, The Queens Medical Research Institute, Edinburgh, United Kingdom ¤ Current address: MRC Centre for Reproductive Health, University of Edinburgh, The Queens Medical Research Institute, Edinburgh strated that breast-fed but not formula-fed sons from mothers exposed to dioxin after the accident in Seveso had permanently reduced sperm quality [5]. In utero exposure to TCDD also impaired prostate development in many mammals including rats and mice. However, data on testicular development are debated [6–8]. In rat females, it has also been shown that TCDD exposure in utero is associated with malformations of external genitalia, reduced fertility, and disruption of estrus cycles and inhibition of ovulation [9,10]. PLoS ONE \| www.plosone.org ation: Magre S, Rebourcet D, Ishaq M, Wargnier R, Debard C, et al. (2012) Gender Differences in Transcriptional Signature of Developing owing Embryonic Exposure to 2,3,7,8-TCDD. PLoS ONE 7(7): e40306. doi:10.1371/journal.pone.0040306 Introduction Dioxins, which refer to a family of structurally and chemically related polychlororinated dibenzo-p-dioxins (PCDDs) and diben- zofurans (PCDFs) are lipophilic chemicals resistant to degradation and categorized as persistent organic pollutants, with 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) being the most toxic dioxin. Dioxins are suspected of interfering with the endocrine systems of humans and wildlife [1–3] causing a broad spectrum of adverse effects including developmental and reproductive toxicity in the offspring of laboratory animals, and perhaps in humans. These disorders are of very high concern, because they occur at much lower doses than those causing wasting syndrome or carcinogen- esis [4]. Moreover, dioxins tend to accumulate in the food chain, essentially fatty food including breast milk, and may also cross the placental barrier. These data emphasize that foetuses and neonates are vulnerable populations. For example, it was recently demon- Most of the toxic effects of TCDD are mediated through the binding and activation of the aryl hydrocarbon receptor (AHR) and subsequent alteration of target gene expression. The classic battery of dioxin-responsive genes exhibit dioxin response elements in their promoter moiety and includes phase I and phase II enzymes of the detoxification machinery, such as the cytochrome P450 (Cyps) 1a1 and 1b1. However, a recent study pointed out that 65% of the gene expression responses elicited by PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40306 July 2012 \| Volume 7 \| Issue 7 \| e40306 1 PLoS ONE \| www.plosone.org Transcriptomic TCDD Signature in Gonads Table 1. List of primers used in the study. List of primers Accession number Forward 5’-3’ Reverse 5’-3’ size (bp) Ahrr NM_001024285.1 CAGCAACATGGCTTCTTTCA GAAGCACTGCATTCCAGACA 172 Cyp1a1 NM_012540.2 CAAGAGCTGCTCAGCATAGTC GCTCAATGAGGCTGTCTGTG 229 Cyp1b1 NM_012940.1 GCAGCCGCCTTCCTGGTAGC CCACGCGCCCTGTCCCTACT 116 Nqo1 NM_017000.3 TGCTTTCAGTTTTCGCCTTT GAGGCCCCTAATCTGACCTC 122 Cyp19a1 NM_017085.2 TGTTGTTGGTGACAGAGACA AAG CAAGTCCACGACAGGCTGATA 103 Star NM_031558.2 GTCATCAGAGCTGAACACGG TGGCGAACTCTATCTGGGTC 163 Art2b NM_198735.2 TGTGGTTCTCCCCAGTCTTC CTCCTTGGCCTCCCTTTAAC 104 Fgf13 NM_053428.1 TGTATCGTCAACCCCAGTCA GCCACTGTTCCACAGTTCCT 139 Gzmf NM_153466.1 CAAATGTCCGTCGATGTCAC CCCTTGTACGCAGCCTGTAT 158 Hpgds NM_031644.2 AGAGCGGACGTTCAATGACT GGTGCTGCAGATATCCCAAT 139 Hprt1 NM_012583.2 AGGACCTCTCGAAGTGT ATTCAAATCCCTGAAGTACTCAT 111 doi:10.1371/journal.pone.0040306.t001 doi:10.1371/journal.pone.0040306.t001 elucidate target pathways involved in the aetiology of TCDD and related compounds toxicity in liver and kidney [15]. Using this strategy, we focused our study on developing gonads, testes and ovaries, in rats. The age of 5 days was chosen for males because this time period coincides with germ cell reentry into mitosis, the set-up of the spermatogonial program including stem cell self- renewal and the maturation of the somatic cell lineages, i.e. Materials and Methods Figure 1. TCDD-regulated genes detected by microarrays in ovaries and testes. Genes were selected on the basis of a p- value,0.05 and a 1.5 fold change factor of mRNA expression in response to TCDD. The Venn diagram illustrates the repartition between genes up- and down-regulated in testes or ovaries, and the 7 genes both regulated in testes and ovaries are listed. doi:10.1371/journal.pone.0040306.g001 Introduction the Sertoli and Leydig cells [16]. Regarding females, we concentrated on prepubertal period and specifically infantile period (from 7 to 20 days of age) when the ovary as well as the pituitary displayed an intense endocrine activity with high levels of estradiol and gonadotropins [17,18]. Real-time PCR was further used not only to validate microarray data generated but also to gain more insight into the kinetics of regulation of the identified genes along with crucial developmental periods. In parallel, the expression level of the classical battery of detoxifying genes was surveyed. TCDD do not involve direct AhR binding to a Xenobiotic Response Element (XRE) [11]. It suggested that expression of genes lacking a XRE element reflected indirect AhR-mediated signalling, and that such changes in expression could be attributed to latent secondary effects. It could as well suggest that non- consensus XRE element may confer TCDD inducibility as shown recently with PAI-1 [12]. These two mechanisms may well explain the pleiotropic nature of TCDD toxicity illustrated with the large panel of target genes identified that include for example genes involved in cellular growth, differentiation and inflammation [13,14]. Global gene expression technology provides a comprehensive strategy whereby critical AHR-regulated genes apart from the classic battery of genes can be identified, and has been used to Overall, our results evidenced that both sex gonads responded differently to TCDD exposure with respect to enzymes of the detoxifying machinery. We illustrated that inflammatory pathway is one pathway activated by TCDD in gonads. We identified several new genes targeted by TCDD including Fgf13 in testis and one gene, Ptgds2/Hpgds regulated in the two sex gonads. Experimental Design p g Time pregnant Sprague-Dawley females of embryonic day 12 were purchased from Janvier’s Breeding (Le Genest, France). They were housed individually in plastic cages with food (Altromin 1310; Genestil, Royaucourt, France) and water provided ad libitum at 23uC and a 12:12 photoperiod. Animals were randomly assigned to treatment groups. Dams were allowed 3-day acclima- tization and were given one oral dose of 2,3,7,8-TCDD (ref ED- 901-C) (LGC Promochem, Molsheim, France) 200 ng/kg body weight (bw) in sesame oil on embryonic day 15. Control animals received sesame oil. Pups were sacrificed by cervical dislocation under CO2 anesthesia at various ages, at 3, 6, 10, 12, 14 and 25 days for females, and at 5, 28, 40, 67, 145 days for males. Ovaries and testes were dissected and snap-frozen. Pituitaries were collected from female pups of 3, 6, 12 and 14 days of age. In addition, livers were collected at 5 and 28 days for males and 3 and 6 days for females. All organs were kept at 280uC before use. Throughout the manuscript, treated organs refer to organs collected from animals born from TCDD-exposed dams. Control Figure 1. TCDD-regulated genes detected by microarrays in ovaries and testes. Genes were selected on the basis of a p- value,0.05 and a 1.5 fold change factor of mRNA expression in response to TCDD. The Venn diagram illustrates the repartition between genes up- and down-regulated in testes or ovaries, and the 7 genes both regulated in testes and ovaries are listed. doi:10.1371/journal.pone.0040306.g001 Figure 1. TCDD-regulated genes detected by microarrays in ovaries and testes. Genes were selected on the basis of a p- value,0.05 and a 1.5 fold change factor of mRNA expression in response to TCDD. The Venn diagram illustrates the repartition between genes up- and down-regulated in testes or ovaries, and the 7 genes both regulated in testes and ovaries are listed. doi:10.1371/journal.pone.0040306.g001 Figure 1. TCDD-regulated genes detected by microarrays in ovaries and testes. Genes were selected on the basis of a p- value,0.05 and a 1.5 fold change factor of mRNA expression in response to TCDD. The Venn diagram illustrates the repartition between genes up- and down-regulated in testes or ovaries, and the 7 genes both regulated in testes and ovaries are listed. doi:10.1371/journal.pone.0040306.g001 July 2012 \| Volume 7 \| Issue 7 \| e40306 PLoS ONE \| www.plosone.org 2 Transcriptomic TCDD Signature in Gonads Table 2. Experimental Design Sub-list of relevant genes TCDD-regulated in testes. Gene Title Gene Symbol Fold Change ADP-ribosyltransferase 2b Art2b 8.23 granzyme F Gzmf 4.40 aryl-hydrocarbon receptor repressor Ahrr 3.34 ficolin B Fcnb 2.00 peroxisomal biogenesis factor 11 alpha Pex11a 1.87 prostaglandin D2 synthase 2, hematopoietic Ptgds2/Hpgds 1.81 protein kinase (cAMP-dependent, catalytic) inhibitor beta Pkib 1.78 versican Vcan 1.68 fibroblast growth factor 16 Fgf16 1.62 prostaglandin reductase 2 Ptgr2 1.59 glycerol-3-phosphate acyltransferase, mitochondrial Gpam 1.58 spectrin repeat containing, nuclear envelope 1 Syne1 1.56 wingless-type MMTV integration site family, member 4 Wnt4 1.55 Cd44 molecule Cd44 1.51 tyrosine hydroxylase Th 1.51 platelet factor 4 Cxcl4/Pf4 1.32 chemokine (C-C motif) ligand 5 Ccl5/Rantes 21.50 stanniocalcin 1 Stc1 21.52 antisense paternally expressed gene 3 Apeg3 21.53 dipeptidylpeptidase 4 Dpp4 21.64 immunity-related GTPase family, M Irgm 21.73 growth hormone releasing hormone receptor Ghrhr 21.85 EGF-containing fibulin-like extracellular matrix protein 1 Efemp1 21.86 ATP-binding cassette, sub-family B (MDR/TAP), member 1A Abcb1a 21.89 cartilage acidic protein 1 Crtac1 22.05 fibroblast growth factor 13 Fgf13 23.15 SH2 domain protein 1A Sh2d1a 24.00 crystallin, gamma B Crygb 25.17 Genes were identified by the microarray procedure and their fold-change over control is indicated (gonad from untreated dams) (PF4/Cxcl4 was added in the list as a relevant gene even though its fold change is ,1.5). doi:10.1371/journal.pone.0040306.t002 Table 2. Sub-list of relevant genes TCDD-regulated in testes. array procedure and their fold-change over control is indicated (gonad from untreated dams) (PF4/Cxcl4 was added in the list as a change is ,1.5). Genes were identified by the microarray procedure and their fold-change over control is indicated (gonad from untreated dams) (PF4/Cxcl4 was added in the list as a relevant gene even though its fold change is ,1.5). doi:10.1371/journal.pone.0040306.t002 affymetrix.com). The arrays were read with a confocal laser (Genechip scanner 3000, Affymetrix). Then CEL files were generated using the Affymetrix GeneChip Command Console (AGCC) software 3.0. The obtained data were normalized with Affymetrix Expression Console software using MAS5 statistical algorithm. Normalized data were compared and filtered using Partek Genomic Suite software 6.5 (Partek Inc., St. Louis, MO, US). Microarray data are available in the GEO database under the number GSE32890. organs refer to organs collected from animals born from sesame oil exposed dams. Experimental Design Animals were housed and maintained according to published European communities’ guidelines (86/609/CEE) and all the performed experiments on animals were approved by the experimental animal committee of the Paris Rive Gauche site (Agreement A75-13-17, Centre National de la Recherche Scientifique, Paris 7 University, Paris, France). A detailed protocol of TCDD exposure and follow-up of the animals has been published [19]. PLoS ONE \| www.plosone.org RNA Preparation and Microarray Analysis Changes in global gene expression induced by TCDD were analyzed in 5-day old testes and in 14-day old ovaries. In both cases, 3 rats treated in utero by TCDD were compared to 3 rats treated with sesame-oil vehicle. RNA extraction was performed with the RNeasy Mini RNA extraction kit (Qiagen, Courtaboeuf France). A principal component analysis (PCA) of the complete list of genes present in the chip and two sample t-test were performed between TCDD samples and control samples for ovaries and for testes. Only genes showing a variation of at least 1.5-fold and a p- value less than 0.05 were retained. Then, a gene was considered as differentially expressed between groups only if the detected signal was above the background for at least one of the compared groups. David functional annotation clustering (http://david.abcc.ncifcrf. gov/conversion.jsp) was performed to identify enrichment in biological functions and pathways. Promoter sequences (650pb) of the regulated genes were extracted from Rat RGSC 3.4 assembly Microarray analysis has been performed in the genomic and microgenomic core facility profileXpert (Bron, France) as described previously [20,21], using a high-density oligonucleotide array (GeneChip Rat Genome 230 2.0 array, Affymetrix, Santa Clara, CA, USA), and following Affymetrix protocol (http://www. PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40306 3 Transcriptomic TCDD Signature in Gonads Table 3. Sub-list of characterized genes TCDD-regulated in ovaries. RNA Preparation and Microarray Analysis Gene Title Gene Symbol Fold Change ubiquitin-like modifier activating enzyme 7 Uba7 21.73 Translocase of outer mitochondrial membrane 34 Tomm34 21.74 ATP-binding cassette, sub-family G (WHITE), member 1 Abcg1 21.76 family with sequence similarity 92, member A1 Fam92a1 21.78 solute carrier organic anion transporter family, member 1a4 Slco1a4 21.81 period homolog 3 (Drosophila) Per3 21.83 basic helix-loop-helix family, member e41 Bhlhe41 21.85 Fc receptor-like S, scavenger receptor Fcrls 21.97 phosphodiesterase 10A Pde10a 21.98 spastic paraplegia 3A homolog (human) Spg3a 22.14 nucleoporin 62 C-terminal like Nup62cl 22.47 Genes were identified by the microarray procedure and their fold-change over control is indicated (gonad from untreated dams). doi:10.1371/journal.pone.0040306.t003 Table 3. Cont. Table 4. David functional analysis. ovarian genes cluster enrichment scores p-value Examples of genes up-regulated (34 identified; 4 unknwon) immune response, positive regulation of cell differentiation and developmental processes 2.43 4.7.1024 Ccl5, Adam10 positive regulation of response to stimulus 1.93 5.1024 Ccl5, Ccl6, Adam10 chemotaxis and chemokine activity 1.7 3.3.1024 Ccl5, Ccl6, Pf4 response to steroid hormone stimulus and organic substance 1.56 1.2.1022 Adam10, Ccl5, Cyp1b1 down-regulated (56 identified; 19 unknown) behavior 1.4 2.7.1022 S100a4, Amph, Cck Summary of the analysis using the list of genes regulated by TCDD in ovary. doi:10.1371/journal.pone.0040306.t004 Summary of the analysis using the list of genes regulated by TCDD in ovary. doi:10.1371/journal.pone.0040306.t004 electrophoresis. All samples were run in quadriplicate reactions (duplicate of two dilutions). Quantification of gene expression was performed using the Relative Quantification Software (Roche using BioMart, and firstly analyzed for pattern matching using Common TFs from the Genomatix software package (Genomatix software suite v2.5, Mu¨nchen, Germany). Briefly, the sequences were scanned for matches to Transcription Factor binding sites and only matrices displaying an enrichment p-value ,0.05 were considered enriched in the promoters of interest compared to the rest of the vertebrate promoter sequences. Table 5. Expression levels of classical genes targeted by TCDD. ovary Testis control TCDD Fold- Change control TCDD Fold- change Ahrr 18 79 4.2* 14 48 3.34* Cyp1a1 nd nd nd nd nd nd Cyp1a2 nd nd nd nd nd nd Cyp1b1 1419 2300 1.63* 1079 1315 1.22 Cyp2s1 66 55 0.83 65 54 0.83 Nqo1 350 470 1.34* 303 444 1.46 Gsta1 13200 12800 0.96 7705 7652 0.99 Aldh3a1 nd nd nd nd nd nd Ugt1a6 210 273 1.3 381 436 1.14 Data were obtained using ovaries of 14 days of age and testes of 5 days of age. RNA Preparation and Microarray Analysis g g Gene Title Gene Symbol Fold Change aryl-hydrocarbon receptor repressor Ahrr 4.20 acyl-CoA thioesterase 1 Acot1 2.94 chemokine (C-C motif) ligand 5 Ccl5 2.50 Zinc finger protein 212 Zfp212 2.42 prolylcarboxypeptidase (angiotensinase C) Prcp 2.42 leucine rich repeat and Ig domain containing 1 Lingo1 2.22 prostaglandin D2 synthase 2, hematopoietic Ptgds2/Hpgds 2.00 claudin 15 Cldn15 1.98 leucine rich repeat containing 4C Lrrc4c 1.95 ras-related C3 botulinum toxin substrate 2 Rac2 1.79 reprimo-like Rprml 1.78 TBC1 domain family, member 10C Tbc1d10c 1.76 RT1 class II, locus Ba RT1-Ba 1.73 histocompatibility 2, class II antigen E alpha H2-Ea 1.73 granzyme K Gzmk 1.71 ADAM metallopeptidase domain 10 Adam10 1.70 carbonic anhydrase 5b, mitochondrial Car5b 1.69 complement component 1, q subcomponent, beta polypeptide C1qb 1.69 cystatin F (leukocystatin) Cst7 1.69 chemokine (C-C motif) ligand 6 Ccl6 1.67 C1q and tumor necrosis factor related protein 3 C1qtnf3 1.66 cytochrome P450, family 1, subfamily b, polypeptide 1 Cyp1b1 1.63 HEAT repeat containing 6 Heatr6 1.60 S100 calcium-binding protein A4 S100a4 1.58 platelet factor 4 Pf4/Cxcl4 1.57 potassium inwardly rectifying channel, subfamily J, member 11 Kcnj11 1.52 aldo-keto reductase family 1, member C-like 1 Akr1cl1 1.51 leucine rich repeat containing 61 Lrrc61 21.51 isoleucyl-tRNA synthetase 2, mitochondrial Iars2 21.51 CAP-GLY domain containing linker protein 1 Clip1 21.51 amphiphysin Amph 21.52 ELMO/CED-12 domain containing 2 Elmod2 21.54 tripartite motif-containing 2 Trim2 21.54 zinc finger, DHHC-type containing 1 Zdhhc1 21.54 proline-rich transmembrane protein 1 Prrt1 21.55 cytoplasmic linker associated protein 1 Clasp1 21.55 parathyroid hormone receptor 1 Pthr1 21.55 mutY homolog (E. coli) Mutyh 21.56 pinin, desmosome associated protein Pnn 21.58 UDP-N-acteylglucosamine pyrophosphorylase 1-like 1 Uap1l1 21.61 cholecystokinin Cck 21.61 sodium channel, voltage-gated, type VII, alpha Scn7a 21.61 phosphatidylinositol 4-kinase type 2 beta Pi4k2b 21.61 apoptotic chromatin condensation inducer 1 Acin1 21.62 potassium channel tetramerisation domain containing 13 Kctd13 21.64 neuronal pentraxin 1 Nptx1 21.64 mitogen-activated protein kinase 11 Mapk11 21.68 ATP-binding cassette, sub-family C (CFTR/MRP), member 5 Abcc5 21.70 purinergic receptor P2X, ligand-gated ion channel, 3 P2rx3 21.71 S100 calcium binding protein B S100b 21.72 July 2012 \| Volume 7 \| Issue 7 \| e40306 PLoS ONE \| www.plosone.org Transcriptomic TCDD Signature in Gonads Table 3. Cont. RNA Preparation and Microarray Analysis p,0.05; nd, not detectable. doi:10.1371/journal.pone.0040306.t005 Table 5. Expression levels of classical genes targeted by TCDD. PLoS ONE \| www.plosone.org p doi:10.1371/journal.pone.0040306.t005 Global Analysis of Testes and Ovaries Transcriptomic D t The total amount of genes expressed in ovaries and testes was approximately of 65% of the rat genome, indicating that an average of 20,000 genes is expressed in the gonads. PCA analysis did not allow segregating genes between treated-testes and control testes, or between treated-ovaries and control ovaries. It indicated that the sample to sample variation within the group (male or female) was bigger than the variation due to the treatment (not shown). A two sample t-test was next performed and a list of 113 and 56 differentially expressed genes showing a variation of 1.5- fold and a p-value less than 0.05 came out in ovary and testis, respectively. Of the 113 genes responding to TCDD in ovaries, 38 were up-regulated and 75 down-regulated. In testes, 27 were up- regulated and 29 down-regulated (Fig. 1). About, half of them are not fully characterized and are identified by their Affymetrix probeset ID. Some of them are transcribed locus or expressed sequence tags (ESTs). A sub-list of characterized genes differen- tially expressed in response to TCDD, in testes and in ovaries is given in Tables 2 and 3, respectively. We also identified a total of 7 genes common to testes and ovaries if cross-comparing the list of regulated genes that exhibited at least a 1.5 fold change over its respective control. There were Ahr repressor (Ahrr), prostaglandin D2 synthase 2, hematopoietic (Ptgds2/Hpgds), the chemokines Rantes/Ccl5 and Pf4/Cxcl4, and 3 uncharacterized genes which were not further studied (Fig.1, Tables 2, 3). David functional annotation clustering indicated one cluster with enrichment score of 1.61 and a p-value of 4.9.1023 using the total list of testis genes. It corresponded to immune and defense response with 6 transcripts including Ccl5 and Pf4. No cluster came out if only the list of the up or down-regulated genes was considered. Using the list of the up-regulated genes in the ovary, 4 clusters came out with enrichment score comprised between 1.56 and 2.43 and corresponding to immune response, positive regulation of response to stimulus, chemotaxis and chemokine activity, and response to steroid hormone stimulus. In addition, KEGG pathway pointed to the chemokine signaling pathway associating Ccl5, Ccl6 and Pf4. A single cluster with an enrichment score of 1.4 and corresponding to behavior came out when uploading the list of down-regulated genes (Table 4). Figure 3. Reverse Transcription and Real-time RT-PCR Reverse transcription was carried out with 250 ng of total RNA recovered from testes (of 5, 28, 40, 67, 145 days of age), ovaries (of 3, 6, 10, 12, 14, 25 days of age), pituitaries (of females aged of 3, 6, 12, 14 days of age) or livers (of 5 and 28 days for males and 3 and 6 days for females), 400 ng oligo-dT primers (Qiagen) and Superscript II reverse transcriptase (Invitrogen, France). Real- time RT-PCR (QRT-PCR) was performed in a LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) using the LightCycler 480 SYBR Green I Master mix according to the manufacturer’s protocol. Primers used are listed in table 1. They were tested before use for specificity and efficacy. Amplification conditions were as following: 10 min at 95uC followed by 40 cycles of denaturation (10 sec at 95uC), annealing (10 sec at 60uC), and extension (10 sec at 72uC) with single acquisition of fluorescence at the end of each extension step. The specificity of PCR products was confirmed by analysis of the melting curve and agarose gel PLoS ONE \| www.plosone.or PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40306 5 Figure 2. Ahrr, Cyp1a1, Cyp1b1 and Nqo1 gene expression levels in testes and ovaries of rats exposed in utero recovered from males aged from 5 to 145 days (A) and ovaries were recovered from females aged from 3 to 25 days (B). Testes detectable Cyp1a1 levels (A). Levels were normalized using Hprt. Values are the mean 6 SEM of n = 4 to 6 animals (n = 2 for ovaries). p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g002 Transcriptomic TCD Transcriptomic TCDD Signature in Gonads Figure 2. Ahrr, Cyp1a1, Cyp1b1 and Nqo1 gene expression levels in testes and ovaries of rats exposed in utero to TCDD. Testes were recovered from males aged from 5 to 145 days (A) and ovaries were recovered from females aged from 3 to 25 days (B). Testes samples did not have detectable Cyp1a1 levels (A). Levels were normalized using Hprt. Values are the mean 6 SEM of n = 4 to 6 animals (n = 2 for 3- and 25-day control ovaries). p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g002 July 2012 \| Volume 7 \| Issue 7 \| e40306 PLoS ONE \| www.plosone.org 6 Transcriptomic TCDD Signature in Gonads Figure 3. Global Analysis of Testes and Ovaries Transcriptomic D t Ahrr, Cyp1a1, Cyp1b1 and Nqo1 gene expression levels in pituitaries of rats exposed in utero to TCDD. Pituitaries were recovered from females aged from 6 and 14 days of age. Levels were normalized using Hprt. Values are the mean 6 SEM of n = 3 to 5 animals. p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g003 Reverse Transcription and Real-time RT-PCR Ahrr, Cyp1a1, Cyp1b1 and Nqo1 gene express levels in pituitaries of rats exposed in utero to TCDD. Pituita were recovered from females aged from 6 and 14 days of age. Lev were normalized using Hprt. Values are the mean 6 SEM of n = 3 t animals. * p,0.05 versus its time-matched control. (dpn), days postna doi:10.1371/journal.pone.0040306.g003 Figure 3. Ahrr, Cyp1a1, Cyp1b1 and Nqo1 gene ex levels in pituitaries of rats exposed in utero to TCDD. were recovered from females aged from 6 and 14 days of a were normalized using Hprt. Values are the mean 6 SEM of animals. * p,0.05 versus its time-matched control. (dpn), days doi:10.1371/journal.pone.0040306.g003 Reproductive Parameters of the F1 Offspring Reproductive Parameters of the F1 Offspring A follow-up of the female offspring including body weight, fertility assessment and measurement of mRNA levels of some key genes involved in the endocrine function of the ovary during prepubertal period is provided (Supporting Information S1). Except for body weight, no significant differences were obtained. Regarding the male offspring exposed in utero to TCDD, testicular and epididymal weight did not change. Testis histology was grossly normal so were testosterone levels throughout development. A precise description was provided previously [19]. Expression of Genes Belonging to the Classic Battery of TCDD Target Genes To extend microarray data, we first focused on Ahrr strongly induced in both gonads, and other genes belonging to the TCDD- inducible Ahr gene battery. The TCDD battery includes in addition to Ahrr which acts as a dominant negative factor to repress Ahr induced signalling pathways [22,23], four phase I xenobiotic metabolizing enzymes, i.e., Cyp1b1, Cyp1a1, Cyp1a2, and Cyp2s1, and four phase II xenobiotic metabolizing enzymes, i.e., NADP(H) quinone oxidoreductase (Nqo1), glutathione Diagnostics), and data were expressed as a ratio of target gene to the reference gene hypoxanthine phosphoribosyltransferase, HPRT. Statistical analyses were done using Statview 5.0 software package (SAS Institute Inc. Cary, NC 27513). Comparisons between treatments were made by one-way analysis of variance (ANOVA) followed by the post hoc Fisher PLSD test for multiple comparisons. A p value of less than 0.05 was considered significant. PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40306 7 Transcriptomic TCDD Signature in Gonads Figure 4. Gzmf, Art2b, Hpgds and Fgf13 gene expression levels in testes of rats exposed in utero to TCDD. Testes were recovered from males of 5, 28 and 67 days of age. Levels were normalized using Hprt. Values are the mean 6 SEM of n = 4 animals. p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g004 Figure 4. Gzmf, Art2b, Hpgds and Fgf13 gene expression levels in testes of rats exposed in utero to TCDD. Testes were recovered from males of 5, 28 and 67 days of age. Levels were normalized using Hprt. Values are the mean 6 SEM of n = 4 animals. p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g004 transferase a1 (Gsta1), cytosolic aldehyde dehydrogenase-3 (Aldh- 3a1), and UDP glucuronosyltransferase 1a6 (Ugt1a6) (Table 5). Three genes, Cyp1a1, Cyp1a2 and Aldh-3a1 were not found at detectable levels in the gonads. Two genes appeared to be constitutively expressed (Cyp2s1 and Gsta1) or weakly enhanced (Nqo1 and Ugt1a6) after TCDD treatment. Cyp1b1 was significantly up-regulated in ovaries (FC 1.63) but not in testes (FC 1.22 with a p-value of 0.36). Finally, gene coding Ahrr was the most up-regulated gene in both gonads of treated animals (FC 4.2 in ovaries and 3.34 in testes). days. No expression was detected at 14 days consistent with the microarray data (Table 2). Expression of Genes Belonging to the Classic Battery of TCDD Target Genes Gene expression levels of Cyp1b1 and Nqo1 were at detectable levels in control ovaries and roughly constant from 3 to 25 days of age. Treated ovaries had enhanced levels of Cyp1b1 and Nqo1 from 3 to 14 days, also in agreement with the microarray data. No differences were observed at 25 days (Fig. 2). We also studied an additional endocrine organ, the pituitary, and the liver as the primary detoxifying organ. Data synthesized on Fig. 3, indicate a strong and significant enhancement of Ahrr, Cyp1a1, Cyp1b1 and Nqo1 at 6 days of age in pituitary of TCDD- treated animals. Although significant, induction was less pro- nounced at 14 days of age (Fig. 3). As expected, RNA from both male (5 days old) and female (6 days of age) liver showed increased Ahrr and Nqo1 expression levels (data not shown) in treated animals consistent with the induction in Cyp1a1 mRNA levels reported previously [19]. To validate and extend the microarray results, we analyzed the expression of Ahrr and Cyp1b1 as well as Cyp1a1 and Nqo1 using real-time PCR. This was done not only in ovaries at 14 days and in testes at 5 days but also during a period of time extending from 3– 25 days in females and 5–145 days in males to cover critical developmental steps in both gonads. Real-time PCR confirmed the findings of the microarray in gonads (Fig. 2). For example in testes, Ahrr was significantly enhanced at 5 days of age although the fold-change was lower than the one observed using micro- array. In addition, none of the metabolizing enzymes had their gene expression levels altered after TCDD treatment (Fig. 2). Noticeably, Cyp1a1 gene was not expressed in testis, either control or TCDD-treated (not shown). Cyp1b1 and Nqo1 expression levels increased as a function of age from 5 to 145 days (Fig. 2). In contrast, neonate ovaries recovered from the same litters were strongly responsive to TCDD exposure. Expression levels of Ahrr were maximally up-regulated at 3 and 6 days. A significant enhancement was still observed until 14 days, consistent with the microarray data. No effect was observed in ovaries of 25 days. Of note, Ahrr gene expression levels were almost undetectable in control ovaries (Fig. 2). Cyp1a1, the hallmark of TCDD-inducible Ahr gene [24], although not present in control ovaries was highly induced in TCDD-treated ovaries of 3 to 10 days, peaking at 10 Expression of Chemokines in Response to TCDD in Gonads but also in Pituitary and Liver In pituitaries, Gzmf/Nrkp7 and Art2b were present at 3 and 12 days of age and organs from treated animals exhibited levels higher than 2-fold over control levels (Fig. 6). Fgf13 was also present in pituitaries collected from 3 and 12 days old animals, and its expression levels were significantly decreased (a 20% decrease) in treated organs (Fig. 6). Ptgds2/Hpgds was expressed in pituitary but not TCDD-regulated (Fig. 6). Finally, real-time PCR using liver from 3 and 6 day-old animals revealed transcripts for Gzmf/Nrkp7, Art2b and Ptgds2/Hpgds but not Fgf13, with TCDD up-regulation of Gzmf/Nrkp7 and Art2b (p,0.05) at 3 but not at 6 days of age. Ptgds2/Hpgds was not targeted (Fig. 7). Table 6 recapitulates data. Identification of DNA Matrices, Potential Sites for Transcription Factors To find out whether certain transcriptional factor binding site(s) were enriched in the TCDD-regulated genes identified in this study, we selected 650bp promoter of each gene, and searched for DNA matrices families using Genomatix promoter analysis. A total of 25 promoter sequences for testes regulated genes and 54 promoter sequences for ovaries regulated genes could be processed. We observed that the DNA matrices family ETSF was consistently identified in the promoters of all 25 TCDD regulated genes in testes (Table 7) and the 54 regulated in ovaries (Table 8). We also identified 10 other overrepresented matrices families’ specific to either testes or ovaries (Table 8) which may account for the gender differences that we detected in this study. Discussion The objective of our project was to determine the impact of an in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on reproductive function of male and female offspring in the rat with a special emphasis on the immature period, in conditions in which reproductive parameters are grossly normal both in males [19] and in females (this study). To this end, animals were exposed during gestation to low doses to avoid collateral toxic effects. We also considered the TCDD half-life of 3 weeks in rodents, and conclusions published [25] indicating that the majority of occurrences of TCDD in offspring of dosed dams arise from lactational transfer of TCDD. In addition, given the unexpected microarray results highlighting gender difference responses; we developed a real time PCR approach on gonads at various developmental periods of interest. On the one hand, this approach permitted to extend our study on male and female gonad development following an in utero exposure to TCDD. On the other hand, this approach allowed comparing common stages in both sexes (specifically, 5 and 28 days in testes, and 6 and 25 days in ovaries). Figure 5. Hpgds and Fgf13 gene expression levels in ovaries of rats exposed in utero to TCDD. Ovaries were recovered from females of 3 to 25 days of age. Levels were normalized using Hprt. Values are the mean 6 SEM of of n = 4 to 6 animals (n = 2 for 3- and 25- day control ovaries). p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g005 y p doi:10.1371/journal.pone.0040306.g005 Pf4/Cxcl4 (1.81 fold-change, n = 4; p,0.05) versus time- matched controls. Ccl5 but not Pf4/Cxcl4 was also up-regulated (2.12 fold-change, n = 4; p,0.05) in the liver recovered from 28-day old male rats (data not shown). Expression of Chemokines in Response to TCDD in Gonads but also in Pituitary and Liver Consistently with our previous data [19], we observed down- regulation of Ccl5 and upregulation of Pf4/Cxcl4 in the testes of 5-day old rats of TCDD-treated dams (Table 2). In females, microarray studies indicated that both Ccl5 and Pf4/Cxcl4 were significantly up regulated 2.5- and 1.57-fold, respectively (Table 3). In addition, KEGG pathway pointed to the chemokine signaling pathway associating Ccl5, Ccl6 and Pf4/ Cxcl4, in ovary and one enriched cluster comprised of Ccl5 and Pf4/Cxcl4 in testis. To define if chemokines could be targeted by TCDD in other organs, real-time PCR was performed with RNA from pituitary and liver. We found that pituitaries recovered from 14-day-old female rats from TCDD-treated dams had increased Ccl5 (3.26 fold-change, n = 4; p,0.05) and PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40306 8 Transcriptomic TCDD Signature in Gonads Figure 5. Hpgds and Fgf13 gene expression levels in ovaries of rats exposed in utero to TCDD. Ovaries were recovered from females of 3 to 25 days of age. Levels were normalized using Hprt. Values are the mean 6 SEM of of n = 4 to 6 animals (n = 2 for 3- and 25- day control ovaries). p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g005 6, 10 and 14 days of age, nor induced in the ovaries of rats from TCDD-treated dams at these age-time points (not shown). 6, 10 and 14 days of age, nor induced in the ovaries of rats from TCDD-treated dams at these age-time points (not shown). In pituitaries, Gzmf/Nrkp7 and Art2b were present at 3 and 12 days of age and organs from treated animals exhibited levels higher than 2-fold over control levels (Fig. 6). Fgf13 was also present in pituitaries collected from 3 and 12 days old animals, and its expression levels were significantly decreased (a 20% decrease) in treated organs (Fig. 6). Ptgds2/Hpgds was expressed in pituitary but not TCDD-regulated (Fig. 6). Finally, real-time PCR using liver from 3 and 6 day-old animals revealed transcripts for Gzmf/Nrkp7, Art2b and Ptgds2/Hpgds but not Fgf13, with TCDD up-regulation of Gzmf/Nrkp7 and Art2b (p,0.05) at 3 but not at 6 days of age. Ptgds2/Hpgds was not targeted (Fig. 7). Table 6 recapitulates data. 6, 10 and 14 days of age, nor induced in the ovaries of rats from TCDD-treated dams at these age-time points (not shown). PLoS ONE \| www.plosone.org TCDD-regulated Genes in Testis and Comparison with Levels were normalized using Hprt. Values are the mean 6 SEM of n = 4 animals. p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g006 testes while in ovaries, Cyp1a1, Cyp1b1, and Nqo1 exhibited a very high up-regulation in addition to Ahrr. Ahrr functions as a naturally occurring dominant-negative factor [22,23]. Hence, Ahrr is an important determinant of tissue specific responsiveness to TCDD, and strong evidences have been reported on an inverse relationship between Ahrr expression and sensitivity to induction of xenobiotic-metabolizing enzymes caused by TCDD. This is coherent with elevated constitutive levels of Ahrr in testis [26,27]. The data presented herein i.e., high expression of Ahrr and no detectable Cyp1a1, Cyp1b1 and Nqo1 induction in testis are in keeping with these observations. testes while in ovaries, Cyp1a1, Cyp1b1, and Nqo1 exhibited a very high up-regulation in addition to Ahrr. Ahrr functions as a naturally occurring dominant-negative factor [22,23]. Hence, Ahrr is an important determinant of tissue specific responsiveness to TCDD, and strong evidences have been reported on an inverse relationship between Ahrr expression and sensitivity to induction of xenobiotic-metabolizing enzymes caused by TCDD. This is coherent with elevated constitutive levels of Ahrr in testis [26,27]. The data presented herein i.e., high expression of Ahrr and no detectable Cyp1a1, Cyp1b1 and Nqo1 induction in testis are in keeping with these observations. induction was no longer observed at 12 days of age in the ovary while still detected at 14 days in pituitaries (and 25 days, not shown) and 28 days in livers [19], illustrating organ specificity in the timing of the response. Indeed, the other detoxifying genes studied in ovary, i.e., Ahrr, Cyp1b1 and Nqo1 were still enhanced at 12 and 14 days of age. Together, these data are in keeping with previous studies demonstrating enhanced Ahrr and Cyp1a1 in the pituitaries of male rats exposed to an acute-dose of TCDD at the adult age [32]. Regarding the ovary, evidences have been brought indicating enhanced Cyp1a1 in response to dioxin [33]. However, to our knowledge, this is the first report illustrating the TCDD- induction of Ahrr in ovary. Nonetheless, the presence of Ahrr and its up-regulation in TCDD treated testes indicate that the Ahr canonical pathway is active in testis even though the role played by Ahr in testis is misunderstood. Impairment of the urogenital sinus is the major phenotype in Ahr2/2 male mice [8,28]. TCDD-regulated Genes in Testis and Comparison with Two Endocrine Organs (Ovary and Pituitary) and Liver Microarray data were further exploited by real-time PCR to analyse the testis response to TCDD exposure. Genes selected included Art2b, Gzmf/Nrkp7, and Fgf13 because they were among the most regulated genes in testis, and Ptgds2/Hpgds because it was regulated in both sex gonads (Table 2). We observed up-regulation of Gzmf/Nrkp7, Art2b, Ptgds2/Hpgds and down-regulation of Fgf13 (p,0.05) at 5 but not at 28 or 67 days (Fig. 4). The microarray technology is a powerful method allowing full gene analyses of different samples. It is especially fruitful when scarce differences are expected. In the present study, less than 1% of the expressed genes in gonads were found to be altered following embryonic TCDD exposure. Specifically, we identified a total of 113 genes in ovaries and 56 genes in testes differentially regulated by the treatment over the 20,000 genes found expressed in the ovary and testis, respectively. The low number of regulated genes found in testis versus the ovary probably suggested that TCDD may have less deleterious effects in testes than in ovaries. This could result from the lack of response of the TCDD battery of detoxifying genes in testis by comparison with the neonate ovary. Indeed, TCDD only induced gene expression of Ahrr in neonate In ovaries, Ptgds2/Hpgds was present at all ages investigated from 3 to 25 days. Enhanced expression levels of Ptgds2/Hpgds were observed in the ovaries of rats from TCDD-treated dams at 6, 12 and 14 days but not at 3 and 25 days of age (Fig. 5). Fgf13 was present in neonate ovaries (3 and 6 days of age) but its expression levels did not change in treated ovaries (Fig. 5). Art2b and Gzmf/Nrkp7 were neither expressed in the ovary at PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40306 9 Transcriptomic TCDD Signature in Gonads Figure 6. Gzmf, Art2b, Hpgds and Fgf13 gene expression levels in pituitaries of rats exposed in utero to TCDD. Pituitaries were recovered from females of 3 to 12 days of age. Levels were normalized using Hprt. Values are the mean 6 SEM of n = 4 animals. p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g006 Figure 6. Gzmf, Art2b, Hpgds and Fgf13 gene expression levels in pituitaries of rats exposed in utero to TCDD. Pituitaries were recovered from females of 3 to 12 days of age. PLoS ONE \| www.plosone.org TCDD-regulated Genes in Testis and Comparison with It may well be the case for Ptgds2/Hpgds because its induction in ovary was not immediate and could not be detected at 3 days of age, an age at which all the detoxifying genes have been shown to be deregulated. We cannot ascertain that this situation is female- specific because we did not recover males younger than 5 days of age. Ptgds2/Hpgds is a cytosolic protein responsible for the biosynthesis of prostaglandin D2 (PGD2) in immune and inflammatory cells, being widely distributed in antigen presenting cells [38]. Interestingly, very recent data have brought new insight into involvement of Ptgds2/Hpgds in differentiation and function of gonads [39,40]. Ptgds2/Hpgds is expressed in the early embryonic gonad in both sexes and participates to the initial nuclear translocation of the Sox9 protein triggering Sertoli cell differentiation in males [40]. It is also expressed in the adult ovary where it indirectly participates to the regulation of progesterone secretion [39]. Therefore, more studies need to be done regarding to Ptgds2/Hpgds because of the stimulatory effect of PGD2 on steroidogenesis regulation [39]. The 3 other genes, Fgf13, and Gzmf and Art2b, were among the most differentially expressed genes in testis, in response to TCDD. Fgf13 (also called Fgf homologous factor 2) was first shown to be expressed in human fetal and adult brain and in adult kidney [41]. Fibroblast Growth Factors (FGFs) form a large family of conserved signaling proteins with essential develop- mental functions in organ patterning and morphogenesis. Fgf13 belongs to the intracrine FGF family indicating that it is not secreted and that it functions in an FGF receptor-independent manner [42]. In fetal mice, Fgf13 expression was detected within the mesonephros of both sexes at 12.5 embryonic day, and restricted to testis by embryonic day 13.5 [43,44]. In the present study, Fgf13 showed high inhibition in testis microarray and data were validated by real time PCR with gene expression levels halved in testis from TCDD-treated rats. Interestingly, its expression was not regulated in ovaries while down- regulated in pituitary; it may suggest that Fgf13 is under the regulation of transcription factors commonly expressed in testis and pituitary. There is little information regarding Gzmf and Art2b and their possible regulation by TCDD. Granzyme genes are serine proteinases previously shown to be implicated in tissue remodel- ling in the placenta and in the testis [45,46]. TCDD-regulated Genes in Testis and Comparison with Testis alteration was also reported in aged Ahr2/2 mice with reduced testosterone production and sperm numbers [29]. It is of interest that enzymes of the TCDD battery of inducible genes expressed in testis including Cyp1b1 and Nqo1 had their expression levels increasing as a function of time, from infancy to adulthood. It may suggest that these enzymes, which are confined to Leydig cells in testis [30,31], exert a physiological role during development in relation with the endocrine status of the animal. Present data extend our previous study reporting alteration of chemokines in the testes of TCDD-treated rats, with up-regulation of Cxcl4 and down-regulation of Ccl5 [19], and other studies pointing out that various chemokines were targeted by TCDD exposure including Ccl5 in a model of endometriosis [34], Ccl1 [35], Ccl2 [36]. In this study, the chemokine pathway associating Ccl5, Pf4/Cxcl4 and Ccl6 was induced in TCDD-treated ovaries. Therefore, in addition to growth factors and cytokines [37], chemokines might be added to the list of the TCDD-targeted genes, extending the notion that TCDD interacts with the inflammatory pathways. Our results also evidenced a sex specific response of gonads to the TCDD exposure with Fgf13, Art2b, and Gzmf regulated in testes but not in ovaries, and, a gonad gene expression signature with Ptgds2/Hpgds. Indeed, this study demonstrated that Ptgds2/ Hpgds, although expressed in various tissues, was only regulated by TCDD in the gonads. Interestingly, none of these genes, and Ccl5 and Pf4/Cxcl4 exhibited-XRE elements in the 2,0000 bp Our data also illustrated that in addition to liver, which is a primary detoxification organ, endocrine organs such as ovary and pituitary displayed an up-regulation of the expression of genes coding enzymes of the detoxifying machinery. For example, in both ovary and pituitary there was an induction of Cyp1a1 which is the hallmark of TCDD exposure [24]. Nonetheless, Cyp1a1 PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40306 10 Figure 7. Gzmf, Art2b, and Hpgds gene expression lev livers of rats exposed in utero to TCDD. Livers were rec from females of 3 and 6 days of age. Levels were normalized usin Values are the mean 6 SEM of n = 3 to 5 animals. p,0.05 ve time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g007 Transcriptomic TCDD Signature in Gonads Transcriptomic TCDD Signature in Gonads secondary to a primary event, which remains to be defined. PLoS ONE \| www.plosone.org TCDD-regulated Genes in Testis and Comparison with Art2b is an ADP- ribosyl transferase gene, and 5 Arts have been described in the mammalian genome. ADP ribosylation is a reversible post- translational modification that can be used as a mechanism to regulate endogenous functions [47]. The full significance of the alteration of these genes in the testis and pituitary, but not in the ovary recovered from siblings’ warrants further investigation. Interestingly, through cross-comparison between the lists of DNA matrices present in the proximal promoters of 25 genes in testes and 54 genes in ovaries, all regulated by TCDD, we extracted potential families of transcription factors allowing regulation by TCDD in testis and/or ovary. However, the importance of these findings remains to be determined. Figure 7. Gzmf, Art2b, and Hpgds gene expression levels in livers of rats exposed in utero to TCDD. Livers were recovered from females of 3 and 6 days of age. Levels were normalized using Hprt. Values are the mean 6 SEM of n = 3 to 5 animals. p,0.05 versus its time-matched control. (dpn), days postnatal. doi:10.1371/journal.pone.0040306.g007 upstream of the transcription sites, in rats (not shown). This situation contrasted with the mouse orthologs of Art2b, Fgf13, Ptgds2/Hpgds and Ccl5 having XRE elements in the 2,000 bp upstream of the transcription sites (http://drgap.nies.go.jp/pub/ page/element). Nonetheless, these genes may be direct targets if considering that only 1/3 of the gene expression responses elicited by TCDD involves direct AhR binding to a XRE [11]. It may also indicate that the TCDD-induced enhancement of these genes is Overall, our results evidenced that male and female gonads responded differently to TCDD exposure showing, for example, the induction of the canonic battery of genes coding enzymes of the detoxifying machinery in ovaries but not in testes. We illustrated that inflammatory pathway is one of the pathways targeted by TCDD in gonads. Finally, we identified several new genes targeted by TCDD, including Fgf13 in testis and one gene, Ptgds2/Hpgds targeted in testis and ovary. July 2012 \| Volume 7 \| Issue 7 \| e40306 July 2012 \| Volume 7 \| Issue 7 \| e40306 11 PLoS ONE \| www.plosone.org Transcriptomic TCDD Signature in Gonads Table 6. Summary of the effects on Art2b, Fgf13, Gzmf and Ptgds2/Hpgds following TCDD exposure. TCDD-regulated Genes in Testis and Comparison with Testis Ovary Pituitary Liver Ptgds2/ Hpgds up-regulated at 5 but not at 28 and 67 days of age up-regulated at 6, 12 and 14 but not at 3 or 25 days of age expressed but not regulated at 3 and 12 days of age expressed but not regulated at 3 and 6 days of age Fgf13 down-regulated at 5 but not at 28 and 67 days of age expressed but not regulated from 3 to 25 days of age) down-regulated at 3 and 12 days of age not detected at 3 and 6 days of age Gzmf up-regulated at 5 but not at 28 and 67 days of age not detected up-regulated at 3 and 12 days of age up-regulated at 3 but not at 6 days of age Art2b up-regulated at 5 but not at 28 and 67 days of age not detected up-regulated at 3 and 12 days of age up-regulated at 3 but not at 6 days of age doi:10.1371/journal.pone.0040306.t006 Table 6. Summary of the effects on Art2b, Fgf13, Gzmf and Ptgds2/Hpgds following TCDD exposure. Table 7. List of Matrices Family overrepresented in the promoter sequences of the TCDD regulated genes in testes. DNA Matrices Family Transcription factors p-value #sequences V$ETSF Ehf, Elf1,Elf2, Elf3,Elf4, Elf5,Elk1, Elk3,Elk4, Erf, Erg, Ets1, Ets2, Etv1,Etv2, Etv3,Etv4, Etv5,Etv6, Fev,Fli1, Gabpa,Gabpb1, Gabpb1l, Gabpb2, Sfpi1, Spdef, Spib,Spic 0.0127746 25 V$GCMF Gcm1, Gcm2 0.0072138 22 V$MYT1 Myt1, Myt1l,St18 0.0355996 22 V$CDXF Cdx1, Cdx2,Cdx4 0.00668855 21 V$SF1F Nr5a1, Nr5a2 0.00610062 16 V$RREB Rreb1 0.0090441 15 V$GRHL Grhl1, Grhl3 0.0176176 14 V$INSM Insm1 0.0473584 13 V$LTFM Ltf 0.00939393 12 V$NBRE Nr4a1, Nr4a2,Nr4a3 0.0365125 10 V$PAX9 Pax9 0.0173214 8 doi:10.1371/journal.pone.0040306.t007 rices Family overrepresented in the promoter sequences of the TCDD regulated genes in testes. Table 8. List of Matrices Family overrepresented in the promoter sequences of the TCDD regulated genes in ovaries. References (2007) The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein. FEBS Lett 581: 3608– 3615. p p g 7. Foster WG, Maharaj-Briceno S, Cyr DG (2010) Dioxin-induced changes in epididymal sperm count and spermatogenesis. Environ Health Perspect 118: 458–464. 25. Li X, Weber LW, Rozman KK (1995) Toxicokinetics of 2,3,7,8-tetrachlorodi- benzo-p-dioxin in female Sprague-Dawley rats including placental and lactational transfer to fetuses and neonates. Fundam Appl Toxicol 27: 70–76. 8. Ohsako S, Fukuzawa N, Ishimura R, Kawakami T, Wu Q, et al. (2010) Comparative contribution of the aryl hydrocarbon receptor gene to perinatal stage development and dioxin-induced toxicity between the urogenital complex and testis in the mouse. Biol Reprod 82: 636–643. 26. Hahn ME, Allan LL, Sherr DH (2009) Regulation of constitutive and inducible AHR signaling: complex interactions involving the AHR repressor. Biochem Pharmacol 77: 485–497. 9. Gray LE, Ostby JS (1995) In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters reproductive morphology and function in female rat offspring. Toxicol Appl Pharmacol 133: 285–294. 27. Korkalainen M, Tuomisto J, Pohjanvirta R (2004) Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain. Biochem Biophys Res Commun 315: 123–131. 10. Salisbury TB, Marcinkiewicz JL (2002) In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,4,7,8-pentachlorodibenzofuran re- duces growth and disrupts reproductive parameters in female rats. Biol Reprod 66: 1621–1626. 28. Lin TM, Ko K, Moore RW, Simanainen U, Oberley TD, et al. (2002) Effects of aryl hydrocarbon receptor null mutation and in utero and lactational 2,3,7,8- tetrachlorodibenzo-p-dioxin exposure on prostate and seminal vesicle develop- ment in C57BL/6 mice. Toxicol Sci 68: 479–487. 11. Dere E, Lo R, Celius T, Matthews J, Zacharewski TR (2011) Integration of genome-wide computation DRE search, AhR ChIP-chip and gene expression analyses of TCDD-elicited responses in the mouse liver. BMC Genomics 12: 365. 29. Baba T, Shima Y, Owaki A, Mimura J, Oshima M, et al. (2008) Disruption of aryl hydrocarbon receptor (AhR) induces regression of the seminal vesicle in aged male mice. Sex Dev 2: 1–11. 12. Huang G, Elferink CJ (2012) A novel nonconsensus xenobiotic response element capable of mediating aryl hydrocarbon receptor-dependent gene expression. Mol Pharmacol 81: 338–347. 30. Ge RS, Dong Q, Sottas CM, Chen H, Zirkin BR, et al. (2005) Gene expression in rat leydig cells during development from the progenitor to adult stage: a cluster analysis. Biol Reprod 72: 1405–1415. 13. References 18. Meijs-Roelofs HM, Uilenbroek JT, de Jong FH, Welschen R (1973) Plasma oestradiol-17beta and its relationship to serum follicle-stimulating hormone in immature female rats. J Endocrinol 59: 295–304. 1. Poland A, Knutson JC (1982) 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22: 517–554. 1. Poland A, Knutson JC (1982) 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22: 517–554. 19. Rebourcet D, Odet F, Verot A, Combe E, Meugnier E, et al. (2010) The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker. Int J Androl 33: 413–424. 2. Diamanti-Kandarakis E, Bourguignon J, Giudice LC, Hauser R, Prins GS, et al. (2009) Endocrine-disrupting chemicals: an Endocrine Society scientific state- ment. Endocr Rev 30: 293–342. 3. Hotchkiss AK, Rider CV, Blystone CR, Wilson VS, Hartig PC, et al. (2008) Fifteen years after "Wingspread"–environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go. Toxicol Sci 105: 235–259. 20. Wierinckx A, Roche M, Raverot G, Legras-Lachuer C, Croze S, et al. (2011) Integrated genomic profiling identifies loss of chromosome 11p impacting transcriptomic activity in aggressive pituitary PRL tumors. Brain Pathol 21: 533–543. 4. Larsen JC (2006) Risk assessments of polychlorinated dibenzo- p-dioxins, polychlorinated dibenzofurans, and dioxin-like polychlorinated biphenyls in food. Mol Nutr Food Res 50: 885–896. 21. Sabbah M, Prunier C, Ferrand N, Megalophonos V, Lambein K, et al. (2011) CCN5, a novel transcriptional repressor of the transforming growth factor beta signaling pathway. Mol Cell Biol 31: 1459–1469. 5. Mocarelli P, Gerthoux PM, Needham LL, Patterson DG, Limonta G, et al. (2011) Perinatal exposure to low doses of dioxin can permanently impair human semen quality. Environ Health Perspect 119: 713–718. g g p y 22. Abel J, Haarmann-Stemmann T (2010) An introduction to the molecular basics of aryl hydrocarbon receptor biology. Biol Chem 391: 1235–1248. 23. Mimura J, Ema M, Sogawa K, Fujii-Kuriyama Y (1999) Identification of a novel mechanism of regulation of Ah (dioxin) receptor function. Genes Dev 13: 20–25. 6. Bell DR, Clode S, Fan MQ, Fernandes A, Foster PM, et al. (2011) Interpretation of studies on the developmental reproductive toxicology of 2,3,7,8-tetrachloro- dibenzo-p-dioxin in male offspring. Food Chem Toxicol 48: 1439–1447. g p 24. Barouki R, Coumoul X, Fernandez-Salguero P. Acknowledgments We thank the profileXpert transcriptomic platform (Bron, France), and Se´verine Croze and Nicolas Nazaret for helpful assistance in microarray experiments. Supporting Information tomic analysis on 14 dpn ovaries. Values are the mean of the 3 samples analysed by microarray. (d) Real-time RT-PCR measurement of Cyp19a1 and Star genes in ovaries during prepubertal period. Values were normalized using Hprt and are mean 6 SEM of () number of ovaries. No significant differences were observed between control and TCDD- 200 ng treated females assessed from 3 to 25 postnatal days (dpn). (DOC) tomic analysis on 14 dpn ovaries. Values are the mean of the 3 samples analysed by microarray. (d) Real-time RT-PCR measurement of Cyp19a1 and Star genes in ovaries during prepubertal period. Values were normalized using Hprt and are mean 6 SEM of () number of ovaries. No significant differences were observed between control and TCDD- 200 ng treated females assessed from 3 to 25 postnatal days (dpn). (DOC) Supporting Information S1 Reproductive parameters of the female progeny exposed in utero to TCDD. (a) F1 female progeny weight from 5 to14 postnatal days. Values (g) are mean 6 SEM of () number of pups. TCDD-200 ng females were significantly lighter than control females at 4, 7 and 10 postnatal days (* p,0.05). (b) F1 female fertility assessment. Control females (7) and TCDD-200 ng females (5) were mated continuously with males from 2 to 7 months of age. A total of 5 litters was obtained for each female. Newborn pups were sacrificed after 2 days to check viability. For each litter, the mean number of days between beginning of mating and parturition is indicated for the 7 control and 5 TCDD-treated females. We also recorded the mean number of pups of each sex for the 7 control and 5 TCDD-treated females. (c) Expression levels of key genes involved in endocrine function of the ovary. No significant differences were observed between control and TCDD-200 ng treated females assessed through a transcrip- Supporting Information S1 Reproductive parameters of the female progeny exposed in utero to TCDD. (a) F1 female progeny weight from 5 to14 postnatal days. Values (g) are mean 6 SEM of () number of pups. TCDD-200 ng females were significantly lighter than control females at 4, 7 and 10 postnatal days (* p,0.05). (b) F1 female fertility assessment. Control females (7) and TCDD-200 ng females (5) were mated continuously with males from 2 to 7 months of age. TCDD-regulated Genes in Testis and Comparison with DNA Matrices Family Transcription factors p-value #sequences V$ETSF Ehf, Elf1,Elf2, Elf3,Elf4, Elf5,Elk1, Elk3,Elk4, Erf, Erg Ets1, Ets2, Etv1,Etv2, Etv3,Etv4, Etv5,Etv6, Fev, Fli1, Gabpa,Gabpb1, Gabpb1l, Gabpb2, Sfpi1,Spdef, Spib,Spic 0.00212351 54 V$SP1F Cdca7l, Eapp,Klf10, Klf11,Klf5, Sp1,Sp2, Sp3,Sp4, Sp5,Sp6, Sp7,Sp8 0.000099594 46 V$MYBL Myb, Mybl1,Mybl2 0.044378 46 V$IRFF Irf1, Irf2,Irf3, Irf4,Irf5, Irf6,Irf7, Irf8,Irf9, Rnf31 0.0422676 44 V$ZF02 Zbtb7a, Zbtb7b,Zfp148, Zfp202, Zfp219,Zfp281 0.0076816 41 V$EGRF Egr1, Egr2,Egr3, Egr4,Wt1 0.0218414 38 V$MAZF Maz, Zfp278 0.013593 33 V$CTCF Ctcf, Ctcfl,LOC100360757 0.0329843 33 V$PAX2 Pax2 0.0270729 27 V$ZF10 0.0137818 23 V$BTBF Zbtb33 0.0416754 14 doi:10.1371/journal.pone.0040306.t008 trices Family overrepresented in the promoter sequences of the TCDD regulated genes in ovaries. ble 8. List of Matrices Family overrepresented in the promoter sequences of the TCDD regulated genes July 2012 \| Volume 7 \| Issue 7 \| e40306 PLoS ONE \| www.plosone.org 12 Transcriptomic TCDD Signature in Gonads Author Contributions Conceived and designed the experiments: BLMB SM. Performed the experiments: SM DR MI RW CD EM. Analyzed the data: SM BLMB. Wrote the paper: BLMB SM HV JCT. Conceived and designed the experiments: BLMB SM. Performed the experiments: SM DR MI RW CD EM. Analyzed the data: SM BLMB. Wrote the paper: BLMB SM HV JCT. References Kobayashi S, Okamoto H, Iwamoto T, Toyama Y, Tomatsu T, et al. (2008) A role for the aryl hydrocarbon receptor and the dioxin TCDD in rheumatoid arthritis. Rheumatology (Oxford) 47: 1317–1322. 31. Zappa F, Ward T, Butler J, Pedrinis E, McGown A (2001) Overexpression of NAD(P)H:quinone oxidoreductase 1 in human reproductive system. J Histochem Cytochem 49: 1187–1188. 14. Sutter TR, Guzman K, Dold KM, Greenlee WF (1991) Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1 beta. Science 254: 415–418. 32. Huang P, Ceccatelli S, Hakansson H, Grandison L, Rannug A (2002) Constitutive and TCDD-induced expression of Ah receptor-responsive genes in the pituitary. Neurotoxicology 23: 783–793. 33. Valdez KE, Shi Z, Ting AY, Petroff BK (2009) Effect of chronic exposure to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin in female rats on ovarian gene expression. Reprod Toxicol 28: 32–37. 15. Boutros PC, Bielefeld KA, Pohjanvirta R, Harper PA (2009) Dioxin-dependent and dioxin-independent gene batteries: comparison of liver and kidney in AHR- null mice. Toxicol Sci 112: 245–256. rats on ovarian gene expression. Reprod Toxicol 28: 32–37. 34. Yu J, Wang Y, Zhou WH, Wang L, He YY, et al. (2008) Combination of estrogen and dioxin is involved in the pathogenesis of endometriosis by promoting chemokine secretion and invasion of endometrial stromal cells. Hum Reprod 23: 1614–1626. 16. de Kretser DM, Loveland KL, Meinhardt A, Simorangkir, Wreford N (1998) Spermatogenesis. Hum Reprod 13 Suppl 1: 1–8. 17. Dohler KD, Wuttke W (1975) Changes with age in levels of serum gonadotropins, prolactin and gonadal steroids in prepubertal male and female rats. Endocrinology 97: 898–907. 35. N’Diaye M, Le Ferrec E, Lagadic-Gossmann D, Corre S, Gilot D, et al. (2006) Aryl hydrocarbon receptor- and calcium-dependent induction of the chemokine PLoS ONE \| www.plosone.org 13 July 2012 \| Volume 7 \| Issue 7 \| e40306 July 2012 \| Volume 7 \| Issue 7 \| e40306 July 2012 \| Volume 7 \| Issue 7 \| e40306 Transcriptomic TCDD Signature in Gonads CCL1 by the environmental contaminant benzo[a]pyrene. J Biol Chem 281: 19906–19915. 42. Itoh N, Ornitz DM (2011) Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J Biochem 149: 121–130. 36. Vogel CF, Nishimura N, Sciullo E, Wong P, Li W, et al. (2007) Modulation of the chemokines KC and MCP-1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. Arch Biochem Biophys 461: 169–175. 43. 42. Itoh N, Ornitz DM (2011) Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J Biochem 149: 121–130. 47. Glowacki G, Braren R, Cetkovic-Cvrlje M, Leiter EH, Haag F, et al. (2001) Structure, chromosomal localization, and expression of the gene for mouse ecto- mono(ADP-ribosyl)transferase ART5. Gene 275: 267–277. 46. Sferruzzi-Perri AN, Macpherson AM, Roberts CT, Robertson SA (2009) Csf2 null mutation alters placental gene expression and trophoblast glycogen cell and giant cell abundance in mice. Biol Reprod 81: 207–221. References Beverdam A, Koopman P (2006) Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes. Hum Mol Genet 15: 417–431. ( ) p y 37. Haarmann-Stemmann T, Bothe H, Abel J (2009) Growth factors, cytokines and their receptors as downstream targets of arylhydrocarbon receptor (AhR) signaling pathways. Biochem Pharmacol 77: 508–520. 44. Cory AT, Boyer A, Pilon N, Lussier JG, Silversides DW (2007) Presumptive pre- Sertoli cells express genes involved in cell proliferation and cell signalling during a critical window in early testis differentiation. Mol Reprod Dev 74: 1491–1504. 38. Trivedi SG, Newson J, Rajakariar R, Jacques TS, Hannon R, et al. (2006) Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity. Proc Natl Acad Sci U S A 103: 5179–5184. 45. Hirst CE, Buzza MS, Sutton VR, Trapani JA, Loveland KL, et al. (2001) Perforin-independent expression of granzyme B and proteinase inhibitor 9 in human testis and placenta suggests a role for granzyme B-mediated proteolysis in reproduction. Mol Hum Reprod 7: 1133–1142. 39. Farhat A, Philibert P, Sultan C, Poulat F, Boizet-Bonhoure B (2011) Hematopoietic-Prostaglandin D2 synthase through PGD2 production is involved in the adult ovarian physiology. J Ovarian Res 4: 3. 46. Sferruzzi-Perri AN, Macpherson AM, Roberts CT, Robertson SA (2009) Csf2 null mutation alters placental gene expression and trophoblast glycogen cell and giant cell abundance in mice. Biol Reprod 81: 207–221. 40. Moniot B, Farhat A, Aritake K, Declosmenil F, Nef S, et al. (2011) Hematopoietic prostaglandin D synthase (H-Pgds) is expressed in the early embryonic gonad and participates to the initial nuclear translocation of the SOX9 protein. Dev Dyn 240: 2335–2343. 47. Glowacki G, Braren R, Cetkovic-Cvrlje M, Leiter EH, Haag F, et al. (2001) Structure, chromosomal localization, and expression of the gene for mouse ecto- mono(ADP-ribosyl)transferase ART5. Gene 275: 267–277. p y 41. Greene JM, Li YL, Yourey PA, Gruber J, Carter KC, et al. (1998) Identification and characterization of a novel member of the fibroblast growth factor family. Eur J Neurosci 10: 1911–1925. PLoS ONE \| www.plosone.org July 2012 \| Volume 7 \| Issue 7 \| e40306 14
https://openalex.org/W2547382175	http://www.scielo.br/pdf/tce/v24n2/0104-0707-tce-24-02-00381.pdf	es		Ser cuidado por un familiar: sentimientos existenciales de pacientes oncológicos	null	2,015	cc-by	6,368	Original Article - 381 http://dx.doi.org/10.1590/0104-07072015003760013 BEING CARED BY A FAMILY MEMBER: THE EXISTENTIAL FEELINGS OF CANCER PATIENTS1 julia Wakiuchi2, Anna Maria de Oliveira Salimena3, Catarina Aparecida Sales4 Extracted from the master’s dissertation - O cuidado sob o olhar do paciente oncológico: o cotidiano junto à família e a equipe de saúde, presented to the Graduate Nursing Program, Universidade Estadual de Maringá (UEM), 2013. Research financed by the Araucária Foundation for Scientific and Technological Development of Paraná, Brazil. 2 Doctoral student at the Graduate Nursing Program, UEM. Maringá, Paraná, Brazil. E-mail: julia.wakiuchi@gmail.com 3 Ph.D. in Nursing. Associate Professor, Undergraduate and Graduate Nursing Program, Universidade Federal de Juiz de Fora. Juiz de Fora, Minas Gerais, Brazil. E-mail: annasalimena@terra.com.br 4 Ph.D. in Nursing. Professor at the Graduate Nursing Program, UEM. Maringá, Paraná, Brazil. E-mail: casales@uem.br 1 Abstract: The present article aimed to understand the daily life of cancer patients under palliative care while experiencing home care provided by family members. This was a Heideggerian phenomenological study with 20 patients being treated at the primary health care service of Northeast Paraná, Brazil, between November 2012 and February 2013. Data collection was based on the following research guiding question: What has been your experience of being cared for by your family? Phenomenological analysis was conducted by selecting units of meaning from statements and then selecting ontologic themes, namely: “being alone in the presence of the other” and “finding the foundation of care in love.” In conclusion, when based on love and solicitude, home care coupled with palliative practices can give “wings” to those who are suffering and perceive their lives as threatened. Descriptors: Family. Home nursing. Neoplasms. Palliative care. Nursing. SENDO CUIDADO POR UM FAMILIAR: SENTIMENTOS EXISTENCIAIS DE PACIENTES ONCOLÓGICOS RESUMO: Objetivou-se compreender o cotidiano de pacientes com câncer em cuidados paliativos ao vivenciar o cuidado de sua família no domicílio. This was a Heideggerian phenomological study with 20 patients being treated at the primary health care service of Northeast Paraná, Brazil, between November 2012 and February 2013. Para coleta de dados, utilizou-se a questão norteadora: como tem sido sua experiência ao ser cuidado por sua família? A análise fenomenológica foi realizada pela seleção das unidades de sentido de cada depoimento e posterior seleção das temáticas ontológicas, sendo estas: “permanecendo só ante a presença do outro” e “encontrando no amor o fundamento para o cuidado”. Conclui-se que o cuidado domiciliar em consonância com a prática paliativista, quando fundamentado em amor e solicitude, é capaz de dar “asas” àqueles que, no padecimento, visualizaram suas vidas ameaçadas. DESCRITORES: Família. Assistência domiciliar. Neoplasias. Cuidados paliativos Enfermagem. SER CUIDADO POR UN FAMILIAR: SENTIMIENTOS EXISTENCIALES DE PACIENTES ONCOLÓGICOS ResumEN: este estudio tuvo como objetivo comprender el cotidiano de pacientes oncológicos en lo cuidado de la familia en domicilio. Estudio fenomenológico de Heidegger en se encuestó a 20 pacientes de la Atención Primaria a la Salud del Noroeste de Paraná, entre noviembre de 2012 a febrero de 2013. Se utilizó la pregunta guía: ¿Cómo ha sido su experiencia al ser cuidado por su familia?. El análisis fenomenológica se realizó mediante la selección de unidades de cada selección y posterior de las cuestiones ontológicas, que son lo que significa: Permaneciendo solo ante la presencia del otro y Encontrando en el amor el fundamento para el cuidado. Se concluye que el cuidado domiciliar en consonancia con la práctica paliativita, cuando fundamentado en amor y solicitud, es capaz de dar ‘alas’ a aquellos que visualizaron sus vidas amenazadas. DescriPtores: Familia. Asistencia domiciliar. Neoplasias. Cuidados paliativos. Enfermería. Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. - 382 - INTRODUCTION The magnitude of cancer, confirmed by its high morbimortality rates, constitutes a public health problem.1 Despite technological developments related to diagnostic precision and advanced therapeutic procedures, neoplasms are still grim diseases, requiring long-lasting, painful, and frequently debilitating treatments. The seriousness involved in the prognosis for cancer is usually linked to fear and negative associations that have a global effect on the patients’ spheres of existence, especially on their quality of life after initiating therapy.2 Thus, as the experiences involved in the everyday life of individuals with cancer are contextualized, both patients and their family circumstances as a whole have their stability threatened.3 Following this line of thought, it is clear the importance of the type of care provided, as cancer must be faced as much more than the physical manifestations caused by the disease, given the relationship between cancer, suffering, and the deterioration of being. This requires comprehension and emotional support from family members in accordance with the patient’s care needs.4 In order to meet such needs, palliative care should be introduced at the time of cancer diagnosis,5 as it allows patients and their family nuclei to actively participate in the therapeutic process and act as a team when faced with treatment options and care-related decisions.6 However, while receiving home care, patients experience an assortment of feelings that emerge from the patient-caregiver relationship in the context of the daily care routine. The literature has shown that recognizing the diligence and attention provided by family members, counteracts feelings of dependency and obligation felt by chronic patients, in addition to any predicaments that can occur during the care process.7 In the meantime, the everyday life of caregivers and care receivers is marked by an extremely close relationship, which can both strengthen the bonds and reciprocity between them and unleash conflicts and disagreements that will be expressed through the most diverse emotions within this pair.8 Considering that cancer patients experience both their own emotions related to the process of illness and treatment and the distinctive impressions and anxieties that permeate their care, we asked: How do cancer patients feel when receiving home care by their family? Wakiuchi J, Salimena AMO, Sales CA In the Brazilian literature, there is still a lack of studies that address home care for cancer patients.9 Such a gap becomes even more evident when touching on the subject of patients’ feelings and perceptions about home care. Thus, it is important to understand the feelings experienced by patients during the process of home care, as the transformations in their lives caused by cancer require attention and care. With this study, we hoped to have underpinned the importance of the family in home care for oncology patients. Our objective was to sensitize health professionals to the magnitude of such situations and the need for them to constantly increase their knowledge and skills. In so doing, they can provide support in a time in people’s lives usually permeated by doubt and difficulty. Thus, the aim of the present article was to understand the daily life of cancer patients under palliative care while experiencing home care provided by family members. Methodology This was a qualitative study based on existential phenomenology.10 This approach allowed us to focus on the phenomenon in order to understand others in their facticity, considering individuals within their singularities; in other words, as people in their own existential totality.10 The subjects of this study were cancer patients under palliative care under the scope of the Family Health Strategy program (FHS) in a city in the Northeast of the state of Paraná, Brazil. We selected basic health units (BHUs) from the Brazilian National Registry of Health Facilities, and those with the greatest number of FHS teams were chosen for this study. Three BHUs were included, one with four FHS teams and two with five FHS teams. We conducted a survey of all the cancer patients in these FHSs in November 2012 with the help of the FHS teams, community health agents (CHA), and the nurses responsible for each area. Inclusion criteria for the study were: being 18 years or older; residing in one of the areas within the scope of the FHS of Maringá, state of Paraná, Brazil; and having received cancer treatment for over six months. The last criterion aimed to select patients who had already been cared for by the healthcare teams after the onset of cancer and who could express this experience in their Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. Being cared by a family member: the existential feelings... speech. Furthermore, patients had to be in physical and mental conditions to answer questions, a criterion that was assessed together with the FSH teams while selecting participants. Hence, we excluded patients who were tracheotomized and could not respond to the interview orally and patients diagnosed with neurodegenerative diseases or mental disorders as specified in the FHS records, as such pathologies could interfere when trying to answer questions based on their actual experience. After identifying the subjects, we scheduled our first visits together with the CHAs, in order to take advantage of the already-existing bond between them and the patients and to comply with the ethical precepts of anonymity and confidentiality.11 The other meetings were scheduled without the presence of the CHAs and led to empathetic moments in which feelings were shared. Each patient was visited and interviewed approximately three times between November 2012 and February 2013. The researcher used the interrogation method in order to gain closer access to the discourse of the individual experiencing the analyzed situation. In so doing, the interviewer started down a path towards the phenomenon, or what manifests itself.12 To this end, the guiding question of the study was: What has been your experience of being cared for by your family? We also collected sociodemographic data and information on the disease and treatment received. The audio of the interviews was recorded in its entirety; the behaviors of the interviewees, such as pauses, moments of silence, tears, expressions, smiles, and touching were recorded in the field diary so that they could be analyzed together with the speech. The same researcher that conducted the interviews was responsible for their transcription. This was done in order to transmit the greatest possible reliability through the languages used by participants. Data analysis was based on Heidegger’s existential phenomenology, which starts with the ontic and moves toward the ontologic, thus discovering the phenomenon that is unconcealed to the researcher. Therefore, first, we carefully read the interviews in order to separate meaningful excerpts or units that appeared as fundamental structures of the participants’ existence. In other words, we selected excerpts that included the feelings expressed by the interviewees during an ontologic interrogation.13 Next, we analyzed these Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. - 383 - units of meaning to conduct the phenomenological selection of each one, giving rise to ontologic themes.13 These themes were analyzed in light of some ideas from Heidegger’s analytic philosophy, postulates of palliative care, and authors who deal with this field of study. The anonymity of patients was ensured by using pseudonyms from the book “The Diary of Anne Frank”. This book is the true story of a 12-year-old Jewish girl written in the form of a diary while she was in hiding with her family and friends during the Nazi persecution of the Jews.14 Her writings are pertinent to cancer patients, as they originated from a daily life lived in isolation, anguish, sacrifice, and fear, in which her diary was her only confidant. This study abided by the regulatory standards and norms for human research as established in Resolution 196 of October 10, 1996, later substituted by Resolution 466, of December 12, 2012,11 of the Brazilian National Health Council. To this end, subjects were given Informed Consent Forms, which they signed in duplicate. Furthermore, as the current research is affiliated with the project called “The applicability of palliative care in basic health care, promoting improved quality of life for patients with malignant neoplasm, their families, and health professions”, it was also approved by the Permanent Committee of Human Research Ethics of the Universidade Estadual de Maringá, under protocol no. 435/2011. RESULTS AND DISCUSSION Twenty home care cancer patients were included in the results of this study. Of these, 7 were men and 13 were women, all between the ages of 35 and 77. Ten received care from their spouses, four were cared for by their offspring, two by their mothers, two by their sisters, one by a sister-inlaw, and one by an aunt. Cancers were located in the brain, neck, larynx, breasts, lungs, intestines, uterus, prostate, and one was leukemia; of these, five presented metastases. Time of diagnosis ranged from seven months to eight years and all patients were receiving antineoplastic treatment at the time of the interview, i.e., chemotherapy, radiotherapy, or postoperative recovery. Language analysis revealed a mixture of feelings expressed by the patients, which ranged from the desire of being cared for to gratitude for the attention provided by family members. - 384 - Being alone in the presence of the other According to Heidegger’s analytics, our encounter with others is guided by our own beingin-the-world. In daily coexistence, we are placed before others who, far from representing those different fom us, are among those from whom, for the most part, we cannot distinguish ourselves. As such, the human being is also living amid others in the shared world of this encounter.10 In this perspective, encounters with the other take place in the world in which the being-there is not only concerned with the other, but mostly occupied with intra-mundane beings. Thus, “the being-with existentially determines presence, even when the other is not in fact given or perceived. Even the being-alone of presence is a form of being-with in the world. The other can be lacking only in and for a being-with. Being-alone is a deficient mode of being-with; its possibility is a proof for the latter”.10:177 In light of these considerations and under the prism of co-existence, those who provide care frequently deprive themselves of understanding their pre-ontologic constitution and are involved in an unauthentic form of being, hiding in the impersonal in which they usually remain.15 Thus, frequently, it is in such an unauthentic mode that human beings provide care, maintaining a certain distance from the care receiver. Therefore, in this case, caregivers are together-with, but are not open to being-with-one-another, as demonstrated in the speech below. [...] Just last week I spent three days in which I only got up to try to eat something, and even my daughter, on the days I have chemo, when I’m doing worse, she doesn’t come to help [...]. Those who see you can’t know what you’re feeling, so you don’t know what suffering a person is experiencing. Nobody knows. Sometimes I go to my mother’s house and chat with her, like I’m doing here with you, and they think having cancer is easy, that it’s simple (Anne); [...]. I think that instead of pressuring me, saying that I should eat, that I have to gather up my strength, they should stay here with me, because it’s the only way they can take care of me (Henk); My sister-in-law, my sister, they call me, but they don’t come here to give me support. I think the family has to be there, it’s important. So, because of such things, I used to be sad in the beginning, but not anymore. I believe that such things can even make you stronger, because then you know who you can count on (Wessel). Wakiuchi J, Salimena AMO, Sales CA On analyzing the participants’ discourse, they reported experiencing a certain “affective detachment” from their own families. In other words, their family members were close to them, but distant at the same time, as they didn’t effectively take part in their pain. The desire to know how the other is feeling was limited to a superficial perception, which escaped from true openness to the unveiling of what was actually being experienced at that moment. For these individuals, palliative care is but an ideal, as this philosophy seeks to provide individuals with the greatest possible care at home through the support of their families.16 By transposing this reality to Heidegger’s analytics, we understand that, because relationships with the other were based on modes of indifference and/or deficiency, patients felt excluded, as others did not reach them. These attitudes, which are characteristic of average and everyday co-existence, are due to feelings of uncanniness before the mishaps of the other.15 Inasmuch, the being-in-the-world can be alone even when the other or several others are co-present, as these frequently come to their encounter in modes of indifference or uncanniness. The latter is understood as the feeling of not being at home, and it is the most primordial phenomenon, 10 existentially and ontologically. Not recognizing things as they are is a feeling of those who do not belong anywhere. The public nature of the impersonal represses all nonfamiliarity, and being together-with, whether understood or not, is veiled, as if the subject were fleeing from understanding.10 By avoiding what is unpleasant, family members exhaust their possibilities of being-with the patient authentically. The lack of emotional reaction of the other before such lack of devotion and care for the patient show us that even in face of the difficulties of cancer treatment, those who experience the illness remain the foundation of support in their homes. By transcending the barriers imposed by the disease, patients find the possibility of caring for the other when they themselves need care, as portrayed in the following excerpts. [...] I went after everything myself. Since I have always been the soul of this house, my husband was helpless, he didn’t take any action, he was lost. So I went, I thought: ‘I have to go!’ I had my hair cut, because I knew they were going to shave it off. Before going into surgery, I did all the Christmas shopping Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. Being cared by a family member: the existential feelings... so that they would want for nothing. I tidied up the house, the room, so that afterwards I would have clean linen and everything. And up to this day I’m the one who takes the lead, that’s the way it is. (Anne); [...] I didn’t receive ‘care’; nobody took the initiative of helping me. So I have always took care of things myself! I dealt with everything while taking care of myself and the others, who can’t live without me. Of course, there are times when it’s impossible; you have to ask for help. However, each person deals with it in their own way, there are those who become dependent, there are those who put on a strong facade, [there are those] who exaggerate. I have always been like this, alone. (Elli); [...] God is the one taking care of me! [laughter]. It´s God! Because, to tell the truth, it’s not easy [she reflects for a moment and her eyes tear up]. But I am a fighter, you know, I am a strong woman! For me to lose my spirits, it has to be something big [laughter]. I feel that I am the pillar here at home, because if I lose heart, then... (Edith). The participants had experienced in their journey a time in which they could take care of both themselves and others on their own, but at the time of the interview, they had the need to receive care, which was not available at home. This was expressed in the participants’ discourse, which revealed that while they coexisted with the facticity of being the mother responsible for the home and coping with the process of illness and their frailties at the same time, they wished to move from “being-caredwith” to “being cared for.” In the language used by Edith, we noticed that when she perceived herself as unsupported in her fight against cancer, she directed her source of relief and consolation to an intangible plane and turned to the presence of a Supreme Being for comfort. The multidimensional complexity of spirituality makes it a vital element for many patients under palliative care. Its purpose can range from a form of emotional support to a search for meaning, purpose and transcendence in life.17 Based on this analysis, the onset of a disease like cancer, which brings deep changes to the lives of those who live with it, caused in the participants an avalanche of feelings, sometimes related to the search for support, sometimes questioning the authenticity of those who offered a hand. However, transiting in impropriety is not a static process, but a journey in search of authenticity that, within the home, can reach pure palliativism as a form of care, as shown in the next category. Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. - 385 - Finding the foundation of care in love Family care is a multidimensional phenomenon, simultaneously visible and abstract. It involves feelings of affection, harmony, and the necessary responsibility for such a stance.7 The principles of palliative state that the home environment is the best place for patients to enjoy the humanized aspects of care. In their own home, they can organize a more individualized and informal routine capable of providing comfort and quality of life.9 Heidegger conceives care as an ontologic condition that makes existence possible, and as such, is inherent to man in his mode of living.4 When governed by care, a relationship with the other depends on interpellation, i.e., being-there as care can only be open if the other is allowed to unconceal himself and manifest his anguish authentically or inauthentically in different moments of his life. 18 Amid all the chores, relationships, and obligations of each Dasein, the modes of occupation and preoccupation, or solicitude, are in full force. These manifest themselves in the unfolding in which care is formed in average everyday life.19 Authentic care can be manifested through solicitude, which in its own way seeks to anticipate the other in a considerate stance, taking on the responsibility for his care.10 Solicit care paves the way so that the other can take responsibility for him or herself. In other words, authentic care opens up possibilities for being one’s own self.20 Caring for a family member with cancer through authentic solicit manifestations allows caregivers to enter the patient’s existential dimension and become a being-with-the-other.21 Beingwith determines that “the I never has to leave itself to enter the world of the other”,20:168 as it communicates in the shared world of coexistence in an ontologic and existential manner, in which he is already and has always been available to the other. From the moment the family recognizes itself as the center of caregiving and takes full responsibility for this task, it begins a process of positive influence regarding the coping strategy used by the patient, decision making, daily self-care tasks and displays of affection.22 Even though patients did not clearly express such feelings of gratitude to the other, they recognized their importance and held them in esteem. Margot’s discourse is an example of acknowledgement of the immensity of care received. - 386 - [...] I received total support from my family. My aunt, nieces and nephews, and siblings all took turns, everybody called and visited me. Even my sisters-inlaw, which I like so much. So I always felt taken care of. I was happy to see that my family was always by my side, supporting me (Jopie); [...] I found my sisters to be a great source of support. They help me so much! They are so patient! [When her sister enters the room, she gives a coy smile and interrupts her praise]. I think that the fright I gave her made her blood pressure rise and she is already of a certain age. [She is silent for a bit, until her sister leaves] But she has been with me from the beginning, she talks with the doctors, she knew what was happening, both in terms of the disease and the suffering I experienced. Without her, I don’t know... (Margot); My wife does everything for me: food, bath, she tries to please me and everything. Poor thing. She does what she can. I think she helps me too much! Because since I’ve been this way, I’ve changed a lot, I don’t do anything anymore! But she still helps me; she gives me everything I need (Vossen). These statements show that the unconditional support of the family nurtured the patients’ health and wellbeing, reflected in their recognition of the efforts of the other to care for them. For these patients, the care received surpassed expectations and there were no gaps in care. Considering that a family fabric was woven for provision of care, patients did not report moments of loneliness or suffering. Furthermore, they referred to the period of treatment as a time of union, sharing, and love. Family members must take the lead in overcoming the obstacles presented by cancer by acting as the pillar of psychological support, in addition to reorganizing the entire home dynamic in order to adapt to the new conditions imposed by the disease.23 From this perspective, the devotion of these family members to the patient agrees with the concept of dedication, through which the human being emerges as a presence in his own existence and penetrates into the possibility of things, leading one to a primordial comprehension of such things.20 In dedication mode, one gives of one’s self to the world without losing the power-to-be-oneself. Furthermore, individuals expose themselves through this positive care, which builds them up authentically by means of surrender and diligent and affectionate creation.20 The participants’ discourses also portray a co-existence marked by affection, in which a person that takes on the care for another transforms his or her life into surrender to unconcealment. Wakiuchi J, Salimena AMO, Sales CA Such full dedication to the other, by means of minimal gestures, seems to decrease the burden of cancer and the suffering associated with it, as this burden ends up being shared between caregiver and care receiver. My wife does everything for me, everything, because I can’t do a thing. I even feel sorry for her. Even when I put a shirt on, she has to go and get it for me and help me put it on. I can’t do anything; she’s with me all the time. Here at home I have everything, everything I need, I have (Hans); My sister-in-law stopped living her life. She had already interrupted it to live with my father for three months and two months later, she left her life to come live mine, together with me. She goes everywhere with me, to the doctor, tests, chemo, home. She only goes home once I’ve eaten, when she thinks I am ok. And before you know it, she’s back here again [laughter] (Miep); My mother-in-law gave me a lot of support, you know, she comes to my house every day, she supports me in every sense, I think it’s abuse [laughter]. She helps me with work, she helps me with family problems, and with this support, everything is easier (Petronella). Such bonds between patient and caregiver emanate feelings of extreme joy and gratitude, reflected in the facial expressions of participants when they referred to the person who cared for them. In consonance with the patients’ statements, we noticed that the care received was capable of transcending the anguish brought on by cancer and made it so that this time in their lives could manifest itself as the revelation of an unconditional being-with. This mode of care, in its own particular way, reaches the meaning of its act ontologically and understands what is authentically important in the other. Primordially, providing care is the act of truly taking on the possibilities of being and, to this end, allowing to be guided by it and becoming attached; it is a loving attachment.20 Care involves directing both emotional and expressive feelings to the other, and such feelings must focus on developing the caregiver’s awareness and valuing the individuality of care receivers.7 Undoubtedly, family is the most ancient and the strongest institution of care, rooted in a system of socialization and interaction in which the limitation of a member of its bonds not only concerns the whole unit, but also leads them to redirect one of their own to the care of the other.24 Thus, despite the cultural heritage that flaunts women as primordial caregivers, we found Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. Being cared by a family member: the existential feelings... that both men and women had provided care to the interviewed patients. In the case of spouses, these remained faithful to their loved ones and the care provided gave new life to the affection between the couple. When we get married there’s that thing that says: in health and in sickness, in richness and in poor. Look, she has been victorious in this phase; I don’t know if I could have taken what she has been dealing with. She is everything to me. But if you don’t have a partner, you end up dying. I am rich in partner. Not in children, they hang around for an hour, and then they forget. But my partner! And you get so attached after this, it changes everything in life, it changes all the freedom of love, of knowing who is by your side, wow! There’s nothing like it (Harry); [...] my husband is a boyfriend to me, a father, my everything. The hair in his head is a bit white, but he’s my boyfriend! After I got sick, he even wants to cook for me, he makes different kinds of pies; he makes up recipes to make me happy. He looks them up on the Internet and then makes them. I’ll tell you the truth, with everything he does for me, it’s like I don’t even have this disease! I feel so exalted! (Sanne). These statements show how the relationship between man and wife was renewed. The difficulties of living with cancer mobilized feelings, and these feelings were reflected in kindness, affection, and giving of oneself to the other. Moreover, the palliative approach advocates that in order to improve the quality of life of those receiving care, support must be provided primordially through love in the home.2 The possibility of a relationship is the basis of care. When care is interwoven with love, affection, and benevolence, the being-with can form relationships and begin down the path towards the desire to take on this function.20 When care is a consequence of love, it happens naturally. Thus, the attitudes and feelings present in the process of devotion and attention originate from actual desire, making them truly authentic. The discourse of patients who received authentic care from their families expressed the impact that this attitude had on the course of their disease and their lives. Before the difficulties brought on by cancer, they found an opportunity to grow and transcend through care. Their lives gained new meaning coupled to feelings of acknowledgment and gratitude for the attitudes of those who offered care. The current research was limited to the population cared for by the Family Health Teams Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. - 387 - of a municipality known as a reference center for cancer treatment, and therefore, we recognize that our results do not encompass the reality and feelings experienced by all families living with cancer. Due to the particularities of this situation, each family should be analyzed within the context of their care facility. We were not able to include patients who lived in regions not covered by the FHS, even within the municipality in question, as such experiences can be distinctive. Also, these situations should serve as a reference to indicate the need for these places to be included in care coverage. CONCLUSIONS Heidegger’s hermeneutic and analytic movement allowed us to unveil the paths undertaken by care installed in the homes of cancer patients under palliative care. In the beginning of our journey to understand these patients, we found an inauthentic being-with, in which relationships were concealed by distance and self-accommodation. Many remain in this state of uncanniness, and in so doing, avoid paving the way to authenticity. However, before the horizon of possibilities into which man has been thrown, the encounter of solicitude also manifests itself through responsible and individualized attitudes that transform care into an expression of devotion and love. Such circumstances indicated that authentic care is connected to the legitimate desire of executing this function, which is confused with the feelings that one has for the care receiver. In this situation, receiving care becomes the factor that solidifies relationships and the love between caregivers and care receivers. This study showed the magnitude of home care that, in consonance with palliative care, can give “wings” to those who experience suffering and experience a threat to their life. When based on love and solicitude, home care can transform the period of the disease into a time of recognizing oneself and manifesting the best feelings of being alive. Through an act in which one surrenders to another, the burden of the disease can be shared and joy can emerge. It is essential for nursing to value this instance of care, for without the support and information of healthcare teams, living with the disease and its difficulties can become an abstract experience, which does not always contemplate all - 388 - the health and personal needs of an individual. We emphasize the value of the discourse in this study, which indicates the need for patients and families to be oriented so that they can alleviate the reality of cancer and so that healthcare teams, patients, and families can work together. Healthcare teams are also responsible for helping families find stability in home care, a factor that requires proximity with families and with the reality experienced by patients. Active listening, open dialogue, and humanized attitudes align nursing care with the wishes and needs of the patient, as portrayed in this study. It is important to emphasize that as this study was conducted in a city that is a reference for cancer treatment in its region, and whose healthcare teams have their own particular culture and behaviors, this study was restricted to depicting a reality which might not reflect or be applicable to other contexts. Thus, we emphasize that further studies are needed in this area to clarify possible deficiencies in health care for cancer patients and find strategies that can establish a new level of care in oncology nursing. REFERENCES 1. Gomes NS, Silva SR. Avaliação da autoestima de mulheres submetidas à cirurgia oncológica mamária. Texto Contexto Enferm [online]. 2013 [acesso 2014 Dez 01]; 22(2):509-16. Disponível em: http://www.scielo.br/scielo.php?pid=S010407072013000200029&script=sci_arttext 2. Fabricka P, Nowick A. Selected aspects of palliative care and quality of life at the terminal stage of neoplasic disease. Contemp Oncol (Pozn). 2012 Jan; 16(6):506-11. 3. Fernandes AF, Bonfim IM, Araújo IMA, Silva RM, Barbosa ICFJ, Santos MCL. Significado do cuidado domiciliar à mulher mastectomizada. Esc Anna Nery. 2012 Jan-Mar; 16(1):27-33. Wakiuchi J, Salimena AMO, Sales CA 8. Baptista BO, Beuter M, Girardon-Perlini NMO, Brondani CM, Budó MLD, Santos NO. A sobrecarga do familiar cuidador no âmbito domiciliar: uma revisão integrativa da literatura. Rev Gaúcha Enferm. 2012 Mar; 33(1):147-56. 9. Oliveira SG, Quintana AM, Budó MLD, Lüdtke MF, Cassel PA, Wottrich SH. Familiares cuidadores e a terminalidade: tendência da produção científica na área de saúde. Rev Min Enferm. 2011 Out-Dez; 15(4):588-94. 10. Heidegger M. Ser e tempo. Petrópolis (RJ): Vozes; 2012. 11. Ministério da Saúde (BR). Conselho Nacional de Saúde. Resolução nº 466, de 12 de dezembro de 2012. Aprova as diretrizes e normas regulamentadoras de pesquisas envolvendo seres humanos. Diário Oficial da União, Brasília, 13 jun. 2013. 12. Sales CA, Silva VA, Pilger C, Marcon SS. Música na terminalidade humana: concepções dos familiares. Rev Esc Enferm USP. 2011 Mar; 45 (1): 138-45. 13. Josgrilberg RS. A fenomenologia como novo paradigma de uma ciência do existir. In: Porladeck DD. A fenomenologia do cuidar: prática dos horizontes vividos nas áreas da saúde, educacional e organizacional. São Paulo (SP): Vetor; 2004. p. 31-52. 14. Frank OH, Pressler M. O diário de Anne Frank. Rio de Janeiro (RJ): Record; 2013. 15. Martins Filho JRF. Heidegger e a concepção de “outro” em ser e tempo. Aproximação. 2010 JanJun; 3:56-76. 16. Ferris FD, Bruera E, Cherny N, Cummings C, Currow D, Dudgeon D, et al. Palliative cancer care a decade later: accomplishments, the need, next steps. J Clin Oncol. 2009; 27(18):3052-58. 17. Asgeirsdottir GH, Sigurbjörnsson E, Traustadottir R, Sigurdardottir V, Gunnarsdottir S, Kelly E. “To cherish each day as it comes”: a qualitative study of spirituality among persons receiving pallitive care. Support Care Cancer. 2013; 21:1445-51. 18. Oliveira MFV, Carraro TE. Cuidado em Heidegger: uma possibilidade ontológica para enfermagem. Rev Bras Enferm. 2011; 64(2):376-80. 4. Sena ELS, Reis HFT, Carvalho PAL, Souza VS. A intersubjetividade e o conhecimento na perspectiva fenomenológica. Rev Rene. 2011 Jan-Mar; 12(1):181-8. 19. Nogueira RP. Extensão fenomenológica dos conceitos de saúde e enfermidade em Heidegger. Cienc Saúde Colet. 2011; 16(1):259-66. 5. Capello EMCS, Velosa MVM, Salotti SR, Guimarães HCQP. Enfrentamento do paciente oncológico e do familiar/cuidador frente à terminalidade da vida. J Health Sci Inst. 2012; 30(3):235-40. 20. Fernandes MA. O cuidado como amor em Heidegger. Rev Abordagem Gestalt. 2011 Jul-Dez; 17(2):158-71. 6. Kirk RA, Brawley O. Palliative care: a lifeline to quality of life. J Oncol Pract. 2012 Mar; 8(2):128-9. 7. Faber V, Rosanelli CP, Loro MM, Kolankiewicz ACB, Piovesan S, Leite MT. Percepções de doentes crônicos acerca do cuidado prestado por familiares. Ciênc Cuid Saude. 2012 Jul-Set; 11(3):565-72. 21. Sales CA, Matos PCB, Mendonça DPR, Marcon SS. Cuidar de um familiar com câncer: o impacto no cotidiano de vida do cuidador. Rev Eletr Enferm [online]. 2010 [acesso 2014 Dez 01]; 12(4):616-21. Disponível em: http://www.fen.ufg.br/revista/ v12/n4/pdf/v12n4a04.pdf 22. Ferreira AMC. Amor e liberdade em Heidegger. Kriterion. 2011; (123):139-58. Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. Being cared by a family member: the existential feelings... 23. Violim MR, Bringmann PB, Marcon SS, Waidman MAP, Sales CA. O significado de conviver com um familiar com estomia por câncer gastrointestinal. Rev Rene. 2011 Jul-Set; 12(3):510-7. Correspondece: Julia Wakiuchi Avenida Colombo, 5790 87020-270 – Maringá, Paraná, Brazil E-mail: julia.wakiuchi@gmail.com Text Context Nursing, Florianópolis, 2015 Abr-Jun; 24(2): 381-9. - 389 - 24. Rodriguez LH. Los qué, cuándo, por qué y como de la sedación paliativa. Rev Hosp Ital B Aires. 2010 Dez; 30(2):69-76. Received: December 19, 2013 Approved: December 01. 2014
https://openalex.org/W3150128323	https://rodin.uca.es/bitstream/10498/24942/1/2021_343.pdf	English	null	Effects of Osteopathic T9–T10 Vertebral Manipulation in Tonsillitis: A Randomized Clinical Trial	Healthcare	2,021	cc-by	10,409	Article Effects of Osteopathic T9–T10 Vertebral Manipulation in Tonsillitis: A Randomized Clinical Trial Agustín Luceño-Mardones 1,2, Irene Luceño-Rodríguez 3, Elena Sonsoles Rodríguez-López 1,4,* , Jesús Oliva-Pascual-Vaca 1,5,6 , Ignacio Rosety 7 and Ángel Oliva-Pascual-Vaca 1,5 1 Escuela de Osteopatía de Madrid, 28002 Madrid, Spain; agustin@osteopatiaglobal.es (A.L.-M.); joliva5@us.es (J.O.-P.-V.); angeloliva@us.es (Á.O.-P.-V.) 1 Escuela de Osteopatía de Madrid, 28002 Madrid, Spain; agustin@osteopatiaglobal.es (A.L.-M.); joliva5@us.es (J.O.-P.-V.); angeloliva@us.es (Á.O.-P.-V.) 2 Centro Sanitario de Fisioterapia y Osteopatía Agustín Luceño, 10005 Cáceres, Spain 3 Irene Luceño Psycho-Sexology Center, 28200 Madrid, Spain; iluceno@ucm.es 4 Department of Physiotherapy, Universidad Camilo José Cela, 28692 Madrid, Spain 5 Departamento de Fisioterapia, Universidad de Sevilla, 41004 Sevilla, Spain 6 Escuela Universitaria Fco. Maldonado, Osuna, 41640 Sevilla, Spain 7 School of Medicine, University of Cadiz, 11003 Cádiz, Spain; ignacio.rosety@uca.es * Correspondence: esrodriguez@ucjc.edu 1 Escuela de Osteopatía de Madrid, 28002 Madrid, Spain; agustin@osteopatiaglobal.es (A.L.-M.); joliva5@us.es (J.O.-P.-V.); angeloliva@us.es (Á.O.-P.-V.) 2 Centro Sanitario de Fisioterapia y Osteopatía Agustín Luceño, 10005 Cáceres, Spain 3 Irene Luceño Psycho-Sexology Center, 28200 Madrid, Spain; iluceno@ucm.es 4 Department of Physiotherapy, Universidad Camilo José Cela, 28692 Madrid, Spain 5 Departamento de Fisioterapia, Universidad de Sevilla, 41004 Sevilla, Spain 6 Escuela Universitaria Fco. Maldonado, Osuna, 41640 Sevilla, Spain 7 School of Medicine, University of Cadiz, 11003 Cádiz, Spain; ignacio.rosety@uca.es * Correspondence: esrodriguez@ucjc.edu 1 Escuela de Osteopatía de Madrid, 28002 Madrid, Spain; agustin@osteopatiaglobal.es (A.L.-M.); joliva5@us.es (J.O.-P.-V.); angeloliva@us.es (Á.O.-P.-V.) 2 Centro Sanitario de Fisioterapia y Osteopatía Agustín Luceño, 10005 Cáceres, Spain 3 Irene Luceño Psycho-Sexology Center, 28200 Madrid, Spain; iluceno@ucm.es 4 Department of Physiotherapy, Universidad Camilo José Cela, 28692 Madrid, Spain 5 Departamento de Fisioterapia, Universidad de Sevilla, 41004 Sevilla, Spain 6 Escuela Universitaria Fco. Maldonado, Osuna, 41640 Sevilla, Spain 7 School of Medicine, University of Cadiz, 11003 Cádiz, Spain; ignacio.rosety@uca.es * Correspondence: esrodriguez@ucjc.edu * Correspondence: esrodriguez@ucjc.edu Abstract: This study aimed to determine whether osteopathic manipulation of the T9–T10 vertebrae improves the evolution of tonsillitis. A randomized, stratiﬁed, controlled clinical trial with blinded patients, evaluator and data analyst was performed. The patients in the control group (CG) under- went a “sham” manipulation. A high-speed, low-amplitude technique was applied to the T9–T10 vertebrae in the osteopathic manipulative group (OMG) patients. The number of days needed to resolve the tonsillitis was signiﬁcantly lower (p = 0.025) in the OMG (2.03 ± 0.95 days) than the CG (2.39 ± 0.82 days). Citation: Luceño-Mardones, A.; Luceño-Rodríguez, I.; Rodríguez-López, E.S.; Oliva-Pascual-Vaca, J.; Rosety, I.; Oliva-Pascual-Vaca, Á. Effects of Osteopathic T9–T10 Vertebral Manipulation in Tonsillitis: A Randomized Clinical Trial. Healthcare 2021, 9, 394. https://doi.org/ 10.3390/healthcare9040394 Keywords: tonsillitis; osteopathy; manual therapy; physical therapy; spinal manipulation; OMT; otorhinolaryngology; otolaryngology; tonsillectomy Academic Editor: Saleh A. Naser Received: 8 February 2021 Accepted: 25 March 2021 Published: 1 April 2021 Article Effects of Osteopathic T9–T10 Vertebral Manipulation in Tonsillitis: A Randomized Clinical Trial Additionally, the number of episodes of tonsillitis after the treatment decreased signiﬁcantly more in the OMG (0.8 ± 1.88 episodes/year in total) than the CG (2 ± 2.12) (p = 0.005). In the OMG, 60.8% had no recurrences of tonsillitis, compared to 22.5% of the CG, in the following year (χ2 (1) = 15.57, p < 0.001). No patients reported adverse effects. It has been concluded that during an episode of tonsillitis, the number of days to resolution was signiﬁcantly lower after the application of an osteopathic manipulation of the T9–T10 vertebrae, compared to a sham manipulation. The number of subsequent year tonsillitis episodes was greatly reduced in both groups, signiﬁcantly more in the OMG than in the CG patients. Citation: Luceño-Mardones, A.; Luceño-Rodríguez, I.; Rodríguez-López, E.S.; Oliva-Pascual-Vaca, J.; Rosety, I.; Oliva-Pascual-Vaca, Á. Effects of Osteopathic T9–T10 Vertebral Manipulation in Tonsillitis: A Randomized Clinical Trial. Healthcare 2021, 9, 394. https://doi.org/ 10.3390/healthcare9040394 Academic Editor: Saleh A. Naser Received: 8 February 2021 Accepted: 25 March 2021 Published: 1 April 2021 Citation: Luceño-Mardones, A.; Luceño-Rodríguez, I.; Rodríguez-López, E.S.; Oliva-Pascual-Vaca, J.; Rosety, I.; Oliva-Pascual-Vaca, Á. Effects of Osteopathic T9–T10 Vertebral Manipulation in Tonsillitis: A Randomized Clinical Trial. Healthcare 2021, 9, 394. https://doi.org/ 10.3390/healthcare9040394 Academic Editor: Saleh A. Naser Received: 8 February 2021 Accepted: 25 March 2021 Published: 1 April 2021 healthcare healthcare healthcare 1. Introduction Palatal tonsillitis, either acute, recurrent or chronic, is a relatively common disease [1], especially in childhood [1,2]. The treatment of choice is the use of nonsteroidal anti- inﬂammatory drugs (NSAIDs) and/or antibiotics [1,3,4], and in some cases tonsillec- tomy [5–9]. These treatments can present side effects [5,6,10–13] in the short and long term [14]. Each episode of tonsillitis also implies costs related to absence from work and school [1,9,10,15]. With tonsillectomy, adults experience a sore throat for an average of 13 to 17 days [10,12]. In addition to being painful [9,10], the risk of iatrogenic mor- bidity [6,7,9,10,16,17] and mortality [5,6,16] of tonsillectomy must be taken into account. Although tonsillectomies have decreased in recent decades [18] (except for cases of sleep apnea due to hypertrophic tonsils and recurrent tonsillitis) [6,7,19,20], it is still one of the most frequent surgical interventions [6,9,10,15]. Tonsillectomy rates vary greatly between countries [18]. Among alternative and complementary medicine interventions, spinal manipulations have been used to treat tonsillitis. Through occiput-cervical manipulation, a favorable evolution has been obtained in recurrent or chronic childhood tonsillitis, in which Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/healthcare Healthcare 2021, 9, 394. https://doi.org/10.3390/healthcare9040394 Healthcare 2021, 9, 394 2 of 13 a high rate of pathological blockages of joints was also found [21]. This favorable evolution might be due to the fact that spinal manipulation may inﬂuence the biomarkers of local and systemic inﬂammation [22,23]. However, cervical manipulations are considered riskier manipulations, although severe complications are very rare [24–27]. Manipulation of the thoracic vertebrae presents fewer severe adverse events than cervical manipulation [25,27]. Furthermore, the vertebral segments T9 and T10 innervate the adrenal glands [28–31], which produce cortisol, and patients with tonsillitis have been found to have altered cor- tisol levels [32]. This hormone may inﬂuence the immune response [22,33,34], and its levels are modiﬁed after thoracic manipulation [22,23,35,36]. Thus, in a prospective study (75 participants) without a control group, manipulation of the lower thoracic vertebrae (mainly in the T9–T10 segments), obtained 55% resolution sooner than 24 h and 76% sooner than 48h, showing promising results for treating tonsillitis in both children and adults [37]. 2. Materials and Methods A stratiﬁed, randomized placebo trial was conducted with blinded patients, evaluator and data analyst. The study was carried out between 2-21-2014 and 6-30-2018 in the private center of Physiotherapy and Osteopathy of the main researcher of the study, in the city of Cáceres (Spain). The study was prospectively registered (ACTRN12612000068864), it followed the principles of the Declaration of Helsinki in its latest version [38] and was approved by the Ethics Committee of the University of Seville. 2.2. Participants The participants were recruited from a Physiotherapy and Osteopathy Health Center, a pediatrician’s ofﬁce, a General Medicine ofﬁce, and a private clinic (in its Emergency Service and in a General Medicine ofﬁce). Independently of the place where the recruitment took place, the participants were diagnosed by a collaborating physician who also visually measured the tonsil at enrollment in order to classify it in degrees (0-IV) according to the occupation of the oropharyngeal space [2]. Participants of both sexes between 3 and 65 years old diagnosed with acute or recurrent tonsillitis of less than 48 h of evolution, or chronic tonsillitis in the symptomatic phase, were included in the study. Those vacci- nated or treated with immunomodulators (immunoferon and the like) [39] during three previous years to recruitment, any subject who was suffering an episode of pharyngitis or adenoiditis without palatal tonsillitis (i.e., tonsillectomized), being treated with antibiotics immediately before the tonsillitis episode, subjects who did not provide a phone number in the initial questionnaire for control calls and subjects who presented contraindications to the experimental treatment [40,41] were excluded. Before the start of the study, all participants or their parents or guardians in case of minor signed an informed consent form. 1. Introduction The objective of the present study is to determine if osteopathic manipulation of the T9–T10 vertebrae improves the evolution of tonsillitis, under the hypothesis that it produces a decrease of days with symptoms and a decrease in recurrences. In addition, it is intended to analyze the possible inﬂuencing factors. 2.3. Treatment Protocols The same physiotherapist—oste with professional experience of more than 20 years, applied the interventions t groups. This researcher did not participate in the recruitment, randomization, eval nor statistical analysis, being blinded to all of those processes. Regardless of the application of the experimental procedure or placebo, all su continued pharmacological treatment (analgesics, NSAIDs and/or antibiotics) pres by their doctor. Figure 1. Placebo maneuver and its development. Patients in the osteopathic manipulation group (OMG) had a high-speed, low-amplitude technique applied to the T9–T10 vertebrae in a sitting position, with crossed arms, with contact via the therapist’s knees [42–44] (Figure 2). 1, 9, x FOR PEER REVIEW Figure 2. T9–T10 thrust manipulation in sitting position (adult man and 5-year-old girl). 2.4. Evaluations Figure 2. T9–T10 thrust manipulation in sitting position (adult man and 5-year-old girl). Figure 1. Placebo maneuver and its development. Patients in the osteopathic manipulation group (OMG) had a high-speed, low tude technique applied to the T9–T10 vertebrae in a sitting position, with crossed with contact via the therapist’s knees [42–44] (Figure 2). The osteopathic manipulative procedure or the sham manipulation were o Figure 1. Placebo maneuver and its development. Patients in the osteopathic manipulation group (OMG) had a high-speed, low-amplitude technique applied to the T9–T10 vertebrae in a sitting position, with crossed arms, with contact via the therapist’s knees [42–44] (Figure 2). 1, 9, x FOR PEER REVIEW Figure 1. Placebo maneuve Figure 1. Placebo maneuver and its development. Patients in the osteopathic manipulation group (OMG) had a high-speed, low tude technique applied to the T9–T10 vertebrae in a sitting position, with crosse with contact via the therapist’s knees [42–44] (Figure 2). The osteopathic manipulative procedure or the sham manipulation were o Patients in the osteopathic manipulation group (OMG) had a high-speed, low-amplitude technique applied to the T9–T10 vertebrae in a sitting position, with crossed arms, with contact via the therapist’s knees [42–44] (Figure 2). 9, x FOR PEER REVIEW plied once, as soon as possible after the enrollment, always in the first 48 h of th ning of the symptoms of that episode of tonsillitis. The same physiotherapist—o with professional experience of more than 20 years, applied the interventions groups. This researcher did not participate in the recruitment, randomization, ev nor statistical analysis, being blinded to all of those processes. 2.3. Treatment Protocols The subjects were randomly assigned to the study groups by using two computer- generated (Microsoft Excel) tables of sequence of numbers (2:1 ratio experimental/control), one table for those participants treated with antibiotics and the other one for those who were not, in order to avoid between-groups differences related to antibiotic use. The randomization sequence was guarded by an independent collaborator who guaranteed its concealment. To implement random allocation, sequentially numbered opaque sealed envelopes were used. Those researchers and collaborators who recruited the sample were blinded to the number sequence and to the intervention assignment. Additionally, every intervention was blinded for both the participants and evaluators. The patients in the Healthcare 2021, 9, 394 3 of 13 control group (CG) underwent sham manipulation, consisting of a careful 150◦passive ﬂexion of the shoulders, with gentle contact of the osteopath’s knees in the middle thoracic vertebrae, without impulse or causing tension. (Figure 1) intervention was blinded for both the participants and evaluators. The patients in trol group (CG) underwent sham manipulation, consisting of a careful 150° pass ion of the shoulders, with gentle contact of the osteopath’s knees in the middle vertebrae, without impulse or causing tension. (Figure 1) control group (CG) underwent sham manipulation, consisting of a careful 150◦passive ﬂexion of the shoulders, with gentle contact of the osteopath’s knees in the middle thoracic vertebrae, without impulse or causing tension. (Figure 1) intervention was blinded for both the participants and evaluators. The patients in trol group (CG) underwent sham manipulation, consisting of a careful 150° pass ion of the shoulders, with gentle contact of the osteopath’s knees in the middle vertebrae, without impulse or causing tension. (Figure 1) , g p vertebrae, without impulse or causing tension. (Figure 1) ion of the shoulders, with gentle contact of the osteopath’s knees in the middle th vertebrae, without impulse or causing tension. (Figure 1) Figure 1. Placebo maneuver and its development. Patients in the osteopathic manipulation group (OMG) had a high-speed, low- tude technique applied to the T9–T10 vertebrae in a sitting position, with crossed with contact via the therapist’s knees [42–44] (Figure 2). The osteopathic manipulative procedure or the sham manipulation were on plied once, as soon as possible after the enrollment, always in the first 48 h of the ning of the symptoms of that episode of tonsillitis. 2.3. Treatment Protocols Regardless of the application of the experimental procedure or placebo, all continued pharmacological treatment (analgesics, NSAIDs and/or antibiotics) pr by their doctor. Figure 2. T9–T10 thrust manipulation in sitting position (adult man and 5-year-old girl). Figure 2. T9–T10 thrust manipulation in sitting position (adult man and 5-year-old girl). Figure 2. T9–T10 thrust manipulation in sitting position (adult man and 5-year-old girl Figure 2. T9–T10 thrust manipulation in sitting position (adult man and 5-year-old girl). Healthcare 2021, 9, 394 4 of 13 The osteopathic manipulative procedure or the sham manipulation were only applied once, as soon as possible after the enrollment, always in the ﬁrst 48 h of the beginning of the symptoms of that episode of tonsillitis. The same physiotherapist—osteopath, with professional experience of more than 20 years, applied the interventions to both groups. This researcher did not participate in the recruitment, randomization, evaluation nor statistical analysis, being blinded to all of those processes. Regardless of the application of the experimental procedure or placebo, all subjects continued pharmacological treatment (analgesics, NSAIDs and/or antibiotics) prescribed by their doctor. 2.4. Evaluations The main results were the number of days for the total resolution of the symptoms of tonsillitis (fever, sore throat, cough, etc.) and the number of episodes of tonsillitis during the following year. Both were measured by telephone consultation [45–48]. The number of days for the resolution of the symptoms was evaluated seven days after the application of the experimental treatment or the placebo maneuver, and the result was also classiﬁed as excellent (resolution in less than 24 h), good (resolution in less than 48 h), moderate (improvement from the ﬁrst day, but with resolution ≥48 h) or poor (no improvement on the ﬁrst day, with resolution ≥48 h). The number of recurrences during the following year was evaluated through monthly telephone calls for 12 months. In the case of those participants younger than 18 years old, all the information was given by their parents or tutors. As secondary results, the different associations between the variables measured were evaluated. The independent variables were collected through the initial questionnaire ﬁlled out by the patient and the initial clinical examination carried out by the collaborating physician. In addition to the outcome variables, age, gender, season of the year, degree of tonsillar hypertrophy, consumption of NSAIDs, paracetamol and prescribed antibiotics were recorded. Additionally, we also registered the number of episodes in the two years prior to the study, the scheduling of tonsillectomies, the presence of a fever, odynophagia, cough, pultaceous tonsillitis, mucus the previous days, ear pain or infection, habitual nasal voice, nasal voice during the episode, habitual snoring, snoring during the episode or adenitis greater than 2 cm. From the moment that the participants were included in the study, they were adverted about the information they would be asked by phone, so that they had to pay attention and control it. For instance, they were asked to assess any sign of hyperthermia thermo- metrically. In the case of participants younger than 18 years old, all of this was explained to their parents or tutors. 2.5. Statistical Analysis Statistical analysis was carried out with SPSS 22.0 software (SPSS Science, Chicago, IL, USA). The descriptive study of the variables was carried out in tables with mean, standard deviation, 95% conﬁdence interval (CI) for continuous variables and in percentages for qualitative variables. Before carrying out the statistical analysis, the conditions of its application were considered; the Shapiro–Wilk test was used to verify that the sample met normality criteria. The Mann–Whitney U test was used to verify homogeneous distribution between groups when they did not meet normality criteria; otherwise, the T test was used. Chi-square was used for qualitative variables. A least squares estimation was used to quantify the interval of difference between groups. Analysis of variance of repeated measures (ANOVA) with linear model with Bonferroni adjustment was used to test the proﬁle of the change in the result of the number of episodes in the two and one year before and after the two study groups, and the comparison in pairs according to time and group. The global clinical effects for the repeated measures analysis were calculated using the Eta-squared value (η2), categorized as small = 0.01, medium = 0.06 and large = 0.14 [49]. For the analysis of the number of days of resolution, the Mann–Whitney U test was used. Pearson’s chi-square was used for the analysis according to group of the resolution greater Healthcare 2021, 9, 394 5 of 13 he two ccording than 48 h, initial result and at 12 months. The bivariate correlations of the quantitative variables were analyzed using Pearson’s coefﬁcient. A signiﬁcance level p < 0.05 and a conﬁdence level of 95% were established. Finally, participants under 18 years old were considered as children. 0.14 [49]. For the analysis of the number of days of resolution, the Mann–Whitne was used. Pearson’s chi-square was used for the analysis according to group of t lution greater than 48 h, initial result and at 12 months. The bivariate correlation quantitative variables were analyzed using Pearson’s coefficient. A significance l 0.05 and a confidence level of 95% were established. Finally, participants under The sample size calculation was performed (GRANMO v7.12; IMIM Hospital del Mar– Barcelona–Spain) for the proportion of cases resolved in the ﬁrst 48 h after the application of the intervention. An alpha level of 0.05 and a desired power (beta) of 80% with a bilateral contrast were assumed. 3.1. Sample While all of the 40 subjects randomized to CG completed the study, one of the eighty- one subjects randomized to OMG never answered the phone calls, so they were considered as a withdrawal. (Figure 3) p While all of the 40 subjects randomized to CG completed the study, one of the one subjects randomized to OMG never answered the phone calls, so they were ered as a withdrawal. (Figure 3) While all of the 40 subjects randomized to CG completed the study, one of the eighty- one subjects randomized to OMG never answered the phone calls, so they were considered as a withdrawal. (Figure 3) p While all of the 40 subjects randomized to CG completed the study, one of the one subjects randomized to OMG never answered the phone calls, so they were ered as a withdrawal. (Figure 3) Figure 3. Consort flow diagram. Figure 3. Consort ﬂow diagram. Thus, 120 subjects (70 women) aged between 3 and 57 years (23.53 ± 14.84 years) completed this study. No between-groups differences were found for any of the baseline characteristics (p > 0.05 for all variables). (Table 1). Comparing the number of episodes two years before with that of the previous year, there was a signiﬁcant increase in OMG (p = 0.011) and a clear similar trend in CG (p = 0.052). This increase occurred both in children (with a change from 4.79 episodes two years earlier, to 6.10 in the previous year) and in adults (from 4.19, to 4.94). There was a negative correlation between age and the number of episodes in the previous year (r = −0.186; p = 0.042). Tonsillar hypertrophy was more prevalent in males (p = 0.035), in those under 18 years of age (p = 0.009) and among those who snored during the episode (p = 0.047). As for the pediatric sample, when compared with the adult participants, it had a higher male composition (p = 0.033), had a higher prevalence of nasal mucus (p = 0.004) and snoring during episodes (p = 0.018). Figure 3. Consort ﬂow diagram. Thus, 120 subjects (70 women) aged between 3 and 57 years (23.53 ± 14.84 years) completed this study. No between-groups differences were found for any of the baseline characteristics (p > 0.05 for all variables). (Table 1). 2.5. Statistical Analysis Additionally, the sample size was estimated according to an expected proportion of resolutions of 65% in the ﬁrst 48 h in the OMG and a proportion of 36% in the CG. Losses were estimated at 15%. These assumptions generated a sample size of 80 participants in OMG and 40 in CG. old were considered as children. The sample size calculation was performed (GRANMO v7.12; IMIM Hosp Mar–Barcelona–Spain) for the proportion of cases resolved in the first 48 h after th cation of the intervention. An alpha level of 0.05 and a desired power (beta) of 80 a bilateral contrast were assumed. Additionally, the sample size was estimated ac to an expected proportion of resolutions of 65% in the first 48 h in the OMG and a p tion of 36% in the CG. Losses were estimated at 15%. These assumptions generated ple size of 80 participants in OMG and 40 in CG. 3.1. Sample Comparing the number of episodes two years before with that of the previous year, there was a signiﬁcant increase in OMG (p = 0.011) and a clear similar trend in CG (p = 0.052). This increase occurred both in children (with a change from 4.79 episodes two years earlier, to 6.10 in the previous year) and in adults (from 4.19, to 4.94). There was a negative correlation between age and the number of episodes in the previous year (r = −0.186; p = 0.042). Tonsillar hypertrophy was more prevalent in males (p = 0.035), in those under 18 years of age (p = 0.009) and among those who snored during the episode (p = 0.047). As for the pediatric sample, when compared with the adult participants, it had a higher male composition (p = 0.033), had a higher prevalence of nasal mucus (p = 0.004) and snoring during episodes (p = 0.018). Healthcare 2021, 9, 394 6 of 13 Table 1. Baseline characteristics of participants. Table 1. Baseline characteristics of participants. 3.1. Sample SD, standard deviation; NSAID, non-steroidal anti-inﬂammatory drugs. 3.1. Sample Control Control Group Osteopathic Manipulative Group Characteristics Mean/n SD/% Mean/n SD/% p-Value Age (years) (n = 120) 25.30 15.11 22.64 14.73 0.439 N◦episodes 2 years before (n = 116) 4.75 3.95 4.21 3.61 0.435 N◦episodes 1 year before (n = 120) 5.45 4.13 5.16 3.89 0.781 Sex (n =120) Male 17 42.5% 33 41.3% 0.896 Female 23 57.5% 47 58.8% Fever (n =120) No (Temperature <37.5 ◦C) 10 25.0% 21 26.3% 0.883 Yes (Temperature ≥37.5 ◦C) 30 75.0% 59 73.8% Pain or odinophagy (n = 119) No 0 0.0% 4 5.1% 0.148 Yes 40 100.0% 75 94.9% Cough currently (n = 118) No 22 55.0% 32 41.0% 0.149 Yes 18 45.0% 46 49.0% Levels of tonsilar hypertrophy (n = 115) The pillars do not protrude 1 2.6% 5 6.7% 0.760 Occupy less than 25% of the space 9 23.1% 16 21.3% Occupy 25–49% of the space 12 30.8% 20 26.7% Occupy 50–74% 14 35.9% 31 41.3% Occupy >75% 3 7.7% 3 4.0% Mucus days before the episode (n = 119) No 16 40.0% 28 35.4% 0.627 Yes 24 60.0% 51 64.6% Pain or infection of ears (n = 119) No 25 64.1% 47 58.8% 0.575 Yes 14 35.9% 33 42.3% Usual nasal voice (n = 119) No 32 80.0% 58 73.4% 0.429 Yes 8 20.0% 21 26.6% Nasal voice in the episode (n = 119) No 6 15.0% 11 13.9% 0.874 Yes 34 85.0% 68 86.1% Snore usually (n = 117) No 22 56.4% 56 71.8% 0.096 Yes 17 43.6% 22 29.2% Snore during the episode (n = 116) No 11 28.2% 30 39.0% 0.252 Yes 28 71.8% 47 61.0% Take prescribed antibiotics (n = 120) No 18 45.0% 37 46.3% 0.897 Yes 22 55.0% 43 53.8% Take prescribed NSAID (n = 119) No 6 15.0% 22 27.8% 0.119 Yes 34 85.0% 57 72.2% Take prescribed paracetamol (n = 120) No 15 37.5% 38 47.5% 0.298 Yes 25 62.5% 42 52.5% Pultous tonsilitis (n = 117) No 24 61.5% 49 62.8% 0.893 Yes 15 38.5% 29 37.2% Palpable adenitis over 2 cm (n = 116) No 5 13.2% 21 26.9% 0.095 Yes 33 86.8% 57 73.1% Station (n = 120) Spring 8 20.0% 13 16.3% 0.673 Summer 8 20.0% 12 15.0% Autumn 9 22.5% 26 32.5% Winter 15 37.5% 29 36.3% SD, standard deviation; NSAID, non-steroidal anti-inﬂammatory drugs. SD, standard deviation; NSAID, non-steroidal anti-inﬂammatory drugs. 3.2. Intervention Effects on Tonsillitis 3.2. Intervention Effects on Tonsillitis The number of days needed to resolve the tonsillitis was significantly lower (PSest = 0.247; p = 0.025) in the OMG (2.03 ± 0.95 days) than the CG (2.39 ± 0.82 days). In the case of the adults, this significant difference persisted (PSest = 0.263; p = 0.001), with better results for the OMG (2.00 ± 0.81 days) compared to the CG (2.44 ± 0.61 days), but not in the pediatric participants (p = 0.856; OMG 2.00 ± 1.00 days; CG 1.91 ± 0.83 days). p p (p y y ) The 63.75% of the subjects of the OMG resolved the episode in less than 48 h, compared to 42.5% in the CG (Table 2). In the case of adults, 68.6% of the subjects of the OMG resolved p p (p y y ) The 63.75% of the subjects of the OMG resolved the episode in less than 48 h, compared to 42.5% in the CG (Table 2). In the case of adults, 68.6% of the subjects of the OMG resolved Healthcare 2021, 9, 394 7 of 13 the episode in less than 48 h, compared to 33.3% in the CG, showing a signiﬁcant difference with medium effect size, which was not present in children. the episode in less than 48 h, compared to 33.3% in the CG, showing a signiﬁcant difference with medium effect size, which was not present in children. Table 2. Distribution of the results in the ﬁrst week (E = Excellent, G = Good, M = Moderate and P = Poor) in relation to the group to which they belonged (CG = Control Group, EG = Experimental Group) and according to age. 3.2. Intervention Effects on Tonsillitis Results 1st Week Results 1st Week Grouped Group E G M P E + G (Resolution < 48 h) M + P (Resolution ≥48 h) CG 5 (12.5%) 12 (30.0%) 14 (35.0%) 9 (22.5%) 17 (42.5%) 23 (57.5%) EG 20 (25.0%) 31 (38.75%) 11 (13.75%) 18 (22.5%) 51 (63.75%) 29 (36.25%) p value χ2 (3) = 8.35, p = 0.039, V = 0.264 χ2 (1) = 4.90, p = 0.022, V = 0.202 Children CG 4 (30.7%) 4 (30.7%) 4 (30.7%) 1 (7.7%) 8 (61.5%) 5 (38.5%) EG 9 (31%) 7 (24.1%) 5 (17.2%) 8 (27.6%) 16 (55.2%) 13 (44.8%) p value χ2 (3) = 2.57, p = 0.462 χ2 (1) = 0.15, p = 0.700 Adults CG 1 (3.7%) 8 (29.6%) 10 (37%) 8 (29.6%) 9 (33.3%) 18 (66.6%) EG 11 (21.5%) 24 (47%) 6 (11.7%) 10 (19.6%) 35 (68.6%) 16 (31.4%) p value χ2 (3) = 11.23, p = 0.011, V = 0.38 χ2 (1) = 8.94, p = 0.003, V = 0.34 E (resolution in <24 h); G (resolution in <48 h); M (improvement on the ﬁrst day, resolution ≥48 h); P (no improvement on the ﬁrst day, resolution ≥48 h). Table 2. Distribution of the results in the ﬁrst week (E = Excellent, G = Good, M = Moderate and P = Poor) in relation to the group to which they belonged (CG = Control Group, EG = Experimental Group) and according to age. E (resolution in <24 h); G (resolution in <48 h); M (improvement on the ﬁrst day, resolution ≥48 h); P (no improvement on the ﬁrst day, resolution ≥48 h). The number of episodes of tonsillitis after intervention decreased in the CG and in the OMG, as shown in Table 3 and Figure 4. Ten tonsillectomy cases (two from the CG and eight from the OMG), six cases that were vaccinated (three of the CG and three of the OMG) in the following year by their doctor and three cases of the OMG that had not registered the number of episodes of the second previous year were excluded. SD, standard deviation; CI, conﬁdence interval; CG, control group; OMG, osteopathic manipulative group. 3.2. Intervention Effects on Tonsillitis Number of episodes of tonsillitis two years before, one year before and one year after the study, in the CG and the OMG. Figure 4. Number of episodes of tonsillitis two years before, one year before and one year after the study, in the CG and the OMG. Figure 4. Number of episodes of tonsillitis two years before, one year before and one year after the study, in the CG and the OMG. Figure 4. Number of episodes of tonsillitis two years before, one year before and one year after the study, in the CG and the OMG. Figure 4. Number of episodes of tonsillitis two years before, one year before and one year after the study, in the CG and the OMG. In the analysis of the number of post-treatment episodes according to age, the OMG achieved a significant improvement when compared to the control group both in adults The 60.8% of the OMG had no tonsillitis recurrences, against 22.5% of the CG, in the following year (χ2 (1) = 15.57, p < 0.001), as shown in Table 4. (PSest = 0.25; p < 0.001; OMG 0.69 ± 1.61 episodes; CG 2.00 ± 2.07 episodes) and in childre (PSest = 0.29; p = 0.026; OMG 0.92 ± 2.26 episodes; CG 2.00 ± 2.33 episodes). Table 4. Analysis of covariance for efﬁcacy of intervention: tonsillitis recurrence at 12 months. (PSest = 0.25; p < 0.001; OMG 0.69 ± 1.61 episodes; CG 2.00 ± 2.07 episodes) and in children (PSest = 0.29; p = 0.026; OMG 0.92 ± 2.26 episodes; CG 2.00 ± 2.33 episodes). In the supporting repeated measures ANOVA including data points from two years before, one year before and after intervention, we did not find a significant time by group interaction effect (F (2,198) = 1.408, p = 0.247, η2 = 0.014). However, we found a significant time interaction effect (F (2,198) = 82.897, p < 0.001, η2 = 0.456). Specifically, post hoc anal- ysis in both groups showed significant between-time differences (p < 0.001) in the change from two years and one year before intervention compared to after the intervention. Table 4. Analysis of covariance for efﬁcacy of intervention: tonsillitis recurrence at 12 months. Control Group (n = 40) Osteopathic Manual Group (n = 79) Count % Count % p Value (Exact Sig. 3.2. Intervention Effects on Tonsillitis p p y In the analysis of the number of post-treatment episodes according to age, the OMG achieved a signiﬁcant improvement when compared to the control group both in adults (PSest = 0.25; p < 0.001; OMG 0.69 ± 1.61 episodes; CG 2.00 ± 2.07 episodes) and in children (PSest = 0.29; p = 0.026; OMG 0.92 ± 2.26 episodes; CG 2.00 ± 2.33 episodes). p p p In the supporting repeated measures ANOVA including data points from two years before, one year before and after intervention, we did not ﬁnd a signiﬁcant time by group interaction effect (F (2,198) = 1.408, p = 0.247, η2 = 0.014). However, we found a signiﬁcant time interaction effect (F (2,198) = 82.897, p < 0.001, η2 = 0.456). Speciﬁcally, post hoc analysis in both groups showed signiﬁcant between-time differences (p < 0.001) in the change from two years and one year before intervention compared to after the intervention. Furthermore, the number of episodes of tonsillitis after the treatment decreased signiﬁcantly more in the OMG (0.8 ± 1.88 episodes in total) than the CG (2 ± 2.12) (p = 0.005). Table 3. Analysis of covariance for efﬁcacy of intervention: number of episodes of tonsillitis. Control Group (n = 35) Osteopathic Manipulative Group (n = 66) Mean SD (95% CI) Mean SD (95% CI) p-Value (Time) p-Value (Group Time) p- Value (Time) Two years before 4.49 3.09 (3.19–5.78) 4.20 3.73 (3.25–5.14) 0.721 0.247 <0.001 One year before 5.23 4.30 (3.90–6.55) 4.86 3.76 (3.89–5.83) 0.660 After intervention 2 2.12 (1.33–2.66) 0.80 1.88 (0.32–1.28) 0.005 Two years before vs. after intervention −2.48 3.59 (−3.84–−1.12) −3.39 3.15 (−4.38–−2.40) CG p < 0.001; OMG p < 0.001 One year before vs. after intervention −3.22 3.52 (−4.55–−1.89) −4.06 3.06 (−5.02–−3.09) CG p < 0.001; OMG p < 0.001 SD, standard deviation; CI, conﬁdence interval; CG, control group; OMG, osteopathic manipulative group. Analysis of covariance for efﬁcacy of intervention: number of episodes of tonsillitis. Table 3. Analysis of covariance for efﬁcacy of intervention: number of episodes of tonsillitis. SD, standard deviation; CI, conﬁdence interval; CG, control group; OMG, osteopathic manipulative group. 8 of 13 Healthcare 2021, 9, 394 y ter interventio Figure 4. Number of episodes of tonsillitis two years before, one year before and one year after the study, in the CG and the OMG. Figure 4. 3.2. Intervention Effects on Tonsillitis 2-Sided) Chi-Square Recurrence 31 77.5% 31 39.2% <0.001 χ2 (1) = 15.57 No recurrence 9 22.5% 48 60.8% In the CG, there was a signiﬁcant correlation (p = 0.012) between the number of episodes in the previous year and recurrence at 12 months, but not in OMG (p = 0.095). Additionally, there was no correlation between age and the number of episodes in the subsequent year (p = 0.950). Five subjects from the OMG and one from the CG did not end up receiving the planned tonsillectomy, due to the fact that they had presented an average of 10 episodes in the previous year. Five of them were hypertrophic. p p y yp p No patients reported adverse effects, not even soreness, in relation to the manipulation received, neither in the OMG nor in the CG. 4. Discussion 4. Discussion After childhood, there is a natural tendency for the number of episodes to decrease [1,53], but in our study, no such trend was found in the previous two years, neither in children nor in adults. In contrast, the number of episodes of tonsillitis increased from the two years before to the year before participation in the study. This increase was signiﬁcant in the OMG, although not in the CG (p = 0.052), perhaps due to the smaller size of the CG. While the inﬂuence of recall bias cannot be ruled out, this increasing trend in tonsillitis episodes in the previous two years indicates that there was no decreasing trend in tonsillitis episodes, either natural or due to the treatments received. Therefore, the large decrease in episodes in the subsequent year observed in both groups does not seem attributable to the natural course of the disease or to the conventional treatments applied to the subjects in the sample. Furthermore, it should be remembered that vaccination and the consumption of immunomodulators [39] the previous year were considered as exclusion criteria. Although there was a negative correlation between age and the number of episodes in the previous year, this correlation was small (r = −0.186), and there was no correlation with the number of episodes in the following year (p = 0.950). Therefore, we can hypothesize that age had little inﬂuence on the long-term result and, therefore, treatment can be considered from three years of age. y g Several studies on tonsillitis describe a decrease of 0.5 to 1 episode on average in the subsequent year of follow-up in the control group, as shown in a recent clinical practice guideline [2]. In a study with children and adults, the placebo group obtained 51% of the cases without relapses in the following year [4,54], although in our CG there was a 22.5% of cases without relapses. In our sample, the considerable decrease in post-intervention episodes in CG led us to think that there could be a strong placebo component in both study groups [55–57]. In one pediatric study about prophylaxis with cefpodoxime versus placebo [4,58], the number of acute episodes at 12 months was reduced by 84% in the intervention group compared to 15% in the placebo group. In our sample, the reduction in the number of episodes of CG infants was much greater (64% fewer episodes, versus 83% reduction in OMG). 4. Discussion In spite of the importance of results from scientiﬁc research in evidence-based practice for osteopaths [50], there are not many studies about the role of spinal manipulation in tonsillitis, as previously explained [21,37]. Our study’s main objective was to explore if T9–T10 osteopathic manipulation reduces the duration of symptoms as well as the number of recurrences in subjects with tonsillitis. Through a randomized clinical trial, we observed that the number of days of resolution of the episode of tonsillitis was signiﬁcantly lower in the OMG than in the CG. This result was only found in adults but not in children. Likewise, the number of episodes of tonsillitis in the following year decreased signiﬁcantly in both groups, compared to the previous year, but signiﬁcantly more in the T9–T10 manipulation group. In this case, this effect was found in both adults and children. It should be noted that the follow-up comprised one whole year. Furthermore, there were no intergroup differences at baseline for any variable, Healthcare 2021, 9, 394 9 of 13 9 of 13 including taking antibiotics. However, the data obtained about the number of episodes in the previous year and, even more so, two years earlier, may be subject to recall bias. Previous studies reported an overestimation of the frequency of sore throats by parents in retrospective-prospective studies during childhood [51]. What is more, it has to be taken into account that children younger than three were excluded due to the low number of tonsillitis diagnosed in the ﬁrst year of life, which would affect the reported data for the previous years. On the other hand, the great improvement in the number of episodes in the subsequent year in both groups may be inﬂuenced by “attention bias” or Hawthorne effect [52], since the participants received a monthly telephone call from the collaborating nurse, and they knew they belonged to a clinical trial in which they had received treatment. Regarding the weight of the intervention group with a 2:1 randomization, it was due to ethical issues and the existence of indications of promising results for the experimental intervention [37]. Finally, in spite of the fact that the phone follow-up is a reality in both clinical and research settings [45–48], it is a limitation of the study that, in that it is based on subjective outcomes of patients’ interview through telephone. 4. Discussion This makes us suppose a strong placebo effect in pediatric age in our study, which might be increased by the fact that the parents/tutors observed the application of the procedures. Additionally, it could make the parents/tutors more likely to report a positive outcome. What is more, before the treatment, patients generally could already be perceived as very hopeful, possibly because the study center has been involved in research on this issue for years, with hundreds of cases treated in the region, which could favor the placebo effect [59]. To this effect, the aforementioned recall bias [51] can be added, which hypothetically would overestimate the number of episodes in the previous years, while those in the following year would be better quantiﬁed by monthly follow-up calls. In any case, regardless of the possible placebo effect of both groups, the number of post-treatment episodes in the OMG was signiﬁcantly lower compared to the CG. Healthcare 2021, 9, 394 10 of 13 10 of 13 In the same way, although the OMG obtained a signiﬁcantly faster total resolution of symptoms than the CG for the current episode, the good short-term results in a number of CG cases could be partly explained by this placebo effect as well [60]. However, taking up to two days for the episode to evolve, drug treatment and spontaneous remission can also explain these data. A Cochrane analysis [3] showed that spontaneous healing occurred in adults on day three in approximately 40%, and that the antibiotic group healed on average 16 h (0.67 days) earlier than the placebo group (in our sample, 0.36 days formerly the OMG). In clinical practice guidelines [1] it is considered that with adequate therapy, most patients, especially adolescents and adults, are asymptomatic within 48 h (in our study they were 63.75% of the OMG). Although the objective of our study was not to analyze the possible mechanisms of action that could explain the results obtained, we think that the neuroendocrine pathway should be considered. However, we did not evaluate neuroendocrine data, which con- stitutes a limitation of the study. As previously explained, patients with tonsillitis show altered cortisol levels [32], which is known to inﬂuence the immune response [22,33,34]. The adrenal glands, which produce cortisol, are innervated by T9 and T10 levels [28–31]. 4. Discussion Fur- thermore, spinal manipulation may inﬂuence the biomarkers of local and systemic inﬂam- mation [22,23], and speciﬁcally, thoracic manipulation modiﬁes cortisol levels [22,23,35,36]. All of this could inﬂuence the evolution of tonsillitis. The correlation observed in CG between the number of episodes in the previous year and recurrence at 12 months did not occur in the OMG, which leads us to hypothesize that manipulative treatment may be effective regardless of the number of previous episodes. In other words, a high number of episodes in the previous year is a poor prognostic factor for recurrence in the natural course of tonsillitis, but not after the proposed osteopathic treatment. The fact that six planned tonsillectomies were avoided (ﬁve in the OMG), despite having presented 10 episodes on average in the previous year and that ﬁve of them were hypertrophic, suggests that manipulative treatment might be proposed prior to decision making or performance of tonsillectomy, being able to prolong the expectant attitude in some nonurgent cases [2,9,10], even when the number of previous episodes or the tonsil size suggest a possible poor evolution. This is supported by the fact that, without adenoid hypertrophy, the size of the palatine tonsils does not correlate with obstructive sleep apnea syndrome [2]. In our study, no distinction was made between viral and bacterial pharyngo-tonsillitis. However, whether the doctor had prescribed antibiotics or not was controlled, either due to antistreptolysin O titer (ASOT), clinical suspicion of bacterial infection [38] or by conﬁrmation by rapid antigen detection tests of GABHS or pharyngeal exudate culture. In future studies, it would be interesting to perform rapid antigenic detection tests of GABHS, ASOT titer and/or throat swab culture [53] on all participants to ﬁnd out if the results of T9–T10 manipulation are inﬂuenced by the etiology of tonsillitis (viral or bacterial). 5. Conclusions During an episode of tonsillitis, the number of days to resolution was signiﬁcantly lower after the application of an osteopathic manipulation of the T9–T10 vertebrae com- pared to the application of a sham manipulation. In most OMG patients, remission of tonsillitis symptoms was obtained in less than 48 h, but not in CG. In addition, the number of tonsillitis episodes in the following year was greatly reduced in both groups, signiﬁcantly more in OMG than in sham patients. More studies are needed to conﬁrm these results and, if conﬁrmed, to analyze the possible mechanisms of action. 6. Declaration Ethical approval: This clinical trial was approved by the Ethical Committee for exper- imentation of the University of Seville, Seville. Informed consent was obtained from all participants prior to enrollment in the study, and all rights were protected. Transcriptions were anonymized and treated with strictest conﬁdence. All identifying information was removed by giving each participant a unique code that was used to attribute comments Healthcare 2021, 9, 394 11 of 13 11 of 13 during analysis. The protocol was performed following the Ethical Principles for Medical Research in Humans of the Declaration of Helsinki. during analysis. The protocol was performed following the Ethical Principles for Medical Research in Humans of the Declaration of Helsinki. This trial was registered in the ANZCTR (ACTRN12612000068864). This trial was registered in the ANZCTR (ACTRN12612000068864). Author Contributions: Conceptualization, A.L.-M. and Á.O.-P.-V.; methodology, I.R. and Á.O.-P.-V.; formal analysis, E.S.R.-L.; investigation, A.L.-M. and I.L.-R.; resources, A.L.-M. and J.O.-P.-V.; data curation, J.O.-P.-V.; writing—original draft preparation, E.S.R.-L., J.O.-P.-V. and A.L.-M.; writing— review and editing, I.R. and I.L.-R.; supervision, I.R. and Á.O.-P.-V.; project administration, Á.O.-P.-V. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of University of Seville (17/12/2011) Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. References 1. Windfuhr, J.P.; Toepfner, N.; Steffen, G.; Waldfahrer, F.; Berner, R. Clinical practice guideline: Tonsillitis I. diagnostics and nonsurgical management. Eur. Arch. Otorhinolaryngol. 2016, 273, 973–987. [CrossRef] 1. Windfuhr, J.P.; Toepfner, N.; Steffen, G.; Waldfahrer, F.; Berner, R. Clinical practice guideline: T nonsurgical management. Eur. Arch. Otorhinolaryngol. 2016, 273, 973–987. [CrossRef] 2. Mitchell, R.B.; Archer, S.M.; Ishman, S.L.; Rosenfeld, R.M.; Coles, S.; Finestone, S.A.; Friedman, N.R.; Giordano, T.; Hildrew, D.M.; Kim, T.W.; et al. Clinical practice guideline: Tonsillectomy in children (update). Otolaryngol. Head Neck Surg. 2019, 160, S1–S42. [CrossRef] A.; Glasziou, P.P.; Del Mar, C.B. Antibiotics for sore throat. Cochrane Database Syst. Rev. 2013, 2013, CD000023. 3. Spinks, A.; Glasziou, P.P.; Del Mar, C.B. Antibiotics for sore throat. Cochrane Database Syst. Rev. 2013 3. Spinks, A.; Glasziou, P.P.; Del Mar, C.B. Antibiotics for sore throat. Cochrane Database Syst. Rev. 2013, 2013, CD000023. [CrossRef] 4. Munck, H.; Jørgensen, A.W.; Klug, T.E. Antibiotics for recurrent acute pharyngo-tonsillitis: Systematic review. Eur. J. Clin. Microbiol. Infect. Dis. 2018, 37, 1221–1230. [CrossRef] 4. Munck, H.; Jørgensen, A.W.; Klug, T.E. Antibiotics for recurrent acute pharyngo-tonsillitis: Systematic review. Eur. J. Clin. Microbiol. Infect. Dis. 2018, 37, 1221–1230. [CrossRef] 5. Windfuhr, J.P.; Toepfner, N.; Steffen, G.; Waldfahrer, F.; Berner, R. Clinical practice guideline: Tonsillitis II. Surgical management. Eur. Arch. Otorhinolaryngol. 2016, 273, 989–1009. [CrossRef] 6. Mitchell, R.B.; Archer, S.M.; Ishman, S.L.; Rosenfeld, R.M.; Coles, S.; Finestone, S.A.; Friedman, N.R.; Giordano, T.; Hildrew, D.M.; Kim, T.W.; et al. Clinical practice guideline: Tonsillectomy in children (Update)—Executive summary. Otolaryngol. Head Neck Surg. 2019, 160, 187–205. [CrossRef] g 7. Lee, H.S.; Yoon, H.Y.; Jin, H.J.; Hwang, S.H. The safety and efﬁcacy of powered intracapsular tonsillectomy in children: A meta-analysis. Laryngoscope 2018, 128, 732–744. [CrossRef] 8. Kim, J.S.; Kwon, S.H.; Lee, E.J.; Yoon, Y.J. Can intracapsular tonsillectomy be an alternative to classical tonsillectomy? A meta-analysis. Otolaryngol. Head Neck Surg. 2017, 157, 178–189. [CrossRef] Burton, M.J.; Glasziou, P.P.; Chong, L.Y.; Venekamp, R.P. Tonsillectomy or adenotonsillectomy versus non- chronic/recurrent acute tonsillitis Cochrane Database Syst Rev 2014 2014 CD001802 [CrossRef] n, M.J.; Glasziou, P.P.; Chong, L.Y.; Venekamp, R.P. Tonsillectomy or adenotonsillectomy versus non-surgica 9. Burton, M.J.; Glasziou, P.P.; Chong, L.Y.; Venekamp, R.P. Tonsillectomy or adenotonsillectomy versu chronic/recurrent acute tonsillitis. Cochrane Database Syst. Rev. 2014, 2014, CD001802. [CrossRef] c/recurrent acute tonsillitis. Cochrane Database Syst. Rev. 2014, 2014, CD001802. [CrossRef] y 10. Alho, O.-P.; Koivunen, P.; Penna, T.; Teppo, H.; Koskela, M.; Luotonen, J. References Tonsillectomy versus watchful waiting in recurrent streptococcal pharyngitis in adults: Randomised controlled trial. Br. Med. J. 2007, 334, 939–941. [CrossRef] 10. Alho, O.-P.; Koivunen, P.; Penna, T.; Teppo, H.; Koskela, M.; Luotonen, J. Tonsillectomy versus watchful waiting in recurrent streptococcal phar ngitis in adults Randomised controlled trial Br Med J 2007 334 939 941 [CrossRef] 11. Andreou, N.; Hadjisymeou, S.; Panesar, J. Does tonsillectomy improve quality of life in adults? A systematic literature review. J. Laryngol. Otol. 2013, 127, 332–338. [CrossRef] [PubMed] 12. Koskenkorva, T.; Koivunen, P.; Koskela, M.; Niemela, O.; Kristo, A.; Alho, O.-P. Short-term outcomes of tonsillectomy in adult patients with recurrent pharyngitis: A randomized controlled trial. Can. Med Assoc. J. 2013, 185, E331–E336. [CrossRef] [PubMed] 13. Burton, M.J.; Pollard, A.J.; Ramsden, J.D.; Chong, L.Y.; Venekamp, R.P. Tonsillectomy for periodic fever, aphthous stomatitis, 13. Burton, M.J.; Pollard, A.J.; Ramsden, J.D.; Chong, L.Y.; Venekamp, R.P. Tonsillectomy for periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis syndrome (PFAPA). Cochrane Database Syst. Rev. 2014, 11, CD008669. [CrossRef] [PubMed] 14 Byars S G ; Stearns S C ; Boomsma J J Association of long term risk of respiratory allergic and infectious diseases with removal 13. Burton, M.J.; Pollard, A.J.; Ramsden, J.D.; Chong, L.Y.; Venekamp, R.P. Tonsillectomy for periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis syndrome (PFAPA). Cochrane Database Syst. Rev. 2014, 11, CD008669. [CrossRef] [PubMed] 14. Byars, S.G.; Stearns, S.C.; Boomsma, J.J. Association of long-term risk of respiratory, allergic, and infectious diseases with removal , J ; , J ; , J ; g, ; p, y p , p , pharyngitis and cervical adenitis syndrome (PFAPA). Cochrane Database Syst. Rev. 2014, 11, CD008669. [CrossRef] [PubMed] 14. Byars, S.G.; Stearns, S.C.; Boomsma, J.J. Association of long-term risk of respiratory, allergic, and infectious diseases with removal of adenoids and tonsils in childhood. JAMA Otolaryngol. Head Neck Surg. 2018, 144, 594–603. [CrossRef] [PubMed] 15. Senska, G.; Atay, H.; Pütter, C.; Dost, P. Long-term results from tonsillectomy in adults. Dtsch. Aerzteblatt Int. 2015, 112, 849–855. g p y p p pharyngitis and cervical adenitis syndrome (PFAPA). Cochrane Database Syst. Rev. 2014, 11, CD008669. [CrossRef] [PubMed] 14. Byars, S.G.; Stearns, S.C.; Boomsma, J.J. Association of long-term risk of respiratory, allergic, and infectious diseases with removal of adenoids and tonsils in childhood. JAMA Otolaryngol. Head Neck Surg. 2018, 144, 594–603. [CrossRef] [PubMed] of adenoids and tonsils in childhood. JAMA Otolaryngol. Head Neck Surg. 2018, 144, 594–603. y y p g y g g 18. Surgical Operations and Procedures Performed in Hospitals by ICD-9-CM. Available online: https:/ statistics-explained/pdfscache/37391.pdf (accessed on 28 January 2021). References Low morning serum cortisol levels in children with tonsillar hypertrophy and moderate-to-severe OSA. Sleep 2013, 36, 1349–1354. [CrossRef] [PubMed] 33. Oster, H.; Challet, E.; Ott, V.; Arvat, E.; De Kloet, E.R.; Dijk, D.-J.; Lightman, S.; Vgontzas, A.; Van Cauter, E. The functional and clinical signiﬁcance of the 24 h rhythm of circulating glucocorticoids Endocr Rev 2017 38 3 45 [CrossRef] 33. Oster, H.; Challet, E.; Ott, V.; Arvat, E.; De Kloet, E.R.; Dijk, D.-J.; Lightman, S.; Vgontzas, A.; Van Cauter, E. The functional and clinical signiﬁcance of the 24-h rhythm of circulating glucocorticoids. Endocr. Rev. 2017, 38, 3–45. [CrossRef] ster, H.; Challet, E.; Ott, V.; Arvat, E.; De Kloet, E.R.; Dijk, D.-J.; Lightman, S.; Vgontzas, A.; Van Cauter, E. T nical signiﬁcance of the 24-h rhythm of circulating glucocorticoids. Endocr. Rev. 2017, 38, 3–45. [CrossRef] j J g g gniﬁcance of the 24-h rhythm of circulating glucocorticoids. Endocr. Rev. 2017, 38, 3–45. [CrossRef] 34. Lightman, S.L.; Birnie, M.T.; Conway-Campbell, B.L. Dynamics of ACTH and cortisol secretion and implications for disease. Endocr. Rev. 2020, 1–21. [CrossRef] 35. Sampath, K.K.; Katare, R.; Tumilty, S. Stress axis and osteopathy: A dual hormone approach. Int. J. Osteopat. Med. 2019, 1–7. [CrossRef] 36. Sampath, K.K.; Botnmark, E.; Mani, R.; Cotter, J.D.; Katare, R.; Munasinghe, P.E.; Tumilty, S. Neuroendo a thoracic spinal manipulation in healthy men. J. Orthop. Sports Phys. Ther. 2017, 47, 617–627. [CrossRe K.K.; Botnmark, E.; Mani, R.; Cotter, J.D.; Katare, R.; Munasinghe, P.E.; Tumilty, S. Neuroendocrine response ath, K.K.; Botnmark, E.; Mani, R.; Cotter, J.D.; Katare, R.; Munasinghe, P.E.; Tumilty, S. Neuroendocrine resp acic spinal manipulation in healthy men. J. Orthop. Sports Phys. Ther. 2017, 47, 617–627. [CrossRef] g y p c spinal manipulation in healthy men. J. Orthop. Sports Phys. Ther. 2017, 47, 617–627. [CrossRef] 37. Luceño Mardones, A. Tratamiento de la amigdalitis mediante manipulación osteopática de las vértebras T8 a T12 hipomóviles. Rev. Cientíﬁca Ter. Man. Osteopat. 2004, 17, 13–32. ﬁ p 38. World Medical Association. World medical association declaration of Helsinki: Ethical principles for medical research involving human subjects. J. Am. Med. Assoc. 2013, 310, 2191–2194. [CrossRef] 39. Sánchez-Rodríguez, C.; Peraza Cruces, K.R.; Rodrigáñez Riesco, L.; García-Vela, J.; Sanz-Fernández, R. Immunomodulatory effect of Polypodium leucotomos (Anapsos) in child palatine tonsil model. Int. J. Pediatr. Otorhinolaryngol. 2018, 107, 56–61. [CrossRef] [PubMed] 40. Fisher, L.R.; Alvar, B.A.; Maher, S.F.; Cleland, J.A. References [CrossRef] [PubMed] 15. Senska, G.; Atay, H.; Pütter, C.; Dost, P. Long-term results from tonsillectomy in adults. Dtsch. Aerzteblatt Int. 2015, 112, 849–855. [CrossRef] J y g g [ ] [ ] 15. Senska, G.; Atay, H.; Pütter, C.; Dost, P. Long-term results from tonsillectomy in adults. Dtsch. Aerzteblatt Int. 2015, 112, 849–855. [CrossRef] [ ] 16. Greig, S.R. Current perspectives on the role of tonsillectomy. J. Paediatr. Child Health. 2017, 53, 1065–1070. [CrossRef] 17. Amoils, M.; Chang, K.W.; Saynina, O.; Wise, P.H.; Honkanen, A. Postoperative complications in pediatric tonsillectomy and adenoidectomy in ambulatory vs inpatient settings JAMA Otolaryngol Head Neck Surg 2016 142 344 350 [CrossRef] [PubMed] 16. Greig, S.R. Current perspectives on the role of tonsillectomy. J. Paediatr. Child Health. 2017, 53, 1065–1070. [CrossRef] 16. Greig, S.R. Current perspectives on the role of tonsillectomy. J. Paediatr. Child Health. 2017, 53, 1065–1070. [CrossRef] 17. Amoils, M.; Chang, K.W.; Saynina, O.; Wise, P.H.; Honkanen, A. Postoperative complications in pediatric tonsillectomy and adenoidectomy in ambulatory vs inpatient settings. JAMA Otolaryngol. Head Neck Surg. 2016, 142, 344–350. [CrossRef] [PubMed] 18. Surgical Operations and Procedures Performed in Hospitals by ICD-9-CM. Available online: https://ec.europa.eu/eurostat/ statistics-explained/pdfscache/37391.pdf (accessed on 28 January 2021). Healthcare 2021, 9, 394 12 of 13 12 of 13 19. Huang, C.-G.; Huang, Y.-S.; Chen, N.-H.; Li, H.-Y.; Fang, T.-J.; Hsu, J.-F.; Lai, H.-C.; Chen, J.-Y.; Lee, L.-A. Relationships among and predictive values of obesity, inﬂammation markers, and disease severity in pediatric patients with obstructive sleep apnea before and after adenotonsillectomy. J. Clin. Med. 2020, 9, 579. [CrossRef] y 20. Templier, L.; Rossi, C.; Miguez, M.; Pérez, J.D.L.C.; Curto, A.; Albaladejo, A.; Vich, M.L. Combined treatments in children with OSA: A systematic review. J. Clin. Med. 2020, 9, 2387. [CrossRef] 20. Templier, L.; Rossi, C.; Miguez, M.; Pérez, J.D.L.C.; Curto, A.; Albaladejo, A.; Vich, M.L. Combined surgical and orthodontic treatments in children with OSA: A systematic review. J. Clin. Med. 2020, 9, 2387. [CrossRef] 21. Lewit, K. Manipulative Therapy in Rehabilitation of the Locomotor System, 3rd ed.; Butterworth Heinemann: Oxford, UK, 1999; 21. Lewit, K. Manipulative Therapy in Rehabilitation of the Locomotor System, 3rd ed.; Butterworth Heinem pp. 21–23, 282–283. pp 22. Kovanur-Sampath, K.; Mani, R.; Cotter, J.; Gisselman, A.S.; Tumilty, S. Changes in biochemical markers following spinal manipulation-a systematic review and meta-analysis. Musculoskelet. Sci. Pract. 2017, 29, 120–131. [CrossRef] 23. Plaza-Manzano, G.; Molina-Ortega, F.; Lomas-Vega, R.; Martínez-Amat, A.; Achalandabaso-Ochoa, A.; Hita-Contreras, F. References Changes in biochemical markers of pain perception and stress response after spinal manipulation. J. Orthop. Sports Phys. Ther. 2014, 44, 231–239. [CrossRef] 24. Nielsen, S.M.; Tarp, S.; Christensen, R.; Bliddal, H.; Klokker, L.; Henriksen, M. The risk associated with spinal manipulation: An overview of reviews. Syst. Rev. 2017, 6, 64. [CrossRef] 25. Gorrell, L.M.; Engel, R.M.; Brown, B.; Lystad, R.P. The reporting of adverse events following spinal manipulation in randomized clinical trials—A systematic review. Spine J. 2016, 16, 1143–1151. [CrossRef] 26. Kranenburg, H.; Schmitt, M.; Puentedura, E.; Luijckx, G.; van der Schans, C. Adverse events associated with the use of cervical spine manipulation or mobilization and patient characteristics: A systematic review. Musculoskelet. Sci. Pract. 2017, 28, 32–38. [CrossRef] 27. Carnes, D.; Mars, T.S.; Mullinger, B.; Froud, R.; Underwood, M. Adverse events and manual therapy: A systematic review. Man. Ther. 2010, 15, 355–363. [CrossRef] [PubMed] 28. Tomlinson, A.; Coupland, R.E. The innervation of the adrenal gland. IV. Innervation of the rat adrenal medulla from birth to old age. A descriptive and quantitative morphometric and biochemical study of the innervation of chromafﬁn cells and adrenal medullary neurons in Wistar rats. J. Anat. 1990, 169, 209–236. [PubMed] y 29. Kesse, W.K.; Parker, T.L.; Coupland, R.E. The innervation of the adrenal gland. I. The source of pre- and postganglionic nerve ﬁbres to the rat adrenal gland. J. Anat. 1988, 157, 33–41. [PubMed] 30. Mohamed, A.A.; Parker, T.L.; Coupland, R.E. The innervation of the adrenal gland. II. The source of spinal afferent nerve ﬁbres to the guinea-pig adrenal gland. J. Anat. 1988, 160, 51–58. 30. Mohamed, A.A.; Parker, T.L.; Coupland, R.E. The innervation of the adrenal gland. II. The so the guinea-pig adrenal gland. J. Anat. 1988, 160, 51–58. g p g g 31. Testud, L. Tratado de Anatomía Humana (Tomo III); Salvat: Barcelona, Spain, 1978; p. 1131. 32. Malakasioti, G.; Alexopoulos, E.I.; Varlami, V.; Chaidas, K.; Liakos, N.; Gourgoulianis, K.; Kaditis, A.G. Low morning serum cortisol levels in children with tonsillar hypertrophy and moderate-to-severe OSA. Sleep 2013, 36, 1349–1354. [CrossRef] [PubMed] 33 O t H Ch ll t E Ott V A t E D Kl t E R Dijk D J Li ht S V t A V C t E Th f ti l d 32. Malakasioti, G.; Alexopoulos, E.I.; Varlami, V.; Chaidas, K.; Liakos, N.; Gourgoulianis, K.; Kaditis, A.G. References Short-term effects of thoracic spine thrust manipulation, exercise, and education in individuals with low back pain: A randomized controlled trial. J. Orthop. Sports Phys. Ther. 2020, 50, 24–32. [CrossRef] [PubMed] 41. De Oliveira, R.F.; Liebano, R.E.; Costa, L.D.C.M.; Rissato, L.L.; Costa, L.O.P. Immediate effects of region-speciﬁc and non-region- speciﬁc spinal manipulative therapy in patients with chronic low back pain: A randomized controlled trial. Phys. Ther. 2013, 93, 748–756. [CrossRef] [PubMed] [ ] [ ] artman, L. Handbook of Osteopathic Technique, 3rd ed.; Stanley Thornes: Cheltenham, UK, 1997; pp. 116–130. f p q y pp 43. Le Corre, F.; Rageot, E. Manipulaciones Vertebrales; Masson: Barcelona, Spain, 1990; pp. 143–147. 43. Le Corre, F.; Rageot, E. Manipulaciones Vertebrales; Masson: Barcelona, Spain, 1990; pp. 143–147. 44. Maigne, R. Manipulaciones Columna Vertebral y Extremidades; Norma: Madrid, Spain, 1997; p. 54. 45. Brueton, V.C.; Tierney, J.; Stenning, S.; Harding, S.; Meredith, S.; Nazareth, I.; Rait, G. Strategies to improve retention in randomised trials. Cochrane Database Syst. Rev. 2013, MR000032–126. [CrossRef] 46. Elnicki, D.M.; The TELI Group; Ogden, P.; Flannery, M.; Hannis, M.; Cykert, S. Telephone medicine for internists. J. Gen. Intern. Med. 2000, 15, 337–343. [CrossRef] Healthcare 2021, 9, 394 13 of 13 13 of 13 47. Downes, M.J.; Mervin, M.C.; Byrnes, J.M.; Scuffham, P.A. Telephone consultations for general practice: A systematic review. Syst. Rev. 2017, 6, 1–6. [CrossRef] [PubMed] [ ] [ ] 48. Flodgren, G.; Rachas, A.; Farmer, A.J.; Inzitari, M.; Shepperd, S. Interactive telemedicine: Effects on professional practice and health care outcomes. Cochrane Database Syst. Rev. 2015, 2015, CD002098. [CrossRef] 49 Field A Discovering Statistics Using IBM SPSS Statistics 3rd ed ; Sage Publication: London UK 2013 48. Flodgren, G.; Rachas, A.; Farmer, A.J.; Inzitari, M.; Shepperd, S. Interactive telemedicine: Effects on p health care outcomes. Cochrane Database Syst. Rev. 2015, 2015, CD002098. [CrossRef] y g Statistics Using IBM SPSS Statistics, 3rd ed.; Sage Publication: London, UK, 2013. y 49. Field, A. Discovering Statistics Using IBM SPSS Statistics, 3rd ed.; Sage Publication: London, UK, 2013. 50. Fernández-Domínguez, J.C.; Escobio-Prieto, I.; Sesé-Abad, A.; Jiménez-López, R.; Romero-Franco, N.; Oliva-Pascual-Vaca, Á. Health sciences—Evidence based practice questionnaire (HS-EBP): Normative data and differential proﬁles in spanish osteopathic professionals. Int. J. Environ. Res. Public Health 2020, 17, 8454. [CrossRef] [PubMed] 51. Capper, R.; Canter, R.J. How well do parents recognize the difference between tonsillitis and other sore throats? Clin. Otolaryngol. 2001, 26, 458–464. [CrossRef] 52. References McCambridge, J.; Witton, J.; Elbourne, D.R. Systematic review of the Hawthorne effect: New co research participation effects. J. Clin. Epidemiol. 2014, 67, 267–277. [CrossRef] 53. Georgalas, C.C.; Tolley, N.S.; Narula, P.A. Tonsillitis. BMJ Clin Evid. 2014, 2014, 1–14. [CrossRef] 53. Georgalas, C.C.; Tolley, N.S.; Narula, P.A. Tonsillitis. BMJ Clin Evid. 2014, 2014, 1–14. [CrossRef] 54. Lildholdt, T.; Doessing, H.; Lyster, M.; Outzen, K. The natural history of recurrent acute tonsillitis and a clinical trial of azithromycin for antibiotic prophylaxis. Clin. Otolaryngol. Allied Sci. 2003, 28, 371–373. [CrossRef] 55. Cerritelli, F.; Verzella, M.; Cicchitti, L.; D’Alessandro, G.; Vanacore, N. The paradox osteopathy: A systematic review. Medicine 2016, 95, e4728. [CrossRef] 56. Bishop, F.L.; Coghlan, B.; Geraghty, A.W.; Everitt, H.; Little, P.; Holmes, M.M.; Seretis, D.; Lewith, G. What techniques might be used to harness placebo effects in non-malignant pain? A literature review and survey to develop a taxonomy. BMJ Open 2017, 7, 1–12. [CrossRef] 57. Benedetti, F. Placebo and the new physiology of the doctor-patient relationship. Physiol. Rev. 2013, 93, 57. Benedetti, F. Placebo and the new physiology of the doctor-patient relationship. Physiol. Rev. 2013, 93, 1207–1246. [CrossRef] 58. Mora, R.; Salami, A.; Mora, F.; Cordone, M.P.; Ottoboni, S.; Passali, G.C.; Barbieri, M. Efﬁcacy of cefpodoxime in the prophylaxis of recurrent pharyngotonsillitis Int J Pediatr Otorhinolaryngol 2003 67 S225–S228 [CrossRef] 58. Mora, R.; Salami, A.; Mora, F.; Cordone, M.P.; Ottoboni, S.; Passali, G.C.; Barbieri, M. Efﬁcacy of cefpodoxime in the prophylaxis of recurrent pharyngotonsillitis. Int. J. Pediatr. Otorhinolaryngol. 2003, 67, S225–S228. [CrossRef] Mora, R.; Salami, A.; Mora, F.; Cordone, M.P.; Ottoboni, S.; Passali, G.C.; Barbieri, M. Efﬁcacy of cefpodo of recurrent pharyngotonsillitis. Int. J. Pediatr. Otorhinolaryngol. 2003, 67, S225–S228. [CrossRef] 59. Sanders, A.E.; Slade, G.D.; Fillingim, R.B.; Ohrbach, R.; Arbes, S.J.; Tchivileva, I.E. Effect of treatment expectation on placebo response and analgesic efﬁcacy: A secondary aim in a randomized clinical trial. JAMA Netw. Open 2020, 3, e202907. [CrossRef] 60. Olsson, B. Effect of patients’ expectations on recovery from acute tonsillitis. Fam. Pr. 1989, 6, 188–192. [CrossRef] 59. Sanders, A.E.; Slade, G.D.; Fillingim, R.B.; Ohrbach, R.; Arbes, S.J.; Tchivileva, I.E. Effect of treatment expectation on placebo response and analgesic efﬁcacy: A secondary aim in a randomized clinical trial. JAMA Netw. Open 2020, 3, e202907. [CrossRef]
https://openalex.org/W2547287412	http://www.nomos-elibrary.de/10.5771/0506-7286-1999-4-434.pdf	German	null	Zur Entstehung eines Verwaltungsrechts in der Volksrepublik China	Verfassung und Recht in Übersee	1,999	cc-by	9,086	Zur Entstehung eines Verwaltungsrechts in der Volksrepublik China! Zur Entstehung eines Verwaltungsrechts in der Volksrepublik China! Von Ingwer Ebsen 2 Herbert-Krüger-Gedächtnisvorlesung auf der 24. Tagung des Arbeitskreises Überseeische Verfas sungsvergleichung vom 25 - 27. Juni 1 999 in FrankfurtlMain. Der Beitrag beruht u.a. auf Informationen, die der Verfasser 1998 in mehreren rechtsvergleichen den Seminaren zum deutschen und chinesischen Verwaltungsrecht gewonnen hat, welche gemein sam von der Gesellschaft für Technische Zusammenarbeit (GTZ) und der Nationalen Verwal tungshochschule, Peking, veranstaltet worden waren. Ergänzt wurden dieselben durch sehr inten sive Diskussionen während eines Forschungs- und Lehraufenthalts im März 1 999 an der Südzen tralen Universität für Politische Wissenschaft und Recht in Wuhan. Herrn Professor Fang Si-rong von der dortigen Universität habe ich zu danken für geduldiges Erläutern der rechtlichen Hinter gründe für manche verwaltungsprozessuale Einzelheit. 2 Herbert-Krüger-Gedächtnisvorlesung auf der 24. Tagung des Arbeitskreises Überseeische Verfas sungsvergleichung vom 25 - 27. Juni 1 999 in FrankfurtlMain. Der Beitrag beruht u.a. auf Informationen, die der Verfasser 1998 in mehreren rechtsvergleichen den Seminaren zum deutschen und chinesischen Verwaltungsrecht gewonnen hat, welche gemein sam von der Gesellschaft für Technische Zusammenarbeit (GTZ) und der Nationalen Verwal tungshochschule, Peking, veranstaltet worden waren. Ergänzt wurden dieselben durch sehr inten sive Diskussionen während eines Forschungs- und Lehraufenthalts im März 1 999 an der Südzen tralen Universität für Politische Wissenschaft und Recht in Wuhan. Herrn Professor Fang Si-rong von der dortigen Universität habe ich zu danken für geduldiges Erläutern der rechtlichen Hinter gründe für manche verwaltungsprozessuale Einzelheit. Zu den Verhältnissen seit Gründung der Volksrepublik China (VRC) siehe Edgar Tomson I Jyun hsyong Su, Regierung und Verwaltung der Volksrepublik China, Köln 1 972, insbes. S. 1 14 ff.; 156 ff.; 281 ff. Textabdruck der amtlichen deutschen Übersetzung der Verfassung vom 20.9. 1954 bei Tomson I Su (Fn. 2), S. 375 ff. Herbert-Krüger-Gedächtnisvorlesung auf der 24. Tagung des Arbeitskreises Überseeische Verfas sungsvergleichung vom 25 - 27. Juni 1 999 in FrankfurtlMain. 1. Die Tendenz zur Verrechtlichung der chinesischen Administration und ihrer Außenbeziehungen 1. Die Tendenz zur Verrechtlichung der chinesischen Administration und ihrer Außenbeziehungen Es bedarf der Rechtfertigung, bezogen auf ein Land von der "Entstehung eines Verwal tungsrechts" zu sprechen, dessen wesentliche politische Strukturen traditionell eine durchorganisierte und komplexe Verwaltung aufgewiesen haben.2 Selbstverständlich hat es bereits im nach 1 949 aufgebauten und mit der Verfassung von 1 9543 strukturierten System der Volksrepublik China insofern eine Unmenge an "Verwaltungsrecht" gegeben, als die bürokratische Lenkung von Wirtschaft und Bevölkerung durch generelle, staatlicherseits gesetzte Normen stattfand. Seit einiger Zeit gibt es aber etwas für China Neues, nämlich eine Verrechtlichung sowohl der Staatsorganisation als auch der Beziehungen von Staat und privatem Sektor. Als Zäsur kann vereinfachend das Ende der Kulturrevolution und die 2 Herbert-Krüger-Gedächtnisvorlesung auf der 24. Tagung des Arbeitskreises Überseeische Verfas sungsvergleichung vom 25 - 27. Juni 1 999 in FrankfurtlMain. Der Beitrag beruht u.a. auf Informationen, die der Verfasser 1998 in mehreren rechtsvergleichen den Seminaren zum deutschen und chinesischen Verwaltungsrecht gewonnen hat, welche gemein sam von der Gesellschaft für Technische Zusammenarbeit (GTZ) und der Nationalen Verwal tungshochschule, Peking, veranstaltet worden waren. Ergänzt wurden dieselben durch sehr inten sive Diskussionen während eines Forschungs- und Lehraufenthalts im März 1 999 an der Südzen tralen Universität für Politische Wissenschaft und Recht in Wuhan. Herrn Professor Fang Si-rong von der dortigen Universität habe ich zu danken für geduldiges Erläutern der rechtlichen Hinter gründe für manche verwaltungsprozessuale Einzelheit. 2 2 Zu den Verhältnissen seit Gründung der Volksrepublik China (VRC) siehe Edgar Tomson I Jyun hsyong Su, Regierung und Verwaltung der Volksrepublik China, Köln 1 972, insbes. S. 1 14 ff.; 156 ff.; 281 ff. 2 Zu den Verhältnissen seit Gründung der Volksrepublik China (VRC) siehe Edgar Tomson I Jyun hsyong Su, Regierung und Verwaltung der Volksrepublik China, Köln 1 972, insbes. S. 1 14 ff.; 156 ff.; 281 ff. Textabdruck der amtlichen deutschen Übersetzung der Verfassung vom 20.9. 1954 bei Tomson I Su (Fn. 2), S. 375 ff. Textabdruck der amtlichen deutschen Übersetzung der Verfassung vom 20.9. 1954 bei Tomson I Su (Fn. 2), S. 375 ff. 4 5 Zu dessen Grundsatzrede vom 1 8.8. 1980 über die Reform der Führungssysteme in Partei und Staat siehe Robert Heuser, Die Bemühungen um Verwaltungsrecht und Verwaltungsrechtswissen schaft in der Volksrepublik China, DÖV 1 988, S. 17. 5 6 Zu Reformen in Richtung auf eine stärkere Verselbständigung von Rekrutierung, Weiterbildung und Einsatz des Administrativpersonals siehe lohn P. Bums, Chinese Civil Service Reform: the 1 3th Party Congress Proposals, The China Quarterly 1 989, S. 739 ff. ; T. c. Lam / Hon S. Chan, Designing China s Ci viI Service System: General Principles and Realities, in: International Jour nal of Public Administration 1 8 ( 1995), S. 1 297 - 1 332. 1. Die Tendenz zur Verrechtlichung der chinesischen Administration und ihrer Außenbeziehungen 5 "Art. 5 Der Staat wahrt die Einheitlichkeit und die Unverbrüchlichkeit des sozialisti schen Rechtssystems. Keine Gesetze oder Administrativvorschriften oder lokale Vorschriften dürfen der Verfassung widersprechen. Alle Staatsorgane, die Streitkräfte, alle politischen Parteien und öffentlichen Organisationen und alle Unternehmen und Einrichtungen sind an die Verfassung und das Gesetz gebunden. Alle Handlungen, welche die Verfassung und das Gesetz verletzen, sind zu ermitteln. Keine Organisation und kein Individuum hat das Privileg, über der Verfassung oder dem Gesetz zu stehen." Hier dürfte die explizite Einbeziehung "politischer Parteien" und die Verneinung des "Pri vilegs", über dem gesetzten Recht zu stehen, nur als Betonung der Abkehr von dem zuvor praktizierten System verstanden werden können. Die andere Grundentscheidung, welche eine Verrechtlichung der "Außenverhältnisse" des Staates geradezu notwendig machte, ist die - zumindest weitgehende - Trennung eines auch immer mehr die Ökonomie umfassenden privaten Sektors vom politisch-administrati ven System. Diese Entwicklungen sind zwar - im Hinblick auf die recht kurze Zeitspanne seit der Umorientierung - schon bemerkenswert weit gediehen, aber bei weitem noch nicht abgeschlossen. Dazu nur der Hinweis auf den staatseigenen Sektor der Wirtschaft und auf die Rolle der Kommunistischen Partei Chinas (KPC) im politisch-administrativen System. Dennoch kann im Ansatz von einem Prozeß der Ausdifferenzierung von Staat und Gesell schaft gesprochen werden, dessen Motor sicherlich die Ökonomie ist. Die Option für das Recht als Medium staatlicher Steuerung führt dazu, daß - dringlicher als t l htli h R l öti d fü Die Option für das Recht als Medium staatlicher Steuerung führt dazu, daß - dringlicher als Die Option für das Recht als Medium staatlicher Steuerung führt dazu, daß - dringlicher als zuvor - zentrale rechtliche Regelungen nötig werden für: zuvor - zentrale rechtliche Regelungen nötig werden für: zuvor - zentrale rechtliche Regelungen nötig werden für: die Errichtung und Organisation von Behörden, die Begründung und Abgrenzung von Verwaltungskompetenzen, die Gestaltung der Verwaltungsverfahren und die Beziehung zwischen dem Staat und den Rechtssubjekten des privaten bzw. quasi pri vaten Sektors. die Beziehung zwischen dem Staat und den Rechtssubjekten des privaten bzw. quasi pri vaten Sektors. Das sind die typischen Felder von Verwaltungsrecht: das Verwaltungsorganisationsrecht, das materielle Verwaltungsrecht und das Verwaltungsverfahrensrecht. 1. Die Tendenz zur Verrechtlichung der chinesischen Administration und ihrer Außenbeziehungen 434 Verfassung und Recht in Übersee (VRÜ) 32 (1999) Verfassung und Recht in Übersee (VRÜ) 32 (1999) Umorientierung der chinesischen Politik und Wirtschaft durch Deng Xiaoping gewählt werden.4 Umorientierung der chinesischen Politik und Wirtschaft durch Deng Xiaoping gewählt werden.4 Diese Umorientierung brachte auch für die Administration und das Verhältnis von Staat und sich entwickelndem privaten Sektor eine allmähliche Ums teuerung, die sich als Ver bindung von zwei Grundentscheidungen ausdrücken läßt. Die eine ist die Option für das Recht als vorrangiges Medium staatlicher Steuerung. Zwar enthielt auch bereits Art. 1 8 der Verfassung von 1 954 die Pflicht aller in den staatlichen Organen beschäftigten Funktio näre, die Verfassung und das Gesetz zu achten. Jedoch war allgemein klar, daß dies nur nach Maßgabe der Entscheidungen der Organe der Partei auf den jeweiligen Ebenen galt, deren führende Rolle allerdings im Text der Verfassung nur in der Präambel angesprochen war und ist. Auch war die Funktion des Rechts als Steuerungsmedium dadurch relativiert, daß (mit der Kulturrevolution als Höhepunkt dieses Konzepts) zum einen - man könnte sagen "von unten" - die Gesellschaft und der Staat durch Massenkampagnen auf Linie gebracht wurden und zum anderen - "von oben" - die Orientierung und Loyalität der "Kader" durch die entscheidende Rolle der Partei bei Rekrutierung, Ausbildung und Ein satz der Funktionäre in Staat und Wirtschaft gesichert wurden.5 Man könnte sagen, daß insofern im Verhältnis zur Steuerung durch Massenorganisationen, Massenkampagnen und persönliche Loyalität des Herrschaftspersonals die Steuerung durch Recht nur sekundären und instrumentellen Charakter hatte. Demgegenüber dürfte - mit einer gewissen Vorsicht und dem Hinweis, daß es sich um eine längerfristige Entwicklung handelt - das entspre chende Gebot in Art. 5 der Verfassung der Volksrepublik China (VRC)6 schon in seinem Wortlaut die Veränderung ausdrücken. Die Vorschrift lautet: 6 Text der 1 983 vollständig revidierten Verfassung in der erneut geänderten Fassung von 1 993; Laws of the People's Republic of China 1 993, herausgegeben vom Ständigen Ausschuß des Nationalen Volkskongresses, Foreign Language Press, Beijing 1 995. Die Änderungen durch den Nationalen Volkskongreß im März 1 999 sind noch nicht berücksichtigt. Sie betreffen den Gegen stand insofern, als u.a. ein Text aufgenommen wurde, welcher vom Amt für Öffentlichkeitsarbeit folgendermaßen zitiert wurde: "The People's Republic of China exercises the rule of law, building a socialist country governed according to law." (http;//www.prc50.com). Soweit nichts anderes gesagt ist, sind alle Gesetzeszitate eigene Übersetzungen der genannten amtlichen englischspra ehigen Ausgabe. 435 /0506 – "Art. 1. Die Tendenz zur Verrechtlichung der chinesischen Administration und ihrer Außenbeziehungen In all diesen Feldern hat die VRC seit 1 980 erhebliche Mengen an Recht produziert, die selbst dann bemerkens wert sind, wenn man sich auf die heiden Hauptinstrumente zentraler Rechtsetzung konzen triert, nämlich die Gesetze des Nationalen Volkskongresses (NVK) oder seines ihn außer halb der Sitzungs perioden vertretenden Ständigen Ausschusses und die praktisch gesetzes gleichen Verordnungen des Staatsrates als der zentralen Regierung. 436 Verfassung und Recht in Übersee (VRÜ) 32 (1999) Noch eine weitere Option ergibt sich fast notwendig aus den skizzierten Grundentschei dungen für die Verrechtlichung des Verhältnisses von Staat und Gesellschaft, nämlich diejenige für das subjektive öffentliche Recht und damit verbunden diejenige für gerichtli chen Rechtsschutz im Verhältnis zwischen den privaten Rechtssubjekten und der staatli chen Administration. Und auch auf diesem Gebiet hat es eine beachtliche Entwicklung gegeben, die von zunächst eher punktuellen Rechtsschutzmöglichkeiten im Rahmen des Zivilprozeßgesetzes zu einem immer noch enumerativen, aber doch weitgehenden Verwal tungsrechtsschutz mit eigenständigem Verwaltungsprozeßrecht geführt hat. Die folgenden Darlegungen werden sich auf drei Gegenstände konzentrieren, nämlich die Verwaltungsorganisation, den verwaltungsrechtlichen Normenbestand und die Rechtskon trolle des administrativen Handeins. Letztes ist insofern von besonderem Interesse, als hier die Ansätze für die Entwicklung eines Allgemeinen Verwaltungsrechts liegen. Die einzel nen Zweige des Besonderen Verwaltungsrechts tauchen dabei nur im Überblick über wich tige neuere Gesetze auf. 2. Die Grundzüge der Verwaltungsorganisation Die Verwaltungsorganisation ist in den Grundzügen vorgezeichnet in der Verfassung und im Detail ausgeführt in einer Reihe von Organisationsgesetzen. Hervorgehoben seien die folgenden Gesetze in den Fassungen, die sie ab dem Beginn der neuen Entwicklung erhal ten haben: Organisationsgesetz für die regionalen und lokalen Volkskongresse und Volksregierun gen ( 1 979)7 8 Organisationsgesetz für die Volksgerichte ( 1 979)8 Organisationsgesetz für die Volksstaatsanwaltschaften (1 979)9 10 Organisationsgesetz für den nationalen Volkskongreß (1 982) 10 1 1 Organisationsgesetz für den Staatsrat ( 1982)1 1 12 Gesetz über regionale nationale Autonomie ( 1 984) 12 13 Dorfkomiteegesetz (vorläufig) ( 1 987) 13 Dorfkomiteegesetz (vorläufig) ( 1 987) 13 14 Organisationsgesetz für die städtischen Wohnbezirkskomitees (1 989) 14 7 8 Laws of the People's Republic of China 1 979 - 1982, S. 50. Laws of the People's Repub1ic of China 1 979 - 1 982, S. 7 1 . 9 Laws of the People's Repub1ic of China 1 979 - 1982, S. 80. 10 Laws of the Peop1e's Republic of China 1 979 - 1982, S. 334. 1 1 Laws of the People's Republic of China 1979 - 1982, S . 343. 12 Laws of the Peop1e's Republic of China 1 983 - 1986, S. 87. 13 Laws of the People's Republic of China 1 987 - 1989, S. 6 1 . 7 8 Laws of the People's Republic of China 1 979 - 1982, S. 50. Laws of the People's Repub1ic of China 1 979 - 1 982, S. 7 1 . 9 Laws of the People's Repub1ic of China 1 979 - 1982, S. 80. 10 Laws of the Peop1e's Republic of China 1 979 - 1982, S. 334. 1 1 Laws of the People's Republic of China 1979 - 1982, S . 343. 12 Laws of the Peop1e's Republic of China 1 983 - 1986, S. 87. 13 Laws of the People's Republic of China 1 987 - 1989, S. 6 1 . 437 Die gesamte Organisationsstruktur ist gekennzeichnet durch ein nicht aufgelöstes Neben einander von zwei Organisationsprinzipien. Einerseits entdecken wir einen Stufenbau der Verwaltung, der in gewisser Hinsicht an unsere föderale Verwaltungsorganisation unter Einbeziehung der kommunalen Selbstverwaltung erinnert. Es gibt eine im Prinzip fünfstu fige Aufbauorganisation 15, nämlich den Zentralstaat, die Provinzen, autonomen Regionen und zentralstaatsunmittelbaren Großstädte, die Präfekturen und provinzunmittelbaren Städte, die Landkreise und Bezirke der provinzunmittelbaren Städte, die Kleingemeinden und Stadtteile. 15 Dazu in knapper Darstellung Qingkui. Xie, Der Aufbau und die Funktionen des regionalen Ver- waltungssystems in China , in: Siedentopf, Heinrich. Öffnung und Kooperation, Baden-Baden 1 997, S. 47 - 52. 14 15 Laws of the People's Republic of China 1987 - 1 989, S. 327. 2. Die Grundzüge der Verwaltungsorganisation Auf den vier oberen Ebenen gibt es eine im Prinzip einheitliche Grundstruktur. Das jeweils höchste Organ ist der Volkskongreß mit einem Ständigen Ausschuß als seinem alltäglichen Entscheidungsorgan. Vom ihm berufen und ihm verantwortlich ist eine allgemeine Ver waltungsspitze, die jeweilige "Volksregierung" . Sie leitet die eigentliche professionelle Verwaltung mit entsprechend ausdifferenzierten Zweigen von der Kreisebene aufwärts. Auf dieser Ebene wählen die Volkskongresse auch die jeweiligen Gerichtspräsidenten und obersten Staatsanwälte und einige weitere Spitzenbeamte. Insoweit haben wir ein gestuftes System von Selbstverwaltung. Auf der Provinzebene können die Volkskongresse und Volksregierungen auch Rechtsvorschriften erlassen. Andererseits ist der gesamte Staatsapparat hierarchisch organisiert, so daß es durchgängig Weisungsstränge von der Zentralregierung über die Provinzregierungen nach unten gibt. Hierzu sagt die Verfassung, daß alle Staatsorgane dem Prinzip des demokratischen Zentra lismus verpflichtet seien. Für bestimmte Funktionen, z.B. die Rechnungsprüfungs- und die Aufsichtsbehörden ist darüber hinaus die Zustimmung der höheren Ebene zur Ernennung der Behördenleitungen auf der unteren Ebene erforderlich. Insgesamt besteht eine nicht aufgelöste Spannung zwischen den von Rechts wegen vorge sehenen Hierarchiesträngen und den durch die lokalen Rekrutierungs- und Beförderungs mechanismen geschaffenen Loyalitäten. Die hieraus erwachsenden Schwierigkeiten in der Durchsetzung zentraler Vorgaben gegen lokale Interessen und Strukturen dürften einen ganz starken Impuls zur Verrechtlichung der Verwaltungstätigkeit und zur Schaffung von Rechtsdurchsetzungsinstrumentarien darstellen, von denen der gerichtliche Rechtsschutz nur einer ist. 438 Verfassung und Recht in Übersee (VRÜ) 32 (1999) Verfassung und Recht in Übersee (VRÜ) 32 (1999) Verfassung und Recht in Übersee (VRÜ) 32 (1999) Neben dem gerichtlichen Rechtsschutz existieren als vorrangig im öffentlichen Interesse bestehende, aber auch zur Abarbeitung von Beschwerden aus der Bevölkerung verpflichtete Kontrollinstitutionen - jeweils in der genannten Verwaltungsgliederung von der Kreis ebene aufwärts: Neben dem gerichtlichen Rechtsschutz existieren als vorrangig im öffentlichen Interesse bestehende, aber auch zur Abarbeitung von Beschwerden aus der Bevölkerung verpflichtete K t lli tit ti j il i d t V lt li du d K i bestehende, aber auch zur Abarbeitung von Beschwerden aus der Bevölkerung verpflichtete Kontrollinstitutionen - jeweils in der genannten Verwaltungsgliederung von der Kreis ebene aufwärts: t Kontrollinstitutionen - jeweils in der genannten Verwaltungsgliederung von der Kre ebene aufwärts: t die Staatsanwaltschaften, die nicht nur zur Steuerung der Polizei bei der Strafverfol gung, sondern auch zur Rechtskontrolle von Verwaltung und 1ustiz zuständig sind, die Staatsanwaltschaften, die nicht nur zur Steuerung der Polizei bei der Strafverfol gung, sondern auch zur Rechtskontrolle von Verwaltung und 1ustiz zuständig sind, Verwaltungsaufsichtsbehörden, bei welchen die Rechtsaufsicht über die je unteren Verwaltungsebenen liegt, ut Verwaltungsaufsichtsbehörden, bei welchen die Rechtsaufsicht über die je unteren Verwaltungsebenen liegt, ut Rechnungsprüfungsbehörden, welche zur Rechts- und Wirtschaftlichkeitskontrolle der Ausgaben und Einnahmen zuständig sind Rechnungsprüfungsbehörden, welche zur Rechts- und Wirtschaftlichkeitskontrolle der Ausgaben und Einnahmen zuständig sind. Rechnungsprüfungsbehörden, welche zur Rechts- und Wirtschaftlichkeitskontrolle der Ausgaben und Einnahmen zuständig sind. Ausgaben und Einnahmen zuständig sind. Ausgaben und Einnahmen zuständig sind. Die Grundstrukturen der Behördenorganisation sind mit Vereinfachungen in einem Schau bild auf S. 440 dargestellt. Die Grundstrukturen der Behördenorganisation sind mit Vereinfachungen in einem Schau bild auf S. 440 dargestellt. Das Bild ist selbstverständlich unvollständig ohne Berücksichtigung der Rolle der KPc. Die Partei kommt ausdrücklich weder in normativ regelnden Bestimmungen der Verfas sungl6 noch im sonstigen zentralen Organisationsrecht vor, ist aber selbstverständlich auf allen Ebenen eine wesentliche Steuerungskraft, dies aber informal.17 Exemplarisch sei der 1ustizsektor genannt. Von Rechts wegen sind die Gerichte unabhän gig und nur dem Gesetz verpflichtet. Die Gerichtspräsidenten werden vom jeweiligen Volkskongreß gewählt, die den jeweiligen Spruchkörpern vorsitzenden Richter auf seinen Vorschlag ebenfalls. Wesentliche Entscheidungen, gegebenenfalls auch über die Recht sprechung, trifft ein aus Richtern bestehendes, vom Ständigen Ausschuß des Volkskongres ses auf der jeweiligen Ebene berufenes 1ustizkomitee unter Vorsitz des Präsidenten. Für dieses gilt wie im gesamten Staatsapparat das Prinzip des demokratischen Zentralismus. 16 17 18 Lediglich in der Präambel ist mehrfach - auch mit Bezug auf die künftige Entwicklung - von der Führung durch die KPC die Rede. Zur Rolle der KPC bei der Rekrutierung der Mitglieder der Volkskongresse siehe Chih-Yu Shih, The institutionalization of China's People's Congress system: The views of people's deputies, International Politics 33 (1 996), S. 145 ff. Einzelheiten in Art. 1 1 des Organisationsgesetzes für die Volksgerichte der VRC in der Fassung vom September 1983. Verfassung und Recht in Übersee (VRÜ) 32 (1999) 18 Faktisch wird das Gericht darum auch hinsichtlich der Rechtsprechung wie eine Behörde vom Präsidenten geführt. Auf der Ebene jedes Gerichts gibt es auch ein 1ustizkomitee der KPC, welchem der Gerichtspräsident faktisch notwendig angehört und welches mit anderen Institutionen der KPC - ebenfalls nach dem Prinzip des demokratischen Zentralismus - verknüpft ist. Es bedarf keiner großen Phantasie, um sich auszumalen, wie gerade in einem Feld wie dem Rechtsschutz in Verwaltungssachen die oben angesprochene "Spannungs lage" zwischen dem Gesetz als Medium zentraler Steuerung und den Interessen lokaler Exemplarisch sei der 1ustizsektor genannt. Von Rechts wegen sind die Gerichte unabhän gig und nur dem Gesetz verpflichtet. Die Gerichtspräsidenten werden vom jeweiligen Volkskongreß gewählt, die den jeweiligen Spruchkörpern vorsitzenden Richter auf seinen Vorschlag ebenfalls. Wesentliche Entscheidungen, gegebenenfalls auch über die Recht sprechung, trifft ein aus Richtern bestehendes, vom Ständigen Ausschuß des Volkskongres ses auf der jeweiligen Ebene berufenes 1ustizkomitee unter Vorsitz des Präsidenten. Für dieses gilt wie im gesamten Staatsapparat das Prinzip des demokratischen Zentralismus. 18 439 / – Grundzüge der chinesischen BehördenorganisatIon udI ì -- "ll )t \t Nationaler Volks- Slaatsprasident Staatsrat Zentrale MUitar- Oberstes Oberst kongreB Ministerprasident kommission VOllricht schaft Standiger Ausschuß r VIZepremiers ------l staatsrate r------- -Minister I '--:-: Ob- . R--ech - nU- ng- ,p-rQ fer ----- Zentral gefiJhrte Institutionen der VelWa/fungskontroi/e . l - Vetwa/fungsauf3ichtsbehOrden (St.atsrat) I ReohnungsprQfungsbehClden (Sfaat""'t) Staatsenwaltschaf!eil (Oberste Sfaatsanwaltschatr) Vo/ksgariohte (Oberstes VoIësgerioht) I i ProvInzebene: VolkskongreB VolksregIerung: prafekturebene: VolkskongreB VolksregIerung: - J- Provinzgouvemeur oder OberbOrgennelsler \ I. einzelne Verwaitungsableilungan , AufslchtsbehOrden +------------ I Rechnung.prOfung.behOrda ---- ----1-- Volksgertcht Staat '\J prafekt oder OberbQrgermelstar 1 VolksgerIcht Staat f--ݹ einzelne Verwaitungsabtailungen AufsichtsbehOrden +-------------.. ----- I Rechnung.prüfungsbehörde •. -.--. -----.!-- ! I I I I I ! '" , "'- ' VolkskongreB Volksregierung: I Kreischet, Borgennelster oder DIstriktchef I Volksgertcht Staat Kreisebene: .. , • ___ í 'll.'. Gemeindeebene: -ű einzelne VerwaltungsabteIlungen AufsIchtsbehOrden >------... ------ , RechnungsprüfungsbehOrde > -.-J . Selbstverwaltungsorgane der kleinen Gemeinden und der Stadtbezirke https://doi.org/10.5771/0506-7286-1999-4-434, am 24.10.2024, 07:24:24 Open Access – - https://www.nomos-elibrary.de/agb '\J VolksgerIcht meindeebene: . Selbstverwaltungsorgane der kleinen Gemeinden und der Stadtbezirke https://doi.org/10.5771/0506-7286-1999-4-434, am 24.10.2024, 07:24:24 Administrationen und der in ihnen mächtigen Personen sich als ganz konkreter "Druck" auch auf die Richter auswirken kann. 19 Dieses System dürfte in allen wesentlichen Berei chen staatlicher Verwaltung ebenso funktionieren. Immerhin stellt das so skizzierte System eine Abweichung vom bis in die 80er Jahre fak tisch gültigen System einheitlicher Kaderführung durch die Parteiorganisation dar. 19 20 Zur Verschränkung gerade der lokalen Justiz mit der Partei der jeweiligen Ebene und örtlichen Interessen siehe auch Jerome A. Cohen, Reforming China's Civil Procedure: Judging the Courts, The American Journal of Comparative Law 45 (1 997), S. 793 ff. (797 ff.). 21 Dazu Ji, You, China's administrative reform : constructing a new model for a market economy, in: Issues & studies; 34 ( 1998), S. 69 - 1 03. 22 22 Vgl. Pei Minxin, Citizens v. Mandarins: Administrative Litigation in China, in: The China Quar- terly ( 1997), S. 832 ff. (848 f.). 20 21 Vgl. auch oben Fn. 5. Dazu Ji, You, China's administrative reform : constructing a new model for a market economy, in: Issues & studies; 34 ( 1998), S. 69 - 1 03. 22 19 20 21 Zur Verschränkung gerade der lokalen Justiz mit der Partei der jeweiligen Ebene und örtlichen Interessen siehe auch Jerome A. Cohen, Reforming China's Civil Procedure: Judging the Courts, The American Journal of Comparative Law 45 (1 997), S. 793 ff. (797 ff.). Vgl. auch oben Fn. 5. Dazu Ji, You, China's administrative reform : constructing a new model for a market economy, in: Issues & studies; 34 ( 1998), S. 69 - 1 03. 22 Vgl. Pei Minxin, Citizens v. Mandarins: Administrative Litigation in China, in: The China Quar- terly ( 1997), S. 832 ff. (848 f.). Verfassung und Recht in Übersee (VRÜ) 32 (1999) Die Verrechtlichung auch der Verwaltungsorganisation kann geradezu als ein Mittel dafür angesehen werden, eine von der KPC relativ verselbständigte, eigenen Rekrutierungs-, Karriere- und Entscheidungsmustern verpflichtete Staatsbürokratie zu schaffen.20 Schließlich bedarf noch die partielle und relative Verselbständigung des ökonomischen Sektors einiger Erläuterungen. Denn auch sie ist ein wesentlicher Motor der Entwicklung eines Verwaltungsrechts im eingangs genannten Sinne. Vereinfachend können wir drei Bereiche unterscheiden, nämlich die staatseigenen Betriebe, welche den weitaus größten Teilsektor der Wirtschaft darstellen, den sich entwickelnden Sektor echt privater kleinerer Unternehmen und die Joint Ventures mit ausländischen Investoren. Während die echt privaten Unternehmen und auch die Joint Ventures problemlos einen dem Staat gegenüberstehenden privatautonomen Bereich darstellen, dessen Beziehungen zum Staat durch verwaltungsrechtliche Instrumente bestimmt werden wie etwa Genehmi gungserfordernisse, Kontrollbefugnisse und Sanktionen bei Rechtsverstößen, bedarf der Bereich der staatseigenen Betriebe der Erläuterung. Vor der Einführung dessen, was offiziell "sozialistische Marktwirtschaft" heißt, wurden die Betriebe weitgehend wie die Staatsbürokratie geführt. Demgegenüber hat man nun Organisationsformen geschaffen, die im Prinzip zur Trennung der Eigentümer- und UnternehmersteIlung von den staatlichen Hoheitsfunktionen führen?1 Dies ist allerdings erst im Entwicklungsstadium. Auch haben bestimmte Administrationen, insbesondere die Armee, noch direkt geführte und auch fak tisch privilegierte Unternehmen. Dennoch gibt es Indizien, die auf eine tendenzielle Los lösung der staatseigenen Wirtschaft vom hoheitlichen Staat hindeuten. So sind es z.B. gerade die staatseigenen Betriebe, die nach einer Auswertung veröffentlichter Gerichtsfalle aus der ersten Hälfte der 90er Jahre neben den echten Privatunternehmen den größten Teil der Kläger in verwaltungsrechtlichen Prozessen stellten.22 22 Vgl. Pei Minxin, Citizens v. Mandarins: Administrative Litigation in China, in: The China Quar- terly ( 1997), S. 832 ff. (848 f.). 441 5771/0506 – 23 24 Li Xinsheng, A Brief Account of China's Administrative Proceedings System, Typoskript Juni 1 998. Alle diese Gesetze sind in der schon zitierten amtlichen englischen Ausgabe Laws of the People's Republic of China abgedruckt. Soweit ersichtlich gibt es derzeit 8 Bände (1979-1982, 1 983-1986, 1 987-1989, 1 990-1 992 und ab 1 993 bis 1 996 für jedes Jahr einen Band). 3. Schwerpunkte der Gesetzgebung im VerwaItungsrecht Größere Gesetzesvorhaben des besonderen VerwaItungsrechts24 (ohne Wirtschaftsaufsicht, Steuerrecht, Landesverteidigung, Bildungswesen) Waldgesetz (vorläufig) ( 1 979) Wassergesetz (1 988) Umweltschutzgesetz (vorläufig) (1 979) Naturschutzgesetz ( 1988) Meeresumweltschutzgesetz (1 982) Umweltschutzgesetz (1989) Kulturgüterschutzgesetz (1 982) Gesetz über Versammlungen, Aufzüge Landbeschaffungsverordnung ( 1 982) und Demonstrationen ( 1989) Statistikgesetz ( 1 983) Stadtplanungsgesetz ( 1 989) 442 Verfassung und Recht in Übersee (VRÜ) 32 (1999) Verfassung und Recht in Übersee (VRÜ) 32 (1999) Gesetz über Gewässerverschmutzung (1984) Waldgesetz (1 984) Arzneimittelverwaltungsgesetz (1 984) Bodenverwaltungsgesetz (1986) Luftverschmutzungsgesetz (1987) Archivgesetz (1 987) Eisenbahngesetz (1990) Wasser- und Bodenschutzgesetz (199 1 ) Verwaltungsgesetz fü r städtischen Boden (1 994) Volkspolizei gesetz (1 995) Abfallgesetz (1995) Lärmschutzgesetz ( 1996) Gesetz über Gewässerverschmutzung (1984) Waldgesetz (1 984) Arzneimittelverwaltungsgesetz (1 984) Bodenverwaltungsgesetz (1986) Luftverschmutzungsgesetz (1987) Archivgesetz (1 987) Eisenbahngesetz (1990) Wasser- und Bodenschutzgesetz (199 1 ) Verwaltungsgesetz fü r städtischen Boden (1 994) Volkspolizei gesetz (1 995) Abfallgesetz (1995) Lärmschutzgesetz ( 1996) Gesetz über Gewässerverschmutzung (1984) Waldgesetz (1 984) Arzneimittelverwaltungsgesetz (1 984) Bodenverwaltungsgesetz (1986) Luftverschmutzungsgesetz (1987) Archivgesetz (1 987) Eisenbahngesetz (1990) Wasser- und Bodenschutzgesetz (199 1 ) Verwaltungsgesetz fü r städtischen Boden (1 994) Volkspolizei gesetz (1 995) Abfallgesetz (1995) Lärmschutzgesetz ( 1996) Auffällig, wenn auch natürlich auch durch die vorher getroffene Selektion bedingt, ist der große Anteil an Umweltschutzgesetzen. Hier, aber auch in den anderen Bereichen, kann man sich ohne weiteres vorstellen, daß genügend Konfliktstoff im Verhältnis zwischen Bürgern bzw. Unternehmen entstehen kann, um die Gerichte zu beschäftigen. Auch ist nicht überraschend, daß sich durch die Entdeckung typischer, in den verschiedenen Rechts bereichen wiederkehrender Konstellationen ein Trend zu allgemeinen, für die verschiede nen Verwaltungszweige geltenden Regelungen ergibt. 3. Schwerpunkte der Gesetzgebung im VerwaItungsrecht 3. Mit einem gewissen Stolz wurde auf einer Tagung zur Entwicklung des Verwaltungsrechts berichtet, wie die Rechtsetzungsmaschinerie zunehmend auf Touren komme. Bis Ende 1 988 habe es einschließlich Gesetzesänderungen ca. 90 Gesetze des NVK oder seines Ständigen Ausschusses gegeben sowie 500 nationale und 1 000 regional geltende Verord nungen des Staatsrates. Bis Ende 1 997 seien es schon 300 Gesetze und Verordnungen des NVK und des Ständigen Ausschusses sowie 750 nationale und 5.300 regional geltende Verordnungen des Staatsrates gewesen?3 Hinsichtlich des nationalen Rechts nehmen die formellen Gesetze (zumeist erlassen vom Ständigen Ausschuß) also einen zunehmend größeren Raum ein. Welche Felder des Verwaltungsrechts werden erfaßt? Hierzu sollen einige nach subjektiver Einschätzung als exemplarisch und wichtig eingeschätzte Gesetze genannt werden, nur um zu verdeutlichen, in welchen Feldern des besonderen Verwaltungsrechts in jüngerer Zeit Rechtsreformen stattgefunden haben. Ausgelassen wurden dabei insbesondere die Bereiche der Wirtschaftsaufsicht einschließlich der Regelungen über Joint Ventures und das Bank wesen, das Steuerrecht, die Landesverteidigung, das Bildungswesen, und das Recht der Verwaltungsstrafen, welche ein wichtiges Steuerungsinstrument darstellen und faktisch zu einer Vermischung von Strafsanktionen und Verwaltungszwang führen. Die danach verbleibenden größeren Gesetzesvorhaben des besonderen Verwaltungsrechts, die sich aus einer Durchmusterung der amtlichen englischsprachigen Laws of the People's Republic of China bis 1 996 ergeben, sind in der folgenden Übersicht zusammengestellt: 23 24 Größere Gesetzesvorhaben des besonderen VerwaItungsrechts24 (ohne Wirtschaftsaufsicht, Steuerrecht, Landesverteidigung, Bildungswesen) Waldgesetz (vorläufig) ( 1 979) Wassergesetz (1 988) Umweltschutzgesetz (vorläufig) (1 979) Naturschutzgesetz ( 1988) Meeresumweltschutzgesetz (1 982) Umweltschutzgesetz (1989) Kulturgüterschutzgesetz (1 982) Gesetz über Versammlungen, Aufzüge Landbeschaffungsverordnung ( 1 982) und Demonstrationen ( 1989) Statistikgesetz ( 1 983) Stadtplanungsgesetz ( 1 989) Li Xinsheng, A Brief Account of China's Administrative Proceedings System, Typoskript Juni 1 998. Alle diese Gesetze sind in der schon zitierten amtlichen englischen Ausgabe Laws of the People's Republic of China abgedruckt. Soweit ersichtlich gibt es derzeit 8 Bände (1979-1982, 1 983-1986, 1 987-1989, 1 990-1 992 und ab 1 993 bis 1 996 für jedes Jahr einen Band). 25 Laws of the People's Republic of China 1987 - 1989, S. 285. Eine deutsche Fassung, die auf der unmittelbaren Übersetzung aus dem Chinesischen beruht, findet sich bei Robert Heuser, Das Verwaitungsprozeßgesetz der Volksrepublik China, VerwAreh 1 989, S. 437 ff. (447 ff.). 26 Verfassung und Recht in Übersee (VRÜ) 32 (1999) a) Die wesentlichen Rechtsvorschriften für den Schutz subjektiver Rechte gegenüber der Verwaltung An der Spitze dieser Entwicklung steht die Herausbildung einer innerhalb des Justizsystems ausdifferenzierten, mit wenigen Ausnahmen für die gesamte Verwaltung zuständigen Ver waltungsrechtsprechung mit einer eigenen Prozeßordnung, die auch materiell-rechtliche Regelungen enthält - so wie auch in Deutschland die Ausbildung eines allgemeinen Ver waltungsrechts einen engen Bezug zum Rechtsschutz hatte; man denke nur an die Funktion des Verwaltungsaktes, den Rechtsschutz zu strukturieren. Hierzu gehören auch noch die Schaffung eines ebenfalls generell geltenden Systems ver waltungsinternen Rechtsschutzes analog unserer Widerspruchsverfahren sowie die Normie rung von genereller Staatshaftung für Verwaltungsunrecht. Ebenfalls können wegen der Möglichkeit der Beschwerde bei einer eigens auch dafür geschaffenen Instanz die Institu tionen objektiver Rechtskontrolle der Verwaltung dazu gezählt werden. Das sind die Ver waltungsaufsichts- und die Rechnungsprüfungsbehörden. Die insoweit einschlägigen wichtigsten Gesetze und Verordnungen zur allgemeinen Rechtmäßigkeitskontrolle sind in der folgenden Übersicht zusammengefaßt: 443 5771/0506 – Gesetze für Verwaltungsrechtsschutz und Verwaltungskontrolle 25 Verwaltungsprozeßgesetz (1989) 26 Verwaltungsüberprüfungsverordnunêp 990) Staatshaftungsgesetz (1994) Rechnungsprüfungsgesetz (1994)28 Verwaltungsaufsichtsgesetz (1997)29 Der Gegenstandsbereich für Verwaltungsrechtsschutz durch die Verwaltungsrechtsabteilungen der Volksgerichte Der Gegenstandsbereich für Verwaltungsrechtsschutz durch die Verwaltungsrechtsabteilungen der Volksgerichte b) Im Vordergrund der Entwicklung eines Allgemeinen Verwaltungsrechts steht der Schutz subjektiver öffentlicher Rechte durch die Verwaltungsabteilungen der Volksgerichte und durch inneradministrativen Rechtsschutz. Erster Ausgangspunkt ist das Prinzip der Ge setzmäßigkeit der Verwaltung, welches in Art. 5 Abs. 2 der Verfassung ausgedrückt ist, nur als explizite Abkehr von der Vergangenheit verstanden werden kann und in der chinesi schen Diskussion durchaus als Parallele zum Rechtsstaatsprinzip und zur "rule oi law" begriffen wird. 30 Zweiter Ausgangspunkt ist das subjektive öffentliche Recht des Individuums gegenüber dem Staat. Dieses ist anerkannt in Art. 33 S. 3 der Verfassung: "Jeder Bürger hat die Rechte und muß die Pflichten erfüllen, welche durch die Verfassung und das Recht normiert sind." Als Institutionen zur Wahrung subjektiver öffentlicher Rechte dienen das Verfahren inner administrativer Überprüfung und der gerichtliche Rechtsschutz. Beide Verfahren bestehen hinsichtlich der Hoheitsakte, gegen welche Rechtsschutz vorgesehen ist, parallel. Die vor herige Durchführung des Verfahrens inneradministrativer Überprüfung war bis vor kurzem in einer Verordnung des Staatsrates von 1990 geregelt. Seit kürzester Zeit soll diese durch 25 Laws of the People's Republic of China 1987 - 1989, S. 285. Eine deutsche Fassung, die auf der unmittelbaren Übersetzung aus dem Chinesischen beruht, findet sich bei Robert Heuser, Das Verwaitungsprozeßgesetz der Volksrepublik China, VerwAreh 1 989, S. 437 ff. (447 ff.). 26 Englische Fassung von Charles D. Paglee, University of Maryland (http:/www.qisnet/chinalaw). 27 Laws of the People's Republic of China 1 994, S. 4 1 . 28 Laws of the People' s Republic of China 1 994, S. 1 09. 29 Englische Fassung von Charles D. Paglee (Fn. 26). 30 Dazu auch Heuser, ARSP 78 ( 1992), S. 355 ff., 359 ff. 444 ein Gesetz ersetzt worden sein, welches, soweit ersichtlich, allerdings noch nicht auf eng lisch vorliegt. Grundsätzlich, sofern spezialgesetzlich nichts anderes vorgesehen ist, stellt die vorherige Durchführung des inneradministrativen Rechtsschutzverfahrens keine Zuläs sigkeitsvoraussetzung für den gerichtlichen Rechtsschutz dar. Aus diesem Grunde soll auf die Behandlung dieses Verfahrens verzichtet und gleich zum Rechtsschutz nach dem Ver waltungsprozeßgesetz von 1 989 übergegangen werden. ein Gesetz ersetzt worden sein, welches, soweit ersichtlich, allerdings noch nicht auf eng lisch vorliegt. Grundsätzlich, sofern spezialgesetzlich nichts anderes vorgesehen ist, stellt die vorherige Durchführung des inneradministrativen Rechtsschutzverfahrens keine Zuläs sigkeitsvoraussetzung für den gerichtlichen Rechtsschutz dar. 3! Aus diesen Anwendungsfällen wird deutlich, daß das deutsche Wort "Verwaltungsakt" insofern mißverständlich wäre, als der Rechtsschutz nicht nur gegen konkrete Regelungen, sondern auch gegen in Rechte eingreifende Realakte und teilweise sogar gegen schlichtes Unterlassen gegeben ist. Der Gegenstandsbereich für Verwaltungsrechtsschutz durch die Verwaltungsrechtsabteilungen der Volksgerichte Aus diesem Grunde soll auf die Behandlung dieses Verfahrens verzichtet und gleich zum Rechtsschutz nach dem Ver waltungsprozeßgesetz von 1 989 übergegangen werden. Im Verwaltungsprozeßgesetz ist eine begrenzte Rechtsschutzgewährleistung normiert, die zugleich auf ein allgemeines verwaltungsrechtliches System verweist. Art. 2 bestimmt: "Wenn ein Bürger, eine juristische Person oder irgendeine andere Organisation der Ansicht ist, daß seine oder ihre Rechte oder rechtlich geschützten Interessen durch eine konkrete Verwaltungsmaßnahme eines Verwaltungsorgans oder seines Perso nals beeinträchtigt wurden, ist er oder sie berechtigt, hiergegen nach den Bestim mungen dieses Gesetzes vor einem Volks gericht Klage zu erheben." Entsprechend heißt es zur Jurisdiktion der verwaltungsrechtlichen Abteilungen der Volks gerichte in Art. 5 des Gesetzes: Entsprechend heißt es zur Jurisdiktion der verwaltungsrechtlichen Abteilungen der Volks gerichte in Art. 5 des Gesetzes: "In verwaltungsrechtlichen Fällen haben die Volksgerichte die Rechtmäßigkeit kon kreter Verwaltungsmaßnahmen zu prüfen." "In verwaltungsrechtlichen Fällen haben die Volksgerichte die Rechtmäßigkeit kon kreter Verwaltungsmaßnahmen zu prüfen." Der Begriff der "konkreten Verwaltungsmaßnahmen" wird in Art. 1 1 durch einen Katalog von Typen von Verwaltungshandeln definiert3! , welche damit das Feld verwaltungsrechtli chen Rechtsschutzes abstecken. In gewisser Vereinfachung handelt es sich um 8 Gruppen von Maßnahmen. Ergänzt wird die positive Umschreibung des Feldes gerichtlichen Rechts schutzes in Verwaltungssachen durch explizite, in Art. 12 des Gesetzes aufgelistete Aus nahmetatbestände, die demgemäß nicht zu den "verwaltungsrechtlichen Fällen" gehören. Die acht Gruppen von Verwaltungsmaßnahmen und die Ausnahmetatbestände sind in den bei den folgenden Übersichten zusammengestellt: 3! 3! Aus diesen Anwendungsfällen wird deutlich, daß das deutsche Wort "Verwaltungsakt" insofern mißverständlich wäre, als der Rechtsschutz nicht nur gegen konkrete Regelungen, sondern auch gegen in Rechte eingreifende Realakte und teilweise sogar gegen schlichtes Unterlassen gegeben ist. 445 Konkrete gerichtlich angreifbare Verwaltungsmaßnahmen Verwaltungssanktionen einschließlich der Aufhebung zuvor gewährter Begünstigungen Verwaltungszwangsmaßnahmen Eingriffe in jemandes unternehmerische Entscheidungsbefugnisse Ablehnung oder Nichtgewährung beantragter Genehmigungen Ablehnung oder Nichtgewährung beantragter gesetzlich vorgesehener Schutzmaß nahmen für die Person oder das Eigentum des Antragstellers Nichtgewährung einer gesetzlich vorgesehenen Pension Aufforderungen durch Verwaltungsorgane, Pflichten zu erfüllen sonstige Eingriffe in Rechte der Person oder in Eigentum 33 Über entsprechende Bemühungen in der Verwaltungsrechtswissenschaft berichtet Heuser (Fn. 4), S. 18. 32 Einige Einzelheiten auch bei Heuser (Fn. 25). 33 32 Einige Einzelheiten auch bei Heuser (Fn. 25). 33 Über entsprechende Bemühungen in der Verwaltungsrechtswissenschaft berichtet Heuser (Fn. 4), S. 18. Vom Rechtsschutz ausgenommene Verwaltungshandlungen Staatshandlungen in Gebieten wie der nationalen Verteidigung und auswärtigen Ange legenheiten allgemein formulierte und bindende Verwaltungsvorschriften und Verordnungen Belohnungs- und Strafentscheidungen, Ernennungen und Entlassungen durch Ver waltungsorgane gegenüber ihrem Personal konkrete Verwaltungshandlungen, für welche gesetzlich normiert ist, daß die Letztent scheidung bei einem Verwaltungsorgan liegt Staatshandlungen in Gebieten wie der nationalen Verteidigung und auswärtigen Ange legenheiten allgemein formulierte und bindende Verwaltungsvorschriften und Verordnungen Belohnungs- und Strafentscheidungen, Ernennungen und Entlassungen durch Ver waltungsorgane gegenüber ihrem Personal konkrete Verwaltungshandlungen, für welche gesetzlich normiert ist, daß die Letztent scheidung bei einem Verwaltungsorgan liegt Staatshandlungen in Gebieten wie der nationalen Verteidigung und auswärtigen Ange legenheiten allgemein formulierte und bindende Verwaltungsvorschriften und Verordnungen Belohnungs- und Strafentscheidungen, Ernennungen und Entlassungen durch Ver waltungsorgane gegenüber ihrem Personal konkrete Verwaltungshandlungen, für welche gesetzlich normiert ist, daß die Letztent scheidung bei einem Verwaltungsorgan liegt Es fällt auf, daß die Abgrenzung des Rechtsschutzes quer zur auch in China geläufigen Einteilung in Rechtsakte und schlichtem Verwaltungshandeln liegt. Andererseits wird mit dem Abstellen auf konkretes Verwaltungshandeln die auch bei uns gebräuchliche Abgren zung gegenüber abstrakt-generellen Regelungen vorgenommen. Dies wird noch einmal verdeutlicht im Negativkatalog der vom Rechtsschutz ausgeschlossenen Verwaltungs handlungen. In unseren rechtlichen Kategorien läßt sich der Positivkatalog abstrahierend zusammenfassen zu eingreifenden Verwaltungsakten, eingreifenden Verwaltungsakten, Verweigerung und schlichte Nichtgewährung gesetzlich eingeräumter Rechte auf Begünstigungen durch die Verwaltung, Verweigerung und schlichte Nichtgewährung gesetzlich eingeräumter Rechte auf Begünstigungen durch die Verwaltung, Eingriffe in persönliche und Eigentumsrechte durch schlichtes Verwaltungshandeln. 446 Verfassung und Recht in Übersee (VRÜ) 32 (1999) Einige Stichworte zur Ausgestaltung des Rechtsschutzes c) n 32 Zur Ausgestaltung des Rechtsschutzes müssen einige ergänzende Stichworte genügen.32 Diese betreffen zum einen das sich im Rechtsschutzsystem ausdrückende Verhältnis des Betroffenen zur Behörde und zum anderen das justizinterne Verhältnis der Instanzen. Dabei soll nicht eine systematische Übersicht angestrebt, sondern das aus subjektiver Sicht Bemerkenswerte hervorgehoben werden Zur Ausgestaltung des Rechtsschutzes müssen einige ergänzende Stichworte genügen.32 Diese betreffen zum einen das sich im Rechtsschutzsystem ausdrückende Verhältnis des Betroffenen zur Behörde und zum anderen das justizinterne Verhältnis der Instanzen. Dabei soll nicht eine systematische Übersicht angestrebt, sondern das aus subjektiver Sicht Bemerkenswerte hervorgehoben werden aa) Stichworte zum Prozeßrechtsverhältnis (I) Ein wichtiges, den Schutz der subjektiven öffentlichen Rechte verstärkendes Prinzip ist die grundsätzliche Beweislast der Behörden - diese sind regelmäßig als solche die Beklag ten - für das Vorliegen von Eingriffsvoraussetzungen. Art. 32 des Verwaltungsprozeßge setzes bestimmt: "Der Beklagte trägt die Beweislast für die konkrete Verwaltungsmaßnahme, die er getroffen hat, und hat die Beweise vorzulegen sowie die Vorschriften, auf welche die Maßnahme gestützt ist." "Der Beklagte trägt die Beweislast für die konkrete Verwaltungsmaßnahme, die er getroffen hat, und hat die Beweise vorzulegen sowie die Vorschriften, auf welche die Maßnahme gestützt ist." Diese Regelung, welche als Vermutung gegen behördliche Eingriffsbefugnisse gelesen werden könnte, bieten einen Ansatz zur Entwicklung eines Gesetzesvorbehaltes für Ein griffe in subjektive Rechtspositionen?3 Diese Regelung, welche als Vermutung gegen behördliche Eingriffsbefugnisse gelesen werden könnte, bieten einen Ansatz zur Entwicklung eines Gesetzesvorbehaltes für Ein griffe in subjektive Rechtspositionen?3 (2) Ein weiteres Element, welches die Möglichkeiten des Betroffenen erweitert, ist sein grundsätzliches, wenn auch unter dem Vorbehalt abweichender Regelung durch Gesetz oder Verordnung stehendes Wahlrecht zwischen der Vorschaltung eines unserem Wider spruchsverfahren entsprechenden behördlichen Verfahrens der Überprüfung durch die übergeordnete Behörde und der unmittelbaren Klage. Dies bestimmt Art. 37 des Verwal tungsprozeßgesetzes. Man darf davon ausgehen, daß durch das nachgeschaltete gerichtliche Verfahren auch der inneradministrative Beschwerdeweg, den es im Prinzip schon immer gegeben hat, eine andere Bedeutung erlangt hat. (3) Ein Obsiegen des Klägers führt entweder zu voller oder teilweiser Aufhebung einer Maßnahme oder zur Verurteilung zu einer Maßnahme unter Berücksichtigung des Urteils oder zur Verurteilung zu Schadensersatz. Dabei ist zu berücksichtigen, daß nach Art. 1 1 Nr. 3 auch die Verweigerung einer Genehmigung oder Lizenz eine Maßnahme ist. Der Kläger obsiegt nach Art. 54 Nr. 2 bei: 447 5771/0506 – a) mangelnden Beweisen für wesentliche Tatsachen, b) fehlerhafter Rechtsanwendung, c) Verstoß gegen Verfahrensvorschriften, d) Überschreitung der Kompetenz, oder e) Mißbrauch der Kompetenz. a) mangelnden Beweisen für wesentliche Tatsachen, (4) Es ist eine bewußt getroffene Entscheidung, daß die vollzugsfähigen Verwaltungsmaß nahmen grundsätzlich nicht durch die Einlegung von Rechtsbehelfen im Vollzug suspen diert werden. Eine Suspendierung gibt es nach Art. 44 Satz 1 des Gesetzes lediglich für bestimmte Fälle, nämlich neben den sondergesetzlich vorgesehenen und denjenigen, in denen die Behörde selbst eine Aussetzung für erforderlich hält, für den Fall, daß das Ge richt diese zur Vermeidung irreparablen Schadens für geboten hält und die Suspendierung öffentliche Interessen nicht verletzt. Laws of the People's Republic of China 1 983 - 1986, S. 39. aa) Stichworte zum Prozeßrechtsverhältnis (5) Auf allen Stufen des Verfahrens sieht das Gesetz relativ knapp bemessene Fristen vor, die eine zügige Erledigung sichern sollen. Für den Antrag auf inneradministrative Über prüfung gilt grundsätzlich eine Frist von 1 5 Tagen ab Kenntnis von der angegriffenen Verwaltungsmaßnahme (Art. 29 der Verwaltungsüberprüfungs-VO). Binnen 2 Monaten nach Eingang des Überprüfungsantrags hat die zuständige Behörde eine abschließende Entscheidung zu treffen (Art. 38 Abs. 1 des Gesetzes). Hiergegen kann der Betroffene binnen 1 5 Tagen das Gericht anrufen (Art. 38 Abs. 2). Wer nicht den Weg der inneradmini strativen Überprüfung wählt (an diesen ist man 2 Monate gebunden, wenn er einmal be schritten wurde, Art. 30 der Verordnung), sondern direkt das Gericht anruft, hat hierfür eine Frist von 3 Monaten (Art. 39 des Gesetzes). Das Gericht erster Instanz hat eine ab schließende Entscheidung binnen drei Monaten zu treffen; eine Verlängerung dieser Frist hat es beim Oberen Volksgericht zu beantragen; dieses selbst gegebenenfalls beim Obersten Volksgericht (Art. 57). Für Rechtsbehelfe gegen die erstinstanzlichen Urteile gibt es wieder eine Frist von 1 5 Tagen (Art. 58). Die abschließende Entscheidung über den Rechtsbehelf soll binnen 2 Monaten ergehen mit der schon erwähnten Möglichkeit der Verlängerung durch ein Oberes oder das Oberste Volksgericht (Art. 60). (6) Die Umsetzung gerichtlicher Entscheidungen ist in Art. 65 und 66 des Verwaltungspro zeßgesetzes geregelt. Gegenüber nichtstaatlichen Adressaten findet Zwangsdurchsetzung durch Gerichtspersonal statt, zu welchem neben Gerichtsvollziehern auch eine Justizpolizei gehört (Art. 41 des Organisationsgesetzes für die Volksgerichte34). Gegenüber staatlichen Stellen werden Geldforderungen einschließlich Zwangsgeldern, die das Gericht festsetzen kann, durch Anordnung der Abbuchung vom Bankkonto der jeweiligen Behörde durchge setzt. Im übrigen kann das Gericht die jeweils vorgesetzten Stellen und die zur Verwal tungskontrolle zuständigen Behörden zur Durchsetzung auffordern. Allerdings ist die (6) Die Umsetzung gerichtlicher Entscheidungen ist in Art. 65 und 66 des Verwaltungspro zeßgesetzes geregelt. Gegenüber nichtstaatlichen Adressaten findet Zwangsdurchsetzung durch Gerichtspersonal statt, zu welchem neben Gerichtsvollziehern auch eine Justizpolizei gehört (Art. 41 des Organisationsgesetzes für die Volksgerichte34). Gegenüber staatlichen Stellen werden Geldforderungen einschließlich Zwangsgeldern, die das Gericht festsetzen kann, durch Anordnung der Abbuchung vom Bankkonto der jeweiligen Behörde durchge setzt. Im übrigen kann das Gericht die jeweils vorgesetzten Stellen und die zur Verwal tungskontrolle zuständigen Behörden zur Durchsetzung auffordern. Allerdings ist die 34 4 Laws of the People's Republic of China 1 983 - 1986, S. 39. 35 Pei Minxin (Fn. 22), S. 841 ff., erklärt bestimmte Unterschiede im Erfolg von Klägern gegen Behörden und eine gewisse Neigung von Gerichten, eine Verurteilung von Behörden durch Anre gung außergerichtlicher Streitbeilegung zu vermeiden, mit der Einfluß der lokalen Regierungsbe hörden und die relativ besseren Erfolgsaussichten von Klägern in der Berufungsinstanz mit der höheren Professionalität und größeren faktischen Unabhängigkeit der Richter in den höheren Instanzen. aa) Stichworte zum Prozeßrechtsverhältnis Verfassung und Recht in Übersee (VRÜ) 32 (1999) 448 Verfassung und Recht in Übersee (VRÜ) 32 (1999) faktische Durchsetzung der Gesetze gegenüber der Administration wohl eher ein Problem der oben angesprochenen "Spannungslage" gegenüber lokalen Loyalitäten. Deren Über windung dienen Instrumente, die mit der Gerichtsorganisation und der Zuständigkeitsord- 35 nung zu tun haben. bb) bb) Stichworte zur Organisation und Zuständigkeit ( 1 ) Zwar wurde keine eigene auf verwaltungsrechtliche Fälle spezialisierte Gerichtsbarkeit geschaffen, sondern das Prinzip der einheitlichen, für nahezu alle Gerichtsverfahren zu ständigen Volksgerichte beibehalten. Es wurden jedoch eigene verwaltungsrechtliche Abteilungen mit alleiniger Zuständigkeit für Verwaltungsrechtsfalle geschaffen (Art. 3 Abs. 2). ( 1 ) Zwar wurde keine eigene auf verwaltungsrechtliche Fälle spezialisierte Gerichtsbarkeit geschaffen, sondern das Prinzip der einheitlichen, für nahezu alle Gerichtsverfahren zu ständigen Volksgerichte beibehalten. Es wurden jedoch eigene verwaltungsrechtliche Abteilungen mit alleiniger Zuständigkeit für Verwaltungsrechtsfalle geschaffen (Art. 3 Abs. 2). (2) Es gibt eine Reihe von objektiven, nicht von den Parteien abhängigen Instrumenten rechtlicher Überprüfung der gerichtlichen Entscheidungen. Generell haben die Staatsan waltschaften die Aufgabe, von Amts wegen die Rechtmäßigkeit der Gerichtsverfahren in Verwaltungssachen zu überprüfen (Art. 1 0) und Einspruch bei den zuständigen Stellen zu erheben (Art. 64). Entweder auf solchen Einspruch oder von Amts wegen können Gerichts verfahren wieder aufgerollt werden. Der Gerichtspräsident, der einen Rechtsfehler in einer gerichtlichen Entscheidung gefunden hat und eine Wiederaufnahme des Verfahrens für notwendig hält, kann die Sache zur Entscheidung über die Wiederaufnahme dem Justiz komitee seines Gerichts vorlegen (Art. 63 Abs. 1). Entsprechend kann auch ein jeweils übergeordnetes Gericht den Fall entweder selbst erneut verhandeln oder dieses dem unter geordneten Gericht befehlen (Art. 63 Abs. 2). (3) Bemerkenswert ist das Kompetenzverhältnis der Instanzen. Grundsätzlich sind zwei In stanzen gegeben (Art. 6), von denen grundsätzlich die unteren Volksgerichte, also diejeni gen auf Kreisebene, die Eingangsinstanz sind (Art. 1 3). Die mittleren Volksgerichte, d.h. diejenigen auf Präfekturebene, sind erstinstanzlich zuständig für Patent- und Zollsachen, Klagen gegen konkrete Verwaltungsmaßnahmen von Behörden von der Provinzebene aufwärts und für Fälle im Bereich ihrer örtlichen Zuständigkeit, die sie als hinreichend schwerwiegend und kompliziert ansehen (Art. 1 4). Letzeres gilt ebenso für die oberen Volksgerichte, also diejenigen auf Provinzebene (Art. 1 5) und - mit Zuständigkeit für das ganze Land - das oberste Volksgericht (Art. 1 6). Diese einigermaßen vage Zuständigkeits abgrenzung wird ergänzt durch ein Zugriffsrecht von Amts wegen der jeweils höheren (3) Bemerkenswert ist das Kompetenzverhältnis der Instanzen. Grundsätzlich sind zwei In stanzen gegeben (Art. 6), von denen grundsätzlich die unteren Volksgerichte, also diejeni gen auf Kreisebene, die Eingangsinstanz sind (Art. 1 3). Die mittleren Volksgerichte, d.h. 36 37 Siehe Art. 32 des Organisationsgesetzes für den Nationalen Volkskongreß in der Fassung von Dezember 1982. 38 39 Laws of the Peop1e s Republic of China, 1 979 - 1 982, S. 251 . Art. 3 3 des Organisationsgesetzes für die Volksgerichte der VRC i n der Fassung vom September 1983 lautet: "Das Oberste Volksgericht gibt Interpretationen zu Fragen, welche die konkrete Anwendung von Gesetzen und Anordnungen in Gerichtsprozessen betreffen." bb) Stichworte zur Organisation und Zuständigkeit diejenigen auf Präfekturebene, sind erstinstanzlich zuständig für Patent- und Zollsachen, Klagen gegen konkrete Verwaltungsmaßnahmen von Behörden von der Provinzebene aufwärts und für Fälle im Bereich ihrer örtlichen Zuständigkeit, die sie als hinreichend schwerwiegend und kompliziert ansehen (Art. 1 4). Letzeres gilt ebenso für die oberen Volksgerichte, also diejenigen auf Provinzebene (Art. 1 5) und - mit Zuständigkeit für das ganze Land - das oberste Volksgericht (Art. 1 6). Diese einigermaßen vage Zuständigkeits abgrenzung wird ergänzt durch ein Zugriffsrecht von Amts wegen der jeweils höheren 35 449 Gerichte auf Fälle, die bei Gerichten niedrigerer Ebenen ihres Zuständigkeits bereichs anhängig sind, und durch die umgekehrte Kompetenz, Fälle entsprechend nach unten abzu geben. (Art. 23). Hiermit ist den Gerichten auch ein Instrument an die Hand gegeben, in eventuellen Konflikten mit lokalen Interessen oder Machthabern die unteren Gerichts ebenen "aus der Schußlinie" zu nehmen. (4) Dieses System der unmittelbaren, vom Durchlaufen eines Instanzenzuges unabhängigen Verlagerung der "wichtigen" Entscheidungen nach oben, gilt gerade auch für die Rechts fortbildung durch Gesetzesinterpretation. Durch die Möglichkeit, daß das eigentlich zuständige Gericht die Sache nach oben weiterreicht, und die entsprechende Kompetenz der je höheren Gerichte, die Sache an sich ziehen, können im Zusammenwirken der Instan zen "Leitentscheidungen" produziert werden. Praktisch wichtiger ist allerdings ein anderes Instrument der Vereinheitlichung der Gesetzesauslegung. Es gibt eine Hierarchie verbindli cher Gesetzesauslegung vom Ständigen Ausschuß des Nationalen Volkskongresses über den Staatsrat zum Obersten Volksgericht. Schon Art. 67 Nr. 1 und 6 der Verfassung geben dem Ständigen Ausschuß die Kompetenz zur (authentischen) Interpretation von Verfassung und Gesetzen. Hierfür können u.a. das Oberste Volksgericht, die Oberste Staatsanwalt schaft und der Staatsrat Vorlagen unterbreiten.36 Entsprechend interpretiert der Staatsrat die von ihm erlassenen Rechtsvorschriften authentisch. Eine systematische Zusammenfas sung dieses Systems authentischer Interpretation durch das jeweilige Rechtsetzungsorgan findet sich in einer Resolution des Ständigen Ausschusses vom 10. Juni 1 98 1 .37 Auf dieser Grundlage authentischer Interpretation kann das Oberste Volksgericht unabhängig von konkreten RechtsFallen abstrakt gehaltene Interpretationsanweisungen geben, die für die nachgeordneten Gerichte verbindlich sind.38 Auf dieser Rechtsgrundlage werden allge meine Anweisungen erlassen, die - ähnlich wie unsere allgemeinen Verwaltungsvorschrif ten zur Anwendung bestimmter Gesetze, aber eben innerhalb der Justiz - die Rechtsanwen dung steuern.39 Zu diesem System gehört es auch, daß die verbindliche Entscheidung über die Vereinbarkeit von Rechtsvorschriften mit der Verfassung und die Vereinbarkeit lokalen Rechts mit nationalem Recht beim Ständigen Ausschuß des Nationalen Volkskongresses konzentriert ist (Art. 67 Nr. 8 der Verfassung). Verfassung und Recht in Übersee (VRÜ) 32 (1999) bb) Stichworte zur Organisation und Zuständigkeit 36 37 38 39 Siehe Art. 32 des Organisationsgesetzes für den Nationalen Volkskongreß in der Fassung von Dezember 1982. Laws of the Peop1e's Republic of China, 1 979 - 1 982, S. 251 . Art. 3 3 des Organisationsgesetzes für die Volksgerichte der VRC i n der Fassung vom September 1983 lautet: "Das Oberste Volksgericht gibt Interpretationen zu Fragen, welche die konkrete Anwendung von Gesetzen und Anordnungen in Gerichtsprozessen betreffen." Siehe Auch Cohen (Fn. 19), S. 794 f. 450 Verfassung und Recht in Übersee (VRÜ) 32 (1999) Verfassung und Recht in Übersee (VRÜ) 32 (1999) Staatshaftung für rechtswidriges Verwaltungshandeln d) Als Instrument zur Durchsetzung rechtmäßigen Verwaltungshandelns kann auch die Staatshaftung angesehen werden. Staatshaftung ist zum einen im Verwaltungsprozeßgesetz und zum anderen in einem eigenen Staatshaftungsgesetz geregelt. In Art. 67 - 69 des Ver waltungsprozeßgesetzes ist ganz generell bestimmt, daß Bürger, juristische Personen und andere Organisationen, deren Rechte oder rechtlich geschützte Interessen durch konkrete Verwaltungsmaßnahmen eines Verwaltungsorgans verletzt werden, den Ersatz ihres da durch verursachten Schadens verlangen können. Dies kann sowohl unabhängig als auch verbunden mit dem Rechtsschutz gegen das entsprechende Verwaltungshandeln selbst vor die Volks gerichte gebracht werden. Der Schadensersatz geht zu Lasten des Haushalts des Verwaltungsträgers, dem das Organ angehört. Gegen die Bediensteten, welche die Rechts verletzung vorsätzlich oder grob fahrlässig verursacht haben, ist Regreß zu nehmen. Das Staatshaftungsgesetz vom Mai 1994, in Kraft ab 1 9954°, regelt die Staatshaftung unab hängig davon, ob ein "konkretes Verwaltungshandeln" LS.d. Verwaltungsprozeßgesetzes vorliegt. Dafür gilt dieses Gesetz aber nur für bestimmte Rechtsgutverletzungen, nämlich rechtswidrige Eingriffe in die persönliche Freiheit, die körperliche Unversehrtheit und das Eigentum. Dieser Schadensersatz ist auch vorgesehen für unrechtmäßige Strafverfolgungs maßnahmen sowie Strafvollstreckungsmaßnahmen, soweit nachträglich die Unschuld fest gestellt wurde. 40 Laws of the People's Republic of China, 1994, S. 4 1 . e) Die Durchsetzung objektiven Rechts durch Kontrollbehörden Die Tätigkeit der Volksgerichte dient selbstverständlich nicht nur dem individuellen Rechtsschutz, sondern auch der Durchsetzung der als Recht zentral getroffenen Entschei dungen in der Peripherie. Dies dürfte sogar in den Augen Vieler die wichtigere Funktion sein. Wie wichtig die Kontrolle der nachgeordneten Ebenen und die Durchsetzung des objektiven Rechts ist, zeigt der organisatorische Aufwand, der zusätzlich zum gerichtlichen Rechtsschutz hierfür gemacht wird. Die dafür geschaffenen Institutionen haben zwar vor rangig das objektive Recht zu wahren, sind aber über ihre Pflicht, Beschwerden aus der Bevölkerung nachzugehen, auch Rechtsschutzinstitutionen. In jeweils von der Zentralebene bis zur Kreisebene durch Weisungsstränge hierarchischem Aufbau, der allerdings durch die Rekrutierungskompetenz der unteren Ebenen relativiert ist, dienen drei Behördenarten der objektiven Rechtskontrolle, nämlich 40 Laws of the People's Republic of China, 1994, S. 4 1 . 45 1 die Staatsanwaltschaften 41 , 42 die Aufsichtsbehörden 42 und die Rechnungsprüfungsbehörden 43. Die Staatsanwaltschaften sind sowohl gegenüber den Justizbehörden als auch gegenüber der Verwaltung selbständig und unterstehen einem vom Nationalen Volkskongreß gewähl ten und unmittelbar diesem bzw. dem Ständigen Ausschuß verantwortlichen Obersten Staatsanwalt. Neben der Verfolgung von Straftaten obliegt den Staatsanwaltschaften die Kontrolle über die Gerichte und über die Verwaltung, ohne daß allerdings insoweit Wei sungskompetenzen bestehen. Die Sanktionen bestehen in der Möglichkeit, entdeckte Straf taten zu verfolgen, in Berichten an übergeordnete Stellen, und in bestimmten Fällen in der Kompetenz, durch einen Einspruch ein Verfahren zur Prüfung einer Wiederaufnahme in Gang zu setzen. Die Aufsichtsbehörden sind innerhalb der Verwaltung errichtet und unterstehen der jewei ligen Verwaltungsspitze. Die Aufsichtsbehörde des Staatsrates beaufsichtigt sämtliche Zentralbehörden und leitet die Aufsicht auch auf den nachgeordneten Ebenen. Insofern bilden die Aufsichtsbehörden einen eigenständigen hierarchischen Verwaltungszweig. Bürger können sich mit Beschwerden an die Aufsichtsbehörden wenden. Gegenüber den beaufsichtigten Stellen bestehen umfassende Informations- und vorläufige Eingriffskom petenzen. Die Rechnungsprüfungsbehörden bilden - ebenso wie die Aufsichtsbehörden - einen eige nen Verwaltungszweig mit hierarchischer Organisation vom Obersten Rechnungsprüfer, der unmittelbar der Spitze des Staatsrates unterstellt ist, bis hinunter auf die Kreisebene. Sie sind andererseits der Verwaltungsspitze ihrer jeweiligen Ebene verantwortlich. Die Rech nungsprüfungsbehörden beurteilen die Rechtmäßigkeit und Wirtschaftlichkeit der finanz wirksamen Tätigkeiten aller staatlichen Stellen. Sie haben umfassende Kontroll-, aber statt Sanktions- nur Berichtskompetenzen. 41 42 43 Rechtsgrundlage insbesondere das Verwaltungsaufsichtsgesetz vom Mai 1 997; englische Fassung von Charles D. Paglee (Fn. 26). 43 Rechtsgrundlage das Rechnungsprüfungsgesetz vom August 1 994, Laws of the People's Republic of China, 1 994, S. 109. 41 42 Rechtsgrundlagen insbesondere in Art. 1 29 - 1 33 der Verfassung; Organisationsgesetz der Volks- staatsanwaltschaften der Volksrepublik China von 1979 in der Fassung vom September 1 983, Laws of the Peoples Republic of China, 1 983 - 1 986, S. 48. e) Die Durchsetzung objektiven Rechts durch Kontrollbehörden 452 Verfassllilg und Recht in Übersee (VR Ü) 32 (1999) 44 Ein in verschiedenen Hinsichten systematisierender empirischer Bericht über den Verwaltungs- rechtsschutz in den Jahren 1 990 bis 1 996 findet sich bei Pei Minxin (Fn. 22). 45 44 Ein in verschiedenen Hinsichten systematisierender empirischer Bericht über den Verwaltungs- rechtsschutz in den Jahren 1 990 bis 1 996 findet sich bei Pei Minxin (Fn. 22). 45 Heuser (Fn. 4), S.19; siehe aber auch den Bericht über Fallzahlen bei Pei Minxin (Fn. 22), S. 836. 46 Li Xinsheng (Fn. 23). 45 Heuser (Fn. 4), S.19; siehe aber auch den Bericht über Fallzahlen bei Pei Minxin (Fn. 22), S. 836. 46 Li Xinsheng (Fn. 23). 5. Einige Bemerkungen zur weiteren Entwicklung 5. Durch die zwar begrenzte, aber doch recht breite Rechtsschutzgewährung ist die Voraus setzung dafür geschaffen, daß auch das materielle Verwaltungsrecht, nämlich die Kriterien für die Rechtmäßigkeit der konkreten Verwaltungshandlungen, im Laufe der Zeit verfeinert und systematisiert werden. Dies ist in vollem Gange, wobei sich auch im Wissenschafts be trieb eine verwaltungsrechtliche Spezialisierung herausgebildet hat. Relevant ist natürlich, inwiefern der auf dem Papier gegebene Rechtsschutz auch in der Praxis funktioniert.44 Solange noch nicht ein eigenes Verwaltungsprozeßrecht existierte, sondern der Verwaltungsrechtsschutz im Verfahren des Zivilprozesses stattfand, war die Effektivität wohl nicht sehr hoch. Es wird berichtet, die erste erfolgreiche Klage eines Bürgers gegen einen polizeilichen Bußgeldbescheid habe es 1 987 gegeben.45 Nach Inkraft treten des Verwaltungsprozeßgesetzes hat es wohl einen deutlichen Wandel gegeben. Ein chinesischer Wissenschaftler berichtete auf einer Tagung im Jahre 1 998, daß von 1 990 bis 1 998 ca. 340.000 Gerichtsentscheidungen in Verwaltungssachen ergangen seien, von denen ca. 140.000, also gut 41 %, für die Verwaltung negativ gewesen seien.46 Dies belege sowohl die noch erheblichen Defizite hinsichtlich der Rechtmäßigkeit von Verwaltungs handeln als auch die Wirksamkeit des verwaltungsgerichtlichen Rechtsschutzes. Derzeit wird an einem allgemeinen Verwaltungsverfahrensgesetz und an einem Verwal tungsgenehmigungsgesetz gearbeitet, welches für alle möglichen Arten von behördlichen Genehmigungen gelten soll. Beides dürfte zu einer analytischen Durchdringung und Systematisierung sowohl des Verfahrensrechts als auch der Handlungsformen und ihrer Rechtsfolgen beitragen. 453 Administrative Law in the People's Republic of China By Ingwer Ebsen As a strong and weIl organised administration has been characteristic for the People's Republic of China for the past decades, this contribution on the development of "an" administrative law refers only to the changes that followed the end of the cultural revolu tion and the beginning of the reorientation of the Chinese economy. This reorientation had a strong influence on the relationship between the State and the developing private sector: First, the instrument of law was chosen as primary means to shape the new administrative process. The privilege of a political party to be above the law has explicitly been removed. Second, a private sector of the economy developed out of the political-administrative system of the economy. The developments regarding both the legal changes and the inde pendence of the private sector are still in the process. However, some elements of the remarkable changes may already be discussed. The article emphasises the changes within the organisation of the administrative, gives an overview over the areas of administrative law covered during the last two decades and looks at the judicial control of the implementation of administrative law. This last part includes aspects of administrative and judicial procedure, access to courts and liability of the state for acts of public authorities and officials. Finally, a brief outiook on expected developments is given. Can Bartolus Save the Tiger? l Reflections on the use of property rights for Iand-based biodiversity conservation ABSTRACTS Administrative Law in the People's Republic of China By Boudewijn R.A. Bouckaert and Britt Groosman By Boudewijn R.A. Bouckaert and Britt Groosman By Boudewijn R.A. Bouckaert and Britt Groosman By Boudewijn R.A. Bouckaert and Britt Groosman The variety of life on earth (biodiversity) is beIieved to be more at risk now than ever before. The rapid decline of biodiversity is nearly entirely caused by processes induced by humans, such as overexploitation of species, deIiberate habitat destruction, introduction of new and hostile species and ecological mismanagement. This paper aims to investigate whether market solutions operating through specific (private) property rights systems might 43 1
https://openalex.org/W4210598433	https://cp.copernicus.org/preprints/cp-2021-133/cp-2021-133.pdf	English	null	Comment on cp-2021-133	null	2,022	cc-by	13,087	Spring onset and seasonality patterns during the Lateglacial in the eastern Baltic region The comparison of pollen- and chironomid-inferred past temperature estimations with spring onset, growth-degree-days, and plant macrofossil data shows coherent patterns during during the Lateglacial period. The dynamics of the estimated length of the growing season and spring onset, combined with 15 the regional collection of plant macrofossil records, suggest the importance of local settings to species migration. During the Lateglacial warming period (Bølling/Allerød), a notable spring warming and longer growing season was calculated based on micro-phenology, but the treeline did not extend beyond central Estonia. The comparison of pollen- and chironomid-inferred past temperature estimations with spring onset, growth-degree-days, and plant macrofossil data shows coherent patterns during the cooler Older Dryas and warmer Bølling/ Allerød periods while suggesting more complicated climate dynamics and possible 20 warmer episodes during the Younger Dryas cold reversal. the cooler Older Dryas and warmer Bølling/ Allerød periods while suggesting more complicated climate dynamics and possible 20 warmer episodes during the Younger Dryas cold reversal. Spring onset and seasonality patterns during the Lateglacial in the eastern Baltic region Leeli Amon1, Friederike Wagner-Cremer 2, Jüri Vassiljev1, Siim Veski1 1Department of Geology, Tallinn University of Technology, Tallinn, 19086, Estonia 5 2Department of Geosciences and Physical Geography, Utrecht University, Utrecht, 3584, The Netherlands Correspondence to: Leeli Amon (leeli.amon@taltech.ee) Leeli Amon1, Friederike Wagner-Cremer 2, Jüri Vassiljev1, Siim Veski1 1Department of Geology, Tallinn University of Technology, Tallinn, 19086, Estonia 5 2Department of Geosciences and Physical Geography, Utrecht University, Utrecht, 3584, The Netherlands Correspondence to: Leeli Amon (leeli.amon@taltech.ee) Abstract. Spring onset is an important phenological observation that is sensitive to modern climate change and can be traced back in geological time. The Lateglacial (~14500 – 11700 cal yr BP) spring onset and growing season (growth-degree-days) dynamics in the eastern Baltic region were reconstructed using the micro-phenological approach based on the dwarf birch 10 (Betula nana) subfossil leaf cuticles. The presented study sites, Lake Lielais Svetinu (eastern Latvia) and Lake Kosilase (central Estonia), are located ~200 km apart in the region affected by the south-eastern sector of the Scandinavian Ice Sheet. During the Lateglacial period the region and its biota were influenced by the retreating glacier and the different stages of the Baltic Ice Lake. The plant macrofossil data confirms that the study sites were located in different vegetation zones (arctic-to-boreal) during the Lateglacial period. The dynamics of the estimated length of the growing season and spring onset, combined with 15 the regional collection of plant macrofossil records, suggest the importance of local settings to species migration. During the Lateglacial warming period (Bølling/Allerød), a notable spring warming and longer growing season was calculated based on micro-phenology, but the treeline did not extend beyond central Estonia. The comparison of pollen- and chironomid-inferred past temperature estimations with spring onset, growth-degree-days, and plant macrofossil data shows coherent patterns during the cooler Older Dryas and warmer Bølling/ Allerød periods while suggesting more complicated climate dynamics and possible 20 warmer episodes during the Younger Dryas cold reversal. during the Lateglacial period. The dynamics of the estimated length of the growing season and spring onset, combined with 15 the regional collection of plant macrofossil records, suggest the importance of local settings to species migration. During the Lateglacial warming period (Bølling/Allerød), a notable spring warming and longer growing season was calculated based on micro-phenology, but the treeline did not extend beyond central Estonia. https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. 1 Introduction During the past decade the Lateglacial environmental history of this region has been intensely studied (e.g. Stancikaite et al., 2009; Heikkilä et al., 2009; Veski et al., 2012; Druzhinina et al., 2020, Seiriene et al., 2020). The Lateglacial changeable climatic conditions (Rasmussen et al., 2014), available past summer and winter temperature reconstructions (Veski et al., 2015), and the associated arctic-to-boreal vegetation dynamics (Amon et al., vegetation reconstructions based on a variety of biological proxies. The Baltic region was covered by the south-eastern sector 50 of the last Scandinavian Ice Sheet (SIS) (Kalm, 2012). During the past decade the Lateglacial environmental history of this region has been intensely studied (e.g. Stancikaite et al., 2009; Heikkilä et al., 2009; Veski et al., 2012; Druzhinina et al., 2020, Seiriene et al., 2020). The Lateglacial changeable climatic conditions (Rasmussen et al., 2014), available past summer and winter temperature reconstructions (Veski et al., 2015), and the associated arctic-to-boreal vegetation dynamics (Amon et al., 2012; Veski et al., 2012; Amon et al., 2016) and tree-line presence (Amon et al., 2014) provide an excellent case to study the 55 seasonal dynamics of rapid natural climate warming and cooling episodes. Here we apply the microphenological UI proxy to two sites which hold uncommonly continuous and well-preserved B. nana subfossil leaf fragments in order to determine spring onset dates and the thermal properties of the growing season. The first indication that the UI proxy is also applicable in a more continental settings has been provided in experimental studies on B. nana relict stands in Poland (Ercan et al., 2021) and is 2012; Veski et al., 2012; Amon et al., 2016) and tree-line presence (Amon et al., 2014) provide an excellent case to study the 55 seasonal dynamics of rapid natural climate warming and cooling episodes. Here we apply the microphenological UI proxy to two sites which hold uncommonly continuous and well-preserved B. nana subfossil leaf fragments in order to determine spring onset dates and the thermal properties of the growing season. The first indication that the UI proxy is also applicable in a more continental settings has been provided in experimental studies on B. nana relict stands in Poland (Ercan et al., 2021) and is 2012; Veski et al., 2012; Amon et al., 2016) and tree-line presence (Amon et al., 2014) provide an excellent case to study the 55 seasonal dynamics of rapid natural climate warming and cooling episodes. 1 Introduction This proxy makes use of the direct correlation between the lateral epidermal cell expansion that regulates cell size and shape during the maturation of leaves past summer and winter temperatures have been reconstructed more often, the dynamics of the spring season is still 35 underrepresented in the temperature records available so far. The micro-phenological proxy based on the dwarf birch (Betula nana) subfossil leaf cuticle features (Wagner-Cremer et al., 2010) offers a means to shed light on past seasonality patterns as well the dynamics of spring onset and the amount of growing-degree-days (GDD). This proxy makes use of the direct correlation between the lateral epidermal cell expansion that regulates cell size and shape during the maturation of leaves past summer and winter temperatures have been reconstructed more often, the dynamics of the spring season is still 35 underrepresented in the temperature records available so far. The micro-phenological proxy based on the dwarf birch (Betula nana) subfossil leaf cuticle features (Wagner-Cremer et al., 2010) offers a means to shed light on past seasonality patterns as well the dynamics of spring onset and the amount of growing-degree-days (GDD). This proxy makes use of the direct correlation between the lateral epidermal cell expansion that regulates cell size and shape during the maturation of leaves (Wagner-Cremer et al., 2010; Ercan et al., 2020). The longer the growth period available to plants, and the higher the 40 accumulated GDD, the larger and increasingly undulated the epidermal cells grow. Quantified as the Undulation Index (UI) in long-term monitoring studies and validated in free-field growth experiments (Ercan et al., 2021), this proxy has demonstrated asynchronous spring versus summer temperature dynamics during the warming from the Late Pleniglacial to the Bølling/Allerød and during the transition from the Younger Dryas to the Holocene (Wagner-Cremer and Lotter, 2011; (Wagner-Cremer et al., 2010; Ercan et al., 2020). The longer the growth period available to plants, and the higher the 40 accumulated GDD, the larger and increasingly undulated the epidermal cells grow. Quantified as the Undulation Index (UI) in long-term monitoring studies and validated in free-field growth experiments (Ercan et al., 2021), this proxy has demonstrated asynchronous spring versus summer temperature dynamics during the warming from the Late Pleniglacial to the Bølling/Allerød and during the transition from the Younger Dryas to the Holocene (Wagner-Cremer and Lotter, 2011; (Wagner-Cremer et al., 2010; Ercan et al., 2020). 1 Introduction Changing seasonality belongs to the most eminent characteristics of ongoing global climate change. Phenological observations are sensitive and easily obtainable indicators of biospheric changes in response to climate change (Peñuelas and Filella, 2001; 25 Badeck et al., 2004, Cleland et al. 2007). The earlier unfolding of leaves has been observed since the mid-20th century all over the Northern Hemisphere (Ahas et al., 2002; Badeck et al., 2004 and references therein, Menzel et al. 2006, Jeong et al., 2011 and references therein), although the rate of change has decelerated during the last few decades (Fu et al., 2015). The spring phenology is influenced by pre-season temperature that is described as temperature before spring phenological date, winter 1 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. temperature and accumulated precipitation (Wang et al. 2015). The seasonality changes are the subject of intensive discussions, 30 given the strong impact of the earlier spring onset on many sectors including agriculture, ecosystem stability, and vegetation dynamics (e.g. Buermann et al. 2018, Menzel et al. 2020). Palaeoecological studies have the potential to contribute to a better understanding of spatio-temporal seasonality dynamics by reconstructing seasonal temperature changes during the phases of natural climate change that occurred in the recent past. While temperature and accumulated precipitation (Wang et al. 2015). The seasonality changes are the subject of intensive discussions, 30 given the strong impact of the earlier spring onset on many sectors including agriculture, ecosystem stability, and vegetation dynamics (e.g. Buermann et al. 2018, Menzel et al. 2020). Palaeoecological studies have the potential to contribute to a better understanding of spatio-temporal seasonality dynamics by reconstructing seasonal temperature changes during the phases of natural climate change that occurred in the recent past. While past summer and winter temperatures have been reconstructed more often, the dynamics of the spring season is still 35 underrepresented in the temperature records available so far. The micro-phenological proxy based on the dwarf birch (Betula nana) subfossil leaf cuticle features (Wagner-Cremer et al., 2010) offers a means to shed light on past seasonality patterns as well the dynamics of spring onset and the amount of growing-degree-days (GDD). 1 Introduction The longer the growth period available to plants, and the higher the 40 accumulated GDD, the larger and increasingly undulated the epidermal cells grow. Quantified as the Undulation Index (UI) in long-term monitoring studies and validated in free-field growth experiments (Ercan et al., 2021), this proxy has demonstrated asynchronous spring versus summer temperature dynamics during the warming from the Late Pleniglacial to the Bølling/Allerød and during the transition from the Younger Dryas to the Holocene (Wagner-Cremer and Lotter, 2011; Steinthorsdottir and Wagner-Cremer, 2019). During both warming phases, spring onset and GDD accumulation rose more 45 than a century before the summer temperatures started to increase (Steinthorsdottir and Wagner-Cremer, 2019). A systematic application of this proxy to B. nana leaf-bearing lake and peat sequences thus has the potential to add information on the expression of spring season warming over a large geographical range. In the present study, we focus on the seasonality changes in the eastern Baltic region by compiling multiple temperature and Steinthorsdottir and Wagner-Cremer, 2019). During both warming phases, spring onset and GDD accumulation rose more 45 than a century before the summer temperatures started to increase (Steinthorsdottir and Wagner-Cremer, 2019). A systematic application of this proxy to B. nana leaf-bearing lake and peat sequences thus has the potential to add information on the expression of spring season warming over a large geographical range. In the present study, we focus on the seasonality changes in the eastern Baltic region by compiling multiple temperature and In the present study, we focus on the seasonality changes in the eastern Baltic region by compiling multiple temperature and vegetation reconstructions based on a variety of biological proxies. The Baltic region was covered by the south-eastern sector 50 of the last Scandinavian Ice Sheet (SIS) (Kalm, 2012). During the past decade the Lateglacial environmental history of this region has been intensely studied (e.g. Stancikaite et al., 2009; Heikkilä et al., 2009; Veski et al., 2012; Druzhinina et al., 2020, Seiriene et al., 2020). The Lateglacial changeable climatic conditions (Rasmussen et al., 2014), available past summer and winter temperature reconstructions (Veski et al., 2015), and the associated arctic-to-boreal vegetation dynamics (Amon et al., vegetation reconstructions based on a variety of biological proxies. The Baltic region was covered by the south-eastern sector 50 of the last Scandinavian Ice Sheet (SIS) (Kalm, 2012). 1 Introduction Here we apply the microphenological UI proxy to two sites which hold uncommonly continuous and well-preserved B. nana subfossil leaf fragments in order to determine spring onset dates and the thermal properties of the growing season. The first indication that the UI proxy is also applicable in a more continental settings has been provided in experimental studies on B. nana relict stands in Poland (Ercan et al., 2021) and is applied here to a subfossil leaf record from the sediment core of the continental (Eastern Baltic) region for the first time. 60 Compared to the already available alternative temperature proxies for the same sites, this approach uniquely enables the first detailed analysis of seasonality changes during the Lateglacial in the eastern Baltic region. applied here to a subfossil leaf record from the sediment core of the continental (Eastern Baltic) region for the first time. 60 Compared to the already available alternative temperature proxies for the same sites, this approach uniquely enables the first detailed analysis of seasonality changes during the Lateglacial in the eastern Baltic region. 2 2 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. 2 Study area 2012, Amon et al., 2014; Amon et al., 2016). The present study explores the rich subfossil dwarf birch (Betula nana) leaf records from two sites, namely Lake Kosilase and (14500–11700 cal y BP) an important palaeogeographical feature of the deglaciated eastern Baltic region was the formation 80 of ice lakes (Vassiljev and Saarse, 2013; Rosentau et al., 2009; Amon et al., 2014). The vegetation communities during the Lateglacial period in the study region, as described by plant macrofossil and pollen records, spanned from pioneer snow-patch tundra to mixed boreal forests (Amon et al. 2012, Amon et al., 2014; Amon et al., 2016). The present study explores the rich subfossil dwarf birch (Betula nana) leaf records from two sites, namely Lake Kosilase and Lake Lielais Svetinu (L. Svetinu) ~200 km southeast of Lake Kosilase (Fig. 1). 85 Lake Kosilase (58°20.57 N; 25°39. 48 E) is a small (1.8 ha) lake located in central Estonia. Lake L. Svetinu (surface 18.8 ha) is located in eastern Latvia (56°45.5 N; 27°08.8 E). The postglacial sediments of Lake L. Svetinu have been studied using multiple palaeoecological proxies: chronology and vegetation proxies (Veski et al., 2012), phytoplankton (Stivrins et al., 2015), palaeotemperature reconstructions (Veski et al., 2015), and ancient DNA (Kisand et al., 2018). Lake Lielais Svetinu (L. Svetinu) ~200 km southeast of Lake Kosilase (Fig. 1). 85 Lake Kosilase (58°20.57 N; 25°39. 48 E) is a small (1.8 ha) lake located in central Estonia. Lake L. Svetinu (surface 18.8 ha) is located in eastern Latvia (56°45.5 N; 27°08.8 E). The postglacial sediments of Lake L. Svetinu have been studied using multiple palaeoecological proxies: chronology and vegetation proxies (Veski et al., 2012), phytoplankton (Stivrins et al., 2015), palaeotemperature reconstructions (Veski et al., 2015), and ancient DNA (Kisand et al., 2018). 2 Study area The mean value of snow cover duration varies from 75 days in the westernmost islands to more than 130 days in the higher and more forested regions in the north-east and south-east (Jaagus, 1997). A continuous snow cover in Latvia forms between and the prevalence of westerlies (Jaagus et al., 2010). The mean annual temperature in Estonia varies between 4.1-6.5 °C (Riigi 70 Ilmateenistus 2021). The mean annual temperature in Latvia is 7.2 °C, ranging from –1.6 °C (mean temperature in February) to 17.7 °C in July (Krauklis and Draveniece, 2004). The snow cover in Estonia persists from December to late March. The mean value of snow cover duration varies from 75 days in the westernmost islands to more than 130 days in the higher and more forested regions in the north-east and south-east (Jaagus, 1997). A continuous snow cover in Latvia forms between December 8th and 29th, except for the coastal territories in western Latvia where it is established during the first decade of 75 January (Draveniece, 2009) and melts in March/April (Krauklis and Draveniece, 2004). The vegetation types during the Lateglacial period in the eastern Baltics spanned from arctic tundra in N. Estonia to mixed forests in Latvia (Amon et al., 2014; Veski et al., 2012). The vegetation dynamics is influenced by Lateglacial hemispheric climate fluctuations (Rasmussen et al., 2014) as well as by the local factors (Amon et al. 2014). In the Lateglacial period December 8th and 29th, except for the coastal territories in western Latvia where it is established during the first decade of 75 January (Draveniece, 2009) and melts in March/April (Krauklis and Draveniece, 2004). The vegetation types during the Lateglacial period in the eastern Baltics spanned from arctic tundra in N. Estonia to mixed forests in Latvia (Amon et al., 2014; Veski et al., 2012). The vegetation dynamics is influenced by Lateglacial hemispheric climate fluctuations (Rasmussen et al., 2014) as well as by the local factors (Amon et al. 2014). In the Lateglacial period (14500–11700 cal y BP) an important palaeogeographical feature of the deglaciated eastern Baltic region was the formation 80 of ice lakes (Vassiljev and Saarse, 2013; Rosentau et al., 2009; Amon et al., 2014). The vegetation communities during the Lateglacial period in the study region, as described by plant macrofossil and pollen records, spanned from pioneer snow-patch tundra to mixed boreal forests (Amon et al. 2 Study area The topography of the eastern Baltic region has been largely shaped by Pleistocene glaciations, where Weichselian glaciation 65 (SIS) in particular contributed to present day topography. The region is currently situated in the hemiboreal vegetation zone, within the transitional zone between the boreal and nemoral forest zones of Europe. Estonia and Latvia are located between 56°N and 59.5°N on the eastern coast of the Baltic Sea in a transitional area from maritime to continental climate, characterized by a west–east gradient in the continentality of the climate. Climatic conditions are mainly determined by high cyclonic activity The topography of the eastern Baltic region has been largely shaped by Pleistocene glaciations, where Weichselian glaciation 65 (SIS) in particular contributed to present day topography. The region is currently situated in the hemiboreal vegetation zone, within the transitional zone between the boreal and nemoral forest zones of Europe. Estonia and Latvia are located between 56°N and 59.5°N on the eastern coast of the Baltic Sea in a transitional area from maritime to continental climate, characterized by a west–east gradient in the continentality of the climate. Climatic conditions are mainly determined by high cyclonic activity y g y y y g y y and the prevalence of westerlies (Jaagus et al., 2010). The mean annual temperature in Estonia varies between 4.1-6.5 °C (Riigi 70 Ilmateenistus 2021). The mean annual temperature in Latvia is 7.2 °C, ranging from –1.6 °C (mean temperature in February) to 17.7 °C in July (Krauklis and Draveniece, 2004). The snow cover in Estonia persists from December to late March. The mean value of snow cover duration varies from 75 days in the westernmost islands to more than 130 days in the higher and more forested regions in the north-east and south-east (Jaagus, 1997). A continuous snow cover in Latvia forms between December 8th and 29th except for the coastal territories in western Latvia where it is established during the first decade of 75 and the prevalence of westerlies (Jaagus et al., 2010). The mean annual temperature in Estonia varies between 4.1-6.5 °C (Riigi 70 Ilmateenistus 2021). The mean annual temperature in Latvia is 7.2 °C, ranging from –1.6 °C (mean temperature in February) to 17.7 °C in July (Krauklis and Draveniece, 2004). The snow cover in Estonia persists from December to late March. 3 Method 90 The chironomid-based temperature reconstruction from Lake Nakri was published previously (Heiri et al., 2014), as were the pollen-based temperature reconstructions (Veski et al., 2015) and the plant macrofossil data (Veski et al., 2012) from Lake L. Svetinu. 110 100 3 Method 90 nana, GDD5 = 10(2.4232+6.0284log(UIfossil) and Budburst Day of Year = 10(2.3109-(- 1.6888(log(UIfossil)) respectively, following the procedure outlined in detail in Steinthorsdottir and Wagner-Cremer (2019). 100 Budburst dates are commonly used to determine spring onset. Spring onset dates are subsequently expressed as Day-of-Year (DoY) before Mid-summer Warmth (MSW), referring to the number of days before the 15th of July (DoY: 196), which is set as the date for maximum summer warmth in order to facilitate a comparison with summer temperature proxies such as the chironomid-based July temperature reconstructions (Steinthorsdottir and Wagner-Cremer, 2019). This procedure enables the quantification of the time period between spring onset and maximum summer temperatures by calculating the DoY data rather 105 than providing numbers for individual months. Two samples from Lake Kosilase were selected for 14C dates. The selected samples were from the both ends of the ~30-cm- long silty sediment section. The chironomid-based temperature reconstruction from Lake Nakri was published previously (Heiri et al., 2014), as were the pollen-based temperature reconstructions (Veski et al., 2015) and the plant macrofossil data (Veski et al., 2012) from Lake L. Svetinu. 110 circumference (CC, µm), the undulation index (UI, dimensionless) was calculated according to Kürschner (1997). The UI was determined for a minimum of three epidermal pavement cells on at least three leaf fragments per sample. From the UI data, growing degree days above the threshold temperature 5 °C (GDD5) and budburst dates were calculated with the GDD5 and budburst inference models for B. nana, GDD5 = 10(2.4232+6.0284log(UIfossil) and Budburst Day of Year = 10(2.3109-(- 1.6888(log(UIfossil)) respectively, following the procedure outlined in detail in Steinthorsdottir and Wagner-Cremer (2019). 100 Budburst dates are commonly used to determine spring onset. Spring onset dates are subsequently expressed as Day-of-Year (DoY) before Mid-summer Warmth (MSW), referring to the number of days before the 15th of July (DoY: 196), which is set as the date for maximum summer warmth in order to facilitate a comparison with summer temperature proxies such as the chironomid-based July temperature reconstructions (Steinthorsdottir and Wagner-Cremer, 2019). This procedure enables the quantification of the time period between spring onset and maximum summer temperatures by calculating the DoY data rather 105 than providing numbers for individual months. Two samples from Lake Kosilase were selected for 14C dates. The selected samples were from the both ends of the ~30-cm- long silty sediment section. 4 Results Radiocarbon dating results for Lake Kosilase are given in Table 1. The radiocarbon dates were calibrated using Oxcal 4.2.4 (Bronk Ramsey, 2009) and the IntCal13 calibration curve (Reimer et al., 2013). 115 Table 1. 14C dates from Lake Kosilase, Estonia. Core depth, cm Laboratory code Dated material 14C date, yr BP Calibrated, cal yr BP Median calibrated value, cal yr BP 966-967 Poz-104880 Dryas octopetala leaves 11350 ± 70 13070 – 13324 13200 986-987 Poz-104784 Wood 12010 ± 70 13720 - 14080 13870 The mean UI from Lake L. Svetinu subfossil leaf record varies between 1.17 and 1.25 around a total average of 1.2 with an average standard error of 0.03. The UI values of Lake Kosilase subfossil dwarf birch leaves range from mean 1.17 to 1.23 with a total average of 1.21 and a standard error of 0.04 (Fig. 2). The estimated GDD5 range from 674 to 1009 (Fig. 3). The spring 120 onset calculations were based on inferred budburst dates recorded by the fossil leaves and revealed spring onset dates between the 21st of May and June 6th, equivalent to DoY 146 – 157 and translating to 39 – 55 days before MSW (Fig. 3). Table 1. 14C dates from Lake Kosilase, Estonia. Core depth, cm Laboratory code Dated material 14C date, yr BP Calibrated, cal yr BP Median calibrated value, cal yr BP 966-967 Poz-104880 Dryas octopetala leaves 11350 ± 70 13070 – 13324 13200 986-987 Poz-104784 Wood 12010 ± 70 13720 - 14080 13870 The mean UI from Lake L. Svetinu subfossil leaf record varies between 1.17 and 1.25 around a total average of 1.2 with an average standard error of 0.03. The UI values of Lake Kosilase subfossil dwarf birch leaves range from mean 1.17 to 1.23 with a total average of 1.21 and a standard error of 0.04 (Fig. 2). The estimated GDD5 range from 674 to 1009 (Fig. 3). The spring 120 onset calculations were based on inferred budburst dates recorded by the fossil leaves and revealed spring onset dates between the 21st of May and June 6th, equivalent to DoY 146 – 157 and translating to 39 – 55 days before MSW (Fig. 3). average standard error of 0.03. The UI values of Lake Kosilase subfossil dwarf birch leaves range from mean 1.17 to 1.23 with a total average of 1.21 and a standard error of 0.04 (Fig. 2). 3 Method 90 3 3 Method 90 The subfossil leaf material was extracted from the sediments during the plant macrofossil analyses. The sediments were wet- sieved and the remaining material was identified under the stereo- and light microscope. The subfossil B. nana leaves were stored in distilled water at 4 °C until a further analysis with the fluorescence microscope. Leaves with preserved cuticle cells were photographed under the microscope Leica using fluorescent light at 200X magnification (Fig. 2). Epidermal cell properties were measured on digital images using the software ImageJ. From the measured cell area (CA, µm2) and cell 95 3 The subfossil leaf material was extracted from the sediments during the plant macrofossil analyses. The sediments were wet- sieved and the remaining material was identified under the stereo- and light microscope. The subfossil B. nana leaves were stored in distilled water at 4 °C until a further analysis with the fluorescence microscope. Leaves with preserved cuticle cells were photographed under the microscope Leica using fluorescent light at 200X magnification (Fig. 2). Epidermal cell properties were measured on digital images using the software ImageJ. From the measured cell area (CA, µm2) and cell 95 3 The subfossil leaf material was extracted from the sediments during the plant macrofossil analyses. The sediments were wet- sieved and the remaining material was identified under the stereo- and light microscope. The subfossil B. nana leaves were stored in distilled water at 4 °C until a further analysis with the fluorescence microscope. Leaves with preserved cuticle cells were photographed under the microscope Leica using fluorescent light at 200X magnification (Fig. 2). Epidermal cell properties were measured on digital images using the software ImageJ. From the measured cell area (CA, µm2) and cell 95 3 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. circumference (CC, µm), the undulation index (UI, dimensionless) was calculated according to Kürschner (1997). The UI was determined for a minimum of three epidermal pavement cells on at least three leaf fragments per sample. circumference (CC, µm), the undulation index (UI, dimensionless) was calculated according to Kürschner (1997). The UI was determined for a minimum of three epidermal pavement cells on at least three leaf fragments per sample. From the UI data, growing degree days above the threshold temperature 5 °C (GDD5) and budburst dates were calculated with the GDD5 and budburst inference models for B. The mean UI from Lake L. Svetinu subfossil leaf record varies between 1.17 and 1.25 around a total average of 1.2 with an average standard error of 0.03. The UI values of Lake Kosilase subfossil dwarf birch leaves range from mean 1.17 to 1.23 with a total average of 1.21 and a standard error of 0.04 (Fig. 2). The estimated GDD5 range from 674 to 1009 (Fig. 3). The spring 120 onset calculations were based on inferred budburst dates recorded by the fossil leaves and revealed spring onset dates between the 21st of May and June 6th, equivalent to DoY 146 – 157 and translating to 39 – 55 days before MSW (Fig. 3). 4 Results Both lakes are currently surrounded by hemiboreal vegetation, but during the Lateglacial period the floral situation was much more dynamic. During that time Lake L. Svetinu and Lake Kosilase were located in p y g vegetation zones that oscillated north- and southwards, following changing climatic conditions and species migration. The 135 pioneer vegetation phases at both localities were characterized by treeless, Betula nana and Dryas octopetala dominated tundra. During the Allerød, warming permitted the migration of various tree species to eastern Latvia with traces of mixed forests of birch, pine, and aspen, as evident from the macroremain records (Veski et al., 2012). However, the proximity of the retreating glacier and areas submerged by the Baltic Ice Lake hindered the northward migration of trees and the regional vegetation zones that oscillated north- and southwards, following changing climatic conditions and species migration. The 135 pioneer vegetation phases at both localities were characterized by treeless, Betula nana and Dryas octopetala dominated tundra. During the Allerød, warming permitted the migration of various tree species to eastern Latvia with traces of mixed forests of birch, pine, and aspen, as evident from the macroremain records (Veski et al., 2012). However, the proximity of the retreating glacier and areas submerged by the Baltic Ice Lake hindered the northward migration of trees and the regional Lateglacial maximum northern treeline was likely located in southern Estonia (Amon et al., 2014). This implies that even 140 during the warmer phases of the Lateglacial, the surroundings of Lake Kosilase most likely remained treeless and were mainly covered by tundra vegetation. During the Younger Dryas cooling the tree remains disappeared from the records of Lake L. Svetinu, suggesting the re-establishment of tundra vegetation and a southward regression of the regional treeline. Although such vegetation shifts are recognized in many sedimentary records, the driving role of growing season dynamics has so far Lateglacial maximum northern treeline was likely located in southern Estonia (Amon et al., 2014). This implies that even 140 during the warmer phases of the Lateglacial, the surroundings of Lake Kosilase most likely remained treeless and were mainly covered by tundra vegetation. During the Younger Dryas cooling the tree remains disappeared from the records of Lake L. Svetinu, suggesting the re-establishment of tundra vegetation and a southward regression of the regional treeline. 4 Results Although such vegetation shifts are recognized in many sedimentary records, the driving role of growing season dynamics has so far hardly been taken into account, given the lack of temperature records for the early season. Here we have the unique opportunity 145 to synthesise vegetation dynamics and link it to climatic changes through local macroremain analysis, GDD5 and spring onset reconstructions from B. nana leaf remains, and more regional temperature signals for winter and summer deduced from pollen and chironomid data. The length of the growing season is correlated with the UI of both B. nana (Wagner-Cremer et al., 2010) as well as Betula The length of the growing season is correlated with the UI of both B. nana (Wagner-Cremer et al., 2010) as well as Betula pubescens subsp. czerepanovii (Ercan et al., 2019). The estimated length of the Lateglacial growing season in the Baltic region 150 varies between ~670 and 1000 cumulative growing degree days (GDD5) (Fig 3). It is comparable with current Northern and mid-Finnish conditions (Ercan et al., 2019). The GDD5 estimations from the microphenological fossil dataset in Germany (Schleinsee) stop at 14400 cal yr BP, when the present dataset from the Baltic region starts (Steinthorsdottir and Wagner- Cremer, 2019). The GDD5 estimations from Hässeldala (Sweden) (Steinthorsdottir and Wagner-Cremer, 2019) start at 12500 pubescens subsp. czerepanovii (Ercan et al., 2019). The estimated length of the Lateglacial growing season in the Baltic region 150 varies between ~670 and 1000 cumulative growing degree days (GDD5) (Fig 3). It is comparable with current Northern and mid-Finnish conditions (Ercan et al., 2019). The GDD5 estimations from the microphenological fossil dataset in Germany (Schleinsee) stop at 14400 cal yr BP, when the present dataset from the Baltic region starts (Steinthorsdottir and Wagner- Cremer, 2019). The GDD5 estimations from Hässeldala (Sweden) (Steinthorsdottir and Wagner-Cremer, 2019) start at 12500 pubescens subsp. czerepanovii (Ercan et al., 2019). The estimated length of the Lateglacial growing season in the Baltic region 150 varies between ~670 and 1000 cumulative growing degree days (GDD5) (Fig 3). It is comparable with current Northern and mid-Finnish conditions (Ercan et al., 2019). The GDD5 estimations from the microphenological fossil dataset in Germany (Schleinsee) stop at 14400 cal yr BP, when the present dataset from the Baltic region starts (Steinthorsdottir and Wagner- Cremer, 2019). 4 Results The estimated GDD5 range from 674 to 1009 (Fig. 3). The spring 120 onset calculations were based on inferred budburst dates recorded by the fossil leaves and revealed spring onset dates between the 21st of May and June 6th, equivalent to DoY 146 – 157 and translating to 39 – 55 days before MSW (Fig. 3). 4 4 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. 5 Discussion 125 The combination of (palaeo)botanical and phenological records reveals the dynamics of the vegetation, thermal growing season duration, and spring onset during the Lateglacial period in the study area. The captured spring onset signal is probably regional. We have a temporal overlap of the two spring onset records from the study sites ~200 km apart. The sample density in the overlapping part is not high, but the general trend (earlier spring onset around 13900 cal yr BP, later spring onset around 13800 5 Discussion 125 The combination of (palaeo)botanical and phenological records reveals the dynamics of the vegetation, thermal growing season duration, and spring onset during the Lateglacial period in the study area. The captured spring onset signal is probably regional. We have a temporal overlap of the two spring onset records from the study sites ~200 km apart. The sample density in the overlapping part is not high, but the general trend (earlier spring onset around 13900 cal yr BP, later spring onset around 13800 cal yr BP, and earlier again around 13600 cal yr BP) observed in both datasets suggests comparable patterns of the spring onset 130 signal over the entire study region. In the present study, we present spring onset and GDD5 records based on the UI analysis of B. nana leaves from two study localities ~200 km apart (Fig. 1). Both lakes are currently surrounded by hemiboreal vegetation, but during the Lateglacial period the floral situation was much more dynamic. During that time Lake L. Svetinu and Lake Kosilase were located in cal yr BP, and earlier again around 13600 cal yr BP) observed in both datasets suggests comparable patterns of the spring onset 130 signal over the entire study region. In the present study, we present spring onset and GDD5 records based on the UI analysis of B. nana leaves from two study localities ~200 km apart (Fig. 1). 4 Results Comparison of the modern phenological characteristics to the plant macroremain data thus indicates that the major shift between 25 and 56 days, with a mean of 42 days. A modern dataset for B.pendula from several study points in Finnish Lapland 165 indicates that the number of days from spring onset to MSW ranges from 40 to 62 days; average spring onset date is 51 days before MSW (Pudas et al., 2008). The modern observations of the budburst dates of Pinus sylvestris in the Inari region in Finnish Lapland (Salminen & Jalkanen, 2015) suggest the spring onset range of P. sylvestris to be from 50 to 75 days before MSW. Comparison of the modern phenological characteristics to the plant macroremain data thus indicates that the major shift in vegetation composition was related to spring onset dynamics, which might have taken place earlier in the warmer periods 170 of the Lateglacial. The notable shift towards earlier spring onset ~13350-13400 cal yr BP (at the end of GI-1c or Allerød) evident from Lake Kosilase coincides with the findings of pine macrofossils in the sediments of Lake L. Svetinu (Fig 3). It suggests an ameliorating, warmer environment in this region. The microphenology of dwarf birch growing in the tundra/at the verge of the in vegetation composition was related to spring onset dynamics, which might have taken place earlier in the warmer periods 170 of the Lateglacial. The notable shift towards earlier spring onset ~13350-13400 cal yr BP (at the end of GI-1c or Allerød) evident from Lake Kosilase coincides with the findings of pine macrofossils in the sediments of Lake L. Svetinu (Fig 3). It suggests an ameliorating, warmer environment in this region. The microphenology of dwarf birch growing in the tundra/at the verge of the treeline (Kosilase) parallels the warming signal that supported the formation of mixed boreal forests in eastern Latvia, ~200 175 km southwards (L. Svetinu). The regional spring warming ~13350-13400 cal yr BP and its effect on past vegetation may be similar to the modern tundra greening process and earlier spring dates reported by modern phenological observations. The warmer climate, including spring, supported the northward shift of the treeline: pine was present in Latvia; ~13300-13000 cal yr BP the scarce tree-birch treeline (Kosilase) parallels the warming signal that supported the formation of mixed boreal forests in eastern Latvia, ~200 175 km southwards (L. Svetinu). 4 Results The regional spring warming ~13350-13400 cal yr BP and its effect on past vegetation may be similar to the modern tundra greening process and earlier spring dates reported by modern phenological observations. The warmer climate, including spring, supported the northward shift of the treeline: pine was present in Latvia; ~13300-13000 cal yr BP the scarce tree-birch greening process and earlier spring dates reported by modern phenological observations. The warmer climate, including spring, supported the northward shift of the treeline: pine was present in Latvia; ~13300-13000 cal yr BP the scarce tree-birch macrofossils were found in the sediments of Lake Nakri (~100 km southeast of Lake Kosilase, Amon et al., 2012). The plant 180 macrofossil records suggest that the Lateglacial treeline did probably not reach beyond central Estonia (Amon et al., 2014; Amon et al., 2016), although a single poorly preserved, probably tree-birch or hybrid, macrofossil was found in the Lake Kosilase sediments. At the same time, spring in central Estonia was already taking place much earlier as confirmed by the UI of the B. nana leaves and could have been suitable for tree species growth. Thus, the limiting factor for the advancement of macrofossils were found in the sediments of Lake Nakri (~100 km southeast of Lake Kosilase, Amon et al., 2012). The plant 180 macrofossil records suggest that the Lateglacial treeline did probably not reach beyond central Estonia (Amon et al., 2014; Amon et al., 2016), although a single poorly preserved, probably tree-birch or hybrid, macrofossil was found in the Lake Kosilase sediments. At the same time, spring in central Estonia was already taking place much earlier as confirmed by the UI of the B. nana leaves and could have been suitable for tree species growth. Thus, the limiting factor for the advancement of the treeline may have been related to other local conditions in the partially-submerged mosaic landscape, the proximity of the 185 retreating glacier, and the maximum summer temperature. The modern distribution of the treeline-forming tree-birch in Scandinavia follows the 13.2 °C isotherm for summer temperature (Odland, 1996) and the 10–12 °C summer temperature isotherm for tree-line formation (CAVM, 2003). 4 Results The GDD5 estimations from Hässeldala (Sweden) (Steinthorsdottir and Wagner-Cremer, 2019) start at 12500 cal yr BP and span until the start of the Holocene; they are in general lower than the Baltic GDD5 estimations, however both 155 5 5 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. display an interval of higher GDD5 values during the Younger Dryas cooling period. The Holocene onset is more pronounced in the Hässeldala (Swedish) dataset than in the Baltic (Latvian) one. 160 this period, the continuous plant macrofossil records document the persisting local flora and provide evidence for vegetation 160 dynamics. The dominant plant species substituting the dwarf birch during the Allerød in Lake L. Svetinu were tree birch (Betula pendula) and pine (Pinus sylvestris) (Fig 3). The occurrence of these two species provides additional information on the spring conditions through their contemporaneous growth requirements. Phenological observations of B. nana budburst dates from the Kevo research station in Finnish Lapland suggest that the number of days from spring onset to MSW varies between 25 and 56 days, with a mean of 42 days. A modern dataset for B.pendula from several study points in Finnish Lapland 165 indicates that the number of days from spring onset to MSW ranges from 40 to 62 days; average spring onset date is 51 days before MSW (Pudas et al., 2008). The modern observations of the budburst dates of Pinus sylvestris in the Inari region in Finnish Lapland (Salminen & Jalkanen, 2015) suggest the spring onset range of P. sylvestris to be from 50 to 75 days before MSW. Comparison of the modern phenological characteristics to the plant macroremain data thus indicates that the major shift between 25 and 56 days, with a mean of 42 days. A modern dataset for B.pendula from several study points in Finnish Lapland 165 indicates that the number of days from spring onset to MSW ranges from 40 to 62 days; average spring onset date is 51 days before MSW (Pudas et al., 2008). The modern observations of the budburst dates of Pinus sylvestris in the Inari region in Finnish Lapland (Salminen & Jalkanen, 2015) suggest the spring onset range of P. sylvestris to be from 50 to 75 days before MSW. 4 Results The chironomid-based July temperature estimates around Lake Nakri for this period were ~+12,7-12,8 °C (Heiri et al., 2014) and the pollen-based reconstructed mean May-June-July-August temperature the treeline may have been related to other local conditions in the partially-submerged mosaic landscape, the proximity of the 185 retreating glacier, and the maximum summer temperature. The modern distribution of the treeline-forming tree-birch in Scandinavia follows the 13.2 °C isotherm for summer temperature (Odland, 1996) and the 10–12 °C summer temperature isotherm for tree-line formation (CAVM, 2003). The chironomid-based July temperature estimates around Lake Nakri for this period were ~+12,7-12,8 °C (Heiri et al., 2014) and the pollen-based reconstructed mean May-June-July-August temperature the treeline may have been related to other local conditions in the partially-submerged mosaic landscape, the proximity of the 185 retreating glacier, and the maximum summer temperature. The modern distribution of the treeline-forming tree-birch in Scandinavia follows the 13.2 °C isotherm for summer temperature (Odland, 1996) and the 10–12 °C summer temperature isotherm for tree-line formation (CAVM, 2003). The chironomid-based July temperature estimates around Lake Nakri for this period were ~+12,7-12,8 °C (Heiri et al., 2014) and the pollen-based reconstructed mean May-June-July-August temperature 6 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. range was ~+12,2-12,3 °C, which are close to the modern tree-line limits. Assuming that the summer temperature was one of 190 the limiting factors for the northwards migration of the treeline, the results resemble the vegetation response under modern climate conditions. The spring onset thus occurs before a significant rise in the summer temperature. The observed warmer climatic conditions were subsequently disrupted by the onset of the Younger Dryas cold reversal. A similar set – warming of the spring preceding the warming of the summer temperatures – is observed by Steinthorsdottir and Wagner-Cremer, 2019. range was ~+12,2-12,3 °C, which are close to the modern tree-line limits. Assuming that the summer temperature was one of 190 the limiting factors for the northwards migration of the treeline, the results resemble the vegetation response under modern climate conditions. The spring onset thus occurs before a significant rise in the summer temperature. The observed warmer climatic conditions were subsequently disrupted by the onset of the Younger Dryas cold reversal. A similar set – warming of the spring preceding the warming of the summer temperatures – is observed by Steinthorsdottir and Wagner-Cremer, 2019. Lateglacial climate fluctuations and seasonality signal 195 Pollen-based temperature estimations for May-June-July-August (MJJA) and December-January-February (DJF) are available for Lake L. Svetinu in the Lateglacial period (Veski et al., 2015) and provide an indication of the winter and summer conditions prevailing in the studied region. A minor offset between the pollen record and the microphenological dataset is likely related to the different sampling strategies. The pollen samples were taken as 1-cm-thick slices after some interval while the plant macrofossil samples from where the dwarf birch leaves were extracted were 5-cm-thick continuous samples. Additional 200 chironomid-based July temperature estimates from Lake Nakri (Fig. 3) ~90 km north are included to complement the temperature data (Heiri et al., 2014). In the oldest part of the record between 14500-~14000 cal yr BP, assigned to GI-1e (Bølling), all proxies (pollen-based DJF and MJJA temperatures, GDD5, and spring onset estimates (Fig. 3)) follow a comparable pattern of moderate temperatures macrofossil samples from where the dwarf birch leaves were extracted were 5-cm-thick continuous samples. Additional 200 chironomid-based July temperature estimates from Lake Nakri (Fig. 3) ~90 km north are included to complement the temperature data (Heiri et al., 2014). In the oldest part of the record between 14500-~14000 cal yr BP, assigned to GI-1e (Bølling), all proxies (pollen-based DJF and MJJA temperatures, GDD5, and spring onset estimates (Fig. 3)) follow a comparable pattern of moderate temperatures and MJJA temperatures, GDD5, and spring onset estimates (Fig. 3)) follow a comparable pattern of moderate temperatures during all seasons. A short pulse of cold conditions is paced between 13800 and 13950 cal yr BP with only +10°C for 205 MJJA/chironomid July, dropping to -24°C for DJF; GDD5 values and spring onset (Fig. 3) are declining in parallel. Since this cool pulse is of short duration, the temporal resolution of our proxies is not suitable to determine the offsets in the seasonality changes. The common cooling is interpreted to reflect the GI-1d (Older Dryas) phase (Rasmussen et al., 2014). A gradual decrease in spring thermal conditions has been observed earlier in a GDD5 / spring onset reconstruction from the Schleinsee during all seasons. A short pulse of cold conditions is paced between 13800 and 13950 cal yr BP with only +10°C for 205 MJJA/chironomid July, dropping to -24°C for DJF; GDD5 values and spring onset (Fig. 3) are declining in parallel. Lateglacial climate fluctuations and seasonality signal 195 Since this cool pulse is of short duration, the temporal resolution of our proxies is not suitable to determine the offsets in the seasonality changes. The common cooling is interpreted to reflect the GI-1d (Older Dryas) phase (Rasmussen et al., 2014). A gradual decrease in spring thermal conditions has been observed earlier in a GDD5 / spring onset reconstruction from the Schleinsee section in Southern Germany, where the early season cooling during the Bølling culminates in the Older Dryas, after which 210 the conditions improve again towards the Allerød (Wagner-Cremer and Lotter, 2010). Although the Schleinsee site is located much further to the south, the comparable records point towards a dynamic seasonality pattern during this period. The following warm period (Allerød) is characterized by a temperature increase occurring in all proxies. The summer-related proxies reveal peaks at ~13700 cal yr BP with +12 °C pollen MJJA and +13.7 °C chironomid J. This initial peak is followed section in Southern Germany, where the early season cooling during the Bølling culminates in the Older Dryas, after which 210 the conditions improve again towards the Allerød (Wagner-Cremer and Lotter, 2010). Although the Schleinsee site is located much further to the south, the comparable records point towards a dynamic seasonality pattern during this period. The following warm period (Allerød) is characterized by a temperature increase occurring in all proxies. The summer-related proxies reveal peaks at ~13700 cal yr BP with +12 °C pollen MJJA and +13.7 °C chironomid J. This initial peak is followed by a small decrease in the pollen based MJJA and GDD5 / spring onset data which is not picked up by the chironomid record. 215 During the Allerød, B. nana disappears from the record while B. pendula and P. sylvestris become common in the macroremain record. This succession provides additional evidence for stable, mild conditions. During the Younger Dryas, the signals captured by different proxies diverge. At ~12700 cal yr BP, a rapid temperature decline from ~13 to ~10 °C started according to chironomid-based reconstructed estimates although there is a notable variability by a small decrease in the pollen based MJJA and GDD5 / spring onset data which is not picked up by the chironomid record. 215 During the Allerød, B. nana disappears from the record while B. pendula and P. sylvestris become common in the macroremain record. This succession provides additional evidence for stable, mild conditions. 4 Results Lateglacial climate fluctuations and seasonality signal 195 Pollen-based temperature estimations for May-June-July-August (MJJA) and December-January-February (DJF) are available for Lake L. Svetinu in the Lateglacial period (Veski et al., 2015) and provide an indication of the winter and summer conditions prevailing in the studied region. A minor offset between the pollen record and the microphenological dataset is likely related to the different sampling strategies. The pollen samples were taken as 1-cm-thick slices after some interval while the plant Lateglacial climate fluctuations and seasonality signal 195 During the Younger Dryas, the signals captured by different proxies diverge. At ~12700 cal yr BP, a rapid temperature decline from ~13 to ~10 °C started according to chironomid-based reconstructed estimates although there is a notable variability During the Younger Dryas, the signals captured by different proxies diverge. At ~12700 cal yr BP, a rapid temperature decline from ~13 to ~10 °C started according to chironomid-based reconstructed estimates although there is a notable variability between the individual samples (Fig 3). At the same time (~12650 cal yr BP) the winter (DJF) pollen-based temperature 220 estimates start to decrease as well, reaching the minimal temperature at ~12000 cal yr BP (-20 °C). The minimal chironomid- based temperature estimate (+8.6 °C for July T) is reached ~12500 cal yr BP. This cooling is not entirely paralleled by pollen- between the individual samples (Fig 3). At the same time (~12650 cal yr BP) the winter (DJF) pollen-based temperature 220 estimates start to decrease as well, reaching the minimal temperature at ~12000 cal yr BP (-20 °C). The minimal chironomid- based temperature estimate (+8.6 °C for July T) is reached ~12500 cal yr BP. This cooling is not entirely paralleled by pollen- between the individual samples (Fig 3). At the same time (~12650 cal yr BP) the winter (DJF) pollen-based temperature 220 estimates start to decrease as well, reaching the minimal temperature at ~12000 cal yr BP (-20 °C). The minimal chironomid- based temperature estimate (+8.6 °C for July T) is reached ~12500 cal yr BP. This cooling is not entirely paralleled by pollen- between the individual samples (Fig 3). At the same time (~12650 cal yr BP) the winter (DJF) pollen-based temperature 220 estimates start to decrease as well, reaching the minimal temperature at ~12000 cal yr BP (-20 °C). The minimal chironomid- based temperature estimate (+8.6 °C for July T) is reached ~12500 cal yr BP. This cooling is not entirely paralleled by pollen- 7 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. based summer reconstructions and GDD5 / spring onset data: The MJJA pollen-based temperature remains at the previous (Allerød) level, even indicating a small rise between 12500-12100 cal yr BP, only decreasing from 12100 cal yr BP until the onset of the Holocene. Spring season conditions rather follow the declining winter temperatures than the summer signal. Lateglacial climate fluctuations and seasonality signal 195 However, it does not substantially confirm persistent warm summer conditions during the Younger Dryas as the main trend in chironomids, pollen-based DJF temperatures, and spring onset / GDD5 records indicate a cooling period. Global climate simulations for the Bølling, Allerød, and Younger Dryas 240 (Schenk et al., 2018) show that for the Baltic region the month of May is highly important, with colder May temperatures during the Younger Dryas than during the Allerød period. That is in broad accordance with the trend in the spring onset record but punctuated by occasional short phases with earlier budburst in the first part of the Younger Dryas. Modern studies suggest that the ongoing lengthening of the vegetation period is also due to the earlier budburst and spring onset (Jeong et al., 2011). The relevance of the early season temperatures requires proxies that are able to distinguish between the summer/winter 245 temperatures and spring temperatures. Lateglacial climate fluctuations and seasonality signal 195 The 225 GDD5 values decrease from the ~12400 cal yr BP peak values of above 1000 GDD5 to low ~720 GDD5 at 12250 cal yr BP. Unfortunately, we do not have good data coverage for this time interval and, thus, cannot provide a detailed analysis on the validity of the peak values nor the exact timing of the cooling. From 12200 cal yr BP the GDD5 values stabilise on very low values, varying between 700–800 GDD5. The results also indicate very late spring onset during this phase, where the growing season did not start earlier than 39–43 days before MSW (in June). The general pattern during the Younger Dryas in terms of 230 seasonality changes very closely resembles the results obtained for the Swedish Hässeldala section, where GDD5 values ranging from 650–700 were reconstructed, and spring onset occurred ~40 days before MSW (Steinthordottir and Wagner- Cremer, 2019). The strong variability in several proxies during the Younger Dryas cold reversal (Fig 3) points to a notably dynamic season did not start earlier than 39–43 days before MSW (in June). The general pattern during the Younger Dryas in terms of 230 seasonality changes very closely resembles the results obtained for the Swedish Hässeldala section, where GDD5 values ranging from 650–700 were reconstructed, and spring onset occurred ~40 days before MSW (Steinthordottir and Wagner- Cremer, 2019). The strong variability in several proxies during the Younger Dryas cold reversal (Fig 3) points to a notably dynamic temperature system. There is an ongoing debate about warm summers on the northern latitudes during the Younger Dryas 235 cooling (Björck et al., 2002; Schenck et al., 2018). The pronounced variability on chironomid-based dataset, duration of the growing season and spring onset data, and rather stable pollen-based MJJA temperatures may indicate short, occasional phases with possible early springs and warmer summer temperatures. However, it does not substantially confirm persistent warm summer conditions during the Younger Dryas as the main trend in chironomids, pollen-based DJF temperatures, and spring temperature system. There is an ongoing debate about warm summers on the northern latitudes during the Younger Dryas 235 cooling (Björck et al., 2002; Schenck et al., 2018). The pronounced variability on chironomid-based dataset, duration of the growing season and spring onset data, and rather stable pollen-based MJJA temperatures may indicate short, occasional phases with possible early springs and warmer summer temperatures. Author contribution: LA is the main author (conceptualization, investigation, writing). The co-authors contributed: FW (conceptualization, methodology, writing), JV (chronology), SV (past vegetation and climate reconstructions). LA prepared the manuscript with contributions from all co-authors. 265 hors declare that they have no conflict of interest. Acknowledgements: This study was supported by Estonian Research Agency grants MOBTP140 and PRG323. Conclusions - The present study is one of the rare examples of the application of the micro-phenological proxy to the Lateglacial fossil leaf record. By applying a novel microphenological proxy and plant macrofossil analysis to the multi-proxy approach we can constrain early season temperature dynamics rather than traditionally inferred winter or summer temperatures. - The combination of spring onset/GDD5 estimates and plant macrofossil records indicates that the local tree-line dynamics are linked to local rather than regional environmental conditions. The spring onset in central Estonia during the Allerød period took place early and was comparable with the timing in the modern forest species. However, the Lateglacial plant - The combination of spring onset/GDD5 estimates and plant macrofossil records indicates that the local tree-line dynamics are linked to local rather than regional environmental conditions. The spring onset in central Estonia during the Allerød period took place early and was comparable with the timing in the modern forest species. However, the Lateglacial plant macrofossil data of North Estonia displays the lack of any tree remains. Therefore, we could reconstruct a similar situation are linked to local rather than regional environmental conditions. The spring onset in central Estonia during the Allerød period took place early and was comparable with the timing in the modern forest species. However, the Lateglacial plant macrofossil data of North Estonia displays the lack of any tree remains. Therefore, we could reconstruct a similar situation 255 macrofossil data of North Estonia displays the lack of any tree remains. Therefore, we could reconstruct a similar situation 255 8 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. with modern climate change when the spring onset takes place earlier, but the summer temperatures are not yet rising. The explanation for the eastern Baltic Lateglacial situation could be the proximity of the SIS and the Baltic Ice Lake. Th L t l i l lit d i l t d b i i t ti t d ll b d DJF d MJJA with modern climate change when the spring onset takes place earlier, but the summer temperatures are not yet rising. The explanation for the eastern Baltic Lateglacial situation could be the proximity of the SIS and the Baltic Ice Lake. - The Lateglacial seasonality dynamics are evaluated by comparing spring onset estimates and pollen-based DJF and MJJA temperature reconstructions in combination with chironomid-based July temperatures. Conclusions During the GI-1d and the b i i d (B lli /All d) ll i hil h di d i h YD d i The explanation for the eastern Baltic Lateglacial situation could be the proximity of the SIS and the Baltic Ice Lake. - The Lateglacial seasonality dynamics are evaluated by comparing spring onset estimates and pollen-based DJF and MJJA temperature reconstructions in combination with chironomid-based July temperatures. During the GI-1d and the subsequent warming period (Bølling/Allerød) all proxies agree, while the divergence during the YD suggests a dynamic seasonality pattern with possible occasional warm episodes. - The Lateglacial seasonality dynamics are evaluated by comparing spring onset estimates and pollen-based DJF and MJJA temperature reconstructions in combination with chironomid-based July temperatures. During the GI-1d and the subsequent warming period (Bølling/Allerød) all proxies agree, while the divergence during the YD suggests a dynamic seasonality pattern with possible occasional warm episodes. Author contribution: LA is the main author (conceptualization, investigation, writing). The co-authors contributed: FW (conceptualization, methodology, writing), JV (chronology), SV (past vegetation and climate reconstructions). LA prepared the manuscript with contributions from all co-authors. 265 Author contribution: LA is the main author (conceptualization, investigation, writing). The co-authors contributed FW (conceptualization, methodology, writing), JV (chronology), SV (past vegetation and climate reconstructions). LA prepared the manuscript with contributions from all co-authors. 265 The authors declare that they have no conflict of interest. Acknowledgements: This study was supported by Estonian Research Agency grants MOBTP140 and PRG323. References Draveniece, A.: Detecting changes in winter seasons in Latvia: the role of arctic air masses, Boreal Environment Research, 14, 89–99, 2009. 290 Druzhinina, O., Kublitskiy, Y., Stančikaitė, M., Nazarova, L., Syrykh, L., Gedminienė, L., Uogintas, D., Skipityte, R., Arslanov, K., Vaikutienė, G., Kulkova, M. and Subetto, D.: The Late Pleistocene–Early Holocene palaeoenvironmental evolution in the SE Baltic region: a new approach based on chironomid, geochemical and isotopic data from Kamyshovoye Lake, Russia, Boreas, 49, 544– 561, doi: 10.1111/bor.12438, 2020. Druzhinina, O., Kublitskiy, Y., Stančikaitė, M., Nazarova, L., Syrykh, L., Gedminienė, L., Uogintas, D., Skipityte, R., Arslanov, K., Vaikutienė, G., Kulkova, M. and Subetto, D.: The Late Pleistocene–Early Holocene palaeoenvironmental evolution in the SE Baltic region: a new approach based on chironomid, geochemical and isotopic data from Kamyshovoye Lake, Russia, Boreas, 49, 544– 561, doi: 10.1111/bor.12438, 2020. Ercan, F.E., De Boer, H.J. and Wagner-Cremer, F.: A growing degree day inference model based on mountain birch leaf cuticle 295 analysis over a latitudinal gradient in Fennoscandia, The Holocene, 30, 344-349, doi:10.1177/0959683619865605, 2020. Ercan, F.E.Z., Mikola, J., Silfver, T., Myller, K., Vainio, E., et al.: Effects of experimental warming on Betula nana epidermal cell growth tested over its maximum climatological growth range, PLOS ONE 16, e0251625, doi: 10.1371/journal.pone.0251625, 2021. Ercan, F.E., De Boer, H.J. and Wagner-Cremer, F.: A growing degree day inference model based on mountain birch leaf cuticle 295 analysis over a latitudinal gradient in Fennoscandia, The Holocene, 30, 344-349, doi:10.1177/0959683619865605, 2020. Ercan, F.E.Z., Mikola, J., Silfver, T., Myller, K., Vainio, E., et al.: Effects of experimental warming on Betula nana epidermal cell growth tested over its maximum climatological growth range, PLOS ONE 16, e0251625, doi: 10.1371/journal.pone.0251625, 2021. Fu, Y., et al., .: Declining global warming effects on the phenology of spring leaf unfolding, Nature, 526, 104– 107, 300 doi:10.1038/nature15402, 2015. Fu, Y., et al., .: Declining global warming effects on the phenology of spring leaf unfolding, Nature, 526, 104– 107, 300 doi:10.1038/nature15402, 2015. Heikkilä, M., Fontana, S.L., Seppä, H.: Rapid Lateglacial tree population dynamics and ecosystem changes in the eastern Baltic region, Journal of Quaternary Science, 24, 802–815, doi: 10.1002/jqs.1254, 2009. Heiri, O., Brooks, S.J., Renssen, H., Bedford A., Hazekamp, M. et al.: Validation of climate model-inferred regional Heikkilä, M., Fontana, S.L., Seppä, H.: Rapid Lateglacial tree population dynamics and ecosystem changes in the eastern Baltic region, Journal of Quaternary Science, 24, 802–815, doi: 10.1002/jqs.1254, 2009. References 22, 1727-1738, doi: 10.1002/joc.818, 2002. 270 Amon, L., Saarse, L., Vassiljev, J., Heinsalu, A. and Veski S.: Timing of the deglaciation and the late-glacial vegetation development on the Pandivere Upland, North Estonia, Bulletin of the Geological Society of Finland, 88, 69–83, doi: 10.17741/bgsf/88.2.002, 2016. Amon, L., Veski, S. and Vassiljev, J.: Tree taxa immigration to the eastern Baltic region, southeastern sector of Scandinavian glaciation during the Late-glacial period (14,500–11,700 cal. B.P.), Veget Hist Archaeobot, 23, 207–216, 275 doi:10.1007/s00334-014-0442-6, 2014. Amon, L., Veski, S., Heinsalu, A. and Saarse, L.: Timing of Lateglacial vegetation dynamics and respective palaeoenvironmental conditions in southern Estonia: evidence from the sediment record of Lake Nakri, J. Quaternary Sci, 27, 169-180, doi: 10.1002/jqs.1530, 2012. Badeck, F.-W., Bondeau, A., Böttcher, K., Doktor, D., Lucht, W., Schaber, J. and Sitch, S.: Responses of spring phenology to 280 climate change, New Phytologist, 162, 295-309, doi: 10.1111/j.1469-8137.2004.01059.x, 2004. Badeck, F.-W., Bondeau, A., Böttcher, K., Doktor, D., Lucht, W., Schaber, J. and Sitch, S.: Responses of spring phenology to 280 climate change, New Phytologist, 162, 295-309, doi: 10.1111/j.1469-8137.2004.01059.x, 2004. Bronk Ramsey, C.: Bayesian analysis of radiocarbon dates. Radiocarbon, 51(1), 337–360, 2009. Buermann, W., Forkel, M., O’Sullivan, M. et al. Widespread seasonal compensation effects of spring warming on northern plant productivity. Nature 562, 110–114, doi:10.1038/s41586-018-0555-7, 2018. Buermann, W., Forkel, M., O’Sullivan, M. et al. Widespread seasonal compensation effects of spring warming on northern plant productivity. Nature 562, 110–114, doi:10.1038/s41586-018-0555-7, 2018. CAVM Team.:Circumpolar Arctic Vegetation Map. (1:7,500,000 scale), Conservation of Arctic Flora and Fauna (CAFF) Map 285 No. 1. U.S. Fish and Wildlife Service, Anchorage, Alaska, 2003. 9 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. Cleland, E.E., Chuine, I., Menzel, A., Mooney, H.A. and Schwartz, M.D: Shifting plant phenology in response to global change, Trends in Ecology & Evolution, 22, 357-365, doi:10.1016/j.tree.2007.04.003, 2007. Draveniece, A.: Detecting changes in winter seasons in Latvia: the role of arctic air masses, Boreal Environment Research, 14, 89–99, 2009. 290 Druzhinina, O., Kublitskiy, Y., Stančikaitė, M., Nazarova, L., Syrykh, L., Gedminienė, L., Uogintas, D., Skipityte, Cleland, E.E., Chuine, I., Menzel, A., Mooney, H.A. and Schwartz, M.D: Shifting plant phenology in response to global change, Trends in Ecology & Evolution, 22, 357-365, doi:10.1016/j.tree.2007.04.003, 2007. Krauklis, A. and Draveniece, A.: Landscape seasons and air mass dynamics in Latvia. Folia Geographica 12, 16–47, 2004. References Heiri, O., Brooks, S.J., Renssen, H., Bedford A., Hazekamp, M. et al.: Validation of climate model-inferred regional temperature change for late-glacial Europe, Nature communications, 5, 1-7, doi:10.1038/ncomms5914, 2014. 305 temperature change for late-glacial Europe, Nature communications, 5, 1-7, doi:10.1038/ncomms5914, 2014. 305 Jaagus, J., Briede, A., Rimkus, E. and Remm, K.: Precipitation pattern in the Baltic countries under the influence of large- scale atmospheric circulation and local landscape factors, International Journal of Climatology, 30, 705–720 doi: 10.1002/joc.1929, 2010. Jaagus, J.: The impact of climate change on the snowcover pattern in Estonia, Climatic change 36, 65–77, doi: Jaagus, J., Briede, A., Rimkus, E. and Remm, K.: Precipitation pattern in the Baltic countries under the influence of large- scale atmospheric circulation and local landscape factors, International Journal of Climatology, 30, 705–720 doi: 10.1002/joc.1929, 2010. 10.1023/A:1005304720412, 1997. 310 Jeong, S.-J., Ho, C.-H., Gim, H.-J. and Brown, M.E.: Phenology shifts at start vs. end of growing season in temperate vegetation over the Northern Hemisphere for the period 1982–2008, Global Change Biology, 17, 2385-2399doi: 10.1111/j.1365- 2486.2011.02397.x, 2011. Kalm, V.: Ice-flow pattern and extent of the last Scandinavian Ice Sheet southeast of the Baltic Sea, Quaternary Science Kalm, V.: Ice-flow pattern and extent of the last Scandinavian Ice Sheet southeast of the Baltic Sea, Quaternary Science Reviews, 44, 51-59, doi: 10.1016/j.quascirev.2010.01.019, 2012. 315 Reviews, 44, 51-59, doi: 10.1016/j.quascirev.2010.01.019, 2012. 315 Kisand, V., Talas, L., Kisand, A., Stivrins, N., Reitalu, T. et al.: From microbial eukaryotes to metazoan vertebrates: Wide spectrum paleo-diversity in sedimentary ancient DNA over the last ~14,500 years, Geobiology, 16, 628– 639 doi: 10.1111/gbi.12307, 2018. Krauklis, A. and Draveniece, A.: Landscape seasons and air mass dynamics in Latvia. Folia Geographica 12, 16–47, 2004. Reviews, 44, 51-59, doi: 10.1016/j.quascirev.2010.01.019, 2012. 315 Kisand, V., Talas, L., Kisand, A., Stivrins, N., Reitalu, T. et al.: From microbial eukaryotes to metazoan vertebrates: Wide spectrum paleo-diversity in sedimentary ancient DNA over the last ~14,500 years, Geobiology, 16, 628– 639 doi: 10.1111/gbi.12307, 2018. Krauklis, A. and Draveniece, A.: Landscape seasons and air mass dynamics in Latvia. Folia Geographica 12, 16–47, 2004. 10 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. Kürschner, W.M.: The anatomical diversity of recent and fossil leaves of the durmast oak (Quercus petraea Lieblein/Q. References and Kubin, E.: Timing of plant phenophases in Finnish Lapland in 1997-2006, Boreal Environment Research, 13, 31-43, 2008. Rasmussen, S.O., Bigler, M., P. Blockley,S.P., Blunier, T., Buchardt, S.L. et al.: A stratigraphic framework for abrupt climatic changes during the Last Glacial period based on three synchronized Greenland ice-core records: refining and extending the INTIMATE event stratigraphy, Quaternary Science Reviews, 106, 14-28, doi: 10.1016/j.quascirev.2014.09.007, 2014. 335 INTIMATE event stratigraphy, Quaternary Science Reviews, 106, 14-28, doi: 10.1016/j.quascirev.2014.09.007, 2014. 335 Reimer, P.J., Bard, E., Bayliss, A., Beck, J.W., Blackwell, P.G., Bronk Ramsey, C., Grootes, P.M., Guilderson, T.P., Haflidason, H., Hajdas, I., Hatté, C., Heaton, T.J., Hoffmann, D.L., Hogg, A.G., Hughen, K.A., Kaiser, K.F., Kromer, B., Manning, S.W., Niu, M., Reimer, R.W., Richards, D. A., Scott, E.M., Southon, J.R., Staff, R.A., Turney, C.S.M., van der Plicht, J.: IntCal13 and Marine13 Radiocarbon Age Calibration Curves 0-50,000 Years cal BP. Radiocarbon 55(4), 1869−1887, 2013. 340 Riigi ilmateenistus (2021) Kliimakaardid, https://www.ilmateenistus.ee/kliima/kliimakaardid/ last accessed 21.09.2021. Rosentau, A., Vassiljev, J. and Hang, T.: Development of the Baltic Ice Lake in the eastern Baltic, Quaternary International, 206, 16-23, doi: 10.1016/j.quaint.2008.10.005, 2009. Salminen, H. and Jalkanen, R.: Modeling of bud break of Scots pine in northern Finland in 1908–2014, Front. Plant Sci., 6, 021) Kliimakaardid, https://www.ilmateenistus.ee/kliima/kliimakaardid/ last accessed 21.09.2021. Rosentau, A., Vassiljev, J. and Hang, T.: Development of the Baltic Ice Lake in the eastern Baltic, Quaternary International, 206, 16-23, doi: 10.1016/j.quaint.2008.10.005, 2009. Salminen, H. and Jalkanen, R.: Modeling of bud break of Scots pine in northern Finland in 1908–2014, Front. Plant Sci., 6, 104, doi:10.3389/fpls.2015.00104, 2015. 345 104, doi:10.3389/fpls.2015.00104, 2015. 345 Schenk, F., Väliranta, M., Muschitiello, F. Tarasov, L., Heikkilä, M., Björck, S., Brandefelt J., Johansson, A.V., Näslund, J- O. and Wohlfarth, B.: Warm summers during the Younger Dryas cold reversal. Nat Commun 9, 1634 https://doi.org/10.1038/s41467-018-04071-5, 2018. Stančikaitė, M., Kisielienė, D., Moe, D. and Vaikutienė, G.: Lateglacial and early Holocene environmental changes in Schenk, F., Väliranta, M., Muschitiello, F. Tarasov, L., Heikkilä, M., Björck, S., Brandefelt J., Johansson, A.V., Näslund, J- O. and Wohlfarth, B.: Warm summers during the Younger Dryas cold reversal. Nat Commun 9, 1634 https://doi.org/10.1038/s41467-018-04071-5, 2018. Stančikaitė, M., Kisielienė, D., Moe, D. and Vaikutienė, G.: Lateglacial and early Holocene environmental changes in northeastern Lithuania, Quaternary International, 207, 80-92, doi: 10.1016/j.quaint.2008.10.009, 2009. 350 northeastern Lithuania, Quaternary International, 207, 80-92, doi: 10.1016/j.quaint.2008.10.009, 2009. 350 Steinthorsdottir, M. and Wagner-Cremer, F.: Hot summers ahead? References 320 pseudocastanea Goeppert) — implications for their use as biosensors of palaeoatmospheric CO2 levels, Review of Palaeobotany and Palynology, 96, 1-30, doi: 10.1016/S0034-6667(96)00051-6, 1997. Menzel, A., Sparks, T.H., Estrella, N., Koch, E., Aasa, A., Ahas, R., et al.: European phenological response to climate change matches the warming patter, Global Change Biology, 12, 1969-1976, doi: 10.1111/j.1365-2486.2006.01193.x, 2006. Kürschner, W.M.: The anatomical diversity of recent and fossil leaves of the durmast oak (Quercus petraea Lieblein/Q. 320 pseudocastanea Goeppert) — implications for their use as biosensors of palaeoatmospheric CO2 levels, Review of Palaeobotany and Palynology, 96, 1-30, doi: 10.1016/S0034-6667(96)00051-6, 1997. Menzel, A., Sparks, T.H., Estrella, N., Koch, E., Aasa, A., Ahas, R., et al.: European phenological response to climate change matches the warming patter, Global Change Biology, 12, 1969-1976, doi: 10.1111/j.1365-2486.2006.01193.x, 2006. Menzel, A., Sparks, T.H., Estrella, N., Koch, E., Aasa, A., Ahas, R., et al.: European phenological response to climate change matches the warming patter, Global Change Biology, 12, 1969-1976, doi: 10.1111/j.1365-2486.2006.01193.x, 2006. Menzel, A., Yuan, Y., Matiu, M. et al: Climate change fingerprints in recent European plant phenology. Glob Change Biol., 325 26, 2599– 2612, doi:10.1111/gcb.15000, 2020. Odland, A.: Differences in the vertical distribution pattern of Betula pubescens in Norway and its ecological significance, in: Holocene treeline oscillations, dendrochronology and palaeoclimate, edited by: Frenzel, B., Stuttgart, Gustav Fischer, 43-59, 1996. Menzel, A., Yuan, Y., Matiu, M. et al: Climate change fingerprints in recent European plant phenology. Glob Change Biol., 325 26, 2599– 2612, doi:10.1111/gcb.15000, 2020. Odland, A.: Differences in the vertical distribution pattern of Betula pubescens in Norway and its ecological significance, in: Holocene treeline oscillations, dendrochronology and palaeoclimate, edited by: Frenzel, B., Stuttgart, Gustav Fischer, 43-59, 1996. Odland, A.: Differences in the vertical distribution pattern of Betula pubescens in Norway and its ecological significance, in: Holocene treeline oscillations, dendrochronology and palaeoclimate, edited by: Frenzel, B., Stuttgart, Gustav Fischer, 43-59, 1996. Peñuelas, J. and Filella, I.: Responses to a Warming World, Science, 294, 793-795, doi: 10.1126/science.1066860, 2001. 330 Pudas, E., Tolvanen, A., Poikolainen, J., Sukuvaara, T. and Kubin, E.: Timing of plant phenophases in Finnish Lapland in 1997-2006, Boreal Environment Research, 13, 31-43, 2008. Rasmussen, S.O., Bigler, M., P. Blockley,S.P., Blunier, T., Buchardt, S.L. et al.: A stratigraphic framework for abrupt climatic changes during the Last Glacial period based on three synchronized Greenland ice-core records: refining and extending the Pudas, E., Tolvanen, A., Poikolainen, J., Sukuvaara, T. References Multi-decadal spring season warming precedes sudden summer temperature rise in preanthropogenic climate change, GFF, 141, 175-180, doi: 10.1080/11035897.2019.1655791, 2019. 11 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. Stivrins, N., Kołaczek, P., Reitalu, T., Seppä, H. and Veski, S.: Phytoplankton response to the environmental and climatic , , , , , , pp , , y p p variability in a temperate lake over the last 14,500 years in eastern Latvia, Journal of Paleolimnology, 54, 103 – 119, doi: 355 10.1007/s10933-015-9840-8, 2015. Šeirienė, V., Gastevičienė, N., Luoto, T.P., Gedminienė, L. and Stančikaitė, M.: The Lateglacial and early Holocene climate variability and vegetation dynamics derived from chironomid and pollen records of Lieporiai palaeolake, North Lithuania, Quaternary International, doi:10.1016/j.quaint.2020.12.017, 2020. variability in a temperate lake over the last 14,500 years in eastern Latvia, Journal of Paleolimnology, 54, 103 – 119, doi: 355 10.1007/s10933-015-9840-8, 2015. Šeirienė, V., Gastevičienė, N., Luoto, T.P., Gedminienė, L. and Stančikaitė, M.: The Lateglacial and early Holocene climate variability and vegetation dynamics derived from chironomid and pollen records of Lieporiai palaeolake, North Lithuania, Quaternary International, doi:10.1016/j.quaint.2020.12.017, 2020. Wagner-Cremer, F. and Lotter, A.F.: Spring-season changes during the Late Pleniglacial and Bølling/Allerød interstadial, 360 Quaternary Science Reviews, 30, 1825-1828, doi: 10.1016/j.quascirev.2011.05.003, 2011. Wagner-Cremer, F., Finsinger, W. and Moberg, A.: Tracing growing degree-day changes in the cuticle morphology of Betula nana leaves: a new micro-phenological palaeo-proxy, J. Quaternary Sci., 25, 1008-1017 doi: 10.1002/jqs.1388, 2010. Vassiljev, J. and Saarse, L.: Timing of the Baltic Ice Lake in the eastern Baltic, Bulletin of the Geological Society of Finland, Wagner-Cremer, F. and Lotter, A.F.: Spring-season changes during the Late Pleniglacial and Bølling/Allerød interstadial, 360 Quaternary Science Reviews, 30, 1825-1828, doi: 10.1016/j.quascirev.2011.05.003, 2011. Wagner-Cremer, F., Finsinger, W. and Moberg, A.: Tracing growing degree-day changes in the cuticle morphology of Betula nana leaves: a new micro-phenological palaeo-proxy, J. Quaternary Sci., 25, 1008-1017 doi: 10.1002/jqs.1388, 2010. Vassiljev, J. and Saarse, L.: Timing of the Baltic Ice Lake in the eastern Baltic, Bulletin of the Geological Society of Finland, 85, 9-18, doi:10.17741/bgsf/85.1.001, 2013. 365 85, 9-18, doi:10.17741/bgsf/85.1.001, 2013. 365 Wang, C., Cao, R., Chen, J., Rao, Y. and Tang, Y.: Temperature sensitivity of spring vegetation phenology correlates to within- spring warming speed over the Northern Hemisphere, Ecological Indicators, 50, 62-68, doi:10.1016/j.ecolind.2014.11.004, 2015. 380 Figure 1: A. The location of the study sites B. The position of the Scandinavian Ice Sheet, the shoreline of the Baltic Ice Lake and the vegetation zones characterised by dominant plant macroremains ~13300 cal yr BP. The macrofossil data and chronologies for Lake L. Svetinu were published in Veski et al., 2012; for Kosilase the dates are presented in the present paper, plant macrofossil data is unpublished. The chronologies and plant macrofossil data for the other study sites on the map: 1) Nakri (Amon et al., 2012), 2) Prossa (Amon et al., 2014), 3) Kursi (Amon et al., 2016), 4) Udriku (Amon and Saarse 385 2010). The shoreline reconstruction of the Baltic Ice Lake is modified from Vassiljev and Saarse 2013. Figure 2: The results of the undulation index (UI) measurements. The sample cloud represents the measurements, and the dots display the average values per sample. Two sample photos of a Betula nana subfossil leaf and cuticle. References Veski, S., Amon, L., Heinsalu, A., Reitalu, T., Saarse, L., Stivrins, N. and Vassiljev, J.: Lateglacial vegetation dynamics in the eastern Baltic region between 14,500 and 11,400 cal yr BP: A complete record since the Bølling (GI-1e) to the Holocene, 370 Quatern Sci Rev, 40, 39–53, doi: 10.1016/j.quascirev.2012.02.013, 2012. Veski, S., Amon, L., Heinsalu, A., Reitalu, T., Saarse, L., Stivrins, N. and Vassiljev, J.: Lateglacial vegetation dynamics in the eastern Baltic region between 14,500 and 11,400 calyrBP: A complete record since the Bølling (GI-1e) to the Holocene, Quaternary Science Reviews, 40, 39-53. doi: 10.1016/j.quascirev.2012.02.013, 2012. Veski, S., Seppä, H., Stančikaitė, M., Zernitskaya, V., Reitalu, T., Gryguc, G., Heinsalu, A., Stivrins, N., Amon, L., Vassiljev, 375 J. and Heiri, O.: Quantitative summer and winter temperature reconstructions from pollen and chironomid data between 15 and 8 ka BP in the Baltic–Belarus area, Quaternary International, 388, 4-11, doi: 10.1016/j.quaint.2014.10.059, 2015. 380 Figure 1: A. The location of the study sites B. The position of the Scandinavian Ice Sheet, the shoreline of the Baltic Ice Lake and the vegetation zones characterised by dominant plant macroremains ~13300 cal yr BP. The macrofossil data and chronologies for Lake L. Svetinu were published in Veski et al., 2012; for Kosilase the dates are presented in the present paper, plant macrofossil data is unpublished. The chronologies and plant macrofossil data for the other study sites on the map: 1) Nakri (Amon et al., 2012), 2) Prossa (Amon et al., 2014), 3) Kursi (Amon et al., 2016), 4) Udriku (Amon and Saarse 385 2010). The shoreline reconstruction of the Baltic Ice Lake is modified from Vassiljev and Saarse 2013. Figure 2: The results of the undulation index (UI) measurements. The sample cloud represents the measurements, and the dots display the average values per sample. Two sample photos of a Betula nana subfossil leaf and cuticle. Figure 2: The results of the undulation index (UI) measurements. The sample cloud represents the measurements, and the dots display the average values per sample. Two sample photos of a Betula nana subfossil leaf and cuticle. 12 Figure 3: The compilation of Lateglacial vegetation and climate proxies from the study region: selected plant macrofossil records of Lakes L. Svetinu (L.SV) (Veski et al., 2012) and Kosilase (KOS); pollen-based MJJA & DJF temperature estimations of Lake L. Svetinu (Veski et al. 2015), chironomid-based July temperature estimation of Lake Nakri (location No 1 on Fig3) (Heiri et al., 2014), the spring onset and growth-degree-days dynamics. https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. Figure 3: The compilation of Lateglacial vegetation and climate proxies from the study region: selected plant macrofossil records of Lakes L. Svetinu (L.SV) (Veski et al., 2012) and Kosilase (KOS); pollen-based MJJA & DJF temperature estimations of Lake L. Svetinu (Veski et al. 2015), chironomid-based July temperature estimation of Lake Nakri (location No 1 on Fig3) (Heiri et al., 2014), the spring onset and growth-degree-days dynamics. Figure 3: The compilation of Lateglacial vegetation and climate proxies from the study region: selected plant macrofossil records of Lakes L. Svetinu (L.SV) (Veski et al., 2012) and Kosilase (KOS); pollen-based MJJA & DJF temperature estimations of Lake L. Svetinu (Veski et al. 2015), chironomid-based July temperature estimation of Lake Nakri (location No 1 on Fig3) (Heiri et al., 2014), the spring onset and growth-degree-days dynamics. 390 395 13 14 14 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. 400 15 15 https://doi.org/10.5194/cp-2021-133 Preprint. Discussion started: 20 October 2021 c⃝Author(s) 2021. CC BY 4.0 License. 16 16
https://openalex.org/W2904796739	https://www.revistas.usp.br/anaismp/article/download/151039/147925	Portuguese	null	A poeira do progresso pede passagem: imagens de natureza e desenvolvimento na floresta amazônica	Anais do Museu Paulista	2,018	cc-by	7,620	1. Pesquisador e docente do Programa de Pós-Graduação em História das Ciências e da Saúde pela Casa de Oswaldo Cruz (FioCruz). Mestre e Doutor em História das Ciências e da Saúde pela mesma instituição. E-mail: romulopa@hotmail.com A poeira do progresso pede passagem: imagens de natureza e desenvolvimento na floresta amazônica Dust of progress calls for passage: nature and development images in the Amazon Rainforest ANAIS DO MUSEU PAULISTA São Paulo, Nova Série, vol. 26, 2018, p. 1-22. e14 KEYWORDS: Development. Press History. Amazon History. RÔMULO DE PAULA ANDRADE1 Fundação Oswaldo Cruz / Rio de Janeiro, RJ, Brasil RESUMO: Entre 1958 e 1960 foi construído o principal projeto do governo de Juscelino Ku bitschek para a região amazônica – a Transbrasiliana, ou rodovia Belém-Brasília. Contando com a abertura de mais de 2 mil quilômetros de estrada em meio à floresta, a estrada foi alvo de diversas críticas por parte de parlamentares e da imprensa de oposição. Diante dessa questão, os meios de comunicação favoráveis ao governo Kubitschek foram acionados para a produção de matérias favoráveis ao empreendimento. Este artigo tem como principal obje tivo analisar as imagens produzidas por essas reportagens. Por meio delas é possível trazer à luz as relações entre os projetos de desenvolvimento daquele período e a natureza, a maior impactada por eles. Além disso, o artigo tem objetivo de analisar qual era o lugar da região amazônica nas prioridades do governo Kubitschek. PALAVRAS-CHAVE: Desenvolvimento. História da imprensa. Amazônia. ANAIS DO MUSEU PAULISTA São Paulo, Nova Série, vol. 26, 2018, p. 1-22. e14 ABSTRACT: Between 1958 and 1960, the main project of the government of Juscelino Ku bitschek for the Amazon region was built: the Transbrasiliana, or Belém-Brasília Highway. With the opening of more than two thousand kilometers of road in the middle of the Amazon rainforest, the project was criticized by opposition media. Faced with this question, the press in favor of the Kubitschek government was activated to produce materials favorable to the enterprise. This article aims to analyze the images produced by these reports. Through them, it is also possible to analyze the relationships between the development projects of this period and the nature, as well as how the Amazon region was seen by the Kubitschek government. INTRODUÇÃO INTRODUÇÃO O objetivo deste artigo é analisar as imagens produzidas por reportagens que cobriram a construção do maior empreendimento voltado para a região amazônica durante os anos de 1946 a 1964: a construção da Transbrasiliana, ou rodovia Belém- Brasília. Por meio delas é possível compreender o lugar da natureza e da própria região amazônica nos grandes projetos de desenvolvimento do Brasil do pós-Segunda Guerra Mundial. Entre as memórias do governo de Juscelino Kubistchek, a construção da nova capital, Brasília, é a mais lembrada. Porém, junto com a nova capital foi criado também o “cruzeiro rodoviário”, com o objetivo de “integrar o país”, como afirmava a propaganda governamental da época. Entre as estradas construídas, o projeto mais ousado foi a Belém-Brasília, que previa a abertura de mais de 2 mil quilômetros por dentro da floresta amazônica, passando por três estados (Maranhão, Pará e Goiás) e chegando, por fim, à nova capital. Além de ambiciosa, a construção da estrada foi também um projeto polêmico, com diversas denúncias por parte de jornais opositores, bem como reportagens laudatórias produzidas pelos meios de comunicação que apoiavam Juscelino. Somado a isso, a região amazônica passava por uma série de diferentes projetos e perspectivas de mudança, com a Superintendência de Valorização Econômica da Amazônia (SPVEA), primeira agência de desenvolvimento regional do país. As fotografias produzidas e divulgadas pela imprensa do período 2 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 2. Hardman (2005, p. 35). 3. Hardman (2005, p. 150). 4. Borges (2011, p. 31). 5. Cf. Becker (2004). representam uma oportunidade de compreender a articulação entre as perspectivas de natureza por parte do governo, bem como o posicionamento dos meios de comunicação perante os grandes projetos de desenvolvimento. O contexto de produção das imagens é o primeiro assunto a ser tratado no artigo. Por meio delas é possível identificar os aspectos do que seria a “modernidade” propagandeada pelo governo de Juscelino Kubitschek, além da construção de estradas e da relação entre o então presidente e a imprensa da época. 2. Hardman (2005, p. 35). 3. Hardman (2005, p. 150). 4. Borges (2011, p. 31). 5. Cf. Becker (2004). ANNALS OF MUSEU PAULISTA – vol. 26, 2018. INTRODUÇÃO O último projeto voltado para a região amazônica de tamanho vulto e a causar tanto impacto em escritos locais e nacionais tinha sido a construção da ferrovia Madeira-Mamoré no início do século XX que, nas palavras de Francisco Foot Hardman, foi o “espetáculo privilegiado da civilização capitalista na selva”.2 A indústria das estradas de ferro e sua internacionalização representaram, durante a segunda metade do século XIX, um dos fatores básicos para a articulação do mercado mundial.3 Como aponta Borges, a implantação das estradas de ferro no Brasil significou muito mais que uma simples inovação nos meios de transporte e comunicação; foi na verdade o marco de uma mudança na organização econômica: do sistema mercantil-escravista para o modo capitalista de produção.4 Com um contexto completamente diferente, as estradas passaram a representar o “moderno” em contraposição ao “atraso” das ferrovias, muitas desmanteladas e substituídas a partir dos anos 1950. Qual seria o impacto da construção da Belém-Brasília para uma região amazônica vista nas décadas de 1950 e 1960 como “subdesenvolvida” e, na palavra de parlamentares daqueles anos, “atrasada”? Se em fins de século XIX e início do XX a ferrovia era o signo de modernidade e uma possível entrada da Amazônia em determinada “civilização”, nos anos JK a abertura de uma estrada no meio da floresta amazônica seria a oportunidade ideal de retirar a região de estigmas históricos e recorrentes, como o seu “isolamento”, superado pela “integração” promovida pelo cruzeiro rodoviário. Bertha Becker caracteriza esse período como “fase do planejamento regional” da história da Amazônia, que se situa entre 1930 e 1985, sendo reflexo do nacional- desenvolvimentismo vigente, marcado pelo planejamento governamental (mesmo que com poucas ações efetivas), pela modernização de instituições estatais e pela crescente intervenção pública na economia e no território.5 A natureza e a floresta amazônica seriam impedimentos a esse processo; autênticas vilãs que deveriam ser superadas para dar lugar ao progresso e à modernidade, representados pelas máquinas Caterpillar e pelos carros, símbolos do rodoviarismo juscelinista. Analisando as diversas escolas críticas do realismo fotográfico (a ideia de que há uma simples transposição da realidade para a imagem), Mauad destaca que um dos passos para uma perspectiva nessa chave é compreender que entre o objeto e sua representação fotográfica existe uma série de ações convencionadas, 3 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. 6. Mauad (2008, p. 33). 7. Cf. Lacerda (2012). 6. Mauad (2008, p. 33). INTRODUÇÃO cultural e historicamente. Dessa forma, a fotografia em si é uma escolha entre tantas outras possíveis, uma atitude estreita com a visão de mundo do indivíduo responsável pelo “clic”.6 Dessa forma, as máquinas aqui apresentadas são signos de uma ideia de modernidade calcada nos padrões urbano-industriais, relacionando-se ao intenso e dinâmico mercado editorial do período, com o advento de revistas como O Cruzeiro e Manchete. Um reflexo disso é o advento das fotorreportagens, cujas imagens tinham a intenção de destacar os “50 anos em 5” de forma tangível em todos os lugares, até mesmo em terras distantes das sedes dessas revistas e de seus públicos leitores. 7. Cf. Lacerda (2012). Em trabalho sobre o lugar da fotografia nos arquivos, Aline Lacerda destaca que os registros fotográficos são vistos de forma autorreferente, sem ligação evidente com o restante do arquivo ou de sua entidade produtora.7 Dessa forma, como perspectiva metodológica o artigo pretende inserir as imagens, na medida do possível, em seus contextos originais de (re)produção, contando com as legendas que já estavam no suporte. Tais indícios colaboram para uma tentativa de constituir, mesmo que de forma parcial e seletiva, possíveis intenções dos criadores dessas imagens, cujos critérios passavam por escolhas editoriais e pelos objetivos das próprias reportagens. As imagens aqui destacadas foram produzidas a partir das zonas de contato dessas dimensões. Um tema que percorrerá o texto é a oposição, nas fotografias e nas legendas que as acompanham, entre os humanos e a natureza. Vista como vilã de um processo teleológico cujo rumo inexorável seria uma “civilização” aos moldes urbano-industriais, a natureza foi associada a um passado que deveria ser esquecido, suplantado pela estrada, o “novo”. Para a compreensão desses registros, dois pontos serão centrais no artigo: a contextualização das revistas em que as fotos estão inseridas e as percepções sobre a natureza que esses escritos trazem. Inicialmente as (boas) relações estabelecidas entre Juscelino Kubitschek e a imprensa serão privilegiadas. Outro ponto relevante deste texto é demonstrar a importância que a propaganda política tinha (e tem) também em tempos democráticos, ainda que a historiografia, de forma geral, se concentre mais em períodos ditatórias. As redes estabelecidas por JK com entes públicos e privados são uma pequena amostra da complexidade desse período. 6. Mauad (2008, p. 33). 7. Cf. Lacerda (2012). MODERNIDADE E IMPRENSA NOS ANOS JK Quando se analisam os primeiros artigos sobre a figura de Juscelino Kubitschek na imprensa (à época de sua candidatura e vitória), comparados aos do período da construção da nova capital e da estrada, é perceptível a mudança de postura de alguns veículos, bem como a radicalização de outros. Neste artigo nos dedicamos 4 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 8. Cf. Biroli (2004). 9. Cf. Bizello (1995). 10. Cf. Moreira (2003). especialmente aos favoráveis à construção da BR-14, mas breves notas sobre a imprensa durante o governo JK são necessárias para o andamento do trabalho. A Tribuna da Imprensa, dirigida pelo udenista Carlos Lacerda, manteve-se coerente durante todo o governo Juscelino Kubitschek, ao qual sempre se apresentou contrária, desde o início até os grandes projetos do período. A candidatura de JK, então governador de Minas Gerais, foi contestada pela Tribuna, além do Diário Carioca e do Correio da Manhã. Estes dois últimos posteriormente seriam favoráveis ao governante, enquanto o Tribuna continuaria firme em suas críticas ao “presidente varguista”, nas palavras de Carlos Lacerda.8 Eram tempos democráticos e de convívio (sempre tenso) com o contraditório, porém Juscelino possuía bom suporte de grande parte dos meios de comunicação naqueles anos, destacando-se os jornais e as rádios pertencentes a Assis Chateaubriand, o grupo Globo de Roberto Marinho e, enfim, a revista Manchete, do grupo Bloch.9 É possível apontar um desgaste nessa relação no término do governo, com as crescentes denúncias de corrupção dos grandes projetos desenvolvimentistas. Porém, na maior parte dos anos a boa relação do governante com a imprensa possibilitou maior divulgação dos atos da presidência no período, transformando as reportagens, em alguns momentos, quase em uma extensão da propaganda oficial. Um exemplo é a publicação, por parte da editora SPVEA (pertencente ao governo), de artigos de O Globo sobre a Belém-Brasília. Outras reportagens também foram compiladas em livros da mesma editora, dando origem ao curto periódico Cadernos Belém-Brasília, com a publicação de textos do jornal Última Hora e da Manchete. A palavra de ordem nos jornais apoiadores do governo era o confronto entre os dois “Brasis”, onde o “moderno” superaria o “arcaico”, e a ferramenta para isso seria o desenvolvimentismo e os grandes projetos de Juscelino.10 10. Cf. Moreira (2003). A rodovia Belém-Brasília foi um exemplo da divulgação do desenvolvimentismo juscelinista pelos apoiadores do governo. Reverberações de antigos clichês sobre a região amazônica encontram eco nessas reportagens. MODERNIDADE E IMPRENSA NOS ANOS JK Termos como “vazio demográfico” e “natureza perigosa” são bastante recorrentes nos textos. Diante da dificuldade de construir uma estrada em meio à floresta, foram dados contornos épicos à empreitada. Um exemplo é o livro do jornalista Lauro de Azevedo Rolim, Transbrasiliana-um poema brasilista, onde a BR-14 e os profissionais que a idealizaram e construíram se transformam em personagens de uma epopeia, inseridos em um combate entre humano e natureza, do qual o primeiro sairia ganhando, graças às “máquinas do progresso”: Entre a máquina e a selva, o duelo tremendo a que empresta o machado um papel decisivo, abatendo, implacável, os gigantes da mata! Ao furor combativo toda a selva estremece. Entre a máquina e a selva, o duelo tremendo a que empresta o machado um papel decisivo, abatendo, implacável, os gigantes da mata! Ao furor combativo toda a selva estremece. 5 5 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. 11. Rolim (1960, p. 14). 12. Cf. Martins (2007). 13. Pádua (2012, p. 24). 14. Cf. Pádua (2002). 15. Cf. Kossoy (2002). 16. Barthes (2002, p. 132). A fauna, amedrontada, se embrenha Mas na face da terra encantada e ubertosa vai ficando o perfil retilíneo da estrada!11 A natureza, nas palavras desses escritos, era vista como a grande vilã do progresso. É importante, inicialmente, tomar como referência a observação de Martins segundo a qual, em relação à natureza, não existem somente interesses.12 Em qualquer sociedade, a natureza é fonte de valores e representações intrincados, complexos, contraditórios, que nutrem artes, religiões, mitos e saberes. A natureza seria, assim, uma construção cultural, concretizada nas concepções de mundo, sempre com a ideia de oposição ao outro, conforme José Augusto Pádua assinala: 16. Barthes (2002, p. 132). De um lado, a ideia de natureza serve como uma espécie de eixo conceitual que dá sentido ao nosso entendimento do universo. Ela fundamenta a construção conceitual que dá sentido ao nosso entendimento do universo. Ela fundamenta a construção conceitual da experiência de que existe coerência ontológica no mundo em que vivemos. Por sua vez, a imagem de ser humano e de história humana se construiu em grande parte por oposição à natureza: arte versus natureza; ordem social versus natureza; técnica versus natureza; espírito versus natureza etc. MODERNIDADE E IMPRENSA NOS ANOS JK Em outras palavras, um conjunto de oposições que procuram demarcar, por diferenciação ou por identificação, a especificidade do fenômeno humano em relação à natureza.13 Estamos nos referindo aqui a um constructo cultural específico, fruto de prefigurações seculares sobre a região amazônica, que de certa forma radicalizou os discursos anteriores sobre o local da natureza nos projetos governamentais. As alternativas a esse discurso de “destruição” no país datam do período colonial, por mais que as preocupações não fossem necessariamente conservacionistas, para utilizar um termo contemporâneo.14 A construção da Belém-Brasília gerou fotografias de forte caráter simbólico. A produção da obra fotográfica diz respeito ao conjunto de mecanismos internos do processo de construção da representação, concebido conforme certa intenção, construído e materializado estética, ideológica e tecnicamente.15 Essas fotografias se relacionam a uma tradição nacional e internacional de promover uma fixação de memória e, neste caso, se prestam a uma finalidade propagandística, financiada por instituições oficiais interessadas em divulgar certo tipo de progresso. Assim, essas fotos, como afirma Barthes, não seriam meras “cópias” do real, mas sim emanações, trechos, vestígios de um passado já vivido. Não seriam meras reproduções.16 Tais imagens trazem as concepções de mundo dos seus autores e do contexto em que foram produzidas. Na Figura 1 observamos o presidente e o ministro Lúcio Meira em um momento de descanso das obras. Fotografar o presidente no campo onde estariam 6 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ocorrendo as obras, assim como dar destaque ao aperto de mão entre o presidente e os moradores locais (Figura 5), é colocar em cena o principal ator político do projeto, comprometido com as possíveis mudanças que a obra traria. Figura 1 – Jusce Kubitschek e L Meira. Fotógrafo identificado, 19 Fonte: BRASIL. O nejamento transfo a Amazônia. Rio Janeiro: SPVEA, 19 Figura 1 – Juscelino Kubitschek e Lúcio Meira. Fotógrafo não identificado, 1960. Fonte: BRASIL. O pla nejamento transforma a Amazônia. Rio de Janeiro: SPVEA, 1960. Para compreender a mensagem implícita nas fotografias é necessário cotejá- las com os discursos do período, buscando verificar com que substrato do real estamos nos defrontando. Uma possível interpretação se encontra na análise da fala de JK sobre a natureza, na qual ele reafirma as concepções presentes nas peças publicitárias da época da construção e inauguração da BR-14. Realmente alguns episódios dos dois anos do projeto ajudaram a dar os contornos épicos pretendidos pelo governo JK. MODERNIDADE E IMPRENSA NOS ANOS JK No trecho final da estrada alguns acontecimentos 7 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. 17. O episódio da morte de Bernardo Sayão deu origem a uma série de comoções. O engenheiro inaugurou o cemitério de Brasília e teve a memória bastante laurea da, com a publicação de biografias e textos comemo rativos. Para mais informa ções, cf. Silva (2009). 17. O episódio da morte de Bernardo Sayão deu origem a uma série de comoções. O engenheiro inaugurou o cemitério de Brasília e teve a memória bastante laurea da, com a publicação de biografias e textos comemo rativos. Para mais informa ções, cf. Silva (2009). teriam papel fundamental na memória construída sobre a rodovia: a morte de Rui de Almeida, engenheiro responsável pelo desmatamento da frente de Brasília, e principalmente a morte de Bernardo Sayão. O engenheiro, às vésperas do encontro em Ligação (nome criado para homenagear a conexão das duas vias), encontrava- se em uma espreguiçadeira após o almoço, quando o galho de uma árvore, isolada após o desmatamento, se partiu e o atingiu. Sua morte representou a criação do mito em torno do engenheiro, tido como bandeirante moderno pela propaganda presidencial.17 Alguns meses após a morte de Sayão, ocorreu o evento que ligaria os trechos da rodovia, em especial dois: o que viera de Belém e o que tinha origem em Brasília. O ato simbólico que daria concretude a essa definitiva conexão seria a derrubada de um jatobá pelo próprio presidente (Figura 2). A forma como o acontecimento é narrado por Juscelino expõe o papel daquela árvore em particular (e da mata em geral) no seu projeto de governo: impedindo a ligação […] [lá estava] a árvore imensa. Media-a com os olhos. O caule projetava-se contra o céu quase sem galhos e abria-se, lá em cima, a fronde majestosa. […] o que parecia impossível estava acontecendo. […] Num canto, via-se um trator amare lo. Era a arma de que me utilizaria para a batalha contra o último guerreiro. […] sentia-me orgulhoso da tarefa que me fora reservada. Dera a ordem para derrubar a primeira árvore e eu próprio iria fazer a última. […] ouviu-se um estrondo subterrâneo das raízes que se desprendiam. […] O último tamoio caíra, e, com sua morte, desfizera-se a superstição da inviolabilidade da selva.18 Figura 2 – Juscelino Kubitschek e diplomatas de diversos países na construção da Belém-Brasília. 18. Kubitschek (2000, p. 232). 17. O episódio da morte de Bernardo Sayão deu origem a uma série de comoções. O engenheiro inaugurou o cemitério de Brasília e teve a memória bastante laurea da, com a publicação de biografias e textos comemo rativos. Para mais informa ções, cf. Silva (2009). 19. Cf. Worster (1991). 19. Cf. Worster (1991). 20. Pierre (1960, p. 27). 20. Pierre (1960, p. 27). MODERNIDADE E IMPRENSA NOS ANOS JK Fotó grafo não identificado, 1960. Fonte: BRASIL. O planejamento transforma a Amazônia. Rio de Janeiro: SPVEA, 1960. Figura 2 – Juscelino Kubitschek e diplomatas de diversos países na construção da Belém-Brasília. Fotó grafo não identificado, 1960. Fonte: BRASIL. O planejamento transforma a Amazônia. Rio de Janeiro: SPVEA, 1960. 8 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 19. Cf. Worster (1991). 20. Pierre (1960, p. 27). Ora, a natureza não é uma ideia, mas muitas ideias, significados, pensamentos e sentimentos, empilhados uns sobre os outros, frequentemente da forma menos sistemática possível.19 Seria, assim, uma criação da mente humana, e esta, por mais que se esforce para ver o que a natureza é objetivamente, acaba prendendo o olhar nas grades da própria consciência e na rede de significados humanos. Por isso é imprescindível descobrir como uma cultura de determinada época avaliou e representou a natureza e sua paisagem característica. Se por um lado a floresta significa perigo, por outro traz ideias de deslumbramento diante de seu tamanho. Em reportagem sobre a rodovia, que posteriormente seria publicada nos Cadernos Belém-Brasília, Arnaud Pierre, jornalista do Correio da Manhã, afirmou que o que mais o impressionava era a floresta: “a hileia domina de tal modo a todos aqui, principalmente a nós, que tudo observamos para escrever, que se eu não me precatar, ela me descaminha nesta reportagem […] Quero me desvencilhar da floresta de vez”20. 20. Pierre (1960, p. 27). Figura 3 – Bernardo Sayão, Waldir Bouhid e outros na construção da estrada. Fotógrafo não identificado, 1960. Fonte: PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). Figura 3 – Bernardo Sayão, Waldir Bouhid e outros na construção da estrada. Fotógrafo não identificado, 1960. Fonte: PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). 9 9 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. 21. Cf. Corrêa (2013). 22. Cf. Corrêa (2013). 23. Vaitsman (1958, p. 25). 24. Vaitsman (1958, p. 25- 26). Assim como as percepções de natureza, é necessário apontar qual ideia de paisagem esses jornalistas trouxeram para suas reportagens. MODERNIDADE E IMPRENSA NOS ANOS JK Em artigo de 2013, Dora Corrêa fez um balanço historiográfico sobre esse conceito, o que ajuda a descortinar as concepções presentes nessas imagens sobre a Amazônia, vista por tais reportagens como um lugar pronto (e urgente) para a intervenção estatal.21 A partir de um amplo panorama historiográfico, Corrêa destaca três concepções que de certa forma estão presentes na produção das imagens usadas em seu artigo. A primeira analisa a descrição de paisagens de forma ambígua, encarando-a tanto como uma percepção objetiva do real (podendo ser lida sem interferência externa, de forma objetiva) quanto subjetiva, já que a descrição de uma paisagem pode englobar mais que o sentido pelos olhos. A segunda, mais tributária da História Ambiental, entende a paisagem como um conjunto de elementos concretos, sendo ao mesmo tempo a percepção e o percebido. Assim, é uma leitura objetiva do espaço filtrada pela subjetividade. Por fim, inspirada nos estudos de Juan Manuel Gonzalez, há a percepção de que a paisagem é a expressão material de uma ideologia, ou seja, um conjunto de elementos concretos, e que essa ideologia interfere na relação dos humanos com o meio ambiente.22 Dessa forma, é possível destacar que os jornalistas e fotógrafos que produziram essas imagens e reportagens trouxeram consigo concepções históricas sobre a região amazônica que interferiram de forma decisiva na escolha do que clicar ou do que relatar. Maurício Vaitsman, repórter d’O Globo, ressalta em sua reportagem (também transformada em livro) intitulada Brasília e Amazônia que a floresta amazônica durante séculos desafiara o “homem civilizado” com sua “exuberância prodigiosa” e seus “mistérios insondáveis”. Com as máquinas, “espadas do progresso”, os homens estariam descobrindo um “mundo novo”, “desconhecido” e protegido pela “agressividade da natureza”.23 A própria chamada da reportagem afirmava que os trabalhadores estariam diante de um sonho: a violação da selva, uma “barreira para o progresso”, com os pneus dos tratores: 23. Vaitsman (1958, p. 25). 23. Vaitsman (1958, p. 25). A selva amazônica, que desafiava o progresso desde os tempos do Grão-Pará, está sendo finalmente dominada pela máquina e pelo homem. Centenas de quilômetros de modernas rodovias cortam a floresta bruta, transmitindo o fluxo da civilização ao “hinterland” cabo clo, escravizado pelo extrativismo da borracha. Um punhado de homens está mudando a fisionomia da Amazônia para integrá-la na unidade nacional do país.24 De que forma tal discurso se insere nas imagens? 21. Cf. Corrêa (2013). 22. Cf. Corrêa (2013). 23. Vaitsman (1958, p. 25). 24. Vaitsman (1958, p. 25- 26). MODERNIDADE E IMPRENSA NOS ANOS JK Para responder essa pergunta, é necessário conhecer, compreender e interpretar – à luz das evidências históricas, que têm na imagem fotográfica uma das manifestações – os sentidos que os indivíduos, isoladamente ou em grupo, quiseram atribuir 10 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 25. Borges (2008, p. 112). às suas práticas sociais.25 Compreendemos aqui tais imagens relacionadas aos discursos produzidos no período – e não isoladas deles. Dessa forma, as fotografias buscam registrar a floresta dominada pela ação humana. Uma característica comum às três fotos selecionadas é a posição dos personagens perante as árvores (Figuras 1, 2 e 3). De um lado é possível perceber, nas duas primeiras imagens, a representação política do sucesso da obra, com membros do alto escalão governamental, demonstrando o êxito da construção da estrada. Na Figura 3, personagens fundamentais para a obra, como Bernardo Sayão e Lino Teixeira, trazem à luz a questão nacionalista do período, como visto na legenda que os chama de “verdadeiros generais da conquista da Amazônia”. Um dos discursos mais comuns na época (que de certa forma encontra eco nos dias de hoje) é a necessidade de “conquista” da Amazônia para afastá-la da “cobiça internacional”.26 29. Vaitsman (1958, p. 57). Assim, a vitória sobre as condições naturais e o “efetivo domínio” daquelas plagas reforçavam a vitória do Brasil sobre ele mesmo. Rememorando imagens da Marcha para o Oeste, tratava-se do imperialismo brasileiro conquistando a si próprio. A Marcha, programa de colonização criado por Cassiano Ricardo durante o Estado Novo, foi pródiga em ressaltar o “espírito bandeirante” e a “conquista” das terras brasileiras.27 Pouco efetivo, o programa criou ideias-forças que foram apropriadas pelos presidentes tanto do período democrático quanto do período ditatorial pós-1964, em especial na construção da rodovia Transamazônica. Para o historiador Peter Burke, as imagens são testemunhas dos estereótipos (e também das mudanças graduais) pelos quais indivíduos ou grupos veem o mundo social. Dessa forma, as imagens aqui apresentadas representam também a concepção construída por esses agentes políticos e culturais sobre a região amazônica e seu processo de desenvolvimento.28 A Figura 2 não pode ser descolada do texto que a acompanhava. Além do caráter simbólico já referido, a fotografia foi também a celebração do encontro entre o presidente e os embaixadores da Inglaterra, da Alemanha, do Equador e da Tchecoslováquia. A viagem serviria para os representantes de nações amigas do Brasil conhecerem aspectos do país. 26. Um livro símbolo desse ideário é a obra “Amazônia e a Cobiça Internacional”, de Arthur Cézar Ferreira Reis, lançado em 1965. 25. Borges (2008, p. 112). ANNALS OF MUSEU PAULISTA – vol. 26, 2018. 27. Cf. Ricardo (1970); Ve lho (1976); Silva (2009). 25. Borges (2008, p. 112). 26. Um livro símbolo desse ideário é a obra “Amazônia e a Cobiça Internacional”, de Arthur Cézar Ferreira Reis, lançado em 1965. 27. Cf. Ricardo (1970); Ve lho (1976); Silva (2009). 28. Burke (2004, p. 232). 29. Vaitsman (1958, p. 57). 29. Vaitsman (1958, p. 57). 27. Cf. Ricardo (1970); Ve lho (1976); Silva (2009). 25. Borges (2008, p. 112). 30. Cf. Borges (2008). MODERNIDADE E IMPRENSA NOS ANOS JK Assim, JK mostraria a outros países o que seria o desenvolvimento nessas regiões. Na afirmativa de Vaitsman, “quatro embaixadores […] tiveram o privilégio de ver como o homem brasileiro e as máquinas estão afrontando a misteriosa floresta amazônica”.29 Além de possibilitar à comitiva estrangeira conhecer aspectos da cultura local e sua culinária (foi oferecido um banquete em plena picada), esse ato demonstrou a forma como a imagem do país deveria ser levada ao exterior: um lugar que preservava sua cultura tradicional, mas que também possuía características modernas, como demonstrava a construção de estrada. 11 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. 30. Cf. Borges (2008). 31. Vaitsman (1958, p. 50). Retornando ao argumento de Borges, a fotografia pressupõe um jogo de inclusão e exclusão, ou seja, trata-se de escolhas que, como tal, não constituem apenas uma representação do real, mas integram um sistema simbólico pautado por códigos oriundos da cultura que as produz.30 Um aspecto de exclusão notado em todas as fotografias levantadas é a ausência de elementos indígenas. Uma explicação é possível: em grande parte dos textos aqui selecionados eram apontados chistes de membros da oposição ao governo, sugerindo que os jornalistas e políticos seriam atacados por índios e onças. Quando os indígenas surgem nos textos são somente oriundos de “tribos dóceis” ou “devidamente civilizados”, e parte do grupo de mateiros (trabalhadores responsáveis pela abertura das picadas) constituía-se de indígenas “civilizados”.31 No local onde Bernardo Sayão teve sua vida ceifada foi erguida uma cruz, simbolizando e reforçando o mito do “pioneiro” (Figura 4). 31. Vaitsman (1958, p. 50). 31. Vaitsman (1958, p. 50). Figura 4 – Túmulo improvisado de Bernardo Sayão. Fotógrafo não identificado, 1960. PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). p.13 Figura 4 – Túmulo improvisado de Bernardo Sayão. Fotógrafo não identificado, 1960. PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). p.13 Como temos demonstrado, o discurso sobre progresso/desenvolvimento versus natureza baseou-se na antinomia entre esses polos; melhor dizendo, a existência de um teria como pressuposto a inexistência do outro. Rossi tece considerações sobre tal relação: 12 30. Cf. Borges (2008). 31. Vaitsman (1958, p. 50). ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 32. Rossi (2000, p. 96). ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 32. Rossi (2000, p. 96). Essa ideia de progresso que não põe limite às esperanças dos homens, que identifica o progresso como um processo necessário, […] pertence irremediavelmente ao passado, é expressão de um mundo que não é mais o nosso. Nesse mundo, o “sucesso” parece ba seado nas ilimitadas capacidades criativas do homem; a ideia de luta e de conquista se associa ao culto pelo homo faber capaz de domesticar a natureza e de civilizar os povos bárbaros; a sensação de aventura no grande jogo da sociedade e na grande competição entre o homem e a natureza acompanha a fé na continuidade e na eternidade do regnum hominis. A natureza se configura, assim, como uma entidade integralmente dominável.32 O autor ressalta que uma das ideias-forças desse progresso seria identificar, na luta entre humano e natureza, a capacidade de provocar ilimitados melhoramentos e interpretá-la como mais um elemento do progresso. Somada a isso há a percepção sobre as paisagens da Amazônia e as possibilidades, vistas naquele momento histórico como factíveis, de intervenção sobre aquela realidade. Figura 5 – Juscelino Kubitschek cumprimentando populares na construção da estrada. Fotógrafo não identificado, 1960. PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). p.10 Figura 5 – Juscelino Kubitschek cumprimentando populares na construção da estrada. Fotógrafo não identificado, 1960. PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). p.10 Constituem-se assim duas visões sobre a natureza: ela seria simultaneamente objeto de domínio e de reverência; deveria ser “torturada” e “dobrada a serviço do homem”. Não é difícil identificar que uma percepção de vitória rege os objetivos de expor e divulgar as Figuras 6, 8 e 9: máquinas abrindo picadas na mata e a 13 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. 33. Burke (2004, p. 234). abertura de um campo de pouso em meio à floresta amazônica dão o tom épico dessa “luta” narrada pelos meios de comunicação do período. Afinal, se os discursos oficiais do próprio presidente davam conta da intransponibilidade da natureza, nada mais comemorativo que mostrar a primeira clareira aberta na Belém- Brasília. Alguns aspectos da construção da estrada eram realmente impressionantes: várias reportagens descrevem o lançamento de mantimentos e até animais vivos dos helicópteros e aviões para os trabalhadores, como mostra a Figura 7. 34. Kossoy (2001, p. 39). 35. Cf. Vaitsman (1958). 34. Kossoy (2001, p. 39). 35. Cf. Vaitsman (1958). ANAIS DO MUSEU PAULISTA – vol. 26, 2018. As imagens desempenharam, pois, importante papel na “construção cultural” da sociedade, sendo testemunhas dos arranjos sociais e sobretudo das maneiras de ver e pensar o passado.33 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. Figura 6 – Primeira picada na mata. Fotógrafo não iden tificado, 1960. PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). p. 6 Figura 6 – Primeira picada na mata. Fotógrafo não iden tificado, 1960. PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). p. 6 14 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. A fotografia, apesar de conter amplo potencial de informação, não substitui a realidade tal como se deu no passado. Ela apenas traz informações visuais de um fragmento do real, selecionado e organizado estética e ideologicamente.34 Dessa forma, as imagens aqui apresentadas trazem determinada visão sobre a natureza e sua relação com o elemento humano. Em uma primeira leitura, seria possível afirmar que tal concepção constitui-se hegemônica. Porém, em alguns momentos pode-se perceber, mesmo que de forma breve, vozes dissonantes nesse processo. Maurício Vaitsman, em uma de suas reportagens à época da construção da BR-14, apontou vozes contrárias ao desmatamento, que estavam temerosas de que a penetração pela floresta pudesse causar danos irreparáveis, com a exploração indiscriminada das matas.35 Sua posição pessoal, entretanto, era outra. Afirmava também que a colonização do país só poderia ser feita com o sacrifício de boa parte das florestas, e não havia motivos para alarme em excesso com os “pequenos estragos” que a obra teria causado. Figura 7 – Abasteci mento aéreo dos tra balhadores Belém -Brasília. Fotógrafo não identificado, 1959. Fonte: FLO RES, Aluízio. Be lém-Brasília: Man chete assiste à conquista da Ama zônia. Manchete, Rio de Janeiro, ano 10, n. 7, p. 15- 25, 1959. p. 20 15 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. Vaitsman desaconselhou também a adoção de medidas de proteção rigorosas à floresta amazônica, pois “o remédio seria pior do que o mal”.36 Assim, a destruição da floresta seria um sacrifício necessário para o progresso desenvolvimentista. 36. Vaitsman (1958, p. 53). 37. Cf. Vaitsman (1958). 36. Vaitsman (1958, p. 53). 37. Cf. Vaitsman (1958). 38. Cf. Franco (2009). Figura 9 – Abertura de picada na mata. Fotógrafo não identificado, 1959. Fonte: FLORES, Aluízio. Belém-Brasília: Manchete assiste à conquista da Amazônia. Manchete, Rio de Janeiro, ano 10, n. 7, p. 15-25, 1959. p. 20. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. Deixando de lado temporariamente o maniqueísmo que dominava seus escritos – e provavelmente por dever do ofício, já que se tratava do presidente do órgão que publicava suas reportagens – Vaitsman cita também Waldir Bouhid, diretor da SPVEA, que, em palestra aos governadores dos estados pelos quais a rodovia passava, aconselhava que a ocupação das terras próximas à rodovia fosse regulamentada, visando a resguardar as florestas da destruição e permitir uma colonização que pudesse prosperar sem a necessidade de prisão dos mateiros.37 37. Cf. Vaitsman (1958). Figura 8 – Campo de pouso improvisa do no meio da ma ta. Fotógrafo não identificado, 1959. Fonte: FLORES, Alu ízio. Belém-Brasília: Manchete assiste à conquista da Ama zônia. Manchete, Rio de Janeiro, ano 10, n. 7, p. 15-25, 1959. p. 20 Figura 8 – Campo de pouso improvisa do no meio da ma ta. Fotógrafo não identificado, 1959. Fonte: FLORES, Alu ízio. Belém-Brasília: Manchete assiste à conquista da Ama zônia. Manchete, Rio de Janeiro, ano 10, n. 7, p. 15-25, 1959. p. 20 16 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 38. Cf. Franco (2009). 38. Cf. Franco (2009). De todas as fontes analisadas, essa foi a única em que encontramos um diálogo, mesmo que distante, com as concepções de proteção e conservação da natureza veiculadas à época. Não obstante silenciadas por uma ampla propaganda, essas ideias existiam e ocuparam um espaço significativo no debate sobre a agenda pública brasileira a partir dos anos 1920. Um grupo de pesquisadores relevantes naquele período, como Alberto Sampaio, Armando Magalhães, Cândido de Mello Leitão e Frederico Hoehne, vinculava preocupações pontuais, como o estabelecimento de reservas naturais, a um projeto mais amplo de nacionalidade. Assim, conseguiram sensibilizar associações cívicas e obter espaço nas instâncias deliberativas do governo Vargas. Esses pesquisadores engajados na proteção da natureza se apropriaram de tradições de pensamento que envolviam um conhecimento científico do mundo natural e a ideia de que esse mundo deveria ser conservado, por motivos econômicos e estéticos.38 Figura 9 – Abertura de picada na mata. Fotógrafo não identificado, 1959. Fonte: FLORES, Aluízio. Belém-Brasília: Manchete assiste à conquista da Amazônia. Manchete, Rio de Janeiro, ano 10, n. 7, p. 15-25, 1959. p. 20. Figura 9 – Abertura de picada na mata. Fotógrafo não identificado, 1959. Fonte: FLORES, Aluízio. Belém-Brasília: Manchete assiste à conquista da Amazônia. Manchete, Rio de Janeiro, ano 10, n. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 7, p. 15-25, 1959. p. 20. 17 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. ANNALS OF MUSEU PAULISTA – vol. 26, 2018. 39. Cf. Franco (2009). 40. Cf. Franco (2009). 41. Franco (2009, p. 219). 42. Cf. Dean (2010). Um exemplo de pensador brasileiro com ideias que podem ser consideradas “conservacionistas” foi Alberto Torres, visto como um crítico da modernidade porque, em seu modo de entender, o progresso do industrialismo vinha acelerando a exaustão dos recursos naturais do planeta. Para Torres, a chave do progresso estaria no uso previdente dos recursos naturais e no investimento em educação e saúde. Entre os anos 1920 e 1940 os conceitos de proteção, conservação e preservação eram intercambiáveis, indicando que a natureza deveria ser protegida tanto como conjunto de recursos produtivos a ser explorado racionalmente conforme interesse das gerações presentes e futuras, quanto como diversidade biológica a ser objeto da ciência e contemplação estética.39 Organizações civis, como a Sociedade Geográfica do Rio de Janeiro, o Centro Excursionista Brasileiro e a Sociedade dos Amigos das Árvores, assumiram posturas ativas na preservação da natureza. Criticavam o desmatamento, clamavam por reformas na agricultura, defendiam a promulgação de uma lei florestal, distribuíam sementes e ministravam palestras. O ponto culminante dessa mobilização foi a Primeira Conferência Brasileira de Proteção à Natureza, em 1934. A comissão organizadora, formada por funcionários públicos, cientistas, médicos e professores, buscava concretizar suas propostas por meio de pressão e influência pessoal sobre as autoridades públicas que ocupavam posições de destaque naquela época. O entusiasmo presente nas reuniões e deliberações do grupo transformou-se em frustração com a não incorporação das propostas à Constituição brasileira e com a pouca reverberação que essas ideias tiveram na sociedade civil. A razão do fracasso residiria na difusão da ideologia do desenvolvimentismo, que se definia àquela época como corrente hegemônica dentro da articulação política do Estado.40 Essa ideia teria galvanizado todos os componentes do espectro político e de todos os grupos sociais. Assim, o desenvolvimentismo presente na sociedade brasileira fez com que muitos recursos naturais fossem intensivamente explorados e consumidos. 42. Cf. Dean (2010). No plano mais geral da sociedade e das economias nacionais, prevaleceu o projeto polí tico mais amplo do desenvolvimentismo, que se tornou hegemônico até os dias atuais. Esse projeto prioriza o crescimento econômico, mesmo que às custas da devastação da nature za. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. Fosse a iniciativa privada ou o Estado a explorar os recursos naturais, a nossa socieda de e os seus governos apoiaram, e continuam a apoiar o crescimento econômico a qual quer custo.41 Segundo Warren Dean, o desenvolvimento foi imbuído dos valores positivos de independência e autorrealização; enquanto um sistema de crenças, era milenarista: o atraso se encerraria, o tradicionalismo daria lugar à modernização e o país alcançaria o desenvolvimento, que constituía um patamar edênico de civilização.42 Assim, a ideia de desenvolvimento econômico penetraria a 18 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. 43. Cf. Dean (2010). 43. Cf. Dean (2010). consciência da cidadania, justificando cada ato de governo e de extinção da natureza.43 O que presenciamos na análise da Belém-Brasília é que tal discurso de progresso, desenvolvimento e demais sinônimos atinge um clímax no governo JK, em especial porque tais concepções estavam entranhadas nos projetos governamentais, como o Plano de Metas. Conforme destacado no artigo, existiram críticas à construção da rodovia baseadas na preservação da natureza, e não meramente em ideias oposicionistas. Porém, o discurso do desenvolvimento aliado ao progresso imbuía-se de valores com forte penetração social e política. Como uma cristalização desse debate, a Figura 10 e sua legenda demonstram o que seria a desejada “integração” do Norte ao restante do Brasil: na prática, uma anexação do “vazio” e da “terra selvagem”. Por fim, uma constante nas imagens aqui apresentadas é a antinomia entre humano e natureza, além da associação desta com a ideia de “arcaico”, em contraposição ao que seria considerado moderno nos anos JK. A trajetória da Belém-Brasília é uma pequena amostra de um processo extremamente violento de intervenção estatal nos anos 1950, e por mais que vozes dissonantes existissem, elas não tinham reverberação. Posteriormente, nos anos 1970, tal discurso retornaria com contornos igualmente dramáticos, na construção da rodovia Transamazônica. Um objetivo subjacente a este texto foi demonstrar que processos como o aqui descrito (assim como o intenso uso de propaganda política) também caracterizaram o período democrático brasileiro. Figura 10 – A indústria do Sul pede passagem para os mercados do Norte. Fotógrafo não identificado, 1960. Fonte: BARRETO, Roberto Menna. Aventura através do progresso. São Paulo: Abril, 1960. p. 10 Figura 10 – A indústria do Sul pede passagem para os mercados do Norte. Fotógrafo não identificado, 1960. Fonte: BARRETO, Roberto Menna. Aventura através do progresso. São Paulo: Abril, 1960. p. 10 19 19 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. FONTES IMPRESSAS BARRETO, Roberto Menna. Aventura através do progresso. São Paulo: Abril, 1960. BRASIL. O planejamento transforma a Amazônia. Rio de Janeiro: SPVEA, 1960. FLORES, Aluízio. Belém-Brasília: Manchete assiste à conquista da Amazônia. Manchete, Rio de Janeiro, ano 10, n. 7, p. 15-25, 1959. KUBITSCHEK, Juscelino. Por que construí Brasília. Brasília: Senado Federal, 2000. (Brasil 500 Anos). PIERRE, Arnaud. Primeira viagem na Belém-Brasília. Rio de Janeiro: SPVEA, 1960. (Cadernos Belém-Brasília, v. 4). LIVROS, ARTIGOS E TESES BARTHES, Roland. A câmara clara: nota sobre a fotografia. Rio de Janeiro: Nova Fron teira, 2002. BECKER, Bertha K. Amazônia: geopolítica na virada do III Milênio. Rio de Janeiro, Garamond, 2004. BIROLI, Flavia. Liberdade de imprensa: margens e definições para a democracia durante o governo Kubitschek (1956-1960). Revista Brasileira de História, São Paulo, v. 24, n. 47, p. 213-240, 2004. BIZELLO, Maria Leandra. Imagens otimistas: representações do desenvolvimentismo nos documentários de Jean Manzon. 1995. 250 f. Dissertação (Mestrado em Multimeios) – Uni versidade Estadual de Campinas, Campinas, 1995. BORGES, Barsanufo Gomides. Ferrovia e modernidade. Revista UFG, Goiânia, ano 13, n. 11, p. 27-36, dez. 2011. ORGES, Maria Eliza Linhares. História & fotografia. Belo Horizonte: Autêntica, 2008. BURKE, Peter. Testemunha ocular: história e imagem. Bauru: Edusc, 2004. 20 ANAIS DO MUSEU PAULISTA – vol. 26, 2018. VELHO, Otávio Guilherme. Capitalismo autoritário e campesinato. São Paulo: Difel, 1976. WORSTER, Donald. Para fazer História Ambiental. Estudos Históricos, Rio de Janeiro, v. 4, n. 8, p. 1-17, 1991. VELHO, Otávio Guilherme. Capitalismo autoritário e campesinato. São Paulo: Difel, 1976. HO, Otávio Guilherme. Capitalismo autoritário e campesinato. São Paulo: Difel, 1976. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. CORRÊA, Dora Shellard. História ambiental e a paisagem. Halac, Belo Horizonte, v. 2, n. 1, p. 47-69, set. 2012/fev. 2013. DEAN, Warren. A ferro e fogo: a história e a devastação da Mata Atlântica brasileira. São Paulo: Companhia das Letras, 2010. FRANCO, José Luiz de Andrade; DRUMMOND, José Augusto. Proteção à natureza e iden tidade nacional no Brasil, anos 1920-1940. Rio de Janeiro: Editora Fiocruz, 2009. HARDMAN, Francisco Foot. Trem-Fantasma: a ferrovia Madeira-Mamoré e a modernidade na selva. São Paulo: Companhia das Letras, 2005. KOSSOY, Boris. Realidades e ficções na trama fotográfica. São Paulo: Ateliê, 2002. ______. Fotografia & história. São Paulo: Ateliê, 2001. ______. Fotografia & história. São Paulo: Ateliê, 2001. LACERDA, Aline Lopes. A fotografia nos arquivos: produção e sentido de documentos visuais. História, Ciências, Saúde – Manguinhos, Rio de Janeiro, v. 19, n. 1, p. 283-302, jan./mar. 2012. MARTINS, Marcos Lobato. História e meio ambiente. São Paulo: Annablume; Faculdade Pedro Leopoldo, 2007. MAUAD, Ana Maria. Poses e flagrantes: ensaios sobre história e fotografia. Niterói: Editora da UFF, 2008. MOREIRA, Vânia Maria Losada. Os anos JK: industrialização e modelo oligárquico de de senvolvimento rural. In: FERREIRA, Jorge; DELGADO, Lucília de Almeida Neves (Orgs.). O Brasil Republicano: o tempo da experiência democrática – da democratização de 1945 ao golpe civil-militar de 1964. Rio de Janeiro: Civilização Brasileira, 2003. p. 357-376. PÁDUA, José Augusto. As bases teóricas da História Ambiental. In: FRANCO, José Luiz de Andrade et al. História Ambiental: fronteiras, recursos naturais e conservação da na tureza. Rio de Janeiro: Garamond, 2012. p.1-11. ______. Um sopro de destruição: pensamento político e crítica ambiental no Brasil es cravista (1786-1888). Rio de Janeiro: Jorge Zahar, 2002. RICARDO, Cassiano. Marcha para o Oeste: a influência da “bandeira” na formação social e política do Brasil. São Paulo: Edusp; José Olympio, 1970. ROLIM, Azevedo. Transbrasiliana: poema brasilista. Rio de Janeiro, 1960. ROSSI, Paolo. Naufrágios sem espectador: a ideia de progresso. São Paulo: Editora Unesp, 2000. SILVA, Sandro Duarte. A natureza contra o progresso: mitos e narrativas do “destino bandeirante” na expansão desenvolvimentista. Textos de História, Brasília, v. 17, n. 1, p. 85-106, 2009. 21 ANNALS OF MUSEU PAULISTA – vol. 26, 2018. Artigo apresentado em 26/06/2017. Aprovado em 05/04/2018. All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License 22 WORSTER, Donald. Para fazer História Ambiental. Estudos Históricos, Rio de Janeiro, v. 4, n. 8, p. 1-17, 1991. Artigo apresentado em 26/06/2017. Aprovado em 05/04/2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018. ANAIS DO MUSEU PAULISTA – vol. 26, 2018.
https://openalex.org/W1968475524	https://figshare.com/articles/thesis/The_Effects_of_Extremely_Low-Frequency_Magnetic_Fields_on_Mutation_Induction_in_Mice/10127201/1/files/18251297.pdf	English	null	The effects of extremely low frequency magnetic fields on mutation induction in mice	Mutation research	2,015	cc-by	73,464	The Effects of Extremely Low-Frequency Magnetic Fields on Mutation Induction in Mice James William Wilson Extremely low-frequency magnetic fields (ELF-MF) are classified as possibly carcinogenic, despite inconsistent data and no plausible biological mechanism linking their universal exposure with childhood leukaemia and genotoxic effects. Given discrepancies in mutagenic data and widespread public concern over genotoxic effects, this study was designed to provide an in-depth analysis of potential molecular changes induced by ELF-MF exposure in vivo. Seven-week old, BALB/c x CBA/Ca hybrid F1 male mice were exposed to 50 Hz magnetic fields of 10, 100 and 300 T for 2- or 15 hours. Blood and sperm DNA samples were collected 12 weeks post-exposure and mutation induction frequencies established at the Expanded Simple Tandem Repeat (ESTR) Ms6-hm loci using single-molecule PCR (SM-PCR). Likewise, Ms6-hm mutation induction frequencies were established in age-matched sham-treated hybrid males (control group) and those exposed to 1 Gy acute X-rays (positive controls). No significant increases in ESTR mutation frequencies were detected in either tissue at any ELF-MF exposure parameter compared to their sham-treated controls. Whilst a marginally significant increase was observed in the mutation induction frequency of pooled sperm data, these data should be regarded cautiously due to the lack of correlating dose-dependent responses. Conversely, germline and somatic ESTR mutation frequencies were significantly elevated in males exposed to acute 1 Gy X- rays. These data were validated in a high-throughput microarray pilot study, whereby no significant alterations in gene expression in kidney cells of hybrid males were detected following ELF-MF exposure. In contrast, five transcripts were significantly up-regulated in the irradiated males. Ultimately, these findings indicate that, within the analysed range of doses, the in vivo effects of ELF-MF exposure on mutation induction and gene expression are likely to be negligible. This study represents the first methodical attempt to determine mutation frequencies in vivo after continuous exposure to 50 Hz ELF-MFs up to 300 µT. Acknowledgements Statistical analysis of microarray data was assisted with by Professor. Yuri Dubrova. I would like to take this opportunity to express my utmost gratitude to my supervisor Professor Yuri Dubrova, for all his advice, guidance, and support throughout my time in the laboratory and the completion of this thesis. It is also with heartfelt thanks that I would like to acknowledge my self-proclaimed adoptive work parents, Doctors Aneela Majid and Colin Glen. Thanks to Aneela’s encouragement and guidance, I gained experience with having my work reviewed and published, and was able to broadcast my research to the public through GENIE. Throughout the writing-up process, I have grown ever more grateful for this initial support. Similarly, Dr. Colin Glen and Milly Veselis were invaluable in their advice and guidance. Their knowledge and experience of the assay employed within this project thoroughly advanced my understanding and hastened the progress I was able to make. However, and perhaps more importantly, I would like to thank them both for making the working atmosphere in the laboratory an enjoyable and entertaining one; workdays became somewhat duller and their absences towards the end of the project was sorely felt. Finally, a special thanks to my friends and colleagues Ian and Steven for all their mood- lifting stories and support over lunches and coffee breaks, they enabled me to set about my afternoon’s research with increased vigour. Overall, these six people made my time in Leicester a most enjoyable experience and I am most thankful for their unwavering company and support. I would further like to extend my thanks to the EMF Biological Research Fund for their financial support in presenting the Grant that funded this highly interesting project (BRT11/45). Additionally, I am grateful to Dr. Zenon Sienkiewicz and Jackie Haines for their contributions to the design of this project and also for their guidance, participation and support within every aspect of the work undertaken at Public Health England. Through their continuing support, they made my stay and work there a most valued experience. I would further like to extend my thanks to the EMF Biological Research Fund for their financial support in presenting the Grant that funded this highly interesting project (BRT11/45). Additionally, I am grateful to Dr. Acknowledgements Zenon Sienkiewicz and Jackie Haines for their contributions to the design of this project and also for their guidance, ti i ti d t ithi t f th k d t k t P bli H lth I would also like to express my appreciation for my previous G7 lab colleagues: Dr. Ade Adeolou, Dr. Andre Gomez and Dr. Hamdy Abouzeid Ali for all their help, advice and co-operation in the initial phase of my project. Enormous and heartfelt thanks must also go to both my parents who provided me with never-ending mental and physical support throughout the writing of this thesis - not to mention the endless flow of tea and coffee, without which my concentration and writing abilities would have diminished. I cannot imagine how infinitely more difficult completion of this project would have been without their support throughout this entire process. writing abilities would have diminished. I cannot imagine how infinitely more difficult completion of this project would have been without their support throughout this entire process. Finally, to my friend and partner throughout it all, Catherine, my muse, my rock, you gave me the inspiration to keep ploughing forward, and unwavering support when the dark times set in. Your help will never be forgotten. To all of you that assisted me throughout this process, I am eternally grateful. It’s finally done, thank you. To all of you that assisted me throughout this process, I am eternally grateful. Table of Contents Table of Tables ................................................................................................................... i Table of Figures ................................................................................................................. ii List of Abbreviations ........................................................................................................ iii 1 Introduction ............................................................................................................... 1 1.1 Extremely Low-Frequency Fields ....................................................................... 3 1.1.1 Electric Fields .............................................................................................. 4 1.1.2 Magnetic Fields ........................................................................................... 4 1.1.3 Sources ........................................................................................................ 6 1.1.4 Exposure Guidelines ................................................................................. 13 1.2 Childhood Leukaemia ....................................................................................... 15 1.3 Epidemiologic Studies ...................................................................................... 17 1.3.1 Residential Exposure ................................................................................. 17 1.3.2 Appliance Exposure................................................................................... 24 1.4 Brain Cancer ..................................................................................................... 24 1.5 Adult Studies .................................................................................................... 25 1.6 Breast Cancer ................................................................................................... 27 1.7 Cardiovascular Disease ..................................................................................... 28 1.8 Neurodegenerative Disease ............................................................................. 29 1.8.1 Alzheimer’s Disease .................................................................................. 29 1.8.2 Amyotrophic Lateral Sclerosis .................................................................. 30 1.8.3 Parkinson’s Disease ................................................................................... 31 1.9 Experimental Studies ....................................................................................... 32 1.10 Combined ELF and Carcinogen Exposure ......................................................... 38 1.11 Experimental Evidence for Carcinogenicity of ELF-MF .................................... 41 1.12 Classification ..................................................................................................... 42 1.13 Mechanism ....................................................................................................... 45 1.13.1 The Melatonin Hypothesis ........................................................................ 45 1.13.2 Radical Pair Mechanism ............................................................................ 47 1.13.3 Apoptosis .................................................................................................. 51 1.14 Mutation Detection Methods .......................................................................... Acknowledgements 52 1.14.1 Specific-Locus Test .................................................................................... 52 Table of Contents 1.14.2 Dominant Lethal Test ................................................................................ 54 1.14.3 Hypervariable Tandem Repeat Regions.................................................... 55 1.14.4 Methodology ............................................................................................. 72 1.15 Aims and Objectives ......................................................................................... 74 2 Materials and Methods ........................................................................................... 75 2.1 Mice .................................................................................................................. 75 2.2 Exposure Procedure ......................................................................................... 75 2.2.1 Magnetic Field Exposure System .............................................................. 75 2.2.2 ELF-MF Exposure ....................................................................................... 77 2.2.3 X-ray Irradiation ........................................................................................ 77 2.3 Tissues .............................................................................................................. 78 2.4 DNA Extraction ................................................................................................. 80 2.4.1 Blood ......................................................................................................... 80 2.4.2 Sperm ........................................................................................................ 81 2.5 DNA Digestion and Precipitation...................................................................... 82 2.6 DNA Quantification .......................................................................................... 82 2.7 Single-Molecule PCR Optimisation................................................................... 82 2.8 Poisson Analysis at the Single Molecule Level ................................................. 83 2.9 Mutation Scoring .............................................................................................. 83 2.10 Statistical Analysis ............................................................................................ 83 2.11 Ms6-hm Amplification and Autoradiograph Preparation ................................ 84 2.11.1 Polymerase Chain Reaction ...................................................................... 84 2.11.2 Short Agarose Gel Electrophoresis ........................................................... 84 2.11.3 Long Agarose Gel Electrophoresis ............................................................ 85 2.11.4 Southern Blot ............................................................................................ 85 2.11.5 Probe Labelling ......................................................................................... 85 2.11.6 Probe Recovery ......................................................................................... 86 2.11.7 Hybridisation ............................................................................................. 86 2.11.8 Autoradiography ....................................................................................... 87 2.11.9 Mutation Scoring and Sizing of ESTR Mutants ......................................... 87 2.12 Microarray Analysis .......................................................................................... 88 2.12.1 RNA Extraction..........................................................................................88 2.13 RNA Quantification........................................................................................... 89 2.13.1 RNA Integrity Measurement ..................................................................... 89 2.14 One-Colour Microarray-based Gene Expression Analysis ............................... 92 2.14.1 Template total RNA with Spike-In............................................................. 92 2.14.2 Cyanine 3-Labelling ................................................................................... 92 2.14.3 Cy3-RNA Clean-up ..................................................................................... 94 2.15 Quantifying cRNA ............................................................................................. 94 2.16 Hybridisation .................................................................................................... 95 2.17 Washing of Hybridised Arrays .......................................................................... 96 2.18 Array Scanning.................................................................................................. 97 2.19 Data Normalisation & Quality Control ............................................................. 97 3 Results ..................................................................................................................... 98 3.1 Experimental Design ........................................................................................ 98 3.2 Optimisation ................................................................................................... 101 3.3 Poisson Analysis ............................................................................................. 102 3.4 Mutation Scoring ............................................................................................ 104 3.5 Mutation Mosaicism ...................................................................................... 106 3.6 ESTR Mutation Frequencies in Sham-treated Males ..................................... 107 3.7 ESTR Mutation Frequencies in Irradiated Males............................................ 109 3.8 ESTR Mutation Frequencies in Males Exposed to 50 Hz Magnetic Fields ..... 109 3.8.1 Blood ....................................................................................................... 109 3.8.2 Sperm ...................................................................................................... 111 3.8.3 Combined ELF-MF effect ......................................................................... 113 3.9 Mutation Spectrum ........................................................................................ 114 3.9.1 Size Spectra of Ms6-hm Mutations in Blood .......................................... 115 3.9.2 Size Spectra of Ms6-hm Mutations in Sperm ......................................... 117 3.10 Microarray Pilot Study ................................................................................... 119 3.10.1 Experimental Design ............................................................................... 119 3.10.2 Gene Expression Analysis........................................................................ 120 4 Discussion .............................................................................................................. 126 4.1 Summary of Data ............................................................................................ 126 4.2 Epidemiological Data ...................................................................................... 127 4.3 Epidemiological Limitations ........................................................................... 128 4.4 Dose Response ............................................................................................... 131 4.5 Comparative Analysis of Study Results .......................................................... 133 4.5.1 ELF-MF Data ............................................................................................ Acknowledgements 133 4.5.2 Ionising Radiation ................................................................................... 137 4.6 Pilot Study Microarray Summary ................................................................... 139 4.6.1 Comparative Analysis of Microarray Data .............................................. 139 4.7 Study Limitations ............................................................................................ 141 4.8 Conclusion of Findings ................................................................................... 143 4.9 Future Work ................................................................................................... 144 4.9.1 Microarray Analysis ................................................................................ 144 4.9.2 Mutational Analysis ................................................................................ 145 Reference List................................................................................................................ 148 Table of Tables Table 1. The magnetic fields from 50 Hz supplied household appliances ...................... 10 Table 2. The magnetic fields from 60 Hz supplied household appliances ...................... 11 Table 3. UK and USA average power line exposure ........................................................ 12 Table 4. Wire code assessment categories ..................................................................... 19 Table 5. The ELF-MF time-weighted average exposures for electrical occupations ...... 27 Table 6. The IARC classification scale of carcinogenic risk to humans ........................... 44 Table 7. The seven specific-locus test loci ...................................................................... 54 Table 8. Tandem repetitive sequences ........................................................................... 57 Table 9. Doubling dose estimates ................................................................................... 62 Table 10. SM-PCR optimisation ...................................................................................... 83 Table 11. cDNA master mix ............................................................................................. 93 Table 12. Transcription master mix ................................................................................ 93 Table 13. Fragmentation mix .......................................................................................... 96 Table 14. Experimental Design ..................................................................................... 100 Table 15. ESTR mutation frequencies in sham-treated males...................................... 107 Table 16. Statistics for the difference between sham-treated groups ........................ 108 Table 17. ESTR mutation frequencies for all age-matched controls ............................ 109 Table 18. Summary of ESTR mutation data in blood .................................................... 110 Table 19. Summary of ESTR mutation data in sperm ................................................... 112 Table 20. ESTR mutation spectra of control and treated males in blood..................... 115 Table 21. ESTR mutation spectra of control and treated males in sperm .................... 118 Table 22. Summary of up-regulated genes. .................................................................. 123 i i Table of Figures Figure 1. The Electromagnetic Spectrum.......................................................................... 2 Figure 2. A simplified Two-Hit Model for potential CL onset ......................................... 16 Figure 3. The Fenton reaction ......................................................................................... 49 Figure 4. Diagrammatic representation of an insertion mutation caused by replication slippage ........................................................................................................................... 67 Figure 5. Diagrammatic representation of a deletion mutation caused by replication slippage ........................................................................................................................... 68 Figure 6. Spermatogenesis .............................................................................................. 71 Figure 7. Field uniformity measurement ........................................................................ 76 Figure 8. Helmholtz Coil Exposure System ..................................................................... 78 Figure 9. Hematopoiesis ................................................................................................. 79 Figure 10. RNA Electropherogram .................................................................................. 91 Figure 11. SM-PCR optimisation autoradiograph ......................................................... 101 Figure 12. SM-PCR Poisson analyses ............................................................................. 103 Figure 13. Mutation detection at the Ms6-hm ESTR locus ........................................... 105 Figure 14. Mutational mosaicism at the Ms6-hm ESTR locus ...................................... 107 Figure 15. ESTR mutation frequencies at the Ms6-hm locus in sham-treated males ............................................................................................................................. 108 Figure 16. ESTR mutation frequencies in blood of exposed and matched sham-treated males ...................................................................................................... 111 Figure 17. ESTR mutation frequencies in sperm of exposed and matched sham-treated males ...................................................................................................... 113 Figure 18. ESTR mutation frequencies in sham-treated and exposed males ............... 114 Figure 19. Table of Tables Spectra of somatic ESTR mutations in sham-treated controls and exposed male mice ...................................................................................................................... 117 Figure 20. Spectra of germline ESTR mutations in sham-treated controls and exposed male mice ...................................................................................................................... 118 Figure 21. ELF-MF gene expression analysis ................................................................. 121 Figure 22. Ionising radiation gene expression analysis ................................................ 122 Figure 23. Comparison of differentially expressed probes........................................... 124 Figure 24. Probability plot analysis comparing the exposure distributions between ELF- MF and X-ray irradiated samples .................................................................................. 126 ii ii List of Abbreviations List of Abbreviations .OH Hydroxyl radical .OOH Superoxide radicals ~ Approximately µ Micro µT micro Tesla 6-TGr 6-thioguanine-resistant A/m-1 Amperes per metre AC Alternating current AD Alzheimer’s disease ALL Acute lymphoblastic leukaemia ALS Amyotrophic lateral sclerosis AML Acute myeloid leukaemia B Magnetic flux density BM Bone marrow Bp Base pair BP Benzo(a)pyrene BSA Bovine Serum Albumin CA Chromosomal aberrations CAT Catalase CCTV Closed circuit television cGY centi-Gray CI Confidence interval CL Childhood leukaemia Cm Centimetre c-Myc Cellular myelocytomatosis Cps counts per second cRNA Complementary RNA CTP Cytidine triphosphate DB Dilution buffer DC Direct current dCTP Deoxycytidine triphosphate DEPC Diethylpyrocarbonate df degrees of freedom DLT Dominant lethal test DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate DSB Double strand break EDTA ethylene-diamine-tetra-acetic acid EF Electric field EHF Extremely high-frequency ELF Extremely low-frequency ELF-MF Extremely low-frequency magnetic ENU N-ethyl-N-nitrosourea ES-1 cells Human primary fibroblast cells from .OH Hydroxyl radical .OOH Superoxide radicals ~ Approximately µ Micro µT micro Tesla 6-TGr 6-thioguanine-resistant A/m-1 Amperes per metre AC Alternating current AD Alzheimer’s disease ALL Acute lymphoblastic leukaemia ALS Amyotrophic lateral sclerosis AML Acute myeloid leukaemia B Magnetic flux density BM Bone marrow Bp Base pair BP Benzo(a)pyrene BSA Bovine Serum Albumin CA Chromosomal aberrations CAT Catalase CCTV Closed circuit television cGY centi-Gray CI Confidence interval CL Childhood leukaemia Cm Centimetre c-Myc Cellular myelocytomatosis Cps counts per second cRNA Complementary RNA CTP Cytidine triphosphate DB Dilution buffer DC Direct current dCTP Deoxycytidine triphosphate DEPC Diethylpyrocarbonate df degrees of freedom DLT Dominant lethal test DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate DSB Double strand break EDTA ethylene-diamine-tetra-acetic acid EF Electric field EHF Extremely high-frequency ELF Extremely low-frequency ELF-MF Extremely low-frequency magnetic f ENU N-ethyl-N-nitrosourea ES-1 cells Human primary fibroblast cells from iii ESTR Expanded simple tandem repeat eV Electron-volts F Frequency F1 First filial generation F2 Second filial generation FDR False discovery rate G Gauss G Grams GIT Gastrointestinal tract GM Geometric mean GSH Glutathione Gy Gray Gy min-1 Grays per minute H Hours H Magnetic field strength H/m-1 Henry per metre H2O Water H2O2 Hydrogen peroxide HCC High-current configuration HCl Hydrochloric acid HF High frequency HL-60 cells Human promyelocytic leukaemia cells Hm-2 Human microsatellite-2 HPRT hypoxanthine-guanine phosphoribosyl transferase HR-1d cells Human primary fibroblast cells from 42 year old male HRV Heart rate variability HSC Hematopoietic stem cell HSP Heat shock protein Hz Hertz IARC International Agency for Research on Cancer ICNIRP International Commission on Non-Ionising Radiation Protection iPMS isopropyl methanesulfonate J/cm2 Joules per centimetre squared JEM Job exposure matrix Kb Kilobases Km Kilometres kV Kilovolts L Litre LCC Low-current configuration LF Low frequency lincRNAs Long intergenic non-coding ribonucleic acids LTR Long terminal repeat M Molar m- Milli M5s cells Mouse embryonic skin cells MaLR Mammalian apparent LTR-retrotransposon MCF10A cells Human breast epithelial cells iv MeWo cells Cultured human melanoma cells MF Medium Frequency (Electromagnetic Spectrum (P2 MF Magnetic field Mm milli metre MN Micronuclei MNNG N-methyl-N0-nitro-N-nitrosoguanidine MRC-5 cells Human primary foetal lung fibroblast cells Ms6-hm Minisatellite-6-hypermutable MT Mouse transposon N Nano NaAc Sodium acetate NaCl Sodium Chloride NaOH Sodium Hydroxide NEB New England Biolabs No Number nT nano Tesla Nt Nucleotide oC degrees Celsius OHCC Ordinary high-current configuration OLCC Ordinary low-current configuration OR Odds ratio ORR-1 Origin-region repeat 1 P Pico P Probability PAR Population attributable risk PBS Phosphate-buffered saline PCR Polymerase chain reaction PD Parkinson’s disease pmol Pico-mole PMR Proportionate mortality ratios RAT-1 cells Rat fibroblast cells Rcf Relative centrifugal force RF Radiofrequency RIN RNA integrity number RNA Ribonucleic acid RNA-Seq RNA sequencing ROS Reactive oxygen species RPM Radical pair mechanism Rpm Revolutions per minute RR Relative risk RT Room temperature RT-PCR Reverse transcription-polymerase chain reaction s.e. List of Abbreviations Standard error SCE Sister chromatid exchange SDS Sodium dodecyl Sulphate SHF Super high-frequency SI Système International v v SLT Specific-locus test / Russell 7-locus test SM Single-molecule SM-PCR Single-molecule polymerase-chain reaction SOD Superoxide dismutase SP-PCR Small-pool polymerase chain reaction SSB Single-strand breaks SSC Saline-Sodium Citrate T Student t test value T Tesla TAE Tris-acetate-EDTA TBE Tris-Borate-EDTA T-stock mouse Genetic tester stock mouse U Unit UG Underground cables UHF Ultra-high frequency UK United Kingdom UKCCS UK Childhood Cancer Study USA United States of America UV Ultraviolet UVW Human glioma cells V Volts V/m-1 Volts per metre VF Voice frequency VH25 cells Human skin fibroblast cells VHCC Very high-current configuration VHF Very high-frequency VLCC Very low-current configuration VLF Very low-frequency WI-38 cells Embryonic human lung fibroblast cells α-32P-dCTP deoxycytidine triphosphate-alpha-32-phosphate γH2AX Phosphorylated form of histone H2AX δ 4π x 10-7 λ Wavelength χ2 Chi-squared vi 1 Introduction Energy corresponding to the fields’ properties is emitted from the source and propagated through space or an alternative medium by vectors, in the form of time-varying electric (E) and magnetic (M) fields that act as wave-like modes of transport (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013). Owing to the different physical properties of the various electromagnetic fields, the electromagnetic spectrum is sub-divided into two further categories: ionising and non-ionising radiation. Ionising radiation such as X-rays and γ-rays are often referred to in terms of photon energy (eV). Both possess sufficient energy capable of causing either direct or indirect damage to DNA, lipids and protein through both the removal and ionisation of electrons from their orbit and/or the formation of free radicals (Powell & McMillan, 1990; IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2000). Alternatively, fields characterised as static, extremely low-frequency (ELF), radiofrequency (RF), microwaves, infra-red, visible light and ultraviolet fields are known as non-ionising radiation and do not possess the same quantity of energy within a single quantum needed to ionise an atom or molecule. Furthermore, the field strength related to the electric and magnetic components is described by either the frequency (f) as is the case with static and extremely low-frequency fields, or the wavelength (λ). The frequency of a field is measured in hertz (Hz) and depicts the number of sinusoidal cycles the wave completes per second (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013). Alternatively, the field strength for radiofrequency to infra-red fields is characterised by the wavelength (λ), which relates to the distance in metres between successive peaks of the wave (IARC Working Alternatively, fields characterised as static, extremely low frequency (ELF), radiofrequency (RF), microwaves, infra-red, visible light and ultraviolet fields are known as non-ionising radiation and do not possess the same quantity of energy within a single quantum needed to ionise an atom or molecule. Furthermore, the field strength related to the electric and magnetic components is described by either the frequency (f) as is the case with static and extremely low-frequency fields, or the wavelength (λ). The frequency of a field is measured in hertz (Hz) and depicts the number of sinusoidal cycles the wave completes per second (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013). 1 Introduction Human exposure to the numerous forms of electromagnetic radiation is not a new phenomenon, in fact naturally-occurring fields of different strengths are omnipresent within the environment (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2000; IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002; IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013). Encompassed within the electromagnetic spectrum (Figure 1), the various forms of electromagnetic fields are separated according to the quantity of energy they emit and the way in which they interact with physical matter. Energy corresponding to the fields’ properties is emitted from the source and propagated through space or an alternative medium by vectors, in the form of time-varying electric (E) and magnetic (M) fields that act as wave-like modes of transport (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013). Owing to the different physical properties of the various electromagnetic fields, the electromagnetic spectrum is sub-divided into two further categories: ionising and non-ionising radiation. Ionising radiation such as X-rays and γ-rays are often referred to in terms of photon energy (eV). Both possess sufficient energy capable of causing either direct or indirect damage to DNA, lipids and protein through both the removal and ionisation of electrons from their orbit and/or the formation of free radicals (Powell & McMillan, 1990; IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2000). Alternatively, fields characterised as static, extremely low-frequency (ELF), radiofrequency (RF), microwaves, infra-red, visible light and ultraviolet fields are Human exposure to the numerous forms of electromagnetic radiation is not a new phenomenon, in fact naturally-occurring fields of different strengths are omnipresent within the environment (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2000; IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002; IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013). Encompassed within the electromagnetic spectrum (Figure 1), the various forms of electromagnetic fields are separated according to the quantity of energy they emit and the way in which they interact with physical matter. 1 Introduction Alternatively, the field strength for radiofrequency to infra-red fields is characterised by the wavelength (λ), which relates to the distance in metres between successive peaks of the wave (IARC Working 1 Group on the Evaluation of Carcinogenic Risks to Humans, 2013). It should be noted that frequency is inversely proportional to wavelength as illustrated by Equation 1, where c represents the speed of light in a vacuum (3 x 108 m/s) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). Equation 1 Equation 1 𝝀= 𝓬 𝒇 Figure 1. The Electromagnetic Spectrum. The frequency (expressed in hertz (Hz)) increases from left to right and the category to which they correspond is indicated. Electrical appliances operate in the extremely low-frequency range 50 - 60 Hz, depending on the country of residence. The abbreviations VLF, VF, LF, MF, HF, VHF, UHF, SHF and EHF represent very low-frequency, voice frequency, low frequency, medium frequency, high frequency, very high-frequency, ultra-high frequency, super high-frequency and extremely high-frequency. Thereafter, from radiowaves until ultraviolet, categories are referred to by wavelength and expressed in metres. Ionising radiation is referred to in terms of photon energy expressed in electron-volts (eV) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2000). Figure taken from IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013. Exposure of humans to electromagnetic fields has grown considerably. Indeed, since the 19th century public exposure to man-made electromagnetic fields has been steadily increasing in conjunction with advancements in medical and industrial equipment, Equation 1 𝝀= 𝓬 𝒇 Figure 1. The Electromagnetic Spectrum. The frequency (expressed in hertz (Hz)) increases from left to right and the category to which they correspond is indicated. Electrical appliances operate in the extremely low-frequency range 50 - 60 Hz, depending on the country of residence. The abbreviations VLF, VF, LF, MF, HF, VHF, UHF, SHF and EHF represent very low-frequency, voice frequency, low frequency, medium frequency, high frequency, very high-frequency, ultra-high frequency, super high-frequency and extremely high-frequency. Thereafter, from radiowaves until ultraviolet, categories are referred to by wavelength and expressed in metres. Ionising radiation is referred to in terms of photon energy expressed in electron-volts (eV) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2000). Figure taken from IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013. Exposure of humans to electromagnetic fields has grown considerably. Indeed, since the 19th century public exposure to man-made electromagnetic fields has been steadily increasing in conjunction with advancements in medical and industrial equipment, 2 development of telecommunication systems and the growth of electrical power generation and transmission. Thus, the growth of our technology orientated society and increased reliance upon electrical power has not only led to changes in social behaviour, but also constant environmental exposure to a complex mix of electromagnetic fields. Equation 1 Of particular interest to the media (Edwards, 1990), public and scientific communities (Feychting et al., 2005), is exposure to the extremely low- frequency (ELF) region of the electromagnetic spectrum (Figure 1). Primarily classified as corresponding to frequencies from 30-300 Hz, the scientific literature is often expanded to include the frequency range: 0-100 kHz (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). However, frequencies corresponding to ELF are predominantly associated with the generation, transmission and use of electrical energy, whereby weak electric (EF) and magnetic fields (MF) are produced, which oscillate at a frequency of 50 Hz in Europe and Asia and 60 Hz in the USA (Feychting & Ahlbom, 1995). Concern has increased amongst reports of a potential association between extended ELF exposure and a wide range of adverse health effects relating to: adult and childhood cancers (Wertheimer & Leeper, 1979), depression and suicide (Ahlbom, 2001), neurodegenerative diseases (Hakansson et al., 2003) and compromised reproductive capacity (Huuskonen et al., 1998; Ahlbom et al., 2001; Panagopoulos et al., 2013). Indeed, potential adverse health effects were first illustrated in a series of studies conducted by the former Soviet Union in the early 1960’s. Russian substation workers complained of increased headaches and cardiovascular disorders including abnormal heart rates and arrhythmia (reviewed in (Bonnell, 1982; Fatigoni et al., 2005)). However, any potential follow-up studies were postponed under the premise that the problem was too complex to analyse (reviewed by Santini et al., (2009)). It was not until the publication of Wertheimer and Leeper’s (1979) ground-breaking study linking continuous ELF exposure to an increased incidence of childhood leukaemia that extensive investigations started within this area of research, principally in the form of epidemiologic studies. 1.1 Extremely Low-Frequency Fields The fundamentals of electromagnetism as described by James Clerk Maxwell’s 1.1 Extremely Low-Frequency Fields The fundamentals of electromagnetism as described by James Clerk Maxwell’s equations state that the presence of a time-varying electric field produces a time- 3 3 varying magnetic field and vice versa, which then couple together to form a propagating electromagnetic wave. However, the formation of an electromagnetic wave typically only occurs at high frequencies. In contrast, the production of weak electric and magnetic fields which oscillate within the frequency range of ELF, consist of very long wavelengths (6000 kilometres (km) at 50 Hz and 5000 km at 60 Hz), thus the interdependence of the electric and magnetic field components becomes weaker (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013). As a result the EF and MF propagate, in effect, uncoupled as if arising from independent sources (Fatigoni et al., 2005). 1.1.1 Electric Fields 1.1.1 Electric Fields Electric fields arise from the presence of electric charges, regardless of the flow of an electrical current (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). Subsequently, electric fields are related to voltages rather than currents and measured as electric field strength, which is expressed in the Système International (SI) unit volts per metre (V/m-1) (International Commission on Non- Ionizing Radiation Protection, 1998). Furthermore, electric fields are easily perturbed by materials hence buildings are able to reduce the electric field within them from an external source. Overall, this not only makes obtaining a reliable measure of personal exposure difficult but researchers have also noted that any measurement of the extremely low-frequency electric field component is essentially non-existent (Burdak- Rothkamm et al., 2009). Consequently, any epidemiologic studies which attempt to analyse the effects of external power lines are inherently studies exclusively pertaining to magnetic fields (Savitz, 1995). Similarly, Focke et al. (2010) has provided direct experimental evidence ruling out ELF electric fields as a possible carcinogen or contributing factor. The emphasis of experimental investigations has therefore been placed on determining the potential adverse health effects linked to exposure to the ELF-MF component exclusively (Tenforde, 1992). 1.1.2 Magnetic Fields g In contrast, magnetic fields arise from the motion of electric charges or more simply, the flow of electrical current (International Commission on Non-Ionizing Radiation Protection, 1998). For that reason, magnetic fields are representative of the electric 4 current in a system, irrespective of the voltage. Since electrical current is supplied as alternating current (AC), in the form of a sinusoidal wave, the magnitude of the electric current determines the magnetic field (Kheifets & Oksuzyan, 2008). Within the scientific literature the magnitude of a magnetic field is predominantly described as its magnetic flux density (B), expressed in the SI units of tesla (T) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). However it is not uncommon, especially within the earlier studies published in the USA, to see the magnetic flux density displayed as gauss (G). Also since magnetic fields are generated by the movement of charged particles, they are occasionally, although less commonly, displayed as magnetic field strength (H) and expressed as amperes per metre (A/m-1) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). Despite this variation in SI units, these measurements are easily interchangeable with 1 G being equivalent to 10-4 T, while magnetic flux density and magnetic field strength are related by Equation 2 (International Commission on Non-Ionizing Radiation Protection, 1998). Despite this variation in SI units, these measurements are easily interchangeable with 1 G being equivalent to 10-4 T, while magnetic flux density and magnetic field strength are related by Equation 2 (International Commission on Non-Ionizing Radiation Protection, 1998). Equation 2 𝑩= 𝜹𝑯 Where δ represents the constant of proportionality (the magnetic permeability) in a vacuum and air (4π x 10-7) and is expressed in Henry per metre (H/m-1) (International Commission on Non-Ionizing Radiation Protection, 1998). As a conversion 1 A/m-1 equates to 1.25 µT. Furthermore, in contrast to electric fields, magnetic fields are not easily shielded and thus can easily penetrate the body: only ferromagnetic materials such as iron, which are highly susceptibility to magnetisation, can obstruct them (Fatigoni et al., 2005; Miller & Green, 2010). This represents an issue in today’s world in that, not only do ELF fields occur naturally within the environment, but since the first power station arose in 1882 (Kheifets & Oksuzyan, 2008), global industrialisation and the extended use of electricity have resulted in their increase. Consequently, the presence of extremely low-frequency fields is universal, including within households, in schools and at work. As such, extensive research has been conducted to clarify the 5 plethora of links between ELF-MF exposure and adverse biological effects, particularly carcinogenesis. 1.1.3 Sources 1.1.3.1 Naturally Occurring Fields Although representative of a static field, the most recognisable naturally occurring field is the Earth’s magnetic field. Estimated to be around 50 µT, the geomagnetic field is not constant, but instead is subject to continuous fluctuations. Location is instrumental in the determination of total field intensity, at a maximum of 67 µT at the magnetic poles the intensity diminishes towards the equator (30 µT). Additionally, the geomagnetic field is known to fluctuate as a result of lunar and seasonal variations (Barnes & Greenebaum, 2007). Similarly, time-varying magnetic fields in the ELF range are also present throughout the atmosphere as a product of solar and lunar effects on ion currents in the upper atmosphere (Tenforde, 1992). There is, however, also a continuous global presence of extremely low-frequency electromagnetic waves within the geomagnetic field as a result of a number of very low-intensity signals called Schumann resonances. These fields are generated by thunderstorms and lighting discharges within the resonant cavity formed between the Earth and the ionosphere. 1.1.3.1 Naturally Occurring Fields 1.1.3.1 Naturally Occurring Fields Although representative of a static field, the most recognisable naturally occurring field is the Earth’s magnetic field. Estimated to be around 50 µT, the geomagnetic field is not constant, but instead is subject to continuous fluctuations. Location is instrumental in the determination of total field intensity, at a maximum of 67 µT at the magnetic poles the intensity diminishes towards the equator (30 µT). Additionally, the geomagnetic field is known to fluctuate as a result of lunar and seasonal variations (Barnes & Greenebaum, 2007). Similarly, time-varying magnetic fields in the ELF range are also present throughout the atmosphere as a product of solar and lunar effects on ion currents in the upper atmosphere (Tenforde, 1992). There is, however, also a continuous global presence of extremely low-frequency electromagnetic waves within the geomagnetic field as a result of a number of very low-intensity signals called Schumann resonances. These fields are generated by thunderstorms and lighting discharges within the resonant cavity formed between the Earth and the ionosphere. Lightning discharges create electromagnetic standing waves that cover the ELF spectrum with broad peaks of fading amplitude predominantly at a frequency of 7.83 Hz, but also resonance frequencies of 14.1, 20.3, 26.4, and 32.5 ranging up to 250 Hz (Chand et al., 2009). These waves are then transmitted around the globe through reflection between the lower boundary of the ionosphere and the earth’s surface, the same mechanism through which the basis of distance radio communications are formed (Cherry, 2002). As the Schumann resonance forms electromagnetic waves, it can be detected as electric or magnetic micro-pulses. The electric component of which consists of ~0.01 V/m-1, while the magnetic fields amount to between 1 and 10 nT (Funk et al., 2009). Lightning discharges create electromagnetic standing waves that cover the ELF spectrum with broad peaks of fading amplitude predominantly at a frequency of 7.83 Hz, but also resonance frequencies of 14.1, 20.3, 26.4, and 32.5 ranging up to 250 Hz (Chand et al., 2009). These waves are then transmitted around the globe through reflection between the lower boundary of the ionosphere and the earth’s surface, the same mechanism through which the basis of distance radio communications are formed (Cherry, 2002). As the Schumann resonance forms electromagnetic waves, it can be detected as electric or magnetic micro-pulses. 1.1.3.1 Naturally Occurring Fields The electric component of which consists of ~0.01 V/m-1, while the magnetic fields amount to between 1 and 10 nT (Funk et al., 2009). Artificial ELF-MFs however represent strengths many thousands of magnitude greater than those arising naturally (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). Thus, the main sources of human ELF-MF exposure occur both through background fields originating from distant high-voltage overhead and 6 underground power distribution lines and power transmission lines, and also fields resulting from the use of electrical appliances and devices, regardless of whether it be in an occupational (high levels of exposure over a short duration) or residential environment (generally lower exposure over an extended period of time). 1.1.3.2 Artificial Fields 1.1.3.2.1 Background Fields 1.1.3.2.1 Background Fields g Background magnetic fields within residences in many countries generally arise from the distribution lines supplying them electricity. Additionally, background fields can also arise within homes residing in close proximity to high-voltage transmission lines. The magnitudes of these fields will also be larger than those produced from the lower voltage distribution lines, although very few residences are located near such high- voltage transmission lines (Chapter 1.1.3.2.3) (Swanson & Kaune, 1999). Measurements of background fields have been conducted in many countries including Australia (Karipidis, 2015), North America (Linet et al., 1997) and the UK (Coghill et al., 1996), but studies in the rest of Europe often present poor degrees of geographical coverage (Grellier et al., 2014). The background fields recorded in Australia were in general, comparable with the USA (50-60 nT) (Karipidis, 2015). There are, however, differences in background fields between the USA and the UK with background fields within the UK being approximately 1.5 to 1.9 times weaker than those that resonate in North America (Swanson & Kaune, 1999). The daily average background field in the USA was measured over a 24-hour period in eight individual studies as ranging from 47-99 nT (reviewed in Swanson & Kaune, (1999)). However, uncertainties relating to the measurements made within these studies prevented the formation of a single, unambiguous approximation, leaving the authors to estimate that backgrounds fields within the USA typically present a geometric mean (GM) within the range of 60-70 nT (Swanson & Kaune, 1999). However, in a study conducted by Zaffanella, (1992) which is considered the most representative study of background fields within the USA, spot measurements performed in the centre of most rooms in each home yielded a GM field of 56 nT with only 10% of residences registering background levels above 0.2 µT (Kheifets et al., 2005). Conversely, studies conducted in the UK (Merchant et al., 1994; 7 Coghill et al., 1996; Preece et al., 1996) provided a GM of 38 nT, with the accurate value believed to be between 36-39 nT (Swanson & Kaune, 1999). The variation between these countries is likely a consequence of different electrical supply voltages. Since magnetic fields arise from the flow of electrical currents, consumers in the UK use approximately double the final distribution voltage than in the USA (220-240 volts (V) versus 110 volts, respectively), meaning that the currents in the UK are halved for the same power (Merchant et al., 1994). 1.1.3.2.1 Background Fields The background fields which are generated are, in general, uniformly present throughout the home. This was demonstrated in a study by Preece and co-authors, (1997) who failed to illustrate any variation between rooms when the background fields were measured in the centre of every room of 50 residences. In this study measurements were taken with both the power on and off, recording values of 0.017 + 0.003 µT and 0.012 + 0.002 µT respectively (Preece et al., 1997). Yet, since magnetic fields arise from electrical currents they vary proportionally and according to electrical demand. This has led to variations in readings between different times of day (Banks et al., 2002; Karipidis, 2015), and some studies have also illustrated seasonal differences whereby summer fields are in excess of those measured in winter (Banks et al., 2002). Uncertainties remain as to whether this is in fact founded, since other studies failed to illustrate any such differences (Kaune & Zaffanella, 1994; Karipidis, 2015). Alongside ELF-MF exposure from ubiquitous low-intensity background fields, approximately 30-50% of total ELF-MF exposure resides in fields generated through the use of electrical domestic appliances and devices (Kheifets et al., 2005; Repacholi, 2012). Unlike the background fields, ELF-magnetic fields produced by appliances are constantly more intense, yet only experienced intermittently. 1.1.3.2.2 Electrical Appliances During use, each appliance produces an elevated field within its immediate vicinity. As such humans are exposed to the highest magnetic flux densities when in close proximity to domestic appliances, particularly those containing looped heating elements, motors or transformers (Merchant et al., 1994). Magnetic fields produced by appliances are typically localised and decrease as the inverse cube of distance from the source, thus at distances of 1-2 metres they are similar to background fields (Lacy- 1.1.3.2.2 Electrical Appliances 1.1.3.2.2 Electrical Appliances During use, each appliance produces an elevated field within its immediate vicinity. As such humans are exposed to the highest magnetic flux densities when in close proximity to domestic appliances, particularly those containing looped heating elements, motors or transformers (Merchant et al., 1994). Magnetic fields produced by appliances are typically localised and decrease as the inverse cube of distance from the source, thus at distances of 1-2 metres they are similar to background fields (Lacy- 8 Hulbert et al., 1998). Attempts have been made to assess personal exposure to household appliances which are regularly used both in the UK (50 Hz) and North America (60 Hz), as noted in Table 1 and Table 2 respectively. Preece et al. (1997) assessed the magnetic field strength (40-800 Hz) of 806 domestic appliances within 50 UK homes (Table 1) using the Emdex II (electric and magnetic field digital exposure system) to measure the appliance-generated magnetic field. Upon activation of the appliance, measurements were recorded every 3 seconds for 30 seconds at 5, 30, 60 and 100 centimetres from the appliance surface. Similarly, Farag et al. (1998) assessed the magnetic field strength of domestic appliances at various distances up to 120 centimetres in 254 homes (Table 2). The relatively large magnetic fields experienced when in close proximity to domestic appliances have in fact been hypothesised to contribute to an increased risk of hormone-dependent cancers and in particular breast cancer (Stevens, 1987). While studies have provided a weak yet significant link between the use of electric razors and breast cancer (odd ratio, OR = 1.7, 95%, CI: 1.1-2.8) (Zheng et al., 2000b), and meningioma (OR = 10.9, 95%, CI: 2.3-50) (Kleinerman et al., 2005), there has been no association between breast cancer and the use of hair dryers, curling irons, vacuum cleaners or irons (Zheng et al., 2000b). Moreover, little evidence has been provided of an association between brain cancer and either curling irons or microwave ovens (Kleinerman et al., 2005). Electric blankets however represent a further appliance that has been extensively studied in relation to an increased risk in potential adverse health effects (Savitz et al., 1990; Abel et al., 2007). 1.1.3.2.2 Electrical Appliances Comparatively, the magnetic fields emitted by electric blankets are weaker than those of hair dryers and electric razors with residential measurements at a distance of 10 centimetres recording flux densities between 0.1- 2.2 µT (Wilson et al., 1996; Hatch et al., 1998; Lee et al., 2000). However, the exposure from appliances is related to the duration of the dose alongside the distance from the source and, unlike electric blankets, none of the appliances indicated in Tables 1 and 2 are employed over an extended period of time. In contrast, Preston-Martin et al. (1996) reported that 83% of mothers and 84% of children left the blanket on for the (1996) reported that 83% of mothers and 84% of children left the blanket on for the (1996) reported that 83% of mothers and 84% of children left the blanket on for the 9 9 whole night. Therefore, the use of electric blankets represents a sustained source of magnetic field exposure that is within close proximity to the body and thus may provide vital understanding into the association of exposure to magnetic fields with such diseases. Table 1. system supply. Adapted from Preece et al., (1997). The arithmetic means of calculated broadband magnetic fields at 5, 50 and 100 centimetres (cm) from a selected number of household appliances using a 50 Hz power system supply. Adapted from Preece et al., (1997). entimetres (cm) from a selected number of household appliances using a 50 Hz power 1.1.3.2.2 Electrical Appliances The magnetic fields from 50 Hz supplied household appliances Appliance Number of appliances measured Magnetic field at specified distance from the exterior of appliance (µT) 5 cm + S.D 50 cm + S.D 100 cm + S.D Television 73 2.69 + 1.08 0.26 + 0.11 0.07 + 0.04 Kettle 49 2.82 + 1.51 0.05 + 0.06 0.01 + 0.02 Video recorder 42 0.57 + 0.52 0.06 + 0.05 0.02 + 0.02 Vacuum cleaner 42 39.53 + 74.58 0.78 + 0.74 0.16 + 0.12 Hair-dryer 39 17.44 + 15.56 0.12 + 0.10 0.02 + 0.02 Microwave oven 34 27.25 + 16.74 1.66 + 0.63 0.37 + 0.14 Washing machine 34 7.73 + 7.03 0.96 + 0.56 0.27 + 0.14 Iron 33 1.84 + 1.21 0.03 + 0.02 0.01 + 0.00 Clock radio 32 2.34 + 1.96 0.05 + 0.05 0.01 + 0.01 Hi-fi system 30 1.56 + 4.29 0.08 + 0.14 0.02 + 0.03 Toaster 29 5.06 + 2.71 0.09 + 0.08 0.02 + 0.02 Central heating boiler 26 7.37 + 10.10 0.27 + 0.26 0.06 + 0.05 Fridge / freezer 23 0.21 + 0.14 0.05 + 0.03 0.02 + 0.01 Cooker 18 2.27 + 1.33 0.21 + 0.15 0.06 + 0.04 Dishwasher 13 5.93 + 4.99 0.80 + 0.46 0.23 + 0.13 Freezer 13 0.42 + 0.87 0.04 + 0.02 0.01 + 0.01 Oven 13 1.79 + 0.89 0.39 + 0.23 0.13 + 0.09 Electric shower 12 30.82 + 35.04 0.44 + 0.75 0.11 + 0.25 Food processor 10 12.84 + 12.84 0.23 + 0.23 0.04 + 0.04 Extractor fan 9 45.18 + 107.96 0.50 + 0.93 0.08 + 0.14 Hand blender 8 76.75 + 87.09 0.97 + 1.05 0.15 + 0.16 Tumble dryer 7 3.93 + 5.45 0.34 + 0.42 0.10 + 0.10 Computer 6 1.82 + 1.96 0.14 + 0.07 0.04 + 0.02 The arithmetic means of calculated broadband magnetic fields at 5, 50 and 100 centimetres (cm) from a selected number of household appliances using a 50 Hz power Table 1. The magnetic fields from 50 Hz supplied household appliances 10 Table 2. 1.1.3.2.2 Electrical Appliances The magnetic fields from 60 Hz supplied household appliances Appliance Number of appliances measured Exposure (µT) at 10 cm Minimum Median Maximum Dishwasher 12 0.55 2.08 2.2 Tumble dryer 32 0.41 3 16.5 Electric shaver 69 1 2 13 Hair-dryer 117 0.3 1.5 19 Microwave oven 137 0.26 7 44.2 Refrigerator 225 0.065 0.66 3.2 Washing machine 46 0.2 0.69 1.4 Oven 230 0.12 1.21 5.05 Exposure (µT) at 30 cm Vacuum cleaner 34 1.4 5.1 20.5 Exposure (µT) at 50 cm Air conditioner 27 0.025 0.16 0.82 Blender 27 0.21 0.22 0.7 Electric blower 16 0.018 0.13 1.05 Electric can opener 27 0.92 1.45 6.2 Ceiling fan 24 0.02 0.15 1.2 Coffee maker 20 0.05 0.095 0.16 Iron 60 0.052 0.14 0.3 Non-ceiling fan 21 0.015 0.26 3.2 Food processor 24 0.35 1.2 4 Toaster 18 0.042 0.21 0.51 Oven 230 0.025 1.45 The minimum, median and maximum magnetic flux density at 10, 30 and 50 cm from a selected number of household appliances using a 60 Hz power system supply. Values courtesy of Farag et al., (1998). Table 2. The magnetic fields from 60 Hz supplied household appliances Finally, the transmission of electrical power through high-voltage transmission and distribution lines involves the flow of electrical current and ultimately the production of magnetic fields. As a result, exposure to stray fields associated with the generation and transmission of electricity has been a regular occurrence for nearly a century (Jackson, 1992). Finally, the transmission of electrical power through high-voltage transmission and distribution lines involves the flow of electrical current and ultimately the production of magnetic fields. As a result, exposure to stray fields associated with the generation and transmission of electricity has been a regular occurrence for nearly a century (Jackson, 1992). The minimum, median and maximum magnetic flux density at 10, 30 and 50 cm from a selected number of household appliances using a 60 Hz power system supply. Values courtesy of Farag et al., (1998). 1.1.3.2.3 Power Lines The transportation of electricity from the power station into residences occurs via a wide range of voltages and hence currents, which are determined by the distance of transmission (Jackson, 1992). The magnetic field produced and ultimately the population’s exposure is related to the properties of the transmission/distribution line. 11 Long distance transmission is conducted by 500 kilovolts (kV) and 275/400 kV high- voltage transmission lines in the USA and UK respectively. Once within a local region, these voltages are stepped down by transformers and 132 kV, 33 kV and 11 kV distribution lines carry the electricity into the neighbourhoods, where neighbourhood transformers further step down the voltage to residential delivery via domestic service lines (Kaune, 1993; Merchant et al., 1994). In the UK, as with most of Europe, electricity is delivered into homes at 220-240 volts, in comparison to North America (110 V), therefore the currents needed to provide the same power are lower in the UK and Europe (Repacholi, 2012). Similar to electrical appliances, the strongest magnetic fields are in the immediate vicinity of the source (directly beneath the power line) and diminish in relation to distance (Table 3) so that at approximately 100 metres they are typically equivalent to background fields (Draper et al., 2005; Kroll et al., 2010). Table 3. 1.1.3.2.3 Power Lines UK and USA average power line exposure Source of magnetic fields (USA) Field strength (µT) 500 kV high-voltage transmission line Typical average under line 8.7 Typical average 20 m from centre of line 2.9 24 kV overhead and underground distribution lines Typical maximum beneath and over lines 4-7 Typical average beneath and over lines 1-2 Mean exposure to a typical person from all sources of magnetic fields over a 24 hour period <0.05 Source of magnetic fields (UK) Field strength (µT) 275/400 kV high-voltage transmission line Typical average under line 5.7 Typical average 20 m from centre of line 2 132 kV distribution line Typical average under line 0.5-2 Typical average 25 m from centre of line 0.05-0.2 33 kV distribution lines Typical average under line 1.5 Typical average 20 m from centre of line 0.1 400 V service line Typical average under line 0.143-0.215 Typical average 25 m from centre of line 0.039-0.041 Average exposure to a typical person from all sources of magnetic fields over a 24 hour period <0.11 Typical average exposures to sources of power frequency magnetic fields in the USA, (values courtesy of (Repacholi, 2012) and UK, (EMFs.info, 2015). Adapted from (Repacholi, 2012). Table 3. UK and USA average power line exposure 12 It must be noted that while residing in close vicinity of high-voltage power lines will lead to high levels of electric and magnetic fields’ exposure, only a small percentage of the population own homes within these areas. In the UK 0.07% of homes are within 50 metres of high-voltage transmission lines, while 0.21% lies within 100 metres. These figures correlate with those provided in the USA where approximately 1.1% of residences are within 40 metres of high-voltage power lines (EMFs.info, 2015). Moreover, as is illustrated by Table 3, even directly under a typical high-voltage transmission line the average ELF-MF exposure is less than 5% of the International Commission on Non-Ionising Radiation Protection (ICNIRP) guideline limits (Chapter 1.1.4), (International Commission on Non-Ionizing Radiation Protection, 2010). Taken together, these percentages are particularly pertinent following the publication of several studies associating ELF-MF exposure of residences closer to high-voltage power lines with an increased risk of cancer (Coleman et al., 1989; Feychting & Ahlbom, 1993; Draper et al., 2005). 1.1.3.2.3 Power Lines On the other hand, conflicting studies have similarly been published in which residences in close proximity to high-voltage power lines present no significant association with an increased onset of cancer (Olsen et al., 1993; Kleinerman et al., 2000; UK Childhood Cancer Study Investigators, 2000; Pedersen et al., 2014). A further study noted a significant risk of leukaemia attached up to the 1980’s, though from thereon in, risk declined to insignificance throughout subsequent decades (Bunch et al., 2014). Nonetheless, the indication of a potential elevated risk ensures that ELF-MF falls within the scope of the ICNIRP, an organisation established in 1992 to develop universally appropriate guidelines detailing exposure limits to potentially hazardous forms of non-ionising radiation. 1.1.4 Exposure Guidelines It was following a comprehensiv p It was following a comprehensive review of the published literature, and in an attempt to safeguard against any potential adverse health effects relating to both residential and occupational ELF-MF exposure, that the initial ELF-MF exposure guidelines were established in 1998. Within the 100 kHz frequency range, which includes power frequencies 50 Hz and 60 Hz, reference limits of 500 µT and 100 µT were set for occupational workers and the general population respectively (International Commission on Non-Ionizing Radiation Protection, 1998). A safety factor of five was 13 applied to occupational exposure to represent workers’ chronic exposure to stronger fields (International Commission on Non-Ionizing Radiation Protection, 1998). However, several publications further linking ELF-MF exposure to an increased risk of cancer, in addition to numerous biological and genotoxic effects (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002; WHO, 2007), the ICNIRP reconsidered their guideline limits in 2010 and raised them to 200 µT and 1 mT for residential and occupational exposure respectively (International Commission on Non- Ionizing Radiation Protection, 2010). Ultimately, the ICNIRP reasoned that in an overall absence of consistent, replicated experimental research coupled to a lack of a defined causal relationship, meant that any potential observed effect would not be confronted in such basic exposure restrictions. However, an issue still remains in respect to these guidelines in that a considerable number of epidemiologic studies, alongside several pooled and meta-analysis studies have regularly indicated that chronic exposure to 50/60 Hz magnetic fields many times lower (0.4 µT) is associated with an increased risk of numerous health problems, with probably the most consistent link being to an association with childhood leukaemia (Ahlbom et al., 2001). Indeed, the initial link to childhood leukaemia was reported in 1979 when Wertheimer and Leeper published their ground-breaking study in which they developed and employed a non-invasive method as a surrogate of ELF-MF exposure assessment, known as ‘wire codes’ (Wertheimer & Leeper, 1979). Residences were classified based on the observable characteristics of the electrical cables in close proximity, therefore the distance, size and the type of cable were used to categorise homes as possessing either a high-current configuration (HCC) or a low-current configuration (LCC) (Table 4). Ultimately, homes classified as HCC were presumed to be susceptible to stronger background fields than those of LCC. 1.1.4 Exposure Guidelines It was following a comprehensiv Using this surrogate to indirectly calculate the potential current flow, Wertheimer and Leeper (1979) performed a case-control study within the greater Denver area of Colorado during 1976 to 1977. A total of 344 cases were selected through a stringent two-step process of individuals diagnosed with cancer in Colorado before the age of 19 and between the years of 1950 to 1973, who also presented a Colorado birth certificate. The assignment of matched controls however is rather complicated and inadequately presented in the 14 literature (Lagiou et al., 2002). Though a simplified explanation presented in the IARC summary (2002), indicates the matched-controls were assigned through the possession of a Denver-area birth certificate, alongside lists of births within the Colorado area during the time period of 1946-1973. Although relative risks were not presented in their study, Wertheimer and Leeper reported a statistically significant increase in leukaemia mortality among children who lived in close proximity to cables representing high current configurations. Subsequent re-analyses of the data have indicated a three-fold increase in childhood leukaemia (Kheifets et al., 1997; Lacy- Hulbert et al., 1998). 1.2 Childhood Leukaemia The elevated risk to children is especially pertinent as the role of ELF-MF becomes more prominent in modern society because, although cases of childhood cancers are sparse, of those recorded approximately one third relate to childhood leukaemia (CL), making it the most common type of childhood cancer (Linet et al., 1999). Leukaemia is a cancer that arises in the hematopoietic tissue, i.e. bone marrow before entering the blood, occurring as a result of chromosomal alterations and mutations that disturb the normal differentiation of blood progenitor cells into either lymphoid or myeloid progenitor cells (Chiaretti et al., 2014). Subsequently, leukaemia is classified according to the cell of origin, as well as its clinical course (acute or chronic) (Brain et al., 2003). As such, the major morphologic types are acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML), which refers to cancer of lymphoid or myeloid progenitor cells respectively. Acute lymphoblastic leukaemia is the most common form of CL, accounting for around 78% of the diagnosed cases, while AML accounts for the majority of the remaining diagnoses (Belson et al., 2007). The age of onset for ALL follows a bimodal distribution whereby cases are normally reported up to the age of 15 years (Inaba et al., 2013), with the peak incidence occurring in children aged between 2 and 5 years (Linet & Devesa, 1991; Greaves & Wiemels, 2003; Pui et al., 2008). There then follows a steady increase in prevalence throughout life. The risk of AML on the other hand is highest within the first two years immediately after birth before decreasing thereafter (Gurney et al., 1995). Although the knowledge of genetic changes in CL has improved significantly over the years, the precise aetiology of 15 childhood leukaemia remains largely unknown. Less than 5% of cases of ALL are connected with inherited genetic syndromes or ionising radiation (Pui et al., 2008), which has led researchers to hypothesise that development of the disease results from a combination of factors involving genetic susceptibility, environmental factors and chance (Greaves, 2002). This has led to the development of a ‘two-hit’ model (Figure 2), whereby the onset and progression of leukaemia consists of a multistep process (Greaves, 2006). The general model assumes an initial somatic hit, occurring in utero in a foetal hematopoietic cell, probably as a result of chromosome translocations. 1.2 Childhood Leukaemia This initial hit is believed to be a common event that results in the formation and proliferation of a pre-leukaemic clone (Greaves, 1999). Yet, the reasonably low prevalence of the disease illustrates at least a two-stage pathogenesis in which the conversion of the pre-leukaemic clone into the disease requires either an additional pre-natal and/or post-natal genetic or environmental event (Eden, 2010). Such events are rare: only ~1% of individuals possessing pre-leukaemic clones will get an additional genetic hit to create overt leukaemia, thus acting as a rate-determining step in the progression of the disease (Greaves & Wiemels, 2003). Figure 2. A simplified Two-Hit Model for potential CL onset. Illustrating the crucial events hypothetically required to produce the major morphologic types of childhood leukaemia. Adapted from (Greaves, 2006). Figure 2. A simplified Two-Hit Model for potential CL onset. Illustrating the crucial events hypothetically required to produce the major morphologic types of childhood leukaemia. Adapted from (Greaves, 2006). 16 If indeed the two hit model requires an environmental interaction, in spite of the vast number of epidemiologic studies that have researched a multitude of environmental agents in the general population, ionising radiation remains as the only widely accepted environmental causative factor, but still only accounts for a very limited amount of CL cases (Preston et al., 1994; Doll & Wakeford, 1997). Other suspected risk factors include exposure to environmental agents such as benzene, the human T-cell virus and genetic disorders such as Down’s syndrome (reviewed in (Belson et al., 2007; Eden, 2010; Mezei et al., 2014)). Finally, owing to the seminal study by Wertheimer and Leeper (1979), ELF-MF is also hypothesised as a potential environmental exposure responsible for a causative link to CL. Despite the above, the results of subsequent epidemiologic studies conducted over the past three decades have often presented contradictory and inconclusive evidence of a causal relationship between ELF-MF exposure and an increased risk of cancer. A prime example of such inconsistency between studies is highlighted in a study conducted by Fulton et al. (1980), where attempts to replicate the findings of Wertheimer and Leeper (1979) indicating a relationship between childhood leukaemia and electric power line configurations in Rhode Island (OR = 1.09, 95% CI: 0.7-1.6) proved unsuccessful, despite implementing the same wire classification analysis. 1.2 Childhood Leukaemia Such underlying contradictions present within epidemiological studies are predominantly due to the ubiquitous nature of ELF-MFs in the environment, which has consequently resulted in the assessment of ELF-MF becoming especially problematic. Indeed, in most studies it is more important to assign the residence to the correct classification in an attempt to reduce the chance of misclassification rather than extract an exact exposure measurement value (Kheifets & Oksuzyan, 2008). In fact, throughout the years and hence epidemiologic studies, there has been a plethora of exposure matrixes used which generated both positive and negative correlations to childhood leukaemia. 1.3.1 Residential Exposure The most basic and simple of exposure assessments is to measure the distance between the residence and source as it does not take into consideration multiple sources of exposure. Firstly, Coleman et al. (1989) assessed the potential relationship 17 of indirect residential exposure to power frequency magnetic fields from power lines and transformer substations through distance. The authors state that the risk of leukaemia relative to cancer controls when residing within 100 metres was 1.45 (95% CI: 0.54-3.88); within 50 metres the relative risk (RR) increased to 2.0 but the confidence intervals also expanded (95% CI: 0.4-9.0) while residences within 25 metres carried a risk of 1.3 (95% CI: 0.8-2.0) (Coleman et al., 1989). Although, as previously mentioned (Chapter 1.1.3.2.3), approximately only 0.21% of households lie within 100 metres of high-voltage transmission lines in the UK (EMFs.info, 2015). Moreover the authors reported that only 6% of the 771 case subjects lived within 100 metres of an overhead power line while 40% lived within 100 metres of a substation (OR = 0.99) (Coleman et al., 1989). Similarly, subsequent studies have also failed to report a significantly raised odds ratio between childhood cancer and proximity to overhead power lines (Myers et al., 1990; Verkasalo et al., 1993). Alternatively, some evidence of increased risk of all cancers was observed by Olsen and co-authors for calculated exposures of more than 0.4 µT, but not for leukaemia when taken separately (Olsen et al., 1993). The lack of significantly raised odds ratios does not signify a lack of true association, but more so that too few addresses for cases and controls within their study had background levels of magnetic fields exceeding 0.01 µT (Myers et al., 1990). Thus, in an attempt to record more accurate exposures, the ‘wire code’ classification system was further developed, firstly to include four (Wertheimer & Leeper, 1982) and later five (Savitz et al., 1988) sub-classes of home exposure (Table 4). 18 Table 4. 1.3.1 Residential Exposure Wire code assessment categories Type of external power line & distance to residence Wire code category Transmission line 3 phase primary distribution Secondary distribution >2 Thick Thin 1st span >2nd span HCC VHCC <15 m <15 m <15 m <7.5 m OHCC <40 m <40 m <40 m <20 m <15 m LCC OLCC <40 m <40 m <40 m VLCC None of the above and secondary service overhead UG None of the above and secondary service underground Representative of the specifications of the wire codes used as a surrogate index for residential magnetic field exposure assessment. High-current configuration (HCC) and low-current configurations were used in Wertheimer & Leeper, (1979). Subsequent studies expanded the indices to include very high-current configuration (VHCCs), ordinary high-current configuration (OHCCs), ordinary low-current configuration (OLCCs), very low-current configuration (VLCC) and underground cables (UG). Adapted from Kheifets et al., (1997). Table 4. Wire code assessment categories Type of external power line & distance to residence Wire code category Transmission line 3 phase primary distribution Secondary distribution >2 Thick Thin 1st span >2nd span HCC VHCC <15 m <15 m <15 m <7.5 m OHCC <40 m <40 m <40 m <20 m <15 m LCC OLCC <40 m <40 m <40 m VLCC None of the above and secondary service overhead UG None of the above and secondary service underground Table 4. Wire code assessment categories 1.3.1.1 Wire Code Studies Using the modified wire code categories, Savitz et al. (1988) again performed a case- control study in Denver. The odds ratios presented were generally consistent with the corrected odds ratios of Wertheimer and Leeper (1979). However Savitz and co- authors (1988) concluded that their study did not indicate a series of replicated positive studies but rather a body of literature of suggestive, yet inconclusive, results when taken both as an aggregate and individually. Re-analysis of the data using modified wire codes (high-, medium- and low wire codes), further facilitated the production of more precise odds ratio providing an association for leukaemia, 2.9 (CI: 1.5-5.5) (Savitz & Kaune, 1993). Consistent with these earlier studies, London et al. (1991) also observed a statistically significant association between very high exposure classifications and an increased risk for leukaemia. However, in contrast to these earlier studies, McBride et al. (1999) provided little support for an association between ELF-MF exposure and an increased risk of CL, publishing results portraying a statistically non-significant risk associated with exposure pertaining to high wire codes. Similarly, Linet et al. (1997) found no association between residences classified within the highest wire code category in either the modified Wertheimer and Leeper category (Table 4) or the three code’s surrogate employed by Savitz and Kaune (1993). The lack 19 of an association was observed in both the main residence and the one the pregnant mother had resided in (Linet et al., 1997). 1.3.1.2 Field Measurement Studies 1.3.1.2 Field Measurement Studies While the use of wire codes as a surrogate of exposure to determine predicted magnetic field levels eliminates the prospect of socioeconomic bias, no account of exposure from within the home is taken (Kheifets et al., 1997). The development of magnetic field meters further enhanced the accuracy of exposure assessment, allowing for direct measurements of magnetic fields to be undertaken in homes and over extended periods. Importantly, the use of spot measurements does not provide any reference to variability throughout the day or even seasonal variability unless measurements are constantly repeated (Kheifets & Oksuzyan, 2008). A solution is to record measurements over a longer period. These can be either undertaken as personal or location-based measurements, and normally take place over a 24-48 hour period. Tomenius (1986) conducted the first European study in relation to childhood leukaemia and ELF-MF exposure. The analysis was based on residences and their proximity to different types of electrical installations, alongside spot measurements that were taken outside the entrance doors. Statistically significant odds ratios in children exposed to magnetic fields > 0.3 µT were presented for all tumours combined (OR = 2.1), notably however not for leukaemia (OR = 0.3). Similarly, Savitz et al. (1988), reported that measured magnetic fields under low power use conditions (>0.2 µT) when compared to a cut-off of <0.2 µT, had a modest but statistically insignificant association with total cancer incidence (OR = 1.4; CI: 0.63-2.9), observing odds ratios of 1.9 (CI: 0.67-5.6) for leukaemia. Furthermore, the study conducted by London et al. (1991), which was the first to perform long term-exposure (24-hour) measurements of ELF-MF, in addition to spot (single time point) measurements also observed a non- significant elevated odds ratio in association to childhood leukaemia risk (OR = 1.69, 95% CI: 0.71-4.00). Subsequent studies have also found a lack of significant risk of childhood leukaemia (Feychting & Ahlbom, 1993; Coghill et al., 1996; Michaelis et al., 1998; McBride et al., 1999). Equally, there were no associations in the UKCC study (1999), where adjusted odds ratios of 0.92 (95% CI: 0.47-1.79) for acute lymphoblastic 20 leukaemia, 0.90 (0.49–1.63) for all leukaemia and 0.46 (0.11–1.86) for children with mean exposures of >0.2 µT compared with children with mean exposures of <0.1 µT were published (UK Childhood Cancer Study Investigators, 1999). 1.3.1.2 Field Measurement Studies There were however suggestions of a positive association between CL and ELF-MF >0.2 µT (Dockerty et al., 1998), although the study only possessed five cases and one control within this exposure group, and the confidence intervals produced were too wide to form a viable conclusion (CI: 1.1-224). Additionally, data produced by Linet et al. (1997) suggest an increase in risk at fields >0.3 µT, but again the number of cases in which this exposure category was represented was small and, in contrast to Dockerty et al. (1998), no association was witnessed at >0.2 µT. If an effect does in fact exist below 0.2 µT, then it is likely too small to reach consensus in epidemiological studies alone (Greenland et al., 2000). In addition to studies recording spot and long-term measurements, some used the historical loads as recorded by power companies at the time of diagnosis to calculate magnetic field exposure retrospectively (Feychting & Ahlbom, 1993). The use of historical loads to calculate magnetic fields provided some evidence of an increased risk of CL for exposures >0.3 µT (OR = 3.8, 95% CI: 1.4-9.3) (Feychting & Ahlbom, 1993). Likewise, a meta-analysis in 1998 reported a weak elevated risk of leukaemia for children exposed to residential magnetic fields and a positive association between childhood leukaemia and calculated historic fields ≥0.2 μT (OR = 1.90, 95% CI: 1.07- 3.39) (Wartenberg, 1998). However, Tynes and Haldorsen (1997) failed to reproduce such findings, presenting no association between childhood cancer, including leukaemia, and residences near power lines, based on calculations from power line distance and records of load. While, in a further meta-analysis study comprised of risk estimates from 14 case-control studies and one cohort study, no association between childhood leukaemia and calculated magnetic fields from wiring configuration codes could be substantiated, since the heterogeneity amongst individual studies was found to be statistically significant (Angelillo & Villari, 1999). If follows that the considerable variation between studies illustrated throughout this chapter could be partially attributed to the numerous methods of exposure assessment that were employed, but it must also be noted that most studies involved 21 small numbers of cases within the top exposure categories. However, the organisation and review of the existing data may yet provide important insights into the consistency of the results. These analyses have taken form as two separate entities, pooled analysis studies and meta-analyses. 1.3.1.3 Meta- & Pooled Analysis Studies Indeed this led the authors to conclude that, though there was a lack of statistical significance, their results were in line with previous pooled studies. Furthermore, that their lack of significant data does not alter the previous assessment that magnetic fields are possibly carcinogenic (Kheifets et al., 2010). More recently still, Zhao et al. (2014) aimed to further assess the link between childhood leukaemia and magnetic field exposure in a meta-analysis based on circa 25,000 individuals. A meta-analysis, similar to pooled analysis, is a statistical method extensively used in biomedical research, which is specifically useful in providing a more reliable outcome when data from different studies are inconclusive (Lagiou et al., 2002). A single study, taken separately may be either too small or limited in design to generate a conclusive outcome. Thus, by combining the data from similar, previously published studies, to provide a single summary risk estimate, a more reliable representation of the evidence is presented (Vijayalaxmi & Prihoda, 2009). A total of 11,699 cases and 13,194 controls from nine studies between the years of 1997 to 2013 were included under strict selection criteria. Again using <0.1 μT as the reference, a statistically significant association was exhibited between both total childhood leukaemia and more specifically acute lymphocytic leukaemia and magnetic field intensity of > 0.4 μT and, OR = 1.57, 95% CI: 1.03–2.40 and OR = 2.43, 95% CI: 1.30– 4.55, respectively (Zhao et al., 2014). While this indicates some concordance between studies and potentially identifies a very specific exposure limit associated with damage, only a few residences registered average spot- or long-term measurements >0.3 µT (1-4 %) and even fewer (1-2%) >0.4 µT (Ahlbom et al., 2000; Greenland et al., 2000). Considering the small amount of individuals who are in fact exposed to ELF-MFs greater than 0.4 µT, it follows that an estimate of between one and two additional cases (per 500) would arise as a result of artificial ELF-exposure (UK Childhood Cancer Study Investigators, 1999). While the omnipresence of background fields occupy the majority of concern, an b d f d h b d d d l h l h While the omnipresence of background fields occupy the majority of concern, an abundance of studies have been conducted in order to analyse the relationship between ELF-MF exposure from electric blankets and the incidence of several types of cancer. 1.3.1.3 Meta- & Pooled Analysis Studies y Pooled analysis lays the benchmark for synthesising results from multiple studies. Raw data are collected from previous studies which can then be compared free from artefacts introduced by analytical differences for the gleaning of statistically more stable results (Kheifets et al., 2006; Kheifets et al., 2010). However, despite their strengths, results derived from pooled analyses are still prone to the same biases as the original studies were subjected to. Thus in an attempt to provide a more reliable evaluation of inter-study differences in results, two pooled analyses based on studies up to 1999 were published in 2000 (Ahlbom et al., 2000; Greenland et al., 2000). The first was conducted by a team funded through the National Institute of Environmental Health Sciences. Combining studies that used magnetic field measures as their primary analysis, with those that used wire codes, Greenland and co-authors (2000) derived exposure summary estimates from 12 studies based on 2656 cases and 7084 controls. Of those 12, only two (London et al., 1991; Linet et al., 1997) provided more than four cases exposed to >0.4 µT, hence for categorical magnetic field analysis these cases were combined into a single category of exposures > 0.3 µT. A positive association was concluded to exist between ELF-MF and childhood leukaemia when comparing >0.3 µT to exposures <0.1 µT (OR = 1.68, 95% CI: 1.23-2.31) (Greenland et al., 2000). Likewise, a further pooled analysis study showed a two-fold increase in risk of childhood leukaemia during the year prior to diagnosis with exposures > 0.4 µT (RR 2.00, 95% CI: 1.27-3.13) when compared with a reference level exposure of <0.1 µT (Ahlbom et al., 2000). While it is highlighted that exposures >0.4 µT signify a doubling in risk of childhood leukaemia it must also be noted that this exposure category was only representative of 0.8% of subjects (44 cases and 62 controls). A more recent pooled analysis based on primary data from epidemiological studies on residential magnetic fields and childhood leukaemia published post 2000, found a statistical non-significant risk of leukaemia associated with increased exposure( > 0.3 μT when compared to <0.1 μT, OR = 1.44, 95% CI: 0.88-2.36) (Kheifets et al., 2010). However, the overall number 22 of cases and controls were once more small with only 26 cases and 50 controls present within the >0.3 μT category. 1.3.2 Appliance Exposure pp p The use of electric blankets has been investigated in various scenarios, exploring the association attached to childhood leukaemia in direct correlation to the child’s use, while also the mother’s pre- and post-natal use. The majority of studies have obtained similar findings whereby there is no association between both the child’s use and the mother’s pre-/post-natal use of electric blankets and the onset of childhood leukaemia (Savitz et al., 1990; London et al., 1991; Dockerty et al., 1998). Conversely, some also showed that the risk of childhood ALL was significantly increased when mothers had indicated electric blanket use during pregnancy (OR = 1.59, CI: 1.11-2.29) (Hatch et al., 1998). However the lack of dose-response led the authors to conclude that the association found may not reflect MF exposure from electric blankets but with that of external exposure (Hatch et al., 1998). 1.3.1.3 Meta- & Pooled Analysis Studies Unlike the use of surrogates and direct measurements as a method of 23 exposure assessment for background fields, estimation of exposure from appliances is poorly developed, generally consisting of the collection of data on frequency and duration of use via questionnaires (Kheifets & Oksuzyan, 2008). 1.4 Brain Cancer As well as childhood leukaemia, in their original paper Wertheimer and Leeper (1979) also reported that children living close to high-voltage power lines presented increased risk of brain cancer. Brain cancer accounts for around 90% of all nervous system cancers and similar to leukaemia, onset is moderately rare with an annual incidence rate of 6 cases per 100,000 people in the USA (reviewed in Kheifets, (2001)). Brain cancer also correlates to leukaemia in that the causes for onset are likewise poorly understood and known factors, such as certain inherited genetic conditions and ionising radiation, only relate to a small proportion of cases. However, in the wake of additional studies indicating an increased risk of brain cancer in connection to ELF-MF exposure (Tomenius, 1986; Savitz et al., 1988), and in particular two types of malignant gliomas, glioblastoma and astrocytoma, succeeding studies have investigated them further. It originally appeared that the link to childhood brain tumour was more prominent than that of leukaemia (Ahlbom, 1988). Indeed, a more recent study has again published a positive association between high-level exposure (>0.4 µT) and the risk of brain tumours in Japanese children under 15 years of age (Saito et al., 2010). 24 However, the presence of only three cases and one control within this exposure group led to poor statistical precision with hugely variable confidence intervals presented (CI: 1.05-113). In addition, a meta-analysis of childhood brain tumour studies has also indicated a non-statistically significant link when presenting risk estimates with multiple exposure assessment methods (Wartenberg, 1998). The majority of case- control studies however, have failed to provide evidence of a positive association when assessing exposure with a range of methods (Feychting & Ahlbom, 1993; Gurney et al., 1996; Tynes & Haldorsen, 1997; UK Childhood Cancer Study Investigators, 1999), meta-analysis (Mezei et al., 2008) or in conjunction with appliance use either directly by the child or the mother during gestation (Savitz et al., 1990; Preston-Martin et al., 1996). Overall, it can be concluded that there is little to no evidence which currently supports a link between ELF-MF exposure and the development of childhood brain cancer (Kheifets, 2001). 1.5 Adult Studies 1.5 Adult Studies The findings of Wertheimer and Leeper (1979) did not solely relate to childhood diseases as they also studied adults: a follow-up study in 1982 also found adult cancer to be associated with high-current wire configurations that were in close proximity to residences (Wertheimer & Leeper, 1982). In light of these findings, a large number of epidemiological studies were performed to evaluate potential correlation between ELF-MF exposure and cancer. However, the majority of concerns are related to occupational exposure since chronic exposure to levels of ELF-MFs in occupational environments is extensively higher than those in a residential setting. Such occupational studies have used various designs, including population-based case- control studies, occupational cohorts, and death certificate surveys alongside several different exposure assessments, ultimately as a means of measuring either morbidity or mortality. The results of such studies are similarly conflicting, with some studies presenting a potential association between occupations considered to consist of high ELF-MF exposure and both leukaemia and brain cancer. The first occupational study outlined significantly increased mortality rates for total leukaemia occurrences (proportionate mortality ratios, PMR = 1.4, CI: 1.1-1.6), in-particular acute leukaemia along with non-Hodgkin’s lymphomas and malignant brain tumours among male 25 electrical workers (Milham, 1985). The use of job titles taken from a death certificate database was used as a crude method of assessing exposure to magnetic fields. Employment in an ‘electrical’ profession such as an electrical and electronic technician, radio and telegraph operator, electrician, lineman (power and telephone), television and radio repairman, power station operator, welder and flame-cutter, and motion picture projectionist inferred to the researchers a higher than normal exposure to magnetic fields. A significant increase in brain tumour risk (OR = 2.2, CI: 1.1-4.1) also correlated with increasing probability of exposure to magnetic fields in a further case- control study which used occupation to classify exposure to magnetic fields (Lin et al., 1985). In an attempt to resolve the deficiencies in exposure assessment, industry workers wore personal dosimeters and exposures were catalogued. Questionnaires were combined with workplace measurements to establish a job exposure matrix (JEM) (Floderus et al., 1993), which in turn was used to further analyse the link between ELF-MF exposure at work and the risk of leukaemia and brain tumour in central Sweden. 1.5 Adult Studies Jobs with exposures >0.29 µT reported a strong association with all sub-types of leukaemia (OR = 1.6, CI: 1.1-2.4), especially chronic lymphocytic leukaemia, yet not with any other sub-type of leukaemia or with brain cancer (Floderus et al., 1993). However, a further study on cancer mortality, in which the JEM was applied, found no association between an increased risk of either leukaemia or brain cancer mortality amongst electric utility company workers (Sahl et al., 1993). Similarly, a mortality study by Savitz and Loomis (1995) found no association with leukaemia mortality amongst a cohort of electric utility workers, except for those that had worked for more than twenty years as an electrician, in which case there was a 2.5- fold increase in the risk of leukaemia. It is therefore unsurprising that a pooled analysis study including these studies presented no consistent exposure-response relationship or association with leukaemia or brain tumours (Kheifets et al., 1999). electrical workers (Milham, 1985). The use of job titles taken from a death certificate database was used as a crude method of assessing exposure to magnetic fields. database was used as a crude method of assessing exposure to magnetic fields. Employment in an ‘electrical’ profession such as an electrical and electronic technician, radio and telegraph operator, electrician, lineman (power and telephone), television and radio repairman, power station operator, welder and flame-cutter, and motion picture projectionist inferred to the researchers a higher than normal exposure to magnetic fields. A significant increase in brain tumour risk (OR = 2.2, CI: 1.1-4.1) also correlated with increasing probability of exposure to magnetic fields in a further case- control study which used occupation to classify exposure to magnetic fields (Lin et al., 1985). In an attempt to resolve the deficiencies in exposure assessment, industry workers wore personal dosimeters and exposures were catalogued. Questionnaires were combined with workplace measurements to establish a job exposure matrix (JEM) (Floderus et al., 1993), which in turn was used to further analyse the link between ELF-MF exposure at work and the risk of leukaemia and brain tumour in central Sweden. Jobs with exposures >0.29 µT reported a strong association with all sub-types of leukaemia (OR = 1.6, CI: 1.1-2.4), especially chronic lymphocytic leukaemia, yet not with any other sub-type of leukaemia or with brain cancer (Floderus et al., 1993). 1.5 Adult Studies However, a further study on cancer mortality, in which the JEM was applied, found no association between an increased risk of either leukaemia or brain cancer mortality amongst electric utility company workers (Sahl et al., 1993). Similarly, a mortality study by Savitz and Loomis (1995) found no association with leukaemia mortality amongst a cohort of electric utility workers, except for those that had worked for more than twenty years as an electrician, in which case there was a 2.5- fold increase in the risk of leukaemia. It is therefore unsurprising that a pooled analysis study including these studies presented no consistent exposure-response relationship or association with leukaemia or brain tumours (Kheifets et al., 1999). 26 Table 5. The ELF-MF time-weighted average exposures for electrical occupations Occupation Time-weighted average exposure (µT) Lineman (power & telephone) 3.6 Electrician / electrical engineer 1.6 Power station operators 1.4 Welders and flame-cutters 2.0 Motion picture projectionists 0.8 A list of the time-weighted average exposures to ELF-MF for five selected jobs classified as electrical occupations. Adapted from IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, (2002). Originally cited in Portier & Wolf, (1998). Table 5. The ELF-MF time-weighted average exposures for electrical occupations Occupation Time-weighted average exposure (µT) Lineman (power & telephone) 3.6 Electrician / electrical engineer 1.6 Power station operators 1.4 Welders and flame-cutters 2.0 Motion picture projectionists 0.8 he ELF-MF time-weighted average exposures for electrical occupations 1.6 Breast Cancer Similarly, the long-term exposure to ELF-MF has been hypothesised to increase the risk of breast cancer (Stevens, 1987). The idea was based on experimental evidence which suggested that ELF-MF affects the pineal glands production of the hormone melatonin in the same way that light does at night. Melatonin production follows a specific circadian pattern: low levels in daylight and high levels in darkness, and it is believed that high levels of melatonin could provide a protective effect against the development of breast cancer. Thus it is thought 50 and 60 Hz fields suppress melatonin production ultimately affecting oestrogen production by the ovary. Since then, and over the subsequent three decades, a considerable amount of epidemiological studies have analysed a potential association with breast cancer from ELF exposure in residential and occupational environments, including from electric blankets. However, most epidemiological studies have indicated little or no overall effect of ELF-MF exposure risks and similar findings are reported in the majority of studies of bed heating devices (Verkasalo et al., 1996; Li et al., 1997; Coogan & Aschengrau, 1998; Feychting et al., 1998; Schoenfeld et al., 2003; Elliott et al., 2013). Studies have been established to explore both pre-menopausal (Vena et al., 1994) and post-menopausal breast cancer in conjunction with electric blanket use (Vena et al., 1991; McElroy et al., 2001). However, currently no association to breast cancer risk has been reported in either pre- or post-menopausal women using electric blankets (Gammon et al., 1998; Laden et al., 2000; Zheng et al., 2000b; Kabat et al., 2003). An exception is from a Norwegian case-control study (Kliukiene et al., 2004), where exposures were calculated based on nearby power lines. Effects were visible at quite low exposure levels, as any exposure to magnetic fields >0.05 µT was associated with 27 an increased risk of breast cancer. Similarly, the general outcomes from studies of occupational exposure did not indicate any effects either (Coogan & Aschengrau, 1998; Kliukiene et al., 2004; Forssén et al., 2005; McElroy, 2007). Furthermore, the meta- analysis studies (Erren, 2001; Chen et al., 2010) which, between them, summarised the literature available from 1966-2009, including studies analysing residential and occupational exposures, as well as those from electric blankets. In all cases, both studies were in agreement that there was very little association between ELF-MF exposure and a proneness to female breast cancer. 1.6 Breast Cancer Overall the evidence available is consistently negative and does not suggest an increased risk of breast cancer related to ELF-MF exposure. Indeed the evidence is sufficient enough for Feychting (2013) to conclude with confidence that ELF-MFs do not cause breast cancer and that focus should instead switch to developing more promising hypotheses on biophysical mechanisms. Indeed if there truly is a link between residential and occupational exposure to ELF-MF and cancer (excluding leukaemia) the constant presence of inadequate sample sizes results in inconsistent data and lack of a universally accepted conclusion. 1.8 Neurodegenerative Disease 1.8 Neurodegenerative Disease Due to an aging population neurodegenerative diseases are becoming more frequent, thus representing an extensive burden on public health. Since familial cases only account for a small percent of the vast majority of these diseases’ aetiology, it can be safely assumed that lifestyle coupled with environmental factors play an essential role in their onset. Although not well developed, the potential effects of ELF-MF have been extensively researched in numerous epidemiological studies as a potential risk related to neurodegenerative diseases. Research has predominantly investigated the link to Alzheimer’s disease (AD) and motor neuron disease, particularly amyotrophic lateral sclerosis (ALS). The risk of Parkinson’s disease (PD) and dementia in general have also been analysed but to a lesser extent. 1.7 Cardiovascular Disease 1.7 Cardiovascular Disease Reports have also surfaced in the 1960’s linking ELF-MF exposure to cardiovascular irregularities and more recent studies further link exposure with a reversible relationship to reduced heart rate variability (HRV) (Sastre et al., 1998). Although a pooled study conducted within the same institute later failed to replicate such findings (Graham et al., 2000), a hypothesis was established that ELF-MF could affect heart rate variability (Savitz et al., 1999). It was theorised following evidence of an increased mortality from acute myocardial infarction and arrhythmia-related cardiovascular disease, but not chronic coronary heart disease, which was observed in workers employed for longer durations in occupations with elevated ELF magnetic field exposures (Savitz et al., 1999). Subsequent studies, including one directly replicating the original study (Sahl et al., 2002), and others specifically designed to replicate these findings through analysis of various specified endpoints, mostly failed to find any association of increased myocardial infarction morbidity (Ahlbom et al., 2004), arrhythmia (Johansen et al., 2002) or total cardiovascular disease mortality (Mezei et 28 al., 2005; Cooper et al., 2009; Koeman et al., 2013). A solitary study however did evoke some support for the original hypothesis (Savitz et al., 1999), observing a non- significantly increased risk for acute myocardial infarction (Håkansson et al., 2003). However, the only two studies to illustrate an association between occupational exposure and ELF-MF relied on mortality records as a method of exposure assessment. Thus, when taken as a whole, the evidence does not support the notion of ELF-MF as a causative factor in the development of cardiovascular disease (Kheifets et al., 2007). 1.8.1 Alzheimer’s Disease The risk of Alzheimer’s disease was initially outlined to be elevated 3-fold in association with primary occupations of low intensity ELF-MF exposure (Sobel et al., 1995), findings which have later been partially supported by Feychting et al. (1998). Subsequent cohort studies have then provided evidence that long-term occupational exposure to higher ELF-MF intensities (>0.2 µT) also increased risk of AD 2.3-fold (Qiu et al., 2004), alongside that of AD mortality (Savitz et al., 1998; Feychting et al., 2003). Furthermore, age-specific analyses also indicated a stronger association with the onset of AD, with a proclivity for early onset AD mortality (<75) (Feychting et al., 2003). Conversely and in contrast to the data presented by the aforementioned studies, there has also been evidence presented which does not support the proposition that above- average levels of ELF-MF are related to the onset of AD (Johansen, 2000; Noonan et al., 2002). Similarly, Sorahan and Mohammed (2014) also failed to establish a statistically The risk of Alzheimer’s disease was initially outlined to be elevated 3-fold in association with primary occupations of low intensity ELF-MF exposure (Sobel et al., 1995), findings which have later been partially supported by Feychting et al. (1998). Subsequent cohort studies have then provided evidence that long-term occupational exposure to higher ELF-MF intensities (>0.2 µT) also increased risk of AD 2.3-fold (Qiu et al., 2004), alongside that of AD mortality (Savitz et al., 1998; Feychting et al., 2003). Furthermore, age-specific analyses also indicated a stronger association with the onset of AD, with a proclivity for early onset AD mortality (<75) (Feychting et al., 2003). Conversely and in contrast to the data presented by the aforementioned studies there Conversely and in contrast to the data presented by the aforementioned studies, there has also been evidence presented which does not support the proposition that above- average levels of ELF-MF are related to the onset of AD (Johansen, 2000; Noonan et al., 2002). Similarly, Sorahan and Mohammed (2014) also failed to establish a statistically 29 significant trend associated with risk of AD mortality and increased ELF-MF exposure either over recent or primary occupations, or over a lifetime occupation in a cohort of UK electricity supply workers. Again, inconsistencies on the basis of study design, the examination of morbidity or mortality coupled to the standard of exposure assessment has resulted in discrepancies between the data published. 1.8.1 Alzheimer’s Disease Ultimately, the data in which the risk of Alzheimer’s disease is increased due to ELF-MF exposure are weak. 1.8.2 Amyotrophic Lateral Sclerosis 1.8.2 Amyotrophic Lateral Sclerosis Another disease that has been extensively focused upon is motor neuron disease, the most common of which is amyotrophic lateral sclerosis (ALS), a fatal disease which permanently damages the nerve cells in the brain and spinal cord. A hypothesis whereby high occupational ELF-MF exposure increased the risk of ALS was further accentuated by a small case-control study which reported a potential 2.5-fold increase in the risk of ALS in relation to occupational exposure to ELF-MF for more than 20 years (Davanipour et al., 1997). However the authors cautioned that owing to their method of exposure assessment and the small numbers of cases and controls used, their findings required confirmation in larger studies that use quantitative exposure data. Further support for an association was later offered from several well-designed cohort studies (Savitz et al., 1998; Johansen, 2000). Conversely, the results from studies which used the job exposure matrices (JEMs) to estimate MF exposure have been less consistent. Two studies reported ALS was not associated with ELF-MF exposure at any level (Noonan et al., 2002; Feychting et al., 2003), but both studies did in fact report significant associations of ALS attached with a history of workers in electrical professions, (OR = 2.30, 95% CI: 1.29-4.09) and (OR = 1.4, 95% CI: 1.1-1.9) respectively. Vergara and co-authors (2015) also applied JEM to analyse the relationship between occupational exposure to MFs and ALS mortality. Similarly, they did not find increased ALS mortality odds within high MF exposure categories when compared to low exposure but, consistent with previous publications, an association between electrical professions and ALS was observed. More recently, Sorahan and Mohammed (2014) investigated the risks of ALS mortality in a cohort of UK electricity supply workers and, unlike any of the aforementioned studies, failed to establish a 30 statistically significant trend associated with risk of ALS and increased ELF-MF exposure either over a lifetime, recent or primary occupations. Additionally, two meta-analysis studies have analysed the previously reported associations of occupational magnetic field exposure with amyotrophic lateral sclerosis. Zhou et al. (2012) reported significant increases in risk of ALS due to ELF-MF exposure based on 9 case-control studies (RR = 1.39) but not in 8 cohort studies (RR = 1.16, CI: 0.80-1.69). Similar findings are illustrated in the meta-analysis from Vergara et al. 1.8.2 Amyotrophic Lateral Sclerosis (2013), who analysed 12 case-control studies reporting a significant increased risk of 1.38 for ALS, but again a weak associated risk of 1.14 from 9 cohort studies. The fact that ALS associations are stronger in studies which use occupational titles as a means of exposure measurement, rather than MF levels, suggests that magnetic fields are in fact not the explanation for the observed association of job titles with ALS. 1.8.3 Parkinson’s Disease The risk of Parkinson’s disease has been analysed to a lesser extent than AD and ALS because epidemiological studies using either utility cohort studies, (Savitz et al., 1998; Johansen, 2000; Feychting et al., 2003; Roosli et al., 2007; Sorahan & Mohammed, 2014) or a mortality registry study (Davanipour et al., 1997) have failed to provide any evidence of an association between elevated occupational ELF-MF exposure and PD. A meta-analysis study also failed to report any association between MF and PD (Vergara et al., 2013). Conversely, a positive association was observed in three separate methods of exposure assessment performed within one study (Noonan et al., 2002). However, owing to the inconsistent findings of this study, this positive association would need to be independently replicated in order to provide reliable support for an association between occupational ELF-MF exposure and Parkinson’s disease. In conclusion, there have been many studies into exposure to ELF-MF and risks of the collective group of neurodegenerative diseases, including several meta-analyses. In conclusion, there have been many studies into exposure to ELF-MF and risks of the collective group of neurodegenerative diseases, including several meta-analyses. Based on the literature, although largely contradictory and ultimately inconclusive, it is widely accepted that the extent of excess risk for Alzheimer’s, Parkinson’s and motor neurone disease assigned to occupational magnetic field exposure is probably small or, with regard to Parkinson’s disease, most probably nil. Even in the case of AD, where studies ascribed a potential association to mortality, there remains a wide scope for 31 potential publication bias and misclassification of the disease accounting for the observable risk (Johansen, 2000; Vergara et al., 2013). Although epidemiological studies form the main body of evidence linking ELF-MF exposure to potential health effects, interpretation of data is problematic owing to inconsistent and unreliable methods of exposure assessment. In addition to this, any positive association related to an epidemiological study is based upon health risk estimates in human populations heavily biased towards cancer risk, while in vitro and in vivo studies represent a more reliable method of determining any potential genotoxic and carcinogenic effects of ELF-MFs through the use of defined exposure conditions and a variety of genotoxic endpoints. 1.8.3 Parkinson’s Disease Yet, despite the numerous experimental studies performed using these improved parameters, their ability to evaluate whether power-frequency magnetic fields induce DNA damage has thus far failed to provide conclusive evidence for the mutagenic effects of extremely low- frequency magnetic fields (AGNIR, 2001). It is however obstructive that the specific causes for the development of different types of human cancer are still poorly understood. 1.9 Experimental Studies p The development of cancer is a multistep process in which somatic cells acquire mutations in a specific clonal lineage (Loeb et al., 2003). The current understanding in the causation of cancer points to at least a two pathway system, of which neither path is mutually exclusive: a genotoxic pathway and an epigenetic pathway (Vijayalaxmi & Obe, 2005). Yet in contrast to ionising radiation where the effects of exposure have been well established and the subsequent DNA damage has been extensively studied (reviewed in Lara et al., (2015)), there is currently no established mechanistic model for extremely low-frequency magnetic fields. The current epidemiological data however imply that extremely low-frequency magnetic fields may destabilise the genome of exposed cells, although it has been often outlined that ELF-MFs are themselves not genotoxic (Lacy-Hulbert et al., 1998; McCann et al., 1998). The process of a genotoxic pathway therefore assumes that ELF-MF exposure possesses the capability to induce DNA damage in a cell. Unrepaired DNA damage will result in DNA single- (SSBs) and double-strand breaks (DSBs), which are responsible for genomic 32 instability. Moreover, the repair of DSBs is also an error-prone process, and incorrect repair of DSBs can result in the formation of chromosomal aberrations (CAs), micronuclei (MN), sister chromatid exchanges (SCE) and mutations that ultimately lead to carcinogenesis. Correspondingly, their unrepaired presence in germ cells has also been known to result in reproductive and birth defects. As a result, the possible defects to development and spermatogenesis in relation to ELF-MF exposure have also been confronted in various animal models. For the most part however, the results between studies are relatively consistent in illustrating a lack of adverse effects in regards to both development (reviewed in (Juutilainen, 2003; Juutilainen, 2005)) and spermatogenesis (Duan et al., 2014). Conversely, the experimental data produced in relation to the potential genotoxicity of ELF-MF exposure are reminiscent of that produced by epidemiological studies in that it is moderately conflicting. This point was typified by the intra-laboratory inconsistencies published by Luukkonen et al. (2011) and Luukkonen et al. (2014) who in contrast to their earlier study (Luukkonen et al., 2011), more recently illustrated that ELF-MF exposure (100 µT, 24 hours) was able to induce micronuclei in human SH-SY5Y neuroblastoma cells at 8 and 15 days post- exposure (Luukkonen et al., 2014). 1.9 Experimental Studies Despite this, the majority of studies conducted to date have found no consistent evidence of magnetic field induced effects on genotoxicity using an array of field intensities: 0.001-1 mT (Paile et al., 1995; Luceri et al., 2005; Stronati et al., 2004; Testa et al., 2004; Luukkonen et al., 2011; Saha et al., 2014), and 5-400 mT (Antonopoulos et al., 1995; Tateno et al., 1998; Miyakoshi et al., 2000). Yet there have also been several studies, both in vivo and in vitro where positive correlations have been reported; for example a significant three-fold elevation in SCE was observed in mouse m5S cells exposed to 400 mT (50 Hz) for 40 hours (Yaguchi et al., 1999). Indeed one of the first attempts to analyse the direct genotoxic effects of ELF-MF in vivo was a study by Lai and Singh (1997). The authors reported that acute exposure (2 hours) of adult rats (Sprague-Dawley) to a 60 Hz MF at flux densities of 0.1, 0.25 and 0.5 mT, caused a dose-dependent increase in DNA single strand breaks in brain cells harvested at 2 and 4 hours post-exposure, while a dose-dependent increase in DSBs was also witnessed but only at 0.25 and 0.5 mT. However, several attempts have been made to 33 independently validate these findings, using similar exposure conditions and experimental methods (alkaline comet assay performed on whole brain homogenates) but proved unsuccessful (McNamee et al., 2002, 2005). The sensitivity of such assays had previously been reported to equate to 10-50 cGy of X-irradiation in lymphocytes (McNamee et al., 2000). Furthermore, earlier findings by Frazier et al. (1990) contradict those of Lai and Singh (1997) who reported the presence of strand breaks 4 hours after exposure to MF. Frazier and co-authors (1990) assessed the repair kinetics of DNA SSBs in the presence of 60 Hz magnetic field of 1 mT, noting that within 20 minutes of exposure between 50 to 75% of the induced SSBs had been repaired, while the majority of the remaining damage was repaired after 3 hours. However, a more recent study by Lai and Singh (2004) once again illustrated a significant increase in DNA damage in brain cells from whole brain homogenates taken from rats after 24- or 48 hours continuous exposure to 60 Hz, 10 µT magnetic fields (Lai & Singh, 2004). 1.9 Experimental Studies No significant difference in the 34 amount of DNA breaks were detected between continuously- and sham-exposed cells using either the alkaline or neutral comet assay (Ivancsits et al., 2002). However, previous data published by Nordenson et al. (1994) indicated a significant ~3-fold increase of chromosomal aberrations in human amniotic cells exposed to a 50 Hz, 30 µT intermittent field for 72 hours (20 seconds on/20 seconds off). These findings are perhaps unsurprising given that continuous ELF-MF exposure as part of our daily environment is moderately sparse, since different electrical household appliances produce a range of exposure levels over short periods. Similarly, these findings may also suggest that biological and possibly adverse health effects are potentially related to exposure to intermittent magnetic fields (Kheifets et al., 1997). However subsequent attempts to replicate the findings observed by Nordenson and co-authors (1994) have thus far proven unsuccessful, with Galt et al. (1995) illustrating decreased chromosomal aberrations in similarly exposed amniotic cells. Nevertheless, Ivancsits and co-authors (2002) subsequently concentrated on optimising intermittent exposure conditions for maximal effect on DNA strand break levels. As such, intermittent on/off times were increased in 5 minute increments while the other experimental parameters remained constant (50 Hz, 1 mT for 24 hours). The highest level of DNA strand breaks in both the alkaline and neutral comet assays was induced using intermittent exposures of 5 minutes on and 10 minutes off. Furthermore, by using the optimal intermittent exposure conditions and varying the magnetic flux density between 0.02- 2 mT, significant increases were observed in both the alkaline and neutral comet assay at flux densities as low as 70 µT (Ivancsits et al., 2002). Ensuing studies using identical exposure conditions (50 Hz sinusoidal, 1 mT, intermittent 5 min on/10 min off) for a period of 1 to 24 hours also reported an increase in alkaline and neutral comet tail factors in a time-dependent manner, with the largest being at 15 hours (Ivancsits et al., 2003b). The assay levels declined thereafter, without returning to basal levels. Moreover, increasing the magnetic flux density between 0.02-1 mT (15 hours, intermittent 5 min on/10 min off) resulted in a significant increase in DNA SSBs and DSBs at 35 µT (Ivancsits et al., 2003b). Finally following this series of experiments the amount of DNA breaks were detected between continuously- and sham-exposed cells using either the alkaline or neutral comet assay (Ivancsits et al., 2002). 1.9 Experimental Studies independently validate these findings, using similar exposure conditions and experimental methods (alkaline comet assay performed on whole brain homogenates) but proved unsuccessful (McNamee et al., 2002, 2005). The sensitivity of such assays had previously been reported to equate to 10-50 cGy of X-irradiation in lymphocytes (McNamee et al., 2000). Furthermore, earlier findings by Frazier et al. (1990) contradict those of Lai and Singh (1997) who reported the presence of strand breaks 4 hours after exposure to MF. Frazier and co-authors (1990) assessed the repair kinetics of DNA SSBs in the presence of 60 Hz magnetic field of 1 mT, noting that within 20 minutes of exposure between 50 to 75% of the induced SSBs had been repaired, while the majority of the remaining damage was repaired after 3 hours. However, a more recent study by Lai and Singh (2004) once again illustrated a significant increase in DNA damage in brain cells from whole brain homogenates taken from rats after 24- or 48 hours continuous exposure to 60 Hz, 10 µT magnetic fields (Lai & Singh, 2004). Similarly, a dose-dependent increase in DNA damage was reported in three cell lines (HL-60 leukaemia cells, Rat-1 fibroblasts and WI-38 diploid fibroblasts) exposed for 3- 72 hours to 0.5-1.0 mT, 50 Hz ELF-MF, while genotoxicity peaked at 24 and 72 hours (Wolf et al., 2005). These discrepancies illustrate how the comparison of any corresponding data generated from experimental studies is difficult owing to differences between exposure conditions, designs and methods (Ruiz-Gomez & Martinez-Morillo, 2009). Thus a collaborative European Union funded project known as the REFLEX project was established in an attempt to verify the data under controlled and standardised conditions and thereby eliminate any potential inter-laboratory inconsistencies. A series of studies from several laboratories ultimately led to a plethora of publications analysing the genotoxic effects of ELF-MF exposure (Ivancsits et al., 2002, 2003a, 2003b, 2005, Winker et al., 2005). The initial study was established to analyse the effects of continuous exposure to a 50 Hz sinusoidally magnetic field of 1 mT for 24 hours on cultured human diploid fibroblasts (Ivancsits et al., 2002). Discrimination between DNA single- (SSB) and double-strand breaks (DSB) was achieved through the use of two separate assays: the alkaline comet assay detected both SSB and DSB, while the neutral comet assay was used to solely detect DSBs. 1.9 Experimental Studies However, previous data published by Nordenson et al. (1994) indicated a significant ~3-fold increase of chromosomal aberrations in human amniotic cells exposed to a 50 Hz, 30 µT intermittent field for 72 hours (20 seconds on/20 seconds off). These findings are perhaps unsurprising given that continuous ELF-MF exposure as part of our daily environment is moderately sparse, since different electrical household appliances produce a range of exposure levels over short periods. Similarly, these findings may also suggest that biological and possibly adverse health effects are potentially related to exposure to intermittent magnetic fields (Kheifets et al., 1997). However g subsequent attempts to replicate the findings observed by Nordenson and co-authors (1994) have thus far proven unsuccessful, with Galt et al. (1995) illustrating decreased chromosomal aberrations in similarly exposed amniotic cells. Nevertheless, Ivancsits and co-authors (2002) subsequently concentrated on optimising intermittent exposure conditions for maximal effect on DNA strand break levels. As such, intermittent on/off times were increased in 5 minute increments while the other experimental parameters remained constant (50 Hz, 1 mT for 24 hours). The highest level of DNA strand breaks in both the alkaline and neutral comet assays was induced using intermittent exposures of 5 minutes on and 10 minutes off. Furthermore, by using the optimal intermittent exposure conditions and varying the magnetic flux density between 0.02- 2 mT, significant increases were observed in both the alkaline and neutral comet assay at flux densities as low as 70 µT (Ivancsits et al., 2002). Ensuing studies using identical exposure conditions (50 Hz sinusoidal, 1 mT, intermittent 5 min on/10 min off) for a period of 1 to 24 hours also reported an increase in alkaline and neutral comet tail factors in a time-dependent manner, with the largest being at 15 hours (Ivancsits et al., 2003b). The assay levels declined thereafter, without returning to basal levels. Moreover, increasing the magnetic flux density between 0.02-1 mT (15 hours, intermittent 5 min on/10 min off) resulted in a significant increase in DNA SSBs and DSBs at 35 µT (Ivancsits et al., 2003b). 1.9 Experimental Studies Finally following this series of experiments the authors were able to define the optimum conditions at which the maximum effect on SSB- and DSB was evoked: 100 µT, intermittent (5 min on/10 min off), for 15 hours with human fibroblast cells from donors over 40 years of age, produced a ~2-fold 35 increase in DSB and a 4-fold increase in DSB plus SSB (Ivancsits et al., 2003a, 2003b). Although these effects were predominantly seen in human fibroblast cells, similar effects were also observed in human melanocytes and rat granulosa cells; however no detectable damage was witnessed in human lymphocytes, human monocytes or human skeletal muscles cells (Ivancsits et al., 2005). Comparable results were observed by Luceri et al. (2005) who failed to observe an increase in DNA damage in human blood lymphocytes exposed to 50 Hz magnetic fields at 1, 10 or 100 µT for 18 hours and more recently by Focke et al. (2010), who designed a study in an attempt to validate similar previously conducted studies (Ivancsits et al., 2002, 2003a, 2003b, 2005). Focke and co-authors (2010) were able to replicate an ELF-MF exposure dependent increase in comet tail DNA using three cell models of primary human fibroblasts (ES-1, HR-1d and MRC-5) intermittently exposed to 50 Hz ELF-MF at a flux density of 1 mT. However, statistical significance was smaller and with higher experimental variation than those previously presented by Ivancsits and co-authors (2002), while HeLa cancer cells were also not significantly affected (Focke et al., 2010). It could therefore be hypothesised that any observed inter-laboratory inconsistencies could be due to alternate responses to field exposure between cell types. However Kim et al. (2010) illustrated that DSBs were induced not only in normal human fibroblasts, but also HeLa cervical cancer cells, by both a continuous exposure of 6 mT for 30 minutes and an intermittent exposure of 6 mT for 30 minutes every 24 hours over 3 days. Further studies were performed within the REFLEX programme which analysed different genotoxic endpoints. ELF-MF exposure of intermittent fields (5 min on/10 min off) at a flux density of 1 mT for 10 hours resulted in a significant 3-fold increase in micronuclei and a 10-fold increase in chromosomal aberration frequency above sham-exposure levels (Winker et al., 2005). 1.9 Experimental Studies Several subsequent studies have attempted to replicate these positive findings in a bid to finally obtain a general consensus of consistent data, however Scarfi et al. (2005) failed to confirm the REFLEX programme’s findings using the alkaline comet and micronucleus assays, despite using identical fibroblast line and exposure conditions. The reproducibility of data however must be achieved through the use of the most sensitive assay available at the time. The use of the comet assay for measurement of The reproducibility of data however must be achieved through the use of the most sensitive assay available at the time. The use of the comet assay for measurement of 36 DSB is relatively insensitive and also unable to produce exact measurements of DSB yields (Burdak-Rothkamm et al., 2009). Indeed the use of the γH2AX assay in which the sites of DSB are highlighted through the use of a fluorescently-tagged monoclonal antibody represents an advancement in the detection of DSBs over the conventional comet assays (reviewed in Kuo & Yang, (2008)). The formation of γ-H2AX foci which represents the phosphorylated form of histone H2AX is specific to DNA double-strand breaks. Indeed the phosphorylation of H2AX is one of the primary characteristics of DSB in eukaryotes (Rogakou et al., 1998). Therefore Burdak-Rothkamm and co-authors (2009) re-assessed the genotoxic potential of ELF-MF by using the γH2AX assay as well as comparable techniques and parameters: VH25 fibroblast cells exposed to intermittent 50 Hz ELF-MFs at numerous flux densities (50, 100, 500 and 1000 µT) for 15 hours (5 min on/10 min off), alongside a commercial MF exposure system almost identical to that used in the REFLEX programme studies. A 15 hour continuous field was simultaneously analysed for each of the defined magnetic flux densities while a positive control (X-rays) dose-response curve was also defined for both the alkaline comet and γH2AX assays. Yet, despite the fact that the γH2AX method possessed the ability to reliably detect DNA damage equivalent to an X-ray dose of 0.025 Gy, neither the alkaline comet assay nor the gamma γH2AX assay could detect significant damage at the DNA breakage level in the MF exposed fibroblasts and thus confirm the REFLEX programme’s data. Similarly, neither the chromosome aberration nor the micronucleus assay detected any chromosomal damage in the MF exposed fibroblast cells (Burdak- Rothkamm et al., 2009). 1.9 Experimental Studies These findings are in conjunction with those of Scarfi and co- authors (2005) and more recently still Saha and co-authors (2014). Whilst using similar experimental parameters (50 Hz intermittent, (5 min on/10 min off) magnetic field at 300 µT for 15 hours), the findings of Saha et al. (2014) provide further evidence of an inability of ELF-MFs to cause direct DNA damage. Owing to its rapid recruitment to sites following the introduction of DNA DSBs, Saha and co-authors quantified the tumour suppressor p53 binding protein 1 (53BP1) foci numbers as a direct signalling response to the formation of DSBs. The quantification of 53BP1 foci numbers as a 37 equate to approximately 0.233 DSB per cell, which corresponds to an estimated dose of 22 mGy (Saha et al., 2014). Whilst the establishment of the collaborative REFLEX programme further added to the inconsistent evidence regarding ELF-MFs genotoxic potential, the use of meta-analysis studies provides a method capable of counteracting inter-laboratory inconsistencies in data (Vijayalaxmi & Obe, 2005; Ruiz-Gomez & Martinez-Morillo, 2009). Indeed Vijayalaxmi and Obe (2005) utilised such an analysis, pooling together data from 63 in vitro studies conducted over a period of 13 years using human and animal somatic cells to analyse the overall effects of ELF-MF exposure, using single and/or double strand breaks, chromosomal aberrations, micronuclei and sister chromatid exchange as end points to define genetic damage. They found 22% of studies presented an increase in genetic damage in ELF-MF exposed cells. Comparatively, 46% of studies demonstrated no increase in damage while the remaining 32% of studies were inconclusive in their findings. More recently, similar results were observed when analysing solely DNA strand breaks induced by magnetic fields: 37.5% of studies found induction of DNA strand breaks while 62.5% of studies reported no effects (Ruiz-Gomez & Martinez- Morillo, 2009). While ELF-MF could potentially be carcinogenic, the culmination of these findings appears to support the inability of ELF-MF exposure to cause a direct carcinogenic effect and fundamentally any potential role in the initiation of carcinogenesis can be confidently excluded. However, as was previously mentioned, the current While ELF-MF could potentially be carcinogenic, the culmination of these findings appears to support the inability of ELF-MF exposure to cause a direct carcinogenic effect and fundamentally any potential role in the initiation of carcinogenesis can be confidently excluded. 1.9 Experimental Studies However, as was previously mentioned, the current understanding in the causation of cancer points to at least a two pathway system, the first of which being a genotoxic pathway while the second assumes ELF-MF partakes in i i l (Vij l i & Ob 2005) understanding in the causation of cancer points to at least a two pathway system, the first of which being a genotoxic pathway while the second assumes ELF-MF partakes in an epigenetic role (Vijayalaxmi & Obe, 2005). 1.10 Combined ELF and Carcinogen Exposure g p An epigenetic role assumes that isolated ELF-MFs may not be genotoxic, but rather that the effects of ELF-MF exposure could either be enhanced or rather potentiate the effects of environmental carcinogenic agents such as chemicals, ionising radiation or ultraviolet (UV) radiation. Since humans are exposed to various combinations of an array of different carcinogens at changeable levels, exposure to ELF-MF could potentially act in a promotor, co-promotor or enhancer through either simultaneous or 38 sequential exposure, ultimately contributing towards tumour progression (Juutilainen et al., 2000). Indeed a study had previously reported that mice experienced enhanced skin tumorigenesis when exposed to UV radiation in conjunction with magnetic fields (Kumlin et al., 1998). Additionally, evidence was presented that 50/60 Hz magnetic field exposure at flux densities of 50 and 400 mT potentiated a small but significant increase in the X-ray induced DNA strand breaks in human glioma cells (Miyakoshi et al., 1999, 2000). Similar statistically significant enhanced chromatid-type aberrations were observed in mouse m5S cells pre-exposed to mitomycin C or 3 Gy X-rays followed by 60 Hz, 50 mT or 50 Hz, 400 mT for 40 hours (Yaguchi et al., 2000). Moreover, multiple studies signified that cells pre-treated with the tumour initiator benzo(a)pyrene (BP), and then co-exposed with benzene and 0.8-1 mT ELF-MF introduced an increase in the comet tail moment (Moretti et al., 2005), as well as a significant dose dependent increase in the frequency of sister chromatid exchanges and micronuclei formation (Cho & Chung, 2003). A combination of 3 mT ELF-MF and xenobiotic (N-methyl-N0-nitro-N-nitrosoguanidine (MNNG)) induced DNA damage in human leukocytes (Villarini et al., 2006), while exposure to ELF-MF at field strengths as low as 100 µT for 24 hours have been illustrated to increase the menadione-induced DNA damage and MN formation in human neuroblastoma cells (Luukkonen et al., 2011). The majority of studies in which a positive correlation was discovered were as a result of ELF-MF exposure preceding the known carcinogen (Juutilainen et al., 2006). These results indicate that ELF-MF is more likely to function as an enhancer and amplify the initial DNA damage by the known carcinogen instead of acting as the initiator of the original mutagenic event (Cho & Chung, 2003). 1.10 Combined ELF and Carcinogen Exposure However the majority of these co-carcinogenicity studies are either inconclusive or negative in their findings (Juutilainen et al., 2000; Vijayalaxmi & Obe, 2005), despite using numerous cytogenic assays (comet, MN and SCE) and combined treatments with multiple mutagens (mitomycin C or X-rays) at a range of high 50 Hz flux densities (80- 1740 mT) (Maes et al., 2000). Similarly, a recent series of studies performed by the same laboratory demonstrated that 4 or 16 hour exposure to 60 Hz, 1 mT ELF-MF in combination with ionising radiation (1 Gy X-rays) or H2O2 or cellular myelocytomatosis 39 oncogene (c-Myc) activation did not have either an enhancing or synergistic effect on MN formation (Jin et al., 2012) or primary DNA damage (Jin et al., 2014). However, more recently the authors re-analysed the effects of ELF-MF exposure on DNA damage response in combination with the same mutagens through the formation of γ-H2AX foci (Yoon et al., 2014). Similar to their earlier published data (Jin et al., 2012, 2014), 1 mT ELF-MF in combination with 1 Gy X-rays, or H2O2 or c-Myc activation did not potentiate the formation of γ-H2AX foci (Yoon et al., 2014). However, when the field strength was increased to 2 mT over the same duration (6 hours), a combination of ELF-MF and ionising radiation was capable of inducing γ-H2AX, yet both H2O2 and c- Myc remained incapable of this. It should also be noted that a 6 hour exposure of a 2 mT ELF-MF administered alone induced an increase in γ-H2AX, which is in contrast to the earlier data reported by Burdak-Rothkamm and co-authors (2009). The significance of previous findings has been portrayed as uncertain by Mairs et al. (2007) who claim that the determination of ELF-MFs carcinogenic potential to this date had been based chiefly on studies that, while adept at detecting widespread and extreme cellular damage, were unable to expose any potential subtle molecular alterations. In an attempt to detect any small-scale potential genetic damage the authors developed a more sensitive method of DNA-damage detection involving examination of microsatellite DNA sequences. The use of these non-coding DNA sequences in identifying radiation-induced mutations had previously been shown to represent at least a 1000 times more sensitivity than analysis of the hypoxanthine- guanine phosphoribosyl transferase (HPRT) gene (Boyd et al., 2000). 1.10 Combined ELF and Carcinogen Exposure Through the analysis of three types of microsatellite sequence mutation (change in allele size, loss of heterozygosity and allelic imbalance), a 50 Hz magnetic field of 1 mT in strength was shown to increase the mutagenic capacity of 0.3 and 3 Gy γ-irradiation by factors of 2.62 and 2.75 respectively (Mairs et al., 2007). Despite these findings it is essential that the validity of the positive epidemiological results be corroborated with long-term carcinogenicity studies in animals. As the onset of childhood leukaemia potentially represents the sole cancer-associated end-point to date, numerous animal studies have also focussed on the analysis of whether 50/60 Hz 40 ELF-MF exposures can alter the incidence or onset of leukaemia and lymphomas in rodents (reviewed in Boorman et al., (2000)). 1.11 Experimental Evidence for Carcinogenicity of ELF-MF These studies have several advantages over epidemiological studies in that exposure can be directly assessed while in epidemiological studies direct human measurements are often absent. However, such tests then need to be sensitive enough to extrapolate the data and predict accurately the carcinogenic risk to humans. This is generally accepted to be the case as 84% of carcinogenic agents in humans correlate in experimental animal studies. Indeed the correlation could have been higher but seven remaining agents had not been adequately tested (Wilbourn et al., 1986). Additionally, for those exposures which illustrated carcinogenicity in both humans and experimental animals, there is a common target organ in at least one animal study (Wilbourn et al., 1986). Moreover, in animal studies specifically analysing leukaemia and lymphoma, 100% of the chemicals that result in hematopoietic tumours in humans did so in rodents (Boorman et al., 2000). However, animal studies performed on a selection of rodent models have proven consistently negative for 50/60 Hz magnetic field exposures enhancing the risk of leukaemia. Multiple studies have also been conducted including chronic exposure bioassays with exposures for up to two and a half years with flux densities up to 5 mT (Mandeville et al., 1997; Yasui et al., 1997; Boorman et al., 1999; McCormick et al., 1999), initiation and promotion studies (Shen et al., 1997), transgenic models (McCormick et al., 1998) and tumour growth studies (Sasser et al., 1996; Morris et al., 1999; Anderson et al., 2001). 1.11 Experimental Evidence for Carcinogenicity of ELF-MF Consequently, when taken together, these combined animal bioassays and the lack of detectable ELF-MF genotoxic damage in stimulated lymphocytes (Antonopoulos et al., 1995; Paile et al., 1995) weaken the epidemiologically hypothesised association between extremely low-frequency magnetic field exposures and leukaemia in humans (Wertheimer & Leeper, 1979; Ahlbom et al., 2000). This is because any potential link to leukaemia would indicate stem or progenitor cells as a primary target cell for magnetic field exposure. 41 1.12 Classification In spite of the extensive research, there is no widely accepted consensus within the field as to the potential biological and carcinogenic effects of exposure to ELF-MFs. Yet, ultimately in 2002 the International Agency for Research on Cancer (IARC) published a monograph co-authored by an established working group composed of international experts, evaluating the carcinogenic risks to humans of non-ionising radiation. Categorisation of ELF-MF, as with other potentially hazardous biological agents, is evaluated by designated Working Groups in accordance with three separate criteria: a) carcinogenicity in humans, b) carcinogenicity in experimental animals, and c) mechanistic and other relevant data, relating to mechanisms of carcinogenesis. Such experimental evidence is then further evaluated in consideration of all relevant scientific data and classified by the strength of evidence that exposure to the specific biological agent is carcinogenic. This latter assessment relies on whether the evidence of exposure presents: i) sufficient evidence of carcinogenicity, ii) limited evidence of carcinogenicity, iii) inadequate evidence of carcinogenicity, or that iv) evidence suggests lack of carcinogenicity. Once each biological agent has been classified according to the strength of evidence relating to the three study models, the body of evidence is considered as a whole leading to an overall evaluation of the biological agent’s carcinogenicity to humans. The five definitive groups that relate to an agent’s carcinogenicity are described in Table 6. Of particular note is Group 2, where no quantitative measurement is attached to the terms ‘probably’ and ‘possibly’ carcinogenic. These terms are used by the Working Groups to signify the different quantities of published evidence indicating human carcinogenicity. After reviewing all the data from studies up until this point (2002) the conclusion of the Working Groups was three-fold. First that bias, chance or confounding factors could not be confidently ruled out in any observation of a positive association between ELF-MF exposure and the occurrence of cancer, for which a causal relationship was considered. Secondly, that there was inadequate evidence in humans to establish a correlation for carcinogenicity in relation to all other cancers apart from childhood leukaemia; in other words that the available studies provided insufficient consistency, quality and/or statistical power to allow a conclusion regarding a causal association. 42 Finally, they concluded that there was inadequate evidence in animal studies to characterise the carcinogenicity of ELF-MF since the studies could not be interpreted as showing either the presence or absence of a carcinogenic effect. 1.12 Classification Despite this, and due to the limited evidence from the epidemiological studies outlining a potential positive association with childhood leukaemia, the IARC working group classified extremely low-frequency magnetic fields within group 2B, outlining that exposure to ELF-MF is possibly carcinogenic to humans (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). In retrospect, it has been suggested that a potential deciding factor of the classification passed down by the IARC was the persisting lack of an accepted biophysical mechanism through which extremely low-frequency magnetic fields can induce a genotoxic and/or carcinogenic effect. Yet, despite this inability to identify a plausible mechanism, a number of possibilities have been vigorously studied. 43 Table 6. The IARC classification scale of carcinogenic risk to humans Group Explanation Examples (Biological Agent) 1 Agent is carcinogenic to humans  There is sufficient evidence of carcinogenicity in humans OR  Less than sufficient evidence of carcinogenicity in humans, but, sufficient evidence in experimental animal studies and strong evidence of a relevant mechanism of carcinogenicity. - Benzene - Etoposide - Engine exhaust (Diesel) - Formaldehyde - Ionising radiation (all types) 2A Agent is probably carcinogenic to humans  Limited evidence of carcinogenicity in humans and sufficient evidence in experimental animals OR  Inadequate evidence of carcinogenicity in humans and significant evidence in experimental animal studies, coupled to strong evidence that the carcinogenic mechanism resonates in humans. - Acrylamide - Cisplatin 2B Agent is possibly carcinogenic to humans  Limited evidence of carcinogenicity in humans and less than sufficient evidence in experimental animals. OR  Inadequate evidence in humans, less than sufficient evidence in experimental animals but supporting evidence from mechanistic studies - Chloroform - Coffee (Urinary bladder) - Engine exhaust (Gasoline) - Extremely low- frequency magnetic fields - Lead 3 Agent is not classifiable as to carcinogenicity to humans  Inadequate evidence in humans and inadequate or limited in experimental animal studies.  Further research is needed - Chlorinated drinking-water - Diazepam - Extremely low- frequency electric fields - Fluorescent lighting 4 The agent is probably not carcinogenic to humans  Evidence suggests a lack of carcinogenicity in humans and in experimental animal studies. - Caprolactam The current IARC classification categories and the corresponding criteria used to evaluate the carcinogenic potential of biological agents in humans. Category criteria were taken from the IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, (2013). 1.12 Classification The biological agent examples for each classification were taken from IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, (2016), where h i li t f t d th i t l ifi ti l b l t d able 6. The IARC classification scale of carcinogenic risk to humans 44 1.13.1 The Melatonin Hypothesis The first hypothesised mechanism is referred to throughout the literature as the ‘melatonin hypothesis’ (Stevens, 1987; Stevens & Davis, 1996). Melatonin (N-acetyl-5- methoxytryptamine) is a mammalian neuro-hormone, which is synthesised and secreted predominantly by the pineal gland (Lerner et al., 1959). Although the gastrointestinal tract (GIT), lymphocytes and skin also contribute as alternate sources of melatonin (Bubenik, 2002; Carrillo-Vico et al., 2004; Slominski et al., 2008). The synthesis and secretion of melatonin is a biomarker of circadian rhythmicity which is controlled by a day-night cycle and more specifically by light and dark, in that darkness initiates and light terminates its production (Moore et al., 1967; Lewy et al., 1980). The presence of light is detected by non-rod and non-cone receptors in the retina. Once the light intensity declines below a threshold of ~10 lux (Henshaw & Reiter, 2005), a signal is sent via the retinohypothalmic tract to the suprachiasmatic nuclei (Okamura et al., 2002), which in turn prompts the regulation of melatonin production, primarily in the pineal gland through the parenchymatous cells (Singh & Jadhav, 2014). Melatonin plays a key regulatory role in many important physiological processes which occur with a daily or seasonal synchronisation, such as metabolism regulation, reproduction, sleep (reviewed in Singh & Jadhav, (2014)) and the modulation of hormones (reviewed in (Pandi-Perumal el al., 2006; Slominski et al., 2012)). Moreover, a reduced melatonin concentration has been reported post-magnetic field exposure (Welker et al., 1983), while the suppression of melatonin has been linked to various diseases, including cancer (reviewed in Hill et al., (2011)). Therefore the theory originally devised by Stevens (1987) proposing an ELF-MF associated nocturnal reduction in melatonin synthesis represents a tentative explanation as to the potential oncogenic action of magnetic fields (Wertheimer & Leeper, 1979). Ultimately, since the inception of the ‘melatonin hypothesis’, the effects of ELF-MF exposure on melatonin secretion have been extensively studied. The majority of in vivo studies have provided very little evidence to suggest ELF-MF reduces melatonin synthesis. However, whilst covering a variety of exposure parameters, these have predominantly occurred in short-term studies (< 2 weeks of exposure duration) 45 conducted on rodents (Vanderstraeten et al., 2012). The issue with this is therefore two-fold as the majority of studies have been conducted on a nocturnally active species whose anatomical location of the pineal gland is largely different from that of humans (Touitou et al., 2006). 1.13.1 The Melatonin Hypothesis Moreover, that long-term in vivo studies have failed to provide a definitive conclusion is perhaps best highlighted by a series of 6-week studies conducted in male rats, which assessed the effects of ELF-MF exposure on pineal and plasma melatonin concentrations (Kato et al., 1993, 1994a, 1994b, 1994c, 1994d). The initial study displayed significantly reduced pineal and serum melatonin concentrations in male Wistar-King rats post-ELF exposure to a circularly polarised magnetic field up to 250 µT (Kato et al., 1993), although normal production was restored one week following the termination of exposure (Kato et al., 1994d). Similar nocturnal suppression of melatonin levels were also seen in Long-Evan rats (Kato et al., 1994a). However, a subsequent study using the same exposure protocol on Wistar-King rats, but with horizontal or vertical polarised ELF-magnetic fields instead, had no effect on melatonin concentration levels (Kato et al., 1994c). However, the authors failed to present a clear explanation as to how these different polarised power-frequency magnetic fields could evoke such variations in pineal and plasma melatonin concentrations. Further studies investigating the long-term effects of magnetic fields on the pineal and plasma melatonin concentrations were equally inconsistent with no clear justification presented for as to why (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). While some human volunteer studies have produced similarly contradictory results, it must be noted that the majority have reported a lack of an ELF-MF effect upon melatonin concentration following acute exposure of healthy volunteers (reviewed in Touitou et al., (2006)). On the other hand, the effect of chronic exposure in humans has been less comprehensively considered, although those that have been conducted seemingly confirm a lack of effect. This was illustrated in a study of workers exposed to both daily residential and occupational 50 Hz fields over a period of 1-20 years, whereby no change was detected in either the circadian synthesis of melatonin or their plasma melatonin concentration (Touitou et al., 2003). An issue with this study is that melatonin synthesis predominantly occurs during sleep therefore only nocturnal 46 residential exposure principally applies. A pooled analysis of epidemiological risk estimates, based on either nocturnal or 24-hour residential exposure, concluded that there was no difference between the two (Schuz et al., 2007). 1.13.1 The Melatonin Hypothesis Ultimately, in light of the present literature, the melatonin hypothesis stating that disruption of melatonin secretion is a main factor in the carcinogenic effects of magnetic fields is not supported by either epidemiological or experimental data (Lewczuk et al., 2014), and thus should be regarded as negatively corroborated and rebutted (Vanderstraeten et al., 2012). Furthermore, the core of the original hypothesis was conceived on the potential association between ELF-MF and breast cancer (Stevens, 1987), which has also been dismissed (Feychting, 2013). It is also important to note that there is an equal absence of any valid mechanism by which changes in human melatonin concentrations could result in adverse effects on health or well-being. 1.13.2 Radical Pair Mechanism Since it is widely accepted that ELF-MF does not itself possess sufficient energy to directly cause DNA breaks, several reports have suggested that such DNA and cytotoxic damage may result from an indirect secondary process. The most plausible hypothesis through which ELF-MF induced biological effects occur is the radical pair mechanism (RPM) (Timmel et al., 1998; Brocklehurst, 2002; Hore, 2012). Upon formation of a free radical, the unpaired electron within the outermost atomic orbital can be identified as existing in one of two spin states: ↑ or ↓ (Rodgers & Hore, 2009). Free radicals, predominantly consisting of hydrogen peroxide (H2O2), hydroxyl radicals (.OH) and superoxide radicals (.OOH), are created as a by-product of normal aerobic metabolism (Singh & Jadhav, 2014). Collectively known as reactive oxygen species (ROS) they occur through the conversion of molecular oxygen into water and the subsequent release of electrons from the mitochondrial electron transport chain into the respiratory system (Singh & Jadhav, 2014). Owing to the presence of the unpaired electron, free radicals may be highly reactive species either donating or accepting an electron. The formation of a radical pair therefore comprises two transient radicals created in tandem in which their unpaired electron spins are correlated in either an antiparallel (singlet state (S), ↑↓) or parallel (triplet state (T), ↑↑) orientation. The radical pair mechanism 47 therefore hypothesises that magnetic field interaction alters the correlated spin dynamics of radical pairs, though the process through which this occurs is as yet unconfirmed (Hore, 2012). In addition to the RPM, it has been proposed that a weak magnetic field enhances the lifespan of free radicals in cells, while it has been shown that the yield of free radical recombination can alter by 15 to 30% in the presence of a weak magnetic field (Eveson et al., 2000). Similarly, several studies have also shown increased formation of ROS after exposure to ELF-MF (Ciejka et al., 2011; Patruno et al., 2015). This most likely occurs through the Fenton reaction (Lai & Singh, 2004), which is an iron-mediated catalytic process through which hydrogen peroxide is converted into more potent and toxic hydroxyl free radicals (Phillips et al., 2009) (Figure 3). 1.13.2 Radical Pair Mechanism These findings therefore correlate with recent data suggesting iron may accentuate the effects of ELF-magnetic fields (Lai & Singh, 2004), whereby the addition of iron ions as a stimulator of the oxidation procedure alongside exposure to a 7 mT 50 Hz magnetic field, significantly enhanced the number of damaged cells (Zmyslony et al., 2000). Additionally, an observed increase in DNA single strand breaks in rat brain cells following acute 60 Hz magnetic field exposure (Lai & Singh, 1997) was ascribed to an increased presence of iron within brain cells (Singh & Lai, 1998). Likewise both in vitro and in vivo studies have indicated enhanced lipid peroxidation and oxidative damage after ELF-MF exposure (Zmyslony et al., 2004; Yokus et al., 2005). However, similarly to genotoxic studies, others have noted that ELF-MF exposure fails to contribute to intracellular ROS levels (Hong et al., 2012) and ultimately, to the ability to produce significant amounts of oxidative damage in cells (Focke et al., 2010). 48 Figure 3. The Fenton reaction. Hydrogen peroxide in the presence of a transition metal, particularly Fe2+ produces the most potent reactive oxygen species, the hydroxyl radical. Figure 3. The Fenton reaction. Hydrogen peroxide in the presence of a transition metal, particularly Fe2+ produces the most potent reactive oxygen species, the hydroxyl radical. A build-up of intracellular ROS is widely recognised as a causative factor in the oxidative damage of nuclear DNA, membrane lipids and cytosolic proteins. Moreover, the increased formation of reactive oxygen species is believed by many to be the primary oncostatic event in the formation of the majority of cancers (WHO, 2007). Thus enhanced levels, especially in the lymph nodes and bone marrow could subsequently lead to increased carcinogenesis, especially of lymphomas and leukaemia (Simko & Mattsson, 2004). Several cellular defence mechanisms however exist in oxygen-metabolising cells to prevent free radical-induced oxidative damage (Limon- Pacheco & Gonsebatt, 2009). The first of which includes the catalytic removal of free radical and ROS by radical scavengers, peroxidase and thiol-specific antioxidants and antioxidant-related enzymes such as catalase (CAT) and superoxide dismutase (SOD) (Simko & Mattsson, 2004; Hong et al., 2012). Additionally, free radicals can be further reduced by electron donors such as glutathione (GSH) and vitamin C, alongside the presence of proteins such as stress and heat shock proteins which protect against macromolecular damage and the regulation of redox-sensitive signalling pathways (Limon-Pacheco & Gonsebatt, 2009). 1.13.2 Radical Pair Mechanism In fact, in addition to the melatonin hypothesis, melatonin is also a potent receptor, independent scavenger of free radicals and antioxidant, especially hydroxyl and peroxyl radicals (Allegra et al., 2003), while also playing a role in the stimulation of additional antioxidant synthesis (Reiter et al., 2003). As such, melatonin may play a fundamental role in the protection of the cytosolic proteins, membrane lipids and nuclear DNA against oxidative damage not only in adults but also in developing foetuses’ (Garcia et al., 2014). For example, melatonin in animals has been illustrated by numerous studies to be highly protective of the foetus against oxidative damage (reviewed in Henshaw & Reiter, (2005)). This suggestion is of note following the proposed general model pertaining to the onset and progression of childhood leukaemia which assumes that any initial event(s) occurs in utero (Greaves, 1999) (Chapter 1.2). While the same is potentially true in pregnant women where 49 Nakamura et al. (2001) illustrated that serum melatonin possesses a diurnal rhythm which rises substantially following 24 weeks gestation and remains elevated until the full term. Indeed the addition of radical scavengers and antioxidants were able to inhibit an ELF-MF related increase in cell proliferation (Wolf et al., 2005), the MF- induced production of superoxide radical anions (Mannerling et al., 2010) and also prevent the previously observed induction of single- and double-strand breaks in brain cells of rats following ELF-MF exposure (Lai & Singh, 1997). Under normal cellular conditions, ROS and antioxidants exist in equilibrium; however there have been studies which have outlined alteration in the free radical homeostasis in cells following ELF-MF exposure (Lupke et al., 2004; Simko & Mattsson, 2004). An ELF-MF induced imbalance in intracellular ROS concentration, either through excessive production of ROS or deterioration in the cells’ antioxidant defensive capabilities ultimately leads to cellular oxidative stress (Limon-Pacheco & Gonsebatt, 2009). This has been suggested by the studies indicating ELF-MF exposure inhibits melatonin production and the CAT antioxidant defence (Akdag et al., 2010), while also enhancing ROS concentrations (Patruno et al., 2015). However, a study by Hong and co-authors (2012) analysed whether ELF-MF exposure of cultured human breast epithelial MCF10A cells could elicit oxidative stress by measuring intracellular ROS levels as well as antioxidant enzyme activity within these exposed cells. 1.13.2 Radical Pair Mechanism They found that exposure to 60 Hz ELF-MF at 1 mT for four hours did not affect intracellular concentration of ROS in MCF10A cells (Hong et al., 2012). Likewise, SOD activity, which is recognised as the primary defence response to cellular oxidative stress, remained unaffected following exposure (Hong et al., 2012). Oxidative stress has been linked to numerous disorders but is most prominently recognised as the biological hallmark of neurodegeneration as well as the aging process (Hardeland, 2005; Limon-Pacheco & Gonsebatt, 2009). Correspondingly, persistent or excessive cellular oxidative stress may ultimately induce oxidative damage to the macromolecules and lipid peroxidation which, if left unrepaired and subsequent genomic stability fails to be restored, could result in cell death via apoptosis (Kim et al., 2010). Apoptosis is a protective mechanism through which damaged, infected or potentially cancerous cells are removed to maintain normal homeostasis of multicellular organisms (Lacy-Hulbert et al., 1998; Juutilainen et al., 2000). In fact the alteration of the cells redox state to a more oxidative environment 50 Nakamura et al. (2001) illustrated that serum melatonin possesses a diurnal rhythm which rises substantially following 24 weeks gestation and remains elevated until the full term. Indeed the addition of radical scavengers and antioxidants were able to inhibit an ELF-MF related increase in cell proliferation (Wolf et al., 2005), the MF- induced production of superoxide radical anions (Mannerling et al., 2010) and also prevent the previously observed induction of single- and double-strand breaks in brain cells of rats following ELF-MF exposure (Lai & Singh, 1997). Under normal cellular conditions, ROS and antioxidants exist in equilibrium; however there have been studies which have outlined alteration in the free radical homeostasis in cells following ELF-MF exposure (Lupke et al., 2004; Simko & Mattsson, 2004). An ELF-MF induced imbalance in intracellular ROS concentration, either through excessive production of ROS or deterioration in the cells’ antioxidant defensive capabilities ultimately leads to cellular oxidative stress (Limon-Pacheco & Gonsebatt, 2009). This has been suggested by the studies indicating ELF-MF exposure inhibits melatonin production and the CAT antioxidant defence (Akdag et al., 2010), while also enhancing ROS concentrations (Patruno et al., 2015). However, a study by Hong and co-authors (2012) analysed whether ELF-MF exposure of cultured human breast epithelial MCF10A cells could elicit oxidative stress by measuring intracellular ROS levels as well as antioxidant enzyme activity within these exposed cells. 1.13.2 Radical Pair Mechanism They found that exposure to 60 Hz ELF-MF at 1 mT for four hours did not affect intracellular concentration of ROS in MCF10A cells (Hong et al., 2012). Likewise, SOD activity, which is recognised as the primary defence response to cellular oxidative stress, remained unaffected following exposure (Hong et al., 2012). Oxidative stress has been linked to numerous disorders but is most prominently recognised as the biological hallmark of neurodegeneration as well as the aging process (Hardeland, 2005; Limon-Pacheco & Gonsebatt, 2009). Correspondingly, persistent or excessive cellular oxidative stress may ultimately induce oxidative damage to the macromolecules and lipid peroxidation which, if left unrepaired and subsequent genomic stability fails to be restored, could result in cell death via apoptosis (Kim et al., 2010). Apoptosis is a protective mechanism through which damaged, infected or potentially cancerous cells are removed to maintain normal homeostasis of multicellular organisms (Lacy-Hulbert et al., 1998; Juutilainen et al., 2000). In fact the alteration of the cells redox state to a more oxidative environment 50 has been shown to occur immediately prior to the final phase of caspase activation in numerous models of apoptosis (reviewed in Kannan & Jain, (2000)). Yet, while the inhibition of apoptosis is another related candidate mechanism, the data correlating to the effects of ELF-MF on apoptosis remain rather limited and contradictory. 1.14 Mutation Detection Methods As has been highlighted throughout this thesis, advancements in technological capabilities have subsequently led to a now ubiquitous exposure to a range of electromagnetic fields. It has therefore become a necessity to analyse and understand any potential biological effects ELF-MF may have on living organisms. However, over the years, a body of controversial and contradictory literature has been produced in regards, not only to the effects on the health of the general population, but also those caused at the DNA level. It has been suggested that the cause of these findings may reside in the methodologies employed throughout these studies (Mairs et al., 2007). It is reasoned that while the classical methodologies possess the ability to identify intense, widespread cellular DNA damage, they potentially lack the sensitivity required to reveal the more subtle molecular alterations that lead to possibly harmful genetic damage. Instead, an analysis of the rate at which mutations are induced in male mice following ELF-MF exposure could potentially rectify such assay sensitivity limitations. For more than half a century the mouse model has played an authoritative role in the production of germline mutation induction data following exposure to chemical mutagens and ionizing radiation (Shelby, 1996; UNSCEAR, 2001). The use of pedigree- based assays, namely the specific-locus test (SLT) and the dominant lethal test (DLT), are responsible for providing the majority of experimental data. The germline mutation data generated in mice by these assays are of note since they can ultimately be extrapolated and used to assess the genetic risk of human exposure to such mutagens. 1.13.3 Apoptosis p p As discussed above, a number of studies have consistently illustrated that apoptotic pathways were activated following continuous ELF-MF exposure (Simko et al., 1998; Akdag et al., 2013; Kim et al., 2014) at field intensities as low as 14 µT (Kim et al., 2009). Similarly, the induction of apoptosis in both HeLa (human cervical carcinoma) cells and human lung fibroblast cells was illustrated in an additional study by Kim and co-authors (2010), following repetitive 30 minute exposure to an intermittent 60 Hz, 6 mT magnetic field every 24-hours for 3 days (Kim et al., 2010). However, it must be noted that while Simko and co-authors (1998) reported an induction of apoptosis in human squamous cell carcinoma cells, continuous exposure to a range of 50 Hz field intensities (0.1-1 mT) for various time periods (24-, 48- or 72-hours) failed to indicate any statistically significant difference in apoptotic induction in human amniotic cells (Simko et al., 1998). Further contradicting data were presented, demonstrating that ELF-MF exposure had no statistically significant effect on apoptosis induction (Ding et al., 2004), either directly or 24 hours after acute (2-hour) exposure to a 1 mT magnetic field (McNamee et al., 2002), or succeeding exposure to 50 Hz magnetic fields (100 or 300 µT), using an assay which possessed a detection limit of apoptotic frequency equivalent to that induced by exposure to 25 mGy X-rays (Saha et al., 2014). Despite the widespread presence and functions of radicals within biological systems, the conflicting studies make it abundantly clear that further experimental and theoretical work is needed to fine-tune the conditions relating to ELF-MF sensitivity (Messiha et al., 2015). It follows that it is a fallacy to rationalise that biological systems are so fragile that any minor alteration in the homeostasis of intracellular radical concentration arising from interaction with weak magnetic fields could have a genotoxic effect (Hore, 2012). One exception would arise where exposure was in the presence of an amplification mechanism or the exposed system exhibited an already 51 weakened defence system (Eveson et al., 2000). Nevertheless, the radical pair mechanism remains to date the most feasible method through which environmentally weak magnetic fields interact with our biochemical systems. 1.14.1 Specific-Locus Test p The development of the specific locus test (SLT) enables the accurate, non-subjective detection of viable mutations in the germ cells of mice (Searle, 1974). Developed in 1948, the SLT employs the use of a genetic tester- (T) stock mouse, which are homozygous recessive at seven specific loci (Table 7), six of which relate to the pigmentation of the mouse’s coat: agouti, brown, chinchilla at albino, dilute, piebald- 52 spotting and pink-eyed dilution, while the seventh controls ear morphology: short-ear (Russell, 1951; Russell, 1989). The predominant use of the specific-locus T-stock led to the test being commonly referred to as the Russell-7-loci test (Russell, 1951; reviewed in (Russell, 2004)), yet a second tester-stock mouse was constructed consisting of different loci known as the Harwell-test stock (Lyon & Morris, 1966). Ultimately, through the use of seven easily recognisable visible markers, the SLT provides a platform through which the transmission of novel and heritable gene mutations can be scored. Indeed, the main strength of this assay is that it analyses the transferal of novel germline mutations in the offspring of treated parents, which simulates the genetic changes associated with heritable human genetic diseases (reviewed in Yauk et al., (2015)). The assay functions as wild-type parental mice that have either been exposed to mutagens or left unexposed (control) are mated with T-stock mice. Owing to the homozygous recessive nature of the T-stock mice the majority of progeny will appear wild-type. However, a novel mutant allele (either dominant or recessive) which presumably occurred in the germline of the wild-type parent at any of the seven loci before fertilisation would manifest itself as a distinctive phenotypic change in the offspring (Favor, 1999). The scoring of both the exposed and control parental offspring provides an assessment of both the mutagen-induced and spontaneous mutation rates. Thereby, providing an assay through which mutagenesis can be calculated in the germline and potentially extrapolated to represent whole-genome rates (Davis & Justice, 1998). Ultimately, this has led the SLT to become one of the most efficient methods of mutation induction analysis to date, through which the majority of knowledge pertaining to radiation and chemical mutagenesis has been provided (Selby, 1998). However, the SLT is not without its limitations. The main underlying issue with the SLT is that the average spontaneous mutation rates at the seven protein-coding genes are very low (6.6 x 10-6) (Russell & Russell, 1996). 1.14.1 Specific-Locus Test Since the induction of mutations is measured using a pedigree-based analysis, the SLT requires the use of hundreds to thousands of mice in order to accrue sufficient data through which any significant effect on mutation induction can be determined. Furthermore, the seven specific loci employed herein are each 600 to 5500 base pairs in length 53 (Russell, 2004). As such the process of conducting this assay whilst additionally analysing de novo mutants, becomes tremendously expensive and time consuming. Consequently, laboratories around the world have discontinued the routine maintenance of the specific locus tester- (T) stock mouse. Table 7. The seven specific-locus test loci Name Symbol Protein Chromosome Agouti a Melanocortin-receptor antagonist 2 Brown b Tyrosine-related protein 4 Albino c Tyrosinase 7 Pink-eyed dilution p Tyrosinase-routing protein 7 Dilute d Myosin Va 9 Short-ear se Bone morphogenetic protein 9 Piebald spotting s Endothelin receptor, type B 14 Details of the seven loci employed as markers in the specific-locus test (Russell-7 loci), (Modified from Russell, (2004)). Table 7. The seven specific-locus test loci Details of the seven loci employed as markers in the specific-locus test (Russell-7 loci), (Modified from Russell, (2004)). 1.14.2 Dominant Lethal Test The dominant lethal (DL) test is an extensively used test in the analysis of mutagenicity, measuring the genetic changes in germ cells by using embryonic mortality as an endpoint in rodents. The assay is predominantly conducted to evaluate the mutagenic consequences of paternal exposure, whereby males are either exposed to ionising radiation or used as controls and mated with an untreated female (Green et al., 1987). The manifestation of a dominant lethal mutation derived from a solitary parent could, in theory, occur at any period following fertilisation however, this assay is based solely upon the analysis of pre- and post-implantation death of the zygote. As such, females demonstrating signs of mating are sacrificed approximately seventeen days post-conception, upon which the ovaries and uterine contents are assessed. The amount of ovulated eggs is determined via counting corpora lutea, in addition to the number of viable and dead implanted embryos (reviewed in Yauk et al., (2015)) and the number of implants (living vs dead) against the number of eggs ovulated per pregnant female for both the treated and control groups. The dominant lethal effect is then calculated by comparing the data for the treated and control groups against one another. Indeed a true mutagenic effect, as expressed by the DL assay, would equate to a statistically significant increase in dead embryos per pregnant female or a 54 statistically significant decrease in viable embryos per pregnant female when compared to a non-exposed control. The DL method has been instrumental in identifying numerous mutagenic agents since its inception (Luning & Searle, 1971; Searle & Beechey, 1981; Kirk & Lyon, 1984). Yet, since the DL assay is primarily employed to discover mutagens that cause chromosomal aberrations using embryonic death functions as the end-point, the assay fails to provide a definitive outcome, considering that additional genetic events and environmental factors may also heavily influence the occurrence of in utero loss of embryos. Moreover, similar to the SLT, the DL also implements a pedigree-based analysis, which again necessitates the use of a considerable amount of animals, whilst also being expensive and time consuming. Finally, owing to an elevated spontaneous mutation frequency attached to dominant lethal mutations, the DL assay sensitivity regarding the detection of small increases in mutation frequency is restricted (Russell & Matter, 1980). 1.14.2 Dominant Lethal Test Such limitation in sensitivity presented by both methods means that only exposure to relatively high doses of radiation can be analysed in sufficient detail. This proves problematic when attempting to replicate the effects of environmentally low and intermediate dose exposures. Furthermore, in light of progressive societal developments and an increased public and media awareness with regards to animal testing, the use of such numbers of mice is no longer accepted within modern society. Therefore more economical and sensitive experimental techniques were required in order to further progress the analysis of radiation-induced mutation induction. This proves problematic when attempting to replicate the effects of environmentally low and intermediate dose exposures. Furthermore, in light of progressive societal developments and an increased public and media awareness with regards to animal testing, the use of such numbers of mice is no longer accepted within modern society. Therefore more economical and sensitive experimental techniques were required in order to further progress the analysis of radiation-induced mutation induction. 1.14.3 Hypervariable Tandem Repeat Regions The development of any assay would have to include a genetic system that evokes a high rate of spontaneous mutations in order to allow it to be sensitive enough to detect an alteration in DNA structure induced by a dose representative of that within the environment, whilst using a relatively small population sample. Following the successful utilisation of an individual’s unique DNA fingerprint in both forensic science and the determination of family relationships (Jeffreys et al., 1985b; Jeffreys, 1987), alongside eukaryotic genome ubiquity, the use of hypervariable tandem repeat loci was implemented as a biomarker in the detection of mutation induction at the DNA level (Dubrova et al., 1993). The use of hypervariable tandem repeat regions not only 55 provides all the characteristics required to work as an effective system in the analysis of the effects of mutagenic agents but also improves upon the two traditional approaches (SL & DL tests). Firstly, such regions are known to be highly unstable, possessing spontaneous mutation rates in both germline and somatic cells thought to be at least a thousand times higher than in most protein-coding loci (Jeffreys et al., 1985a, 1991; Kelly et al., 1989). Moreover, the majority of repeat regions are primarily located in non-coding regions of most eukaryotic genomes, including human and mouse, so the identification of mutations ought not to be affected by the selection process (Jeffreys et al., 1985a; Kelly et al., 1989; Gibbs et al., 1993; Bois, 2003). Finally, any mutagenic response at these loci manifests as an alteration in repeat copy number, through either an insertion or deletion of repeat sequence, which translates as an easily detectable alteration in allele length. Thus, the detection of de novo mutant alleles occurs through quantifying changes in length relative to progenitor alleles (Jeffreys et al., 1988; Kelly et al., 1989). The resulting differentially sized alleles can easily be resolved through gel electrophoresis and Southern blot hybridisation. Additionally, the application of multi-locus DNA probes can provide simultaneous analysis of multiple loci which further boosts statistical power compared to the previous conventional approaches. Overall, the development and use of these loci has provided a highly informative laboratory-based biomarker in the analysis of the effects of mutagenic agents, allowing the detection of induced mutations at lower dose exposures within smaller population samples (Dubrova et al., 1993, 1998b, 2000a). 1.14.3 Hypervariable Tandem Repeat Regions Table 8. Tandem repetitive sequences Tandem Repetitive Sequence Sequence Size (nt) Number of repeats Total Size Microsatellites 2-5 10-100 - Minisatellites 10-100 Up to 6000 Up to 30 kb ESTRs 4-9 Up to 4000 Up to 20 kb The structural differences tandem repetitive sequences Data compiled from reviews: Table 8. Tandem repetitive sequences Tandem Repetitive Sequence Sequence Size (nt) Number of repeats Total Size Microsatellites 2-5 10-100 - Minisatellites 10-100 Up to 6000 Up to 30 kb ESTRs 4-9 Up to 4000 Up to 20 kb The structural differences tandem repetitive sequences. Data compiled from reviews: (Bois & Jeffreys, 1999; Yauk, 2004; Yauk & Polyzos, 2005; Bouffler et al., 2006; Somers, 2006). The structural differences tandem repetitive sequences. Data compiled from reviews: (Bois & Jeffreys, 1999; Yauk, 2004; Yauk & Polyzos, 2005; Bouffler et al., 2006; Somers, 2006). 1.14.3 Hypervariable Tandem Repeat Regions provides all the characteristics required to work as an effective system in the analysis of the effects of mutagenic agents but also improves upon the two traditional approaches (SL & DL tests). Firstly, such regions are known to be highly unstable, possessing spontaneous mutation rates in both germline and somatic cells thought to be at least a thousand times higher than in most protein-coding loci (Jeffreys et al., 1985a, 1991; Kelly et al., 1989). Moreover, the majority of repeat regions are primarily located in non-coding regions of most eukaryotic genomes, including human and mouse, so the identification of mutations ought not to be affected by the selection process (Jeffreys et al., 1985a; Kelly et al., 1989; Gibbs et al., 1993; Bois, 2003). Finally, any mutagenic response at these loci manifests as an alteration in repeat copy number, through either an insertion or deletion of repeat sequence, which translates as an easily detectable alteration in allele length. Thus, the detection of de novo mutant alleles occurs through quantifying changes in length relative to progenitor alleles (Jeffreys et al., 1988; Kelly et al., 1989). The resulting differentially sized alleles can easily be resolved through gel electrophoresis and Southern blot hybridisation. Additionally, the application of multi-locus DNA probes can provide simultaneous analysis of multiple loci which further boosts statistical power compared to the previous conventional approaches. Overall, the development and use of these loci has provided a highly informative laboratory-based biomarker in the analysis of the effects of mutagenic agents, allowing the detection of induced mutations at lower dose exposures within smaller population samples (Dubrova et al., 1993, 1998b, 2000a). Tandem repeat sequences can be broadly divided into different classes according to their differing properties: total sequence size, length of repeat unit sequence and total number of repeats (Table 8). In fact, expanded simple tandem repeat (ESTR) loci were initially categorised as minisatellites, owing to the possession of some similar characteristics. However, subsequent characterisation of the loci has determined that ESTR regions are currently located solely within the mouse genome and are separate to the less hypervariable true minisatellite loci encompassed within the mouse genome (Kelly et al., 1989; Gibbs et al., 1993; Bois et al., 1998a, 1998b). As such, to date there is no animal model in which the mutation induction at minisatellites can be 56 appropriately studied, nor is there any such equivalent for a human ESTR model at this time. 1.14.3.1 Minisatellites Minisatellites typically contain GC-rich repeat sequences that range from 10 base pairs to those over 100 base pairs in length (Table 8). Although extensively researched in humans (Jeffreys et al., 1985b, 1999; Jeffreys, 1987), DNA fingerprint analysis has shown that they are omnipresent throughout the genome of most organisms and higher eukaryotes (Burke & Bruford, 1987; Jeffreys et al., 1987; Gilbert et al., 1990; Andersen & Nilsson-Tillgren, 1997; Bois et al., 1998a). Shown to exhibit high levels of germline mutation rates (up to 15% per gamete) (Vergnaud et al., 1991), minisatellites have thus been successfully utilised as a tool for monitoring DNA mutation following exposure to environmental doses of mutagenic agents in both human (Dubrova et al., 1996, 1997, 2002; Kodaira et al., 1995, 2004; Satoh et al., 1996; Livshits et al., 2001; Kiuru et al., 2003), and animal populations (Yauk & Quinn, 1996; Yauk et al., 2000). Indeed such innovation was instigated by Dubrova et al. in 1993 whose pioneering paper implemented the use of previously classified hypervariable minisatellite loci, before reclassification to ESTR loci, to demonstrate a significant increase in the germline mutation rate of offspring following acute parental exposure to 0.5 and 1 Gy of γ-rays in mice (Dubrova et al., 1993). Although still applying a pedigree-based approach, the frequency in which mutations are induced following irradiation was calculated using substantially reduced numbers of animals (~250) when compared to traditional approaches (~1000’s). Two additional studies similarly employed what later transpired to also be ESTR loci to illustrate a statistically significant increase in the induction of germline mutations at the Ms6-hm hypervariable mouse minisatellite locus following parental exposure at up to 3 Gy γ-rays (Sadamoto et al., 1994; Fan et 57 al., 1995). Minisatellite loci have however been applied to the analysis of Herring gulls inhabiting a widely industrialised region of Hamilton harbour (Yauk & Quinn, 1996). Herring gulls residing within Hamilton harbour presented statistically significant differences in minisatellite mutation rates when compared to gulls from three seemingly non-industrialised control rural areas. Although it has since been demonstrated that these studies actually employed ESTR loci as opposed to minisatellites, such an extended analysis initially confirmed that hypervariable minisatellite loci can be used successfully as an efficient biomarker in the analysis of genetic damage in germline cells. 1.14.3.1 Minisatellites Consequently, the unstable GC-rich minisatellite loci were thereby implemented in the analysis of mutation induction in human populations following chronic low-dose exposure to environmental mutagens (Kodaira et al., 1995; Dubrova et al., 1996, 1997, 2002; Satoh et al., 1996; Kodaira et al., 2004). Indeed, unstable GC-rich minisatellite loci were initially implemented following the Chernobyl incident, to analyse the germline mutation rates of Belarusian families inhabiting a rural area of the Mogilev district heavily affected by the incident’s radioactive fallout. An initial 2-fold increase in minisatellite mutation rate was observed within families exposed to high levels of caesium-137 surface contamination (Dubrova et al., 1996). Yet insufficient evidence was presented to dispel concerns that the increased mutation rates were instead attributed to sampling errors, such as the use of Caucasian British families as the control families. In an attempt to authenticate the original findings, several follow-up studies were produced (Dubrova et al., 1997, 2002). Firstly, Dubrova and co-authors (1997) extended the analysis to include an additional 48 Belarusian families as well as a further five minisatellite loci. The inclusion of the extra analysis parameters produced an almost identical 1.9-fold increase in mutation rate, yet regardless of this, the control group was still that of British Caucasians. Their third study was then expanded further to study the germline mutation rate in exposed Ukrainian families from other rural areas of Ukraine contaminated with radionuclides, post-Chernobyl (Dubrova et al., 2002). Within this study, however, the control group consisted of matched Ukrainian families. Similarly, a statistically significant 1.6-fold increase in the frequency of minisatellite mutations was observed in those Ukrainian families residing within areas of Ukraine presenting high 58 levels of surface radionuclide contamination. In addition, upon re-analysis against the matched Ukrainian control families, the exposed families from Belarus exhibited a statistically significant 1.7-fold increase in germline mutations (Dubrova et al., 2002). Although cumulatively the three individual studies present compelling evidence of elevated minisatellite mutation rates within the Chernobyl area, similar studies of minisatellite mutation rate analysis among children of Chernobyl clean-up workers in Ukraine (Livshits et al., 2001), and Estonia (Kiuru et al., 2003), provide contradicting findings. Interestingly, neither of these two studies reported a statistical difference in mutation rate, despite the Estonian study employing the identical eight minisatellite loci as Dubrova and co-authors (1997, 2002). 1.14.3.1 Minisatellites Furthermore, it was calculated that the clean-up workers were exposed to average doses of radiation less than 0.25 Gy (Pitkevitch et al., 1997), which represents a value lower than the calculated doubling dose of 0.33 Gy in mice (Dubrova et al., 1998a). Similar non-significant mutation rates were presented in studies of Hiroshima and Nagasaki atomic bomb survivors and their offspring, in which the same eight minisatellite loci were again employed (Kodaira et al., 1995, 2004; Satoh et al., 1996). The detection of alterations in germline mutation rates within human populations is particularly problematic, relying heavily on the extrapolation of estimations from experimental systems relating to the genetic effects of radiation and other mutagens. Thus, inevitably, the use of animal studies were required in order to elucidate the processes involved in induced tandem repeat mutation, including the mechanisms involved, as well as to resolve the aforementioned inconsistencies in the human investigations. 1.14.3.2 Expanded Simple Tandem Repeats (ESTRs) 1.14.3.2 Expanded Simple Tandem Repeats (ESTRs) As mentioned previously (Chapter 1.14.3), the ESTR regions of mouse DNA containing alleles from the unstable mouse loci Ms6-hm and Hm-2 were initially classified as hypervariable mouse minisatellites (Kelly et al., 1989; Gibbs et al., 1993; Bois et al., 1998b) and have been implemented to demonstrate a significant increase in the germline mutation rate of offspring following acute parental exposure to 0.5 and 1 Gy of γ-rays in mice (Dubrova et al., 1993). However, it has subsequently transpired that the loci are present only in rodent DNA and thus were renamed as expanded simple 59 tandem repeat (ESTR) loci to distinguish them from what is deemed ‘true’ minisatellites in the mouse genome (Kelly et al., 1989; Gibbs et al., 1993; Bois et al., 1998b). The confusion originally stemmed from ESTR loci exhibiting exceedingly high rates of spontaneous mutation in the paternal germline, similar to that of GC-rich human minisatellites. Yet, the occurrence of spontaneous mutations is extremely rare at human minisatellite loci owing to a high level of somatic tissue stability (Jeffreys et al., 1988, 1994). In contrast, ESTR loci provide equally high somatic cell instability (Kelly et al., 1989; Gibbs et al., 1993; Hardwick et al., 2009), similar to that exhibited by microsatellites. Expanded simple tandem repeats consist of long (>1 kb), homologous, non-coding sequences comprised of short tandem repeat arrays (4-10 bp) (Kelly et al., 1989; Gibbs et al., 1993; Bois et al., 1998b). The complexity of the repeat units can also be used to distinguish between loci. Whilst ESTRs comprise a sole repeat unit array, this is not the case for minisatellite loci, which exhibit a large proportion of variant repeats. Similarly, the single repeat unit array contained in the ESTR is generally found to be smaller than in minisatellite loci (Table 8). Additionally, ESTR repeat unit arrays are also deemed too large to be microsatellite loci (Ellegren, 2004). Currently, the only mouse ESTR loci characterised in detail include the Ms6-hm and the Hm-2 (Kelly et al., 1989; Gibbs et al., 1993), both of which originated from highly expanded transcript dispersed repeats in the mouse genome (Kelly, 1994). Ms6-hm and Hm-2 expanded from independent sequences representative of long terminal repeats (MT and ORR-1 respectively), which belonged to the Mammalian-apparent long terminal repeat (LTR)-retrotransposon (MaLR) superfamily (Kelly, 1994). 1.14.3.2 Expanded Simple Tandem Repeats (ESTRs) Ms6-hm is the locus most predominantly used owing to its characterisation as the most unstable locus in the mouse genome (Hardwick et al., 2009). However, both represent efficient in vivo biomarkers in the detection of germline mutation induction, expressing mutation rates of between 1.7-3.6% per gamete (Bois et al., 1998b). Both loci also express similarly high levels of spontaneous somatic mutation with rates of up to 2.8% and 20% for Ms6-hm and Hm-2, respectively (Kelly et al., 1989; Gibbs et al., 1993). Originally detected as a hypervariable locus used in DNA fingerprinting laboratory mice (Jeffreys et al., 1987), the Ms6-hm locus consists of (GGGCA)n 60 pentamer repeats and is mapped on chromosome 4. The length of the GGGCA repeat however varies between the different strains of mice, although most alleles contain more than 400 repeat units (Kelly et al., 1989; Gibbs et al., 1993). The Hm-2 locus, on the other hand, consists of (GGCA)n and is mapped on chromosome 9 (Kelly et al., 1989), and shows sequence similarity to that of the (GGCAGA)n repeat array exhibited by the MMS10 repeat family (Bois et al., 1998b). Following the successful application of ESTR loci as an analysis tool in the evaluation of germline genetic effects (Dubrova et al., 1993; Sadamoto et al., 1994; Fan et al., 1995), the majority of studies investigating germline mutation induction have implemented the use of unstable mouse ESTR loci. The high frequencies of spontaneous and induced mutations at mouse ESTR loci allows the analysis of mutation induction to be conducted at much lower doses of exposure than those previously accessible with the standard approaches. Similarly, because germline mutation induction at ESTR loci typically ranges between two- to four-fold increases, they can be evaluated using much smaller numbers of animals (Table 9) (Dubrova et al., 1998a). 1.14.3.2.1 Doubling Dose Such a significant improvement in sensitivity, compared to the more traditional approaches, provided a more robust estimate of the doubling dose without the need for extrapolating data from high doses of radiation. More importantly though is that the doubling dose attained using ESTR loci is exceedingly similar to estimates previously acquired using the more traditional approaches of mutation detection in mice, specifically the SLT (Russell 7-locus test) (0.34 Gy) (Searle, 1974). 1.14.3.2 Expanded Simple Tandem Repeats (ESTRs) A robust linear dose-response curve was generated following a dose range of 0-1 Gy acute exposure to X-rays in pre-meiotic spermatogonia (Figure 6), culminating in an estimated doubling dose for ESTR mutation of 0.33 Gy (Dubrova et al., 1998a). The doubling dose is used in animal test systems to denote the dose of radiation capable of inducing a mutation frequency corresponding to twice the overall spontaneous mutation frequency per generation (Selby, 1998). Likewise, it can also signify an assay’s relative sensitivity in times of inter-assay comparisons (Somers, 2006). Owing to the SLT’s previous standing as the most efficient method with which to score protein-coding gene mutations in mice (Searle, 1974), the similarities in doubling doses are of 61 significant importance, implying a potential correlation between mutations at tandem repeat DNA and those at coding sequences. These studies taken in conjunction therefore demonstrate a correlation between mutations in tandem repeat sites and other mutation assays insofar as an increase in tandem repeat mutations causes an identical increase in mutation frequency at other sites. Table 9. Doubling dose estimates Method Spontaneous mutation rate Total number of animals tested Exposure doses used (Gy) Doubling dose (Gy) Russell 7- locus 7.95 x 10-6 1,051,869 3, 6, 6.7 0.34 (95%, CI: 0.22-0.50) 0.38 (+ 0.05)* Dominant Lethal 8.11 x 10-6 225,017 6, 12 0.17 (95%, CI: 0.00-0.59) ESTR (2 loci) 5.56 x 10-2 252 0.5, 1 0.33 (95%, CI: 0.06-0.75) 0.41 (+0.11)* Comparison of the doubling dose estimates for acute exposure to ionising radiation of mice spermatogonia (Table adapted from Dubrova et al., (1998a). * re-assessed value (Dubrova, 2005). Comparison of the doubling dose estimates for acute exposure to ionising radiation of mice spermatogonia (Table adapted from Dubrova et al., (1998a). * re-assessed value (Dubrova, 2005). Comparison of the doubling dose estimates for acute exposure to ionising radiation of mice spermatogonia (Table adapted from Dubrova et al., (1998a). * re-assessed value (Dubrova, 2005). 1.14.3.2.2 ESTR Mutation Detection Studies 1.14.3.2.2 ESTR Mutation Detection Studies Subsequently numerous studies have shown that ESTR loci can be implemented successfully to provide an exceedingly sensitive system through which germline and somatic mutation rates can be analysed following exposure to: (a) ionising radiation (Dubrova et al., 1993, 1998a , 2000, 2000a; Barber et al., 2002, 2006; Dubrova & Plumb, 2002; Yauk et al., 2002; Dubrova, 2005; Mughal et al., 2012). Of particular note is the study by Mughal et al. 1.14.3.2 Expanded Simple Tandem Repeats (ESTRs) (2012) in which paternal exposure to doses of acute γ-rays as low as 50 cGy resulted in significant ~2-fold increase in the frequency of ESTR mutation. (b) Chemical mutagens (Vilarino-Guell et al., 2003), which included the treatment of male mice with several doses of known mutagenic alkylating agents: ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), or alternatively the topoisomerase II inhibitor etoposide and (c) Anticancer drugs (Barber et al., 2000; Glen et al., 2008). It should be noted however that the study by Barber and co-authors (2000) failed to observe any change on ESTR mutation rates at any stage during spermatogenesis at which the cisplatin was administered. These findings are somewhat unexplainable considering cisplatin is widely known to propagate a host of DNA-damaging events, notably chromosomal aberrations occurring in mouse spermatocytes and spermatogonial stem cells. The dose of cisplatin that was 62 administered (10 mg/kg) is perhaps notable in that similar studies employing both the dominant lethal and specific locus tests also failed to display any effect at such a dose (Katoh et al., 1990; Witt & Bishop, 1996). One hypothesis for these negative results is that of potential cell killing inadvertently affecting the amount of mutants available for detection (Barber et al., 2000), owing to the administered dose of cisplatin (10 mg/kg) being twenty times that of the doubling dose for chromosome abnormalities (0.5 mg/kg) (Somers, 2006). Glen and co-authors (2008) also utilised ESTR loci alongside a novel methodology known as single-molecule PCR (reviewed later in Chapter 1.14.4.1) to analyse the rate of male germline mutation induction for four commonly used anticancer drugs: bleomycin, cyclophosphamide, mitomycin C and procarbazine. Each chemotherapy drug was applied at clinically relevant doses for humans, which were calculated in accordance to Food and Drug administration guidelines. In addition, ENU was also administered as an assay positive control following previous reports of significant increases in mutation frequencies (Vilarino-Guell et al., 2003). Significant increases in sperm ESTR mutation frequencies were presented for each of the four anticancer drugs (Glen et al., 2008). 1.14.3.2 Expanded Simple Tandem Repeats (ESTRs) Such sensitivity of ESTR loci signified a potential useful tool for studies of environmental pollution thus, lastly but of equal importance, is their implementation in the analysis of (d) environmental mutagens, including ambient urban/industrial air pollution (Somers et al., 2002, 2004; Yauk et al., 2008; Zhou et al., 2009), diesel exhaust particles (Ritz et al., 2011) and sidestream tobacco smoke (Yauk et al., 2007; Marchetti et al., 2011). A similar detection of de novo mutations in ESTRs has also been observed in vitro, signifying a correlation between the mutation responses of cultured embryonic fibroblasts and germ cells and somatic cells in vivo following treatment with chemical mutagens (Polyzos et al., 2006a, 2006b). There are several benefits to the use of ESTR loci in studies of this kind: whilst enabling the identification of direct parental exposure to mutagenic agents, the use of ESTR loci further facilitates the ability to identify the transmission of radiation-induced somatic genome mutations through the germline to first generation (F1) offspring (Dubrova et al., 2000). Indeed, significant increases in mutation rate were detected at ESTR loci in first generation (F1) offspring following parental exposure to ionising radiation 63 (Dubrova et al., 2000; Mughal et al., 2012), anticancer drugs (Glen & Dubrova, 2012), and chemical mutagens (Dubrova et al., 2008). Continuation studies confirmed those previous findings, while also expanding the analysis to illustrate that ESTR mutation rates remain elevated in three separate unexposed second generation (F2) offspring (Barber et al., 2002), and that ESTR transgenerational genomic instability also manifests in somatic tissues (Barber et al., 2006). In contrast, several studies have reported that maternal in utero exposure fails to illicit any effect on transgenerational instability (Barber et al., 2009; Abouzeid Ali et al., 2012). Likewise, the frequency of ESTR mutation of unexposed first generation offspring remained similar to control levels following the administration of a parental methyl-donor deficient diet (Voutounou et al., 2012). 1.14.3.2.3 Mechanisms of Mutation at ESTR Loci 1.14.3.2.3 Mechanisms of Mutation at ESTR Loci Although a mutation response at ESTR loci consists of either an insertion or deletion of repeat sequence, translating to alterations in the allele length, the mechanisms through which this transpires remain poorly understood. Following differences in somatic stability, it was theorised that ESTR mutations arise through a different mechanistic process to that of unstable GC-rich human minisatellites (Kelly et al., 1989, 1991; Gibbs et al., 1993; Bois et al., 1998b). The high frequency of germline mutations at human minisatellites arise through complex meiotic recombination events, however ESTR loci display equal levels of somatic mutation induction, while human minisatellites do not (Jeffreys et al., 1988, 1994). Moreover, a study by Barber et al. (2000) provided further confirmation that increased mutation induction at ESTR loci are not caused by genome-wide elevations in meiotic recombination. Since meiotic recombination is often connected with chromosomal crossover, the authors measured both crossover frequency and germline ESTR mutation frequency following acute exposure to 1 Gy X-rays or 10 mg/kg of cisplatin to ascertain whether mutation induction at ESTR loci is attributed to meiotic recombination (Barber et al., 2000). Cisplatin is a commonly used anticancer drug which is known to produce a variety of DNA adducts, particularly 1, 2-intrastrand meiotic crossovers (Hanneman et al., 1997). Although the authors detected a 3-fold increase in ESTR mutation frequency after examining 25 sites located on six separate chromosomes when compared to the controls, no overall alteration in crossover frequencies was identified. This therefore Cisplatin is a commonly used anticancer drug which is known to produce a variety of DNA adducts, particularly 1, 2-intrastrand meiotic crossovers (Hanneman et al., 1997). Although the authors detected a 3-fold increase in ESTR mutation frequency after examining 25 sites located on six separate chromosomes when compared to the controls, no overall alteration in crossover frequencies was identified. This therefore 64 suggests that ESTR mutation occurs in the absence of meiotic-specific DSBs and meiotic recombination (Barber et al., 2000). Further mechanistic insights of ESTR mutation induction were provided following exposure to both ionising radiation (Sadamoto et al., 1994; Fan et al., 1995; Dubrova et al., 1998a, 1998b, 2000a; Yauk et al., 2002) and chemical mutagens (Vilarino-Guell et al., 2003; Glen et al., 2008). 1.14.3.2.3 Mechanisms of Mutation at ESTR Loci Mutation spectra data generated from studies suggest that both induced and spontaneous ESTR mutations in germline or somatic tissues arise through similar mechanisms (Yauk et al., 2002; Hardwick et al., 2009). Moreover, these data further suggest that the detected increases in ESTR mutation rates are not the direct result of mutagens targeting ESTR loci. It was therefore theorised that ESTR mutations arise through a replication or repair based process involving DNA polymerase slippage during DNA replication, or through DNA polymerase pausing to repair the DNA (Yauk et al., 2002; Hardwick et al., 2009). An explanation that was supported by data compiled from the use of DNA repair-deficient male mice (Barber et al., 2004). The high spontaneous mutation rates exhibited by ESTR loci are potentially linked to either the size of their repeat length (up to 4000 base-pairs) (Bois et al., 1998a, 1998b), or the formation of hairpin structures among the repeat region of ESTR loci during DNA replication, repair and recombination (Weitzmann et al., 1998; Fukuda et al., 2002). A hypothesis supported by data in which observed mutation rates correlate to the size of ESTR allele (Bois et al., 2001; Dubrova, 2005). The representation of these aforementioned hairpin structures instigate DNA polymerase stalling/replication pausing and essentially promote the occurrence of DNA polymerase slippage events (Kang et al., 1995). DNA polymerase stalling ensues following the delayed repair of DNA damage thus, in an attempt to fix DNA damage while crossing an ESTR locus, a polymerase slippage event occurs causing the formation of hairpins among repeat sequences in the ESTR arrays. Upon replication re-initiation these are subsequently transformed into an insertion or deletion of sequence and are detected as length changes (Figures 4 & 5) (Kang et al., 1995). Indeed, a replication-based process similar to mechanisms evoked in microsatellite instability (Ellegren, 2004) represents the most feasible method through which ESTR mutations occur. Furthermore, the proposed 65 replication-based method also provides a conceivable explanation regarding how mutation frequencies remain elevated at ESTR loci across multiple generations (Dubrova et al., 2000; Barber et al., 2002, 2006). replication-based method also provides a conceivable explanation regarding how mutation frequencies remain elevated at ESTR loci across multiple generations (Dubrova et al., 2000; Barber et al., 2002, 2006). replication-based method also provides a conceivable explanation regarding how mutation frequencies remain elevated at ESTR loci across multiple generations (Dubrova et al., 2000; Barber et al., 2002, 2006). replication-based method also provides a conceivable explanation regarding how mutation frequencies remain elevated at ESTR loci across multiple generations (Dubrova et al., 2000; Barber et al., 2002, 2006). replication-based method also provides a conceivable explanation regarding how mutation frequencies remain elevated at ESTR loci across multiple generations (Dubrova et al., 2000; Barber et al., 2002, 2006). 66 Figure 4. Diagrammatic representation of an insertion mutation caused by replication slippage. Strand mispairing (DNA polymerase slippage) during DNA replication on the daughter strand (backward slippage) results in an insertion mutation. Diagram modified from (Kang et al., 1995; Ellegren, 2004). Figure 4. Diagrammatic representation of an insertion mutation caused by Figure 4. Diagrammatic representation of an insertion mutation caused by replication slippage. Strand mispairing (DNA polymerase slippage) during DNA replication on the daughter strand (backward slippage) results in an insertion mutation. Diagram modified from (Kang et al., 1995; Ellegren, 2004). replication slippage. Strand mispairing (DNA polymerase slippage) during DNA replication on the daughter strand (backward slippage) results in an insertion mutation. Diagram modified from (Kang et al., 1995; Ellegren, 2004). 67 Figure 5. Diagrammatic representation of a deletion mutation caused by replication slippage. Strand mispairing (DNA polymerase slippage) during DNA replication in the parent strand (forward slippage) results in a deletion mutation. Diagram modified from (Ellegren, 2004). Figure 5. Diagrammatic representation of a deletion mutation caused by replication slippage. Strand mispairing (DNA polymerase slippage) during DNA replication in the parent strand (forward slippage) results in a deletion mutation. Diagram modified from (Ellegren, 2004). 68 In addition to the process through which ESTR mutations occur, the stage of development at which ESTR mutations arise is also of equal mechanistic importance. Data from numerous studies, analysing the ESTR mutation rate following acute irradiation to X-rays at various stages of spermatogenesis, have shown that rates are significantly elevated only in samples taken at 4, 5, or 6 and above weeks, post- treatment (Dubrova et al., 1998a, 1998b; Barber et al., 2000). These time points represent spermatogonia derived from mid and early pachytene, type B spermatogonia and As spermatogonia, which are indicative of pre-meiotic spermatogonia (Searle, 1974). replication-based method also provides a conceivable explanation regarding how mutation frequencies remain elevated at ESTR loci across multiple generations (Dubrova et al., 2000; Barber et al., 2002, 2006). In contrast, no measurable alterations in the frequency of ESTR mutations were exhibited in post-meiotic spermatids procured from non- dividing sperm cells of irradiated adult mice analysed either 1 week (Barber et al., 2009) or 3 weeks (Dubrova et al., 1998a, 1998b; Barber et al., 2000) after exposure (Figure 6). A collective analysis of these data therefore indicates a proclivity for all radiation-induced ESTR mutations to arise at every stage of spermatogenesis preceding metaphase I (Barber et al., 2000). In addition to induced mouse germline mutations at the ESTR loci, a study by Hardwick and co-authors (2009) investigated the spontaneous mutation process through the measurement of age-related changes in mutation frequencies for male mice aged 12, 26, 48 and 96 weeks. The authors highlighted an abundance of ESTR mutations accumulating in the sperm of older male mice, whereby the number of age-related ESTR mutations correlate with that of cell divisions occurring in the tissue. Considering that spermatogenesis is a continual process throughout adulthood and possesses an exceedingly high proliferation activity of stem cells (de Rooij & Russell, 2000), these findings seem to indicate that both induced and spontaneous ESTR mutation rates correlate to the proliferation rate of the tissues, whereby the age-related increases in ESTR mutation frequency can be explained by an accumulation of replication-driven mutations. Consequently, tissues which manifest a low mitotic index therein present lower ESTR mutation rates. Indeed, similar observations also correspond to induced and spontaneous ESTR t ti i ti ll ESTR t ti t i DNA l t k f b Indeed, similar observations also correspond to induced and spontaneous ESTR mutations in somatic cells. ESTR mutation rates in DNA samples taken from bone marrow and brain cells were similarly measured for age-related changes (Hardwick et al., 2009), in addition to those induced by radiation (Barber et al., 2009). The relatively 69 small changes in mutation rate exhibited in bone marrow cells following acute exposure to X-rays (Barber et al., 2009), coupled with the significant elevations correlating with age, again correspond to the cellular replication proficiency of the tissue, given that the percentage of actively dividing stem cells in mouse bone marrow is very low (∼9.1/105 cells in BALB/c mice) (Muller-Sieburg & Riblet, 1996). 1.14.4 Methodology gy Although most experimental data regarding mutagen-induced tandem repeat mutations in the germline were generated using ESTR loci, the early studies relied upon a pedigree-based approach to determine in vivo germline mutation rates in offspring of test males (Dubrova et al., 1993, 1998a, 1998b, 2000a; Vilarino-Guell et al., 2003). Pedigree-based studies however do not represent the most efficient method of mutation detection, especially in human minisatellite studies where mutation rates are in the region of 1% per gamete (Bois & Jeffreys, 1999). Similar limitations persist in animal studies, including extended experimental timescales through mating and birthing processes and, although drastically reduced, the issue of using an excessive number of animals still continued. These issues were overcome through the progressive development of PCR methodologies, initially through small-pool PCR (SP- PCR) (Jeffreys et al., 1994) and later single-molecule PCR (SM-PCR) (Yauk et al., 2002). Such methodological advancements in mutation screening provide more robust estimations regarding the effect of mutagens on individual mutation rates. Both supply the capability to screen an unrestricted amount of novel germline and somatic alleles, taken directly from genomic DNA of the same exposed individual in a single experiment. Consequently, the number of animals required to undergo analysis has drastically reduced, such that mutation frequencies can now be generated in response to lower exposure doses (Dubrova et al., 2000a; Glen et al., 2008; Mughal et al., 2012), and by using just five animals per exposure parameter (Yauk et al., 2002). replication-based method also provides a conceivable explanation regarding how mutation frequencies remain elevated at ESTR loci across multiple generations (Dubrova et al., 2000; Barber et al., 2002, 2006). Similarly, ESTR mutation frequencies calculated from the brains of males irradiated in utero were significantly elevated (Barber et al., 2009), whilst no discernible change in mutation frequency appeared in non-proliferating adult brains either induced or in conjunction with aging (Hardwick et al., 2009). Since the mitotic proliferation capabilities in an adult brain are low to non-existent (Gross, 2000), with brain cells only undergoing mitotic proliferation in utero, these data further signify such an association. Overall, these findings taken together constitute further evidence that ESTR mutations arise almost entirely in replication-proficient cells, through a replication dependent mechanism predominantly expected to be replication slippage. 70 Figure 6. Spermatogenesis. Originating in the males testes, spermatogenesis represents the production of sperm and occurs continuously from puberty onwards (six weeks after birth in mice), taking approximately 35 days. Male germ cells identified as spermatogonia undergo many mitotic cell divisions generating diploid primary spermatocytes. These cells then undergo meiosis I producing secondary spermatocytes. Each secondary spermatocyte undergoes meiosis II to produce haploid spermatids which subsequently mature into spermatozoa (reviewed in Cooke & Saunders, (2002)). Figure adapted from Berne et al., (2005), 2n denotes diploid cells while n denotes those which are haploid. Figure 6. Spermatogenesis. Originating in the males testes, spermatogenesis represents the production of sperm and occurs continuously from puberty onwards (six weeks after birth in mice), taking approximately 35 days. Male germ cells identified as spermatogonia undergo many mitotic cell divisions generating diploid primary spermatocytes. These cells then undergo meiosis I producing secondary spermatocytes. Each secondary spermatocyte undergoes meiosis II to produce haploid spermatids which subsequently mature into spermatozoa (reviewed in Cooke & Saunders, (2002)). Figure adapted from Berne et al., (2005), 2n denotes diploid cells while n denotes those which are haploid. 71 The Effects of ELF-MF and 1 Gy Acute X-ray Exposure on ESTR Mutation Spectrum This study aims to establish whether ELF-MF or 1 Gy acute X-rays alters the spectra of ESTR mutation in the mouse germline and somatic tissues. Such analysis can provide further evidence into the mechanisms involved in the generation of induced and spontaneous mutations at these loci. The Effects of ELF-MF on Mutation Induction Given the profound differences in the mutagenic data generated following ELF-MF irradiation, this study was specifically designed to evaluate the current poorly understood genetic effects of ELF-MF exposure on ESTR mutation induction in vivo. By establishing the effects of ELF-MF doses currently within the accepted reference limits for a 50 Hz magnetic field (International Commission on Non-Ionizing Radiation Protection, 2010), in both the mouse germline (sperm) and somatic tissues (blood) of directly exposed male mice, the data generated can provide an experimentally based assessment of the effects of ELF-MF exposure on mutation induction. Additionally, the pattern of ESTR mutation induction will be analysed in the germline and somatic tissues of male mice exposed to 1 Gy acute X-rays, and the mutagenic effects compared against those of ELF-MF exposure. 1.15 Aims and Objectives The Effects of ELF-MF on Mutation Induction 1.14.4.1 PCR Methodologies g Such improved characteristics are accomplished through the bulk dilution of genomic DNA (Jeffreys et al., 1994; Yauk et al., 2002). Initially, SP-PCR amplifies multiple aliquots, each containing approximately 10-100 individual progenitor molecules (Jeffreys et al., 1994). While SP-PCR has been used successfully in attaining several reliable estimates of mutation rates in the regions of 10-3 and 10-4, the majority of mutant alleles present indistinguishable length changes which, in the presence of multiple progenitor molecules become difficult to quantify (Jeffreys et al., 1994). Similarly, the existence of PCR artefacts resulting from PCR amplification errors further hinder the detection of genuine mutants expressing such low mutation rates. 72 Further refinement of the methodology culminated in the production of single- molecule PCR (Yauk et al., 2002), whereby multiple reactions are established, each containing on average at least one amplifiable ESTR molecule. Although this process requires the preparation of more aliquots than SP-PCR, the amplification of a single progenitor molecule is advantageous in that de novo mutant ESTR alleles are more readily detected. Moreover, data obtained from ESTR analysis have been shown to correlate with those generated by the previous techniques (Dubrova et al., 1998a), thus proving that investigation through ESTR loci provides a reliable representation of mutation induction throughout the genome. Initially implemented by Yauk and co-authors in 2002, SM-PCR has since been used in conjunction with ESTRs to directly measure in vivo mutation frequencies in both germline and somatic tissues following exposure to ionising radiation (Abouzeid Ali et al., 2012; Mughal et al., 2012), and anticancer drugs (Glen et al., 2008). Therefore, through implementing this system within the current study, we have evaluated the in vivo effects of exposure to extremely low-frequency (50 Hz) magnetic fields on mutation induction in the germline and somatic tissues of BALB/c x CBA/Ca F1 hybrid male mice. 73 The Effects of ELF-MF and 1 Gy Acute X-ray Exposure on the Genome-wide Pattern of Gene Expression Finally, this study aims to analyse and compare the genome-wide pattern of gene expression in the ELF-MF and X-ray irradiated males. Acting as a pilot study, it aims to validate the results obtained by the ESTR assay, while also providing a broader insight into the effects of long-term acute exposure to ionising radiation. 74 2.1 Mice CBA/Ca males were mated with BALB/c females to produce a BALB/c x CBA/Ca hybrid male mice F1 generation (Harlan, Bicester, UK). The BALB/c x CBA/Ca F1 hybrid males were irradiated at 7 weeks old at the Public Health England, Centre for Radiation, Chilton, UK. Somatic and germline tissues were collected from the hybrid males 12 weeks post-irradiation at Public Health England, UK. Prior to, and following exposures all mice were maintained in conventional housing with a light/dark cycle of 12/12 hours. Rooms were maintained at 21 ± 2ºC, 45-56% relative humidity with approximately 20 air changes per hour. All animal procedures were approved by the Local Ethical Review Committee and performed under the Animals (scientific procedures) Act 1986. 2.2.1 Magnetic Field Exposure System g p y The magnetic fields were generated using an exposure system (Figure 8) consisting of a function generator (Hewlett Packard 3324A), a power amplifier (Kepco BOP-50-2M) and a pair of horizontal Helmholtz coils. The horizontal coils (overall diameter of 54 cm) and vertical coils (overall diameter of 99 cm) were manufactured by the Clarendon Laboratory, (University of Oxford, UK.). Each coil consisted of 150 turns of 20 standard wire gauge (equivalent to 0.914 mm) copper wire wound on a frame made from Tufnol resin. An earthed screen was wound around all coils to prevent stray electric fields. Upon field initiation, the required field intensity was stabilised within 60-120 seconds (Figure 7). The magnetic field uniformity was measured using a 3-axis fluxgate magnetometer (MAG-03 MC, Bartington Instruments, UK) connected to a digital voltmeter (Solatron SL 6071). The Bartington fluxgate magnetometer was calibrated annually using equipment traceable to national standards. A personal computer running custom-made software ensured the pre-set field intensity (flux density) was maintained at ± 5% of the nominal at all times; the flux 75 density for each axis of the field was also recorded at approximately twenty second intervals and any deviation then corrected. Mapping revealed that the generated field was homogenous to ± 5% within the centre of the coils occupied by the mice. Figure 7. Field uniformity measurement. 50 Hz Extremely low-frequency magnetic field uniformity for an exposure dose of 300 µT. The cages were placed 50 cm from th Figure 7. Field uniformity measurement. 50 Hz Extremely low-frequency magnetic field uniformity for an exposure dose of 300 µT. The cages were placed 50 cm from the base of the coil system ensuring that mice were centred in the most homogenous part of the field (plotted area ‘0’). Field measurements were also taken at 6 and 12 cm above ground level. a) Field uniformity measured from left to right. b) Field uniformity measured from back to front. Figure 7. Field uniformity measurement. 50 Hz Extremely low-frequency magnetic Figure 7. Field uniformity measurement. 50 Hz Extremely low-frequency magnetic field uniformity for an exposure dose of 300 µT. The cages were placed 50 cm from the base of the coil system ensuring that mice were centred in the most homogenous part of the field (plotted area ‘0’). Field measurements were also taken at 6 and 12 cm above ground level. a) Field uniformity measured from left to right. 2.2.1 Magnetic Field Exposure System b) Field uniformity measured from back to front. Figure 7. Field uniformity measurement. 50 Hz Extremely low-frequency magnetic field uniformity for an exposure dose of 300 µT. The cages were placed 50 cm from the base of the coil system ensuring that mice were centred in the most homogenous part of the field (plotted area ‘0’). Field measurements were also taken at 6 and 12 cm above ground level. a) Field uniformity measured from left to right. b) Field uniformity measured from back to front. 76 During exposures, groups of five mice were housed in square, acrylic cages (30 x 30 x 14 cm; Vet Tech Solutions Ltd, Congleton, UK). The cages were placed 50 cm from the base of the coil system ensuring that mice were centred in the most homogenous part of the field. Each cage was provided with commercial, dust-free bedding material and with free access to water and standard diet (SDS RM3; Lillico, UK). Mice were placed within the cages immediately before exposure and returned to their home cages after exposure. Animals were observed remotely during exposure using CCTV. 2.2.2 ELF-MF Exposure p Exposures were performed using a vertical 50 Hz magnetic field at 10, 100 or 300 T for 2 or 15 hours; the static (or DC) field was maintained at 42.7 µT, the average value of the static field in the laboratory containing the exposure system. A further two groups of 5 sham-exposed control mice were treated identically but without exposure to 50 Hz magnetic fields. Treatments commenced at 09:30 for the 2 hour exposure periods and at 17:00 for the 15 hour exposures. Sham exposed control animals were treated identically but without explicit 50 Hz magnetic field exposure. The average background 50 Hz magnetic was less than 0.01 µT when measured over 24 hours using an EMDEX II magnetic field dose meter. 2.2.3 X-ray Irradiation y Groups of five mice were given whole-body, acute irradiation of 1 Gy of 250 kV X-rays, delivered at a dose rate of 0.5 Gy min-1 as a positive control using a commercial, small animal exposure system (HS System; AGO X-rays Ltd, Aldermaston, UK). During exposure, mice were group housed in corrugated-plastic cages (31 x 27 x 14.5cm). One further group of 5 sham-exposed control animals were again treated identically, but without exposure to the whole-body, acute irradiation 1 Gy of X-rays. 77 Figure 8. Helmholtz Coil Exposure System. The Helmholtz coil exposure system showing one of the acrylic exposure cages containing bedding, food and water. Figure 8. Helmholtz Coil Exposure System. The Helmholtz coil exposure system showing one of the acrylic exposure cages containing bedding, food and water. 2.3 Tissues Blood and testes were taken 12 weeks post-irradiation and DNA samples were extracted from both tissues and purified. Blood and testes were taken 12 weeks post-irradiation and DNA samples were extracted from both tissues and purified. Sperm samples are used to estimate the frequency of ESTR mutation induction and were taken at 12 weeks after exposure to ensure that the sperm samples were derived from exposed As spermatogonia and stem cells (Searle, 1974) (Figure 6), which is necessary since previous studies have illustrated that ESTR mutation is a replication- based process involving polymerase slippage (Barber et al., 2004, 2009; Shanks et al., 2008; Hardwick et al., 2009). Thus, the mutagenic effects of ELF-MF irradiation should lead to the accumulation of ESTR mutations in tissues with a high mitotic index, such as spermatogonia and stem cells. In order to establish the genetic effects of ELF-MF exposure on mutation induction within the somatic tissues of mice, DNA samples were extracted from blood samples taken from the same irradiated animals 12 weeks after exposure. 78 The lifespan of specific mature blood cell types is relatively short with approximately 1% of red blood cells being removed each day. Therefore the regeneration and replacement of adult blood cells represents a continuous process throughout life, originating with hematopoietic stem cells (HSC), located mainly in the bone marrow (BM) niche of adult mammals and occurring through a series of progenitor stages. Hematopoietic stem cells are multi-potent and thus exist as the hierarchy of progenitor cells. They possess the ability to differentiate into multi-lineage progenitors and precursors, ultimately facilitating the emergence of more than ten different individual adult blood cell lineages, each of which possesses unique functions. In addition to their role in replenishing individual blood lineages, immortal HSCs also maintain the blood system through the production of extra HSCs through their ability to self-renew (Orkin & Zon, 2008). Figure 9. Hematopoiesis. A basic schematic illustrating the process through which blood and immune cells are formed through the differentiation and proliferation of multi-potent hematopoietic stem cells. Figure adapted from He et al., (2014). The presence of HSCs and their exposure to hemopoietic growth factors prompt the original division and differentiation into common myeloid and lymphoid progenitors, while at least one daughter cell remains an HSC, allowing the process to continue. 2.3 Tissues These progenitors provide blood precursors programmed for uni-lineage differentiation resulting in the production of megakaryocytes/thrombocytes, erythrocytes, myeloblast cells (basophil, neutrophil and eosinophil), monocyte/macrophage, and lymphocytes. Figure 9. Hematopoiesis. A basic schematic illustrating the process through which Figure 9 Hematopoiesis A basic schematic illustrating the process through which Figure 9. Hematopoiesis. A basic schematic illustrating the process through which Figure 9. Hematopoiesis. A basic schematic illustrating the process through which blood and immune cells are formed through the differentiation and proliferation of multi-potent hematopoietic stem cells. Figure adapted from He et al., (2014). The presence of HSCs and their exposure to hemopoietic growth factors prompt the original division and differentiation into common myeloid and lymphoid progenitors, while at least one daughter cell remains an HSC, allowing the process to continue. These progenitors provide blood precursors programmed for uni-lineage differentiation resulting in the production of megakaryocytes/thrombocytes, erythrocytes, myeloblast cells (basophil, neutrophil and eosinophil), monocyte/macrophage, and lymphocytes. Figure 9. Hematopoiesis. A basic schematic illustrating the process through which blood and immune cells are formed through the differentiation and proliferation of multi-potent hematopoietic stem cells. Figure adapted from He et al., (2014). The presence of HSCs and their exposure to hemopoietic growth factors prompt the original division and differentiation into common myeloid and lymphoid progenitors, while at least one daughter cell remains an HSC, allowing the process to continue. These progenitors provide blood precursors programmed for uni-lineage differentiation resulting in the production of megakaryocytes/thrombocytes, erythrocytes, myeloblast cells (basophil, neutrophil and eosinophil), monocyte/macrophage, and lymphocytes. 79 2.4 DNA Extraction All DNA extractions were performed in a category II laminar flow hood (Walker Safety Cabinets Ltd, Glossop, UK) in order to minimise the risk of contamination by foreign DNA molecules. 2.4.1 Blood Blood DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Southampton, UK). The blood samples (300 μL) were added to 900 μL Cell Lysis Solution (Promega, Southampton, UK) and inverted 5-6 times to mix before being left to incubate for 10 minutes at room temperature (RT). The samples were centrifuged (30 seconds, 13,200 revolutions per minute (rpm)), (Eppendorf, Hamburg, Germany) to separate the red blood cells and cell debris from the white blood cells. The supernatant was removed without disturbing the pellet. The process of cell lysis was repeated until the pellet contained only white blood cells (white pellet). Upon generation of the white pellet, the white blood cells were re-suspended at 40 Hz on the vortex mixer (ZX3, VELP Scientifica, Italy). 300 μL of Nuclei Lysis Solution (Promega, Southampton, UK) was added and the samples mixed in order to lyse the white blood cells. If cell clumps were still visible after mixing then the samples were incubated at 37oC in a circulating water bath (Grant, Cambridge, UK) until the clumps were disrupted. Although, if the cell clumps remained after an hour then an additional 100 μL Nuclei Lysis Solution was added and the incubation was repeated. Once the cell clumps had dissipated then 100 μL Protein Precipitation Solution (Promega, Southampton, UK) was added and the sample mixed on the vortex (40 Hz, 10-20 seconds). Samples were then centrifuged (3 minutes, 13,200 rpm) to remove the proteins. The supernatant was transferred to a new Eppendorf tube containing 300 μL single molecule-PCR clean isopropanol and gently mixed. The mixture was then centrifuged (3 minutes, 13,200 rpm) to separate the DNA. The supernatant was again removed and the pellet was washed with 300 μL 70% ethanol before centrifugation occurred again (3 minutes, 13,200 rpm). The supernatant was once more removed and the pellet left to air dry for 15 minutes in the laminar flow hood. The pellet was finally re-suspended in 50 μL DNA Rehydration Solution (Promega, Southampton, UK) and the DNA left to rehydrate overnight (4oC). 80 2.4.2 Sperm 2.4.2 Sperm Sperm DNA was extracted from the testes of the hybrid F1 male mice. Each DNA extraction was performed on individual testicles and DNA from both testes combined for each animal. Each testicle was placed in a sterile Petri dish and the excess fat removed before being finely chopped. The disaggregated testicle was mixed with 1 mL phosphate-buffered saline (PBS) and transferred through a size 80 mesh (Sigma-Aldrich Company Ltd, Poole, UK) into an Eppendorf tube. Then 5 μL of the PBS/tissue solution was transferred to a haemocytometer and visualised under a light microscope (Olympus) to determine a sperm to somatic tissue ratio. The Eppendorf tubes were centrifuged (2 minutes, 13,200 rpm) and the supernatant removed. Afterwards, they were centrifuged (30 seconds, 13,200 rpm) and the remaining PBS removed again. Then 1 mL 1x Saline-Sodium Citrate (SSC) was added and the samples mixed on the vortex (40 Hz) in order to re-suspend the pellet before 10 μL, 10% Sodium dodecyl Sulphate (SDS), (Fisher Scientific, Loughborough, UK) was added. The samples were centrifuged (2 minutes, 13,200 rpm) to produce a pure white pellet. The supernatant was removed and the samples centrifuged again (30 seconds, 13,200 rpm) and the residual liquid removed. Then 960 μL, 0.2x SSC was added and the pellet re-suspended using the vortex mixer (40 Hz). The samples were once more visualised under the light microscope to ensure that no somatic cells were present. Then 70 μL β-Mercaptoethanol (Sigma-Aldrich Company Ltd, Poole, UK), 100 μL, 10% SDS and 20 μL, 25 mg/mL Proteinase K (Sigma-Aldrich Company Ltd, Poole, UK) were added and the tubes incubated at 37oC in a circulating water bath for between 2-3 hours. After incubation, the samples were transferred to QiagenMaXtract tubes (Qiagen, Crawley, UK) and the DNA extracted in 1 mL Phenol/Chloroform mix (Sigma- Aldrich Company Ltd, Poole, UK) before centrifugation (5 minutes, 3,100 rpm). The supernatant was removed and an additional 1 mL of Chloroform was added followed again by centrifugation (5 minutes, 3,100 rpm). The DNA was transferred into a new 15 mL tube (Corning Incorporated, USA) containing 2-3 volumes of 100% ethanol and 250 μL, 10% 3M NaAc (pH 5.2). The DNA was then transferred again to an Eppendorf tube containing 500 μL 80% ethanol and centrifuged (30 seconds, 13,200 rpm). 2.5 DNA Digestion and Precipitation Approximately 5 μg of each DNA sample was digested with 1x NEB buffer 2, 20 ng Bovine Serum Albumin (BSA) and 25 U MseI restriction enzyme (New England Biolabs (NEB), Hitchin, UK) for 2 hours at 37oC in a circulating water bath. MseI cleaves outside the ESTR array and distal to the PCR primer sites used for PCR amplification, therefore it was used to render genomic DNA fully soluble prior to dilution. DNA was precipitated in 40 μL, 100% ethanol and 10%, 3 M NaAc (pH5.2) and frozen (>2 hours, - 80oC). The tubes were centrifuged (20 minutes, 13,200 rpm) before being rotated 180o and centrifuged again (20 minutes, 13,200 rpm). The supernatant was removed and the DNA pellet washed with 50 μL, 80% ethanol and centrifuged (30 seconds, 13,200 rpm). The supernatant was removed and the pellet air dried for 15 minutes. The pellet was then re-suspended in 50 μL SM-PCR clean ultra-pure water. The yield of digested DNA was measured. 2.4.2 Sperm The 80% ethanol was removed and the DNA left to air-dry for approximately 15 minutes in the 81 laminar flow hood. Finally, the DNA was re-suspended in 50 μL SM-PCR clean ultra- pure H2O (ultra-pure water that has been UV irradiated). 2.6 DNA Quantification Q The digested DNA concentration was estimated using an ND-1000 Spectrophotometer (NanoDrop Technologies, Ringmer, UK). When possible the DNA samples were diluted in a separate Eppendorf tube to a concentration of approximately 10 ng/μL using SM- PCR clean ultra-pure water. 2.7 Single-Molecule PCR Optimisation g p The 10 ng/μL DNA stock was diluted in order to establish a concentration whereby one haploid genome and thus one amplifiable ESTR molecule was present in each reaction. To achieve this, the stock DNA was diluted with dilution buffer (DB) (5 mM Tris-HCl (pH 7.5), 5 μg/mL Salmon sperm DNA) to a variety of concentrations (Table 10). Eight reactions, each containing 1 μL diluted DNA were established for their respective concentrations. They were then all amplified (PCR), blotted and an autoradiograph image obtained. 82 Table 10. SM-PCR optimisation DNA Concentration Volume DNA (stock) : Volume DB (μl) 500 pg 1 (10 ng) : 19 100 pg 2 (500 pg) : 8 50 pg 1 (500 pg) : 9 20 pg 2 (500 pg) : 48 10 pg 10 (20 pg) : 10 5 pg 5 (20 pg) : 5 2 pg 1 (20 pg) : 9 The DNA concentrations for SM-PCR optimisation and the volumes of DNA and dilution buffer (DB) used. Table 10. SM-PCR optimisation 2.8 Poisson Analysis at the Single Molecule Level Using the Hi Fidelity PCR system (Roche) with Betaine, the concentrations obtained from the optimisation assays were used to establish a new set of data. 10 μL reactions were set up for the single-molecule concentration in 96 reactions, which were then amplified, blotted and an autoradiograph produced (Figure 11). 2.8 Poisson Analysis at the Single Molecule Level 2.8 Poisson Analysis at the Single Molecule Level Using the Hi Fidelity PCR system (Roche) with Betaine, the concentrations obtained from the optimisation assays were used to establish a new set of data. 10 μL reactions were set up for the single-molecule concentration in 96 reactions, which were then amplified, blotted and an autoradiograph produced (Figure 11). The amounts of amplifiable molecules were noted and their location on the PCR plate identified. In order to collate robust estimates of individual ESTR mutation frequencies in each animal, an average of approximately 150 amplifiable molecules were required to be obtained and analysed for each tissues for each individual mouse (Barber et al., 2006, Glen et al., 2008, Glen & Dubrova, 2012). Therefore, DNA amplification was performed until a sufficient number of Ms6-hm molecules were ascertained. 2.9 Mutation Scoring g Positive PCR products were resolved on 40 x 20 cm agarose gels. Longer gels were used to ensure the progenitor alleles separated sufficiently thereby providing an appropriate resolution in order to detect the Ms6-hm mutants. 2.10 Statistical Analysis y ESTR mutation frequencies, 95% confidence intervals and standard errors were estimated from the Poisson distribution using a modified version of an approach previously described (Zheng et al., 2000a). To assist data analysis, a database was compiled for each animal representing the individual exposure parameters using Microsoft Excel 2010. Adobe Photoshop CS4 was employed in the preparation of figures included in this thesis. Statistical analysis was performed using software written 83 using Microsoft Basic by Professor Y.E Dubrova alongside programs STATISTICA and SYSTAT. 2.11.1 Polymerase Chain Reaction Since single-molecule PCR involves the dilution of bulk genomic DNA and the amplification of multiple samples, each containing approximately one amplifiable ESTR molecule, the Ms6-hm ESTR locus was amplified in 10 μL reactions. The Ms6-hm ESTR locus consists of a pentanucleotide repeat GGGCA (Kelly et al., 1989; Gibbs et al., 1993), therefore amplification was performed using 0.4 μM flanking primers HM1.1F (5′ - AGA GTT TCT AGT TGC TGT GA - 3′) and HM1.1R (5′ - ATG CCT TAG AAC TGA CTC TC - 3′) primers (Sigma-Aldrich, Poole, U.K.), 10x buffer (Roche Molecular Biochemicals, USA), 200 μM dNTPs (Roche), 1 M Betaine, 3.4 μL SM-PCR clean ultra-pure water and 0.035 U μl-1 of the Expand High Fidelity PCR System (Roche, Mannheim, Germany). Amplification was carried out in 0.2 ml thin-walled PCR tube strips consisting of 8 wells (Thermo Scientific, Ashford, UK). Using a PCT-225 Tetrad PCR machine (Bio-Rad, Hemel Hempstead, UK), the PCR program consisted of heated lids to denature the DNA for 3 minutes at 96°C before 29 cycles of, 96°C for 20 seconds, 58°C for 30 seconds and 68°C for 3 minutes were performed. The program terminated with a final cycle of 68 °C for 10 minutes. Since single-molecule PCR involves the dilution of bulk genomic DNA and the amplification of multiple samples, each containing approximately one amplifiable ESTR molecule, the Ms6-hm ESTR locus was amplified in 10 μL reactions. The Ms6-hm ESTR locus consists of a pentanucleotide repeat GGGCA (Kelly et al., 1989; Gibbs et al., 1993), therefore amplification was performed using 0.4 μM flanking primers HM1.1F (5′ - AGA GTT TCT AGT TGC TGT GA - 3′) and HM1.1R (5′ - ATG CCT TAG AAC TGA CTC TC - 3′) primers (Sigma-Aldrich, Poole, U.K.), 10x buffer (Roche Molecular Biochemicals, USA), 200 μM dNTPs (Roche), 1 M Betaine, 3.4 μL SM-PCR clean ultra-pure water and 0.035 U μl-1 of the Expand High Fidelity PCR System (Roche, Mannheim, Germany). Amplification was carried out in 0.2 ml thin-walled PCR tube strips consisting of 8 wells (Thermo Scientific, Ashford, UK). Using a PCT-225 Tetrad PCR machine (Bio-Rad, Hemel Hempstead, UK), the PCR program consisted of heated lids to denature the DNA for 3 minutes at 96°C before 29 cycles of, 96°C for 20 seconds, 58°C for 30 seconds and 68°C for 3 minutes were performed. The program terminated with a final cycle of 68 °C for 10 minutes. 2.11.4 Southern Blot Gels were removed from their running tanks and inverted into plastic trays. They were rinsed with depurinating solution (0.25 M HCl, 2 x 5 minutes), denaturing solution (0.5M NaOH and 1 M NaCl, 2 x 10 minutes) and finally in neutralising solution (0.5M Tris and 3M NaCl, 2 x 5 minutes). DNA was transferred to a MAGNA nylon transfer membrane (Gilson Scientific Ltd, Luton, UK) by capillary transfer method (Southern, 1975). The MAGNA nylon transfer membrane had been soaked in 2x SSC (0.3M NaCl and 0.03M Na-citrate) prior to transfer and 20x SSC was used as the transfer buffer while 3MM Whatmann filter paper (Munktell, Falun, Sweden) was used to facilitate uptake of DNA to the membrane. After a minimum of 5 hours the nylon membrane was removed and rinsed in 2x SSC. The membrane was dried (10 minutes, 80°C) before the DNA was covalently cross-linked to the membrane using a UV crosslinker (Hoefer) at 7x104 J/cm2. 2.11.3 Long Agarose Gel Electrophoresis 2.11.3 Long Agarose Gel Electrophoresis 2.11.3 Long Agarose Gel Electrophoresis This type of gel electrophoresis was used for mutation scoring analysis. Prior to electrophoresis the gel tanks require balancing to ensure that the DNA fragment does not electrophorese out of the gel and into the running buffer. 1% agarose gels, 40 x 20 cm in size were produced through the addition of 6 g of Seakem Le Agarose and 600 mL 1x TBE buffer (44.5 mM Tris-borate pH 8.3, 1 mM EDTA and 0.5 μg/mL Ethidium Bromide). Each gel consisted of 1 row of 27 wells. 5 μL of each positive PCR reaction was loaded alongside 1 μg, 1 kb ladder and 200 ng, 200 bp ladder (Promega, Southampton, UK), which were loaded to determine the migration distance and the size of the DNA fragments, respectively. Gel electrophoresis occurs in 1x TBE buffer running buffer at 200 volts for approximately 24 hours; until the 1.6 kb band of the 1kb ladder had migrated off the bottom of the gel. This type of gel electrophoresis was used for mutation scoring analysis. P This type of gel electrophoresis was used for mutation scoring analysis. Prior to electrophoresis the gel tanks require balancing to ensure that the DNA fragment does not electrophorese out of the gel and into the running buffer. 1% agarose gels, 40 x 20 cm in size were produced through the addition of 6 g of Seakem Le Agarose and 600 mL 1x TBE buffer (44.5 mM Tris-borate pH 8.3, 1 mM EDTA and 0.5 μg/mL Ethidium Bromide). Each gel consisted of 1 row of 27 wells. 5 μL of each positive PCR reaction was loaded alongside 1 μg, 1 kb ladder and 200 ng, 200 bp ladder (Promega, Southampton, UK), which were loaded to determine the migration distance and the size of the DNA fragments, respectively. Gel electrophoresis occurs in 1x TBE buffer running buffer at 200 volts for approximately 24 hours; until the 1.6 kb band of the 1kb ladder had migrated off the bottom of the gel. 2.11.2 Short Agarose Gel Electrophoresis This type of gel electrophoresis was used in the separation of DNA products for the SM-optimisation and Poisson analysis. 1% agarose gels, 25 x 24 cm in size were produced through the addition of 3 g of Seakem Le Agarose (Lonza, Rockland, Maine, USA) and 300 mL 0.5x TBE buffer (22.5 mM Tris-borate (pH 8.3) and 0.5 mM EDTA). Each gel consisted of 4 rows of 26 wells into which 5 μL of DNA was loaded, alongside 2 wells of 200 ng of 1 kb DNA ladder (Invitrogen, Paisley, UK) per row. The DNA samples each had 5 μL loading dye (5x TAE, 12.5% Ficoll 400, (Sigma-Aldrich Company Ltd, Poole, UK) and 0.1% Bromophenol blue added prior to loading to enable the DNA fragments to be observed. The gels were electrophoresed in 0.5x TBE running buffer, at 200 volts for 1.25 hours; until the blue loading dye had migrated three quarters of the way down the well track. 84 2.11.6 Probe Recovery 2.11.6 Probe Recovery Incubation was stopped by adding 70 μL Oligo-stop solution (5M NaCl, 1M Tris (pH 7.5), 0.5M EDTA, 10% SDS, 10 mM dCTP, H2O). The labelled DNA was then precipitated with 90 μg salmon sperm DNA, 30 μL, 2M NaAc (pH 5.6) and 425 μL, 100% ethanol. The solution was centrifuged (3 minutes, 13,200 rpm) and the supernatant discarded into a beaker of detergent. The pellet was washed with 80% ethanol and centrifuged (1 minute, 13,200 rpm) before the supernatant was again removed into a beaker of detergent. The pellet was re-suspended in 600 μL ultra-pure water. The appropriate labelled ladders, (30 μL, 1 kb for short gel, 30 μL, both 1 kb and 200 bp for long gels) were added to the labelled Ms6-hm probe which was boiled (6 minutes, 100oC) to denature the DNA prior to hybridisation. The probes were finally added to ~10 mL Church buffer (0.5 M Na-Phosphate, 7% SDS and 1 mM EDTA (Fisher Scientific, Loughborough, UK) and then to the hybridisation bottles containing the membranes. 2.11.5 Probe Labelling g Double stranded Ms6-hm probe DNA was produced via PCR using the primers HMA (5′ - GGGCA GGGCA GGGCA GGGCA GG - 3′) and HMB (5′ - TGCCC TGCCC TGCCC TGCCC - 3′), (Sigma-Aldrich, Poole, U.K.). The Ms6-hm probe (20 ng), the 1 kb (40 ng) and the 200 bp (40 ng) ladder probes were all labelled with 1.5 μL α-32P-dCTP (PerkinElmer, Austria), for both the short and long agarose gel membranes. 85 Ms6-hm DNA (20 ng) was added to H2O to make a volume of 30 μL. The tubes were boiled for 6 minutes to denature the DNA. Then 12 μg BSA, 6 μL Oligo-labelling buffer, 8.16 U Klenow DNA polymerase (United States Biochemical Corp (USB), Cleveland, USA) and 1.5 μL of α-32P-dCTP were added. The tubes were incubated for a minimum of 5 hours at room temperature (RT). 2.11.7 Hybridisation y The DNA-bound MAGNA nylon transfer membranes were rolled between two nylon meshes and placed into the hybridisation bottles along with ~15 mL Church buffer(0.5M Na Phosphate, 7% SDS, 1 mM EDTA). The membranes were then pre- hybridised (20 minutes, 65°C) in a Mini 10 hybridisation oven (Thermo Scientific, Ashford, UK). Upon recovery of the probes (Probe Recovery section) the Church buffer was removed and the labelled DNA probes were added to ~10 mL Church buffer and then to the hybridisation bottles containing the membranes. The probes were then left to hybridise to the DNA-bound membranes (>5 hours, 65°C) in the Mini 10 hybridisation oven. 86 Once the labelled probes had hybridised to the DNA-bound nylon membrane the unbound probes were removed through washing. Phosphate wash solution (0.04M Na- Phosphate, 0.5% SDS) was added to the hybridisation bottle firstly for 1 minute (RT) before being removed and secondly for 10 minutes (65°C). High stringency wash solution (0.1x SSC and 0.01% SDS) was then used for further 10 minute washes (65°C) until the radioactivity levels were ~10 counts per second (cps), (Mini 900 Ratemeter, Thermo Scientific, Ashford, UK). 2.11.8 Autoradiography g p y The membranes were then removed from the hybridisation bottles and washed (~500 mL, 2x SSC) and covered in Saran wrap. The membrane was taped, DNA side up, into an autoradiograph cassette (Genetic Research Instrumentation GRI, Braintree, UK) containing an intensifying screen and a Fuji Rx100 X-ray film inserted. The radioactivity level of the membrane determined the length of time for which the cassette was stored at -80oC, (>50cps = 2-3 hours or <50cps = overnight). The films were developed using manual dip tanks instead of a developing machine in order to maintain control over the amount of film development. The cassettes were removed from the -80oC freezer and placed into a drying oven for 10 minutes. Films were removed from the cassette and initially placed into the Developing solution (RG- X-ray developer, Champion Photochemistry, Malaysia) before the developing reaction was terminated by the Stop solution (13.5 litres H2O, 135 mL Acetic acid (Acros Organics, Geel, Belgium) and finally the image was fixed onto the film by placing it into the Fixer solution (RG-X-ray fixer, Champion Photochemistry, Malaysia) until the film appeared clear. 2.11.9 Mutation Scoring and Sizing of ESTR Mutants 2.11.9 Mutation Scoring and Sizing of ESTR Mutants All de novo ESTR mutants pertaining to each tissue, exposure dose and time point, along with the age-matched sham-treated controls and X-ray irradiated males were reliably scored twice through two independent markers. Mutants were scored by eye as novel DNA fragments demonstrating a shift of at least 1 mm relative to the progenitor allele. Smaller length-changes however were not scored as they could not be reliably scored consistently. The parental and mutant allele sizes were estimated in All de novo ESTR mutants pertaining to each tissue, exposure dose and time point, 87 accordance with the method employed by Southern (1979) with the 200 bp DNA step ladder (Promega, UK) used as a reference. accordance with the method employed by Southern (1979) with the 200 bp DNA step ladder (Promega, UK) used as a reference. 2.12.1 RNA Extraction Two kidneys per animal (5x biological replicates per group), of the indicated treatments were collected and fast frozen in liquid nitrogen before being stored at - 80oC. In all instances, RNA samples were extracted individually from two kidneys taken from each animal and pooled. RNA extractions were performed using a modified protocol of the TRIzol reagent (SIGMA-ALDRICH, St Louis, USA) method, whereby the TRIzol reagent is a mixture of guanidine thiocyanate and phenol in a monophase solution which dissolves DNA, RNA and protein upon homogenisation or lysis of tissue (Chomczynski & Sacchi, 1987, 2006). Prior to homogenisation of tissues, the working area and Diethylpyrocarbonate (DEPC)-treated sterilised homogeniser probe were cleaned with RNaseZap (Ambion, Life Technologies Ltd, Paisley, UK) to ensure RNase-free working conditions. The homogeniser probe was assembled into the Multi-Gen 7 homogenizer (PRO Scientific Inc., Oxford, CT, USA) and cooled in dry ice for 30-40 seconds prior to use. The kidneys were retrieved onto dry ice and transferred individually into a sterile 15 mL centrifuge tube containing 7 mL TRIzol reagent, (10-100 mg of tissue per mL TRIzol reagent). Upon addition, the kidney was homogenised for 20 seconds and left for 5 minutes at room temperature. After incubation, 1.4 mL 1-bromo-3-chloro-propane (Sigma B9673), (200 µL per mL TRIzol reagent), was added to lessen genomic DNA contamination. The TRIzol lysate was then vigorously mixed on the vortex at full speed for 30 seconds, and left for a further 3 minutes at room temperature. Afterwards, the contents of the tube were aliquoted into 8 labelled 1.5 mL RNase-free Eppendorf tubes and phase- separated through centrifugation (10 minutes, 12000 relative centrifugal force (rcf), 4oC, cooled at 4 oC before use). The RNA containing upper aqueous phase was transferred to a 1.5 mL RNase-free Eppendorf tube containing 560 µL isopropanol (Sigma), (1 µL per 1 µL aqueous phase), while the DNA and protein containing phases; Prior to homogenisation of tissues, the working area and Diethylpyrocarbonate (DEPC)-treated sterilised homogeniser probe were cleaned with RNaseZap (Ambion, Life Technologies Ltd, Paisley, UK) to ensure RNase-free working conditions. The homogeniser probe was assembled into the Multi-Gen 7 homogenizer (PRO Scientific Inc., Oxford, CT, USA) and cooled in dry ice for 30-40 seconds prior to use. The kidneys were retrieved onto dry ice and transferred individually into a sterile 15 mL centrifuge tube containing 7 mL TRIzol reagent, (10-100 mg of tissue per mL TRIzol reagent). 2.13 RNA Quantification Q After extraction, 5 µL of each RNA sample was transferred to a new Eppendorf tube and the total RNA concentration quantified using an ND-1000 Spectrophotometer (Thermo Fisher Scientific, USA). 2.12.1 RNA Extraction Upon addition, the kidney was homogenised for 20 seconds and left for 5 minutes at room temperature. After incubation, 1.4 mL 1-bromo-3-chloro-propane (Sigma B9673), (200 µL per mL TRIzol reagent), was added to lessen genomic DNA contamination. The TRIzol lysate was then vigorously mixed on the vortex at full speed for 30 seconds, and left for a further 3 minutes at room temperature. Afterwards, the contents of the tube were aliquoted into 8 labelled 1.5 mL RNase-free Eppendorf tubes and phase- separated through centrifugation (10 minutes, 12000 relative centrifugal force (rcf), 4oC, cooled at 4 oC before use). The RNA containing upper aqueous phase was transferred to a 1.5 mL RNase-free Eppendorf tube containing 560 µL isopropanol (Sigma), (1 µL per 1 µL aqueous phase), while the DNA and protein containing phases; 88 interphase and the organic phase were discarded. The RNA isolates were inverted 20 times to mix and left at room temperature for 10 minutes. They were then centrifuged (10 minutes, 12000 rcf, 4 oC) to form an RNA pellet. The samples were collected on ice, the supernatant discarded and the RNA pellet washed with 1 mL, (-20 oC), 75% RNase- free ethanol (3:1 ethanol/DEPC water). Samples were again centrifuged (5 minutes, 12000 rcf, 4 oC) and the RNA pellet was again collected on ice before the ethanol was discarded and the wash repeated. After centrifugation (5 minutes, 12000 rcf, 4 oC), the samples were left in the centrifuge (4 oC) for a further 5 minutes before collection, again on ice. The ethanol was removed completely and the RNA pellet air dried for 5 minutes on ice. The RNA pellets were re-suspended in 80-150 µL nuclease-free water (1/1000 final, DEPC/H2O), depending on their size. Upon complete re-suspension the RNA sample was temporarily pooled into a single solution to measure the concentration and integrity. Afterwards, each RNA sample was stored at -80oC. 2.13.1 RNA Integrity Measurement g The integrity of the RNA samples was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Wokingham, UK) using an Agilent RNA 6000 Nano Kit (Agilent Technologies, Inc. Waldbronn, Germany). Each Agilent RNA 6000 Nano Kit contains multiple RNA Nano Chips, each of which is equipped with an interconnected set of micro-channels that is used for size-dependant separation of the nucleic acid fragments as they are driven through it electrophoretically. Each RNA sample’s integrity is evaluated through the production of a RNA Integrity Number (RIN). The RIN index provides a quantitative value ranging from 1 to 10 that facilitates the assessment of the RNA integrity through classification of the eukaryotic total RNA (Schroeder et al., 2006). The resulting electropherogram exhibits at least two distinct peaks representing the 18S and 28S ribosomal RNA (Figure 10). Prior to evaluation, all Agilent RNA 6000 Nano Reagents equilibrated to room temperature for 30 minutes, while each RNA sample was diluted to 250 ng/µL using 89 RNase-free water. The RNA ladder had been previously heat denatured (70oC, 2 minutes) and stored at -80oC. Prior to the preparation of the gel matrix, the RNA 6000 Nano gel matrix, required filtering, 550 µL of was pipetted into a spin filter and centrifuged (1500 rcf, 10 minutes, room temperature). Thereafter, 65 µL of the filtered gel was aliquoted into a 1.5 mL RNase-free Eppendorf tube whilst the remaining filtered gel was stored at 4oC for up to 4 weeks. The RNA 6000 Nano dye concentrate was then vortexed for 10 seconds and centrifuged before 1 µL was added to the 65 µL filtered gel. The subsequent gel-dye solution was mixed on a vortex and centrifuged (13000 rcf, 10 minutes, room temperature) and stored at room temperature while a new RNA 6000 Nano chip was added to the chip priming station. 9 µL of the gel-dye mix was added to the Nano chip into the well-marked (G). The chip priming station was then closed and the syringe plunger pressed down for exactly 30 seconds before being released. Five seconds after release the plunger was replaced to the starting position and the chip priming station opened once more. A further 9 µL of the gel-dye mix was then added into the two wells marked (G) of the Nano chip and the remaining gel-dye solution discarded. 2.14.1 Template total RNA with Spike-In After extraction, each sample was diluted to a total RNA concentration 40 ng/µL and 1.5 µL transferred to a new 1.5 mL RNase-free microcentrifuge tube, on ice, ready for amplification and labelling. Before use the RNA Spike-In One Colour Mix (Agilent Technologies Inc, TX 78612, USA) was vigorously mixed on a vortex mixer and heated in a circulating water bath (37oC, 5 minutes). Upon removal the RNA Spike Mix was once again vigorously mixed on a vortex mixer and briefly centrifuged ready to prepare the serial dilutions for Cyanine 3- labelling. The RNA Spike Mix was serial diluted in 1.5 mL RNase-free microcentrifuge tubes as per the manufacturer’s instructions, (1st 1:20, 2nd 1:25, 3rd 1:20, 4th 1:4), whereby the fourth dilution is at a 40000-fold final dilution. 2 µL of the fourth dilution was added to a new 1.5 mL RNase-free microcentrifuge tube containing 1.5 µL total RNA, resulting in a total volume of 3.5 µL and 25 ng total RNA. The first dilution of the spike mix was stored for up to 2 months at -80oC while the others were discarded. 2.13.1 RNA Integrity Measurement Next, 5 µL of RNA 6000 Nano maker was pipetted into all 12 sample wells in addition to the ladder well. RNA samples were denatured (70 oC, 2 minutes) immediately prior to loading, where 1 µL of denatured RNA sample was added to each of the 12 sample wells and 1 µL of the prepared ladder was pipetted into the ladder well. If any wells remained unused a further 1 µL of the Nano Marker was added. Once all the wells had been loaded, the chip was removed from the priming station and placed in the adapter of the IKA vortexer. The chip was then vortexed for 1 minute at 2400 rpm and placed into the Agilent 2100 Bioanalyzer for scanning. 90 Figure 10. RNA Electropherogram. An electropherogram illustrating the total RNA peaks of the successful sample X-1.10. The RIN index for the sample shown is 9.7. Characteristic regions for ribosomal peaks and the lower marker are displayed. Figure 10. RNA Electropherogram. An electropherogram illustrating the total RNA peaks of the successful sample X-1.10. The RIN index for the sample shown is 9.7. Characteristic regions for ribosomal peaks and the lower marker are displayed. 91 2.14 One-Colour Microarray-based Gene Expression Analysis Through the use of T7 RNA Polymerase Blend and Cyanine 3-CTP Dye Pack (Agilent Technologies Inc, TX 78612, USA), the Agilent Low Input QuickAmp Labelling Kit (Agilent Technologies Inc, TX 78612, USA) generates fluorescently labelled cRNA. The gene expression in experimental and control samples was measured through the hybridisation of the fluorescent cRNA onto Agilent SurePrint G3 format 8x60k microarray slides (Agilent Technologies Inc, TX 78612, USA). One-color arrays result in absolute fluorescence intensities, which are assumed to be monotonically (if not linearly) related to the abundance of mRNA species complementary to the probes on the array. 2.14.2 Cyanine 3-Labelling 0.8 µL T7 Primer Mix was added to 1 µL nuclease-free water and the resulting 1.8 µL solution aliquoted into the tube containing the total RNA and RNA spike-in forming a total volume of 5.3 µL. The primer and template mix was denatured in a circulating water bath (65oC, 10 minutes), collected on ice and incubated for 5 minutes. While the primer-template mix was incubating on ice, the 5x First Strand Buffer was pre-warmed in a heat block (80oC, 4 minutes) to ensure adequate re-suspensions of the buffer 92 components. Afterwards, the 5x First Strand Buffer was briefly mixed on the vortex and centrifuged. Upon re-suspension, the 5x First Strand Buffer was added along with the other components in Table 11 to form the cDNA master mix. components. Afterwards, the 5x First Strand Buffer was briefly mixed on the vortex and centrifuged. Upon re-suspension, the 5x First Strand Buffer was added along with the other components in Table 11 to form the cDNA master mix. Table 11. cDNA master mix Components Volume (µL) per reaction 5x First Strand Buffer 2 0.1 M DTT 1 10 mM dNTP Mix 0.5 Affinity Script RNase Block Mix 1.2 Total Volume 4.7 The components and required volume of each required in the cDNA master mix for each RNA sample. The components and required volume of each required in the cDNA master mix for each RNA sample. 4.7 µL of cDNA master mix was added to each sample tube, bringing the total volume to 10 µL, and mixed. The samples were then incubated in a circulating water bath (40oC) for two hours. After the incubation period, the samples were moved to another circulating water bath at 70oC for a further 15 minutes and finally incubated on ice for 5 minutes. While the samples were incubating on ice, the Transcription Master Mix was prepared (Table 12). Table 12. Transcription master mix Components Volume (µL) per reaction Nuclease-free water 0.75 5x Transcription Buffer 3.2 0.1 M DTT 0.6 NTP Mix 1 T7 RNA Polymerase Blend 0.21 Cyanine 3-CTP 0.24 Total Volume 6 The components and required volume of each required in the Transcription master mix for each RNA sample. Table 12. Transcription master mix 6 µL of transcription master mix was added to each sample tube resulting in a total volume of 16 µL for each reaction. 2.14.3 Cy3-RNA Clean-up 2.14.3 Cy3-RNA Clean-up Prior to the first use of Buffer RPE 4 volumes (44 mL) 100% ethanol was added to produce a working solution. Upon removal from the water bath, on ice, 84 µL RNase-free water was added to each cRNA sample ensuring a total starting volume of 100 µL. Afterwards, 350 µL Buffer RLT was added and the solutions well mixed before 250 µL 100% ethanol was added to the diluted RNA and mixed by pipetting the solution up and down. The sample was then transferred to an RNeasy Mini spin column which had been supplied with a 2 mL collection tube attached and centrifuged (30 seconds, 13 rcf, 4oC). The flow-through was discarded along with the collection tube, and the spin column replaced back into a new 2 mL collection tube before 500 µL Buffer RPE was added to the spin column. The samples were again centrifuged (30 seconds, 13 rcf, 4oC) and the flow-through once more discarded, however the spin column was replaced back into the same collection tube before a further 500 µL Buffer RPE was added to the column. Samples were again centrifuged (1 minute, 13 rcf, 4oC) and the flow-through and collection tube again discarded. The spin column was transferred to a new collection and spun again (30 seconds, 13 rcf, 4oC) in order to remove any Buffer RPE potentially left on or near the rim of the column alongside any left on the outside of the column. Again the collection tube was discarded and the spin column placed into a fresh 1.5 mL collection tube. The purified cRNA samples were eluted through the addition of 30 µL RNase-free water directly onto the RNeasy filter membrane. The samples were left for 1 minute on ice before centrifuged (30 seconds, 13 rcf, 4oC). The RNeasy spin column was discarded and the cRNA sample placed on ice ready for quantification. 2.14.2 Cyanine 3-Labelling The samples were mixed and once again incubated in a circulating water bath (40oC) for two hours. Following incubation, the newly amplified, cyanine 3-labelled was purified using Qiagen RNeasy Mini Kit (Qiagen Inc., Valencia CA) and the manufacturer’s recommended protocol. 93 2.15 Quantifying cRNA 2.15 Quantifying cRNA Dye incorporation and cRNA yield for all cRNA samples were quantified using the NanoDrop ND-1000 UV-VIS Spectrophotometer. Again, 5 µL of each cRNA sample was removed into a new sterile RNase-free Eppendorf tube and the NanoDrop software initialised. The microarray measurement tab was selected and the RNA-40 sub-tab was selected as the sample type. The instrument was initially blanked with 1.5 µL of the same nuclease-free water used to elute the cRNA samples. Thereafter, 1.5 µL of each sample was loaded on the instrument sample 94 loading area and the following categories recorded; Cyanine 3 dye concentration (pmol/µL), RNA absorbance ratio (260 nm/280 nm) and cRNA concentration (ng/µL). loading area and the following categories recorded; Cyanine 3 dye concentration (pmol/µL), RNA absorbance ratio (260 nm/280 nm) and cRNA concentration (ng/µL). Through the use of these three categories and Equations 3 and 4, the cRNA yield (µg) and specific activity of each sample was determined. Equation 4 The Agilent SurePrint G3 format 8x60k slides are formatted with 8 sub-arrays printed on each 1-inch x 3-inch glass slide. The minimum cRNA yield (µg) loaded onto each slide was 0.825 while the minimum specific activity (pmol Cy3 per µg cRNA) was 6. Any samples that did not meet these requirements were not loaded onto the slide. µg of cRNA = Concentration of cRNA x 30 µL (elution volume) 1000 µg of cRNA 2.16 Hybridisation y Hybridisation was performed using the Agilent Gene Expression Hybridisation Kit (Agilent Technologies Inc, TX 78612, USA) according to the manufacturer’s protocol. Firstly, 500 µL, nuclease-free water was pipetted into the supplied vial containing lyophilized 10x Gene Expression Blocking Agent and gently mixed on the vortex until the pellet was re-suspended. Failure to re-suspend the pellet resulted in the mixture being heated for 5 minutes at 37oC to facilitate re-suspension while the addition of the Agilent-CGHBlock blocking buffer was required to minimize background signal on the array. Using the components of the Agilent High-RPM Gene Expression Hybridisation Kit (Table 13), the following master mix was prepared to hybridise 600 ng of Cy3- labelled cRNA on the SurePrint G3 format 8x60k slide. 95 Table 13. Fragmentation mix Components Volume (µL) / mass per reaction Cyanine 3-labelled cRNA 600 ng 10x Gene Expression Blocking Agent 5 Nuclease-free water Bring volume to 24 25x Fragmentation Buffer 1 Total Volume 25 The fragmentation mix for each sample undergoing hybridisation to a SurePrint G3 format 8x60k slide. Table 13. Fragmentation mix After addition, each sample was incubated for exactly 30 minutes in a circulating water bath (60oC) in order to fragment the RNA. Immediately after 30 minutes the samples were cooled on ice for 1 minute and 25 µL 2x Hi-RPM Hybridization Buffer added to terminate the fragmentation reaction. The samples were carefully mixed through pipetting up and down in an attempt to avoid the introduction of bubbles, centrifuged (13000 rcf, 1 minute, room temperature) and 40 µL of each sample immediately loaded onto their respective Agilent SureHyb Gasket Slide well (Agilent Technologies Inc, TX 78612, USA). Upon completion, the SurePrint G3 microarray slide was slowly lowered on top of the SureHyb gasket slide ensuring that the slides ‘active side’ was facing towards the gasket slide. The SureHyb chamber cover was applied to sandwich the slides and assembly clamp secured. The assembly chamber was then placed into the rotisserie of the hybridisation oven and hybridisation was performed for 17 hours at 65oC rotating at 10 rpm according to the company’s recommendations. 2.17 Washing of Hybridised Arrays 2.17 Washing of Hybridised Arrays To reduce the potential of array wash artefacts, 2 mL Triton X-102 (10%) was added to the Gene Expression Wash Buffers prior to their first use. Furthermore, to ensure optimal wash conditions the Gene Expression Wash Buffer 2 was pre-warmed at 37oC in a circulating water bath for 17 hours. Once hybridisation was complete, the assembly chamber was removed from the hybridization oven, disassembled and the array-gasket sandwich quickly transferred to slide-staining dish 1, where it was completely submerged in Gene Expression Wash Buffer 1 (room temperature). Keeping the microarray slide submerged with the numeric barcode facing upwards the array-gasket sandwich was carefully pried apart. Keeping the barcode at the top, the slide was transferred into a slide rack positioned in Keeping the barcode at the top, the slide was transferred into a slide rack positioned in 96 the slide-staining dish 2, again containing the Gene Expression Wash Buffer 1 and agitated vigorously for 1 minute. Immediately prior to the conclusion of the wash the slide-staining dish 3 was filled with the pre-warmed the Gene Expression Wash Buffer 2 (37oC). The slide rack was then transferred to slide-staining dish 3 and again agitated vigorously for 1 minute. The slide was then slowly removed from the slide-rack minimising the potential of any wash solution remaining on the slide and transferred with the Agilent barcode facing upwards to a slide holder again for scanning. 2.18 Array Scanning y g The slides were scanned and fluorescent signals detected using the Agilent High Resolution C Scanner (G2539A) according to the manufacturer’s protocol. Raw microarray image files were created and the resulting TIFF images were processed using Agilent’s Feature Extraction Software Version 10.5.1.1 (protocol: GE1_105_Dec08). 2.19 Data Normalisation & Quality Control 2.19 Data Normalisation & Quality Control Information from probe features was extracted from the microarray scan data thus allowing the measurement of gene expression, while the software also generated QC reports using the protocols specific for the microarray assay. The microarray data were analysed using GeneSpring software, version 12.6 (Agilent Technologies). To enable inter-array comparisons between Agilent one-colour microarrays signals, the raw signal intensity values for each set of one-colour microarrays were log transformed (Log2) and globally normalised using the 75th percentile of all non-control probes on the microarray, as specified by the manufacturer. Statistical significance of a probes’ expression was later determined using P values calculated by unpaired t test analysis and corrected for multiple-testing with Benjamini-Hochberg false discovery rate (FDR) of P<0.05. Of those probes exhibiting a differential expression of statistical significance (FDR value <0.05), only those presenting a fold increase or decrease of two or more in expression when compared to sham-treated controls were classified as differentially expressed. 97 3.1 Experimental Design Universal exposure to extremely low-frequency magnetic fields within the developed world, in conjunction with often contradictory data, has culminated in a considerable amount of concern regarding the effects of ELF-MF on biological systems. Thus, in view of the conflicting results, it was the aim of this study to provide an in depth analysis of potential molecular changes induced by ELF-MF exposure in the germline and somatic tissues of male mice. Mutation frequencies were evaluated in both the somatic and germline tissues of BALB/c x CBA/Ca F1 hybrid male mice, using a highly sensitive technique that employs the use of extremely unstable expanded simple tandem repeat (ESTR) loci. The use of ESTR loci provides a highly sensitive biomarker for monitoring mutation induction, which has previously been successfully utilised in the analysis of ionising radiation (Dubrova et al., 1993; Mughal et al., 2012), chemical mutagens (Vilarino-Guell et al., 2003) and anticancer drugs (Glen et al., 2008). Germline and somatic mutation frequencies were detected at the Ms6-hm ESTR locus using single-molecule PCR (SM- PCR). This approach involves the bulk dilution of genomic DNA in order to isolate and amplify approximately one ESTR molecule per reaction (Yauk et al., 2002). The utilisation of SM-PCR therefore offered several advantages to mutation screening, in providing unlimited access to de novo mutant alleles from a single individual, and the direct analysis of multiple sample equivalents from both the somatic and germline cells of a single individual. As a result SM-PCR provides a more robust estimate of individual mutation rates in both somatic and germline cells, while dramatically reducing the number of mice needed to do so. Mutation analysis was performed in BALB/c x CBA/Ca F1 hybrid male mice compared to males of an inbred strain in an attempt to more accurately score Ms6-hm mutants. As in previous studies (Yauk et al., 2002; Glen et al., 2008; Abouzeid Ali et al., 2012; Glen & Dubrova, 2012; Mughal et al., 2012; Voutounou et al., 2012), de novo ESTR mutants were to be identified as novel DNA fragments demonstrating a shift of at least 1 mm relative to the progenitor allele. Inbred strains present Ms6-hm heterozygosity owing to the presence of two very similar-sized progenitor alleles which is problematic when 98 identifying mutants presenting such minimal shifts in distance. 3.1 Experimental Design In contrast, the use of BALB/c x CBA/Ca F1 hybrid male mice within the current study substantially facilitated the process of ESTR mutation scoring. Such mice possess two differently-sized Ms6-hm alleles, which have been consistently and readily amplified across previous experiments (Yauk et al., 2002; Glen et al., 2008; Glen & Dubrova, 2012; Voutounou et al., 2012). As such, F1 hybrid male mice provided a well-spaced banding pattern, through which length-change mutations were easily scored at both the BALB/c- (~ 2.5 kb) and CBA/Ca- derived alleles (~3.3 kb), in addition to the unambiguous establishment of the mutant bands allelic origin. Using this system, seven-week old male BALB/c x CBA/Ca hybrid mice were exposed to 10, 100 and 300 T for 2 or 15 hours (Table 14). Blood and sperm DNA samples were extracted 12 weeks post-exposure and the frequencies at which mutations were induced at the Ms6-hm locus calculated. Tissue samples were also collected from additional age-matched sham-treated hybrid males (control group) alongside those exposed to 1 Gy acute X-rays (positive controls). Given the results of previous studies (Dubrova et al., 1998a; Mughal et al., 2012), such an acute exposure (1 Gy X-rays) causes a statistically significant increase in the frequency of Ms6-hm mutants. Thus, it was used as a positive control to validate the SM-PCR technique. According to the results of previous studies (Yauk et al., 2002; Barber et al., 2009; Hardwick et al., 2009), ESTR mutation induction occurs mostly, if not solely, in replication-proficient mitotic cells. The extraction of DNA samples 12 weeks post-exposure therefore ensured that the evaluated sperm was derived from exposed As spermatogonia (Searle, 1974). Likewise, the extremely high-turnover of hematopoietic tissues in mice ensured the majority of nucleated blood cells were also derived from exposed replication-proficient stem cells (Metcalf, 1988). An acute exposure of 2- or 15 hours to a continuous 50 Hz magnetic field was applied due to several studies previously observing a dose-dependent increase in DNA strand breaks both in vitro and in vivo following acute (2 hour) exposure (Lai & Singh, 1997; Ivancsits et al., 2002, 2003b; Focke et al., 2010), with the response plateauing at an exposure of 15 hours (Ivancsits et al., 2002). 3.1 Experimental Design Similarly, the field’s magnetic flux densities employed in the present study (10, 100 or 300 µT) have previously illustrated 99 genotoxic properties in cells exposed both in vivo and in vitro (Lai & Singh, 1997, 2004; Ivancsits et al., 2003b). While these flux densities are comparatively high in relation to household levels associated with CL (< 0.1 µT) and those capable of small but significant increase in DNA breakage (0.035 µT) (Ivancsits et al., 2003b), these values are in accordance with the recommended International Commission on Non-Ionising Radiation reference limits associated with the general public (International Commission on Non-Ionizing Radiation Protection, 1998; International Commission on Non-Ionizing Radiation Protection, 2010). Moreover, any confirmed adverse effects even witnessed at 100 µT would have profound implications for risk assessment, including the need to re-examine the exposure parameters for ELF-MFs. genotoxic properties in cells exposed both in vivo and in vitro (Lai & Singh, 1997, 2004; Ivancsits et al., 2003b). While these flux densities are comparatively high in relation to household levels associated with CL (< 0.1 µT) and those capable of small but Thus, using single-molecule PCR, the frequency of mutations at the Ms6-hm mouse ESTR locus was established in sperm and blood samples exposed to 50 Hz magnetic fields of 10, 100 or 300 T for 2 or 15 hours, and measured against those in age- matched sham-treated and irradiated (acute 1 Gy X-rays) BALB/c x CBA/Ca hybrid F1 males. Table 14. Experimental Design Number Analysed Group Duration Blood Sperm Sham-treated 2 hours 5 4 15 hours 5 5 10 µT 2 hours 5 5 15 hours 5 5 100 µT 2 hours 5 5 15 hours 5 5 300 µT 2 hours 5 5 15 hours 5 5 X-ray sham-treated - 5 5 X-ray acute 1 Gy - 5 5 Total - 50 49 Total number of animals analysed 99 The doses of ELF-MF, X-ray and sham-treatments analysed in the study, the duration for which they were exposed, the tissues analysed and the number of mice analysed for each group. Table 14. Experimental Design Table 14. Experimental Design 99 100 3.2 Optimisation Since de novo mutant alleles are more readily detectable as single molecules, the optimal concentration at which the amplification of, on average, a single Ms6-hm molecule occurs was determined for each individual sample. Multiple DNA stock dilutions containing concentrations of 100 pg/µL, 50 pg/µL, 20 pg/µL, 10 pg/µL, 5 pg/µL and 2 pg/µL were amplified and assessed by autoradiograph. The concentration at which at least one amplifiable molecule is present was identified as the concentration at which ~50% of the eight PCR reactions yielded a positive result (Figure 11). However, in the instances when the optimum concentration fell between two different concentrations the median value would be implemented. For example, when a concentration of 20 pg/µL presented six positive reactions and 10 pg/µL only two, the single molecule equivalent concentration would be ~15 pg/µL. Figure 11. SM-PCR optimisation autoradiograph. Multiple dilutions on sample DNA were performed to the indicated concentrations, and subjected to Southern blot analysis. Dark bands indicated the presence of DNA. 1kb DNA step ladder (Invitrogen) was used as an identification method. A 5 pg/µL optimum concentration is indicated and was subsequently used for Poisson analysis. Figure 11. SM-PCR optimisation autoradiograph. Multiple dilutions on sample DNA were performed to the indicated concentrations, and subjected to Southern blot analysis. Dark bands indicated the presence of DNA. 1kb DNA step ladder (Invitrogen) was used as an identification method. A 5 pg/µL optimum concentration is indicated and was subsequently used for Poisson analysis. Once the optimal concentration had been determined for each individual sample, Poisson analysis was performed in order to establish the mean number of amplifiable molecules per reaction. 101 3.3 Poisson Analysis y DNA samples were diluted to their corresponding optimal concentrations and amplified out in 96 separate reactions (Figure 12), after which, PCR products were detected once more by Southern blot hybridisation. Poisson distribution (Equation 5, 6) was then used to calculate the mean number of amplifiable molecules per reaction. This value was subsequently multiplied by the total number of reactions to provide an estimate for the total number of amplifiable molecules per reaction. Equation 5 𝑃= 𝑛− 𝑘 Equation 6 𝜆 = − ln (𝑃) Where: Equation 5 𝑃= 𝑛− 𝑘 Equation 6 𝜆 = − ln (𝑃) Where: Equation 6 𝜆 = − ln (𝑃) Where: Where: 𝑃 : Frequency of negative reactions n- : Number of negative reactions 𝜆 : Mean number of amplifiable molecules per reaction 𝜆 : Mean number of amplifiable molecules per reaction In order to collate robust estimates of individual ESTR mutation frequencies in each animal, the process of mutation scoring requires that between 90 and 150 individual alleles be procured for each animal (Barber et al., 2006, Glen et al., 2008, Glen & Dubrova, 2012). Therefore aliquots of each DNA sample continued to be amplified in 96 reactions and analysed until a sufficient number of positive reactions were acquired to enable mutation scoring to be accurately performed. 102 Figure 12. SM-PCR Poisson analyses. SM-PCR and Southern blot analysis of the DNA samples at the estimated single-molecule concentration. ESTR arrays in 96 reaction per plate were amplified. The single alleles were identified as positive reactions (bro arrows) and used in subsequent mutation scoring. 1 kb DNA step ladder (Invitrogen was used as an identification method as in Figure 11. Figure 12. SM-PCR Poisson analyses. SM-PCR and Southern blot analysis of the DNA samples at the estimated single-molecule concentration. ESTR arrays in 96 reactions per plate were amplified. The single alleles were identified as positive reactions (brown arrows) and used in subsequent mutation scoring. 1 kb DNA step ladder (Invitrogen) was used as an identification method as in Figure 11. Figure 12. SM-PCR Poisson analyses. SM-PCR and Southern blot analysis of the DNA samples at the estimated single-molecule concentration. ESTR arrays in 96 reactions per plate were amplified. The single alleles were identified as positive reactions (brown arrows) and used in subsequent mutation scoring. 1 kb DNA step ladder (Invitrogen) was used as an identification method as in Figure 11. 103 3.4 Mutation Scoring PCR analysis was conducted on multiple samples, each containing at least one amplifiable molecule (indicated by the brown arrows, Figure 12). The ESTR mutation frequencies were determined for DNA samples of 7 week old male BALB/c x CBA/Ca hybrid mice exposed to 10, 100 and 300 T for 2 or 15 hours, extracted from blood and sperm 12-weeks post-exposure. To compare the effects of ELF-MF, tissue samples were also collected from additional age-matched sham-treated hybrid males (control group) in addition to hybrid males irradiated with an acute dose of 1 Gy X-rays (positive controls). In order to provide a higher resolution for mutation scoring, PCR products were resolved on a 40 cm-long agarose gel and detected again by Southern blot hybridisation. As mutation analysis was performed in BALB/c x CBA/Ca F1 hybrid male mice, mutations were scored at both the smaller BALB/c-derived allele (~ 2.5 kb) and the larger (~3.3 kb) CBA/Ca allele. Similar to previous studies of mutation detection at ESTR loci (Yauk et al., 2002; Glen et al., 2008; Abouzeid Ali et al., 2012; Glen & Dubrova, 2012; Mughal et al., 2012; Voutounou et al., 2012), only novel DNA fragments demonstrating a shift of at least 1 mm comparative to the progenitor allele were scored as de novo ESTR mutants (Figure 13). Given that the Ms6-hm locus consists of a pentamer repeat sequence (GGGCA)n (Kelly et al., 1989), a 1 mm shift in distance equates to either a gain (insertion) or loss (deletion) of three repeat units for the CBA/Ca allele and two repeat units for the smaller BALB/c allele. Smaller length- changes however were not scored as they could not be consistently or reliably scored. All mutants pertaining to each tissue, exposure dose and time point, along with the age-matched sham-treated controls and X-ray irradiated males were reliably scored through two independent markers. Thereafter, the frequencies of ESTR mutations (μ) were calculated for each group (Equation 7), dividing the number of mutants, m by the total number of amplifiable DNA molecules, n. The standard error of mutation frequency, seμ was estimated using Equation 7: 104 Equation 7 𝑠𝑒𝜇= 𝜇√(𝑠𝑒𝑛 𝑛) 2 + 1 𝑚 Equation 7 𝑠𝑒𝜇= 𝜇√(𝑠𝑒𝑛 𝑛) 2 + 1 𝑚 𝑠𝑒𝜇= 𝜇√(𝑠𝑒𝑛 𝑛) 2 + 1 𝑚 where sen is the standard error of the number of amplifiable DNA molecules. The Student’s t test was used to compare the frequency of ESTR mutation in exposed and sham treated mice. Figure 13. Mutation detection at the Ms6-hm ESTR locus. Southern blot analysis of the CBA/CA and BALB/c alleles at the Ms6-hm locus. Single molecule, positive reactions were identified and electrophoresed on a 40 x 20 cm, 1% agarose gel. Bands assigned as mutants are indicated by arrows (CBA/Ca mutants, shifts of 1 & 2 millimetres represent insertions of repeats; BALB/c mutant is indicative of a deletion). 200 bp DNA step ladder (Promega) is used as a side lane reference. Figure 13. Mutation detection at the Ms6-hm ESTR locus. Southern blot analysis of the CBA/CA and BALB/c alleles at the Ms6-hm locus. Single molecule, positive reactions were identified and electrophoresed on a 40 x 20 cm, 1% agarose gel. Bands assigned as mutants are indicated by arrows (CBA/Ca mutants, shifts of 1 & 2 millimetres represent insertions of repeats; BALB/c mutant is indicative of a deletion). 200 bp DNA step ladder (Promega) is used as a side lane reference. 105 * Number of amplifiable molecules (± standard error (s.e.)) is given in brackets. 3.5 Mutation Mosaicism Due to the extremely high spontaneous mutation rate demonstrated by the Ms6-hm and Hm-2 ESTR loci, the occurrence of both germline and somatic mutational mosaicism is especially prominent, with at least 3% of mice displaying an additional non-parental mutant allele in up to 60% of somatic cells (Yauk et al., 2002). Mosaics represent a cluster of identical mutations detected in multiple germline and somatic DNA samples which have arisen in the same cell during the first few cell divisions following fertilisation (Figure 14), (Kelly et al., 1989; Gibbs et al., 1993). Hence the presence of mosaics is not indicative of separate mutational events but rather the propagation of a single mutational event. In previous studies, typically those implementing a pedigree-based approach (Dubrova et al., 1998a; Barber et al., 2009), the presence of mosaics have presented issues during the scoring of mutations as the statistical power is too low to facilitate the detection of mutation rate heterogeneity between individuals (Barber et al., 2009). Furthermore, it has been previously proposed that the presence of mosaics implies that robust estimates of mutation frequency cannot be derived from single animals. Instead, at least three mice must be analysed to test for inter-animal rate homogeneity and therefore lack of mosaicism (Yauk et al., 2002). Thus, to prevent mosaicism compromising the scoring of mutations in this study, blood and sperm samples of five BALB/c x CBA/Ca F1 hybrid male mice were analysed for each ELF-field flux density (10 µT, 100 µT or 300 µT) for 2 or 15 hours, in addition to those from the control groups (Table 14). While, as in previous studies (Dubrova et al., 1998a, 2000a; Voutounou et al., 2012), all cases of mosaicism within de novo mutations detected in multiple DNA samples, were collectively recorded and scored as a single independent mutation event. Moreover, the presence of a third non-parental allele within somatic samples was disregarded and not included in the mutation analysis. 106 Figure 14. Mutational mosaicism at the Ms6-hm ESTR locus. Southern blot analysis of the CBA/CA and BALB/c alleles at the Ms6-hm locus. An autoradiograph representing 4 identical mosaic mutants at the CBA/CA allele and two mutants at the BALB/c (mutants indicated by arrows). 200 bp DNA step ladder (Promega) is used as a side lane reference. Figure 14. Mutational mosaicism at the Ms6-hm ESTR locus. Southern blot analysis of the CBA/CA and BALB/c alleles at the Ms6-hm locus. 3.5 Mutation Mosaicism An autoradiograph representing 4 identical mosaic mutants at the CBA/CA allele and two mutants at the BALB/c (mutants indicated by arrows). 200 bp DNA step ladder (Promega) is used as a side lane reference. Figure 14. Mutational mosaicism at the Ms6-hm ESTR locus. Southern blot analysis of the CBA/CA and BALB/c alleles at the Ms6-hm locus. An autoradiograph representing 4 identical mosaic mutants at the CBA/CA allele and two mutants at the BALB/c (mutants indicated by arrows). 200 bp DNA step ladder (Promega) is used as a side lane reference. 3.6 ESTR Mutation Frequencies in Sham-treated Males 3.6 ESTR Mutation Frequencies in Sham-treated Males Initially, the ESTR mutation frequencies were established in the blood and sperm DNA samples of the three groups of age-matched sham-treated male mice. A summary of the mutational data for both blood and sperm is presented in Table 15. Additionally, the mean ESTR mutation frequencies calculated in blood and sperm are presented by Figure 15. 3.6 ESTR Mutation Frequencies in Sham treated Males Initially, the ESTR mutation frequencies were established in the blood and sperm DNA samples of the three groups of age-matched sham-treated male mice. A summary of the mutational data for both blood and sperm is presented in Table 15. Additionally, the mean ESTR mutation frequencies calculated in blood and sperm are presented by Figure 15. Table 15. ESTR mutation frequencies in sham-treated males Group Number of mutations* Frequency ± s.e. Sham-treated (Blood) 2 h 25 (608 ± 27) 0.0411 ± 0.0084 15 h 28 (738 ± 30) 0.0379 ± 0.0073 X-rays 32 (683 ± 29) 0.0468 ± 0.0085 All sham-treated 85 (2030 ± 50) 0.0419 ± 0.0047 Sham-treated (Sperm) 2 h 22 (479 ± 24) 0.0460 ± 0.0101 15 h 36 (731 ± 30) 0.0507 ± 0.0054 X-rays 36 (646 ± 28) 0.0557 ± 0.0096 All sham-treated 94 (1856 ± 48) 0.0507 ± 0.0054 * Number of amplifiable molecules (± standard error (s e )) is given in brackets 107 Figure 15. ESTR mutation frequencies at the Ms6-hm locus in sham-treated males. Mutations were scored in both tissues of each indicated sham-treated group. Standard error bars are shown. Figure 15. ESTR mutation frequencies at the Ms6-hm locus in sham-treated males. Figure 15. ESTR mutation frequencies at the Ms6-hm locus in sham-treated males. Mutations were scored in both tissues of each indicated sham-treated group. Standard error bars are shown. Figure 15. ESTR mutation frequencies at the Ms6-hm locus in sham-treated males. Mutations were scored in both tissues of each indicated sham-treated group. Standard error bars are shown. A pairwise comparison performed between all the age-matched sham-treated control male mice in both tissues, determined that the frequency of ESTR mutation did not significantly differ between all sham-treated groups in either tissue (Table 16; blood, 0.43 <P < 0.77; sperm, 0.48 < P 0.80). Therefore, the age-matched sham-treated data were combined to form a single control group for each tissue. 3.6 ESTR Mutation Frequencies in Sham-treated Males 3.8 ESTR Mutation Frequencies in Males Exposed to 50 Hz Magnetic Fields 3.8.1 Blood Upon establishing the baseline mutation frequencies and aggregating the blood and sperm data of sham-treated controls, the frequency of ESTR mutation induction was next established in blood samples of MF-exposed male mice. Five BALB/c x CBA/Ca F1 Table 17. ESTR mutation frequencies for all age-matched controls Group No mutations* Frequency ± s.e. Ratio to control t Probability Sham-treated (Blood) X-rays 32 (683 ± 29) 0.0468 ± 0.0085 All sham-treated 85 (2030 ± 50) 0.0419 ± 0.0047 X-rays, 1Gy 54 (706 ± 30) 0.0764 ± 0.0109 1.63 2.14† 0.2275 1.83 2.92‡ 0.0245 Sham-treated (Sperm) X-rays 36 (646 ± 28) 0.0557 ± 0.0096 All sham-treated 94 (1856 ± 48) 0.0507 ± 0.0054 X-rays, 1Gy 75 (615 ± 27) 0.1220 ± 0.0151 2.19 3.71† 0.0014 2.41 4.46‡ 5.99 x 10-5 The total number of unique mutations and the subsequent mutation frequencies in blood and sperm for the sham-treated male mice as well as those exposed to 1 Gy of acute X-rays. Number of amplifiable molecules (± s.e.) is given in brackets. † Student’s test and Bonferroni corrected probability for difference from mutation frequency in the exposure-matched control group. ‡ Student’s test and Bonferroni corrected probability for difference from mutation frequency in the total control group (all sham- treated). 3.6 ESTR Mutation Frequencies in Sham-treated Males A highly significant 2.4-fold elevation in the frequency of Ms6-hm mutants was identified in sperm of irradiated males. Meanwhile, in blood, a less pronounced but still significant 1.8-fold increase was exhibited in these males following exposure. 3.7 ESTR Mutation Frequencies in Irradiated Males Five additional hybrid age-matched male mice were irradiated with an acute dose of 1 Gy X-rays in order to validate the SM-PCR technique (positive control). The frequency of ESTR mutation was significantly elevated in both the germline and somatic tissue of males exposed to 1 Gy of acute X-rays (Table 17). A highly significant 2.4-fold elevation in the frequency of Ms6-hm mutants was identified in sperm of irradiated males. Meanwhile, in blood, a less pronounced but still significant 1.8-fold increase was exhibited in these males following exposure. 3.7 ESTR Mutation Frequencies in Irradiated Males Five additional hybrid age-matched male mice were irradiated with an acute dose of 1 Gy X-rays in order to validate the SM-PCR technique (positive control). The frequency of ESTR mutation was significantly elevated in both the germline and somatic tissue of males exposed to 1 Gy of acute X-rays (Table 17). A highly significant 2.4-fold elevation in the frequency of Ms6-hm mutants was identified in sperm of irradiated males. Meanwhile, in blood, a less pronounced but still significant 1.8-fold increase was exhibited in these males following exposure. 3.7 ESTR Mutation Frequencies in Irradiated Males Table 17. ESTR mutation frequencies for all age-matched controls Group No mutations Frequency ± s.e. Ratio to control t Probability Sham-treated (Blood) X-rays 32 (683 ± 29) 0.0468 ± 0.0085 All sham-treated 85 (2030 ± 50) 0.0419 ± 0.0047 X-rays, 1Gy 54 (706 ± 30) 0.0764 ± 0.0109 1.63 2.14† 0.2275 1.83 2.92‡ 0.0245 Sham-treated (Sperm) X-rays 36 (646 ± 28) 0.0557 ± 0.0096 All sham-treated 94 (1856 ± 48) 0.0507 ± 0.0054 X-rays, 1Gy 75 (615 ± 27) 0.1220 ± 0.0151 2.19 3.71† 0.0014 2.41 4.46‡ 5.99 x 10-5 The total number of unique mutations and the subsequent mutation frequencies in blood and sperm for the sham-treated male mice as well as those exposed to 1 Gy of acute X-rays. Number of amplifiable molecules (± s.e.) is given in brackets. † Student’s test and Bonferroni corrected probability for difference from mutation frequency in the exposure-matched control group. ‡ Student’s test and Bonferroni corrected probability for difference from mutation frequency in the total control group (all sham- treated). 3.6 ESTR Mutation Frequencies in Sham-treated Males Such aggregation of all control data led to an increase in statistical power when comparing the mutation frequencies for all exposed males against those of the sham-treated males. Table 16. Statistics for the difference between sham-treated groups Sperm Blood Sham-treated 2 h 15 h X-rays 2 h 15 h X-rays 2 h - t = 0.25 t = 0.70 - t = 0.48 t = 0.29 15 h P = 0.8026 - t = 0.51 P = 0.6313 - t = 0.79 X-rays P = 0.4841 P = 0.6010 - P = 0.7719 P = 0.4297 - The Student’s test (t) and probability (P) for the difference between the frequencies of ESTR mutation at the Ms6-hm locus in DNA samples extracted from blood and sperm of male mice that were sham-treated for either 2 hours, 15 hours or under the same conditions as the sample group exposed to 1 Gy X-rays. Table 16. Statistics for the difference between sham-treated groups Sperm Blood Sham-treated 2 h 15 h X-rays 2 h 15 h X-rays 2 h - t = 0.25 t = 0.70 - t = 0.48 t = 0.29 15 h P = 0.8026 - t = 0.51 P = 0.6313 - t = 0.79 X-rays P = 0.4841 P = 0.6010 - P = 0.7719 P = 0.4297 - Table 16. Statistics for the difference between sham-treated groups The Student’s test (t) and probability (P) for the difference between the frequencies of ESTR mutation at the Ms6-hm locus in DNA samples extracted from blood and sperm of male mice that were sham-treated for either 2 hours, 15 hours or under the same conditions as the sample group exposed to 1 Gy X-rays. The Student’s test (t) and probability (P) for the difference between the frequencies of ESTR mutation at the Ms6-hm locus in DNA samples extracted from blood and sperm of male mice that were sham-treated for either 2 hours, 15 hours or under the same conditions as the sample group exposed to 1 Gy X-rays. 108 3.7 ESTR Mutation Frequencies in Irradiated Males Five additional hybrid age-matched male mice were irradiated with an acute dose of 1 Gy X-rays in order to validate the SM-PCR technique (positive control). The frequency of ESTR mutation was significantly elevated in both the germline and somatic tissue of males exposed to 1 Gy of acute X-rays (Table 17). 3.8.1 Blood Upon establishing the baseline mutation frequencies and aggregating the blood and sperm data of sham-treated controls, the frequency of ESTR mutation induction was next established in blood samples of MF-exposed male mice. Five BALB/c x CBA/Ca F1 hybrid male mice were analysed for each magnetic field flux density (10, 100 or 300 µT) at both time points of exposure (2 hours or 15 hours). The ESTR mutation data 109 regarding the blood samples are summarised in Table 18, while Figure 16 presents the frequency of ESTR mutations in blood for all males exposed to ELF-MFs and the matched sham-treated males. matched sham-treated males. Table 18. Summary of ESTR mutation data in blood Group No mutations Frequency ± s.e. Ratio to control t Probability Sham-treated 2 h 25 (608 ± 27) 0.0411 ± 0.0084 15 h 28 (738 ± 30) 0.0379 ± 0.0073 All sham- treated 85 (2030 ± 50) 0.0419 ± 0.0047 – – – ELF-exposed 10 μT, 2 h 24 (623 ± 27) 0.0385 ± 0.0080 0.94 0.22† 1 0.92 0.36‡ 1 10 μT, 15 h 26 (575 ± 26) 0.0452 ± 0.0091 1.19 0.62† 1 1.08 0.33‡ 1 100 μT, 2 h 26 (527 ± 25) 0.0494 ± 0.0100 1.20 0.63† 1 1.18 0.68‡ 1 100 μT, 15 h 30 (597 ± 27) 0.0503 ± 0.0095 1.33 1.03† 1 1.20 0.80‡ 1 300 μT, 2 h 23 (624 ± 27) 0.0369 ± 0.0079 0.90 0.37† 1 0.88 0.55‡ 1 300 μT, 15 h 27 (709 ± 30) 0.0381 ± 0.0075 1.00 0.01† 1 0.91 0.43‡ 1 All ELF-MF, 2h 73 (1744 ± 46) 0.0412 ± 0.0049 0.98 0.11 1 All ELF-MF, 15h 83 (1881 ± 48) 0.0441 ± 0.0050 1.05 0.33 1 All ELF-MF 156 (3655 ± 67) 0.0427 ± 0.0035 1.02 0.14 0.8887 * Number of amplifiable molecules (± s.e.) is given in brackets. † Student’s test and Bonferroni corrected probability for difference from mutation frequency in the exposure-matched control group. ‡ Student’s test and Bonferroni corrected probability for difference from mutation frequency in the total control group (all sham-treated). Taken together, these data demonstrate that the frequency of ESTR mutation in the somatic tissue of all males exposed to magnetic fields did not significantly differ from that of their equivalent sham-treated control groups. 3.8.1 Blood Furthermore, a comparison of ESTR mutation frequencies of all exposed males against the aggregated sham-treated group are in accordance with those of their age-matched equivalent, and again fail to reveal any significant differences. Table 18. Summary of ESTR mutation data in blood Group No mutations* Frequency ± s.e. Ratio to control t Probability Sham-treated 2 h 25 (608 ± 27) 0.0411 ± 0.0084 15 h 28 (738 ± 30) 0.0379 ± 0.0073 All sham- treated 85 (2030 ± 50) 0.0419 ± 0.0047 – – – ELF-exposed 10 μT, 2 h 24 (623 ± 27) 0.0385 ± 0.0080 0.94 0.22† 1 0.92 0.36‡ 1 10 μT, 15 h 26 (575 ± 26) 0.0452 ± 0.0091 1.19 0.62† 1 1.08 0.33‡ 1 100 μT, 2 h 26 (527 ± 25) 0.0494 ± 0.0100 1.20 0.63† 1 1.18 0.68‡ 1 100 μT, 15 h 30 (597 ± 27) 0.0503 ± 0.0095 1.33 1.03† 1 1.20 0.80‡ 1 300 μT, 2 h 23 (624 ± 27) 0.0369 ± 0.0079 0.90 0.37† 1 0.88 0.55‡ 1 300 μT, 15 h 27 (709 ± 30) 0.0381 ± 0.0075 1.00 0.01† 1 0.91 0.43‡ 1 All ELF-MF, 2h 73 (1744 ± 46) 0.0412 ± 0.0049 0.98 0.11 1 All ELF-MF, 15h 83 (1881 ± 48) 0.0441 ± 0.0050 1.05 0.33 1 All ELF-MF 156 (3655 ± 67) 0.0427 ± 0.0035 1.02 0.14 0.8887 * † Table 18. Summary of ESTR mutation data in blood * Number of amplifiable molecules (± s.e.) is given in brackets. † Student’s test and Bonferroni corrected probability for difference from mutation frequency in the exposure-matched control group. ‡ Student’s test and Bonferroni corrected probability for difference from mutation frequency in the total control group (all sham-treated). Taken together, these data demonstrate that the frequency of ESTR mutation in the somatic tissue of all males exposed to magnetic fields did not significantly differ from that of their equivalent sham-treated control groups. Furthermore, a comparison of ESTR mutation frequencies of all exposed males against the aggregated sham-treated group are in accordance with those of their age-matched equivalent, and again fail to reveal any significant differences. 110 Figure 16. ESTR mutation frequencies in blood of exposed and matched sham- treated males. SM-PCR and Southern blot analysis were performed on DNA extracted from the blood of exposed CBA/CA x BALB/c male mice. Mutations were scored for each of the indicated exposure doses/time periods. 3.8.1 Blood Standard error bars are shown. Figure 16. ESTR mutation frequencies in blood of exposed and matched sham- Figure 16. ESTR mutation frequencies in blood of exposed and matched sham- treated males. SM-PCR and Southern blot analysis were performed on DNA extracted from the blood of exposed CBA/CA x BALB/c male mice. Mutations were scored for each of the indicated exposure doses/time periods. Standard error bars are shown. Figure 16. ESTR mutation frequencies in blood of exposed and matched sham- treated males. SM-PCR and Southern blot analysis were performed on DNA extracted from the blood of exposed CBA/CA x BALB/c male mice. Mutations were scored for each of the indicated exposure doses/time periods. Standard error bars are shown. Thereafter, the effects of both 2 hours and 15 hours exposure to 50 Hz magnetic fields of 10 µT, 100 µT or 300 µT in strength were established in the germline of BALB/c x CBA/Ca F1 hybrid male mice. 3.8.2 Sperm p Again mutations were scored in five hybrid male mice per group. The ESTR mutation data for sperm are summarised in Table 19. Again mutations were scored in five hybrid male mice per group. The ESTR mutation data for sperm are summarised in Table 19. While, the frequency of ESTR mutation in sperm of all exposed males exceeds that illustrated in blood, no significant differences were revealed between ESTR mutation frequencies of the exposed males and their equivalent sham-treated counterparts (Figure 17; 0.52 < P ≤ 1 for all pair wise comparisons). These findings were again corroborated through a comparison of mutation frequencies against the aggregated sham-treated control data. Again, the frequency of ESTR mutation in the germline 111 tissue of all males exposed to magnetic fields did not significantly differ from that in the total control group (0.45< P ≤ 1 for all pair wise comparisons). Table 19. Summary of ESTR mutation data in sperm Group No mutations* Frequency ± s.e. Ratio to control t Probability Sham-treated 2 h 22 (479 ± 24) 0.0460 ± 0.0101 15 h 36 (731 ± 30) 0.0507 ± 0.0054 All sham-treated 94 (1856 ± 48) 0.0507 ± 0.0054 – – – ELF-exposed 10 μT, 2 h 45 (639 ± 29) 0.0704 ± 0.0110 1.53 1.64† 0.7091 1.39 1.62‡ 0.7378 10 μT, 15 h 38 (621 ± 27) 0.0612 ± 0.0103 1.24 0.90† 1 1.21 0.91‡ 1 100 μT, 2 h 49 (852 ± 33) 0.0575 ± 0.0085 1.25 0.87† 1 1.13 0.68‡ 1 100 μT, 15 h 59 (829 ± 32) 0.0712 ± 0.0097 1.45 1.71† 0.6125 1.40 1.85‡ 0.4508 300 μT, 2 h 59 (833 ± 33) 0.0708 ± 0.0096 1.54 1.78† 0.5271 1.40 1.83‡ 0.4718 300 μT, 15 h 52 (855 ± 33) 0.0609 ± 0.0088 1.24 0.95† 1 1.20 0.99‡ 1 All ELF-MF, 2h 153 (2324 ± 55) 0.0658 ± 0.0055 1.30 1.96 0.1002 All ELF-MF, 15h 146 (2280 ± 54) 0.0640 ± 0.0055 1.26 1.74 0.1638 All ELF-MF 299 (4604 ± 76) 0.0649 ± 0.0039 1.28 2.15 0.0316 * Number of amplifiable molecules (± s.e.) is given in brackets. † Student’s test and Bonferroni corrected probability for difference from mutation frequency in the exposure-matched control group. ‡ Student’s test and Bonferroni corrected probability for difference from mutation frequency in the total control group (all sham-treated). 3.8.2 Sperm Overall, the frequency of ESTR mutation in the germline and blood of all exposed males did not significantly differ from that in their equivalent sham-treated groups (Figures 16, 17; 0.52 < P ≤ 1 for all pair wise comparisons). Nor was there any significant difference when the frequency of ESTR mutation in the blood and sperm of all exposed males were compared to that of an aggregated total control group. Overall, the frequency of ESTR mutation in the germline and blood of all exposed males did not significantly differ from that in their equivalent sham-treated groups (Figures 16, 17; 0.52 < P ≤ 1 for all pair wise comparisons). Nor was there any significant difference when the frequency of ESTR mutation in the blood and sperm of all exposed males were compared to that of an aggregated total control group. 112 Figure 17. ESTR mutation frequencies in sperm of exposed and matched sham- treated males. SM-PCR and Southern blot analysis were performed on DNA extracted from the sperm of exposed CBA/CA x BALB/c male mice. Mutations were scored for each of the indicated exposure doses/time periods. Standard error bars are shown. Figure 17. ESTR mutation frequencies in sperm of exposed and matched sham- Figure 17. ESTR mutation frequencies in sperm of exposed and matched sham- treated males. SM-PCR and Southern blot analysis were performed on DNA extracted from the sperm of exposed CBA/CA x BALB/c male mice. Mutations were scored for each of the indicated exposure doses/time periods. Standard error bars are shown. treated males. SM-PCR and Southern blot analysis were performed on DNA extracted from the sperm of exposed CBA/CA x BALB/c male mice. Mutations were scored for each of the indicated exposure doses/time periods. Standard error bars are shown. 3.8.3 Combined ELF-MF effect To increase the statistical power of the present findings, all the ESTR mutation data for all exposed mice were pooled to include all exposure conditions (field flux density and exposure length), for each individual tissue (Tables 18, 19). Additionally, in the present study, the ESTR mutation frequency of the pooled data was analysed according to the length of exposure for each tissue to determine any cumulative effects of exposure for either 2- or 15 hours. Upon aggregation of all blood data, the ESTR mutation frequency did not significantly differ and was found to be only marginally elevated when compared to that in sham- treated animals (P = 0.8887). While analysis of pooled data for each individual exposure length (2- or 15 hours) also presented similar findings, analysis of the aggregated sperm data illustrated a marginally significant elevation in the total frequency of ESTR mutation (P = 0.0316). Meanwhile, separate analysis of each individual exposure length revealed virtually no discernible difference in total mutation 113 frequencies between males exposed for 15 hours and those irradiated for a duration of 2 hours (Figure 18; 2 hours, P = 0.1002; 15 hours, P = 0.1638). Figure 18. ESTR mutation frequencies in sham-treated and exposed males. A comparison of the average fold induction of ESTR mutation frequencies for both blood and sperm of exposed mice relative to the control. Standard errors are shown. Figure 18. ESTR mutation frequencies in sham-treated and exposed males. A comparison of the average fold induction of ESTR mutation frequencies for both blood and sperm of exposed mice relative to the control. Standard errors are shown. Figure 18. ESTR mutation frequencies in sham-treated and exposed males. A comparison of the average fold induction of ESTR mutation frequencies for both blood and sperm of exposed mice relative to the control. Standard errors are shown. 3.9 Mutation Spectrum Following ESTR mutation frequency analysis, the spectra of length-changes were next examined in each of the identified induced and spontaneous Ms6-hm mutants. Following ESTR mutation frequency analysis, the spectra of length-changes were next examined in each of the identified induced and spontaneous Ms6-hm mutants. According to previous studies, Ms6-hm mutants exhibit similar mutational spectra irrespective of whether mutations arose spontaneously or were induced by ionising radiation (Yauk et al., 2002) or chemical mutagens (Glen et al., 2008). Similarly, no significant differences in the size distribution of mutants were found to arise between Ms6-hm mutations in the germline or somatic tissues taken from 8-12 week old male mice (Yauk et al., 2002; Barber et al., 2006, 2009; Hatch et al., 2007). These data therefore suggest that mutations arising in germline or somatic tissues, either spontaneously or induced, do so through a similar mutation process previously theorised to be through replication slippage. According to previous studies, Ms6-hm mutants exhibit similar mutational spectra irrespective of whether mutations arose spontaneously or were induced by ionising radiation (Yauk et al., 2002) or chemical mutagens (Glen et al., 2008). Similarly, no significant differences in the size distribution of mutants were found to arise between Ms6-hm mutations in the germline or somatic tissues taken from 8-12 week old male mice (Yauk et al., 2002; Barber et al., 2006, 2009; Hatch et al., 2007). These data therefore suggest that mutations arising in germline or somatic tissues, either spontaneously or induced, do so through a similar mutation process previously theorised to be through replication slippage. 114 Therefore, to further substantiate the aforementioned working hypothesis, the spectra for all 757 de novo ESTR mutant molecules recovered in blood and sperm were analysed. As in previous studies (Yauk et al., 2002; Glen et al., 2008; Abouzeid Ali et al., 2012; Voutounou et al., 2012), analysis involved the categorisation of Ms6-hm mutants as either insertions or deletions, as well as the determination of the number of repeat units gained or lost at each locus. As with mutational analysis, those mutants exhibiting mosaicism were treated as a single mutational event and subsequently analysed as one. All Ms6-hm mutants identified in blood samples exhibiting repeat unit insertion following ELF-MF exposure were aggregated, irrespective of exposure parameters, as were those presenting repeat unit deletions. The incidence of unique mutations involving gain and loss of repeat units at the Ms6- hm locus detected in DNA samples extracted from blood of control and exposed male mice. The chi-square test and corresponding P values for differences between groups are also shown. 3.9 Mutation Spectrum These measures were undertaken to increase the statistical power in the analysis of the distribution of repeat units gained or lost by the identified Ms6-hm mutants following ELF-MF exposure. 3.9.1 Size Spectra of Ms6-hm Mutations in Blood The mutation distributions were initially analysed for blood and a summary of the mutation spectrums for the 291 de novo mutations detected is presented in Table 20. 3.9.1 Size Spectra of Ms6-hm Mutations in Blood The mutation distributions were initially analysed for blood and a summary of the mutation spectrums for the 291 de novo mutations detected is presented in Table 20. 3.9.1 Size Spectra of Ms6-hm Mutations in Blood The progenitor allele was assumed to be the parental allele closest in size to the mutant allele. Kruskal-Wallis test, P = 0.0819. Figure 19. Spectra of somatic ESTR mutations in sham-treated controls and exposed male mice. The progenitor allele was assumed to be the parental allele closest in size to the mutant allele. Kruskal-Wallis test, P = 0.0819. Figure 19. Spectra of somatic ESTR mutations in sham-treated controls and exposed male mice. The progenitor allele was assumed to be the parental allele closest in size to the mutant allele. Kruskal-Wallis test, P = 0.0819. Figure 19. Spectra of somatic ESTR mutations in sham-treated controls and exposed male mice. The progenitor allele was assumed to be the parental allele closest in size to the mutant allele. Kruskal-Wallis test, P = 0.0819. 3.9.1 Size Spectra of Ms6-hm Mutations in Blood Table 20. ESTR mutation spectra of control and treated males in blood Group Gains (%) Losses (%) Total Control 62 (72.94%) 23 (27.06%) 85 MF 98 (64.47%) 54 (35.53%) 152 X-rays 40 (74.07%) 14 (25.93%) 54 Total 200 (68.73%) 91 (31.27%) 291 Chi-square all χ2 df=2, = 2.70 P = 0.2592 Chi-square MF χ2 df=1, = 1.78 P = 0.1819 Chi-square X-rays χ2 df=1, = 0.02 P = 0.8829 The incidence of unique mutations involving gain and loss of repeat units at the Ms6- hm locus detected in DNA samples extracted from blood of control and exposed male mice. The chi-square test and corresponding P values for differences between groups are also shown. Individual analysis into the distribution of mutant alleles (gain vs loss of repeat units) for either ELF-MF or X-ray samples revealed no significant differences to those exhibited by the sham-treated controls (Table 20; ELF-MF, P = 0.1819; X-rays, P = 0.8829). Likewise, although demonstrating a slight visible preference towards the gain of repeat units, there was no significant difference in the combined distributions of Individual analysis into the distribution of mutant alleles (gain vs loss of repeat units) for either ELF-MF or X-ray samples revealed no significant differences to those exhibited by the sham-treated controls (Table 20; ELF-MF, P = 0.1819; X-rays, P = 0.8829). Likewise, although demonstrating a slight visible preference towards the gain of repeat units, there was no significant difference in the combined distributions of 115 mutations involving gain or loss of repeat units between the irradiated (MF & X-ray) and control groups (P = 0.2592) in blood samples. Upon characterising the distribution of the identified Ms6-hm mutants, the spectra of length-changes involved in both mutant additions and deletions were next considered (Figure 19). The same process was followed as in Yauk et al. (2002), in which mutations involving insertions or deletions of similar lengths were grouped (+ 2-5 repeats, + 6-9 repeats, + 10-13, + 14-29 and + 30+ repeats). The spectra of size changes in Ms6-hm mutants were compared between blood samples taken from unexposed sham-treated, ELF-MF exposed and X-ray irradiated male mice. The combined distributions of length- changes at ESTR loci did not differ significantly (P = 0.0819), indicating that there is no divergence between the insertion and deletion of repeat units. 116 Figure 19. Spectra of somatic ESTR mutations in sham-treated controls and exposed male mice. 3.9.2 Size Spectra of Ms6-hm Mutations in Sperm The distribution and spectra of length-changes for all 466 Ms6-hm germline mutations were also defined. A summary of the frequency distribution for gains and losses of Ms6-hm repeat units is presented in Table 21. Comparable to blood samples, there were no significant differences in the combined distributions of mutations involving gain or loss of repeat units in sperm between the irradiated (ELF-MF & X-ray) and control groups (P = 0.5541). Furthermore, the spectra of length-changes in the sperm Ms6-hm mutants did not significantly differ between all groups either (Figure 20; P = 0.8438). 117 Table 21. ESTR mutation spectra of control and treated males in sperm Group Gains (%) Losses (%) Total Control 52 (55.32%) 42 (44.68%) 94 MF 183 (61.62%) 114 (38.38%) 297 X-rays 45 (60.00%) 30 (40.00%) 75 Total 280 (60.09%) 186 (39.91%) 466 Chi-square all χ2 df=2, = 1.18 P = 0.5541 Chi-square MF χ2 df=1, = 1.18 P = 0.2772 Chi-square X-rays χ2 df=1, = 0.37 P = 0.5409 The incidence of unique mutations involving gain and loss of repeat units at the Ms6- hm locus detected in DNA samples extracted from sperm of control and exposed male mice. The chi-square test and corresponding P values for differences between groups are also shown. Table 21. ESTR mutation spectra of control and treated males in sperm Group Gains (%) Losses (%) Total Control 52 (55.32%) 42 (44.68%) 94 MF 183 (61.62%) 114 (38.38%) 297 X-rays 45 (60.00%) 30 (40.00%) 75 Total 280 (60.09%) 186 (39.91%) 466 Chi-square all χ2 df=2, = 1.18 P = 0.5541 Chi-square MF χ2 df=1, = 1.18 P = 0.2772 Chi-square X-rays χ2 df=1, = 0.37 P = 0.5409 Table 21. ESTR mutation spectra of control and treated males in sperm The incidence of unique mutations involving gain and loss of repeat units at the Ms6- hm locus detected in DNA samples extracted from sperm of control and exposed male mice. The chi-square test and corresponding P values for differences between groups are also shown. Figure 20. Spectra of germline ESTR mutations in sham-treated controls and exposed male mice. The progenitor allele was assumed to be the parental allele closest in size to the mutant allele. Kruskal-Wallis test, P = 0.8438. Figure 20. Spectra of germline ESTR mutations in sham-treated controls and exposed male mice. The progenitor allele was assumed to be the parental allele closest in size to the mutant allele. 3.9.2 Size Spectra of Ms6-hm Mutations in Sperm Kruskal-Wallis test, P = 0.8438. Overall, the data presented in the current study therefore indicate that neither the frequency distribution nor the spectra of length-changes in Ms6-hm mutants significantly alter from those found in age-matched sham-treated controls, in either the germline or somatic tissues of hybrid male mice exposed to a 50 Hz magnetic field at intensities of 10, 100 and 300 T for 2- and 15 hours. Likewise, acute irradiation to 1 118 Gy X-rays does not significantly change frequency distribution, or the spectra of length- changes in Ms6-hm mutants from those found in either germline or somatic tissues of age-matched sham-treated controls. The data from this present study are therefore in accordance with those previously presented (Yauk et al., 2002; Dubrova, 2005), and provide further evidence as to the presence of a similar mutational process within both the germline and soma in irradiated male mice (1 Gy acute X-rays). 3.10.1 Experimental Design p g While the present analysis of ESTR mutation frequencies failed to detect any significant changes between ELF-MF exposed and sham-treated males, ELF-MF represents a weak environmental stimulus incapable of ionising molecules, therefore any potential impact of ELF-MF on human health is likely complex. Thus the use of microarray technology to analyse gene expression profiles, following ELF-MF exposure, may not only identify prospective gene candidates but also provides an additionally informative endpoint in the series of ELF-MF studies. Considering this, this present pilot study was designed and initiated in an attempt to validate the mutation induction data. Gene expression profiles pertaining to ELF-MF and age-matched sham-treated BALB/c x CBA/Ca F1 hybrid male mice were compared in parallel with those from X-ray irradiated males using microarray expression data. Five BALB/c x CBA/Ca F1 hybrid male mice were exposed for 15 hours to a magnetic flux density of 300 µT or whole-body acute irradiation of 1 Gy X-rays and their kidney samples taken 12 weeks post-exposure. Kidney samples were also taken from an additional five sham-treated BALB/c x CBA/Ca F1 hybrid male mice 12 weeks post mock-exposure and used as control samples for data analysis. Indeed, the kidney represents a transcriptionally rich tissue in which to analyse the effects of ELF-MF on gene expression as it is a highly specialised organ with a great diversity of functions and cell types essential in numerous hormonal and homeostatic regulatory functions. Moreover, high quality RNA extraction and gene pattern analyses have previously been illustrated in non-exposed offspring of irradiated CBA/Ca and BALB/c male mice (Gomes et al., 2015) as well as in CBA/Ca and BALB/c mice subjected to a methyl-donor deficient diet (Glen et al., 2015). RNAs were therefore extracted from both kidneys in 119 each mouse per exposure group and quality analysis performed (Chapter 2.13.1, Figure 10). Additionally, each individual RNA sample from the five male mice was subjected to three independent biological replicates while, to minimize inter-experiment variation, microarrays were performed in duplicate for each experimental group. This preliminary study implemented a one-colour microarray-based gene expression analysis, whereby cyanine 3-labelled target sequences were used to measure gene expression profiles of MF-exposed and X-ray irradiated BALB/c x CBA/Ca F1 hybrid males, with the highly sensitive Agilent SurePrint G3 Mouse GE 8x60K microarrays (Agilent Technologies, Santa Clara, CA, USA). 3.10.1 Experimental Design Each Agilent SurePrint G3 Mouse GE 8x60K microarray slide was printed with eight identical arrays containing 39,430 Entrez Gene RNAs and 16,251 lincRNAs (long intergenic non-coding RNAs). Gene expression profiles were analysed using a one-colour microarray analysis which utilises an intensity-based platform, through which the intensities of each individual probe were generated in each array experiment. The use of a one-colour microarray analysis offered several advantages in that it allowed for inter-array comparisons of the measured gene expression, while the use of only cyanine 3 dye was more robust compared to the more degradation-prone cyanine 5 dye. 3.10.2.1 ELF-MF Gene Expression Analysis 3.10.2.1 ELF MF Gene Expression Analysis The pattern of gene expression was initially established in the ELF-MF exposed males (300 µT, 15 hours). Microarray analysis was visualised primarily through volcano plots following the employment of the Student’s t test with false discovery rate (FDR) parameters of P <0.05 and identifying only those transcripts expressing a fold change of >2. The primary data did not reveal any differentially expressed loci following exposure to 300 µT for 15 hours compared with the sham-treated controls (Figure 21). 120 Figure 21. ELF-MF gene expression analysis. Volcano plot analysis exhibiting the differential pattern of gene expression in BALB/c x CBA/Ca F1 hybrid male mice following 15 hour exposure to 300 µT compared to their age-matched sham-treated controls. The log2 fold-change values are plotted on the x-axis of the volcano plots and are compared to the negative log10 raw P values on the y-axis. Figure 21. ELF-MF gene expression analysis. Volcano plot analysis exhibiting the Figure 21. ELF-MF gene expression analysis. Volcano plot analysis exhibiting the differential pattern of gene expression in BALB/c x CBA/Ca F1 hybrid male mice following 15 hour exposure to 300 µT compared to their age-matched sham-treated controls. The log2 fold-change values are plotted on the x-axis of the volcano plots and are compared to the negative log10 raw P values on the y-axis. 3.10.2.2 Ionising Radiation Gene Expression Analysis g p y Using the same two-fold cut-off criteria, the pattern of expression of protein-coding genes (mRNA) and long intergenic non-coding RNAs (lincRNAs) were analysed 12- weeks post whole-body acute 1 Gy X-ray exposure (positive control). In contrast to ELF-MF gene expression profile analysis, RNA samples extracted from irradiated kidney cells revealed five significantly up-regulated loci (P < 0.05) when compared with sham- treated controls (shown in red, Figure 22). The five significantly up-regulated loci identified within the positive control experiment of this pilot study relate to the two genes; Il12rb2 and Ltb4r2, in addition to three lincRNAs and are summarised in Table 22. 121 Figure 22. Ionising radiation gene expression analysis. Volcano plot analysis exhibiting the differential pattern of gene expression in BALB/c x CBA/Ca F1 hybrid male mice following whole-body acute 1 Gy X-ray exposure compared to their age-matched sham-treated controls. The log2 fold-change values are plotted on the x-axis of the volcano plots and are compared to the negative log10 raw P values on the y-axis. Genes with an absolute differential expression fold-change >2, alongside a raw P value <0.05 are shown in red. Figure 22. Ionising radiation gene expression analysis. Volcano plot analysis exhibitin Figure 22. Ionising radiation gene expression analysis. Volcano plot analysis exhibiting the differential pattern of gene expression in BALB/c x CBA/Ca F1 hybrid male mice following whole-body acute 1 Gy X-ray exposure compared to their age-matched sham-treated controls. The log2 fold-change values are plotted on the x-axis of the volcano plots and are compared to the negative log10 raw P values on the y-axis. Genes with an absolute differential expression fold-change >2, alongside a raw P value <0.05 are shown in red. 122 Table 22. Summary of up-regulated genes Probe Name Gene Symbol Description Fold Change Log Fold Change Regulation P (Corr) * A_30_P01018642 lincRNA:chr17:14320464-14320975 forward strand 4.73 2.24 Up 0.0384 A_55_P1970489 Il12rb2 Mus musculus interleukin 12 receptor, beta 2 (Il12rb2), mRNA [NM_008354] 6.25 2.64 Up 0.0384 A_51_P282211 Ltb4r2 Mus musculus leukotriene B4 receptor 2 (Ltb4r2), mRNA [NM_020490] 7.51 2.91 Up 0.0072 A_30_P01025210 lincRNA:chr8:44232164-44267061 reverse strand 5.55 2.47 Up 0.0454 A_30_P01030493 lincRNA:chr10:39659425-39674200 forward strand 5.33 2.42 Up 0.0323 The details of expression changes exhibited by the five up-regulated probes following whole body acute exposure to 1 Gy X-rays. * Probability values were corrected for multiple testing using the Benjamini-Hochberg False Discovery Rate method. Table 22. 3.10.2.2 Ionising Radiation Gene Expression Analysis Summary of up-regulated genes The details of expression changes exhibited by the five up-regulated probes following whole body acute exposure to 1 Gy X-rays. * Probability values were corrected for multiple testing using the Benjamini-Hochberg False Discovery Rate method. 123 Next, the normalised Cy-3 intensity values were compared for each of the five up- regulated probes between each X-ray irradiated and sham-treated male mice. These data confirmed an absence of outliers, illustrating that each probe was persistently up- regulated in each of the five X-ray irradiated mice, and in contrast to the corresponding expression in the sham-treated males (Figure 23). Figure 23. Comparison of differentially expressed probes. Line plot analysis illustrating the normalised signal intensities (log2 values) of each up-regulated probe in each individual sham-treated and X-ray irradiated male mouse. Figure 23. Comparison of differentially expressed probes. Line plot analysis illustrating the normalised signal intensities (log2 values) of each up-regulated probe in each individual sham-treated and X-ray irradiated male mouse. Figure 23. Comparison of differentially expressed probes. Line plot analysis illustrating the normalised signal intensities (log2 values) of each up-regulated probe in each individual sham-treated and X-ray irradiated male mouse. Figure 23. Comparison of differentially expressed probes. Line plot analysis illustrating the normalised signal intensities (log2 values) of each up-regulated probe in each individual sham-treated and X-ray irradiated male mouse. Finally, a probability plot was employed as an alternative exposure metric to confirm the prior analyses identifying the absence of statistically significant exposure effect in the ELF-MF exposed males, in addition to the significant upregulation of five transcripts in males irradiated with 1 Gy acute X-rays (Figure 24). Probability plots represent a useful method in preliminary microarray data analyses as it allows the correction for the large number of comparisons, thereby offering an informative and functionally useful interpretation for the thousands of genes being studied (Wartenberg et al., 1994). Normally implemented to evaluate how well a data set matches a specific distribution, in this current study the probability plot has been utilised to represent a visual aid in the confirmation of genes with differential expression levels. Owing to such a large number of comparisons, genes whose 124 expression levels vary between the control and treatment groups can be identified as the points that deviate significantly from the otherwise linear relationship. 3.10.2.2 Ionising Radiation Gene Expression Analysis Figure 24. Probability plot analysis comparing the exposure distributions between ELF-MF and X-ray irradiated samples. The P-values are ranked and then log- transformed (-log10) in order of rank (x-axis). These are compared to the negative log10 transformed corrected P values plotted on the y-axis. 3.10.2.2 Ionising Radiation Gene Expression Analysis For the two datasets considered here (ELF-MF exposure and ionising radiation) (Figure 24), it is illustrated that no single gene was differentially expressed 12 weeks following a 15 hour, 300 µT ELF-MF exposure. It follows that such an expression is likely representative of background global levels of exposure. Comparatively, following exposure to 1 Gy X-rays, Figure 24 not only illustrates the remnants of a global exposure response in the same transcripts 12 weeks post irradiation, but the expression levels of five transcripts have deviated significantly from the linear relationship expressed by the other transcripts, corresponding to those transcripts whose expression levels were significantly up-regulated in Figure 22. Such a visual representation of the data therefore confirms the robustness of the previous SM-PCR analyses, and moreover, that the effects of ELF-MF on gene expression are negligible compared to those following exposure to 1 Gy X-rays. expression levels vary between the control and treatment groups can be identified as the points that deviate significantly from the otherwise linear relationship. For the two datasets considered here (ELF-MF exposure and ionising radiation) (Figure 24), it is illustrated that no single gene was differentially expressed 12 weeks following a 15 hour, 300 µT ELF-MF exposure. It follows that such an expression is likely representative of background global levels of exposure. Comparatively, following exposure to 1 Gy X-rays, Figure 24 not only illustrates the remnants of a global exposure response in the same transcripts 12 weeks post irradiation, but the expression levels of five transcripts have deviated significantly from the linear relationship expressed by the other transcripts, corresponding to those transcripts whose expression levels were significantly up-regulated in Figure 22. Such a visual representation of the data therefore confirms the robustness of the previous SM-PCR analyses, and moreover, that the effects of ELF-MF on gene expression are negligible compared to those following exposure to 1 Gy X-rays. 125 Figure 24. Probability plot analysis comparing the exposure distributions between ELF-MF and X-ray irradiated samples. The P-values are ranked and then log- transformed (-log10) in order of rank (x-axis). These are compared to the negative log10 transformed corrected P values plotted on the y-axis. Figure 24. Probability plot analysis comparing the exposure distributions between ELF-MF and X-ray irradiated samples. The P-values are ranked and then log- transformed (-log10) in order of rank (x-axis). These are compared to the negative log10 transformed corrected P values plotted on the y-axis. 4.1 Summary of Data y In this thesis, the frequency of germline and somatic ESTR mutation induction was analysed following 2- or 15 hour exposures to 50 Hz magnetic fields of 10, 100 or 300 µT. The data presented from the studies within this thesis demonstrate that in neither tissue does the frequency of ESTR mutation induction of all exposed male mice significantly differ from that in their equivalent sham-treated groups (Figures 16, 17). Moreover, a lack of significant difference in ESTR mutation frequency remained in both the germline and somatic tissues of exposed mice when compared to that of an aggregated group of sham-treated controls. In contrast, the frequency of ESTR mutation was significantly elevated in both the germline and somatic tissue of males exposed to 1 Gy of acute X-rays (Table 17). 126 These data were in part confirmed by an additional pilot study in which high- throughput microarray analysis further demonstrated the lack of any significant alteration in gene expression in kidney cells of BALB/c x CBA/Ca hybrid F1 males following a 15 hour exposure to 50 Hz magnetic fields of 300 µT (Figure 21). Overall, these studies represent, to the best of my knowledge, the first methodical attempt to determine the in vivo frequency with which mutation induction occurs in the germline and somatic tissues of male mice after continuous exposure to 50 Hz ELF- MFs up to a dose of 300 μT (Wilson et al., 2015). 4.2 Epidemiological Data p g Throughout this thesis, a constant emphasis has been placed on how exposure to ELF- MF background environmental fields is unavoidable. The development of electrical technology and expansion of their use within modern society has led to an increase in both residential and occupational exposure to ELF-MFs. Owing to such continuous worldwide ELF-MF exposure, it is therefore important to analyse and understand the effects of ELF-MF on living organisms. As such, over the years, the effects of ELF-MF have been extensively studied in regards to both their impact on general health and at the DNA level, using an array of techniques, doses, measurement strategies and systems. Yet, this has resulted in a contradicting body of literature. Thus, an issue still remains as to the potential adverse biological/genotoxic effects of residential and/or occupational ELF-MF exposure. It was therefore owing to this discrepancy that the present study was designed in order to investigate the possible mutagenic potential of low dose ELF-MF exposure in vivo. This was achieved through the analysis of mutation induction frequency in both the somatic and germline tissues of BALB/c x CBA/Ca hybrid F1 males, following exposure to 10, 100 and 300 T for 2 or 15 hours. These designated field strengths incorporate a spectrum of doses specifically chosen to encompass the International Commission on Non-Ionising Radiation’s recommended exposure limits pertaining to the general public, which is set at 200 μT for 50 Hz magnetic fields (International Commission on Non-Ionizing Radiation Protection, 2010). Moreover, they are inclusive of field strengths to which exposure has previously resulted in DNA damage in vitro (intermittent exposure, > 35 µT, (Ivancsits et al., 2003b)). However, it is of note that these field strengths are substantially larger than 127 those associated with the average household exposure in both the UK and USA (AGNIR, 2001), in addition to those reported in epidemiological studies associated with a diverse array of cancers, and in particular childhood leukaemia (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). Indeed such data generated by epidemiological studies associating ELF-MF with an increased risk of childhood leukaemia are chiefly responsible for ELF-MFs group 2B classification as possibly carcinogenic (Chapter 1.12) (Ahlbom et al., 2000; Greenland et al., 2000; IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). 4.2 Epidemiological Data It is owing to such classification that the study of the health effects related to ELF-MF exposure continues to occur. Thus as epidemiological studies are the principal reference in classification, the evidence generated through these studies warrants further discussion. 4.3 Epidemiological Limitations It is important to note that while the majo p g It is important to note that while the majority of epidemiological studies compensate for outside influences within their analyses, there is potential to attribute some causal role to that of possible confounding factors and/or selection bias in the designation of cases and/or controls. Yet, probably with the exception of traffic density, most studies sufficiently account for potential confounding factors and thus the impact upon the observed results from possible confounders is likely to be negligible (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). The same however is unlikely to be true for potential selection bias, as a review of the epidemiological literature by Mezei and Kheifets (2006) identified evidence to suggest it played a role both for and against an association between ELF-MF and childhood leukaemia. One of the main problems with epidemiological studies is with the measurement of ELF-MF exposures. Since these investigations are almost exclusively conducted retrospectively, assessment of human health effects and in particular the study of chronic diseases in relation to ELF-MF exposure proves problematic since direct measurements of exposure during the aetiologically relevant period cannot be recreated. 128 The early studies were based upon exposure estimates taken from wire codes rather than from measured values of direct personal exposures. The use of calculated field measurements using wire codes and distance has potential to counteract such fluctuations in exposure as the distance and wiring to residential areas remains stable over extended time periods. However, studies which rely on the use of calculated fields exclude all other sources of exposure which ultimately leads to an underestimation of exposure, while only a few report adequate exposure parameters. Moreover, the use of wire codes also has potential to introduce selection bias in terms of socioeconomic status in studies where controls were selected through random-digit dialling (Savitz et al., 1988; London et al., 1991; Linet et al., 1997). The existence of publication bias will further have an influence over the amount of data available for meta-analysis studies. However, these studies evaluated the influence of socioeconomic status on the observed results and each concluded that it was not a potential confounder. 4.3 Epidemiological Limitations It is important to note that while the majo In fact, the difficulties lie in the selection and recruitment of cases and controls since the average home is exposed to an average field strength of <1 µT (UK Childhood Cancer Study Investigators, 1999), with a fraction of homes with average fields above certain thresholds for example, 1% to 2% of homes in the United Kingdom and 10% in the United States presenting fields of >0.2 micro T (Kheifets et al., 2005). Therefore the designation of cases and controls at exposures above these limits proves difficult. Studies have consistently struggled to find sufficient cases to form a viable conclusion of effect, such as Dockerty et al. (1998), who identified suggestions of a positive association between CL and ELF-MF >0.2 µT (Dockerty et al., 1998), although the study only possessed five cases and one control within this exposure group, and the confidence intervals produced were too wide to form a viable conclusion (CI: 1.1-224). Likewise in meta-analysis studies, as of the 12 studies included in the Greenland et al. (2000) study, only two (London et al., 1991; Linet et al., 1997) provided more than four cases exposed to >0.4 µT. Moreover, while it was highlighted by Ahlbom et al. (2000) that exposures >0.4 µT signify a doubling in risk of childhood leukaemia, it must also be noted that this exposure category was only representative of 0.8% of subjects (44 cases and 62 controls). In fact, the difficulties lie in the selection and recruitment of cases and controls since the average home is exposed to an average field strength of <1 µT (UK Childhood Cancer Study Investigators, 1999), with a fraction of homes with average fields above certain thresholds for example, 1% to 2% of homes in the United Kingdom and 10% in the United States presenting fields of >0.2 micro T (Kheifets et al., 2005). Therefore the designation of cases and controls at exposures above these limits proves difficult. 129 As study design progressed, many studies still only invoked direct measurements such as spot measurements, despite the fact that these only assess exposure at one time point. Whilst this type of exposure assessment is able to offer a quantitative estimate of the field strength to which a household is exposed, such studies are supremely limited. Firstly, spot measurements exclusively measure background fields, failing to account for exposure metrics when using or within the vicinity of in use appliances. 4.3 Epidemiological Limitations It is important to note that while the majo Secondly, exposure assessment of this kind is unrepresentative of long-term exposure to magnetic fields as it fails to account for the seasonal and long-term fluctuations in ELF-MF field strength. Taken together, these limitations suggest that the use of spot measurements in the retrospective assessment of ELF-MF exposure does not provide sufficient information regarding the aetiologically relevant period in order to sufficiently evaluate the association between exposure to ELF-MF and the increased incidence of childhood leukaemia. While 24-hour measurements represent an improvement over spot measurement, they too fail to account for fluctuations in field strength. Overall, direct measurement studies possess limitations in their evaluation of long-term exposure assessment, which may potentially lead to instances of exposure misclassification. It should however be noted that it is unlikely the subsequent association with disease status is due to the misclassifications of exposure. Indeed such misclassifications more prominently bias in favour of the null and are thus representative of an underestimation of the exposure effect (Rothman et al., 2008). As study design progressed, many studies still only invoked direct measurements such as spot measurements, despite the fact that these only assess exposure at one time point. Whilst this type of exposure assessment is able to offer a quantitative estimate of the field strength to which a household is exposed, such studies are supremely limited. Firstly, spot measurements exclusively measure background fields, failing to account for exposure metrics when using or within the vicinity of in use appliances. Evidence from epidemiological studies suggests that the presence of any potential association between childhood leukaemia and ELF-MF occurs through post-natal exposures of >0.4 µT. Yet since epidemiological studies are limited in their analysis, scientific evidence is still lacking as to the effect of exposures greater than this. Moreover, whilst there have been positive epidemiological associations, uncertainties remain as to whether the association between ELF-MF and childhood leukaemia is causal. Yet, given how few cases and controls are actually exposed to ELF-MFs greater than 0.4 µT, it is roughly estimated that between one and two additional cases (per 500) would arise as a result of artificial ELF-exposure (UK Childhood Cancer Study Investigators, 1999; Kroll et al., 2010). Further analysis by Teepen and Dijck (2012) also Evidence from epidemiological studies suggests that the presence of any potential association between childhood leukaemia and ELF-MF occurs through post-natal exposures of >0.4 µT. 4.3 Epidemiological Limitations It is important to note that while the majo Yet since epidemiological studies are limited in their analysis, scientific evidence is still lacking as to the effect of exposures greater than this. Moreover, whilst there have been positive epidemiological associations, uncertainties remain as to whether the association between ELF-MF and childhood leukaemia is causal. Yet, given how few cases and controls are actually exposed to ELF-MFs greater than 0.4 µT, it is roughly estimated that between one and two additional cases (per 500) would arise as a result of artificial ELF-exposure (UK Childhood Cancer Study Investigators, 1999; Kroll et al., 2010). Further analysis by Teepen and Dijck (2012) also 130 estimated that ELF-MF exposure potentially only contributes an overall population attributable risk (PAR) percentage of 1.9(%). Taken together these statistical analyses indicate that exposure to ELF-MF is unlikely to significantly impact upon the incidence of childhood leukaemia if indeed causality is established. In light of the present findings, it is therefore posited that the classification of ELF-MF exposure as possibly carcinogenic represents a conservative and premature public-health decision. This decision has continued to drive an increasing number of studies to attempt to validate potential causality between ELF- MF exposure and childhood leukaemia as indicated by epidemiological studies. However, such causality cannot be validated without establishing either a dose- dependent response in vitro and in vivo or the existence of a plausible biophysical mechanism through which extremely low-frequency magnetic fields are proven to possess carcinogenic characteristics. 4.4 Dose Response p As such, this current study investigated the presence of a dose-dependent response to ELF-MF exposure. While analysis of pooled sperm data for each individual exposure length (2 hours or 15 hours) illustrated a marginally significant elevation in the total frequency of ESTR mutation when compared to that in sham-treated animals (Figure 18, Table 19), these data do not however present any evidence to indicate a dose- dependent increase within the exposed male mice. The lack of evidence with regards to an ELF-MF dose-response is seemingly in contrast to several earlier studies (Lai & Singh, 1997; Ivancsits et al., 2002, 2003b; Wolf et al., 2005; Focke et al., 2010). However, all of these studies also provided evidence of ELF-MF exposure possessing genotoxic capabilities. In the study by Lai and Singh (1997) brain cells of adult Sprague-Dawley rats were analysed 2 and 4 hours after acute (2 hours) exposure to a 60 Hz magnetic field at flux densities of 0.1, 0.25 and 0.5 mT. Using the previously employed micro-gel electrophoresis assay (Lai & Singh, 1996), a significant dose-dependent increase in DNA single-strand breaks was observed at all flux densities in which the average DNA migration lengths increased in conjunction with flux density. A similar dose-dependent response was also witnessed in double-strand breaks, however only following 131 exposure to 0.25 and 0.5 mT (Lai & Singh, 1997). Similar dose-responses were identified in ensuing studies, although these studies implemented intermittent exposures of ELF-MFs. Progressive intermittent ELF-MF exposures (20-1000 µT, 5 min on/10 min off) of cultured human fibroblast cells for a period of 1 to 24 hours resulted in both a dose-dependent and time-dependent increase in DNA single- and double- strand breaks as measured by alkaline and neutral comet tail factors (Ivancsits et al., 2003b). DNA damage increased in a time-dependent manner, although it peaked at 15 hours with the assay levels declining thereafter without returning to basal levels. Likewise, an increasing scale of magnetic flux densities from 20 to 1000 µT induced a dose-dependent significant increase in single- and double-strand breaks from a magnetic flux density of 35 µT (Ivancsits et al., 2003b). Additionally, Wolf et al. 4.4 Dose Response (2005) reported a dose-dependent increase in DNA damage, detected as DNA strand breaks and measured by the comet assay, in three separate cell lines (HL-60 leukaemia cells, Rat-1 fibroblasts and human diploid fibroblasts (WI-38 cells) exposed for 3-72 hours to 0.5-1.0 mT, 50 Hz ELF-MF, where genotoxicity peaked at 24 and 72 hours (Wolf et al., 2005). Finally, Focke and co-authors (2010) were able to replicate an ELF-MF exposure dependent increase in comet tail DNA in three cell models of primary human fibroblasts (ES-1, HR-1d and MRC-5) intermittently exposed to 50 Hz ELF-MF at a flux density of 1 mT. However, statistical significance was smaller and with higher experimental variation than those previously presented by Ivancsits et al. (2002). exposure to 0.25 and 0.5 mT (Lai & Singh, 1997). Similar dose-responses were identified in ensuing studies, although these studies implemented intermittent exposures of ELF-MFs. Progressive intermittent ELF-MF exposures (20-1000 µT, 5 min on/10 min off) of cultured human fibroblast cells for a period of 1 to 24 hours resulted in both a dose-dependent and time-dependent increase in DNA single- and double- strand breaks as measured by alkaline and neutral comet tail factors (Ivancsits et al., 2003b). DNA damage increased in a time-dependent manner, although it peaked at 15 hours with the assay levels declining thereafter without returning to basal levels. Likewise, an increasing scale of magnetic flux densities from 20 to 1000 µT induced a dose-dependent significant increase in single- and double-strand breaks from a magnetic flux density of 35 µT (Ivancsits et al., 2003b). Additionally, Wolf et al. (2005) reported a dose-dependent increase in DNA damage, detected as DNA strand breaks and measured by the comet assay, in three separate cell lines (HL-60 leukaemia cells, Rat-1 fibroblasts and human diploid fibroblasts (WI-38 cells) exposed for 3-72 hours to 0.5-1.0 mT, 50 Hz ELF-MF, where genotoxicity peaked at 24 and 72 hours (Wolf et al., 2005). Finally, Focke and co-authors (2010) were able to replicate an ELF-MF exposure dependent increase in comet tail DNA in three cell models of primary human fibroblasts (ES-1, HR-1d and MRC-5) intermittently exposed to 50 Hz ELF-MF at a flux density of 1 mT. However, statistical significance was smaller and with higher experimental variation than those previously presented by Ivancsits et al. (2002). exposure to 0.25 and 0.5 mT (Lai & Singh, 1997). 4.4 Dose Response Similar dose-responses were identified in ensuing studies, although these studies implemented intermittent exposures of ELF-MFs. Progressive intermittent ELF-MF exposures (20-1000 µT, 5 min on/10 min off) of cultured human fibroblast cells for a period of 1 to 24 hours resulted in both a dose-dependent and time-dependent increase in DNA single- and double- strand breaks as measured by alkaline and neutral comet tail factors (Ivancsits et al., 2003b). DNA damage increased in a time-dependent manner, although it peaked at 15 hours with the assay levels declining thereafter without returning to basal levels. y g g Likewise, an increasing scale of magnetic flux densities from 20 to 1000 µT induced a dose-dependent significant increase in single- and double-strand breaks from a magnetic flux density of 35 µT (Ivancsits et al., 2003b). Additionally, Wolf et al. (2005) reported a dose-dependent increase in DNA damage, detected as DNA strand breaks and measured by the comet assay, in three separate cell lines (HL-60 leukaemia cells, Rat-1 fibroblasts and human diploid fibroblasts (WI-38 cells) exposed for 3-72 hours to 0.5-1.0 mT, 50 Hz ELF-MF, where genotoxicity peaked at 24 and 72 hours (Wolf et al., 2005). Finally, Focke and co-authors (2010) were able to replicate an ELF-MF exposure dependent increase in comet tail DNA in three cell models of primary human fibroblasts (ES-1, HR-1d and MRC-5) intermittently exposed to 50 Hz ELF-MF at a flux density of 1 mT. However, statistical significance was smaller and with higher experimental variation than those previously presented by Ivancsits et al. (2002). Ultimately, since there are currently neither consistent laboratory evidence in support of a dose response, nor any viable biophysical mechanism through which extremely low-frequency magnetic fields can biologically interact with and give rise to mutations in mammalian tissues, the presence and shape of a dose response cannot be accurately ascertained. Moreover, without the foundation of dose-effect relationships, the validity of the causal effect of extremely low-frequency magnetic field irradiations on any increased risk of cancerous diseases, as portrayed by epidemiological studies, cannot be explained by any scientific reasoning. 132 4.5.1 ELF-MF Data 4.5.1 ELF-MF Data It is not solely the presence of dose-dependent responses to ELF-MF exposure which is seemingly lacking conclusive supporting data; the absence of viable data replication, both between and within laboratories, has contributed to the contradictory nature of available data regarding this subject. While the majority of studies have been unable to confirm the genotoxic nature of magnetic field exposure, conclusions of such nature have been questioned with arguments pertaining to the lack of assay sensitivity in studies where an absence of ELF-MFs carcinogenic potential has been determined (Mairs et al., 2007). It follows that, while the classic methods provide the sensitivity capable of identifying extensive and severe cellular DNA damage, they may lack the relevant sensitivity required to reveal subtle molecular alterations that lead to potentially harmful genetic damage (Mairs et al., 2007). In light of this, previous studies have called for the implementation of sensitive high-throughput methods in the detection of ELF-MFs potential genotoxic effects (Burdak-Rothkamm et al., 2009). Indeed it has been illustrated throughout that the use of hypervariable tandem repeat loci located within the mammalian genome has been previously employed as a successful biomarker in the detection of induced germline mutations in humans and animals following environmental exposure to radioactive and chemical pollutants (Chapters 1.14 to 1.14.3.2.2) (reviewed in Yauk, (2004)). Therefore, by having recourse to ESTRs in the current study, a highly sensitive in vivo system was employed in order to assess any mutagenic effects of ELF-MFs at levels comparable to those populations are exposed to. The data presented through the implementation of this system are in line with the majority of previous findings in which ELF-MF exposure does not significantly alter the frequency of mutation induction in vivo. Yet further comparison has proven difficult as there are a limited number of in vivo studies which have been conducted to date. Not only that but studies in which mutational analysis was conducted post-ELF-MF exposure are fewer still. Moreover, the majority of mutation analysis studies that have been performed have investigated the biological effects of ELF-MF at high flux densities (Miyakoshi et al., 1996, 1997; 133 Mairs et al., 2007), and are thus neither representative of those flux densities employed within this study, nor of true environmental ELF-MF exposures. 4.5.1 ELF-MF Data 134 The alteration in allele size directly correlates to the measurement of mutation induction conducted within this thesis, whereby any alteration was due to an addition or deletion of repeat units. The authors reported that exposure induced an additional 0.011 mutations/locus/cell when compared to the sham-exposed cells, equating to a 3.75-fold increase in mutation induction (Mairs et al., 2007). Since the ESTR loci used within this project are also likely to represent a class of highly expanded microsatellites (Hardwick et al., 2009), the data presented by the current thesis’ study, in which an absence of mutation induction was found after ELF-MF exposure, are in direct contrast to the data presented by Mairs et al. (2007).Though it should be noted that the field strength to which the glioma cell line were exposed was approximately three times higher than the largest employed within the present study. However, the present study was also designed, in part, in comparison to those previously conducted via the REFLEX programme (Ivancsits et al., 2002, 2003b; Winker et al., 2005), in which exposure to intermittent and continuous magnetic field exposures resembling doses near the IARC environmental safe-guards resulted in an increase in DSBs. As has been mentioned, the data of this current study are in contrast to those previously presented by the REFLEX programme (Ivancsits et al., 2002, 2003b; Winker et al., 2005). Likewise are those of Burdak-Rothkamm et al. (2009), whose analysis of the in vitro effects of similar intermittent exposure doses (50-1000 µT) failed to identify any measureable alterations in the amount of double-strand DNA breaks and sister chromatid exchanges. Despite the γH2AX method employed in their study possessing the ability to reliably detect DNA damage equivalent to an X-ray dose of 0.025 Gy, neither the alkaline comet assay, nor the γH2AX assay could detect significant damage at the DNA breakage level in the MF exposed fibroblasts and thus confirm the REFLEX programme’s data. Similarly, a more recent in vivo study by Saha et al. (2014), in which similar exposure parameters to those implemented in this current study were utilised (continuous 2 hour exposure to 100 µT and 15 hours exposure to 300 µT, 50 Hz MF) and failed to provide any evidence of mutagenesis following 50 Hz magnetic field exposure. In the study conducted by Saha et al. (2014), 53BP1 foci were used as a direct signalling response to the formation of DSBs. 4.5.1 ELF-MF Data A series of studies investigated the effect of ELF-MF (50 Hz at 400 mT) exposure on the induction of 6-thioguanine-resistant (6-TGr) mutations in the HPRT gene of cultured human melanoma (MeWo) cells (Miyakoshi et al., 1996, 1997). An increase in induced 6-thioguanine-resistant (6-TGr) mutations was observed between 4- and 10 hours after a 2 hour exposure (Miyakoshi et al., 1997). Similarly, in a further study, the authors also reported that exposure to a 50 Hz magnetic field at 400 mT for up to 20 hours induced a 6-fold increase in 6-thioguanine-resistant (6-TGr) mutations as compared to the sham-treated control (Miyakoshi et al., 1996). Furthermore, the frequency of mutations increased in a time-dependent manner until becoming, in their opinion, saturated after 10 hours (Miyakoshi et al., 1996). Conversely, an additional study in which a 12 hour exposure to a 60 Hz, 0.7 mT magnetic field did not enhance the mutation frequency in Chinese hamster ovary cells exposed in the absence of ionising radiation (Walleczek et al., 1999); the latter findings are more in line with those from this present study. However, it has been concluded that the analysis of alteration in hypervariable tandem repeat DNA sequences such as microsatellite and minisatellite may provide a radically enhanced analysis of mutation frequencies when compared to the HPRT locus (Ogheri et al., 1995). The use of these non-coding DNA sequences in identifying radiation- induced mutations has previously been shown to represent at least a 1000 times more sensitivity than analysis of the HPRT gene, which is renowned as the current benchmark against which other methods of DNA mutation detection are measured (Boyd et al., 2000). Indeed the use of hypervariable tandem repeats within this present study to analyse the mutagenic potential of ELF-MF is not a novel idea as a former study conducted by Mairs and co-authors (2007) previously used microsatellite DNA sequences. Mairs and co-authors, (2007) studied the in vitro effects of ELF-MF. UVW human glioma cells were exposed to a 1 mT, 50 Hz magnetic field for 12 hours after which the analysis of mutation induction at microsatellite loci was conducted. DNA damage was determined through the analysis of three types of microsatellite sequence mutation inductions (change in allele size, loss of heterozygosity and allelic imbalance). 4.5.1 ELF-MF Data The use of 53BP1 foci represents a sensitive monitor of DSB formation and repair, with nearly 135 100% efficiency. Pregnant C57BL/6 mice were exposed to a continuous 50 Hz magnetic field of 100 µT for 2 hours or to a continuous or an intermittent (5 min on/10 min off) field of 300 µT for 15 hours. Through this process, Saha et al. (2014) were able to provide further evidence that continuous exposures to either a 2 hour, 100 µT, or 15 hour, 300 µT, 50 Hz magnetic field does not cause direct DNA damage. Therefore these data, while using a different detection method, implement similar exposure conditions in vivo to this present study and further provide no evidence that the frequency in which mutations are induced is affected following exposure to 50 Hz magnetic fields up to a dose of 300 μT, ultimately strengthening these findings. Moreover, data from previous ESTR studies have provided an ESTR mutation rate with a detection sensitivity cut-off equivalent to an exposure to 0.5 Gy of acute X-/γ-rays (Dubrova et al., 1998a; Mughal et al., 2012). Consequently, to findings herein, it is posited that the mutagenic effects on the public of MF exposure are only likely to cause ESTR mutations equivalent to 0.5 Gy of X-rays, if any at all. In fact, the study conducted by Saha et al. (2014) provided evidence of increased assay detection sensitivity equivalent to 10 mGy X-rays, therefore suggesting a lower threshold. Therefore these data, while using a different detection method, implement similar exposure conditions in vivo to this present study and further provide no evidence that the frequency in which mutations are induced is affected following exposure to 50 Hz magnetic fields up to a dose of 300 μT, ultimately strengthening these findings. Moreover, data from previous ESTR studies have provided an ESTR mutation rate with a detection sensitivity cut-off equivalent to an exposure to 0.5 Gy of acute X-/γ-rays (Dubrova et al., 1998a; Mughal et al., 2012). Consequently, to findings herein, it is posited that the mutagenic effects on the public of MF exposure are only likely to cause ESTR mutations equivalent to 0.5 Gy of X-rays, if any at all. In fact, the study conducted by Saha et al. (2014) provided evidence of increased assay detection sensitivity equivalent to 10 mGy X-rays, therefore suggesting a lower threshold. 4.5.1 ELF-MF Data What is abundantly clear from reviewing all the literature in this area is that any ELF- MF induced biological effects on DNA damage or gene expression, are subject to a range of factors and characteristics including, but not limited to, the frequency of emittance and the intensity, duration and mode of said exposure (Ivancsits et al., 2002). However, contradictory results regarding genotoxic potential of extremely low- frequency magnetic fields are common within the literature; this may be due to the differences in cellular targets examined by individual studies. Ivancsits et al. (2005) reported that six types of cultured cells derived from different tissues induced different genotoxic responses when exposed to 1 mT intermittent ELF-MFs. Of the six, there were three responder cell types consisting of: human fibroblasts, human melanocytes and rat granulosa cells, and three non-responder types: human lymphocytes, human monocytes and human skeletal muscle cells. Additionally, Luceri et al. (2005) failed to observe an increase in DNA damage in human blood lymphocytes exposed to 50 Hz magnetic fields at 1, 10 or 100 µT for 18 hours. 136 These contradictory data are the product of exposure to several different cell lines and organisms which evoked different responses. These discrepancies occur since most of these aforementioned studies employ transformed cells known to exhibit inherent genetic instability. Similarly, primary cell cultures may prove unreliable since, when reaching maturation, they may present high background damage. Therefore the uses of in vivo analyses and of similar exposure parameters, both in the present study and within the Saha et al. (2014) study, overcome these limitations and ultimately start to provide a clear and concise conclusion regarding the effects of ELF-MF on biological systems which were previously unavailable. 4.5.2 Ionising Radiation 4.5.2 Ionising Radiation Within this current study, the use of 1 Gy of acute X-rays was applied as a positive control in order to validate the SM-PCR technique. Additionally, the analysis of ESTR mutation induction has, to date, predominantly been studied in the germline of male mice following exposure to ionising radiation (Dubrova et al., 1993, 1998a, 1998b, 2000b; Sadamoto et al., 1994; Fan et al., 1995; Niwa et al., 1996; Dubrova & Plumb, 2002; Yauk et al., 2002; Dubrova, 2005). However, such effects in somatic tissues are yet to be sufficiently established. Initial data correspond to similar mechanisms, as in the germline, whereby the rate of mutation induction was elevated in stem cells 10 and 20 weeks post-irradiation, in a stage-specific manner (Dubrova et al., 1998a, 1998b). Moreover, relatively small changes in mutation rates were exhibited in bone marrow cells following acute exposure to X-rays, while no discernible change in mutation frequency appeared in non-proliferating adult brains (Barber et al., 2009). Since the mitotic proliferation capabilities in an adult brain are low to non-existent (Gross, 2000), with brain cells only undergoing mitotic proliferation in utero, these data further signify that ESTR mutations arise almost entirely in replication-proficient cells (Barber et al., 2009). Similarly, data from numerous studies analysing the ESTR mutation rate following acute irradiation to X-rays at various stages of spermatogenesis have previously shown that all radiation-induced ESTR mutations tend to arise at every stage of spermatogenesis preceding metaphase I (Barber et al., 2000). That is, mutation rates are significantly elevated only in spermatogonia derived from mid and early 137 pachytene, type B spermatogonia and As spermatogonia (Dubrova et al., 1998b; Barber et al., 2000), which are indicative of samples taken from offspring conceived 4, 5, or 6 and above weeks post-irradiation, representing pre-meiotic spermatogonia (Searle, 1974). In contrast, no measurable alterations in the frequency of ESTR mutations were exhibited in post-meiotic spermatids procured from non-dividing sperm cells of irradiated adult mice analysed either one week (Barber et al., 2009) or three weeks (Dubrova et al., 1998b; Barber et al., 2000) after exposure. Since hematopoietic tissues in mice undergo a vast turnover (Metcalf, 1988), the design of this current study, in which nucleated blood cells were sampled 12 weeks post-exposure, ensured that the majority of sampled nucleated blood cells originated from exposed replication-proficient stem cells. 4.5.2 Ionising Radiation As indicated by Table 17, exposure to 1 Gy of acute X-rays causes a statistically significant increase in both the germline and somatic tissues of irradiated male mice. Yet, while the spontaneous mutation frequencies remain relatively similar in both blood and sperm (t=1.23; P=0.2188), the radiation-induced ESTR mutation frequency in sperm is significantly higher than that in blood (t=2.45; P=0.0144), (Figure 18). A statistically significant 2.4-fold increase was shown in the germline ESTR mutation frequency of irradiated males, while a significant though less distinct 1.8-fold increase was reported in the blood of these same male mice. The reasoning for such a less-pronounced increase in ESTR mutation induction in the hematopoietic stem cells, compared to the spermatogonial cells, is as yet unknown and should warrant further analysis in the future. A potential rationale however may be related to the cellular replication proficiency of the two tissues, such that the percentage of actively dividing hematopoietic stem cells in mouse bone marrow is very low (~9.1/105 cells in BALB/c mice) (Muller-Sieburg & Riblet, 1996), when compared to that of sperm. Within the context of the analysis of ESTR mutation induction in the somatic tissues of male mice exposed to ionising radiation, the current findings represent the initial analysis of ESTR mutation induction in blood cells of male mice exposed to ionising radiation. Moreover, these findings contribute further support to the original 138 hypothesis whereby mutations arising in germline or somatic tissues, either spontaneously or induced, do so through a similar mutation process (Hardwick et al., 2009). 4.6 Pilot Study Microarray Summary In addition to the evaluation of the rate at which mutations are induced following ELF- MF exposure, the present study also initiated a pilot study in which high-throughput microarray analysis was used to assess the pattern of gene expression in vivo following exposure to a 50 Hz magnetic field (300 µT) for 15 hours. This preliminary study implemented a one-colour microarray-based gene expression analysis, whereby the highly sensitive Agilent SurePrint G3 Mouse GE 8x60K microarrays were used in the analysis of kidney samples taken from five BALB/c x CBA/Ca F1 hybrid male mice following a 15 hour exposure to a 50 Hz magnetic field of 300 µT. The primary data did not reveal any differentially expressed loci following exposure to 300 µT for 15 hours compared with their sham-treated controls (Figure 21). 4.6.1 Comparative Analysis of Microarray Data 4.6.1 Comparative Analysis of Microarray Data Several hypotheses have been proposed regarding potential mechanisms through which ELF-MF ultimately interacts with biological systems, including the production of free-radicals (Chapter 1.13.2, reviewed in Simko & Mattsson, (2004)). One proposal by Phillips (1993) referred to an ELF-MF mediated alteration in gene expression. Following this proposal, several studies performed small-scale analysis studies on a small proportion of various genes with a potential to be affected. Loberg et al. (2000) performed a small-scale analysis of the transcript levels of 588 cancer-related genes in HL-60 and mammary epithelial cells after exposure to a 60 Hz magnetic field of 10 µT or 1 mT for 24 hours. Likewise, Coulton et al. (2004) analysed the pattern of gene expression in HSP27, HSP70A or HSP70B of human leukocytes. Neither study reported significant alterations in the expression profiles in any of the 588 cancer-related genes (Loberg et al., 2000), nor that 50 Hz magnetic fields up to 100 µT altered the pattern of gene expression in HSP27, HSP70A or HSP70B of human leukocytes (Coulton et al., 2004). However, the effect of human exposure on health appears to be multifaceted. While the absence of biological mechanism and failure to establish a dose-response means 139 that there is a lack of starting point at which to initiate investigations. Therefore, the use of whole-genome screening methods, like microarray gene expression analyses possess the potential to globally analyse genes, and could identify viable gene candidates which respond to ELF-MF exposure. The issue is that, similar to mutation analyses studies, there remains as yet a limited number of large-scale gene expression analyses evaluating the biological effects of ELF-MF exposure. The present pilot study invokes the use of a genome-wide platform of investigation in an attempt to gather preliminary data as to whether 50 Hz magnetic fields are able to interact with biological systems in vivo. Previous attempts have been made to study the global gene-expression effects of ELF- MF, although these have occurred in vitro. Luceri et al. (2005) assessed the variation in overall gene expression profiles in human lymphocytes cells and Saccharomyces cerevisiae after exposure to 1, 10 or 100 µT for 18 hours. By using DNA microarray in the holistic analysis of the yeast and human genomes, an exposure to ELF-MFs ranging from 1-100 µT for a period of 18 hours was not seen to render a change in any gene expression profiles. 4.6.1 Comparative Analysis of Microarray Data Although the yeast genome was analysed in full, the study of the human genome was limited in its design with only 13971 oligonucleotides represented on the array (Luceri et al., 2005). More recently, a study by Henderson et al. (2006) employed the use of microarrays containing approximately thirty thousand oligonucleotides in their analysis of the transcriptional response in human primary vascular endothelial cells. Neither continuous exposure of a 50 Hz MF representative of occupational (700 µT) and domestic (10 µT) environments for a period of 24 hours, nor intermittent exposure (700 µT, 60 min on/30 min off) over a 24 hour cycle gave rise to an effect on the expression of any genes analysed which could be replicated. Similarly, Chen et al. (2012) analysed the gene expression response in Saccharomyces cerevisiae exposed to a 50 Hz magnetic field of 0.4 mT for 6 hours, the use of which was attempting to mimic the 1998 ICNIRP occupational exposure guideline. Despite three genes being discovered to be up-regulated by at least 1.3 fold, upon further analysis with real-time reverse transcription-polymerase chain reaction (RT-PCR), the microarray detected changes were unconfirmed. After six independent replications real-time RT-PCR analysis failed to confirm any significant change in any of the three 140 gene expression levels, with all three gene candidates presenting fold changes of approximately 1.0. While these three studies were performed in vitro, the exposure doses to which these cells were subject to are approximately equivalent to the one used in the present study (300 µT for 15 hours), and thus the preliminary data generated from this pilot study are in conjunction with those previously conducted (Luceri et al., 2005, Henderson et al., 2006; Chen et al., 2012). Taken together, the data from these separately conducted global analysis studies on gene expression profiles suggest that 50 Hz magnetic fields of up to 400 µT do not significantly alter the pattern of gene expression profiles in human lymphocytes, yeast cells or in vivo. Moreover, additional evidence from the study by Loberg et al. (2000) confirms that no significant changes were identified in the expression of genes related to the development of cancer. 4.6.1 Comparative Analysis of Microarray Data The results of this present study are in conjunction with, and further add to, the growing body of evidence which suggests that there is no causality between changes in gene expression and exposure to 50 Hz magnetic fields, nor do such magnetic fields possess any ability to interact with biological systems. 4.7 Study Limitations 4.7 Study Limitations The analysis of continuous exposure is viable as it allows the prolonged exposure to ELF-MF experienced by households inhabiting in close proximity to high-voltage power lines to be mimicked. However, only 1.1% of homes in the USA reside within 40 meters of high-voltage power lines, while in the UK this figure is further reduced to 0.07% of homes situated within 50 metres (EMFs.info, 2015). Moreover, whilst we are continually exposed to ELF-MF throughout domestic life, there are fluctuations in intensity dependent upon our location, the time of day or the season of the year. Whilst subsequent studies have not confirmed such findings (Burdak-Rothkamm et al., 2009; Saha et al., 2014), it was previously demonstrated that exposure to intermittent magnetic fields are not continuous, and result in a significant induction of DSB formation (Ivancsits et al., 2002, 2003b; Winker et al., 2005). Therefore, it is probable that the use of intermittent fields similar to those employed in previous studies (Ivancsits et al., 2002, 2003a, 2003b, 2005; Winker et al., 2005; Burdak-Rothkamm et al., 2009; Saha et al., 2014), would not only have allowed for a more realistic y The analysis of continuous exposure is viable as it allows the prolonged exposure to ELF-MF experienced by households inhabiting in close proximity to high-voltage power lines to be mimicked. However, only 1.1% of homes in the USA reside within 40 meters of high-voltage power lines, while in the UK this figure is further reduced to 0.07% of homes situated within 50 metres (EMFs.info, 2015). Moreover, whilst we are continually exposed to ELF-MF throughout domestic life, there are fluctuations in intensity dependent upon our location, the time of day or the season of the year. 141 mimicking of global exposure but would have allowed data between studies to be more accurately compared. Finally, while the use of non-coding repeat sequences currently represents the best laboratory model for studying mutagen-induced tandem repeat instability in the germline, this is mostly due to a lack of a more biologically-relevant model. Whilst the mouse genome does encompass the characteristic true minisatellite loci, the germline mutation rates exhibited by these loci (< 10-3 per generation) cannot be considered hypervariable (Bois et al., 1998a). 4.7 Study Limitations As such, to date there is no animal model in which the mutation induction at minisatellites can be appropriately studied, nor is there any such equivalent for a human ESTR model at this time. The issue lies in the fact that the mutational mechanism through which ESTR loci are processed is fundamentally different to that of human minisatellites (Kelly et al., 1989; Gibbs et al., 1993; Bois et al., 1998b). The high frequency germline mutations exhibited by human minisatellites arise through complex meiotic recombination events, whilst ESTR instability arises through a replication or repair-based process involving DNA polymerase slippage during DNA replication, or through DNA polymerase pausing to repair the DNA following the formation of secondary structures (Barber et al., 2004; Hardwick et al., 2009). Moreover, human minisatellites do not exhibit the same levels of somatic instability as ESTR loci (Jeffreys et al., 1988, 1994). This may cast doubts over the biological relevance of ESTR assays and their substitution for human minisatellite instability (reviewed in (Bouffler et al., 2006; Singer & Yauk, 2010; Yauk et al., 2015)). However, the dose-response of ESTR mutation induction has been shown to correlate to that of other classical mutation scoring assays (Dubrova et al., 1998a; Barber et al., 2006). Additionally, not only is there an increasing amount of evidence of tandem repeat loci partaking an essential role in genome structure and function, but also evidence of an association with genes and location in and/or near promoter sites (Armour, 2006; Mirkin, 2007). This equates to an expanding amount of evidence associating mutations at tandem repeat loci with that of human genetic diseases, some of which even causally (Mirkin, 2007). However, despite these findings, it would be premature to view these data as anything more than comparative until such a time where either a more biologically relevant However, despite these findings, it would be premature to view these data as anything more than comparative until such a time where either a more biologically relevant 142 animal model for minisatellite studies is produced, or until the presence of human ESTRs are confirmed and revealed to respond in the same biological manner to mutagen exposure. 4.8 Conclusion of Findings 4.8 Conclusion of Findings In summary, this present study represents, to the best of my knowledge, the first methodical attempt to determine the in vivo frequency at which mutation induction occurs in the germline and somatic tissues of male mice after continuous exposure to 50 Hz ELF-MFs of doses up 300 μT (Wilson et al., 2015). This study was established in response to the repeatedly inconsistent data and absence of any independent replication which have frequented this area of scientific investigation. Thus, through the implementation of a highly sensitive biomarker within this study which has previously been applied with success in the detection of mutation induction within proliferating mouse tissues, no significant increases in the frequency of ESTR mutation in blood were detected following exposure to 50 Hz magnetic fields. Additionally, whilst a marginally significant increase was observed in the frequency of induced germline mutations, these data should be regarded cautiously due to the lack of an evident correlating dose response. Yet, it must be stressed that whilst causality could be inferred from a simultaneous increase of both disease risk and exposure dose, an absence of a dose response does not de facto indicate the lack of a causal relationship. However, considering these findings and the absence of any significant alteration in gene expression profiles in mouse kidney cells after a continuous 15 hour exposure to 300 μT, it can be concluded that where individuals have experienced mutagenic effects as a result of ELF-MF exposure, then such effects equate to that of less than 1 Gy X- rays in germline cells. Moreover, it can also be posited that such mutagenic effects arising from exposure observed in somatic tissues are likely to be negligible (Wilson et al., 2015). Nonetheless, while these findings will lend themselves to the assumption that observation of any of the adverse health effects stated within the literature are unlikely, it is too early to disregard the possibility of such an association because of the 143 complex and diverse nature in the causation of human disease, as illustrated by the onset of childhood leukaemia. As such, it follows that the biological effects of ELF-MF and any link it may have to adverse health effects should be approached cautiously as lacking scientific validation. 4.8 Conclusion of Findings This should remain so until either the detection of a persistent biophysical mechanism, or an irrefutable biological response is witnessed consistently within independent laboratories in response to ELF-MF exposure. Yet until such a time, it remains that such adverse health effects will continue to be considered with uncertainty by certain groupings within the scientific community. 4.9.1 Microarray Analysis y y Since ELF-MF is representative of such a weak stimulus, the additional approach of global gene expression analysis could potentially identify responding gene candidates and ultimately determine a potential biological mechanism. While microarray analyses are capable of detecting changes in expression equivalent to a two-fold or greater increase, they are limited in terms of detecting minor changes. Therefore, a further study would aim to continue the gene expression analysis and implement the use of a more powerful method, such as RNA-sequencing (RNA-Seq) (Wang et al., 2009, Fonseca et al., 2014), to initially confirm that the lack of microarray detected gene alterations in response to ELF-MF exposure are indeed accurate and not in fact false negatives. RNA-Seq represents a more powerful alternative to transcriptome profiling than microarrays, utilising next generation sequencing technology to provide a high-throughput and quantitative analysis, through sequencing the whole transcriptome at the single-nucleotide level. The implementation of such a sequencing based method to directly determine the cDNA sequence allows the investigation of mRNAs, small RNAs and non-coding RNAs expression levels, and provides a more precise measurement and discovery of novel and rare transcripts alongside their splice isoforms. Ultimately, the use of RNA-Seq allows for the detection of global gene expression patterns that were previously inaccessible using hybridisation-based methods such as microarrays. Indeed, unlike hybridisation-based approaches, RNA-Seq allows the detection of transcripts beyond 144 those corresponding to existing genomic sequences. This particular property makes RNA-Seq a seemingly attractive analysis tool for non-model organisms with genomic sequences that are yet to be determined. Additionally, in contrast to microarray methods, RNA-Seq presents minimal to no background and signal saturation, thereby providing a higher sensitivity for genes expressed either at low or very high levels. It follows that RNA-Seq presents a higher dynamic range than that of DNA microarrays (one hundred fold to a few hundred fold) (Wang et al., 2009). Finally, there is no amplification step allowing RNA-Seq analysis to be performed using less RNA A comparative study of genome-wide gene expression analysis using the same RNA samples with a RNA-Seq and Agilent 4x44 K oligonucleotide microarray platforms validated the performance metrics of both assays using quantitative real-time PCR discovered that RNA-Seq outperformed Agilent in all categories: sensitivity (78% vs 51%), specificity (75% vs 38%), precision (76% vs 46%), accuracy (77% vs 44%) and false discovery rate (24% vs 54%) (Nault et al., 2015). 4.9.1 Microarray Analysis A further study similarly measured expression levels for 48 genes using RNA-Seq, microarray and real-time PCR methods. In accordance with Nault et al. (2015), Li and co-authors (2016) discovered a consistent level of expression between RNA-Seq and real-time PCR for 40 of the 48 genes (83.3%). Yet conversely, only 18 of the 48 genes (37.5%) were observed to demonstrate consistent expression levels when comparing microarray and real-time PCR approaches (Li et al., 2016). The use of RNA-Seq therefore provides a beneficial accompaniment to the microarray-based gene expression analysis. Similarly, the use of RNA-Seq should also provide new insights into the effects of exposure to ionising radiation on gene expression. 4.9.2 Mutational Analysis y Although in this present study it has been demonstrated that the frequencies of ESTR mutation in germline and somatic tissues of male mice exposed for 15 hours do not exceed those following a shorter 2 hour exposure, further analysis of the genotoxic effects of long-term exposure to magnetic fields would prove informative. Indeed, a recent in vivo study analysed the long-term (28 day) exposure to 200 μT of 50 Hz magnetic field, finding that the yield of micronucleated polychromatic erythrocytes were significantly increased in mice (Alcaraz et al., 2014). Such findings are further 145 supported by other previous publications (reviewed in Udroiu et al., (2010)), and seem to indicate that ELF-MFs only induce noticeable genotoxic effects after long-term exposure. Therefore future work could involve the in vivo examination of mice exposed for a longer period than the 15 hours implemented in this present study. The use of the same highly sensitive ESTR loci biomarker in addition to extended exposure duration would ultimately provide information as to the risk of ELF-MF exposure over a lifetime. The use of intermittent exposure parameters is more representative of worldwide exposure, as one’s exposure to ELF-MF is constantly fluctuating, with the field undergoing change depending on the varying use of household appliances. Also, some experimental studies in which a genotoxic effect was observed occurred after exposure to intermittent ELF-MF exposure as opposed to continuous exposure. Whilst this has not been confirmed by others (Burdak-Rothkamm et al., 2009; Saha et al., 2014), the use of ESTR loci in the analysis of intermittent ELF-MF will provide the foundations to unequivocally conclude either finding. It follows that future studies should be designed as a way of validating whether alterations in gene expression response, alongside any induced DNA damage, are dependent on either continuous or intermittent exposure. Furthermore, if indeed the genotoxic effect is determined by such exposure parameters, then an explanation will need to be determined as to why intermittent and not continuous exposures should induce such effects. Additionally, while there are currently numerous hypotheses relating to the mechanism through which ELF-MF may possess genotoxic and carcinogenic potential, the radical pair mechanism remains to date the most feasible method through which environmentally weak magnetic fields could potentially interact with mammalian biochemical systems. 4.9.2 Mutational Analysis Yet, it follows that it is a fallacy to rationalise that biological systems are so fragile that any minor alteration in the homeostasis of intracellular radical concentration arising from interaction with weak magnetic fields could have a genotoxic effect. One exception would arise where ELF-MF exposure was administered in the presence of an amplification agent or the exposed system exhibited an already weakened defence system. While this current study was not designed to further the available data or add to the understanding of any mechanism involved with ELF-MF 146 exposure in itself, the widespread presence and functions of radicals within biological systems coupled to the presence of conflicting data within the literature, make it abundantly clear that further experimental and theoretical work is needed in this field. Such future studies should ultimately be performed in vivo in order to fine-tune the conditions relating to ELF-MF sensitivity. These future analyses could finally determine whether exposure to ELF-MFs, within the range of doses used in the current study, can alter the balance of melatonin and free radicals, since as of yet, no appropriate comparison can be made. A final point should be made that there is a need for more in vitro studies which analyse the effects on human cells, not only to perpetuate the investigation into potential health risks, but also to better understand the occurrence of organism mutations and their biological interactions with ELF-MF exposure. Of particular note, the potential genotoxic role of free radicals and their relationship with the cell during and after exposure are yet to be clarified. Ultimately, in a field where the amount of in vivo studies are minimal, the development of a novel mouse model representative of childhood ALL presents an ideal opportunity to explore any effects induced by environmental exposures and the mechanics of any such occurrences (Li et al., 2013). 147 Reference List Abel, E.L., Hendrix, S.L., McNeeley, G.S., O'Leary, E.S., Mossavar-Rahmani, Y., Johnson, S.R., Kruger, M., 2007. Use of electric blankets and association with prevalence of endometrial cancer. European Journal of Cancer Prevention : The Official Journal of the European Cancer Prevention Organisation (ECP). 16, 243-250. Abouzeid Ali, H.E., Barber, R.C., Dubrova, Y.E., 2012. The effects of maternal irradiation during adulthood on mutation induction and transgenerational instability in mice. Mutation Research. 732, 21-25. (AGNIR) Advisory Group on Non-ionising Radiation. 2001. ELF Electromagnetic Fields and the Risk of Cancer. Report of an Advisory Group on Non-ionising Radiation. Documents of the NRPB 12 (1). pp. 5-179. Ahlbom, A., 2001. Neurodegenerative diseases, suicide and depressive symptoms in relation to EMF. Bioelectromagnetics. Suppl 5, S132-43. Ahlbom, A., 1988. A review of the epidemiologic literature on magnetic fields and cancer. Scandinavian Journal of Work, Environment & Health. 14, 337-343. Ahlbom, A., Day, N., Feychting, M., Roman, E., Skinner, J., Dockerty, J., Linet, M., McBride, M., Michaelis, J., Olsen, J.H., Tynes, T., Verkasalo, P.K., 2000. A pooled analysis of magnetic fields and childhood leukaemia. British Journal of Cancer. 83, 692- 698. Ahlbom, A., Feychting, M., Gustavsson, A., Hallqvist, J., Johansen, C., Kheifets, L., Olsen, J.H., Medicinska och farmaceutiska vetenskapsområdet, Uppsala universitet, 2004. Occupational Magnetic Field Exposure and Myocardial Infarction Incidence. Epidemiology. 15, 403-408. Ahlbom, I.C., Cardis, E., Green, A., Linet, M., Savitz, D., Swerdlow, A., ICNIRP (International Commission for Non-Ionizing Radiation Protection) Standing Committee on Epidemiology, 2001. Review of the epidemiologic literature on EMF and Health. Environmental Health Perspectives. 109 Suppl 6, 911-933. Akdag, M.Z., Dasdag, S., Ulukaya, E., Uzunlar, A.K., Kurt, M.A., Taskin, A., 2010. Effects of extremely low-frequency magnetic field on caspase activities and oxidative stress values in rat brain. Biological Trace Element Research. 138, 238-249. Akdag, M.Z., Dasdag, S., Uzunlar, A.K., Ulukaya, E., Oral, A.Y., Celik, N., Aksen, F., 2013. Can safe and long-term exposure to extremely low frequency (50 Hz) magnetic fields affect apoptosis, reproduction, and oxidative stress? International Journal of Radiation Biology. 89, 1053-1060. 148 Alcaraz, M., Olmos, E., Alcaraz-Saura, M., Achel, D.G., Castillo, J., 2014. Effect of long- term 50 Hz magnetic field exposure on the micronucleated polychromatic erythrocytes of mice. Electromagnetic Biology and Medicine. 33, 51-57. Allegra, M., Reiter, R.J., Tan, D.X., Gentile, C., Tesoriere, L., Livrea, M.A., 2003. The chemistry of melatonin's interaction with reactive species. Journal of Pineal Research. 34, 1-10. Andersen, T.H. & Nilsson-Tillgren, T., 1997. Reference List A fungal minisatellite. Nature. 386, 771. Anderson, L.E., Morris, J.E., Miller, D.L., Rafferty, C.N., Ebi, K.L., Sasser, L.B., 2001. Large granular lymphocytic (LGL) leukemia in rats exposed to intermittent 60 Hz magnetic fields. Bioelectromagnetics. 22, 185-193. Angelillo, I.F. & Villari, P., 1999. Residential exposure to electromagnetic fields and childhood leukaemia: a meta-analysis. Bulletin of the World Health Organization. 77, 906-915. Antonopoulos, A., Yang, B., Stamm, A., Heller, W.-., Obe, G., 1995. Cytological effects of 50 Hz electromagnetic fields on human lymphocytes in vitro. Mutation Research Letters. 346, 151-157. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. Banks, R.S., Thomas, W., Mandel, J.S., Kaune, W.T., Wacholder, S., Tarone, R.E., Linet, M.S., 2002. Temporal trends and misclassification in residential 60 Hz magnetic field measurements. Bioelectromagnetics. 23, 196-205. Barber, R., Plumb, M., Smith, A.G., Cesar, C.E., Boulton, E., Jeffreys, A.J., Dubrova, Y.E., 2000. No correlation between germline mutation at repeat DNA and meiotic crossover in male mice exposed to X-rays or cisplatin. Mutation Research. 457, 79-91. Barber, R., Plumb, M.A., Boulton, E., Roux, I., Dubrova, Y.E., 2002. Elevated mutation rates in the germ line of first- and second-generation offspring of irradiated male mice. Proceedings of the National Academy of Sciences of the United States of America. 99, 6877-6882. Barber, R.C., Hardwick, R.J., Shanks, M.E., Glen, C.D., Mughal, S.K., Voutounou, M., Dubrova, Y.E., 2009. The effects of in utero irradiation on mutation induction and transgenerational instability in mice. Mutation Research. 664, 6-12. Barber, R.C., Hickenbotham, P., Hatch, T., Kelly, D., Topchiy, N., Almeida, G.M., Jones, G.D., Johnson, G.E., Parry, J.M., Rothkamm, K., Dubrova, Y.E., 2006. Radiation-induced transgenerational alterations in genome stability and DNA damage. Oncogene. 25, 7336-7342. 149 Barber, R.C., Miccoli, L., van Buul, P.P., Burr, K.L., van Duyn-Goedhart, A., Angulo, J.F., Dubrova, Y.E., 2004. Germline mutation rates at tandem repeat loci in DNA-repair deficient mice. Mutation Research. 554, 287-295. Barnes, F.S. & Greenebaum, B., 2007. Bioengineering and biophysical aspects of electromagnetic fields. [e-book]. 3rd ed. Boca Raton, Fla; London: CRC Press. Belson, M., Kingsley, B., Holmes, A., 2007. Risk Factors for Acute Leukemia in Children: A Review. Environmental Health Perspectives. 115, 138-145. Berne, R.M., Levy, M.N., Stanton, B.A. and Koeppen, B.M., 2005. Berne and Levy principles of physiology. 4th ed. Philadelphia, Pa: Elsevier Mosby. Bois, P. & Jeffreys, A.J., 1999. Minisatellite instability and germline mutation. Cellular and Molecular Life Sciences : CMLS. 55, 1636-1648. Bois, P., Stead, J.D., Bakshi, S., Williamson, J., Neumann, R., Moghadaszadeh, B., Jeffreys, A.J., 1998a. Isolation and characterization of mouse minisatellites. Genomics. 50, 317-330. Bois, P., Williamson, J., Brown, J., Dubrova, Y.E., Jeffreys, A.J., 1998b. A novel unstable mouse VNTR family expanded from SINE B1 elements. Genomics. 49, 122-128. Bois, P.R., 2003. Hypermutable minisatellites, a human affair? Genomics. 81, 349-355. Bois, P.R., Southgate, L., Jeffreys, A.J., 2001. Length of uninterrupted repeats determines instability at the unstable mouse expanded simple tandem repeat family MMS10 derived from independent SINE B1 elements. Mammalian Genome : Official Journal of the International Mammalian Genome Society. 12, 104-111. Bonnell, J.A., 1982. Bouffler, S.D., Bridges, B.A., Cooper, D.N., Dubrova, Y., McMillan, T.J., Thacker, J., Wright, E.G., Waters, R., 2006. Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers. Radiation Research. 165, 249-268. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. Effects of electric fields near power-transmission plant. Journal of the Royal Society of Medicine. 75, 933-941. Boorman, G.A., McCormick, D.L., Findlay, J.C., Hailey, J.R., Gauger, J.R., Johnson, T.R., Kovatch, R.M., Sills, R.C., Haseman, J.K., 1999. Chronic toxicity/oncogenicity evaluation of 60 Hz (power frequency) magnetic fields in F344/N rats. Toxicologic Pathology. 27, 267-278. Boorman, G.A., Rafferty, C.N., Ward, J.M., Sills, R.C., 2000. Leukemia and lymphoma incidence in rodents exposed to low-frequency magnetic fields. Radiation Research. 153, 627-636. Bouffler, S.D., Bridges, B.A., Cooper, D.N., Dubrova, Y., McMillan, T.J., Thacker, J., Bouffler, S.D., Bridges, B.A., Cooper, D.N., Dubrova, Y., McMillan, T.J., Thacker, J., Wright, E.G., Waters, R., 2006. Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers. Radiation Research. 165, 249-268. 150 Boyd, A. Livingstone, L. E. Wilson, E. M. Marshall, A. G. McCluskey, R. J. Mairs,T.E.Wheldon, M., 2000. Dose-response relationship for radiation-induced mutations at micro- and minisatellite loci in human somatic cells in culture. International Journal of Radiation Biology. 76, 169-176. Boyd, M., Livingstone, A., Wilson, L.E., Marshall, E.M., McCluskey, A.G., Mairs, R.J., Wheldon, T.E., 2000. Dose-response relationship for radiation-induced mutations at micro- and minisatellite loci in human somatic cells in culture. International Journal of Radiation Biology. 76, 169-176. Brain, J.D., Kavet, R., McCormick, D.L., Poole, C., Silverman, L.B., Smith, T.J., Valberg, P.A., Van Etten, R.A., Weaver, J.C., 2003. Childhood leukemia: electric and magnetic fields as possible risk factors. Environmental Health Perspectives. 111, 962-970. Brocklehurst, B., 2002. Magnetic fields and radical reactions: recent developments and their role in nature. Chemical Society Reviews. 31, 301-311. Bubenik, G.A., 2002. Gastrointestinal melatonin: localization, function, and clinical relevance. Digestive Diseases and Sciences. 47, 2336-2348. Bunch, K.J., Keegan, T.J., Swanson, J., Vincent, T.J., Murphy, M.F., 2014. Residential distance at birth from overhead high-voltage powerlines: childhood cancer risk in Britain 1962-2008. British Journal of Cancer. 110, 1402-1408. Burdak-Rothkamm, S., Rothkamm, K., Folkard, M., Patel, G., Hone, P., Lloyd, D., Ainsbury, L., Prise, K.M., 2009. DNA and chromosomal damage in response to intermittent extremely low-frequency magnetic fields. Mutation Research. 672, 82-89. Burke, T. & Bruford, M.W., 1987. DNA fingerprinting in birds. Nature. 327, 149-152. Carrillo-Vico, A., Calvo, J.R., Abreu, P., Lardone, P.J., Garcia-Maurino, S., Reiter, R.J., Guerrero, J.M., 2004. Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. 18, 537-539. Chand, R., Israil, M., Rai, J., 2009. Schumann resonance frequency variations observed in magnetotelluric data recorded from Garhwal Himalayan region India. Annales Geophysicae. 27, 3497-3507. Chen, C., Ma, X., Zhong, M., Yu, Z., 2010. Extremely low-frequency electromagnetic fields exposure and female breast cancer risk: a meta-analysis based on 24,338 cases and 60,628 controls. Breast Cancer Research and Treatment. 123, 569-576. Chen, G., Lu, D., Chiang, H., Leszczynski, D., Xu, Z., 2012. Using model organism Saccharomyces cerevisiae to evaluate the effects of ELF-MF and RF-EMF exposure on global gene expression. Bioelectromagnetics. 33, 550-560. 151 Cherry, N., 2002. Schumann Resonances, a plausible biophysical mechanism for the human health effects of Solar. Natural Hazards. 26, 279-331. Cherry, N., 2002. Schumann Resonances, a plausible biophysical mechanism for the human health effects of Solar. Natural Hazards. 26, 279-331. Chiaretti, S., Gianfelici, V., Ceglie, G., Foa, R., 2014. Genomic characterization of acute leukemias. Medical Principles and Practice : International Journal of the Kuwait University, Health Science Centre. 23, 487-506. Cho, Y.H. & Chung, H.W., 2003. The effect of extremely low frequency electromagnetic fields (ELF-EMF) on the frequency of micronuclei and sister chromatid exchange in human lymphocytes induced by benzo(a)pyrene. Toxicology Letters. 143, 37-44. Chomczynski, P. & Sacchi, N., 2006. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nature Protocols. 1, 581-585. Chomczynski, P. & Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry. 162, 156-159. Ciejka, E., Kleniewska, P., Skibska, B., Goraca, A., 2011. Effects of extremely low frequency magnetic field on oxidative balance in brain of rats. Journal of Physiology and Pharmacology : An Official Journal of the Polish Physiological Society. 62, 657-661. Coghill, R.W., Steward, J., Philips, A., 1996. Extra low frequency electric and magnetic fields in the bedplace of children diagnosed with leukaemia: a case-control study. European Journal of Cancer Prevention : The Official Journal of the European Cancer Prevention Organisation (ECP). 5, 153-158. Coleman, M.P., Bell, C.M., Taylor, H.L., Primic-Zakelj, M., 1989. Leukaemia and residence near electricity transmission equipment: a case-control study. British Journal of Cancer. 60, 793-798. Coogan, P. & Aschengrau, A., 1998. Exposure to Power Frequency Magnetic Fields and Risk of Breast Cancer in the Upper Cape Cod Cancer Incidence Study. Archives of Environmental Health: An International Journal. 53, 359-367. Cooke, H.J. & Saunders, P.T., 2002. Mouse models of male infertility. Nature Reviews.Genetics. 3, 790-801. Coulton, L.A., Harris, P.A., Barker, A.T., Pockley, A.G., 2004. Effect of 50 Hz electromagnetic fields on the induction of heat-shock protein gene expression in human leukocytes. Radiation Research. 161, 430-434. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. Cooper, A.R., Van Wijngaarden, E., Fisher, S.G., Adams, M.J., Yost, M.G., Bowman, J.D., 2009. A Population-Based Cohort Study of Occupational Exposure to Magnetic Fields and Cardiovascular Disease Mortality. Annals of Epidemiology. 19, 42-48. Coulton, L.A., Harris, P.A., Barker, A.T., Pockley, A.G., 2004. Effect of 50 Hz electromagnetic fields on the induction of heat-shock protein gene expression in human leukocytes. Radiation Research. 161, 430-434. 152 Davanipour, Z., Sobel, E., Bowman, J.D., Qian, Z., Will, A.D., 1997. Amyotrophic lateral sclerosis and occupational exposure to electromagnetic fields. Bioelectromagnetics. 1 28-35. Davanipour, Z., Sobel, E., Bowman, J.D., Qian, Z., Will, A.D., 1997. Amyotrophic lateral sclerosis and occupational exposure to electromagnetic fields. Bioelectromagnetics. 18, 28-35. Davis, A.P. & Justice, M.J., 1998. An Oak Ridge legacy: the specific locus test and its role in mouse mutagenesis. Genetics. 148, 7-12. de Rooij, D.G. & Russell, L.D., 2000. All you wanted to know about spermatogonia but were afraid to ask. Journal of Andrology. 21, 776-798. Ding, G.R., Nakahara, T., Hirose, H., Koyama, S., Takashima, Y., Miyakoshi, J., 2004. Extremely low frequency magnetic fields and the promotion of H2O2-induced cell death in HL-60 cells. International Journal of Radiation Biology. 80, 317-324. Dockerty, J.D., Elwood, J.M., Herbison, G.P., 1998. Electromagnetic Field Exposures and Childhood Cancers in New Zealand. Cancer Causes & Control. 9, 299-309. Doll, R. & Wakeford, R., 1997. Risk of childhood cancer from fetal irradiation. The British Journal of Radiology. 70, 130-139. Draper, G., Vincent, T., Kroll, M.E., Swanson, J., 2005. Childhood cancer in relation to distance from high voltage power lines in England and Wales: a case-control study. BMJ (Clinical Research Ed.). 330, 1290. Duan, W., Liu, C., Wu, H., Chen, C., Zhang, T., Gao, P., Luo, X., Yu, Z., Zhou, Z., 2014. Effects of exposure to extremely low frequency magnetic fields on spermatogenesis in adult rats. Bioelectromagnetics. 35, 58-69. Dubrova, Y.E., 2005. Radiation-induced mutation at tandem repeat DNA Loci in the mouse germline: spectra and doubling doses. Radiation Research. 163, 200-207. Dubrova, Y.E., Grant, G., Chumak, A.A., Stezhka, V.A., Karakasian, A.N., 2002. Elevated minisatellite mutation rate in the post-chernobyl families from ukraine. American Journal of Human Genetics. 71, 801-809. Dubrova, Y.E., Hickenbotham, P., Glen, C.D., Monger, K., Wong, H.P., Barber, R.C., 2008. Paternal exposure to ethylnitrosourea results in transgenerational genomic instability in mice. Environmental and Molecular Mutagenesis. 49, 308-311. Dubrova, Y.E., Jeffreys, A.J., Malashenko, A.M., 1993. Mouse minisatellite mutations induced by ionizing radiation. Nature Genetics. Dubrova, Y.E., Nesterov, V.N., Krouchinsky, N.G., Ostapenko, V.A., Vergnaud, G., Giraudeau, F., Buard, J., Jeffreys, A.J., 1997. Further evidence for elevated human Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. 5, 92-94. Dubrova, Y.E., Nesterov, V.N., Krouchinsky, N.G., Ostapenko, V.A., Neumann, R., Neil, D.L., Jeffreys, A.J., 1996. Human minisatellite mutation rate after the Chernobyl accident. Nature. 380, 683-686. Dubrova, Y.E., Nesterov, V.N., Krouchinsky, N.G., Ostapenko, V.A., Vergnaud, G., Giraudeau, F., Buard, J., Jeffreys, A.J., 1997. Further evidence for elevated human 153 minisatellite mutation rate in Belarus eight years after the Chernobyl accident. Mutation Research. 381, 267-278. Dubrova, Y.E., Plumb, M., Brown, J., Boulton, E., Goodhead, D., Jeffreys, A.J., 2000a. Induction of minisatellite mutations in the mouse germline by low-dose chronic exposure to gamma-radiation and fission neutrons. Mutation Research. 453, 17-24. Dubrova, Y.E., Plumb, M., Brown, J., Fennelly, J., Bois, P., Goodhead, D., Jeffreys, A.J., 1998a. Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation. Proceedings of the National Academy of Sciences of the United States of America. 95, 6251-6255. Dubrova, Y.E., Plumb, M., Brown, J., Jeffreys, A.J., 1998b. Radiation-induced germline instability at minisatellite loci. International Journal of Radiation Biology. 74, 689-696. Dubrova, Y.E., Plumb, M., Gutierrez, B., Boulton, E., Jeffreys, A.J., 2000. Transgenerational mutation by radiation. Nature. 405, 37. Dubrova, Y.E. & Plumb, M.A., 2002. Ionising radiation and mutation induction at mouse minisatellite loci. The story of the two generations. Mutation Research. 499, 143-150. Eden, T., 2010. Aetiology of childhood leukaemia. Cancer Treatment Reviews. 36, 286- 297. Edwards, R., 1990. Electric jobs 'carry high cancer risk'. The Guardian (1959-2003), pp.20. Ellegren, H., 2004. Microsatellites: simple sequences with complex evolution. Nature Reviews.Genetics. 5, 435-445. Elliott, P., Shaddick, G., Douglass, M., de Hoogh, K., Briggs, D.J., Toledano, M.B., 2013. Adult Cancers Near High-voltage Overhead Power Lines. Epidemiology. 24, 184-190. (EMFs.info)Electric and magnetic fields and health. 2015. EMFs.info Sources. [Online] Available from: http://www.emfs.info/sources/. [Accessed: 02/03/2015]. Erren, T.C., 2001. A meta-analysis of epidemiologic studies of electric and magnetic fields and breast cancer in women and men. Bioelectromagnetics. Suppl 5, S105-19. Eveson, R.W., Timmel, C.R., Brocklehurst, B., Hore, P.J., McLauchlan, K.A., 2000. The effects of weak magnetic fields on radical recombination reactions in micelles. International Journal of Radiation Biology. 76, 1509-1522. Fan, Y.J., Wang, Z., Sadamoto, S., Ninomiya, Y., Kotomura, N., Kamiya, K., Dohi, K., Kominami, R., Niwa, O., 1995. Dose-response of a radiation induction of a germline mutation at a hypervariable mouse minisatellite locus. International Journal of Radiation Biology. 68, 177-183. 154 Farag, A.S., Dawoud, M.M., Selim, S.Z., Cheng, T.C., Marcus, A.M., Penn, D., 1998. Forssén, U.M., Rutqvist, L.E., Ahlbom, A., Feychting, M., 2005. Occupational magnetic fields and female breast cancer: a case-control study using Swedish population registers and new exposure data. American Journal of Epidemiology. 161, 250-259. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. Electromagnetic fields in the home. Electric Power Systems Research. 45, 73-89. Farag, A.S., Dawoud, M.M., Selim, S.Z., Cheng, T.C., Marcus, A.M., Penn, D., 1998. Electromagnetic fields in the home. Electric Power Systems Research. 45, 73-89. Fatigoni, C., Dominici, L., Moretti, M., Villarini, M., Monarca, S., 2005. Genotoxic effects of extremely low frequency (ELF) magnetic fields (MF) evaluated by the Tradescantia- micronucleus assay. Environmental Toxicology. 20, 585-591. Favor, J., 1999. Mechanisms of mutation induction in germ cells of the mouse as assessed by the specific locus test. Mutation Research. 428, 227-236. Feychting, M. & Ahlbom, A., 1995. Childhood leukemia and residential exposure to weak extremely low frequency magnetic fields. Environmental Health Perspectives. 103 Suppl 2, 59-62. Feychting, M. & Ahlbom, A., 1993. Magnetic fields and cancer in children residing near Swedish high-voltage power lines. American Journal of Epidemiology. 138, 467-481. Feychting, M., Ahlbom, A., Kheifets, L., 2005. EMF and health. Annual Review of Public Health. 26, 165-189. Feychting, M., 2013. Invited commentary: extremely low-frequency magnetic fields and breast cancer--now it is enough. American Journal of Epidemiology. 178, 1046. Feychting, M., Forssén, U., Rutqvist, L.E., Ahlbom, A., 1998. Magnetic Fields and Breast Cancer in Swedish Adults Residing near High-Voltage Power Lines. Epidemiology. 9, 392-397. Feychting, M., Jonsson, F., Pedersen, N.L., Ahlbom, A., 2003. Occupational Magnetic Field Exposure and Neurodegenerative Disease. Epidemiology. 14, 413-419. Feychting, M., Pedersen, N.L., Svedberg, P., Floderus, B., Gatz, M., 1998. Dementia and occupational exposure to magnetic fields. Scandinavian Journal of Work, Environment & Health. 24, 46-53. Floderus, B., Persson, T., Stenlund, C., Wennberg, A., Ost, A., Knave, B., 1993. Occupational exposure to electromagnetic fields in relation to leukemia and brain tumors: a case-control study in Sweden. Cancer Causes & Control : CCC. 4, 465-476. Focke, F., Schuermann, D., Kuster, N., Schar, P., 2010. DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure. Mutation Research. 683, 74-83. Fonseca, N.A., Marioni, J., Brazma, A., 2014. RNA-Seq gene profiling-a systematic empirical comparison. PloS One. 9, e107026. Forssén, U.M., Rutqvist, L.E., Ahlbom, A., Feychting, M., 2005. Occupational magnetic fields and female breast cancer: a case-control study using Swedish population registers and new exposure data. American Journal of Epidemiology. 161, 250-259. 155 Frazier, M.E., Reese, J.A., Morris, J.E., Jostes, R.F., Miller, D.L., 1990. Exposure of mammalian cells to 60-Hz magnetic or electric fields: analysis of DNA repair of induced, single-strand breaks. Bioelectromagnetics. 11, 229-234. Gomes, A.M., Barber, R.C., Dubrova, Y.E., 2015. Paternal irradiation perturbs the expression of circadian genes in offspring. Mutation Research. 775, 33-37. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. Frazier, M.E., Reese, J.A., Morris, J.E., Jostes, R.F., Miller, D.L., 1990. Exposure of mammalian cells to 60-Hz magnetic or electric fields: analysis of DNA repair of induced, single-strand breaks. Bioelectromagnetics. 11, 229-234. Fukuda, H., Katahira, M., Tsuchiya, N., Enokizono, Y., Sugimura, T., Nagao, M., Nakagama, H., 2002. Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1. Proceedings of the National Academy of Sciences of the United States of America. 99, 12685-12690. Funk, R.H., Monsees, T., Ozkucur, N., 2009. Electromagnetic effects - From cell biology to medicine. Progress in Histochemistry and Cytochemistry. 43, 177-264. Galt, S., Wahlström, J., Hamnerius, Y., Holmgvist, D., Johannesson, T., 1995. Study of effects of 50 Hz magnetic fields on chromosome aberrations and the growth-related enzyme ODC in human amniotic cells. Bioelectrochemistry and Bioenergetics. 36, 1-8. Gammon, M.D., Schoenberg, J.B., Britton, J.A., Kelsey, J.L., Stanford, J.L., Malone, K.E., Coates, R.J., Brogan, D.J., Potischman, N., Swanson, C.A., Brinton, L.A., 1998. Electric blanket use and breast cancer risk among younger women. American Journal of Epidemiology. 148, 556. Garcia, J.J., Lopez-Pingarron, L., Almeida-Souza, P., Tres, A., Escudero, P., Garcia-Gil, F.A., Tan, D.X., Reiter, R.J., Ramirez, J.M., Bernal-Perez, M., 2014. Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: a review. Journal of Pineal Research. 56, 225-237. Gibbs, M., Collick, A., Kelly, R.G., Jeffreys, A.J., 1993. A tetranucleotide repeat mouse minisatellite displaying substantial somatic instability during early preimplantation development. Genomics. 17, 121-128. Gilbert, D.A., O'Brien, S.J., Lehman, N., Wayne, R.K., 1990. Genetic fingerprinting reflects population differentiation in the California Channel Island fox. Nature. 344, 764-767. Glen, C.D. & Dubrova, Y.E., 2012. Exposure to anticancer drugs can result in transgenerational genomic instability in mice. Proceedings of the National Academy of Sciences of the United States of America. 109, 2984-2988. Glen, C.D., McVeigh, L.E., Voutounou, M., Dubrova, Y.E., 2015. The effects of methyl- donor deficiency on the pattern of gene expression in mice. Molecular Nutrition & Food Research. 59, 501-506. Glen, C.D., Smith, A.G., Dubrova, Y.E., 2008. Single-molecule PCR analysis of germ line mutation induction by anticancer drugs in mice. Cancer Research. 68, 3630-3636. Gomes, A.M., Barber, R.C., Dubrova, Y.E., 2015. Paternal irradiation perturbs the expression of circadian genes in offspring. Mutation Research. 775, 33-37. 156 Graham, C., Cook, M.R., Sastre, A., Gerkovich, M.M., Kavet, R., 2000. Cardiac autonomic control mechanisms in power-frequency magnetic fields: a multistudy analysis. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2000. Ionizing radiation, Part I, X- and gamma- radiation and neutrons. IARC Monographs on the Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. Environmental Health Perspectives. 108, 737-742. Graham, C., Cook, M.R., Sastre, A., Gerkovich, M.M., Kavet, R., 2000. Cardiac autonomic control mechanisms in power-frequency magnetic fields: a multistudy analysis. Environmental Health Perspectives. 108, 737-742. Greaves, M., 2006. Infection, immune responses and the aetiology of childhood leukaemia. Nature Reviews.Cancer. 6, 193-203. Greaves, M., 2002. Childhood leukaemia. BMJ (Clinical Research Ed.). 324, 283-287. Greaves, M., 1999. Molecular genetics, natural history and the demise of childhood leukaemia. European Journal of Cancer (Oxford, England : 1990). 35, 1941-1953. Greaves, M.F. & Wiemels, J., 2003a. Origins of chromosome translocations in childhood leukaemia. Nature Reviews.Cancer. 3, 639-649. Green, S., Lavappa, K.S., Manandhar, M., Sheu, C.J., Whorton, E., Springer, J.A., 1987. A guide for mutagenicity testing using the dominant lethal assay. Mutation Research. 189, 167-174. Greenland, S., Sheppard, A.R., Kaune, W.T., Poole, C., Kelsh, M.A., 2000. A pooled analysis of magnetic fields, wire codes, and childhood leukemia. Epidemiology. 11, 624- 634. Grellier, J., Ravazzani, P., Cardis, E., 2014. Potential health impacts of residential exposures to extremely low frequency magnetic fields in Europe. Environment International. 62, 55-63. Gross, C.G., 2000. Neurogenesis in the adult brain: death of a dogma. Nature Reviews.Neuroscience. 1, 67-73. Gurney, J.G., Mueller, B.A., Davis, S., Schwartz, S.M., Stevens, R.G., Kopecky, K.J., 1996. Childhood brain tumor occurrence in relation to residential power line configurations, electric heating sources, and electric appliance use. American Journal of Epidemiology. 143, 120. Gurney, J.G., Severson, R.K., Davis, S., Robison, L.L., 1995. Incidence of cancer in children in the United States. Sex-, race-, and 1-year age-specific rates by histologic type. Cancer. 75, 2186-2195. Hakansson, N., Gustavsson, P., Johansen, C., Floderus, B., 2003. Neurodegenerative diseases in welders and other workers exposed to high levels of magnetic fields. Epidemiology. 14, 420-6. Håkansson, N., Gustavsson, P., Sastre, A., Floderus, B., 2003. Occupational exposure to extremely low frequency magnetic fields and mortality from cardiovascular disease. American Journal of Epidemiology. 158, 534-542. 157 Hanneman, W.H., Legare, M.E., Sweeney, S., Schimenti, J.C., 1997. Cisplatin increases meiotic crossing-over in mice. Proceedings of the National Academy of Sciences of the United States of America. 94, 8681-8685. Hanneman, W.H., Legare, M.E., Sweeney, S., Schimenti, J.C., 1997. Cisplatin increases meiotic crossing-over in mice. Proceedings of the National Academy of Sciences of the United States of America. 94, 8681-8685. Hardeland, R., 2005. Antioxidative protection by melatonin: multiplicity of mechanisms from radical detoxification to radical avoidance. Endocrine. 27, 119-130. Hardeland, R., 2005. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. IARC Monographs on the 158 Evaluation of Carcinogenic Risks to Humans / World Health Organization, International Agency for Research on Cancer. 75 Pt 1, 1-448. Evaluation of Carcinogenic Risks to Humans / World Health Organization, International Agency for Research on Cancer. 75 Pt 1, 1-448. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002. Non- ionizing radiation, Part 1: static and extremely low-frequency (ELF) electric and magnetic fields. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans / World Health Organization, International Agency for Research on Cancer. 80, 1-395. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2013. Non- ionizing radiation, Part 2: Radiofrequency electromagnetic fields. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans / World Health Organization, International Agency for Research on Cancer. 102, 1-460. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2016. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans / World Health Organization, International Agency for Research on Cancer - List of Classifications, Volumes 1-115. [Online]. [Accessed 23 March 2016]. Available from: http://monographs.iarc.fr/ENG/Classification/latest_classif.php Inaba, H., Greaves, M., Mullighan, C.G., 2013. Acute lymphoblastic leukaemia. Lancet. 381, 1943-1955. Inaba, H., Greaves, M., Mullighan, C.G., 2013. Acute lymphoblastic leukaemia. Lancet. 381, 1943-1955. International Commission on Non-Ionizing Radiation Protection, 2010. Guidelines for limiting exposure to time-varying electric and magnetic fields (1 Hz to 100 kHz). Health Physics. 99, 818-836. International Commission on Non-Ionizing Radiation Protection, 2010. Guidelines for limiting exposure to time-varying electric and magnetic fields (1 Hz to 100 kHz). Health Physics. 99, 818-836. International Commission on Non-Ionizing Radiation Protection, 1998. "Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz)". Health Physics. 74, 494-522. Ivancsits, S., Diem, E., Jahn, O., Rudiger, H.W., 2003a. Age-related effects on induction of DNA strand breaks by intermittent exposure to electromagnetic fields. Mechanisms of Ageing and Development. 124, 847-850. Ivancsits, S., Diem, E., Jahn, O., Rudiger, H.W., 2003b. Intermittent extremely low frequency electromagnetic fields cause DNA damage in a dose-dependent way. International Archives of Occupational and Environmental Health. 76, 431-436. Ivancsits, S., Diem, E., Pilger, A., Rudiger, H.W., Jahn, O., 2002. Induction of DNA strand breaks by intermittent exposure to extremely-low-frequency electromagnetic fields in human diploid fibroblasts. Mutation Research. 519, 1-13. Ivancsits, S., Pilger, A., Diem, E., Jahn, O., Rudiger, H.W., 2005. Cell type-specific genotoxic effects of intermittent extremely low-frequency electromagnetic fields. Mutation Research. Ivancsits, S., Pilger, A., Diem, E., Jahn, O., Rudiger, H.W., 2005. Cell type-specific genotoxic effects of intermittent extremely low-frequency electromagnetic fields. Mutation Research. 583, 184-188. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. Antioxidative protection by melatonin: multiplicity of mechanisms from radical detoxification to radical avoidance. Endocrine. 27, 119-130. Hardwick, R.J., Tretyakov, M.V., Dubrova, Y.E., 2009. Age-related accumulation of mutations supports a replication-dependent mechanism of spontaneous mutation at tandem repeat DNA Loci in mice. Molecular Biology and Evolution. 26, 2647-2654. Hatch, E.E., Linet, M.S., Kleinerman, R.A., Tarone, R.E., Severson, R.K., Hartsock, C.T., Haines, C., Kaune, W.T., Friedman, D., Robison, L.L., Wacholder, S., 1998. Association between childhood acute lymphoblastic leukemia and use of electrical appliances during pregnancy and childhood. Epidemiology. 9, 234-245. Hatch, T., Derijck, A.A., Black, P.D., van der Heijden, G.W., de Boer, P., Dubrova, Y.E., 2007. Maternal effects of the scid mutation on radiation-induced transgenerational instability in mice. Oncogene. 26, 4720-4724. He, Y., Jiang, X., Chen, J., 2014. The role of miR-150 in normal and malignant hematopoiesis. Oncogene. 33, 3887-3893. Henderson, B., Kind, M., Boeck, G., Helmberg, A., Wick, G., 2006. Gene expression profiling of human endothelial cells exposed to 50-Hz magnetic fields fails to produce regulated candidate genes. Cell Stress & Chaperones. 11, 227-232. Henshaw, D.L. & Reiter, R.J., 2005. Do magnetic fields cause increased risk of childhood leukemia via melatonin disruption? Bioelectromagnetics. Suppl 7, S86-97. Hill, S.M., Blask, D.E., Xiang, S., Yuan, L., Mao, L., Dauchy, R.T., Dauchy, E.M., Frasch, T., Duplesis, T., 2011. Melatonin and associated signaling pathways that control normal breast epithelium and breast cancer. Journal of Mammary Gland Biology and Neoplasia. 16, 235-245. Hong, M.N., Han, N.K., Lee, H.C., Ko, Y.K., Chi, S.G., Lee, Y.S., Gimm, Y.M., Myung, S.H., Lee, J.S., 2012. Extremely low frequency magnetic fields do not elicit oxidative stress in MCF10A cells. Journal of Radiation Research. 53, 79-86. Hore, P.J., 2012. Are biochemical reactions affected by weak magnetic fields? Proceedings of the National Academy of Sciences of the United States of America. 109, 1357-1358. Hore, P.J., 2012. Are biochemical reactions affected by weak magnetic fields? Proceedings of the National Academy of Sciences of the United States of America. 109, 1357-1358. Huuskonen, H., Lindbohm, M.L., Juutilainen, J., 1998. Teratogenic and reproductive effects of low-frequency magnetic fields. Mutation Research. 410, 167-183. Huuskonen, H., Lindbohm, M.L., Juutilainen, J., 1998. Teratogenic and reproductive effects of low-frequency magnetic fields. Mutation Research. 410, 167-183. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2000. Ionizing radiation, Part I, X- and gamma- radiation and neutrons. Johansen, C., 2000. Exposure to electromagnetic fields and risk of central nervous system disease in utility workers. Epidemiology. 11, 539-543. Jin, Y.B., Kang, G.Y., Lee, J.S., Choi, J.I., Lee, J.W., Hong, S.C., Myung, S.H., Lee, Y.S., 2012. Effects on micronuclei formation of 60-Hz electromagnetic field exposure with ionizing radiation, hydrogen peroxide, or c-Myc overexpression. International Journal of Radiation Biology. 88, 374-380. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. 583, 184-188. 159 Jackson, J.D., 1992. Are the stray 60-Hz electromagnetic fields associated with the distribution and use of electric power a significant cause of cancer? Proceedings of the National Academy of Sciences of the United States of America. 89, 3508-3510. Jeffreys, A.J., 1987. Highly variable minisatellites and DNA fingerprints. Biochemical Society Transactions. 15, 309-317. Jeffreys, A.J., Barber, R., Bois, P., Buard, J., Dubrova, Y.E., Grant, G., Hollies, C.R., May, C.A., Neumann, R., Panayi, M., Ritchie, A.E., Shone, A.C., Signer, E., Stead, J.D., Tamaki, K., 1999. Human minisatellites, repeat DNA instability and meiotic recombination. Electrophoresis. 20, 1665-1675. Jeffreys, A.J., Royle, N.J., Wilson, V., Wong, Z., 1988. Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature. 332, 278-281. Jeffreys, A.J., Tamaki, K., MacLeod, A., Monckton, D.G., Neil, D.L., Armour, J.A., 1994. Complex gene conversion events in germline mutation at human minisatellites. Nature Genetics. 6, 136-145. Jeffreys, A.J., Turner, M., Debenham, P., 1991. The efficiency of multilocus DNA fingerprint probes for individualization and establishment of family relationships, determined from extensive casework. American Journal of Human Genetics. 48, 824- 840. Jeffreys, A.J., Wilson, V., Kelly, R., Taylor, B.A., Bulfield, G., 1987. Mouse DNA 'fingerprints': analysis of chromosome localization and germ-line stability of hypervariable loci in recombinant inbred strains. Nucleic Acids Research. 15, 2823- 2836. Jeffreys, A.J., Wilson, V., Thein, S.L., 1985a. Hypervariable 'minisatellite' regions in human DNA. Nature. 314, 67-73. Jeffreys, A.J., Wilson, V., Thein, S.L., 1985a. Hypervariable 'minisatellite' regions in human DNA. Nature. 314, 67-73. Jeffreys, A.J., Wilson, V., Thein, S.L., 1985b. Individual-specific 'fingerprints' of human DNA. Nature. 316, 76-79. Jeffreys, A.J., Wilson, V., Thein, S.L., 1985b. Individual-specific 'fingerprints' of human DNA. Nature. 316, 76-79. Jin, Y.B., Choi, S.H., Lee, J.S., Kim, J.K., Lee, J.W., Hong, S.C., Myung, S.H., Lee, Y.S., 2014. Absence of DNA damage after 60-Hz electromagnetic field exposure combined with ionizing radiation, hydrogen peroxide, or c-Myc overexpression. Radiation and Environmental Biophysics. 53, 93-101. Jin, Y.B., Kang, G.Y., Lee, J.S., Choi, J.I., Lee, J.W., Hong, S.C., Myung, S.H., Lee, Y.S., 2012. Effects on micronuclei formation of 60-Hz electromagnetic field exposure with ionizing radiation, hydrogen peroxide, or c-Myc overexpression. International Journal of Radiation Biology. 88, 374-380. Johansen, C., 2000. Exposure to electromagnetic fields and risk of central nervous system disease in utility workers. Epidemiology. 11, 539-543. Johansen, C., 2000. Exposure to electromagnetic fields and risk of central nervous system disease in utility workers. Epidemiology. Armour, J.A., 2006. Tandemly repeated DNA: why should anyone care? Mutation Research. 598, 6-14. 11, 539-543. 160 Johansen, C., Feychting, M., Møller, M., Arnsbo, P., Ahlbom, A., Olsen, J.H., 2002. Risk of severe cardiac arrhythmia in male utility workers: a nationwide Danish cohort study. American Journal of Epidemiology. 156, 857-861. Johansen, C., Feychting, M., Møller, M., Arnsbo, P., Ahlbom, A., Olsen, J.H., 2002. Risk of severe cardiac arrhythmia in male utility workers: a nationwide Danish cohort study. American Journal of Epidemiology. 156, 857-861. Juutilainen, J., 2003. Developmental effects of extremely low frequency electric and magnetic fields. Radiation Protection Dosimetry. 106, 385-390. Juutilainen, J., Kumlin, T., Naarala, J., 2006. Do extremely low frequency magnetic fields enhance the effects of environmental carcinogens? A meta-analysis of experimental studies. International Journal of Radiation Biology. 82, 1-12. Juutilainen, J., Lang, S., Rytomaa, T., 2000. Possible cocarcinogenic effects of ELF electromagnetic fields may require repeated long-term interaction with known carcinogenic factors. Bioelectromagnetics. 21, 122-128. Juutilainen, J., 2005. Developmental effects of electromagnetic fields. Bioelectromagnetics. 26, S107-S115. Juutilainen, J., 2005. Developmental effects of electromagnetic fields. Bioelectromagnetics. 26, S107-S115. Kabat, G.C., O'Leary, E.S., Schoenfeld, E.R., Greene, J.M., Grimson, R., Henderson, K., Kaune, W.T., Gammon, M.D., Britton, J.A., Teitelbaum, S.L., Neugut, A.I., Leske, M.C., EBCLIS Group, 2003. Electric Blanket Use and Breast Cancer on Long Island. Epidemiology. 14, 514-520. Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., Wells, R.D., 1995. Pausing of DNA synthesis in vitro at specific loci in CTG and CGG triplet repeats from human hereditary disease genes. The Journal of Biological Chemistry. 270, 27014-27021. Kannan, K. & Jain, S.K., 2000. Oxidative stress and apoptosis. Pathophysiology : The Official Journal of the International Society for Pathophysiology / ISP. 7, 153-163. Karipidis, K.K., 2015. Survey of residential power-frequency magnetic fields in Melbourne, Australia. Radiation Protection Dosimetry. 163, 81-91. Kato, M., Honma, K., Shigemitsu, T., Shiga, Y., 1994a. Circularly polarized 50-Hz magnetic field exposure reduces pineal gland and blood melatonin concentrations of Long-Evans rats. Neuroscience Letters. 166, 59-62. Kato, M., Honma, K., Shigemitsu, T., Shiga, Y., 1994b. Circularly polarized, sinusoidal, 50 Hz magnetic field exposure does not influence plasma testosterone levels of rats. Bioelectromagnetics. 15, 513-518. Kato, M., Honma, K., Shigemitsu, T., Shiga, Y., 1994c. Horizontal or vertical 50-Hz, 1- microT magnetic fields have no effect on pineal gland or plasma melatonin concentration of albino rats. Neuroscience Letters. 168, 205-208. Kato, M., Honma, K., Shigemitsu, T., Shiga, Y., 1994d. Recovery of nocturnal melatonin concentration takes place within one week following cessation of 50 Hz circularly polarized magnetic field exposure for six weeks. Bioelectromagnetics. 15, 489-492. 161 Kato, M., Honma, K., Shigemitsu, T., Shiga, Y., 1993. Effects of exposure to a circularly polarized 50-Hz magnetic field on plasma and pineal melatonin levels in rats. Bioelectromagnetics. 14, 97-106. Katoh, M.A., Cain, K.T., Hughes, L.A., Foxworth, L.B., Bishop, J.B., Generoso, W.M., 1990. Female-specific dominant lethal effects in mice. Mutation Research. 230, 205- 217. Kaune, W.T., 1993. Assessing human exposure to power-frequency electric and magnetic fields. Environmental Health Perspectives. 101 Suppl 4, 121-133. Kaune, W.T. & Zaffanella, L.E., 1994. Assessing historical exposures of children to power-frequency magnetic fields. Journal of Exposure Analysis and Environmental Epidemiology. 4, 149-170. Kelly, R., Bulfield, G., Collick, A., Gibbs, M., Jeffreys, A.J., 1989. Characterization of a highly unstable mouse minisatellite locus: evidence for somatic mutation during early development. Genomics. 5, 844-856. Kelly, R., Gibbs, M., Collick, A., Jeffreys, A.J., 1991. Juutilainen, J., 2005. Developmental effects of electromagnetic fields. Bioelectromagnetics. 26, S107-S115. Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events. Proceedings.Biological Sciences / the Royal Society. 245, 235-245. Kelly, R.G., 1994. Similar origins of two mouse minisatellites within transposon-like LTRs. Genomics. 24, 509-515. Kheifets, L.I., 2001. Electric and magnetic field exposure and brain cancer: a review. Bioelectromagnetics. Suppl 5, S120. Kheifets, L.I., Kavet, R., Sussman, S.S., 1997. Wire codes, magnetic fields, and childhood cancer. Bioelectromagnetics. 18, 99-110. Kheifets, L., Ahlbom, A., Crespi, C.M., Draper, G., Hagihara, J., Lowenthal, R.M., Mezei, G., Oksuzyan, S., Schuz, J., Swanson, J., Tittarelli, A., Vinceti, M., Wunsch Filho, V., 2010. Pooled analysis of recent studies on magnetic fields and childhood leukaemia. British Journal of Cancer. 103, 1128-1135. Kheifets, L., Mezei, G., Greenland, S., 2006. Comment concerning "Childhood leukemia and residential magnetic fields: are pooled analyses more valid than the original studies?" (Bioelectromagnetics 27:1-7 [2006]). Bioelectromagnetics. 27, 674-5; discussion 675-6. Kheifets, L. & Oksuzyan, S., 2008. Exposure assessment and other challenges in non- ionizing radiation studies of childhood leukaemia. Radiation Protection Dosimetry. 132, 139-147. 162 Kheifets, L., Repacholi, M., Saunders, R., van Deventer, E., 2005. The sensitivity of children to electromagnetic fields. Pediatrics. 116, 303-13. Kheifets, L.I., Gilbert, E.S., Sussman, S.S., Guenel, P., Sahl, J.D., Savitz, D.A., Theriault, G., 1999. Comparative analyses of the studies of magnetic fields and cancer in electric utility workers: studies from France, Canada, and the United States. Occupational and Environmental Medicine. 56, 567-574. Kheifets, L., Ahlbom, A., Johansen, C., Feychting, M., Sahl, J., Savitz, D., 2007. Extremely low-frequency magnetic fields and heart disease. Scandinavian Journal of Work, Environment & Health. 33, 5-12. Kim, H.S., Park, B.J., Jang, H.J., Ipper, N.S., Kim, S.H., Kim, Y.J., Jeon, S.H., Lee, K.S., Lee, S.K., Kim, N., Ju, Y.J., Gimm, Y.M., Kim, Y.W., 2014. Continuous exposure to 60 Hz magnetic fields induces duration- and dose-dependent apoptosis of testicular germ cells. Bioelectromagnetics. 35, 100-107. Kim, J., Ha, C.S., Lee, H.J., Song, K., 2010. Repetitive exposure to a 60-Hz time-varying magnetic field induces DNA double-strand breaks and apoptosis in human cells. Biochemical and Biophysical Research Communications. 400, 739-744. Kim, Y.W., Kim, H.S., Lee, J.S., Kim, Y.J., Lee, S.K., Seo, J.N., Jung, K.C., Kim, N., Gimm, Y.M., 2009. Effects of 60 Hz 14 microT magnetic field on the apoptosis of testicular germ cell in mice. Bioelectromagnetics. 30, 66-72. Kirk, K.M. & Lyon, M.F., 1984. Kliukiene, J., Tynes, T., Andersen, A., 2004. Residential and occupational exposures to 50-Hz magnetic fields and breast cancer in women: a population-based study. American Journal of Epidemiology. 159, 852-861. American Journal of Epidemiology. 159, 852-861. Juutilainen, J., 2005. Developmental effects of electromagnetic fields. Bioelectromagnetics. 26, S107-S115. Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages. Mutation Research. 125, 75-85. Kiuru, A., Auvinen, A., Luokkamaki, M., Makkonen, K., Veidebaum, T., Tekkel, M., Rahu M., Hakulinen, T., Servomaa, K., Rytomaa, T., Mustonen, R., 2003. Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers. Radiation Research. 159, 651-655. Kleinerman, R.A., Kaune, W.T., Hatch, E.E., Wacholder, S., Linet, M.S., Robison, L.L., Niwa, S., Tarone, R.E., 2000. Are children living near high-voltage power lines at increased risk of acute lymphoblastic leukemia? American Journal of Epidemiology. 151, 512-515. Kleinerman, R.A., Linet, M.S., Hatch, E.E., Tarone, R.E., Black, P.M., Selker, R.G., Shapiro, W.R., Fine, H.A., Inskip, P.D., 2005. Self-reported electrical appliance use and risk of adult brain tumors. American Journal of Epidemiology. 161, 136-146. Kleinerman, R.A., Linet, M.S., Hatch, E.E., Tarone, R.E., Black, P.M., Selker, R.G., Shapiro, W.R., Fine, H.A., Inskip, P.D., 2005. Self-reported electrical appliance use and risk of adult brain tumors. American Journal of Epidemiology. 161, 136-146. Kliukiene, J., Tynes, T., Andersen, A., 2004. Residential and occupational exposures to 50-Hz magnetic fields and breast cancer in women: a population-based study. American Journal of Epidemiology. 159, 852-861. Kliukiene, J., Tynes, T., Andersen, A., 2004. Residential and occupational exposures to 50-Hz magnetic fields and breast cancer in women: a population-based study. American Journal of Epidemiology. 159, 852-861. 163 Kodaira, M., Izumi, S., Takahashi, N., Nakamura, N., 2004. No evidence of radiation effect on mutation rates at hypervariable minisatellite loci in the germ cells of atomic bomb survivors. Radiation Research. 162, 350-356. Kodaira, M., Izumi, S., Takahashi, N., Nakamura, N., 2004. No evidence of radiation effect on mutation rates at hypervariable minisatellite loci in the germ cells of atomic bomb survivors. Radiation Research. 162, 350-356. Kodaira, M., Satoh, C., Hiyama, K., Toyama, K., 1995. Lack of effects of atomic bomb radiation on genetic instability of tandem-repetitive elements in human germ cells. American Journal of Human Genetics. 57, 1275-1283. Koeman, T., Slottje, P., Kromhout, H., Schouten, L.J., Goldbohm, R.A., van den Brandt, P.A., Vermeulen, R., 2013. Occupational exposure to extremely low-frequency magnetic fields and cardiovascular disease mortality in a prospective cohort study. Occupational and Environmental Medicine. 70, 402. Kroll, M.E., Swanson, J., Vincent, T.J., Draper, G.J., 2010a. Childhood cancer and magnetic fields from high-voltage power lines in England and Wales: a case-control study. British Journal of Cancer. 103, 1122-1127. Juutilainen, J., 2005. Developmental effects of electromagnetic fields. Bioelectromagnetics. 26, S107-S115. Kumlin, T., Kosma, V.M., Alhonen, L., Janne, J., Komulainen, H., Lang, S., Rytomaa, T., Servomaa, K., Juutilainen, J., 1998. Effects of 50 Hz magnetic fields on UV-induced skin tumourigenesis in ODC-transgenic and non-transgenic mice. International Journal of Radiation Biology. 73, 113-121. Kuo, L.J. & Yang, L.X., 2008. Gamma-H2AX - a novel biomarker for DNA double-strand breaks. In Vivo. 22, 305-309. Lacy-Hulbert, A., Metcalfe, J.C., Hesketh, R., 1998. Biological responses to electromagnetic fields. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. 12, 395-420. Laden, F., Neas, L.M., Tolbert, P.E., Holmes, M.D., Hankinson, S.E., Spiegelman, D., Speizer, F.E., Hunter, D.J., 2000. Electric blanket use and breast cancer in the Nurses' Health Study. American Journal of Epidemiology. 152, 41-49. Lagiou, P., Tamimi, R., Lagiou, A., Mucci, L., Trichopoulos, D., 2002. Is epidemiology implicating extremely low frequency electric and magnetic fields in childhood leukemia? Environmental Health and Preventive Medicine. 7, 33-39. Lai, H. & Singh, N.P., 1997. Melatonin and N-tert-butyl-alpha-phenylnitrone block 60- Hz magnetic field-induced DNA single and double strand breaks in rat brain cells. Journal of Pineal Research. 22, 152. Lai, H. & Singh, N.P., 2004. Magnetic-field-induced DNA strand breaks in brain cells of the rat. Environmental Health Perspectives. 112, 687-694. Lai, H. & Singh, N.P., 1997. Acute exposure to a 60 Hz magnetic field increases DNA strand breaks in rat brain cells. Bioelectromagnetics. 18, 156-165. 164 Lai, H. & Singh, N.P., 1996. Single- and double-strand DNA breaks in rat brain cells after acute exposure to radiofrequency electromagnetic radiation. International Journal of Radiation Biology. 69, 513-521. Lai, H. & Singh, N.P., 1996. Single- and double-strand DNA breaks in rat brain cells after acute exposure to radiofrequency electromagnetic radiation. International Journal of Radiation Biology. 69, 513-521. Lara, P.C., Lopez-Penalver, J.J., Farias Vde, A., Ruiz-Ruiz, M.C., Oliver, F.J., Ruiz de Almodovar, J.M., 2015. Direct and bystander radiation effects: a biophysical model and clinical perspectives. Cancer Letters. 356, 5-16. Lee, G.M., Neutra, R.R., Hristova, L., Yost, M., Hiatt, R.A., 2000. The use of electric bed heaters and the risk of clinically recognized spontaneous abortion. Epidemiology. 11, 406-415. Lerner, A.B., Case, J.D., Mori, W., Wright, M.R., 1959. Melatonin in peripheral nerve. Nature. 183, 1821. Lewczuk, B., Redlarski, G., Zak, A., Ziolkowska, N., Przybylska-Gornowicz, B., Krawczuk, M., 2014. Influence of electric, magnetic, and electromagnetic fields on the circadian system: current stage of knowledge. BioMed Research International. 2014, 169459. Linet, M.S. & Devesa, S.S., 1991. Descriptive epidemiology of childhood leukaemia. British Journal of Cancer. 63, 424-429. Juutilainen, J., 2005. Developmental effects of electromagnetic fields. Bioelectromagnetics. 26, S107-S115. Lewy, A.J., Wehr, T.A., Goodwin, F.K., Newsome, D.A., Markey, S.P., 1980. Light suppresses melatonin secretion in humans. Science. 210, 1267-1269. Li, C.Y., Theriault, G., Lin, R.S., 1997. Residential exposure to 60-Hertz magnetic fields and adult cancers in Taiwan. Epidemiology. 8, 25-30. Li, J., Hou, R., Niu, X., Liu, R., Wang, Q., Wang, C., Li, X., Hao, Z., Yin, G., Zhang, K., 2016. Comparison of microarray and RNA-Seq analysis of mRNA expression in dermal mesenchymal stem cells. Biotechnology Letters. 38, 33-41. Li, M., Jones, L., Gaillard, C., Binnewies, M., Ochoa, R., Garcia, E., Lam, V., Wei, G., Yang, W., Lobe, C., Hermiston, M., Passegue, E., Kogan, S.C., 2013. Initially disadvantaged, TEL-AML1 cells expand and initiate leukemia in response to irradiation and cooperating mutations. Leukemia. 27, 1570-1573. Limon-Pacheco, J. & Gonsebatt, M.E., 2009. The role of antioxidants and antioxidant- related enzymes in protective responses to environmentally induced oxidative stress. Mutation Research. 674, 137-147. Limon-Pacheco, J. & Gonsebatt, M.E., 2009. The role of antioxidants and antioxidant- related enzymes in protective responses to environmentally induced oxidative stress. Mutation Research. 674, 137-147. Lin, R.S., Dischinger, P.C., Conde, J., Farrell, K.P., 1985. Occupational exposure to electromagnetic fields and the occurrence of brain tumors. An analysis of possible associations. Journal of Occupational Medicine.: Official Publication of the Industrial Medical Association. 27, 413. Linet, M.S. & Devesa, S.S., 1991. Descriptive epidemiology of childhood leukaemia. British Journal of Cancer. 63, 424-429. 165 Linet, M.S., Ries, L.A., Smith, M.A., Tarone, R.E., Devesa, S.S., 1999. Cancer surveillance series: recent trends in childhood cancer incidence and mortality in the United States. Journal of the National Cancer Institute. 91, 1051-1058. Linet, M.S., Hatch, E.E., Kleinerman, R.A., Robison, L.L., Kaune, W.T., Friedman, D.R., Severson, R.K., Haines, C.M., Hartsock, C.T., Niwa, S., Wacholder, S., Tarone, R.E., 1997. Residential Exposure to Magnetic Fields and Acute Lymphoblastic Leukemia in Children. The New England Journal of Medicine. 337, 1-8. Livshits, L.A., Malyarchuk, S.G., Kravchenko, S.A., Matsuka, G.H., Lukyanova, E.M., Antipkin, Y.G., Arabskaya, L.P., Petit, E., Giraudeau, F., Gourmelon, P., Vergnaud, G., Le Guen, B., 2001. Children of chernobyl cleanup workers do not show elevated rates of mutations in minisatellite alleles. Radiation Research. 155, 74-80. Loberg, L.I., Engdahl, W.R., Gauger, J.R., McCormick, D.L., 2000. Expression of cancer- related genes in human cells exposed to 60 Hz magnetic fields. Radiation Research. 153, 679-684. Loeb, L.A., Loeb, K.R., Anderson, J.P., 2003. Multiple mutations and cancer. Juutilainen, J., 2005. Developmental effects of electromagnetic fields. Bioelectromagnetics. 26, S107-S115. Loeb, L.A., Loeb, K.R., Anderson, J.P., 2003. Multiple mutations and cancer. Proceedings of the National Academy of Sciences of the United States of America. 100, 776-781. Luukkonen, J., Liimatainen, A., Juutilainen, J., Naarala, J., 2014. Induction of genomic instability, oxidative processes, and mitochondrial activity by 50Hz magnetic fields in human SH-SY5Y neuroblastoma cells. Mutation Research. 760, 33-41. , , , , , , p Proceedings of the National Academy of Sciences of the United States of America. 100 776-781. London, S.J., Thomas, D.C., Bowman, J.D., Sobel, E., Cheng, T.C., Peters, J.M., 1991. Exposure to residential electric and magnetic fields and risk of childhood leukemia. American Journal of Epidemiology. 134, 923-937. Luceri, C., De Filippo, C., Giovannelli, L., Blangiardo, M., Cavalieri, D., Aglietti, F., Pampaloni, M., Andreuccetti, D., Pieri, L., Bambi, F., Biggeri, A., Dolara, P., 2005. Extremely low-frequency electromagnetic fields do not affect DNA damage and gene expression profiles of yeast and human lymphocytes. Radiation Research. 164, 277- 285. Luning, K.G. & Searle, A.G., 1971. Estimates of the genetic risks from ionizing irradiation. Mutation Research. 12, 291-304. Lupke, M., Rollwitz, J., Simkó, M., 2004. Cell Activating Capacity of 50 Hz Magnetic Fields to Release Reactive Oxygen Intermediates in Human Umbilical Cord Blood- derived Monocytes and in Mono Mac 6 Cells. Free Radical Research. 38, 985-993. Luukkonen, J., Liimatainen, A., Hoyto, A., Juutilainen, J., Naarala, J., 2011. Pre-exposure to 50 Hz magnetic fields modifies menadione-induced genotoxic effects in human SH- SY5Y neuroblastoma cells. PloS One. 6, 18021. Luukkonen, J., Liimatainen, A., Juutilainen, J., Naarala, J., 2014. Induction of genomic instability, oxidative processes, and mitochondrial activity by 50Hz magnetic fields in human SH-SY5Y neuroblastoma cells. Mutation Research. 760, 33-41. Luukkonen, J., Liimatainen, A., Juutilainen, J., Naarala, J., 2014. Induction of genomic instability, oxidative processes, and mitochondrial activity by 50Hz magnetic fields in human SH-SY5Y neuroblastoma cells. Mutation Research. 760, 33-41. 166 Lyon, M.F. & Morris, T., 1966. Mutation rates at a new set of specific loci in the mouse. Genetical Research. 7, 12-17. Lyon, M.F. & Morris, T., 1966. Mutation rates at a new set of specific loci in the mouse. Genetical Research. 7, 12-17. Maes, A., Collier, M., Vandoninck, S., Scarpa, P., Verschaeve, L., 2000. Cytogenetic effects of 50 Hz magnetic fields of different magnetic flux densities. Bioelectromagnetics. 21, 589-596. Mairs, R.J., Hughes, K., Fitzsimmons, S., Prise, K.M., Livingstone, A., Wilson, L., Baig, N., Clark, A.M., Timpson, A., Patel, G., Folkard, M., Angerson, W.J., Boyd, M., 2007. Microsatellite analysis for determination of the mutagenicity of extremely low- frequency electromagnetic fields and ionising radiation in vitro. Mutation Research. 626, 34-41. Mandeville, R., Franco, E., Sidrac-Ghali, S., Paris-Nadon, L., Rocheleau, N., Mercier, G., Desy, M., Gaboury, L., 1997. Evaluation of the potential carcinogenicity of 60 Hz linear sinusoidal continuous-wave magnetic fields in Fischer F344 rats. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. 11, 1127-1136. McElroy, J., 2007. Occupational exposure to electromagnetic field and breast cancer risk in a large, population-based, case-control study in the United States. J Occup Environ Med. 49, 266-274. , , , , , , p Proceedings of the National Academy of Sciences of the United States of America. 100 776-781. Mandeville, R., Franco, E., Sidrac-Ghali, S., Paris-Nadon, L., Rocheleau, N., Mercier, G., Desy, M., Gaboury, L., 1997. Evaluation of the potential carcinogenicity of 60 Hz linear sinusoidal continuous-wave magnetic fields in Fischer F344 rats. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. 11, 1127-1136. Mannerling, A.C., Simko, M., Mild, K.H., Mattsson, M.O., 2010. Effects of 50-Hz magnetic field exposure on superoxide radical anion formation and HSP70 induction in human K562 cells. Radiation and Environmental Biophysics. 49, 731-741. Mannerling, A.C., Simko, M., Mild, K.H., Mattsson, M.O., 2010. Effects of 50-Hz magnetic field exposure on superoxide radical anion formation and HSP70 induction in human K562 cells. Radiation and Environmental Biophysics. 49, 731-741. Marchetti, F., Rowan-Carroll, A., Williams, A., Polyzos, A., Berndt-Weis, M.L., Yauk, C.L., 2011. Sidestream tobacco smoke is a male germ cell mutagen. Proceedings of the National Academy of Sciences of the United States of America. 108, 12811-12814. McBride, M.L., Gallagher, R.P., Thériault, G., Armstrong, B.G., Tamaro, S., Spinelli, J.J., Deadman, J.E., Fincham, S., Robson, D., Choi, W., 1999. Power-frequency electric and magnetic fields and risk of childhood leukemia in Canada. American Journal of Epidemiology. 149, 831. McCann, J., Dietrich, F., Rafferty, C., 1998. The genotoxic potential of electric and magnetic fields: an update. Mutation Research. 411, 45-86. McCormick, D.L., Boorman, G.A., Findlay, J.C., Hailey, J.R., Johnson, T.R., Gauger, J.R., Pletcher, J.M., Sills, R.C., Haseman, J.K., 1999. Chronic toxicity/oncogenicity evaluation of 60 Hz (power frequency) magnetic fields in B6C3F1 mice. Toxicologic Pathology. 27, 279-285. McCormick, D.L., Ryan, B.M., Findlay, J.C., Gauger, J.R., Johnson, T.R., Morrissey, R.L., Boorman, G.A., 1998. Exposure to 60 Hz magnetic fields and risk of lymphoma in PIM transgenic and TSG-p53 (p53 knockout) mice. Carcinogenesis. 19, 1649-1653. McElroy, J., 2007. Occupational exposure to electromagnetic field and breast cancer risk in a large, population-based, case-control study in the United States. J Occup Environ Med. 49, 266-274. McElroy, J., 2007. Occupational exposure to electromagnetic field and breast cancer risk in a large, population-based, case-control study in the United States. J Occup Environ Med. 49, 266-274. 167 McElroy, J.A., Newcomb, P.A., Remington, P.L., Egan, K.M., Titus-Ernstoff, L., Trentham- Dietz, A., Hampton, J.M., Baron, J.A., Stampfer, M.J., Willett, W.C., 2001. Electric Blanket or Mattress Cover Use and Breast Cancer Incidence in Women 50-79 Years of Age. Epidemiology. 12, 613-617. Milham, S., 1985. Mortality in Workers Exposed to Electromagnetic Fields. Environmental Health Perspectives. 62, 297-300. , , , , , , p Proceedings of the National Academy of Sciences of the United States of America. 100 776-781. McElroy, J.A., Newcomb, P.A., Remington, P.L., Egan, K.M., Titus-Ernstoff, L., Trentham- Dietz, A., Hampton, J.M., Baron, J.A., Stampfer, M.J., Willett, W.C., 2001. Electric Blanket or Mattress Cover Use and Breast Cancer Incidence in Women 50-79 Years of Age. Epidemiology. 12, 613-617. McNamee, J.P., Bellier, P.V., Chauhan, V., Gajda, G.B., Lemay, E., Thansandote, A., 2005. Evaluating DNA damage in rodent brain after acute 60 Hz magnetic-field exposure. Radiation Research. 164, 791-797. McNamee, J.P., Bellier, P.V., McLean, J.R., Marro, L., Gajda, G.B., Thansandote, A., 2002. DNA damage and apoptosis in the immature mouse cerebellum after acute exposure to a 1 mT, 60 Hz magnetic field. Mutation Research. 513, 121-133. McNamee, J.P., McLean, J.R., Ferrarotto, C.L., Bellier, P.V., 2000. Comet assay: rapid processing of multiple samples. Mutation Research. 466, 63-69. Merchant, C.J., Renew, D.C., Swanson, J., 1994. Exposures to power-frequency magnetic fields in the home. Journal of Radiological Protection. 14, 77. Messiha, H.L., Wongnate, T., Chaiyen, P., Jones, A.R., Scrutton, N.S., 2015. Magnetic field effects as a result of the radical pair mechanism are unlikely in redox enzymes. Journal of the Royal Society, Interface / the Royal Society. 12,: 2014.1155. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Mezei, G., Cher, D., Kelsh, M., Edinboro, C., Chapman, P., Kavet, R., 2005. Occupational Magnetic Field Exposure, Cardiovascular Disease Mortality, and Potential Confounding by Smoking. Annals of Epidemiology. 15, 622-629. Mezei, G., Gadallah, M., Kheifets, L., 2008. Residential Magnetic Field Exposure and Childhood Brain Cancer: A Meta-Analysis. Epidemiology. 19, 424-430. Mezei, G. & Kheifets, L., 2006. Selection bias and its implications for case-control studies: a case study of magnetic field exposure and childhood leukaemia. International Journal of Epidemiology. 35, 397-406. Mezei, G., Sudan, M., Izraeli, S., Kheifets, L., 2014. Epidemiology of childhood leukemia in the presence and absence of Down syndrome. Cancer Epidemiology. 38, 479-489. Michaelis, J., Schüz, J., Meinert, R., Zemann, E., Grigat, J., Kaatsch, P., Kaletsch, U., Miesner, A., Brinkmann, K., Kalkner, W., Kärner, H., 1998. Combined Risk Estimates for Two German Population-Based Case-Control Studies on Residential Magnetic Fields and Childhood Acute Leukemia. Epidemiology. 9, 92-94. Milham, S., 1985. Mortality in Workers Exposed to Electromagnetic Fields. Environmental Health Perspectives. 62, 297-300. 168 Miller, A.B. & Green, L.M., 2010. Electric and magnetic fields at power frequencies. Chronic Diseases in Canada. 29 Suppl 1, 69-83. Mirick, D.K. & Davis, S., 2008. Melatonin as a biomarker of circadian dysregulation. Cancer Epidemiology, Biomarkers & Prevention : A Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. 17, 3306-3313. Mirkin, S.M., 2007. Expandable DNA repeats and human disease. Nature. 447, 932- 940. Miyakoshi, J., Kitagawa, K., Takebe, H., 1997. Mutation induction by high-density, 50- Hz magnetic fields in human MeWo cells exposed in the DNA synthesis phase. International Journal of Radiation Biology. 71, 75-79. Miyakoshi, J., Koji, Y., Wakasa, T., Takebe, H., 1999. Long-term exposure to a magnetic field (5 mT at 60 Hz) increases X-ray-induced mutations. Journal of Radiation Research. 40, 13-21. Miyakoshi, J., Yamagishi, N., Ohtsu, S., Mohri, K., Takebe, H., 1996. Increase in hypoxanthine-guanine phosphoribosyl transferase gene mutations by exposure to high-density 50-Hz magnetic fields. Mutation Research. 349, 109-114. Miyakoshi, J., Yoshida, M., Shibuya, K., Hiraoka, M., 2000. Exposure to strong magnetic fields at power frequency potentiates X-ray-induced DNA strand breaks. Journal of Radiation Research. 41, 293-302. Moore, R.Y., Heller, A., Wurtman, R.J., Axelrod, J., 1967. Visual pathway mediating pineal response to environmental light. Science. 155, 220-223. Moretti, M., Villarini, M., Simonucci, S., Fatigoni, C., Scassellati-Sforzolini, G., Monarca, S., Pasquini, R., Angelucci, M., Strappini, M., 2005. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Effects of co-exposure to extremely low frequency (ELF) magnetic fields and benzene or benzene metabolites determined in vitro by the alkaline comet assay. Toxicology Letters. 157, 119-128. Morris, J.E., Sasser, L.B., Miller, D.L., Dagle, G.E., Rafferty, C.N., Ebi, K.L., Anderson, L.E., 1999. Clinical progression of transplanted large granular lymphocytic leukemia in Fischer 344 rats exposed to 60 Hz magnetic fields. Bioelectromagnetics. 20, 48-56. Mughal, S.K., Myazin, A.E., Zhavoronkov, L.P., Rubanovich, A.V., Dubrova, Y.E., 2012. The dose and dose-rate effects of paternal irradiation on transgenerational instability in mice: a radiotherapy connection. PloS One. 7, 41300. Muller-Sieburg, C.E. & Riblet, R., 1996. Genetic control of the frequency of hematopoietic stem cells in mice: mapping of a candidate locus to chromosome 1. The Journal of Experimental Medicine. 183, 1141-1150. 169 Myers, A., Clayden, A.D., Cartwright, R.A., Cartwright, S.C., 1990. Childhood cancer and overhead powerlines: a case-control study. British Journal of Cancer. 62, 1008-1014. Nakamura, Y., Tamura, H., Kashida, S., Takayama, H., Yamagata, Y., Karube, A., Sugino, N., Kato, H., 2001. Changes of serum melatonin level and its relationship to feto‐ placental unit during pregnancy. Journal of Pineal Research. 30, 29-33. Nault, R., Fader, K.A., Zacharewski, T., 2015. RNA-Seq versus oligonucleotide array assessment of dose-dependent TCDD-elicited hepatic gene expression in mice. BMC Genomics. 16, 373-015-1527-z. Niwa, O., Fan, Y.J., Numoto, M., Kamiya, K., Kominami, R., 1996. Induction of a germline mutation at a hypervariable mouse minisatellite locus by 252Cf radiation. Journal of Radiation Research. 37, 217-224. Noonan, C.W., Reif, J.S., Yost, M., Touchstone, J., 2002. Occupational exposure to magnetic fields in case-referent studies of neurodegenerative diseases. Scandinavian Journal of Work, Environment & Health. 28, 42-48. Nordenson, I., Mild, K.H., Andersson, G., Sandstrom, M., 1994. Chromosomal aberrations in human amniotic cells after intermittent exposure to fifty hertz magnetic fields. Bioelectromagnetics. 15, 293-301. Ogheri, S., Rampazzo, C., Celotti, L., 1995. Mutagenic effects at hprt locus and in minisatellite sequences induced in V79 cells by treatments with UV and methyl-nitro- nitroso guanidine. Mutation Research. 348, 193-199. Okamura, H., Yamaguchi, S., Yagita, K., 2002. Molecular machinery of the circadian clock in mammals. Cell and Tissue Research. 309, 47-56. Olsen, J.H., Nielsen, A., Schulgen, G., 1993. Residence near high voltage facilities and risk of cancer in children. BMJ. 307, 891-895. Orkin, S.H. & Zon, L.I., 2008. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 132, 631-644. Paile, W., Jokela, K., Koivistoinen, A., Salomaa, S., 1995. Pandi-Perumal, S.,R., Srinivasan, ,V., Maestroni, G.J.,M., Cardinali, D.,P., Poeggeler, ,B. Hardeland, ,R., 2006. Melatonin. FEBS Journal. 273, 2813-2838. Preece, A.W., Kaune, W., Grainger, P., Preece, S., Golding, J., 1997. Magnetic fields from domestic appliances in the UK. Physics in Medicine and Biology. 42, 67-76. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Effects of 50 Hz sinusoidal magnetic fields and spark discharges on human lymphocytes in vitro. Bioelectrochemistry and Bioenergetics. 36, 15-22. Panagopoulos, D.J., Karabarbounis, A., Lioliousis, C., 2013. ELF alternating magnetic field decreases reproduction by DNA damage induction. Cell Biochemistry and Biophysics. 67, 703-716. Pandi-Perumal, S.,R., Srinivasan, ,V., Maestroni, G.J.,M., Cardinali, D.,P., Poeggeler, ,B., Hardeland, ,R., 2006. Melatonin. FEBS Journal. 273, 2813-2838. 170 Patruno, A., Tabrez, S., Pesce, M., Shakil, S., Kamal, M.A., Reale, M., 2015. Effects of extremely low frequency electromagnetic field (ELF-EMF) on catalase, cytochrome P450 and nitric oxide synthase in erythro-leukemic cells. Life Sciences. 121, 117-123. Patruno, A., Tabrez, S., Pesce, M., Shakil, S., Kamal, M.A., Reale, M., 2015. Effects of extremely low frequency electromagnetic field (ELF-EMF) on catalase, cytochrome P450 and nitric oxide synthase in erythro-leukemic cells. Life Sciences. 121, 117-123. Pedersen, C., Raaschou-Nielsen, O., Rod, N.H., Frei, P., Poulsen, A.H., Johansen, C., Schuz, J., 2014. Distance from residence to power line and risk of childhood leukemia: a population-based case-control study in Denmark. Cancer Causes & Control : CCC. 25, 171-177. Phillips, J.L., 1993. Effects of electromagnetic field exposure on gene transcription. Journal of Cellular Biochemistry. 51, 381-386. Phillips, J.L., Singh, N.P., Lai, H., 2009. Electromagnetic fields and DNA damage. Pathophysiology : The Official Journal of the International Society for Pathophysiology / ISP. 16, 79-88. Pitkevitch, V.A., Ivanov, V.K., Tsyb, A.F., Maksyoutov, M.A., Matiash, V.A., Shchukina, N.V., 1997. Exposure levels for persons involved in recovery operations after the Chernobyl accident. Statistical analysis based on the data of the Russian National Medical and Dosimetric Registry (RNMDR). Radiation and Environmental Biophysics. 36, 149-160. Polyzos, A., Parfett, C., Healy, C., Douglas, G., Yauk, C., 2006a. A single-molecule PCR approach to the measurement of induced expanded simple tandem repeat instability in vitro. Mutation Research. 594, 93-100. Polyzos, A., Parfett, C., Healy, C., Douglas, G.R., Yauk, C.L., 2006b. Instability of expanded simple tandem repeats is induced in cell culture by a variety of agents: N- Nitroso-N-ethylurea, benzo(a)pyrene, etoposide and okadaic acid. Mutation Research. 598, 73-84. Portier C, Wolfe M, editors. NIEHS. 1998. Assessment of Health Effects from Exposure to Power-Line Frequency Electric and Magnetic Fields. NIH publication no. 98-3981. [Accessed 13 August 2015]. Available: http://www.niehs.nih.gov/health/assets/docs_a_e/assessment_of_health_effects_fro m_exposure_to_powerline_frequency_electric_and_magnetic_fields.pdf. Powell, S. & McMillan, T.J., 1990. DNA damage and repair following treatment with ionizing radiation. Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology. 19, 95-108. Powell, S. Preece, A.W., Grainger, P., Golding, J., Kaune, W., 1996. Domestic magnetic field exposures in Avon. Physics in Medicine and Biology. 41, 71-81. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. & McMillan, T.J., 1990. DNA damage and repair following treatment with ionizing radiation. Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology. 19, 95-108. Preece, A.W., Grainger, P., Golding, J., Kaune, W., 1996. Domestic magnetic field exposures in Avon. Physics in Medicine and Biology. 41, 71-81. Preece, A.W., Kaune, W., Grainger, P., Preece, S., Golding, J., 1997. Magnetic fields from domestic appliances in the UK. Physics in Medicine and Biology. 42, 67-76. 171 Preston, D.L., Kusumi, S., Tomonaga, M., Izumi, S., Ron, E., Kuramoto, A., Kamada, N., Dohy, H., Matsuo, T., Matsui T [corrected to Matsuo,T.], 1994. Cancer incidence in atomic bomb survivors. Part III. Leukemia, lymphoma and multiple myeloma, 1950- 1987. Radiation Research. 137, S68-97. Preston, D.L., Kusumi, S., Tomonaga, M., Izumi, S., Ron, E., Kuramoto, A., Kamada, N., Dohy, H., Matsuo, T., Matsui T [corrected to Matsuo,T.], 1994. Cancer incidence in atomic bomb survivors. Part III. Leukemia, lymphoma and multiple myeloma, 1950- 1987. Radiation Research. 137, S68-97. Preston-Martin, S., Gurney, J.G., Pogoda, J.M., Holly, E.A., Mueller, B.A., 1996. Brain tumor risk in children in relation to use of electric blankets and water bed heaters. Results from the United States West Coast Childhood Brain Tumor Study. American Journal of Epidemiology. 143, 1116-1122. Pui, C.H., Robison, L.L., Look, A.T., 2008. Acute lymphoblastic leukaemia. Lancet. 371, 1030-1043. Qiu, C., Fratiglioni, L., Karp, A., Winblad, B., Bellander, T., 2004. Occupational Exposure to Electromagnetic Fields and Risk of Alzheimer's Disease. Epidemiology. 15, 687-694. Reiter, R.J., Tan, D.X., Mayo, J.C., Sainz, R.M., Leon, J., Czarnocki, Z., 2003. Melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans. Acta Biochimica Polonica. 50, 1129-1146. Repacholi, M., 2012. Concern that "EMF" magnetic fields from power lines cause cancer. The Science of the Total Environment. 426, 454-458. Ritz, C., Ruminski, W., Hougaard, K.S., Wallin, H., Vogel, U., Yauk, C.L., 2011. Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation. Mutation Research. 712, 55-58. Rodgers, C.T. & Hore, P.J., 2009. Chemical magnetoreception in birds: the radical pair mechanism. Proceedings of the National Academy of Sciences of the United States of America. 106, 353-360. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., Bonner, W.M., 1998. DNA double- stranded breaks induce histone H2AX phosphorylation on serine 139. The Journal of Biological Chemistry. 273, 5858-5868. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Roosli, M., Lortscher, M., Egger, M., Pfluger, D., Schreier, N., Lortscher, E., Locher, P., Spoerri, A., Minder, C., 2007. Mortality from neurodegenerative disease and exposure to extremely low-frequency magnetic fields: 31 years of observations on Swiss railway employees. Neuroepidemiology. 28, 197-206. Rothman K. J., Greenland S., Lash T. L. Validity in epidemiologic studies. In: Rothman K. J., editor; Greenland S., editor; Lash T. L., editor. Modern Epidemiology 3rd edition. Philadelphia, PA: Lippincott Williams & Wilkins; 2008c. pp. 128–147. Ruiz-Gomez, M.J. & Martinez-Morillo, M., 2009. Electromagnetic fields and the induction of DNA strand breaks. Electromagnetic Biology and Medicine. 28, 201-214. 172 Russell, L.B., 2004. Effects of male germ-cell stage on the frequency, nature, and spectrum of induced specific-locus mutations in the mouse. Genetica. 122, 25-36. Russell, L.B. & Matter, B.E., 1980. Whole-mammal mutagenicity tests: evaluation of five methods. Mutation Research. 75, 279-302. Russell, L.B. & Russell, W.L., 1996. Spontaneous mutations recovered as mosaics in the mouse specific-locus test. Proceedings of the National Academy of Sciences of the United States of America. 93, 13072-13077. Russell, W.L., 1989. Reminiscences of a mouse specific-locus test addict. Environmental and Molecular Mutagenesis. 14 Suppl 16, 16-22. Russell, W.L., 1951. X-ray-induced mutations in mice. Cold Spring Harbor Symposia on Quantitative Biology. 16, 327-336. Sadamoto, S., Suzuki, S., Kamiya, K., Kominami, R., Dohi, K., Niwa, O., 1994. Radiation induction of germline mutation at a hypervariable mouse minisatellite locus. International Journal of Radiation Biology. 65, 549-557. Saha, S., Woodbine, L., Haines, J., Coster, M., Ricket, N., Barazzuol, L., Ainsbury, E., Sienkiewicz, Z., Jeggo, P., 2014. Increased apoptosis and DNA double-strand breaks in the embryonic mouse brain in response to very low-dose X-rays but not 50 Hz magnetic fields. Journal of the Royal Society, Interface / the Royal Society. 11, 20140783. Sahl, J.D., Kelsh, M.A., Smith, R.W., Aseltine, D.A., 1994. Exposure to 60 Hz magnetic fields in the electric utility work environment. Bioelectromagnetics. 15, 21-32. Sahl, J.D., Kelsh, M.A., Greenland, S., 1993. Cohort and Nested Case-Control Studies of Hematopoietic Cancers and Brain Cancer among Electric Utility Workers. Epidemiology. 4, 104-114. Sahl, J., Mezei, G., Kavet, R., McMillan, A., Silvers, A., Sastre, A., Kheifets, L., 2002. Occupational magnetic field exposure and cardiovascular mortality in a cohort of electric utility workers. American Journal of Epidemiology. 156, 913-918. Santini, M.T., Rainaldi, G., Indovina, P.L., 2009. Cellular effects of extremely low frequency (ELF) electromagnetic fields. International Journal of Radiation Biology. 85, 294-313. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Saito, T., Nitta, H., Kubo, O., Yamamoto, S., Yamaguchi, N., Akiba, S., Honda, Y., Hagihara, J., Isaka, K., Ojima, T., Nakamura, Y., Mizoue, T., Ito, S., Eboshida, A., Yamazaki, S., Sokejima, S., Kurokawa, Y., Kabuto, M., 2010. Power-Frequency Magnetic Fields and Childhood Brain Tumors: A Case-Control Study in Japan. Journal of Epidemiology. 20, 54-61. Santini, M.T., Rainaldi, G., Indovina, P.L., 2009. Cellular effects of extremely low frequency (ELF) electromagnetic fields. International Journal of Radiation Biology. 85, 294-313. 173 Sasser, L.B., Morris, J.E., Miller, D.L., Rafferty, C.N., Ebi, K.L., Anderson, L.E., 1996. Exposure to 60 Hz magnetic fields does not alter clinical progression of LGL leukemia in Fischer rats. Carcinogenesis. 17, 2681-2687. Sastre, A., Cook, M.R., Graham, C., 1998. Nocturnal exposure to intermittent 60 Hz magnetic fields alters human cardiac rhythm. Bioelectromagnetics. 19, 98-106. Satoh, C., Takahashi, N., Asakawa, J., Kodaira, M., Kuick, R., Hanash, S.M., Neel, J.V., 1996. Genetic analysis of children of atomic bomb survivors. Environmental Health Perspectives. 104 Suppl 3, 511-519. Savitz, D.A., John, E.M., Kleckner, R.C., 1990. Magnetic field exposure from electric appliances and childhood cancer. American Journal of Epidemiology. 131, 763. Savitz, D.A., Liao, D., Sastre, A., Kleckner, R.C., Kavet, R., 1999. Magnetic field exposure and cardiovascular disease mortality among electric utility workers. American Journal of Epidemiology. 149, 135. Savitz, D.A., 1995. Exposure assessment strategies in epidemiological studies of health effects of electric and magnetic fields. The Science of the Total Environment. 168, 143- 153. Savitz, D.A., Checkoway, H., Loomis, D.P., 1998. Magnetic field exposure and neurodegenerative disease mortality among electric utility workers. Epidemiology (Cambridge, Mass.). 9, 398-404. Savitz, D.A., John, E.M., Kleckner, R.C., 1990. Magnetic field exposure from electric appliances and childhood cancer. American Journal of Epidemiology. 131, 763-773. Savitz, D.A. & Kaune, W.T., 1993. Childhood cancer in relation to a modified residential wire code. Environmental Health Perspectives. 101, 76-80. Savitz, D.A. & Loomis, D.P., 1995. Magnetic field exposure in relation to leukemia and brain cancer mortality among electric utility workers. American Journal of Epidemiology. 141, 123. Savitz, D.A., Wachtel, H., Barnes, F.A., John, E.M., Tvrdik, J.G., 1988. Case-control study of childhood cancer and exposure to 60-Hz magnetic fields. American Journal of Epidemiology. 128, 21-38. Scarfi, M.R., Sannino, A., Perrotta, A., Sarti, M., Mesirca, P., Bersani, F., 2005. Evaluation of genotoxic effects in human fibroblasts after intermittent exposure to 50 Hz electromagnetic fields: a confirmatory study. Radiation Research. 164, 270-276. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Schoenfeld, E.R., O'Leary, E.S., Henderson, K., Grimson, R., Kabat, G.C., Ahnn, S., Kaune, W.T., Gammon, M.D., Leske, M.C., EBCLIS Group, 2003. Electromagnetic fields and breast cancer on Long Island: a case-control study. American Journal of Epidemiology. 158, 47-58. 174 Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M., Lightfoot, S., Menzel, W., Granzow, M., Ragg, T., 2006. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology. 7, 3. Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M., Lightfoot, S., Menzel, W., Granzow, M., Ragg, T., 2006. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology. 7, 3. Schuz, J., Svendsen, A.L., Linet, M.S., McBride, M.L., Roman, E., Feychting, M., Kheifets, L., Lightfoot, T., Mezei, G., Simpson, J., Ahlbom, A., 2007. Nighttime exposure to electromagnetic fields and childhood leukemia: an extended pooled analysis. American Journal of Epidemiology. 166, 263-269. Searle, A.G., 1974. Mutation Induction in Mice. Advances in Radiation Biology. 4, 131- 207. Searle, A.G. & Beechey, C.V., 1981. The effects of radiation dose-rate and quality on the induction of dominant lethals in mouse spermatids. Mutation Research. 81, 403- 410. Selby, P.B., 1998. Discovery of numerous clusters of spontaneous mutations in the specific-locus test in mice necessitates major increases in estimates of doubling doses. Genetica. 102-103, 463-487. Shanks, M., Riou, L., Fouchet, P., Dubrova, Y.E., 2008. Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline. Mutation Research. 641, 58-60. Shelby, M.D., 1996. Selecting chemicals and assays for assessing mammalian germ cell mutagenicity. Mutation Research. 352, 159-167. Shen, Y.H., Shao, B.J., Chiang, H., Fu, Y.D., Yu, M., 1997. The effects of 50 Hz magnetic field exposure on dimethylbenz(alpha)anthracene induced thymic lymphoma/leukemia in mice. Bioelectromagnetics. 18, 360-364. Simko, M., Kriehuber, R., Weiss, D.G., Luben, R.A., 1998. Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines. Bioelectromagnetics. 19, 85-91. Simko, M. & Mattsson, M.O., 2004. Extremely low frequency electromagnetic fields as effectors of cellular responses in vitro: possible immune cell activation. Journal of Cellular Biochemistry. 93, 83-92. Singer, T.M. & Yauk, C.L., 2010. Germ cell mutagens: risk assessment challenges in the 21st century. Environmental and Molecular Mutagenesis. 51, 919-928. Singh, M. & Jadhav, H.R., 2014. Melatonin: functions and ligands. Drug Discovery Today. 19, 1410-1418. Singh, N. & Lai, H., 1998. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. 60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells. Mutation Research. 400, 313-320. 175 Slominski, R.M., Reiter, R.J., Schlabritz-Loutsevitch, N., Ostrom, R.S., Slominski, A.T., 2012. Melatonin membrane receptors in peripheral tissues: distribution and functions. Molecular and Cellular Endocrinology. 351, 152-166. Slominski, R.M., Reiter, R.J., Schlabritz-Loutsevitch, N., Ostrom, R.S., Slominski, A.T., 2012. Melatonin membrane receptors in peripheral tissues: distribution and functions. Molecular and Cellular Endocrinology. 351, 152-166. Slominski, A., Tobin, D.J., Zmijewski, M.A., Wortsman, J., Paus, R., 2008. Melatonin in the skin: synthesis, metabolism and functions. Trends in Endocrinology and Metabolism: TEM. 19, 17-24. Sobel, E., Davanipour, Z., Sulkava, R., Erkinjuntti, T., Wikstrom, J., Henderson, V.W., Buckwalter, G., Bowman, J.D., Lee, P.J., 1995. Occupations with exposure to electromagnetic fields: a possible risk factor for Alzheimer's disease. American Journal of Epidemiology. 142, 515. Somers, C.M., 2006. Expanded simple tandem repeat (ESTR) mutation induction in the male germline: lessons learned from lab mice. Mutation Research. 598, 35-49. Somers, C.M., McCarry, B.E., Malek, F., Quinn, J.S., 2004. Reduction of particulate air pollution lowers the risk of heritable mutations in mice. Science. 304, 1008-1010. Somers, C.M., Yauk, C.L., White, P.A., Parfett, C.L., Quinn, J.S., 2002. Air pollution induces heritable DNA mutations. Proceedings of the National Academy of Sciences of the United States of America. 99, 15904-15907. Sorahan, T. & Mohammed, N., 2014. Neurodegenerative disease and magnetic field exposure in UK electricity supply workers. Occupational Medicine. 64, 454. Southern, E.M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology. 98, 503-517. Southern, E.M., 1979. Measurement of DNA length by gel electrophoresis. Analytical Biochemistry. 100, 319-323. Stevens, R.G., 1987. Electric power use and breast cancer: a hypothesis. American Journal of Epidemiology. 125, 556-561. Stevens, R.G. & Davis, S., 1996. The melatonin hypothesis: electric power and breast cancer. Environmental Health Perspectives. 104 Suppl 1, 135-140. Stronati, L., Testa, A., Villani, P., Marino, C., Lovisolo, G.A., Conti, D., Russo, F., Fresegna, A.M., Cordelli, E., 2004. Absence of genotoxicity in human blood cells exposed to 50 Hz magnetic fields as assessed by comet assay, chromosome aberration, micronucleus, and sister chromatid exchange analyses. Bioelectromagnetics. 25, 41-48. Swanson, J. & Kaune, W.T., 1999. Comparison of residential power-frequency magnetic fields away from appliances in different countries. Bioelectromagnetics. 20, 244-254. 176 Tateno, H., Iijima, S., Nakanishi, Y., Kamiguchi, Y., Asaka, A., 1998. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. No induction of chromosome aberrations in human spermatozoa exposed to extremely low frequency electromagnetic fields. Mutation Research. 414, 31-35. Teepen, J.C. & van Dijck, J.A., 2012. Impact of high electromagnetic field levels on childhood leukemia incidence. International Journal of Cancer.Journal International Du Cancer. 131, 769-778. Tenforde, T.S., 1992. Biological interactions and potential health effects of extremely- low-frequency magnetic fields from power lines and other common sources. Annual Review of Public Health. 13, 173-196. Testa, A., Cordelli, E., Stronati, L., Marino, C., Lovisolo, G.A., Fresegna, A.M., Conti, D., Villani, P., 2004. Evaluation of genotoxic effect of low level 50 Hz magnetic fields on human blood cells using different cytogenetic assays. Bioelectromagnetics. 25, 613- 619. Timmel, C.R., Till, U., Brocklehurst, B., Mclauchlan, K.A., Hore, P.J., 1998. Effects of weak magnetic fields on free radical recombination reactions. Molecular Physics: An International Journal at the Interface between Chemistry and Physics. 95, 71-89. Tomenius, L., 1986. 50-Hz electromagnetic environment and the incidence of childhood tumors in Stockholm County. Bioelectromagnetics. 7, 191-207. Touitou, Y., Bogdan, A., Lambrozo, J., Selmaoui, B., 2006. Is melatonin the hormonal missing link between magnetic field effects and human diseases? Cancer Causes & Control : CCC. 17, 547-552. Touitou, Y., Lambrozo, J., Camus, F., Charbuy, H., 2003. Magnetic fields and the melatonin hypothesis: A study of workers chronically exposed to 50-Hz magentic fields. American Journal of Physiology. 53, R1529. Tynes, T. & Haldorsen, T., 1997. Electromagnetic fields and cancer in children residing near Norwegian high-voltage power lines. American Journal of Epidemiology. 145, 219- 226. Udroiu, I., Giuliani, L., Ieradi, L.A., 2010. Genotoxic properties of extremely low frequency electromagnetic fields. In: Giuliani, L., Soffritti, M. (eds). Non-thermal Effects and Mechanisms of Interaction between Electromagnetic Fields and Living Matter. National Institute for the Study and Control of Cancer and Environmental Diseases, Bernardino Ramazzini, Bologna: Mattioli 1885, 123-134. (UKCC) UK Childhood Cancer Study Investigators, 1999. Exposure to power-frequency magnetic fields and the risk of childhood cancer. UK Childhood Cancer Study Investigators. Lancet. 354, 1925-1931. (UKCC) UK Childhood Cancer Study Investigators, 2000. Childhood cancer and residential proximity to power lines. British Journal of Cancer. 83, 1573-1580. (UKCC) UK Childhood Cancer Study Investigators, 2000. Childhood cancer and residential proximity to power lines. British Journal of Cancer. 83, 1573-1580. 177 (UNSCEAR) United Nations Scientific Committee on the Effects of Atomic Radiation. 2001. Hereditary Effects of Radiation. [Online]. Report to the General Assembly, with Scientific Annex. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. New York: United Nations. [01/09/2014]. Available from: http://www.unscear.org/docs/reports/2001/2001Annex_pages%208-160.pdf Vanderstraeten, J., Verschaeve, L., Burda, H., Bouland, C., de Brouwer, C., 2012. Health effects of extremely low-frequency magnetic fields: reconsidering the melatonin hypothesis in the light of current data on magnetoreception. Journal of Applied Toxicology : JAT. 32, 952-958. Vanderstraeten, J., Verschaeve, L., Burda, H., Bouland, C., de Brouwer, C., 2012. Health effects of extremely low-frequency magnetic fields: reconsidering the melatonin hypothesis in the light of current data on magnetoreception. Journal of Applied Toxicology : JAT. 32, 952-958. Vena, J.E., Freudenheim, J.L., Marshall, J.R., Laughlin, R., Swanson, M., Graham, S., 1994. Risk of premenopausal breast cancer and use of electric blankets. American Journal of Epidemiology. 140, 974. Vena, J.E., Graham, S., Hellmann, R., Swanson, M., Brasure, J., 1991. Use of electric blankets and risk of postmenopausal breast cancer. American Journal of Epidemiology. 134, 180. Vergara, X., Kheifets, L., Greenland, S., Oksuzyan, S., Cho, Y., Mezei, G., 2013. Occupational Exposure to Extremely Low-Frequency Magnetic Fields and Neurodegenerative Disease: A Meta-Analysis. Journal of Occupational and Environmental Medicine. 55, 135-146. Vergara, X., Kheifets, L., Greenland, S., Oksuzyan, S., Cho, Y., Mezei, G., 2013. Occupational Exposure to Extremely Low-Frequency Magnetic Fields and Vergara, X., Mezei, G., Kheifets, L., 2015. Case-control study of occupational exposure to electric shocks and magnetic fields and mortality from amyotrophic lateral sclerosis in the US, 1991-1999. Journal of Exposure Science & Environmental Epidemiology. 25, 65-71. Vergnaud, G., Mariat, D., Apiou, F., Aurias, A., Lathrop, M., Lauthier, V., 1991. The use of synthetic tandem repeats to isolate new VNTR loci: cloning of a human hypermutable sequence. Genomics. 11, 135-144. Verkasalo, P.K., Pukkala, E., Hongisto, M.Y., Valjus, J.E., Jarvinen, P.J., Heikkila, K.V., Koskenvuo, M., 1993. Risk of cancer in Finnish children living close to power lines. BMJ. 307, 895-899. Verkasalo, P.K., Pukkala, E., Kaprio, J., Heikkila, K.V., Koskenvuo, M., 1996. Magnetic fields of high voltage power lines and risk of cancer in Finnish adults: nationwide cohort study. BMJ. 313, 1047-1051. Vijayalaxmi & Obe, G., 2005. Controversial cytogenetic observations in mammalian somatic cells exposed to extremely low frequency electromagnetic radiation: a review and future research recommendations. Bioelectromagnetics. 26, 412-430. Vijayalaxmi & Prihoda, T.J., 2009. Genetic damage in mammalian somatic cells exposed to extremely low frequency electro-magnetic fields: a meta-analysis of data from 87 publications (1990-2007). International Journal of Radiation Biology. 85, 196-213. Vijayalaxmi & Prihoda, T.J., 2009. Yauk, C.L., Aardema, M.J., Benthem, J., Bishop, J.B., Dearfield, K.L., DeMarini, D.M., Dubrova, Y.E., Honma, M., Lupski, J.R., Marchetti, F., Meistrich, M.L., Pacchierotti, F., Stewart, J., Waters, M.D., Douglas, G.R., 2015. Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays. Mutation Research.Genetic Toxicology and Environmental Mutagenesis. 783, 36-54. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Genetic damage in mammalian somatic cells exposed to extremely low frequency electro-magnetic fields: a meta-analysis of data from 87 publications (1990-2007). International Journal of Radiation Biology. 85, 196-213. 178 Vilarino-Guell, C., Smith, A.G., Dubrova, Y.E., 2003. Germline mutation induction at mouse repeat DNA loci by chemical mutagens. Mutation Research. 526, 63-73. Vilarino-Guell, C., Smith, A.G., Dubrova, Y.E., 2003. Germline mutation induction at mouse repeat DNA loci by chemical mutagens. Mutation Research. 526, 63-73. Villarini, M., Moretti, M., Scassellati-Sforzolini, G., Boccioli, B., Pasquini, R., 2006. Effects of co-exposure to extremely low frequency (50 Hz) magnetic fields and xenobiotics determined in vitro by the alkaline comet assay. The Science of the Total Environment. 361, 208-219. Voutounou, M., Glen, C.D., Dubrova, Y.E., 2012. The effects of methyl-donor deficiency on mutation induction and transgenerational instability in mice. Mutation Research. 734, 1-4. Walleczek, J., Shiu, E.C., Hahn, G.M., 1999. Increase in radiation-induced HPRT gene mutation frequency after nonthermal exposure to nonionizing 60 Hz electromagnetic fields. Radiation Research. 151, 489-497. Wang, Z., Gerstein, M., Snyder, M., 2009. RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews.Genetics. 10, 57-63. Wartenberg, D., 1998. Residential magnetic fields and childhood leukemia: a meta- analysis. American Journal of Public Health. 88, 1787-1794. Wartenberg, D., Ehrlich, R., Lilienfeld, D., 1994. Environmental tobacco smoke and childhood asthma: comparing exposure metrics using probability plots. Environmental Research. 64, 122-135. Weitzmann, M.N., Woodford, K.J., Usdin, K., 1998. The mouse Ms6-hm hypervariable microsatellite forms a hairpin and two unusual tetraplexes. The Journal of Biological Chemistry. 273, 30742-30749. Welker, H.A., Semm, P., Willig, R.P., Commentz, J.C., Wiltschko, W., Vollrath, L., 1983. Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content of the rat pineal gland. Experimental Brain Research. 50, 426-432. Wertheimer, N. & Leeper, E., 1982. Adult cancer related to electrical wires near the home. International Journal of Epidemiology. 11, 345-355. Wertheimer, N. & Leeper, E., 1979. Electrical wiring configurations and childhood cancer. American Journal of Epidemiology. 109, 273-284. (WHO) World Health Organization. 2007. Extremely Low Frequency Fields. [Online]. Environmental Health Criteria Monograph No.238. WHO Spain: WHO Press. [26/01/2015]. Available from: http://www.who.int/peh- emf/publications/elf_ehc/en/index.html. Wilbourn, J., Haroun, L., Heseltine, E., Kaldor, J., Partensky, C., Vainio, H., 1986. Response of experimental animals to human carcinogens: an analysis based upon the IARC Monographs programme. Carcinogenesis. 7, 1853-1863. 179 Wilson, B.W., Lee, G.M., Yost, M.G., Davis, K.C., Heimbigner, T., Buschbom, R.L., 1996. Magnetic field characteristics of electric bed-heating devices. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. Bioelectromagnetics. 17 174-179. Wilson, B.W., Lee, G.M., Yost, M.G., Davis, K.C., Heimbigner, T., Buschbom, R.L., 1996. Magnetic field characteristics of electric bed-heating devices. Bioelectromagnetics. 17, 174-179. Wilson, J.W., Haines, J., Sienkiewicz, Z., Dubrova, Y.E., 2015. The effects of extremely low frequency magnetic fields on mutation induction in mice. Mutation Research. 773, 22-26. Winker, R., Ivancsits, S., Pilger, A., Adlkofer, F., Rudiger, H.W., 2005. Chromosomal damage in human diploid fibroblasts by intermittent exposure to extremely low- frequency electromagnetic fields. Mutation Research. 585, 43-49. Witt, K.L. & Bishop, J.B., 1996. Mutagenicity of anticancer drugs in mammalian germ cells. Mutation Research. 355, 209-234. Wolf, F.I., Torsello, A., Tedesco, B., Fasanella, S., Boninsegna, A., D'Ascenzo, M., Grassi, C., Azzena, G.B., Cittadini, A., 2005. 50-Hz extremely low frequency electromagnetic fields enhance cell proliferation and DNA damage: possible involvement of a redox mechanism. Biochimica Et Biophysica Acta. 1743, 120-129. Yaguchi, H., Yoshida, M., Ding, G.R., Shingu, K., Miyakoshi, J., 2000. Increased chromatid-type chromosomal aberrations in mouse m5S cells exposed to power-line frequency magnetic fields. International Journal of Radiation Biology. 76, 1677-1684. Yaguchi, H., Yoshida, M., Ejima, Y., Miyakoshi, J., 1999. Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells. Mutation Research. 440, 189-194. Yasui, M., Kikuchi, T., Ogawa, M., Otaka, Y., Tsuchitani, M., Iwata, H., 1997. Carcinogenicity test of 50 Hz sinusoidal magnetic fields in rats. Bioelectromagnetics. 18, 531-540. Yauk, C., Polyzos, A., Rowan-Carroll, A., Somers, C.M., Godschalk, R.W., Van Schooten, F.J., Berndt, M.L., Pogribny, I.P., Koturbash, I., Williams, A., Douglas, G.R., Kovalchuk, O., 2008. Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location. Proceedings of the National Academy of Sciences of the United States of America. 105, 605-610. Yauk, C.L., 2004. Advances in the application of germline tandem repeat instability for in situ monitoring. Mutation Research. 566, 169-182. Yauk, C.L., Aardema, M.J., Benthem, J., Bishop, J.B., Dearfield, K.L., DeMarini, D.M., Dubrova, Y.E., Honma, M., Lupski, J.R., Marchetti, F., Meistrich, M.L., Pacchierotti, F., Stewart, J., Waters, M.D., Douglas, G.R., 2015. Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays. Mutation Research.Genetic Toxicology and Environmental Mutagenesis. 783, 36-54. 180 Yauk, C.L., Berndt, M.L., Williams, A., Rowan-Carroll, A., Douglas, G.R., Stampfli, M.R., 2007. Mainstream tobacco smoke causes paternal germ-line DNA mutation. Cancer Research. 67, 5103-5106. Yauk, C.L., Dubrova, Y.E., Grant, G.R., Jeffreys, A.J., 2002. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus. Mutation Research. 500, 147-156. Yauk, C.L., Fox, G.A., McCarry, B.E., Quinn, J.S., 2000. Induced minisatellite germline mutations in herring gulls (Larus argentatus) living near steel mills. Mutation Research. 452, 211-218. Yauk, C.L. & Quinn, J.S., 1996. Multilocus DNA fingerprinting reveals high rate of heritable genetic mutation in herring gulls nesting in an industrialized urban site. Proceedings of the National Academy of Sciences of the United States of America. 93, 12137-12141. Yauk, C. & Polyzos, A., 2005. Tandem repeat DNA: applications in mutation analysis. Environmental Mutagen Research. 27, 93-98. Yokus, B., Cakir, D.U., Akdag, M.Z., Sert, C., Mete, N., 2005. Oxidative DNA damage in rats exposed to extremely low frequency electromagnetic fields. Free Radical Research. 39, 317-323. Yoon, H.E., Lee, J.S., Myung, S.H., Lee, Y.S., 2014. Increased gamma-H2AX by exposure to a 60-Hz magnetic fields combined with ionizing radiation, but not hydrogen peroxide, in non-tumorigenic human cell lines. International Journal of Radiation Biology. 90, 291-298. Zaffanella, L.E. (1992). Survey of residential magnetic field sources. EPRI Report. United States. http://www.epri.com/abstracts/Pages/ProductAbstract.aspx?ProductId=TR- 100194, Last visited 8/12/2015 Zhao, L., Liu, X., Wang, C., Yan, K., Lin, X., Li, S., Bao, H., Liu, X., 2014. Magnetic fields exposure and childhood leukemia risk: a meta-analysis based on 11,699 cases and 13,194 controls. Leukemia Research. 38, 269-274. Zhao, L., Liu, X., Wang, C., Yan, K., Lin, X., Li, S., Bao, H., Liu, X., 2014. Magnetic fields exposure and childhood leukemia risk: a meta-analysis based on 11,699 cases and 13,194 controls. Leukemia Research. 38, 269-274. Zheng, N., Monckton, D.G., Wilson, G., Hagemeister, F., Chakraborty, R., Connor, T.H., Siciliano, M.J., Meistrich, M.L., 2000a. Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy. Environmental and Molecular Mutagenesis. 36, 134-145. Zheng, T., Holford, T.R., Mayne, S.T., Owens, P.H., Zhang, B., Boyle, P., Carter, D., Ward, B., Zhang, Y., Zahm, S.H., 2000b. Exposure to electromagnetic fields from use of electric blankets and other in-home electrical appliances and breast cancer risk. American Journal of Epidemiology. 151, 1103-1111. Zheng, T., Holford, T.R., Mayne, S.T., Owens, P.H., Zhang, B., Boyle, P., Carter, D., Ward, B., Zhang, Y., Zahm, S.H., 2000b. Exposure to electromagnetic fields from use of electric blankets and other in-home electrical appliances and breast cancer risk. American Journal of Epidemiology. 151, 1103-1111. Metcalf, D., 1988. The molecular control of blood cells. Harvard University Press. 181 Zhou, H., Chen, G., Chen, C., Yu, Y., Xu, Z., 2012. Association between extremely low- frequency electromagnetic fields occupations and amyotrophic lateral sclerosis: a meta-analysis. PloS One. 7, 48354. Zhou, Y., Liu, Y., Qiu, W., Zeng, J., Chen, X., Zhou, H., Li, A., Zhou, J., 2009. Exposure to residential indoor air induces heritable DNA mutations in mice. Journal of Toxicology and Environmental Health.Part A. 72, 1561-1566. Zmyslony, M., Palus, J., Jajte, J., Dziubaltowska, E., Rajkowska, E., 2000. DNA damage in rat lymphocytes treated in vitro with iron cations and exposed to 7 mT magnetic fields (static or 50 Hz). Mutation Research. 453, 89-96. Zmyslony, M., Rajkowska, E., Mamrot, P., Politanski, P., Jajte, J., 2004. The effect of weak 50 Hz magnetic fields on the number of free oxygen radicals in rat lymphocytes in vitro. Bioelectromagnetics. 25, 607-612. 182
https://openalex.org/W2973163466	https://rua.ua.es/dspace/bitstream/10045/96151/1/2019_Galiano-Garrigos_etal_Sustainability.pdf	English	null	Energy Efficiency and Economic Viability as Decision Factors in the Rehabilitation of Historic Buildings	Sustainability	2,019	cc-by	14,929	Received: 2 August 2019; Accepted: 6 September 2019; Published: 10 September 2019 Abstract: The restoration of historical buildings often implies a change in the main use of the building so that it can once again become a part of people’s lives. Among the interventions needed to adapt the buildings to their new purpose, improving the energy performance is always a challenge due to their particular construction solutions and the inﬂuence that these improvements can have on their protected elements. The regulations in force in European Union (EU) member states leave a gap in how the energy performance evaluations in these types of buildings can be deﬁned, and even exclude them from the process. However, rehabilitation of buildings is always seen as an opportunity, because it allows the building to once again be useful to society and play an important role in people’s lives. At the same time, it can also improve their performance and allow beneﬁts to be gained from their use through a reduction in maintenance costs. In the rehabilitation process, the economic viability of the renovation plays a fundamental role which must be compared, in the case of protected buildings, to its impact on the architecture of the building. Since 2002, the EU has issued directives with the aim that countries should deﬁne objective methods to improve the energy performance of buildings and, in recent times, methods that demonstrate the amortization of such improvements. Within the process of implementing the new methodologies adapted to the EPBD, Spain was one of the last EU countries to deﬁne a process for the energy assessment of existing buildings, introducing an analysis of the economic viability of the construction improvements suggested in the process. The objective of this research was to describe the decision-making process during the evaluation of the feasibility of introducing construction improvements to the energy performance of two catalogued historic buildings located in a warm climate. The estimated energy consumption was evaluated, the net present value (NPV) and the payback period of the investment calculated, and the results obtained were compared with the real energy consumption. At the end of the process, it can be said that the methodologies adopted in Spain offer results that can lead designers to make wrong decisions that may affect the protected heritage values of these buildings. Keywords: rehabilitation; energy efﬁciency; economic viability; life cycle cost; NPV Energy Efﬁciency and Economic Viability as Decision Factors in the Rehabilitation of Historic Buildings Antonio Galiano-Garrigós 1,* , Ángel González-Avilés 1, Carlos Rizo-Maestre 1 and MªDolores Andújar-Montoya 2 Antonio Galiano-Garrigós 1,* , Ángel González-Avilés 1, Carlos Rizo-Maestre 1 and MªDolores Andújar-Montoya 2 1 Department of Architectural Constructions, University of Alicante, 03690 Alicante, Spain 2 Building Sciences and Urbanism Department, University of Alicante, 03690 Alicante, Spain * Correspondence: antonio.galiano@ua.es and MªDolores Andújar-Montoya 2 1 Department of Architectural Constructions, University of Alicante, 03690 Alicante, Spain 2 Building Sciences and Urbanism Department, University of Alicante, 03690 Alicante, Spain * Correspondence: antonio.galiano@ua.es 1 Department of Architectural Constructions, University of Alicante, 03690 Alicante, Spain 2 Building Sciences and Urbanism Department, University of Alicante, 03690 Alicante, Spain * Correspondence: antonio.galiano@ua.es Received: 2 August 2019; Accepted: 6 September 2019; Published: 10 September 2019 sustainability sustainability sustainability sustainability Sustainability 2019, 11, 4946; doi:10.3390/su11184946 www.mdpi.com/journal/sustainability 1. Introduction The overall value of a building is not solely limited to its economic, artistic, technical, or historical merits. A building forms part of an urban network where many activities take place at the same time, creating strong links to the subjective part of the inhabitants’ lives. Some of the oldest or most used buildings become anchor points for those who live close by, and reinforce the need to maintain history and tradition to create a healthy urban environment. Historical legacy is necessary for citizens’ lives and allows them to face challenges in the future. The loss of a building with historical, cultural, and artistic value is not only a material fact, but more importantly, results in the loss of Sustainability 2019, 11, 4946; doi:10.3390/su11184946 www.mdpi.com/journal/sustainability 2 of 27 Sustainability 2019, 11, 4946 collective identity, which is never to be restored. When these buildings reach the end of their lifespan, sometimes a change of use needs to be integrated in the urban network again. The adaptation of the building to the new purpose implies the maintenance of the building’s cultural value, as well as the introduction of construction and conditioned systems that were not used before. The evaluation of the viability of these construction improvements must be done from two points of view—economic viability, and the impact on the architecturally protected elements. The analysis of the economic viability of a rehabilitation operation to stop the abandonment and ruin of a building and to be able to update its operation implies that a cost is established for the investment that must include the study costs, the construction costs, and the future operational costs that will occur during the new life cycle of the building after rehabilitation. The technical decisions used for the renovation may lead to an increase in construction costs, but at the same time, to long-term savings in the maintenance or operation of the building. This variable allows a comparative analysis of the possible advantages of using higher-quality products, which are initially more expensive compared to a less signiﬁcant investment in terms of subsequent costs. When the viability of an intervention is measured based on energy savings, the savings achieved in energy consumption must be signiﬁcant during the useful life of the building since energy savings are directly related to ﬁnancial savings that could also be promoted as an indicator of sustainability. 1. Introduction Currently, within the initiatives of the European Union (EU) is the energy rehabilitation of existing buildings as a measure to reduce the energy bill and improve user comfort. In the EU, 38.7% of energy consumption is accounted for by residential, commercial, and institutional buildings [1]. In terms of electricity consumption, this percentage is close to 70% [2]. These ﬁgures take on importance when the built inventory shows signs of aging, as only 1% of the buildings have been built since 2006 [3]. Among the built inventory that is the object of renovation work are the buildings that, due to their architectural characteristics, are catalogued and subject to protection. These buildings are often outside the scope of the regulations for the evaluation of the energy performance of buildings [4], and it is up to the architect and the competent administration to deﬁne the scope of the renovation. An analysis of the investment in this type of building then becomes a complex process where there is no standardized method and where subjective factors come into play, limiting decision-making and the adoption of known and viable solutions. At the same time, there is not a uniﬁed method which can be used to approach a building restoration, as every building has its speciﬁc protection level and uniqueness. Therefore, there is a chance to explore the viability of interventions over catalogued buildings where an improvement in energy performance is planned and where there are not ofﬁcial methods to be applied over the process. For the explanation of the present investigation, the article is divided as follows: Section 2 introduces the regulatory framework. Related research is analyzed at Section 3. Two case studies are presented in Section 4. Section 5 presents the research methodology. The results obtained in the energy analysis of buildings are described in Section 6. Section 7 describes the results obtained from the calculation of the net present value (NPV). Section 8 describes decision-making at the end of the project. The discussion of the results and comparison with the actual performance of the building are analyzed in Section 9. Finally, Section 10 describes the conclusions drawn. 2.2. Evaluation of the Energy Performance of Existing Buildings The process of adaptation of the different EU member countries to the EPBD mainly occurred between 2005 and 2013. Those particular countries located in southern Europe, where a warm climate prevails in a large part of its territory and the widespread use of air-conditioning systems in private homes has been relatively recent, were the last to adapt to the EPBD. Among these countries is Spain, which, in 2013, adopted a methodology for evaluating the energy performance of existing buildings [16]. Within the scope of the approved methods, both the EPBD and the most ofﬁcial methods provide that, in major interventions (1000 m2 is deﬁned as the limit quantity), energy efﬁciency can be improved if the intervention is technically, functionally, and economically feasible. However, the directive allows for certain exceptions. Interventions in heritage and historical buildings with a certain degree of protection are exempted from compliance with the EPBD if the intervention could unacceptably alter the monument. 2.1. Assessing the Energy Performance of Buildings in the EU 2.1. Assessing the Energy Performance of Buildings in the EU In 2002, the EU published the Energy Performance Building Directive (EPBD) [5] as the tool that is described in the Agreement of the European Union [6] for promoting initiatives to reduce energy costs. In the EPBD, the EU obliges the EU members to adopt measures for improving the energy performance of buildings. Previously, the EU had designated the European Committee for Normalization (CEN) [7] as responsible for developing the general regulations in Europe. Therefore, in 2006, the CEN provided a group of regulations for evaluating the energy performance of buildings and deﬁning 3 of 27 Sustainability 2019, 11, 4946 indicators of the sustainability and efﬁciency of buildings. These documents were gathered under an “umbrella document” [8] that includes UNE EN 13790 [9], entitled “Methodology for evaluating energy performance in buildings, energy for heating and cooling in buildings”; EN 15603 [10], entitled “Overall energy use and deﬁnition of energy ratings”; and EN 15217 [11], entitled “Energy efﬁciency in buildings. Methodologies to express energy efﬁciency and energy certiﬁcations in buildings ” [11]. g g p gy y gy g Despite the EU’s efforts to homogenize the way member countries treat the assessment of the energy performance of buildings, EU analyzes have shown a wide variety of situations that dispute the claim that there is a standard procedure in Europe [12,13]. This situation is further complicated by the analysis of the methodologies adopted in southern European countries where warm climates prevail and, therefore, experience higher energy needs in summer. The results obtained when analysing buildings offer random situations, as the use of natural ventilation, thermal inertia, and the need for a more permeable envelope make the assessment more complex, and no adequate response can be obtained from ofﬁcial methods [14]. Neither has the appearance of new simulation tools linked to Building Information Modelling (BIM) work environments managed to improve the decision-making process, since the particularities of the performance of buildings in hot climates mean that the results are not comparable with the real performance of the building [15]. 2.3. EPBD Update The analysis of the various methods demonstrated that most of the countries used the EN ISO 13790 as a basis for calculating energy need in buildings [4]. Moreover, there is a lack of homogeneity in the way the methods evaluate the building geometry [13] in the material properties and constructive systems, which depend on software databases and the heat production systems. The methods for calculating the energy certiﬁcation are mainly based on the EN 15217 standard with the indicators that are deﬁned in EN 15603. Most of the EU members certify residential buildings via a ﬁxed reference method, while tertiary buildings, with fewer buildings for comparison, used to be certiﬁed via a variable method, although some authors state that this method causes designers to adopt incorrect decisions [17]. Nevertheless, the EU member governments’ priorities inﬂuence the limits and the method to be used [18]. In 2010, the EU published an update [19], in which the diversity and lack of unity among methodologies for evaluating the energy performance of buildings throughout Europe were discussed. It also identiﬁed some key ways to improve the methods, such as considering thermal inertia in the procedures, especially in the methods to be applied in countries with warm climates. In the other recommendations, the most important change came from the need to develop a recovery-of-investment method for the adopted energy-saving measures in the buildings. Sustainability 2019, 11, 4946 4 of 27 This strategy was reinforced in 2018 when the EU forced the EU members to design methods to facilitate a proﬁtable economic transformation of existing buildings with an almost zero-energy supply [20]. Therefore, since 2010, the EU has promoted the veriﬁcation of the economic feasibility of interventions in buildings to improve their energy performance, and since 2018, this initiative has reached existing buildings. However, at the EU level, there is no minimum level of return on investment for any type of building [21]. 2.4. Calculation of the NPV To complement Directive 2010/31/UE, the UE published the Delegated Regulation (UE) 244/2012 [22] that developed the above-mentioned directive and obliges the EU members to deﬁne methods for evaluating energy performance in buildings that could guarantee the economic efﬁciency of the adopted measures. These regulations had to be adopted by all EU members by the beginning of 2013. This regulation introduced the NPV as a comparative value of construction improvements. The purpose of the NPV was to determine the viability of an investment on the basis of the result obtained by comparing the investment initially made with the savings obtained, also taking into account the maintenance carried out over time. A positive NPV indicates a proﬁtable investment that increases as the NPV increases. The regulation offers two methods of global cost calculations as the NPV for any type of improvement intervention: the global cost at the ﬁnancial level, and the global cost at the macroeconomic level. The difference between the two methods focuses on the exclusion in the second method of all applicable taxes and subsidies, and on the consideration instead of the costs of greenhouse gas emissions. The overall cost at the ﬁnancial level is calculated using the following formula: Cg (τ) = CI + ∑ j τ ∑ i=1 (Ca (j) ∗Rd (i)) −Vf,τ(j) ! 2.4. Calculation of the NPV • τ is the calculation period; • τ is the calculation period; • τ is the calculation period; C ( ) i th ll t ( f i t th i iti l d i th l l ti i d) p • Cg (τ) is the overall cost (referring to the initial year τ0 during the calculation period); • CI are the initial investment costs of the measure or set of measures j; j • Ca(j) is the annual cost during year i of the measure or set of measures j; • Vf,τ (j) is the residual value of the measure or set of measures j at the end of the calculation period (updated to the initial value τ0); p • Rd is the update factor applicable to each year based on the update rate r, which must be deﬁned by each member state, calculated according to the formula: p • Rd is the update factor applicable to each year based on the update rate r, which must be deﬁned by each member state, calculated according to the formula: Rd (p) = 1 1 + r 100 p Rd (p) = 1 1 + r 100 p • where p is the number of years from the initial year and r is the actual update rate. • where Cc,i (j) is the carbon cost of the measure or set of measures j during year i. • where Cc,i (j) is the carbon cost of the measure or set of measures j during year i. Complementary to the regulation, the EU publishes a series of reports that deﬁne the conditions for an optimal calculation of the global cost by deﬁning the future energy prices in each country [23]. In addition, it deﬁnes the amortization periods for each type of construction improvement—for example, the recovery periods for thermal insulation, windows, carpentry, and installations. These reports also set out the cost of CO2 emissions, the annual discount rate, and the ﬁnal energy conversion factors into primary energy, and energy conversion into CO2 emissions. As seen in the formulas, the cost optimization methodology is, in the ﬁrst moment, technologically neutral, as it does not favour any technological solution to the detriment of others. However, in deep analysis of both methods and the report, those technological solutions with low maintenance costs and with low CO2 emissions are expected to obtain better results. Maintenance costs act negatively in the calculation through the analyzed period, while CO2 emission costs grows because of political strategies. • where p is the number of years from the initial year and r is the actual update rate. • where p is the number of years from the initial year and r is the actual update rate. In the case of a construction renovation to improve the energy efﬁciency of an existing building being studied, it is necessary to take the initial year as the moment in which work begins; use the calculation period of depreciation indicated by each country; and consider the types of costs and energy prices in the long-term. The regulation also completes the methodological framework by deﬁning an estimated life cycle of buildings, setting update rates, and deﬁning the costs of energy carriers, products, systems, maintenance, operation, and labor. At the energy level, the regulation requires member countries to deﬁne primary energy conversion factors and an estimate of future energy prices. For calculation Sustainability 2019, 11, 4946 5 of 27 purposes, the regulation deﬁnes a calculation period of 30 years for public and residential buildings and 20 years for commercial and non-residential buildings. purposes, the regulation deﬁnes a calculation period of 30 years for public and residential buildings and 20 years for commercial and non-residential buildings. As an alternative, we found the global cost at the macroeconomic level. As indicated above, this cost must consider the impact of greenhouse gas emissions and is calculated using the following formula: Cg (τ) = CI + ∑ j τ ∑ i=1 (Ca (j) ∗Rd (i)) + Cc,i (j)) −Vf,τ(j) ! 2.5. Regulatory Situation in Spain This tool, based on the DOE-2 calculation engine, has proved difﬁcult to manage and is characterized by offering results that do not help in the decision-making process during the project phase because it favors buildings with complicated geometry over buildings with an efﬁcient design [17]. At the same time, this program follows the line of methods approved in other southern European countries that do not offer robust results in the process of studying construction improvements, and hampers the decision-making process by introducing construction improvements in the design [30]. Uniﬁed LIDER-CALENER Tool (HULC). This tool, based on the DOE-2 calculation engine, has proved difﬁcult to manage and is characterized by offering results that do not help in the decision-making process during the project phase because it favors buildings with complicated geometry over buildings with an efﬁcient design [17]. At the same time, this program follows the line of methods approved in other southern European countries that do not offer robust results in the process of studying construction improvements, and hampers the decision-making process by introducing construction improvements in the design [30]. The process of evaluation and energy certiﬁcation of existing buildings is performed from two tools called CE3 and CE3X, both developed under the calculation engine, DOE-2. The ﬁrst one is characterized by its simplicity and limitations in the introduction of building information. The second tool allows introduction of the geometry of the building, but without allowing its modeling. At the same time, CE3X allows, for the ﬁrst time in Spain, the possibility of studying construction improvements to improve the energy performance of buildings, as well as analysis of the return on investment over time. The process for justifying energy demand in residential buildings is deﬁned, according to DB HE, by a maximum energy need per m2 of surface area. In the case of other-use buildings, the energy demand is made through a percentage improvement in the energy need of a reference building, called the object building, which the calculation program deﬁnes. This object building is deﬁned as a building that has the same geometry and orientation and that at least meets the energy demand parameters of the climatic zone. For the energy certiﬁcation scale, Spain adopted a comparative framework between the options offered by EN 15217. Thus, certiﬁcation is made after a comparison between buildings that have the same use and are in the same climate. 2.5. Regulatory Situation in Spain As mentioned above, the process of adopting national methodologies for assessing the energy performance of buildings in southern Europe is slower than in northern countries. This situation also extends to the evaluation of existing buildings, with Spain becoming one of the last countries to enact a law accommodating this certiﬁcation in 2013. Spain has not been a pioneer in the implementation of measures to reduce energy consumption in its construction market. The ﬁrst regulation published in Spain that sought to improve the energy performance of residential buildings was the NBE CT-79 [24], which deﬁned a method based on the calculation of a building form factor, combined with a minimum transmittance value (U-Value). The adaptation to the EPBD 2002/91/CE in Spain was carried out through the Technical Building Code [25], which has its origin in the Law on Building Management [26]. Within this document, the Basic Energy Saving Document (DB HE) (Government of Spain, 2017) [27] is the part of the CTE that deﬁnes the requirements for evaluating the energy performance of buildings. This document deﬁnes its scope in all types of new buildings and in existing buildings subject to major actions, but it also excludes protected buildings, leaving the improvement measures to be taken at the discretion of the designer and administration responsible for the conservation of the buildings. This exclusion follows the line of other methodologies approved in Europe where the degree to which the monument is affected by the construction improvements can condition its application. To obtain energy certiﬁcation for new buildings, Spain published its ﬁrst law in 2007 (Spain, 2007) [28], although a fairly long moratorium period was allowed until the start-up of the certiﬁcate registration ofﬁces. In 2013, there was an update of the procedure for the energy assessment and certiﬁcation of buildings, and a procedure was deﬁned for the ﬁrst time for the energy certiﬁcation of existing buildings, becoming mandatory in the processes of renting or selling existing buildings. In 2017, the procedures were updated, including the limit values for obtaining the different energy certiﬁcations [29]. Until September 2018, the process for evaluating the energy performance of new buildings was obligatorily carried out through a tool developed by the Spanish government, called LIDER, and energy certiﬁcation was achieved through a tool called CALENER, which, in 2013 became the 6 of 27 Sustainability 2019, 11, 4946 Uniﬁed LIDER-CALENER Tool (HULC). 2.5. Regulatory Situation in Spain However, as it happens while calculating the energy demand, a distinction is made between buildings intended for housing and buildings for other uses. This separation occurs because it is impossible to make a comparison between buildings in which there is a large difference in the envelope and in the size of the air-conditioning installations. Residential buildings are compared with the performance of other buildings in the built inventory within a period. In the case of non-residential buildings, as there are not enough repetitive patterns to establish homogeneous groups of buildings for comparison, it was decided to compare the building subject to certiﬁcation with a ﬁctitious building, called the reference building, which must have the same geometry, orientation, uses and zoning, solar control elements, and construction that meets, at a minimum, the energy demand for the purposes of air-conditioning equipment and the production of domestic hot water. This system has proved to be imprecise, given the difﬁculties encountered by architects in interpreting the results of the program and, therefore, in decision-making in the project phase [17]. 3. Related Literature Research shows that the lack of interest in efﬁcient buildings, as in Spain, is related to a lack of importance of energy performance in terms of investment [31], as well as to a lack of information and adequate regulations [32]. This situation conﬂicts with the forecasts made by the United Nations when it stated that there has been an increase of 5 EJ in the ﬁnal energy demand of buildings between 2010 and 2016. This is a consequence of the fact that energy efﬁciency efforts have not kept pace with the increase in the surface area used. It should be borne in mind that more than half of the buildings that will exist in 40 years will be constructed over the next 20 years, and two-thirds of them will be in countries that currently do not have adequate energy building codes. Energy consumption and CO2 emissions account for 36% of global ﬁnal energy use and 39% of energy-related CO2 emissions when upstream power generation is included. Progress toward sustainable buildings and construction is advancing, but improvements are still not keeping up with a growing building sector and rising demand for energy services. The energy intensity per square meter (m2 ) of the global buildings sector needs to improve, on average, by 30% by 2030 (compared to 2015) to be on track to meet global climate ambitions set forth in the Paris Agreement [33]. The technical decisions made for rehabilitation can lead to an increase in the construction costs and long-term savings in the maintenance or operation of a building. This variable allows a comparative analysis of the possible advantages of using higher-quality products, which are initially more expensive compared to a less signiﬁcant investment in terms of subsequent costs. In public residential buildings, it has been determined that seeking lower energy savings and emissions reductions has the relevant advantage of being sustainable from a ﬁnancial point of view, compared to an intervention that seeks maximum energy savings [34]. In residential buildings, taking into account the energy contained in the materials, rehabilitation with the substitution of windows, improvement of insulation, and substitution of installations can lead to energy and pollution savings of 60% [35]. 2.6. Objective of the Research The adoption of non-transparent methods for evaluating the energy performance of buildings has made decision-making for a renovation difﬁcult in Spain. The lack of information in the project’s process about the savings that will be obtained may make the promotor reconsider the need to renovate an existing building. This situation may worsen when such construction improvements can inﬂuence the architectural protection of the building. The lack of a scope, and therefore of a speciﬁc methodology for listed buildings makes it necessary to establish a relationship between the impact of the construction improvements on the architecture and the energy savings that these may have. At the same time, it is necessary to evaluate the performance that the methods for evaluating the energy performance of new and existing buildings may have on this type of building. It is therefore in the interest of this research to evaluate the recovery of investment in construction that improves the energy performance of existing buildings that also have some type of protection, 7 of 27 Sustainability 2019, 11, 4946 and to evaluate the inﬂuence of current methodologies in Spain in the decision-making process during the project drafting phase. 3. Related Literature In addition, several studies indicate that the rehabilitation of a building represents potential energy savings compared to other types of interventions [36], since emissions related to the construction phase can be up to 12 times higher for the new construction than in the rehabilitation scenario [37]. ISO 15686-5 [38] deﬁnes the life cycle cost (LCC) as a tool or technique to evaluate the total costs that arise during the entire useful life of a renovation, including the installation, use, maintenance, and loss of value stages. Analyzing from the LCC, the process of renovating an existing building allows decisions to be made during the construction project to optimize future costs and plan maintenance budgets. Another advantage is the possibility of choosing between several alternatives, studying each of them separately to facilitate the choice of the most cost-effective option for the whole life cycle [39]. The evaluation of the economic viability of an intervention can be supported by different types of IT tools. Building Energy Modeling (BEM), used in early design phases, can be useful for measuring achievable energy and environmental savings [40], assisting in decision-making in the project phase. Computational Fluid Dynamics (CFD) simulation [41] also makes it possible to evaluate the viability of passive, and therefore, low-cost energy-saving measures. Some studies state that 86.5% of the experts studied consider that they would use Life Cycle Assessment (LCA) if integrated with Building Information Modelling (BIM). With automation, design experts can compare their options in real time, design to reduce emissions, and ﬁnd optimal solutions at every stage of the design [21]. At the same time, it is considered that energy performance is a very important aspect of the building life cycle. There are management tools that cover the simulation of the complete building life cycle assessments (LCAs). The development of new software provides new measures for obtaining more efﬁcient buildings, even in the early stages of design [42]. Moreover, operating, maintenance, 8 of 27 Sustainability 2019, 11, 4946 and replacement costs of buildings make up more than 80% of total life cycle costs [43] and can be a very useful tool for big infrastructure management when combined with BIM tools [44]. Some authors consider that conventional energy efﬁciency technologies can be used to decrease energy use in new commercial buildings by 20–30% on average, and up to over 40% for some building types and locations. 3. Related Literature These reductions can often be performed at negative life-cycle costs because the improved efﬁciencies allow for the installation of smaller, cheaper HVAC equipment. These improvements not only save money and energy, but can also reduce a building’s carbon footprint by 16% on average. Costs on carbon emissions from energy use increases the return on energy efﬁciency investments because energy is more expensive, making some cost-ineffective projects economically feasible [45]. One of the main drawbacks in ofﬁcial methods for assessing the energy performance of buildings in hot climates is the lack of consideration of traditional measures to limit energy demand in buildings. Thermal inertia, cross-ventilation, or sun control have been traditional tools in hot climates. Passive energy rehabilitation, based on the improvement of the envelope and the implementation of solar control elements, especially beneﬁts buildings located in warm climates as opposed to measures aimed at replacing thermal installations with others of greater efﬁciency. Numerous research studies highlight the importance of reducing energy demand through design and passive building solutions [46]. The process of simulating the energy performance of buildings is also confronted with the human factor when comparing the results of the evaluation with the reality of the use of the building. Several researchers have demonstrated the high impact on the variation in energy consumption after identifying and classifying occupant behavior with energy consumption results and temporary use data [47,48]. The inﬂuence of occupants on variations in energy consumption is estimated to be between 10–25% for residential buildings and between 5–30% for commercial buildings [49]. On the other hand, post-execution studies have also shown that there are conﬂicts between the real cases and the simulations carried out with the programs and the methods for evaluating the energy performance of buildings [15]. Conﬂicts between simulations and real cases have also been detected when quantifying amortization periods for the application of energy-efﬁciency improvement measures [46]. This situation can lead to erroneous decision-making when some authors argue that the time needed to amortize the energy consumption of new construction versus rehabilitation is 60 years [50]. The levels of comfort and security demanded by users are another variable that has indirect costs associated with the investment made and that can condition the cost of maintaining the building. 3. Related Literature In Spain, several studies have been carried out on this effect, as a result of which, in 2012, 9% of Spanish households declared themselves incapable of maintaining their home at an adequate temperature and almost 17% spent energy disproportionately [51]. In tertiary buildings, such as bank branches, the average ﬁnal energy consumption of heating, ventilation, and air-conditioning accounts for 48% of the total consumption of the building [1]. From the point of view of the amortization of the investment through energy savings, it should be noted that energy savings are directly related to ﬁnancial savings, and this should be promoted as an indicator of sustainability so that energy savings should be signiﬁcant during the useful life of the building. It is therefore necessary to create new evaluation and assessment systems using tools integrated with quality models throughout the life of the building and all its elements [52,53]. On the other hand, there are contradictory opinions about life-cycle analyses (LCA). Some authors consider that the LCA of buildings is less advanced than in other industries, but researchers are working to enhance the possibilities of adopting LCA as a decision-making support tool within the design stage. At the same time, a full LCA of a product provides useful and accurate information, but it is costly and time-consuming, and using generic data and information in a specialized application could lead to a wrong choice. Nevertheless, LCA is considered a powerful tool for the evaluation of Sustainability 2019, 11, 4946 9 of 27 environmental impacts of buildings. It has the potential to make a strong contribution to the goal of sustainable development [54]. However, it is important to note that the relationship between the life cycle, energy efﬁciency, and heritage conservation has still been poorly explored [55]. Among the reasons for the lack of research in this ﬁeld is the lack of an initiative at the European level to address this type of building, and this is denounced by some authors [56]. However, it is recognized that the analysis of improvements in protected buildings offers advantages in their energy performance, and can even change users’ habits [57]. Among the research on LCA in protected buildings, some authors argue that LCA for protected buildings should not have a maximum time limit for calculating the return on investment [58]. 3. Related Literature The lack of regulations to be applied over this type of intervention could be compensated for by the integration of an analysis of energy performance based on a methodology and the decision-making process about the impact of the construction improvements on the architecture, which is something subjective. This integrated treatment is being explored by some authors [59]. This opinion is also reinforced when some authors state that an analysis with an appropriate method will allow the value of construction to improve [60]. Finally, the extensive literature review carried out by Cabeza et al. [61], stated that the ﬁrst approach for improving energy performance in listed buildings came from the improvement of the building envelope. This improvement must be done by analyzing the impact over the building’s protected values. Among all the available construction improvements, it showed promising potentiality for the improvement of energy efﬁciency and indoor wellbeing in historical buildings by means of implementing operational control solutions, as they minimize any invasive impact on construction, which means betting on technology applied over heritage buildings. 4. Research Method obtained were compared with the real consumption of one of the buildings, evaluating the inﬂuence that the ofﬁcial methodologies had on the decision-making process. The following steps have been taken in this research: The following steps have been taken in this research: he following steps have been taken in this research: The following steps have been taken in this research: 1. Deﬁnition of case studies. These buildings are two rehabilitation cases actually executed and in which the decision-making process has been affected by the methodology for evaluating the energy performance of buildings. 1. Deﬁnition of case studies. These buildings are two rehabilitation cases actually executed and in which the decision-making process has been affected by the methodology for evaluating the energy performance of buildings. 2. Construction improvements are proposed in both buildings with the aim of reducing energy demand and CO2 emissions. The impact on the architecture and protected elements of both construction solutions is evaluated initially. 3. Evaluation of the energy performance of the buildings. The energy performance of the buildings is assessed at two levels: in its original state, and once the construction improvements have been made. The evaluation is carried out with the ofﬁcial tools in force in Spain, HULC, and CE3X. At the same time, energy certiﬁcation is obtained. 4. Assessment of the feasibility of the investment in terms of savings from energy efﬁ improvements under the Regulation 244/2012 and through the CE3X program. p g g p g 5. Discussion of the results. The software used during the evaluation of the energy performance and the obtention of the energy certiﬁcations, as mentioned above, were HULC (to be applied for new buildings) and CE3X (to be applied for existing buildings). HULC is based on the DOE-2 calculation engine, and it permits the volumetric deﬁnition of the building in deﬁning all the construction materials and systems to obtain the energy need. To obtain the energy supply, it allowed one to completely deﬁne all the HVAC systems of the building. The energy certiﬁcation, as they are tertiary buildings, is obtained by comparing the analyzed building with an object building that met, at a minimum, the energy consumption and CO2 emission levels deﬁned by the Spanish government. 4. Research Method As mentioned in the literature review, there is a clear interest in testing the LCA methods as a tool valid for the evaluation of the economic viability for interventions in listed buildings. There is also a clear interest in the evaluation of the energy savings because of improvements in the building’s envelope and the evaluation of the impact over the protected building values. Therefore, the aim of the method followed in this research was to analyze the economic viability of undertaking construction that improves the energy performance of buildings catalogued in their refurbishment process. For this purpose, two case studies have been chosen, located in southeastern Spain, characterized by its warm and dry climate. These two buildings were chosen as the authors participated in the full refurbishment process, and there was an interest in learning about the recovery of the investment at the end of the process. These buildings had a unique geometry, and according to the design recommendations, they should be extremely efﬁcient in a warm climate, although they cannot be considered as models for any generic thermal considerations. The thickness of their enclosures, the size of the windows, and the available cross-ventilation made us assume good energy performance. Although the buildings fall outside the scope of the CTE, both buildings were analyzed with the regulations and tools in force in Spain for the evaluation of the energy performance of buildings with the aim of evaluating their impact on this type of building. With these tools, an iterative process was proposed where calculation examples were developed in both case studies to support the decision-making process and to know of the impact that construction measures to improve the energy performance of buildings may have. At the same time, a preliminary assessment was made of the impact that the improvements may have on the protected parts of the buildings. As a ﬁnal part of the research, the impact of construction improvements on buildings was evaluated by analyzing the recovery of the investment, comparing the information obtained from the methods approved in Spain with those deﬁned by the EU in Regulation 244/2012. Finally, the results Sustainability 2019, 11, 4946 10 of 27 obtained were compared with the real consumption of one of the buildings, evaluating the inﬂuence that the ofﬁcial methodologies had on the decision-making process. 4. Research Method As the energy certiﬁcation in Spain uses the primary energy, energy supply, and CO2 emissions as indicators to evaluate the energy performance in buildings, it was possible to obtain these factors after the calculation. CE3X works under the same calculation engine, but it is a simpler program as it simpliﬁes the data introduction. It does not allow for volumetric deﬁnition, and the building geometry is introduced in a very basic way. Nevertheless, the building envelope and conditioning systems can be completely deﬁned. The energy certiﬁcation is obtained in the same way as HULC. The main difference between both programs come from the simpliﬁcations and databases used in the calculation process. The different procedure used to introduce the building geometry, with a different certiﬁcation scale by climate zone, building typology, and new or existing buildings is, according to what it is stated by the Spanish government [62], the origin of the slightly different yielded results. 5. Case Studies The two case studies chosen for this research are two protected buildings that were under a rehabilitation process and where the authors of this research participated as consultors. As both rehabilitations implied a change in the building’s use, during the process of drafting the rehabilitation project, the possibility of adopting construction solutions to improve their energy performance was assessed, and the impact that the proposed methodology can have on the decision-making process was demonstrated. At the same time, it was also interesting to know the energy performance of the two buildings with a particular geometry that included passive solutions as thermal inertia and ventilation. The buildings are located in the municipality of Orihuela in southeastern Spain, which is characterized by a vast heritage that requires urgent interventions. Both buildings are catalogued in the Orihuela Urban Master Plan with different levels of protection. In both cases, the buildings were facing serious maintenance problems and had begun the process of structural collapse. 11 of 27 Sustainability 2019, 11, 4946 Given the importance that both buildings had for the neighbors, an integral rehabilitation was proposed that included the reconstruction of some elements. On the other hand, a change of use was also considered, as the former use was not possible again and the neighborhood pursued other types of services. As part of the decision-making process, construction improvements were assessed with the aim of improving the energy performance of both buildings. These construction improvements were based on the introduction of thermal insulation on the outside of the enclosure involved an architectural modiﬁcation that required authorization to a greater or lesser extent. 5.1. The Chapel of the Holy Sepulcher The Chapel of the Holy Sepulcher is a building built during the 18th century whose deterioration and lack of prolonged maintenance caused a process of structural collapse. At that time, it was acquired by the Orihuela City Council, which began a process of reconstruction and rehabilitation that was based on the recovery of two annex buildings that collaborated structurally in the stability of the nave of the Chapel and the collapsed parts of the vault, shown in Figure 1. The Chapel had the maximum level of protection at the city’s catalogue. This level implies the global protection of façades, roofs, and interiors, and that authorization is needed prior to any modiﬁcation. The objective of the rehabilitation was to turn it into a community center for the neighborhood where it was located, which also identiﬁed with this building. The Chapel of the Holy Sepulcher has a surface area of 150.00 m2, and as shown in Figure 2, it is characterized by its construction using thick masonry load-bearing walls, and by its absence of holes in the façade. This typology is typical in southeastern Spain for buildings of religious use, and adapts perfectly to the prevailing climate in the area since thermal inertia and solar control, along with adequate ventilation are traditional solutions for conditioning buildings in warm climates. During the project phase, it was considered that thermal inertia provided by the thick masonry walls completed with proper ventilation would be enough to guarantee the interior comfort conditions. Nevertheless, the option of adding insulation on the building envelope on the foundation and at the roof was considered. The construction solution for the façade, which has the greatest impact on the level of protection of the building, was based on the use of an External Thermal Insulation Control System (ETICS) based on the thermal insulation of extruded polystyrene covered by a monolayer mortar. The cost calculations in construction improvement are described in Table 1. The use of the DB HE over the building implied the accomplishment of the basic parameters deﬁned by this regulation. At the DB HE-1, the building must accomplish the deﬁned U-values to be applied over the climate zone where the building is located. It also needs to demonstrate that the energy loss by thermal bridges is under a certain value, and that at any point, it does not reached the dew-point at any point of the building envelope. 5.1. The Chapel of the Holy Sepulcher Table 1. Calculation of the cost of installing the insulation system. Description €/m2 m2 € Exterior insulation in masonry factory façade s 21.01 447.09 9393.36 Horizontal thermal insulation of foundations 18.03 162.74 2934.20 Outdoor insulation on roofs 55.88 255.54 14,279.58 TOTAL 26,607.14 Table 1. Calculation of the cost of installing the insulation system. 12 of 27 12 of 27 Sustainability 2019, 11, 4946 Figure 1. Image of the Chapel of the Holy Sepulcher. Figure 2. Section and plan of the Chapel of the Holy Sepulcher. Figure 1. Image of the Chapel of the Holy Sepulcher. Figure 1. Image of the Chapel of the Holy Sepulcher. Figure 2. Section and plan of the Chapel of the Holy Sepulcher. 5.2. The Cisterns of Hurchillo 5.2. The Cisterns of Hurchillo The Cisterns of Hurchillo are from the 19th century. Its function was to contain the water to be channeled to the town of Hurchillo, in Orihuela. This building was just as unfortunate as the Chapel, and the process of degradation culminated in its abandonment. This building was included at the catalogue of protected heritage buildings of the city, but with a lower level of protection. In this case, only the main façade to the street and the external shape of the cisterns needed authorization to be modiﬁed. The aim of the rehabilitation was to reuse the cisterns for the local cultural center, recovering both cisterns and adding an annexed service building, as shown in Figure 3. In this case, the geometry of the building is much more complex, since it is a semi-buried structure. The building has an area of 236.00 m2, of which a large part is buried, as shown in Figure 4. In addition to the high thermal inertia provided by the land, there is also a construction made with containing walls of masonry and very thick concrete. At the level of the enclosure, only the roofs and one of the facades are in contact with the outside environment. As was the case with the Chapel of the Holy Sepulcher, an improvement of the thermal envelope was proposed by introducing thermal insulation in contact with the ground, but especially in the area of the roof by means of an ETICS system, given that the Cisterns originally only had plaster on the outside of the curved surface of the vault. The insulation system was completed with a reinforced ventilation system that guaranteed the proper comfort conditions in the interior. The cost calculations in construction improvement are described in Table 2. Sustainability 2019, 11, 4946 13 of 27 The use of the DB HE over the building implied, as in the case of the Chapel, the accomplishment of the minimum requirements to guarantee proper building behaviour from a thermal point of view. The use of the DB HE over the building implied, as in the case of the Chapel, the accomplishment of the minimum requirements to guarantee proper building behaviour from a thermal point of view. e eq e e s o g ee p ope g e o o e po o e Figure 3. Floor plan of the Cisterns. Figure 4. Section plan of the Cisterns. Table 2. 5.2. The Cisterns of Hurchillo Calculation of the cost of installing the insulation system. Description €/m2 m2 € Exterior insulation in masonry factory façades 21.01 278.30 5847.08 Horizontal thermal insulation of foundations 18.03 235.70 4249.67 Outdoor insulation on curved roofs 36.23 381.50 13,821.75 TOTAL 23,918.50 3. Preliminary Study of the Impact of Construction Improvements on Protected Buildings The implementation of an insulation system in the Chapel of the Holy Sepulcher both inside an Figure 3. Floor plan of the Cisterns. Figure 3. Floor plan of the Cisterns. Figure 4. Section plan of the Cisterns. Figure 4. Section plan of the Cisterns. Figure 4. Section plan of the Cisterns. Figure 4. Section plan of the Cisterns. 5.3. Preliminary Study of the Impact of Construction Improvements on Protected Buildings The implementation of an insulation system in the Chapel of the Holy Sepulcher both inside and outside was complicated by the geometry and elements existing in the building. The baroque and Sustainability 2019, 11, 4946 14 of 27 neoclassical buildings in this area of Spain are characterized by a profusion of moldings and pictorial decorations that prevent the installation of an insulation system on the interior. In the case of the exterior, the execution of the ETICS system means an increase in the envelope, coming into conﬂict with decorative elements and historic materials existing on the exterior of the envelope, as shown in Figure 5. At the level of the roofs, conﬂicts arise with the roof system because there are both vaults and domes. The authorities in charge of heritage buildings protection were very reluctant to any intervention that modiﬁed the façades both inside and outside. They would only be taken them into consideration if very big beneﬁts could be obtained during energy simulation. The impact of the construction improvements on the Cisterns was initially minor, given the absence of both interior and exterior decorative elements, as shown in Figure 6. The original image of the buried cisterns where the vault was simply ﬁnished with a mortar allowed this image to be recovered, provided that the execution problems of rendering on a curved surface were solved. In this case, the authorities were open to the introduction of new construction elements if the original shape of the buried cisterns was maintained. Figure 5. Detail of the main façade and example of the interior paintings of the Chapel of the Holy Sepulcher. Figure 5. Detail of the main façade and example of the interior paintings of the Chapel of the Holy Sepulcher. Figure 6. External images of the Cisterns. Figure 6. External images of the Cisterns. 15 of 27 Sustainability 2019, 11, 4946 6. Modeling the Energy Performance of Buildings As indicated above, the DB HE in force in Spain, in its scope of application, exempts existing buildings with recognized historical or architectural value from complying with its prescriptions when the necessary solutions could unacceptably alter their character or appearance, and when the application of these solutions does not lead to an effective improvement in the features related to the basic “energy-saving” requirement, as well as when the solutions are not technically or economically viable. y However, this document also indicates that every effort should be made to improve the performance of the building on the condition that the solutions to be adopted can never worsen the original performance of the building. To carry out this research, we decided to apply the two recognized methods developed by the Spanish Government for evaluating the energy performance of buildings, which were the only tools available until September 2018. In this way, the energy performance of both buildings was simulated, and the corresponding energy certiﬁcation obtained with the HULC and CE3X programs. Two calculations are performed: a ﬁrst calculation without the installation of thermal insulation, and a second calculation with the installation of this improvement. 6.1. Chapel of the Holy Sepulcher Calculation of the energy performance of the Chapel of the Holy Sepulcher with CE3X. Table 4. Calculation of the energy performance of the Chapel of the Holy Sepulcher with CE3X. CE3X Energy Need for Heating kWh/Year Energy Need for Cooling KWh/Year Final Energy KWh/Year Primary Energy KWh/Year CO2 Emissions KgCO2 CO2 Emissions KgCO2/m2 Energy Certiﬁcation No insulation 16,230.00 3240.00 9133.54 30,570.00 5175.00 34.50 C With insulation 10,599.00 3310.50 6978.33 23,356.50 3957.00 26.38 B Improvement (%) 34.70% −2.16% 23.60% 23.60% 23.29% 23.29% 6.1. Chapel of the Holy Sepulcher As shown in Tables 3 and 4, the consideration of a façade insulation system in the building improves the energy performance of the building. However, a paradoxical situation arises when the greatest demand for energy occurs in winter, as it should have occurred in summer because it is a building located in a warm climate. This situation is due to the tremendous efﬁciency of the building in front of a warm climate, given the absence of important openings to the outside and the absence of thermal insulation. On the other hand, this same absence penalizes the demand for heating. It is also worth noting that, although both programs share the same calculation engine, DOE-2, the results are manifestly different. Furthermore, in the case of CE3X, the introduction of higher thermal insulation results in an increased demand for cooling energy. This situation usually occurs in warm climates when excess thermal insulation is detected in the building envelope. It should be noted that this improvement was suggested by the program itself. Table 3. Calculation of the energy performance of the Chapel of the Holy Sepulcher with HULC. Table 3. Calculation of the energy performance of the Chapel of the Holy Sepulcher with HULC. HULC Energy Need for Heating kWh/Year Energy Need for Cooling KWh/Year Final Energy KWh/Year Primary Energy KWh/Year CO2 Emissions KgCO2 CO2 Emissions KgCO2/m2 Energy Certiﬁcation No insulation 22,975.40 3965.30 18,872.30 63,165.70 18,509.60 67.60 C With insulation 19,126.80 3032.30 17,275.40 57,820.80 16,946.80 60.90 B Improvement (%) 16.75% 23.53% 8.46% 8.46% 8.44% 8.44% Table 3. Calculation of the energy performance of the Chapel of the Holy Sepulcher with HULC. HULC Energy Need for Heating kWh/Year Energy Need for Cooling KWh/Year Final Energy KWh/Year Primary Energy KWh/Year CO2 Emissions KgCO2 CO2 Emissions KgCO2/m2 Energy Certiﬁcation No insulation 22,975.40 3965.30 18,872.30 63,165.70 18,509.60 67.60 C With insulation 19,126.80 3032.30 17,275.40 57,820.80 16,946.80 60.90 B Improvement (%) 16.75% 23.53% 8.46% 8.46% 8.44% 8.44% Table 4. Calculation of the energy performance of the Chapel of the Holy Sepulcher with CE3X. CE3X Energy Need for Heating kWh/Year Energy Need for Cooling KWh/Year Final Energy KWh/Year Primary Energy KWh/Year CO2 Emissions KgCO2 CO2 Emissions KgCO2/m2 Energy Certiﬁcation No insulation 16,230.00 3240.00 9133.54 30,570.00 5175.00 34.50 C With insulation 10,599.00 3310.50 6978.33 23,356.50 3957.00 26.38 B Improvement (%) 34.70% −2.16% 23.60% 23.60% 23.29% 23.29% Table 4. 6.3. Conclusions to the Energy Evaluation The analysis of Tables 7 and 8 allows us to conclude that both buildings adapt to a warm climate as their design minimizes the energy demand for refrigeration. On the other hand, this design penalizes the demand for heating to not be able to count on contributions for sunshine and due to the absence of thermal insulation. This situation is one of the problems presented by the tools authorized in Spain since, as mentioned above, they do not consider thermal inertia in the calculation. The results obtained also reinforce what has been indicated above regarding the lack of homogeneity of the results obtained when using different calculation tools. Although HULC and CE3X share the calculation engine, the simpliﬁcations and databases of the two tools make the results different and offer different reductions in energy consumption when construction improvements are introduced. Table 7. Comparison of energy demand and consumption per m2 of built area for the Chapel of the Holy Sepulcher. Holy Sepulcher Energy Need for Heating kWh/Year Energy Need for Cooling KWh/Year Final Energy KWh/Year Primary Energy KWh/Year No insulation HULC 153.17 26.44 125.82 421.10 With insluation HULC 127.51 20.22 115.17 385.47 No insulation CE3X 108.20 21.60 60.89 203.80 With insulation CE3X 70.66 22.07 46.52 155.71 Table 7. Comparison of energy demand and consumption per m2 of built area for the Chapel of the Holy Sepulcher. Table 8. Comparison of energy demand and consumption per m2 of constructed area for the Cisterns. Cisterns of Hurchillo Energy Need for Heating kWh/Year Energy Need for Cooling KWh/Year Final Energy KWh/Year Primary Energy KWh/Year No insulation HULC 110.11 26.38 68.16 228.14 With insluation HULC 82.02 21.52 58.28 195.07 No insulation CE3X 60.30 15.60 41.98 140.50 With insulation CE3X 46.93 11.66 34.97 117.03 Table 8. Comparison of energy demand and consumption per m2 of constructed area for the Cisterns. 6.2. The Cisterns of Hurchillo 6.2. The Cisterns of Hurchillo As reﬂected in Tables 5 and 6, and as with the results obtained in the Chapel of the Holy Sepulcher, the building had a greater demand for energy in winter, which is related to the lack of thermal insulation and the absence of openings to the outside. This situation penalizes this demand, favoring a very low refrigeration demand. Sustainability 2019, 11, 4946 16 of 27 Table 5. Calculation of the energy performance of the Cisterns with HULC. HULC Energy Need for Heating kWh/Year Energy Need for Cooling KWh/Year Final Energy KWh/Year Primary Energy KWh/Year CO2 Emissions KgCO2 CO2 Emissions KgCO2/m2 Energy Certiﬁcation No insulation 25,985.60 6225.40 16,086.30 53,840.90 15,781.90 45.50 C With insulation 19,357.60 5079.40 13,754.60 46,036.60 13,520.90 38.20 B Improvement (%) 25.51% 18.41% 14.49% 14.49% 14.33% 14.33% Table 6. Calculation of the energy performance of the Cisterns with CE3X. CE3X Energy Need for Heating kWh/Year Energy Need for Cooling KWh/Year Final Energy KWh/Year Primary Energy KWh/Year CO2 Emissions KgCO2 CO2 Emissions KgCO2/m2 Energy Certiﬁcation No insulation 14,230.80 3681.60 9906.70 33,158.00 5616.80 23.80 B With insulation 11,075.48 2751.76 8251.90 27,619.08 4679.88 19.83 B Improvement (%) 22.17% 25.26% 16.70% 16.70% 16.68% 16.68% 6 3 C l i t th E E l ti Table 5. Calculation of the energy performance of the Cisterns with HULC. Table 6. Calculation of the energy performance of the Cisterns with CE3X. 6.3. Conclusions to the Energy Evaluation 7. Modeling the Recovery of Investment As outlined above, since 2010, the EU has issued directives in which, due to the lack of a sustainability standard in Europe, it urges member countries to develop methods that guarantee the return on investment in construction improvements to obtain energy savings. This ﬁrst request is based on the Delegated Regulation 244/2012, as explained above. This research proposes a comparison between the method deﬁned by this regulation and that offered by the CE3X program, which is the Sustainability 2019, 11, 4946 17 of 27 ﬁrst approximation of a return on investment in improving the energy performance of buildings in Spain. Both methods are based on the NPV calculation. 7.1. Calculation of NPV through the Delegated Regulation 244/2012 The Delegated Regulation 244/2012 is based on a document drawn up by each country that deﬁnes amortization periods, ﬁnancial and revaluation discounts, as well as an estimate of energy prices and CO2 emissions over the calculation period [23]. The information contained in this document was used as the basis for the calculation of NPV in both case studies. Among the factors deﬁned by the regulation is the calculation period. The regulation indicates 20 years for public buildings, which is increased to 30 years for residential buildings. This calculation period contrasts with the useful life deﬁned for the renovated construction elements, which are as follows: • Insulation: 50 years • Windows: 20 years y • Installations: according to values indicated in Annex A of the standard UNE-EN 15459. y • Installations: according to values indicated in Annex A of the standard UNE-EN 15459 The document also deﬁnes the following energy prices for Spain, for which it makes two estimates, among which the one described in Table 9 was chosen as it deﬁnes higher prices. In addition, as seen in Table 10, a forecast of the cost of CO2 emissions was made, demonstrating a clear intention to penalize them over time. Table 9. Forecasts of energy prices deﬁned by the government of Spain. Table 9. Forecasts of energy prices deﬁned by the government of Spain. Cost [€/kWh] 2012 2020 2025 2030 2035 2040 2045 Boilers Oil 0.096 0.133 0.158 0.186 0.241 0.311 0.404 Gas 0.068 0.108 0.130 0.156 0.207 0.278 0.371 LPG 0.115 0.159 0.188 0.222 0.287 0.373 0.484 Electricity 0.209 0.276 0.303 0.319 0.344 0.370 0.399 Biomass 0.046 0.052 0.056 0.060 0.065 0.070 0.075 Table 10. Forecasts of the cost of CO2 emissions. Table 10. Forecasts of the cost of CO2 emissions. Year Emissions Cost CO2 [ €/ton] From 2012 to 2020 18.60 From 2021 to 2025 22.50 From 2026 to 2030 40.50 From 2031 to 2035 56.30 From 2036 to 2040 58.50 From 2041 to 2045 57.40 From 2046 to 2050 56.30 One of the factors that conditions the viability of the renovation is the updating factor. It is the annual beneﬁt to be obtained by the realization of the energy improvement. This factor changes depending on the calculation method used and the building’s type of use, as shown in Table 11. Table 11. Updated factors deﬁned for the different types of calculations and uses of the building. p yp Financial Calculations: Macroeconomic Calculations: New buildings: 7% 3% Existing buildings: 10% 4% 18 of 27 Sustainability 2019, 11, 4946 Finally, for the calculation, the following factors were considered for the conversion of ﬁnal energy into primary energy and the conversion of energy into CO2 emissions, which are described in Table 12. Finally, for the calculation, the following factors were considered for the conversion of ﬁnal energy into primary energy and the conversion of energy into CO2 emissions, which are described in Table 12. Table 12. Conversion factors to primary energy and CO2 emissions. Table 12. Conversion factors to primary energy and CO2 emissions. y • Installations: according to values indicated in Annex A of the standard UNE-EN 15459. Energy Source Conversion Factors to Primary Energy Emission Conversion Factors [kWh/kWh] [tCO2 /kWh] Biomass 0.250 0 Electricity 2.464 0.000399 Gas 1.070 0.000201 Diesel for Heating 1.120 0.000263 LPG 1.050 0.000234 The process followed for the evaluation of the improvements during the project phase ﬁrst considers the energy performance of the building in a stage prior to refurbishment, so that its enclosures are non-insulated. Once the results have been obtained, the performance is evaluated considering the installation of the thermal insulation system. In the calculation process, the difference between the energy consumption before and after the rehabilitation were the annual beneﬁts with which we compared the cost of the installation of the construction improvement. During the process, and in accordance with what has been indicated above, the increase in the cost of energy must have been considered in accordance with what has been indicated by the government of Spain. As seen in Figures 7–10, the letter “r” is the minimum annual beneﬁt (%) that must be considered to understand the renovation as viable. This performance or update factor is the one previously deﬁned as conditioned by the type of calculation and by the building’s type of use in Table 12. 7.1.1. Calculation of the NPV for the Chapel of the Holy Sepulcher 7.1.1. Calculation of the NPV for the Chapel of the Holy Sepulcher Assuming an initial cost of the investment in the installation of the building insulation system and considering the primary energy consumption deﬁned above, the following values of both ﬁnancial and macroeconomic NPV were obtained from the results of the HULC and CE3X programs, described in Figures 7 and 8. Figure 7. Calculation of the NPV at the ﬁnancial level with energy-savings data obtained with HULC and CE3X. Figure 7. Calculation of the NPV at the ﬁnancial level with energy-savings data obtained with HULC and CE3X. 19 of 27 Sustainability 2019, 11, 4946 Figure 8. Calculation of the macroeconomic NPV with energy-savings data obtained with HULC and CE3X. Figure 8. Calculation of the macroeconomic NPV with energy-savings data obtained with HULC and CE3X. Figure 9. Calculation of the NPV at the ﬁnancial level with energy-savings data obtained with HULC and CE3X. Figure 9. Calculation of the NPV at the ﬁnancial level with energy-savings data obtained with HULC and CE3X Figure 9. Calculation of the NPV at the ﬁnancial level with energy-savings data obtained with HULC and CE3X. Figure 10. Calculation of the macroeconomic NPV with energy-savings data obtained with HULC and CE3X. Figure 10. Calculation of the macroeconomic NPV with energy-savings data obtained with HULC and CE3X. 7.2. Calculation of the NPV by Program CE3X The CE3X program offers the possibility of performing an NPV calculation through a series of standardized improvements within the program. Data entry is limited to the choice of the construction improvement, the introduction of the cost of the improvement, and the deﬁnition of the annual cost/beneﬁt factor. The result obtained is the period of time needed to recover the investment. Thus, the program indicates that for the Chapel of the Holy Sepulcher, the amortization period would be 51.50 years, and for the Cisterns, 52.10 years. 7.1.2. Calculation of NPV for the Cisterns 7.1.2. Calculation of NPV for the Cisterns Following the same criteria, for the Hurchillo Cisterns, the following results were obtained from the energy consumption offered by both programs and shown in Figures 9 and 10. Sustainability 2019, 11, 4946 20 of 27 20 of 27 7.3. Comparative Results of the NPV Calculation The calculation of the value of the NPV, as indicated in Table 13, shows that regardless of the method and the program used, in no case is the investment recovered in the calculation period deﬁned by the delegated regulation and by the Spanish ofﬁcial methods, although in the case of the Cisterns, the values are closer to a positive result. The reason why it is not possible to recover the investment through energy savings by increasing the insulation system is because both buildings are very efﬁcient in their geometry and signiﬁcantly reduce energy demand in the most demanding period, which is the summer. To increase energy savings, it would be necessary to undertake modiﬁcations to conditioning systems and introduce systems that penalize primary energy consumption less. These savings should be 12,500 KWh/year in the Chapel of the Holy Sepulcher, which represents an increase of 232% in the best of cases. In the case of the Cisterns, the savings would be viable from 8400 KWh/year, which means an increase of 7.1%. Table 13. Comparison of NPV values obtained. Financial VAN HULC FINANCIAL VAN CE3X VAN Macro HULC VAN Macro CE3X CE3X Period Holy Sepulcher −15,180.54 −11,186.83 −19,994.73 −5932.78 51.50 Cisterns of Hurchillo −7234.08 −12,077.12 −2055.05 −10,123.58 52.10 Table 13. Comparison of NPV values obtained. In view of these results, more simulations are carried out, among which it is obtained that by reducing the update rate as shown in Figures 11 and 12, it is possible to obtain a positive NPV in both cases but with different tools. This reduction could be considered acceptable since both buildings are publicly owned and their heritage value means that both buildings will be maintained over time. In view of these results, more simulations are carried out, among which it is obtained that by reducing the update rate as shown in Figures 11 and 12, it is possible to obtain a positive NPV in both cases but with different tools. This reduction could be considered acceptable since both buildings are publicly owned and their heritage value means that both buildings will be maintained over time. Figure 11. Calculation of the ﬁnancial NPV with energy-savings data obtained with HULC and CE3X with a beneﬁt of 5% for the Chapel. Figure 11. Calculation of the ﬁnancial NPV with energy-savings data obtained with HULC and CE3X with a beneﬁt of 5% for the Chapel. 7.3. Comparative Results of the NPV Calculation 21 of 27 Sustainability 2019, 11, 4946 Figure 12. Calculation of the ﬁnantial NPV with energy-savings data obtained with HULC and CE3X with a beneﬁt of 5% for the Cisterns. Figure 12. Calculation of the ﬁnantial NPV with energy-savings data obtained with HULC and CE3X with a beneﬁt of 5% for the Cisterns. On the other hand, to make an amortization increasing the term, the analysis becomes complicated due to the signiﬁcant increase in the costs of the CO2 published by the government of Spain and the inﬂuence of the energy data obtained, as shown in Figures 13 and 14. On the other hand, to make an amortization increasing the term, the analysis becomes complicated due to the signiﬁcant increase in the costs of the CO2 published by the government of Spain and the inﬂuence of the energy data obtained, as shown in Figures 13 and 14. Figure 13. Comparison between ﬁnancial and macroeconomic NPV with energysavings data obtained from CE3X during 50 years for the Chapel. Figure 14. Comparison between ﬁnancial and macroeconomic NPV with energysavings data obtained from CE3X during 50 years for the Cisterns. Figure 13. Comparison between ﬁnancial and macroeconomic NPV with energysavings data obtained from CE3X during 50 years for the Chapel. Figure 13. Comparison between ﬁnancial and macroeconomic NPV with energysavings data obtained from CE3X during 50 years for the Chapel. from CE3X during 50 years for the Chapel. Figure 14. Comparison between ﬁnancial and macroeconomic NPV with energysavings data obtained from CE3X during 50 years for the Cisterns. Figure 14. Comparison between ﬁnancial and macroeconomic NPV with energysavings data obtained from CE3X during 50 years for the Cisterns. Figure 14. Comparison between ﬁnancial and macroeconomic NPV with energysavings data obtained from CE3X during 50 years for the Cisterns. Sustainability 2019, 11, 4946 22 of 27 9. Discussion of Results and Comparison with Actual Consumption of the Cisterns of Hurchillo The commissioning of the building made it possible to know the real energy consumption of the building during two years of its operation and to know the energy savings obtained, which can be observed in Table 14. As it is shown in Table 15, the analysis of the real data with the ones obtained from the simulation shows that the results yielded by the simulation programs are far from the real building performance. This implies that the energy savings are not real, so the NPV calculation had been guided in the wrong direction while developing the construction project. It is not possible to recoup the investment based on energy savings obtained by improvements to the building envelope alone. The energy savings were already shown to be low in the calculation, especially in the cooling demand, but when studying the actual data, these savings were even lower. Table 14. Real energy consumption of the Cisterns during 24 months. 2017–2018 Energy Consumption kWh Cost € 2018–2019 Energy Consumption kWh Cost € June 311.00 166.06 June 980.00 25317 July 322.00 194.12 July 769.00 211.04 August 483.00 206.96 August 325.00 171.74 September 953.00 258.58 September 547.00 170.74 October 549.00 164.60 October 1174.00 288.54 November 826.00 240.32 November 761.00 220.16 December 944.00 233.71 December 724.00 205.69 January 972.00 263.78 January 688.00 192.79 February 999.00 271.75 February 1546.00 394.29 March 882.00 277.86 March 891.00 243.57 April 1001.00 252.19 April 749.00 208.36 May 981.00 251.73 May 956.00 250.34 9223.00 2781.66 10,110.00 2810.43 Table 15. Comparison between the simulated and the real energy consumption. Cisterns of Hurchillo HULC CE3X REAL Energy consumption without insulation 53,840.90 33,158.00 Energy consumptio with insulation 46,036.60 27,619.08 9666.50 Table 14. Real energy consumption of the Cisterns during 24 months. Table 15. Comparison between the simulated and the real energy consumption. At the end of the research and with the results obtained, it can be stated that the application of the Spanish ofﬁcial methods to evaluate the energy performance of new and existing buildings on protected buildings follows the same line of lack of homogeneity defended by several authors in the evaluation of these methodologies applied to other types of buildings. Although the software may share a calculation engine, the application of different programs on the same building does not offer comparable results, something already concluded by other investigations [15]. 8. Decision-Making at the End of the Project Once these results had been analyzed, decisions were made with the support of the administration responsible for the conservation of the buildings, and it was decided to undertake the renovation to improve the enclosure only in the Cisterns, shown in Figures 15 and 16. This decision was based not only on the results obtained in terms of savings and recovery of the investment that were close to positive under the NPV point of view, but also on a lesser impact on the protected heritage elements of the building. In the case of the Chapel, the results were not robust enough to justify such a deep intervention in highly protected elements, so it was decided not to be executed. Figure 15. Image of the Cisterns during the installation of the thermal insulation. Figure 16. Image of the interior of the Cisterns. Figure 15. Image of the Cisterns during the installation of the thermal insulation. Figure 15. Image of the Cisterns during the installation of the thermal insulation. Figure 16. Image of the interior of the Cisterns. Figure 16. Image of the interior of the Cisterns. Sustainability 2019, 11, 4946 23 of 27 9. Discussion of Results and Comparison with Actual Consumption of the Cisterns of Hurchillo Furthermore, although it is claimed that an LCA can help in the decision-making process [39], an inadequate tool can lead to erroneous decisions being made. The calculation of the energy demand for cooling conﬁrms the geometric uniqueness of the buildings and their adaptation to the climatic zone in which they are located. This adaptation contrasts with the high demand for heating, which is penalized by the lack of voids and the absence of thermal insulation in the original state. The installation of an external thermal insulation does not signiﬁcantly inﬂuence the results, increasing the demand for refrigeration in one case. Therefore, the difﬁculties defended by some authors in the process of evaluating the energy performance of buildings with a singular geometry are still maintained [63]. The marked differences between theoretical and actual consumption are based on the unsuitability of the methods to work with passive measures to improve the energy performance of buildings, Sustainability 2019, 11, 4946 24 of 27 including thermal inertia. The importance of rehabilitation with passive measures is therefore demonstrated in this type of building [46]. At the same time, the inﬂuence of the human factor on buildings is conﬁrmed as a factor to be taken into account in the methodologies for evaluating the energy performance of buildings, as it can distort the results obtained [47]. The results obtained to calculate the NPV indicate in all the assumptions that trying to recover the investment in construction improvements to reduce energy demand is not feasible in this type of building. As indicated above, the low demand for refrigeration does not allow great savings in energy consumption. This result is considered reasonable given the location and unique geometry of both buildings. It is then conﬁrmed that, to be able to recoup the investment through energy-saving measures, the savings must be signiﬁcant [53]. This situation also reafﬁrms what has been explored by other authors who afﬁrm that to act in protected historic buildings, methods are needed that combine the analysis of energy performance with subjective decision-making [59,60]. 9. Discussion of Results and Comparison with Actual Consumption of the Cisterns of Hurchillo It was not really conﬁrmed whether the study period should be as long as possible because of the political decisions of the government related to the CO2 emissions cost [58], and that it was needed to explore the improvement of energy efﬁciency and indoor wellbeing in historical buildings by means of implementing operational control solutions, as they minimize any invasive impact on construction [61]. 10. Conclusions This research conﬁrms that methods for energy assessment of buildings can condition the decision-making process in a project. The lack of homogeneity in the results made it difﬁcult to analyze the decisions adopted in the project and, therefore, made it difﬁcult to evaluate the impact that a construction improvement may have on the energy performance of a building. It was also conﬁrmed that methods based on a percentage of improvement over a reference building can penalize buildings with an efﬁcient design that are adapted to the climate in which they are located. On the other hand, the inadequacy of using the methods developed for new buildings on old buildings was demonstrated due to the design particularities of old buildings. This research demonstrates the validity of the NPV calculation as a measure to evaluate the economic viability of an intervention, but these data must be supported by robust results from the evaluation of the building energy performance. In addition, the savings obtained should come from other types of construction improvements that are not penalized for their CO2 emissions. Finally, we demonstrated the need for the development of methodologies that allow a cross-sectional analysis of these types of buildings and support an objective decision-making process during intervention. Author Contributions: A.G.-G. was in charge of the restoration of both buildings. The energy assessment and analysis of the LCA was carried out by all the authors during the construction and the analyzed building life period. The manuscript has been developed via collaboration among all authors. Funding: This reseach received no external funding. Funding: This reseach received no external funding. Acknowledgments: The authors of this paper thank the Municipality of Orihuela for providing the information about the projects that allowed this research. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References 1. Munarim, U.; Ghisi, E. Environmental feasibility of heritage buildings rehabilitation. Renew. Sustain. Energy Rev. 2016, 58, 235–249. [CrossRef] 2. Bajenaru, N.; Damian, A.; Frunzulica, R. Evaluation of the Energy Performance for a nZEB Ofﬁce Building under Speciﬁc Climatic Conditions. Energy Procedia 2016, 85, 26–34. [CrossRef] 3. Sigmund, Z. Sustainability in architectural heritage: Review of policies and practices. Organ. Technol. Manag. Constr. Int. J. 2016, 8, 1411–1421. [CrossRef] 3. Sigmund, Z. Sustainability in architectural heritage: Review of policies and practices. Organ. Technol. Manag. Constr. Int. J. 2016, 8, 1411–1421. [CrossRef] 25 of 27 Sustainability 2019, 11, 4946 4. Galiano-Garrigós, A. Comparative Analysis of Methodologies for Evaluating and Certifying the Energy Performance of Buildings in the European Union (Análisis Comparado de las Metodologías de Evaluación y Certiﬁcación del Comportamiento Energético de los Ediﬁcios en la Unión Europea); University of Alicante: Alicante, Spain, 2013. . European Parlament and Council of the European Comunity. EPBD European Directive for Energy Perform in Buildings; European Parlament and Council of the European Comunity: Brussels, Belgium, 2002. 6. European Union. Treaty on European Union; European Parlament and Council of the European Comission: Brussels, Belgium, 1986. 7. European Parlament and Council of the European Comunity. European Directive 98/34/CE; European Parlament and Council of the European Comunity: Brussels, Belgium, 1998. 8. CEN. European Committee for Standardization—TR 15615 Explanation of the General Relationship between Various European Standards and the Energy Performance of Buildings Directive (EPBD)—Umbrella Document; European Committee for Standardization: Brussels, Belgium, 2008. 9. CEN. European Committee for Standardization—EN ISO 13790. Energy performance of buildings Calculation of Energy Use for Space Heating and Cooling; European Committee for Standardization: Brussels, Belgium, 2008. 10. CEN. European Committee for Standardization—EN ISO 15603 Energy Performance of Buildings—Overall Energy 9. CEN. European Committee for Standardization—EN ISO 13790. Energy performance of buildings Calculation of Energy Use for Space Heating and Cooling; European Committee for Standardization: Brussels, Belgium, 2008. 10. CEN. European Committee for Standardization—EN ISO 15603 Energy Performance of Buildings—Overall Energy Use and Deﬁnition of Energy Ratings; European Committee for Standardization: Brussels, Belgium, 2008. 11. CEN. European Committee for Standardization—EN ISO 15217. Energy Performance of Buildings. Methods for Expressing Energy Performance and for Energy Certiﬁcation of Buildings; European Committee for Standardization: Brussels, Belgium, 2011. 12. Maldonado, E. Concerted Action. Supporting Transposition and Implementation of the Directive 2002/91/EC (EPBD); European Commission: Brussels, Belgium, 2010. 13. Spiekman, M.; Dijk, D.V. Comparing Energy Performance requirements over Europe. References ASIEPI—Assessment and Improvement of the EPBD Impact; European Comission Brussels, Belgium, 2008. 14. Galiano, A.; Echarri, V. Analysis of the quality of public urban space through a graphical analysis method. WIT Trans. Ecol. Environ. 2014, 191, 1623–1636. 15. Galiano-Garrigós, A.; García-Figueroa, A.; Rizo-Maestre, C.; González-Avilés, Á. Evaluation of BIM energy performance and CO2 emissions assessment tools: A case study in warm weather. Build. Res. Inf. 2019, 47, 787–812. [CrossRef] 16. Government of Spain. Royal Decree 235/2013, of 5 April, Approving the Basic Procedure for the Certiﬁcation of the Energy Performance of Buildings; Ministry of the Presidency: Madrid, Spain, 2013. 17. Casals, X.G. Analysis of building energy regulation and certiﬁcation in Europe: Their role, limitations and differences. Energy Build. 2006, 38, 381–392. [CrossRef] 8. Economidou, M. Energy performance requirements for buildings in Europe. REHVA J. 2012, 2012, 16–2 19. European Parlament and Council of the European Comunity. Directive 2010/31/EU of the European Parlament and of the Council of 19 May 2010 on the Energy Performance of Buildings; European Parlament and Council of the European Comunity: Brussels, Belgium, 2010. 20. European Parlament and Council of the European Comunity. Directive (EU) 2018/844 of the European Parliament and of the Council of 30 May 2018 Amending Directive 2010/31/EU on the Energy Performance of Buildings and Directive 2012/27/EU on Energy Efﬁciency; European Parlament and Council of the European Comunity: Brussels, Belgium, 2018. 21. Bruce-Hyrkäs, T.; Pasanen, P.; Castro, R. Overview of Whole Building Life-Cycle Assessment for Green Building Certiﬁcation and Ecodesign through Industry Surveys and Interviews. Procedia CIRP 2018, 69, 178–183. [CrossRef] 22. EU Commission. Commission Delegated Regulation (EU) No 244/2012 of 16 January 2012 Supplementing Directive 2010/31/EU of the European Parliament and of the Council on the Energy Performance of Buildings by Establishing a Comparative Methodology Framework for Calculating; EU Commission: Brussels, Belgium, 2012. 23. Minitry of Development. Report on Cost Optimal Calculations and Comparison with the Current and Future Energy; Ministry of Development: Madrid, Spain, 2012. 24. Government of Spain. NBE CT-79. Thermal Conditions in Buildings (Condiciones Térmicas en los Ediﬁcios); Government of Spain: Madrid, Spain, 1979. 25. CTE. Spanish Building Technical Code; CTE: Madrid, Spain, 2006. 25. CTE. Spanish Building Technical Code; CTE: Madrid, Spain, 2006. 25. CTE. Spanish Building Technical Code; CTE: Madrid, Spain, 2006. 26 Government of Spain LEY 38/1999 de 5 de Noviembre de Ordenación de la Ediﬁcación; Government of Spain: 26. Government of Spain. References LEY 38/1999, de 5 de Noviembre, de Ordenación de la Ediﬁcación; Government of Spain: Madrid, Spain, 1999. Sustainability 2019, 11, 4946 26 of 27 26 of 27 27. Government of Spain. CTE (Technical Building Code in Spain) Basic Document, Energy Saving DB-HE; CTE: Madrid, Spain, 2017. 28. Government of Spain. Real Decreto 47/2007, de 19 de enero, por el que se Aprueba el Procedimiento Básico para la Certiﬁcación de Eﬁciencia Energética de Ediﬁcios de Nueva Construcción; Ministry of the Presidency: Madrid, Spain, 2007. 29. Órgano Ministerio de la Presidencia y para las Administraciones Territoriales. Real Decreto 564/2017, de 2 de junio, por el que se Modiﬁca el Real Decreto 235/2013, de 5 de Abril, por el que se Aprueba el Procedimiento Básico para la Certiﬁcación de la Eﬁciencia Energética de los Ediﬁcios; Ministry of the Presidency: Madrid, Spain, 2017. 30. Yilmaz, Z. Evaluation of energy efﬁcient design strategies for different climatic zones: Comparison of thermal performance of buildings in temperate-humid and hot-dry climate. Energy Build. 2007, 39, 306–316. [CrossRef] 31. Williams, B.E.B.E. Overcoming Barriers to Energy Efﬁciency for Rental Housing. Ph.D. Thesis, Massachusetts Institute of Technology, Cambridge, MA, USA, 2008. 32. Baek, C.; Park, S. Policy measures to overcome barriers to energy renovation of existing buildings. Renew. Sustain. Energy Rev. 2012, 16, 3939–3947. [CrossRef] 33. UN Environment and International Energy Agency. Global Status Report: Towards a Zero-Emission, Efﬁcient, and Resilient Buildings and Construction Sector; UN Environment and International Energy Agency: Paris, France, 2017. 34. Bianco, V.; Righi, D.; Scarpa, F.; Tagliaﬁco, L.A. Modeling energy consumption and efﬁciency measures in the Italian hotel sector. Energy Build. 2017, 149, 329–338. [CrossRef] 35. De Luxán, M.; Vázquez Espí, M.; Barbero, M.; Román E., Gómez G. Actuaciones con Criterios de Sostenibilidad en la Rehabilitación de Viviendas en el Centro de Madrid; Empresa Municipal de la Vivienda y Suelo de Madrid: Madrid, Spain, 2009; p. 235. 36. Berg, F.; Flyen, A.C.; Godbolt, A.L.; Broström, T. User-driven energy efﬁciency in historic buildings: A review. J. Cult. Herit. 2017, 28, 188–195. [CrossRef] 37. Berg, F.; Fuglseth, M. Life cycle assessment and historic buildings: Energy-efﬁciency refurbishment versus new construction in Norway. J. Archit. Conserv. 2018, 24, 152–167. [CrossRef] 8. International Organization for Standardization. ISO 15686-5:2017—Buildings and Constructed Assets—Ser Life Planning—Part 5: Life-Cycle Costing; ISO: Geneva, Switzerland, 2017. 39. Ost, C.; Van Droogenbroeck, N. Report on Economics of Conservation. References An Appraisal of Theories, Principles and Methods, Center for Economic Research; ICOMOS: Brussels, Belgium; Economics Committee: Brussels, Belgium, 1998. 40. Abo-Elmagd, M.; Saleh, A.; Aﬁﬁ, G. Evaluation of radon related parameters in environmental samples from Jazan city, Saudi Arabia. J. Radiat. Res. Appl. Sci. 2018, 11, 104–110. [CrossRef] 41. Barrionuevo, A.S. Análisis de estrategias bioclimáticas aplicadas a ediﬁcaciones nZEB; University of Miguel Hernández: Elche, Spain, 2019; Volume 4, p. 4. 42. Jalaei, F.; Jrade, A. Integrating Building Information Modeling (BIM) and energy analysis tools with green building certiﬁcation system to conceptually design sustainable buildings. J. Inf. Technol. Constr. 2014, 19, 494–519. , 43. Boussabaine, H.A.; Kirkham, R.J. Whole Life-Cycle Costing; Blackwell Publishing Ltd.: Oxford, UK, 2004. 4. Galiano-Garrigós, A.; Andújar-Montoya, M.D. Building information modelling in operations of maintena at the university of alicante. Int. J. Sustain. Dev. Plan. 2018, 13, 1–11. [CrossRef] 45. Kneifel, J. Life-cycle carbon and cost analysis of energy efﬁciency measures in new commercial buildings. Energy Build. 2010, 42, 333–340. [CrossRef] 46. Luxán García de Diego, M. D.; Gómez Munoz, G.M.; Román López, M.E. Cuentas Energéticas no Habituales en Ediﬁcación Residencial. Inf. Constr. 2016, 67, 125–134. [CrossRef] 47. Diao, L.; Sun, Y.; Chen, Z.; Chen, J. Modeling energy consumption in residential buildings: A bottom-up analysis based on occupant behavior pattern clustering and stochastic simulation. Energy Build. 2017, 147, 47–66. [CrossRef] 48. Hong, T.; Taylor-Lange, S.C.; D’Oca, S.; Yan, D.; Corgnati, S.P. Advances in research and applications of energy-related occupant behavior in buildings. Energy Build. 2016, 116, 694–702. [CrossRef] 49. Zhang, Y.; Bai, X.; Mills, F.P.; Pezzey, J.C. Rethinking the role of occupant behavior in building energy performance: A review. Energy Build. 2018, 172, 279–294. [CrossRef] 27 of 27 Sustainability 2019, 11, 4946 50. Power, A. Does demolition or refurbishment of old and inefﬁcient homes help to increase our environmental, social and economic viability? Energy Policy 2008, 36, 4487–4501. [CrossRef] 51. Tirado Herrero, S.; Jiménez Meneses, L.; López Fernández, J.L.; Martín García, J.; Perero-Van-Hove, E. Pobreza Energética en España. Análisis de Tendencias; Asociación de Ciencias Ambientales: Madrid, Spain, 2014; p. 175. 51. Tirado Herrero, S.; Jiménez Meneses, L.; López Fernández, J.L.; Martín García, J.; Perero-Van-Hove, E. Pobreza Energética en España. Análisis de Tendencias; Asociación de Ciencias Ambientales: Madrid, Spain, 2014; p. 175. 52. Langdon, D. Literature Review of Life Cycle Costing (LCC) and Life Cycle Assessment (LCA); Davis Langdon p Energética en España. References Análisis de Tendencias; Asociación de Ciencias Ambientales: Madrid, Spain, 2014; p. 175. 52. Langdon, D. Literature Review of Life Cycle Costing (LCC) and Life Cycle Assessment (LCA); Davis Langdon Management Consulting: London, UK, 2006. 52. Langdon, D. Literature Review of Life Cycle Costing (LCC) and Life Cycle Assessment (LCA); Davis Langdon Management Consulting: London, UK, 2006. 53. Langdon, D. Life Cycle Costing (LCC) as a Contribution to Sustainable Construction: A Common Methodology; Davis Langdon Management Consulting: London, UK, 2007. 54. Khasreen, M.M.; Banﬁll, P.F.; Menzies, G.F. Life-cycle assessment and the environmental impact of buildings: A review. Sustainability 2009, 1, 674–701. [CrossRef] 55. Fouseki, K.; Cassar, M. Energy Efﬁciency in Heritage Buildings—Future Challenges and Research Needs. Hist. Environ. Policy Pract. 2014, 5, 95–100. [CrossRef] 56. Ferreira, J.V.; Domingos, I. Assessment of Portuguese thermal building legislation in an energetic and environmental perspective. Energy Build. 2011, 43, 3729–3735. [CrossRef] 57. Sousa, G.; Jones, B.M.; Mirzaei, P.A.; Robinson, D. A review and critique of UK housing stock energy models, modelling approaches and data sources. Energy Build. 2017, 151, 66–80. [CrossRef] 58. Zamperini, E.; Cinieri, V. Lifecycle oriented approach for sustainable preservation of historical built heritage. In Proceedings of the International Conference Built Heritage 2013 Monitoring Conservation Management, Milan, Italy, 18–20 November 2013. 59. Meola, C.; Maio, R.D.; Roberti, N.; Carlomagno, G.M. Application of infrared thermography and geophysical methods for defect detection in architectural structures. Eng. Fail. Anal. 2005, 12, 875–892. [CrossRef] 60. Tadeu, S.; Rodrigues, C.; Tadeu, A.; Freire, F.; Simões, N. Energy retroﬁt of historic buildings: Environmental assessment of cost-optimal solutions. J. Build. Eng. 2015, 4, 167–176. [CrossRef] 61. Cabeza, L.; de Gracia, A.; Pisello, A. Integration of renewable technologies in historical and heritage buildings: A review. Energy Build. 2018, 177, 96–111. [CrossRef] 62. Ministerio para la Transición Ecológica–Energía. Análisis de Precisión–Ediﬁcios Nuevos. Available online: https://energia.gob.es/es-es/Paginas/index.aspx (accessed on 15 May 2019). 63. Casals, X.G. Problems of Variable References in the Energy Certiﬁcation of Buildings; Conama 9; Congreso Nacional del Medio Ambiente: Madrid, Spain, 2009. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2792364692	https://www.dora.lib4ri.ch/psi/islandora/object/psi%3A4831/datastream/PDF/Huang-2018-Exploration_of_PM2.5_sources_on-%28published_version%29.pdf	English	null	Exploration of PM&lt;sub&gt;2.5&lt;/sub&gt; sources on the regional scale in the Pearl River Delta based on ME-2 modeling	Atmospheric chemistry and physics	2,018	cc-by	16,919	Correspondence: Ling-Yan He (hely@pku.edu.cn) Correspondence: Ling-Yan He (hely@pku.edu.cn) Received: 11 March 2018 – Discussion started: 20 March 2018 Revised: 23 July 2018 – Accepted: 24 July 2018 – Published: 16 August 2018 Received: 11 March 2018 – Discussion started: 20 March 2018 Revised: 23 July 2018 – Accepted: 24 July 2018 – Published: 16 August 2018 Abstract. The Pearl River Delta (PRD) of China, which has a population of more than 58 million people, is one of the largest agglomerations of cities in the world and had se- vere PM2.5 pollution at the beginning of this century. Due to the implementation of strong pollution control in recent decades, PM2.5 in the PRD has continuously decreased to relatively lower levels in China. To comprehensively under- stand the current PM2.5 sources in the PRD to support fu- ture air pollution control strategies in similar regions, we performed regional-scale PM2.5 ﬁeld observations coupled with a state-of-the-art source apportionment model at six sites in four seasons in 2015. The regional annual average PM2.5 concentration based on the 4-month sampling was de- termined to be 37 µgm−3, which is still more than 3 times the WHO standard, with organic matter (36.9 %) and SO2− 4 (23.6 %) as the most abundant species. A novel multilin- ear engine (ME-2) model was ﬁrst applied to a comprehen- sive PM2.5 chemical dataset to perform source apportion- ment with predetermined constraints, producing more envi- ronmentally meaningful results compared to those obtained using traditional positive matrix factorization (PMF) model- ing. The regional annual average PM2.5 source structure in the PRD was retrieved to be secondary sulfate (21 %), vehi- cle emissions (14 %), industrial emissions (13 %), secondary nitrate (11 %), biomass burning (11 %), secondary organic aerosol (SOA, 7 %), coal burning (6 %), fugitive dust (5 %), ship emissions (3 %) and aged sea salt (2 %). Analyzing the spatial distribution of PM2.5 sources under different weather conditions clearly identiﬁed the central PRD area as the key emission area for SO2, NOx, coal burning, biomass burning, industrial emissions and vehicle emissions. Correspondence: Ling-Yan He (hely@pku.edu.cn) It was further es- timated that under the polluted northerly air ﬂow in winter, local emissions in the central PRD area accounted for ap- proximately 45 % of the total PM2.5, with secondary nitrate and biomass burning being most abundant; in contrast, the regional transport from outside the PRD accounted for more than half of PM2.5, with secondary sulfate representing the most abundant transported species. 1 Introduction With China’s rapid economic growth and urbanization, air pollution has become a serious problem in recent decades. Due to its smaller size, ﬁne particulate matter (PM2.5) can carry toxic chemicals into human lungs and bronchi, caus- ing respiratory diseases and cardiovascular diseases that can harm human health (Sarnat et al., 2008; Burnett et al., 2014). In particular, long-term exposure to high concentrations of ﬁne particulate matter can also lead to premature death (Lelieveld et al., 2015). The Chinese government has at- tached great importance to improving air quality and issued the “Air Pollution Prevention and Control Action Plan” in September 2013, clearly requiring the concentration levels of ﬁne particulate matter in a few key regions, including the Pearl River Delta (PRD), to drop by 2017 from 15 % to 25 % of their values in 2012. The PRD is one of the fastest-growing regions in China and the largest urban agglomeration in Exploration of PM2.5 sources on the regional scale in the Pearl River Delta based on ME-2 modeling Xiao-Feng Huang1, Bei-Bing Zou1, Ling-Yan He1, Min Hu2, André S. H. Prévôt3, and Yuan-Hang Zhang2 1Key Laboratory for Urban Habitat Environmental Science and Technology, School of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen, 518055, China 2State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing, 100871, China 3Paul Scherrer Institute (PSI), 5232 Villigen-PSI, Switzerland Atmos. Chem. Phys., 18, 11563–11580, 2018 https://doi.org/10.5194/acp-18-11563-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. 2.1 Sampling and chemical analysis Samples were collected every other day during a 1-month- long period for each season in 2015, and Table 2 contains the detailed sampling information for reference. Each sam- pling period lasted for 24 h at each site. The sampling sites of University Town (UT) and Dapeng (DP) used Thermo 2300 PM2.5 samplers (Thermo Fisher Scientiﬁc Inc., Waltham, Massachusetts, USA, with a ﬂow rate of 16.7 L min−1 for two channels and a ﬂow rate of 10.0 L min−1 for the other two channels), while those in Modiesha (MDS), Heshan (HS), Qi’ao Island (QA) and Doumen (DM) used TH-16A PM2.5 samplers (Tianhong Corp., Wuhan, China, with a ﬂow rate of 16.7 L min−1 for four channels). Prior to the sam- pling campaigns, the six instruments sampled in parallel three times, and each time lasted for 12 h. The standard de- viation of the PM2.5 mass concentrations obtained by the six samplers in each parallel sampling was within 5 %. The all sample boxes were then sealed with Paraﬁlm, stored in an ice-packed cooler during transportation, and stored un- der freezing temperatures before analysis. A total of 362 valid samples (15–16 samples at each site for each season) were collected in this study. In addition, to track the possi- ble contamination caused by the sampling treatment, a ﬁeld blank sample was collected at each site for each season. The PM2.5 mass can be obtained based on the difference in the weight of the Teﬂon ﬁlter before and after sampling in a clean room at conditions of 20 ◦C and 50 % relative humidity, ac- cording to the Quality Assurance and Quality Control pro- cedures of the National Environmental Protection Standard (NEPS; MEE, 2013b). The Teﬂon ﬁlters were analyzed for their major ion contents (SO2− 4 , NO− 3 , NH+ 4 and Cl−) via an ion chromatography system (ICS-2500, Dionex; Sunnyvale, California, USA), following the guidelines of NEPS (MEE, 2016a, b). The metal element contents (23 species) were an- alyzed via an inductively coupled plasma mass spectrometer (ICP-MS, auroraM90; Bruker, Germany), also following the guidelines of NEPS (MEE, 2013a). 2.1 Sampling and chemical analysis The PRD is located in south central Guangdong Province. Based on the layout of the cities in the PRD, six sampling sites were selected to represent urban, suburban, and back- ground sites. Detailed descriptions of these sampling sites are listed in Table 1, and their locations are shown on the regional map in Fig. 1. In recent years, the receptor model method (commonly, positive matrix factorization, PMF) in the PRD was applied to perform the source apportionment of PM2.5, which was carried out in several major cities, including Guangzhou (Gao et al., 2013; Liu et al., 2014; Wang et al., 2016), Shenzhen (X. F. Huang et al., 2014), Dongguan (Wang et al., 2015; Zou et al., 2017) and Foshan (Tan et al., 2016). However, the above source apportionment studies only fo- cused on part of PM2.5 (e.g., organic matter) or a single city in the PRD (e.g., Shenzhen and Dongguan), lacking the extensive representation of the PRD region in terms of si- multaneous sampling in multiple cities. Since the lifetime of PM2.5 in the surface layer of the atmosphere is days to weeks and the cities in the PRD are closely linked, the transport of PM2.5 between cities is speciﬁcally noteworthy (Hagler et al., 2006). Conversely, although the PMF model has been successfully applied to source apportionment in the PRD, the apportionment with PMF has high rotational ambigu- ity and can output non-meaningful or mixed factors. Under such conditions, the multilinear engine (ME-2) model can guide the rotation toward a more objective optimal solution by utilizing a priori information (i.e., predetermined factor proﬁles). In recent years, ME-2, initiated and controlled via the Source Finder (SoFi) written by the Paul Scherrer Insti- tute, was successfully developed to apportion the sources of organic aerosols (Canonaco et al., 2013). The novel ME-2 model has become a widely used and successful source anal- ysis technique (e.g., Crippa et al., 2014; Fröhlich et al., 2015; Visser et al., 2015; Elser et al., 2016; Reyes-Villegas et al., 2016). The key challenges in running ME-2 are the con- struction of the appropriate constraint source proﬁles and the determination of factor numbers, and PMF could serve as the ﬁrst step when using ME-2 for the determination of the a pri- ori information needed. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD the world; it includes the cities of Guangzhou, Shenzhen, Zhuhai, Dongguan, Foshan, Huizhou, Zhongshan, Zhaoqing, and Jiangmen and contains more than 58 million people. The PM2.5 concentration in this region reached a high level of 58 µgm−3 in 2007 (Nanfang Daily, 2016); however, the air quality has signiﬁcantly improved due to the implementa- tion of strict air pollution control measures, which were im- plemented earlier here than in other regions in China. The annual average concentration of PM2.5 in the PRD dropped to 34 µgm−3 in 2015 (Ministry of Environmental Protection, 2016). elemental carbon (EC), organic mass (OM), inorganic ions and metal elements) to identify the sources of bulk PM2.5 on the regional scale of the PRD; then, the spatial locations of the sources were systematically explored using the analysis of weather conditions. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 11564 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD X. F. Huang et al.: Source apportionment of PM2.5 in PRD Table 1. Description of the sampling sites in the PRD. Site Site code Coordinates Site description Doumen DM Lat: 22.23◦N Suburban Contains industrial areas Long: 113.30◦E Qi’ao Island QA Lat: 22.43◦N Background An area for ecotourism Long: 113.63◦E Heshan HS Lat: 22.73◦N Suburban Contains industrial areas and farmlands Long: 112.93◦E Modiesha MDS Lat: 23.11◦N Urban Contains dense urban trafﬁc Long: 113.33◦E University town UT Lat: 22.59◦N Urban Contains urban trafﬁc Long: 113.98◦E Dapeng DP Lat: 22.63◦N Background An area for ecotourism Long: 114.41◦E Figure 1. Spatial distribution of the sampling sites in the PRD. Table 1. Description of the sampling sites in the PRD. Figure 1. Spatial distribution of the sampling sites in the PRD. Figure 1. Spatial distribution of the sampling sites in the PRD. back trajectories of the air masses obtained using the NOAA HYSPLIT model (Fig. S1 in the Supplement) revealed that the air masses originated from the northern inland in winter, from the northern inland and the South China Sea in spring, from the South China Sea in summer, and from the northeast coast and the northern inland in fall. Nevada, USA), following the IMPROVE protocol (Chow et al., 1993). The overall OM was estimated as 1.8× OC. In a previous aerosol mass spectrometer (AMS) measurement for PM1, the OM / OC ratio was measured to be 1.6 for an urban atmosphere (He et al., 2011) and 1.8 for a rural atmosphere (Huang et al., 2011). We adopted a uniform OM / OC ratio of 1.8 in this study because it is assumed that the mass dif- ference between PM1 and PM2.5 may mostly contain aged regional aerosol with higher OM / OC. Changes in meteorological conditions with the seasons have signiﬁcant inﬂuences on the air quality in the PRD (Hagler et al., 2006). The same type of weather is often re- peated. Physick and Goudey (2001) classiﬁed the weather over the region surrounding Hong Kong into seven cate- gories based on surface pressure patterns, i.e., as northerly (winter monsoon), northeasterly (winter monsoon), easterly or southeasterly, trough, southerly or southwesterly (sum- mer monsoon), and cyclonic 1 and cyclonic 2 weather types. The PRD region, including Hong Kong, has nearly similar weather patterns and similar meteorological conditions. In 2.1 Sampling and chemical analysis The Quartz ﬁlters were analyzed for organic carbon (OC) and EC contents using an OC–EC analyzer (2001A, Desert Research Institute, Reno, Accurately understanding the regional characteristics of PM2.5 sources in the PRD can certainly guide the regional joint prevention and control of PM2.5 in this region and pro- vide useful references for future air pollution control strate- gies in China. Thus, in this study, the PM2.5 mass and chemi- cal compositions were measured during four seasons in 2015 at six sites in the PRD, which basically represent the pollu- tion level of the PRD on a regional scale rather than on a city scale. For the ﬁrst time, the novel ME-2 model via the SoFi was applied to a comprehensive chemical dataset (including Atmos. Chem. Phys., 18, 11563–11580, 2018 www.atmos-chem-phys.net/18/11563/2018/ 11565 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Table 1. Description of the sampling sites in the PRD. Site Site code Coordinates Site description Doumen DM Lat: 22.23◦N Suburban Contains industrial areas Long: 113.30◦E Qi’ao Island QA Lat: 22.43◦N Background An area for ecotourism Long: 113.63◦E Heshan HS Lat: 22.73◦N Suburban Contains industrial areas and farmlands Long: 112.93◦E Modiesha MDS Lat: 23.11◦N Urban Contains dense urban trafﬁc Long: 113.33◦E University town UT Lat: 22.59◦N Urban Contains urban trafﬁc Long: 113.98◦E Dapeng DP Lat: 22.63◦N Background An area for ecotourism Long: 114.41◦E Figure 1. Spatial distribution of the sampling sites in the PRD. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Atmos. Chem. Phys., 18, 11563–11580, 2018 www.atmos-chem-phys.net/18/11563/2018/ Table 3. Sampling days categorized as southerly ﬂow and northerly ﬂow days. Table 3. Sampling days categorized as southerly ﬂow and northerly ﬂow days. Table 3. Sampling days categorized as southerly ﬂow and northerly ﬂow days. Southerly Wind speed PM2.5 Northerly Wind speed PM2.5 ﬂow (m s−1) (µgm−3) ﬂow (m s−1) (µgm−3) 1 Jul 2015 2.6 16 18 Jan 2015 2.3 78 3 Jul 2015 3.6 17 20 Jan 2015 1.5 82 15 Jul 2015 1.9 17 3 Feb 2015 2 75 23 Jul 2015 2.6 12 7 Feb 2015 1.7 101 25 Jul 2015 2 13 9 Feb 2015 2.2 75 29 Jul 2015 1.3 12 where eij are the elements of the residual matrix E, and uij are the errors/uncertainties of the measured species xij. this study, the daily weather types during the observation pe- riod (excluding rainy days) were also classiﬁed into seven categories based on surface pressure patterns. However, ac- cording to the surface horizontal wind vectors, the PRD was mostly impacted by two types of airﬂow, i.e., southerly ﬂow and northerly ﬂow. Southerly ﬂow, including the south- easterly and southerly or southwesterly (summer monsoon) weather types, was relatively clean and originated from the ocean (e.g., Figs. S2 and S4). Northerly ﬂow, including the northerly (winter monsoon) and northeasterly (winter mon- soon) weather types, was relatively polluted and originated from the north mainland (e.g., Figs. S3 and S5). Southerly ﬂow and northerly ﬂow appeared with the highest frequency in the PRD (i.e., above 80 %), followed by cyclone (10 %), easterly (2 %) and trough (2 %). In this study, southerly ﬂow days (PM2.5 ≤17 µgm−3; see Table 3) were selected to bet- ter reﬂect the local source regions in the PRD, and northerly ﬂow days (PM2.5 ≥75 µgm−3; see Table 3) were selected to better understand the pollution accumulation process and re- gional transport characteristics of pollutants in the PRD. The sampling days for southerly ﬂow and northerly ﬂow are listed in Table 3. X = G × F + E (1) Q = Xn i=1 Xm j=1 eij uij 2 (2) (1) (2) (2) The multilinear engine (ME-2) was later developed by Paatero (1999) based on the PMF algorithm. In contrast to an unconstrained PMF analysis, ME-2 can utilize the con- straints (i.e., predetermined factor proﬁles) provided by the user to enhance the control of rotation for a more objective solution. Table 3. Sampling days categorized as southerly ﬂow and northerly ﬂow days. One or more factor proﬁles can be expediently input into ME-2, and the output proﬁles are allowed to vary from the input proﬁles to some extent. When using ME-2 model- ing, the mixed factors can usually be better resolved. In this study, both PMF and ME-2 models were run for the datasets observed in the PRD. We ﬁrst need to deter- mine the species input into the models. Species that may lead to high species residuals or lower R2 values between measured and model-predicted or non-meaningful factors, such as those that fulﬁlled the following criteria, were not included: (1) species that were below detection in more than 40 % of samples, (2) species that yielded R2 values of less than 0.4 in interspecies correlation analysis, and (3) species that had little implication for pollution sources and lower concentrations. Therefore, 18 species were input into the models; these species accounted for 99.6 % of the total mea- sured species and included OM, EC, SO2− 4 , NO− 3 , NH+ 4 , Cl−, K, Ca, Na, Mg, Al, Zn, Fe, Cd, V, Ni, Ti and Pb. 2.2 Meteorological conditions and weather classiﬁcation The meteorological conditions during the observation period, shown in Table 2, indicated that the PRD region experienced a hot and humid summer and a cool and dry winter, while spring and fall were two transition seasons. Furthermore, the Atmos. Chem. Phys., 18, 11563–11580, 2018 www.atmos-chem-phys.net/18/11563/2018/ 11566 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Table 2. General meteorological conditions during the observation period in the PRD. Mean temp. Rainfall Mean RH Mean wind Predominant (◦C) (mm) (%) speed (m s−1) wind direction Winter (10 Jan–9 Feb) 17 35 63 % 2.1 ENE Spring (2 Apr–30 Apr) 23 61 72 % 1.8 SSW Summer (1 Jul–29 Jul) 29 244 74 % 2.1 SW Fall (11 Oct–10 Nov) 25 92 68 % 1.7 NNE Table 3. Sampling days categorized as southerly ﬂow and northerly ﬂow days. Southerly Wind speed PM2.5 Northerly Wind speed PM2.5 ﬂow (m s−1) (µgm−3) ﬂow (m s−1) (µgm−3) 1 Jul 2015 2.6 16 18 Jan 2015 2.3 78 3 Jul 2015 3.6 17 20 Jan 2015 1.5 82 15 Jul 2015 1.9 17 3 Feb 2015 2 75 23 Jul 2015 2.6 12 7 Feb 2015 1.7 101 25 Jul 2015 2 13 9 Feb 2015 2.2 75 29 Jul 2015 1.3 12 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11566 Table 2. General meteorological conditions during the observation period in the PRD. Mean temp. Rainfall Mean RH Mean wind Predominant (◦C) (mm) (%) speed (m s−1) wind direction Winter (10 Jan–9 Feb) 17 35 63 % 2.1 ENE Spring (2 Apr–30 Apr) 23 61 72 % 1.8 SSW Summer (1 Jul–29 Jul) 29 244 74 % 2.1 SW Fall (11 Oct–10 Nov) 25 92 68 % 1.7 NNE ble 2. General meteorological conditions during the observation period in the PRD. 2.3 Input data matrices for source apportionment modeling The uncertainties of SO2− 4 , NH+ 4 and all metal elements, which have scaled residuals larger than ±3 due to the small analytical uncertainties, need to be increased to re- duce their weights in the solution (Norris and Duvall, 2014). In addition, the uncertainties of EC caused by pyrolyzed car- bon (PC) and the uncertainties of OM, NO− 3 and Cl−due to semi-volatility under high ambient temperatures should also be taken into account (Cao et al., 2018). In this study, more reasonable source proﬁles can be obtained when further in- creasing the estimated uncertainties (uc) of all species by a factor of 2. where uf is the relative error of the sampling volume, ur is the relative error of the repeatability analysis of the standard species, and ue is the relative error of the ion extraction of multiple samples. When the concentration of the species is below the detection limit (DL), the concentration values were replaced by 1/2 of the DL, and the corresponding uncertain- ties were set at 5/6 of the DL. Missing values were replaced by the geometric mean of the species with corresponding uncertainties of 4 times their geometric mean (Polissar et al., 1998). The uncertainties of SO2− 4 , NH+ 4 and all metal elements, which have scaled residuals larger than ±3 due to the small analytical uncertainties, need to be increased to re- duce their weights in the solution (Norris and Duvall, 2014). In addition, the uncertainties of EC caused by pyrolyzed car- bon (PC) and the uncertainties of OM, NO− 3 and Cl−due to semi-volatility under high ambient temperatures should also be taken into account (Cao et al., 2018). In this study, more reasonable source proﬁles can be obtained when further in- creasing the estimated uncertainties (uc) of all species by a factor of 2. SoFi is a user-friendly interface developed by PSI for ini- tiating and controlling ME-2 (Canonaco et al., 2013), and it can conveniently constrain multiple factor proﬁles. Although the U.S. EPA PMF v5.0 can also use some a priori informa- tion (such as the ratio of elements in factor) to control the rotation after the base run, it is not able to use multiple con- strained factor proﬁles to control the rotation (Norris and Du- vall, 2014). Therefore, SoFi is a more convenient and pow- erful tool to establish various constrained factors for source apportionment modeling. 2.3 Input data matrices for source apportionment modeling PMF is a multivariate factor analysis tool widely used for aerosol source apportionment. The PMF algorithm groups the measured matrix X (Eq. 1) into two nonnegative constant matrices G (factor time series) and F (factor proﬁles), and E denotes the model residuals (Paatero and Tapper, 1994). The entries in G and F are ﬁtted using a least-squares algorithm that iteratively minimizes the object function Q in Eq. (2), The application of PMF or ME-2 also depends on the esti- mated realistic uncertainty (uij) of the individual data point of an input matrix, which determines the Q value in Eq. (2). Therefore, the estimation of uncertainty is an important com- Atmos. Chem. Phys., 18, 11563–11580, 2018 www.atmos-chem-phys.net/18/11563/2018/ X.-F. Huang et al.: Source apportionment of PM2.5 in PRD X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11567 Table 4. The constraints of factor species for ME-2 modeling. Factors OM EC Cl− NO− 3 SO2− 4 NH+ 4 Ca Ti V Ni Zn Cd Pb Na Mg Al K Fe Secondary sulfate – 0 0 0 – – 0 0 0 0 0 0 0 0 0 0 0 0 Secondary nitrate – 0 0 – 0 – 0 0 0 0 0 0 0 0 0 0 0 0 Sea salt 0 0 – – – 0 – 0 0 0 0 0 0 – – 0 – 0 Fugitive dust 0 0 0 0 0 0 – – 0 0 0 0 0 – – – – – 11567 Table 4. The constraints of factor species for ME-2 modeling. ponent of the application of these models. There are many sources of uncertainty, including sampling, handling, trans- port, storage, preparation and testing (Leiva et al., 2012). In this study, the sources of uncertainty that contributed little to the total uncertainty could be neglected, such as replacing ﬁl- ters, sample transport and sample storage under strict quality assurance and quality control. Therefore, we ﬁrst considered the uncertainties introduced by sampling and analysis pro- cesses, such as sampling volume, repeatability analysis and ion extraction. The species uncertainties uij are estimated using Eq. (3), where uc is the error fraction of the species, which is estimated using the relative combined error formula Eq. (4) (BIPM et al., 2008). proximately symmetrically distributed between −3 and +3 (Fig. S6) and the most interpretable factor proﬁles (Fig. S7). 2.3 Input data matrices for source apportionment modeling Using the same species concentra- tion matrix and uncertainties matrix, we ran the ME-2 model via SoFi for 9–12 factors with the four factors constrained as described above, as shown in Table 4. The following consid- erations were used. Secondary sulfate and secondary nitrate factors should theoretically not contain species from primary particulates, but they may contain secondary organic matter related to the secondary conversion process of SO2 and NOx (He et al., 2011; Z. B. Yuan et al., 2006; X. F. Huang et 2.3 Input data matrices for source apportionment modeling The model-input total mass of the 18 species and the model- reconstructed total mass of all the factors showed a high cor- relation (R2 = 0.97, slope = 1.01) (Fig. S8). The factor of biomass burning was not extracted in the eight-factor solu- tion, while the factor of fugitive dust was separated into two non-meaningful factors when more factors were set to run PMF. For the nine-factor solution of secondary sulfate-rich aerosol, secondary nitrate-rich aerosol, aged sea salt, fugi- tive dust, biomass burning, vehicle emissions, coal burning, industrial emissions and ship emissions, the source judgment based on tracers for each factor was identical to that of the ME-2 results detailed in Sect. 3.2. However, in Fig. S7, some factors seemed to be mixed by some unexpected components and were thus overestimated. For example, the secondary sulfate-rich and secondary nitrate-rich factors of PMF had certain species from primary particulates, such as EC, Zn, Al, K and Fe, among which EC had obvious percentage ex- plained variation (EV) values, i.e., the percent of a species apportioned to the factor, of 18.7 % and 9.7 %; the EV value of OM in the sea salt factor (which was theoretically negligi- ble) had a high value of 6.4 %, and OM accounted for 37 % of the total mass of this factor; the EV value of SO2− 4 in the fugitive dust factor (which was theoretically negligible) had a high value of 8.6 %, and the SO2− 4 concentration accounted for 26 % of the total mass of this factor. uij = uc × xij, (3) uc = q u2 f + u2 r + u2 e, (4) (3) (4) where uf is the relative error of the sampling volume, ur is the relative error of the repeatability analysis of the standard species, and ue is the relative error of the ion extraction of multiple samples. When the concentration of the species is below the detection limit (DL), the concentration values were replaced by 1/2 of the DL, and the corresponding uncertain- ties were set at 5/6 of the DL. Missing values were replaced by the geometric mean of the species with corresponding uncertainties of 4 times their geometric mean (Polissar et al., 1998). 2.4 Constraint setup in ME-2 modeling In this study, the U.S. EPA PMF v5.0 was applied with the concentration matrix and uncertainties matrix described above to identify the PM2.5 sources. After examining a range of factor numbers from 3 to 12, the nine-factor solution out- put by the PMF base run (Qtrue / Qexp = 2.5) was found to be the optimal solution, with the scaled residuals ap- Atmos. Chem. Phys., 18, 11563–11580, 2018 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD OM 36.9 % EC 6.6 % SO₄²⁻ 23.6 % Ca 0.6 % Al 0.6 % K 1.5 % Na 1.1 % Fe 0.7 % NO₃⁻ 9.3 % NH₄⁺ 10.9 % Cl⁻ 0.9 % Others 6.2 % Mn 0.05 % Cu 0.06 % Zn 0.47 % Mg 0.15 % Pb 0.11 % Ti 0.05 % V 0.03 % Cr 0.02 % Co 0.001 % Ni 0.01 % As 0.005 % Se 0.008 % Mo 0.002 % Cd 0.003 % Tl 0.0007 % Th 0.0002 % U 0.0001 % Ba 0.02 % Trace elements 1.0 % PM2.5 = 37 μg m ³ - Trace elements 1.0 % PM2.5 = 37 μg m ³ - Figure 2. Chemical compositions of 4-month average PM2.5 in the PRD region. with sea salt to displace Cl−(Huang et al., 2006); thus, NO− 3 was also not constrained in the sea salt factor. al., 2014). Therefore, the contributions of the species from primary particulates were constrained to zero in the input secondary aerosol factors, while others were not constrained. In addition, the factors of sea salt and fugitive dust in pri- mary aerosols could be understood based on the abundance of species in seawater and the upper crust (Mason, 1982; Tay- lor and Mclennan, 1995). As seen in Table S1 in the Supple- ment, the abundances of Cl−, Na+, SO2− 4 , Mg2+, Ca2+ and K+ in sea salt were relatively high, as were the abundances of Al, Fe, Ca, Na, K, Mg and Ti in fugitive dust. There- fore, these high-abundance species were not constrained in the sea salt and fugitive dust factors, while the other species (with abundances of less than 0.1 % in the particulates) were constrained to zero (Table 4). In addition, HNO3 might react X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11568 1568 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Table 5. The comparison of the major chemical compositions of PM2.5 in typical cities (unit: µgm−3). Cities Periods PM2.5 OC EC SO2− 4 NO− 3 NH+ 4 References Zhuhai (DM) Jan 2015–Nov 2015 35 6.4 2.3 8.1 4.4 3.6 This study Zhuhai (QA) 37 7.2 2.2 9.9 3.5 4.4 Jiangmen (HS) 47 9.0 2.8 9.8 5.6 5.0 Guangzhou (MDS) 41 9.3 2.7 9.2 3.7 4.6 Shenzhen (UT) 37 7.8 3.0 8.0 2.6 3.7 Shenzhen (DP) 28 6.2 1.8 8.0 1.1 3.3 Hong Kong (urban) Oct 2002–Jun 2003 34.3 6.6 1.9 9.3 1.0 2.5 Hagler et al. (2006) Shenzhen (urban) 47.1 11.1 3.9 10.0 2.3 3.2 Guangzhou (urban) 70.6 17.6 4.4 14.7 4.0 4.5 Beijing Jun 2014–Apr 2015 99.5 15.5 6.2 14.3 17.9 11.5 Huang et al. (2017) Shanghai Sep 2013–Aug 2014 94.6 9.89 1.63 14.5 18.0 8.13 Ming et al. (2017) Chengdu, Sichuan Oct 2014–Jul 2015 67.0 10.9 3.6 11.2 9.1 7.2 Wang et al. (2018) Paris, France Sep 2009–Sep 2010 14.8 3.0 1.4 2.0 2.9 1.4 Bressi et al. (2013) London, UK Dec 2003–Apr 2005 31.0 5.6 1.6 2.8 3.5 2.1 Rodríguez et al. (2007) Los Angeles, US 2002–2013 17.1 2.2 1.3 2.7 4.9 0.1 Hasheminassab et al. (2014) Santiago, Chile Mar 2013–Oct 2013 40 12.1 4.3 1.9 7.1 3.3 Villalobos et al. (2015) Chuncheon, Korea Jan 2013–Dec 2014 34.6 9.0 1.6 3.9 2.8 2.0 Cho et al. (2016) OM 36.9 % EC 6.6 % SO₄²⁻ 23.6 % Ca 0.6 % Al 0.6 % K 1.5 % Na 1.1 % Fe 0.7 % NO₃⁻ 9.3 % NH₄⁺ 10.9 % Cl⁻ 0.9 % Others 6.2 % Mn 0.05 % Cu 0.06 % Zn 0.47 % Mg 0.15 % Pb 0.11 % Ti 0.05 % V 0.03 % Cr 0.02 % Co 0.001 % Ni 0.01 % As 0.005 % Se 0.008 % Mo 0.002 % Cd 0.003 % Tl 0.0007 % Th 0.0002 % U 0.0001 % Ba 0.02 % Trace elements 1.0 % PM2.5 = 37 μg m ³ - Figure 2. Chemical compositions of 4-month average PM2.5 in the PRD region. Table 5. The comparison of the major chemical compositions of PM2.5 in typical cities (unit: µgm−3). 3.1 Spatiotemporal variations in PM2.5 in the PRD The 4-month average PM2.5 concentration for all six sites in the PRD was 37 µgm−3, which was slightly higher than the Grade II national standards for air quality (with an an- nual mean of 35 µgm−3). The chemical compositions of PM2.5 in the PRD are shown in Fig. 2. OM had the high- est contribution of 36.9 %, suggesting severe organic pollu- tion in the PRD. Other important components included SO2− 4 Atmos. Chem. Phys., 18, 11563–11580, 2018 www.atmos-chem-phys.net/18/11563/2018/ www.atmos-chem-phys.net/18/11563/2018/ www.atmos-chem-phys.net/18/11563/2018/ 11568 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Table 5. The comparison of the major chemical compositions of PM2.5 in typical cities (unit: µgm−3). Cities Periods PM2.5 OC EC SO2− 4 NO− 3 NH+ 4 References Zhuhai (DM) Jan 2015–Nov 2015 35 6.4 2.3 8.1 4.4 3.6 This study Zhuhai (QA) 37 7.2 2.2 9.9 3.5 4.4 Jiangmen (HS) 47 9.0 2.8 9.8 5.6 5.0 Guangzhou (MDS) 41 9.3 2.7 9.2 3.7 4.6 Shenzhen (UT) 37 7.8 3.0 8.0 2.6 3.7 Shenzhen (DP) 28 6.2 1.8 8.0 1.1 3.3 Hong Kong (urban) Oct 2002–Jun 2003 34.3 6.6 1.9 9.3 1.0 2.5 Hagler et al. (2006) Shenzhen (urban) 47.1 11.1 3.9 10.0 2.3 3.2 Guangzhou (urban) 70.6 17.6 4.4 14.7 4.0 4.5 Beijing Jun 2014–Apr 2015 99.5 15.5 6.2 14.3 17.9 11.5 Huang et al. (2017) Shanghai Sep 2013–Aug 2014 94.6 9.89 1.63 14.5 18.0 8.13 Ming et al. (2017) Chengdu, Sichuan Oct 2014–Jul 2015 67.0 10.9 3.6 11.2 9.1 7.2 Wang et al. (2018) Paris, France Sep 2009–Sep 2010 14.8 3.0 1.4 2.0 2.9 1.4 Bressi et al. (2013) London, UK Dec 2003–Apr 2005 31.0 5.6 1.6 2.8 3.5 2.1 Rodríguez et al. (2007) Los Angeles, US 2002–2013 17.1 2.2 1.3 2.7 4.9 0.1 Hasheminassab et al. (2014) Santiago, Chile Mar 2013–Oct 2013 40 12.1 4.3 1.9 7.1 3.3 Villalobos et al. (2015) Chuncheon, Korea Jan 2013–Dec 2014 34.6 9.0 1.6 3.9 2.8 2.0 Cho et al. (2016) OM 36.9 % EC 6.6 % SO₄²⁻ 23.6 % Ca 0.6 % Al 0.6 % K 1.5 % Na 1.1 % Fe 0.7 % NO₃⁻ 9.3 % NH₄⁺ 10.9 % Cl⁻ 0.9 % Others 6.2 % Mn 0.05 % Cu 0.06 % Zn 0.47 % Mg 0.15 % Pb 0.11 % Ti 0.05 % V 0.03 % Cr 0.02 % Co 0.001 % Ni 0.01 % As 0.005 % Se 0.008 % Mo 0.002 % Cd 0.003 % Tl 0.0007 % Th 0.0002 % U 0.0001 % Ba 0.02 % Trace elements 1.0 % PM2.5 = 37 μg m ³ - Figure 2. Chemical compositions of 4-month average PM2.5 in the PRD region. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD X.-F. Huang et al.: Source apportionment of PM2.5 in PRD OM EC SO₄²¯ NO₃¯ Cl¯ Metal elements Others NH₄⁺ MDS ( 41) HS (47) DM (35) QA (37) UT(37) DP(28) (a) 40 % 6 % 29 % 4 % 12 % 0.3% 5 % 4 % 33 % 7 % 23 % 13 % 10 % 1.2 % 7 %6 % 35 % 6% 27 % 10 % 12 % 0.7 % 6 %3 % 38 % 8% 21 % 7 % 10 % 0.6 % 5 %10 % 41 % 7 % 22 % 9 % 11 % 1.0 % 4 %5 % 35 % 6 % 21 % 12 % 11 % 1.3% 6 % 8 % 0 0.5 1 1.5 2 2.5 3 Winter Spring Summer Fall OM EC SO₄²ˉ NO₃ˉ NH₄⁺ Clˉ Metal elements OM EC SO₄²¯ NO₃¯ Cl¯ Metal elements Others NH₄⁺ MDS ( 41) HS (47) DM (35) QA (37) UT(37) DP(28) (a) (b) 40 % 6 % 29 % 4 % 12 % 0.3% 5 % 4 % 33 % 7 % 23 % 13 % 10 % 1.2 % 7 %6 % 35 % 6% 27 % 10 % 12 % 0.7 % 6 %3 % 38 % 8% 21 % 7 % 10 % 0.6 % 5 %10 % 41 % 7 % 22 % 9 % 11 % 1.0 % 4 %5 % 35 % 6 % 21 % 12 % 11 % 1.3% 6 % 8 % Season ⁄ 4-month average Figure 3. The spatial distributions of (a) and seasonal variations in (b) the PM2.5 chemical compositions in the PRD. Sizes of the pie charts indicate the concentrations of PM2.5 at the six sites, with the detailed numbers (unit: µgm−3) in brackets. (a) 0 0.5 1 1.5 2 2.5 3 Winter Spring Summer Fall OM EC SO₄²ˉ NO₃ˉ NH₄⁺ Clˉ Metal elements (b) Season ⁄ 4-month average Figure 3. The spatial distributions of (a) and seasonal variations in (b) the PM2.5 chemical compositions in the PRD. Sizes of the pie charts indicate the concentrations of PM2.5 at the six sites, with the detailed numbers (unit: µgm−3) in brackets. (Huang et al., 2017) in northern China, Shanghai (Ming et al., 2017) in eastern China, and Chengdu (Wang et al., 2018) in western China. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD However, the PM2.5 concentrations in ur- ban Guangzhou and Shenzhen observed in this study were clearly higher than those in famous megacities in developed countries, such as Paris (Bressi et al., 2013), London (Ro- dríguez et al., 2007) and Los Angeles (Hasheminassab et al., 2014), while they were similar to those of Santiago (Vil- lalobos et al., 2015) and Chuncheon (Cho et al., 2016). It should be highlighted that the higher concentration of SO2− 4 in the urban atmosphere of the PRD is one of the major rea- sons leading to the higher degree of PM2.5 pollution in the PRD compared to that in developed cities. Table 5 summarizes some previous studies that used sim- ilar ﬁlter-sampling and analytical methods to allow for a better comparison with this study. In 2002–2003, Hagler et al. (2006) also conducted observations and analysis of PM2.5 in the PRD and Hong Kong region, nearly 12 years before this study, as shown in Table 5. Compared with Hagler’s re- sults, the PM2.5 concentrations in this study decreased by 42 % in Guangzhou (MDS) and 21 % in Shenzhen (UT), es- pecially OC, EC and SO2− 4 , which decreased signiﬁcantly by 20 %–47 %, indicating that the measures taken to desul- furize coal-ﬁred power plants, improve the fuel standards of motor vehicles, and phase-out older and more polluting vehicles have played important roles in improving the air quality in the PRD region (People’s Government of Guang- dong Province, 2012). Compared with the PM2.5 concen- trations reported by other cities in China in recent years, the PM2.5 concentrations in urban Guangzhou and Shenzhen in this study were 39 %–63 % lower than those in Beijing X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11569 (23.6 %), NH+ 4 (10.9 %), NO− 3 (9.3 %), EC (6.6 %) and Cl− (0.9 %). The major metallic components included K (1.5 %), Na (1.1 %), Fe (0.7 %), Al (0.6 %), and Ca (0.6 %), and trace elements accounted for 1.0 %. Figure 3a shows the spatial distribution of the PM2.5 and chemical components among the six sites. The PM2.5 pollution level in the PRD was dis- tinctly higher in the northwestern hinterland (HS and MDS) and lower in the southern coastal areas (DM and DP). The DP background site had little local emissions and was hardly in- ﬂuenced by the emissions from the PRD under both southerly ﬂow and northerly ﬂow. Thus, DP air pollution reﬂects the large-scale regional air pollution. The average PM2.5 con- centration at DP was as high as 28 µgm−3, indicating that the PRD had a large amount of air pollution transported from outside this region. At the background DP site, the fractions of Cl−and NO− 3 in PM2.5 were the lowest of the six sites, i.e., 0.3 % and 3.9 %, respectively, suggesting that they had dominantly local sources in the PRD. The highest concen- tration level of PM2.5 was observed at HS (suburban), which was inﬂuenced by the pollution transport of Foshan (indus- trial city) and Guangzhou (metropolis) under the northeast- ern wind, which is the most frequent wind in the PRD. The back trajectories of the air masses (Fig. S1) show that the northern monsoon prevails in winter and the southern mon- soon prevails in summer in the PRD. Under the winter mon- soon, the air masses mostly came from inland and carried higher concentrations of air pollutants. However, under the summer monsoon, the air masses largely originated from the South China Sea and were clean. In addition, the frequent rainfall and higher planetary boundary layer (PBL) in sum- mer in the PRD also favored the dispersion and removal of air pollutants (X. F. Huang et al., 2014). Figure 3b shows that the normalized seasonal variations in the major compo- nents in PM2.5 in the PRD were evidently higher in winter and lower in summer, which is consistent with the seasonal variations in the monsoon and other meteorological factors as mentioned above. 3.2 Source apportionment of PM2.5 using ME-2 The solutions of 9–12 factors of the ME-2 were modeled with the four factors constrained in Table 4, using the SoFi tool, an implementation of ME-2 (Canonaco et al., 2013). Again, the nine-factor solution provided the most reasonable source proﬁles since uninterpretable factors were produced www.atmos-chem-phys.net/18/11563/2018/ www.atmos-chem-phys.net/18/11563/2018/ Atmos. Chem. Phys., 18, 11563–11580, 2018 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11570 Table 6. Comparison of the results of source apportionment of PM2.5 in the PRD. Cities Periods Model Results References Shenzhen Jan–Nov 2015 ME-2 Secondary sulfate (21 %), secondary nitrate (8 %) and SOA (7 %), This study (Four seasons) vehicle emissions (17 %), industrial emissions (11 %), biomass burning (9 %), coal burning (3 %), fugitive dust (6 %), ship emissions (3 %), and aged sea salt (1 %). Shenzhen Jan–Dec 2009 PMF Secondary sulfate (30.0 %), vehicular emissions (26.9 %), X. F. Huang et al. (2014) (Four seasons) biomass burning (9.8), secondary nitrate (9.3 %), high chloride (3.8 %), heavy oil combustion (3.6 %), sea salt (2.6 %), dust (2.5 %), metallurgical industry (2.1 %). Guangzhou Jan–Nov 2015 ME-2 Secondary sulfate (23 %), secondary nitrate (11 %), SOA (7 %), This study (Four seasons) vehicle emissions (18 %), industrial emissions (11 %), biomass burning (8 %), coal burning (6 %), fugitive dust (3 %), ship emissions (2 %) and aged sea salt (1 %). Guangzhou Jan–Dec 2014 PMF Secondary sulfate and biomass burning (38 %), ship emissions (17 %), Tao et al. (2017) (Four seasons) coal combustion (15 %), trafﬁc emissions (10 %), secondary nitrate and chloride (12 %), soil dust (7 %). Guangzhou Jan–Feb 2015 ME-2 Secondary sulfate (20 %), secondary nitrate (16 %), SOA (8 %), This study (Winter) vehicle emissions (11 %), industrial emissions (13 %), biomass burning (6 %), coal burning (9 %), fugitive dust (2 %), ship emissions (1 %) and aged sea salt (1 %). Guangzhou Jan 2013 ME-2 Secondary inorganic-rich aerosol (59.0 %), R. Huang et al. (2014) (Winter) secondary organic-rich aerosol (18.1 %), trafﬁc (8.6 %), coal burning (3.4 %), biomass burning (6.7 %), cooking (0.8 %), dust-related aerosol (3.4 %). Dongguan Dec 2013–Nov 2014 PMF Secondary sulfate (20 %), secondary nitrate (8 %), SOA (10 %), Zou et al. (2017) (Four seasons) vehicle emissions (21 %), industrial emissions (7 %), biomass burning (11 %), coal burning (5 %), fugitive dust (8 %), ship emissions (6 %). Dongguan Feb 2010–Dec 2012 PMF Secondary sulfate (27 %), secondary nitrate (19 %), Wang et al. www.atmos-chem-phys.net/18/11563/2018/ (2015) (Four seasons) industrial emissions (15 %), biomass burning (9 %) and coal combustion (9 %); ship emissions and sea salt, vehicle exhaust, plastic burning and dust no more than 7 %. Table 6. Comparison of the results of source apportionment of PM2.5 in the PRD. large EV values of OM and EC, as well as a certain range of EV values of Fe and Zn, which were related to tires and the brake wear of motor vehicles (Z. Yuan et al., 2006; He et al., 2011). Therefore, this factor was identiﬁed as vehicle emissions. (7) The seventh factor had a high EV value of Cl−and certain concentrations of OM, EC, SO2− 4 and NO− 3 , implying a combustion source. This factor was identiﬁed as coal burning, which was a major source of Cl−in the PRD (Wang et al., 2015). (8) The eighth factor had large EV val- ues of Zn, Cd, and Pb and certain concentrations of OM and EC. Zn, Cd and Pb had high enrichment factors (Table S2) of 821, 4121 and 663, respectively, and were thus consid- ered to be related to industrial emissions (Wang et al., 2015). (9) The last factor had large EV values of V and Ni. V and Ni were predominantly derived from heavy oil combustion, and they had high enrichment factors (Table S2) of 64 and 89, respectively. Heavy oil was related to ship emissions in the PRD (Chow and Watson, 2002; X. F. Huang et al., 2014). Although these nine factors of the ME-2 modeling generally showed high correlations (R2 = 0.81–0.97) with the corre- (e.g., a high Ti factor) when more factors were set to run ME-2. Based on the EV and the contributed concentrations of species in each factor shown in Fig. 4, the sources of PM2.5 can be judged as follows: (1) the ﬁrst factor was explained as secondary sulfate rich, which had large EV values of SO2− 4 and NH+ 4 . (2) The second factor was explained as secondary nitrate rich, which had signiﬁcant EV values of NO− 3 and NH+ 4 . (3) The third factor was related to sea salt due to the large EV values and concentrations of Na and Mg. However, the low Cl−concentration and high SO2− 4 concentration im- plied that SO2− 4 replaced Cl−during the sea salt aging pro- cess. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 OM EC Cl¯ NO₃¯SO₄²¯ NH₄⁺ Ca Ti V Ni Zn Cd Pb Na Mg Al K Fe Secondary sulfate-rich 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 Secondary nitrate-rich 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 Aged sea salt 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 Fugitive dust 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 Biomass burning 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 Vehicle emissions 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 Coal burning 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 Industrial emissions 0 25 50 75 100 0.0001 0.001 0.01 0.1 1 OM EC Cl¯ NO₃¯SO₄²¯ NH₄⁺ Ca Ti V Ni Zn Cd Pb Na Mg Al K Fe Ship emissions Concentration (μg m ³) % of species Concentration of species % of species - Figure 4. The factor proﬁles and explained variations in the ME-2 modeling. Figure 4. The factor proﬁles and explained variations in the ME-2 modeling. the secondary sulfate-rich factor was considered to repre- sent LV-OOA, while the high OM concentration in the sec- ondary nitrate-rich factor was considered to represent SV- OOA (Z. B. Yuan et al., 2006; He et al., 2011). Therefore, it should be acknowledged that mixed secondary factors can- not be solved even using ME-2. However, the contribution time series of LV-OOA (or SV-OOA) can be extracted based on the contribution time series of the secondary sulfate-rich factor (or the secondary nitrate-rich factor) and the mass per- centage of OM in this factor, leaving the remaining mass as the “pure” secondary sulfate (or secondary nitrate). There- fore, a new SOA factor can be reasonably estimated by LV- OOA + SV-OOA. sponding factors of the PMF modeling in terms of time se- ries, it is easy to see that the ME-2 modeling provided a better Qtrue / Qexp ratio (1.2) than that of the PMF model- ing (Qtrue / Qexp = 2.5), indicating that the species residuals were decreased in the ME-2 modeling, and the EV values of tracers (e.g., SO2− 4 , NO− 3 , OM, EC, Cl−, V, Ni, Pb and Cd) were assigned to factors more intensively. www.atmos-chem-phys.net/18/11563/2018/ Therefore, this factor was identiﬁed as aged sea salt (Z. Yuan et al., 2006). (4) The fourth factor was identiﬁed as fugitive dust due to its signiﬁcant EV values of Al, Ca, Mg and Fe. In this study, the undetermined mass of O and Si in this factor was compensated for using the elemental abun- dance in dust particles in Table S1 (Taylor and Mclennan, 1995). (5) The ﬁfth factor was identiﬁed as biomass burning due to its signiﬁcant characteristic value of K (Yamasoe et al., 2000). (6) The sixth factor had high concentrations and Atmos. Chem. Phys., 18, 11563–11580, 2018 www.atmos-chem-phys.net/18/11563/2018/ 11571 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Therefore, it is concluded that the source apportionment results of the ME-2 modeling were more environmentally meaningful and statis- tically better than those of the PMF modeling. In this study, secondary organic aerosol (SOA) did not appear as a single factor, even if we run the ME-2 with 10 or more factors. SOA can usually be described by low- volatility oxygenated organic aerosol (LV-OOA) and semi- volatile oxygenated organic aerosol (SV-OOA), based on the volatility and oxidation state of organics (Jimenez et al., 2009). In previous studies (e.g., He et al., 2011; Lanz et al., 2007; Ulbrich et al., 2009), the time series of LV- OOA and SV-OOA were highly correlated with those of sulfate and nitrate, respectively, implying that LV-OOA and sulfate (or SV-OOA and nitrate) cannot be separated eas- ily in cluster analysis, especially when there is no effective tracer of SOA. In this study, the high OM concentration in Figure 5 shows the 4-month average contributions of the PM2.5 sources in the PRD in 2015 based on the source appor- tionment of ME-2. The total secondary aerosols accounted for 39 % of PM2.5 in the PRD, which were secondary sul- fate (21 %), secondary nitrate (11 %) and SOA (7 %). How- ever, the identiﬁed primary particulates contributed 54 % of PM2.5, which comprised vehicle emissions (14 %), indus- trial emissions (13 %), biomass burning (11 %), coal burn- ing (6 %), fugitive dust (5 %), ship emissions (3 %) and aged sea salt (2 %). The unidentiﬁed sources, including both the X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Secondary sulfate 21 % Secondary nitrate 11 % Aged sea salt 2 % Fugitive dust 5 % Biomass burning 11 % Vehicle emissions 14 % Coal burning 6 % Industrial emissions 13 % Ship emissions 3 % SOA 7 % Other 7 % PM2.5 = 37 μg m ³ - Figure 5. The 4-month average contributions of PM2.5 sources in the PRD. Secondary sulfate 21 % Secondary nitrate 11 % Aged sea salt 2 % Fugitive dust 5 % Biomass burning 11 % Vehicle emissions 14 % Coal burning 6 % Industrial emissions 13 % Ship emissions 3 % SOA 7 % Other 7 % PM2.5 = 37 μg m ³ - (X. F. Huang et al., 2014; Zou et al., 2017), the more pol- luted continental air mass in the winter monsoon made its concentrations in winter much higher than in summer. The semi-volatile secondary ammonium nitrate was also signif- icantly affected by seasonal ambient temperatures. In con- trast, the average contributions of aged sea salt and ship emis- sions for the whole region displayed few seasonal variations, consistent with the fact that the emissions were from local surrounding sea areas. Previous studies of the source apportionment of bulk PM2.5 in the PRD have mainly focused on Guangzhou, Dongguan and Shenzhen, as seen in Table 6. It can be seen that in those studies, PM2.5 was apportioned to six to nine sources and that secondary sulfate was the prominent source, although the results of different studies exhibited certain dif- ferences due to the use of different models or data inputs. Compared with the study of X. F. Huang et al. (2014) in Shenzhen in 2009, the contributions of secondary sulfate and vehicle emissions in Shenzhen in this study were obviously lower due to power plant desulfurization and motor vehicle oil upgrades in recent years (People’s Government of Shen- zhen Municipality, 2013). Compared with previous studies in Guangzhou, this study attained more PM2.5 sources, which can more clearly describe the source structure of PM2.5 in this region, especially industrial emissions (11 %). The PRD region has experienced a high degree of industrialization; thus, industrial sources should be a major source, contribut- ing 8.1 % of PM2.5 reported by the Guangzhou Environ- mental Protection Bureau (2017), similar to our results. Tao et al. (2017) apportioned PM2.5 to six sources using PMF in Guangzhou, including some mixed sources. 3.3 Spatiotemporal variations in sources in the PRD The spatial distributions of the PM2.5 sources among the six sites are shown in Fig. 6a. Secondary sulfate represented the largest fraction (31 %) of PM2.5 at DP, indicating that it was a major air pollutant in the air mass transported to the PRD. Vehicle emissions also contributed relatively highly to ur- ban sites (18 % in MDS and 17 % in UT). Industrial emis- sions, biomass burning, secondary nitrate and coal burning contributed larger fractions of PM2.5 at HS, which could be attributed to both strong local sources (e.g., the surround- ing township factories and farmlands) and regional trans- port from upwind cities at this site. Fugitive dust, which is primarily related to construction activities, was relatively high at DM (9 %). The contributions of ship emissions and aged sea salt were the highest at QA due to the site being located on Qi’ao Island in the Pearl River estuary, which records the greatest impact from the sea. SOA contributed similar amounts (7 %–8 %) at all sites. It should be noted that, although QA was a background site without local anthro- pogenic sources, its PM2.5 level was moderate in the PRD, indicating that QA was impacted by severe regional transport from the surrounding cities. residual from ME-2 and the unmeasured species, accounted for 7 %. residual from ME-2 and the unmeasured species, accounted for 7 %. www.atmos-chem-phys.net/18/11563/2018/ www.atmos-chem-phys.net/18/11563/2018/ Atmos. Chem. Phys., 18, 11563–11580, 2018 11572 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD X.-F. Huang et al.: Source apportionment of PM2.5 in PRD For exam- ple, ship emissions in Tao’s study may not actually repre- sent a primary source due to the signiﬁcant contribution of some secondary inorganics and sea salt in the source proﬁle; thus, they obtained a signiﬁcantly higher contribution (17 %) than that in our study. Ship emissions were unidentiﬁed in R. Huang’s study (2014) in Guangzhou. Figure 5. The 4-month average contributions of PM2.5 sources in the PRD. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11573 25 % 11 % 3 % 6 % 12 % 14 % 5 % 12 % 4 % 8 % 16 % 15 % 2 % 9 % 13 % 14 % 8 % 13 % 3 % 7 % MDS ( 41) HS (47) UT(37) DP(28) Secondary nitrate Aged sea salt Fugitive dust Biomass burning Vehicle emissions Coal burning Industrial emissions Ship emissions SOA Others Secondary sulfate (a) (b) Winter Spring Summer Fall Concentration (μg m ³) 5 % 2 % 4 % 8 % 2 % 4% 8 % 31 % 14 % 11 % 11 % 21 % 8 % 1 % 6 % 9 % 17 % 3 % 11 % 3% 7 % 14 % 23 % 11 % 1 % 3 % 8 % 18 % 6 % 11 % 2% 7 % 10 % 16 % 14 % 1 % 3 % 14 % 12 % 8 % 17 % 2% 7 % 6 % DM (35) QA (37) 0 2 4 6 8 10 12 - Figure 6. The spatial distributions of (a) and seasonal variations in (b) PM2.5 sources in the PRD. Sizes of the pie charts indicate the concentrations of PM2.5 at the six sites, with the detailed numbers (unit: µgm−3) in brackets. 25 % 11 % 3 % 6 % 12 % 14 % 5 % 12 % 4 % 8 % 16 % 15 % 2 % 9 % 13 % 14 % 8 % 13 % 3 % 7 % MDS ( 41) HS (47) UT(37) DP(28) Secondary nitrate Aged sea salt Fugitive dust Biomass burning Vehicle emissions Coal burning Industrial emissions Ship emissions SOA Others Secondary sulfate (a) 5 % 2 % 4 % 8 % 2 % 4% 8 % 31 % 14 % 11 % 11 % 21 % 8 % 1 % 6 % 9 % 17 % 3 % 11 % 3% 7 % 14 % 23 % 11 % 1 % 3 % 8 % 18 % 6 % 11 % 2% 7 % 10 % 16 % 14 % 1 % 3 % 14 % 12 % 8 % 17 % 2% 7 % 6 % DM (35) QA (37) Figure 6. The spatial distributions of (a) and seasonal variations in (b) PM2.5 sources in the PRD. 3.4 Identiﬁcation of high-emission areas in the PRD in typical meteorological conditions Figure 7 shows the contributions of PM2.5 sources under southerly ﬂow and northerly ﬂow conditions in the PRD, based on the classiﬁcation of weather types in Sect. 2.2. Southerly ﬂow primarily originated from the South China Sea and carried clean ocean air masses to the PRD with over- all PM2.5 values of 15 µgm−3. As shown in Fig. 7, secondary sulfate (19 %), vehicle emissions (15 %) and biomass burn- ing (11 %) had higher contributions under southerly ﬂow. In contrast, in northerly ﬂow, the level of PM2.5 (82 µgm−3) was 4.5 times higher than that of southerly ﬂow due to the transport of polluted air masses southward from the north- ern mainland. Under northerly ﬂow, secondary sulfate (18 %) and biomass burning (10 %) were still the major sources, but secondary nitrate became the dominant source of PM2.5, ac- Figure 6b shows the seasonal variations in the major sources of PM2.5 in the PRD. The contributions of most sources were higher in winter and lower in summer, e.g., secondary sulfate, secondary nitrate, fugitive dust, biomass burning, vehicle emissions, coal burning, industrial emis- sions and SOA; these sources were greatly inﬂuenced by the seasonal variations in monsoon, rainfall and PBL, as dis- cussed in Sect. 3.1. For example, although secondary sulfate was proven to be a typical regional pollutant in the PRD Atmos. Chem. Phys., 18, 11563–11580, 2018 www.atmos-chem-phys.net/18/11563/2018/ X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Atmos. Chem. Phys., 18, 11563–11580, 2018 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Sizes of the pie charts indicate the concentrations of PM2.5 at the six sites, with the detailed numbers (unit: µgm−3) in brackets. The inﬂuences of ship emissions exhibited large differ- ences among the six sites, showing signiﬁcant local charac- teristics. In addition, the ship emissions have similar aver- age concentrations in the summer southerly ﬂow and winter northerly ﬂow, also reﬂecting the emissions of local ports in the PRD region. The concentrations of ship emissions were the highest at DP under southerly ﬂow, mainly due to the im- pact of vessels in the upwind Yantian port, while they were the highest at QA under northerly ﬂow, primarily due to the effects of the upwind Nansha port, as shown in Fig. 9. The Yantian port and Nansha port are among the 10 largest ports in the world (Hong Kong Marine Department, 2012). counting for 20 % of PM2.5. In addition, industrial emissions also exhibited a relatively high contribution (14 %). The spatial distributions of the PM2.5 sources under southerly ﬂow and northerly ﬂow are shown in Fig. 8. The high-emission areas for different sources identiﬁed by the discussion below are marked on the map in Fig. 9. The aver- age concentration levels of aged sea salt were similar in the summer southerly ﬂow and the winter northerly ﬂow, reﬂect- ing local release of sea salt. The spatial distribution of aged sea salt among the different sites was a complex result of the site locations relative to the sea and meteorological con- ditions, e.g., wind and tide. A relatively high level of aged sea salt was observed at Qi’ao Island (QA), especially in the northerly ﬂow, which can be attributed to the fact that the QA site was surrounded by the sea and had lower wind speeds in the northerly ﬂow (in Table 3). The contributions of fugitive dust also exhibited signif- icant differences among the six sites, which are consistent with local construction activities. DM is located in a newly developed zone that has experienced relatively high levels of fugitive dust during southerly ﬂow and northerly ﬂow due X.-F. Huang et al.: Source apportionment of PM2.5 in PRD The contributions of PM2.5 sources under southerly ﬂow and northerly ﬂow conditions in the PRD. to active construction activities. Sample records indicate that the high value of fugitive dust at UT under southerly ﬂow may be related to its surrounding short-term road construc- tion project, while the high value at QA under northerly ﬂow may be related to the reconstruction project of the adjacent Nansha port (Guangzhou Municipal People’s Government, 2015). to active construction activities. Sample records indicate that the high value of fugitive dust at UT under southerly ﬂow may be related to its surrounding short-term road construc- tion project, while the high value at QA under northerly ﬂow may be related to the reconstruction project of the adjacent Nansha port (Guangzhou Municipal People’s Government, 2015). Motor vehicles are a common source of air pollution in the highly urbanized and industrialized PRD region. The aver- age concentration of vehicle emissions during northerly ﬂow was nearly 3-fold that during southerly ﬂow. Under southerly ﬂow, MDS, HS and UT, which are located in the hinterland of the PRD, had much higher levels of vehicle emissions than the other three sites; in particular, the highest level at the urban MDS site was caused by the high density of mo- tor vehicles in Guangzhou. Under northerly ﬂow, the high- est concentration of vehicle emissions was still at the ur- ban MDS site, while QA also recorded a prominent contri- bution of vehicle emissions, which was probably closely re- lated to the container trucks in the neighboring Nansha port. It should be noted that the concentration of vehicle emissions at the background DP site exceeded half the regional aver- age value, approaching 4 µgm−3, thus indicating that vehicle emissions had a signiﬁcant impact on the regional transport of air masses from the north. The average concentration of secondary nitrate during northerly ﬂow in winter was 40 times greater than that during southerly ﬂow in summer; this occurred not only because of the unfavorable conditions of atmospheric diffusion in winter but also due to the high semi-volatility of ammonium nitrate, which cannot stably exist in ﬁne particles in the PRD during hot summer weather (Huang et al., 2006). Under southerly ﬂow conditions, the concentrations of secondary nitrate pre- sented prominent differences among the six sites, showing local characteristics. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD that this area is an intensive industrial area. During northerly air ﬂow in winter, HS and DM had lower concentrations than the four upwind sites, i.e., MDS, QA, UT and especially DP (the background site), indicating that secondary sulfate could mainly be derived from regional transport from outside the PRD in this season. Although the industrial area between HS and MDS could emit signiﬁcant amounts of SO2, the lower temperatures and dry air in winter did not appear to favor the quick conversion of SO2 to secondary sulfate. Since both secondary sulfate and LV-OOA belong to a mixed factor with ﬁxed proportions, the spatial distribution of secondary sulfate also reﬂects the corresponding characteristics of LV-OOA. 0 % 10 % 20 % 30 % 40 % 50 % 60 % 70 % 80 % 90 % 100 % Southerly flow Northerly flow The contribution of sources to total PM2.5 Secondary nitrate Aged sea salt Fugitive dust Biomass burning Vehicle emissions Coal burning Industrial emissions Ship emissions SOA Others Secondary sulfate 18 % 20 % 10 % 4 % 8 % 7 % 14 % 2 % 8 % 8 % 1 % 19 % 3 % 11 % 9 % 15 % 4 % 6 % 7 % 5 % 14 % 7 % Figure 7. The contributions of PM2.5 sources under southerly ﬂow and northerly ﬂow conditions in the PRD. The spatial distributions of coal burning were signiﬁcantly different among the six sites during periods of both south wind and north wind, thus showing conspicuous local char- acteristics. The contribution of coal burning was higher at MDS under southerly ﬂow and higher at HS under northerly ﬂow. Most of the coals in the PRD were consumed by ther- mal power plants, but there were no coal-ﬁred power plants near the urban MDS and background DP sites. Therefore, it is speculated that the high-emission areas of coal burning sources mainly exist in the region between HS and MDS, as shown in Fig. 9. The distribution of coal-ﬁred power plants in Guangdong (Wang et al., 2017) reveal that some important coal-ﬁred power plants are distributed in this region. Addi- tionally, DM also exhibited relatively obvious contributions of coal burning during southerly ﬂow and northerly ﬂow, which is also consistent with the distribution of coal-ﬁred power plants in the vicinity. Figure 7. www.atmos-chem-phys.net/18/11563/2018/ www.atmos-chem-phys.net/18/11563/2018/ Atmos. Chem. Phys., 18, 11563–11580, 2018 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11574 www.atmos-chem-phys.net/18/11563/2018/ X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11575 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 DM QA HS MDS UT DP 0 0.1 0.2 0.3 0 0.2 0.4 0.6 0.8 1 1.2 1.4 DM QA HS MDS UT DP 0 0.5 1 1.5 2 2.5 DM QA HS MDS UT DP 0 0.5 1 1.5 2 2.5 DM QA HS MDS UT DP 0 2 4 6 8 10 DM QA HS MDS UT DP 0 0.5 1 1.5 2 2.5 3 3.5 4 DM QA HS MDS UT DP 0 10 20 DM QA HS MDS UT DP 0 2 4 6 DM QA HS MDS UT DP 0 3 6 9 12 15 DM QA HS MDS UT DP 0 2 4 6 DM QA HS MDS UT DP 0 5 10 15 20 DM QA HS MDS UT DP 0 0.5 1 1.5 2 DM QA HS MDS UT DP 0 5 10 15 20 25 DM QA HS MDS UT DP 0 1 2 3 4 5 DM QA HS MDS UT DP 0 1 2 3 4 5 DM QA HS MDS UT DP 0 1 2 3 4 5 DM QA HS MDS UT DP 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 1 2 3 4 5 6 DM QA HS MDS UT DP 0 1 2 3 4 5 6 0 5 10 15 20 25 DM QA HS MDS UT DP 0 30 60 90 120 150 DM QA HS MDS UT DP 0 5 10 15 20 25 DM QA HS MDS UT DP Avg. = 0.9 Avg. = 1.4 Avg. = 14.9 (LV-OOA 3.5 Avg. = 16.2 (SV-OOA 3.4 Avg. = 3.6 Avg. = 8.1 Avg. = 6.8 Avg. = 6.2 Avg. = 11.4 Avg. = 82 Secondary nitrate Secondary sulfate Aged sea salt Fugitive dust Biomass burning Southerly flow (μg m ³) (a) Northerly flow (μg m ³) (b) Avg. = 1.0 Avg. = 1.1 Avg. = 2.7 (LV-OOA 0.6 Avg. = 0.4 (SV-OOA 0.1 Avg. = 1.3 Avg. = 1.7 μg m ³ Avg. = 2.2 Avg. = 0.6 Avg. = 0.8 Avg. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD = 15 Vehicle emissions Coal burning Industrial emissions Ship emissions PM2.5 Southerly flow (μg m ³) (a) Northerly flow (μg m ³) (b) SV-OOA LV-OOA - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - μg m ³ - ( ( ( ( - - - - Figure 8. The average contributions of PM2.5 sources at six sites in the PRD: (a) those in southerly ﬂow and (b) those in northerly ﬂow. Figure 8. The average contributions of PM2.5 sources at six sites in the PRD: (a) those in southerly ﬂ Figure 8. The average contributions of PM2.5 sources at six sites in the PRD: (a) those in southerly ﬂow and (b) those in northerly ﬂow. ontributions of PM2.5 sources at six sites in the PRD: (a) those in southerly ﬂow and (b) those in northerly ﬂow centration of industrial emissions reached 14-fold that of southerly ﬂow, and the high contributions at background DP suggested that regional transport probably dominated the in- dustrial sources of ﬁne particulate matter in the PRD in win- ter. HS had the highest concentration of industrial emissions during southerly ﬂow and northerly ﬂow conditions, which is consistent with the dense factories present in the surrounding area (Hu, 2004; Environmental Protection Agency of Jiang- men City, 2017). In addition, the contribution of industrial emissions was relatively high at MDS during southerly ﬂow and relatively high at QA during northerly ﬂow, which sup- ports the inference that a high-emission region of industrial sources was located between MDS and QA, as seen in Fig. 9. emission area of biomass burning between MDS and QA, as shown in Fig. 9. Those spatial distribution characteristics of biomass burning were similar to those of industrial emissions in the PRD, suggesting that not only the combustion of resi- dential biomass but also the use of industrial biomass boilers could make important contributions to PM2.5 in the PRD. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD As a summary, the central PRD area, i.e., the middle re- gion in between MDS, HS and QA (the shaded region in Fig. 9), represents the most important pollutant emission area in the PRD; these emissions include SO2, NOx, coal burning, biomass burning, industrial emissions and vehicle emissions, thus leading to high pollution levels in the PRD. Therefore, this area is a key area for pollution control in the PRD. Pri- mary ﬁne particulate matter and SO2 from ship emissions had signiﬁcant impacts on PM2.5 in the southern coastal area of the PRD during summer southerly ﬂow, and special atten- tion must be paid to them. The impacts of biomass burning exhibited relatively large differences among the six sites during both south and north wind conditions, presenting somewhat local characteristics. The suburban HS site had relatively high biomass burning levels during southerly ﬂow and northerly ﬂow, which should be related to the presence of many farmlands in its vicinity and thus the popular events of open burning and residential burning of biomass wastes. The concentrations of biomass burning were relatively high at the urban MDS site during southerly ﬂow and relatively high at the background QA site during northerly ﬂow, implying that there was a high- X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Moreover, the relatively low concentra- tions at the background DP site during northerly ﬂow also indicated that secondary nitrate mainly originated from the interior of the PRD. The spatial distribution characteristics of secondary nitrate were very similar to those of coal burn- ing, with the highest occurring at MDS under southerly ﬂow, the highest occurring at HS under northerly ﬂow and sig- niﬁcantly high values occurring at DM under southerly and northerly ﬂow, showing that the NOx emissions produced by coal burning may be the main reason for the high nitrate lev- els in those areas. Since both secondary nitrate and SV-OOA belong to a mixed factor with ﬁxed proportions, the spatial distribution of secondary nitrate also reﬂects the correspond- ing characteristics of SV-OOA. During southerly air ﬂow, the background DP and QA sites and the urban UT site all recorded similar concentrations of secondary sulfate, suggesting that the secondary sulfate at these sites was dominated by regional transport from the Southern Ocean with heavy vessel transport and had little to do with the urban emissions at UT. Kuang et al. (2015) also found that ship emissions could be a major source of sec- ondary sulfate in the PRD in summer. HS and MDS had sig- niﬁcantly higher concentrations than their upwind site, DM, suggesting that the area between MDS and HS could be a high-SO2-emission area, which is consistent with the fact Under southerly ﬂow, the inﬂuence of industrial emis- sions differed vastly among the six sites, showing obvious local characteristics. Under northerly ﬂow, the average con- Atmos. Chem. Phys., 18, 11563–11580, 2018 www.atmos-chem-phys.net/18/11563/2018/ 3.5 Distinguishing local and regional PM2.5 pollution in the PRD The analyses presented in Sect. 3.4 indicate that the sec- ondary sulfates at the four southern coastal sites (DM, QA, UT and DP) in the PRD were almost entirely derived from X.-F. Huang et al.: Source apportionment of PM2.5 in PRD X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 113°E 114°E 115°E 113°E 114°E 115°E 23°N 22°N 23°N 22°N Northerly flow Southerly flow MDS DP UT HS QA DM Aged sea salt Ship emissions Fugitive dust Vehicle emissions Secondary sulfate Coal burning Secondary nitrate Industrial emissions Biomass burning Key emission area Figure 9. The schematic diagram of high-emission areas in the PRD (map from Google Earth). The white shaded area indicates the key emission area for the multiple sources of SO2, NOx, coal burning, biomass burning, industrial emissions and vehicle emissions and is explained further in the text. 113°E 114°E 115°E 113°E 114°E 115°E 23°N 22°N 23°N 22°N Northerly flow Southerly flow MDS DP UT HS QA DM Aged sea salt Ship emissions Fugitive dust Vehicle emissions Secondary sulfate Coal burning Secondary nitrate Industrial emissions Biomass burning Key emission area 114°E 113°E Figure 9. The schematic diagram of high-emission areas in the PRD (map from Google Earth). The white shaded area indicates the key emission area for the multiple sources of SO2, NOx, coal burning, biomass burning, industrial emissions and vehicle emissions and is explained further in the text. tion emissions in the key source area of the PRD were still crucial in winter but lower than the contribution of the re- gional background. Ignoring natural sources, such as aged sea salt and fugitive dust, under northerly ﬂow, the contribu- tions of other anthropogenic sources to DP were considered to represent regional background pollution (47.5 µgm−3), and the differences in their corresponding source concentra- tions between QA and DP were expected to represent the local emissions of source areas in the PRD. Therefore, the source structures in the regional background air mass and local emissions of heavy pollution sources area in the PRD are shown in Fig. 10. Secondary sulfate and LV-OOA occu- pied the vast majority (45.6 %) of the regional background air mass from the northern mainland, followed by industrial emissions (17.8 %), secondary nitrate and SV-OOA (15.5 %). However, the major sources between the sources output by local emissions from the heavy pollution source area of the PRD were secondary nitrate and SV-OOA (37.3 %), biomass burning (20.6 %), vehicle emissions (14.9 %) and coal burn- ing (11.9 %). X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Therefore, measures implemented for the ef- fective control of PM2.5 in the PRD should focus on local controls and regional joint prevention and control under win- ter northerly ﬂow conditions. the conversion of SO2 from the emissions of ships in the Southern Ocean during southerly ﬂow, contributing approx- imately 20 % of the average PM2.5 (13 µgm−3) at the four sites. Considering that the ship emissions directly contributed approximately 10 % of the average PM2.5 at the four sites, the total ship emissions contributed approximately 30 % of PM2.5 in the southern coastal PRD area and acted as the largest source of PM2.5. Under northerly ﬂow conditions, the background DP site, which was barely affected by pol- lution emissions within the PRD, reﬂected regional transport from the north air mass outside the PRD, while the back- ground QA site reﬂected the superposition effect of regional background pollution and the input of the most serious pol- lution area in the PRD. The consistency of the secondary sulfate concentrations at the background QA and DP sites was interpreted to reﬂect almost the same regional back- ground effect during northerly ﬂow; thus, the differences in the six anthropogenic sources between the two background sites, including secondary nitrate (and SV-OOA), biomass burning, industrial emissions, coal burning, vehicle emis- sions and ship emissions, could be used to trace the inter- nal inputs from the most serious pollution area within the PRD to the downwind area. The internal inputs of six anthro- pogenic sources to the corresponding sources of PM2.5 at the background QA site were 66 %, 67 %, 28 %, 76 %, 59 % and 75 %, and the total internal input of 37.7 µgm−3 accounted for 45 % of PM2.5 at the background QA site (83 µgm−3), showing that the local contributions of anthropogenic pollu- the conversion of SO2 from the emissions of ships in the Southern Ocean during southerly ﬂow, contributing approx- imately 20 % of the average PM2.5 (13 µgm−3) at the four sites. Considering that the ship emissions directly contributed approximately 10 % of the average PM2.5 at the four sites, the total ship emissions contributed approximately 30 % of PM2.5 in the southern coastal PRD area and acted as the largest source of PM2.5. www.atmos-chem-phys.net/18/11563/2018/ www.atmos-chem-phys.net/18/11563/2018/ Atmos. Chem. Phys., 18, 11563–11580, 2018 11576 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 4 Conclusions The PRD is one of the largest agglomerations of cities in the world, and its air quality has largely improved in the past 10 years. To reveal the current PM2.5 pollution characteristics on a regional scale in the PRD, six sampling sites were selected to conduct 4 months (one for each season) of sampling and chemical analysis in 2015; then, the source exploration of PM2.5 was performed using a novel method. The conclusions are described below. Data availability. Datasets are available by contacting the corre- sponding author, Ling-Yan He (hely@pku.edu.cn). Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/acp-18-11563-2018-supplement. 1. The 4-month average PM2.5 concentration for all six sites in the PRD was 37 µgm−3, of which OM, SO2− 4 , NH+ 4 , NO− 3 , EC, metal elements and Cl−contributed 36.9 %, 23.6 %, 10.9 %, 9.3 %, 6.6 %, 6.5 % and 0.9 %, respectively. The spatiotemporal PM2.5 variations were generally characterized as being higher in the northern inland region and higher in winter. Author contributions. X-FH, B-BZ, and L-YH analyzed the data and wrote the paper. L-YH, MH, and Y-HZ designed the study. B- BZ performed the chemical analysis. ASHP helped with the ME-2 running. All authors reviewed and commented on the paper. Competing interests. The authors declare that they have no conﬂict of interest. 2. This study revealed that the ME-2 model produced more environmentally meaningful and statistically robust re- sults of source apportionment than the traditional PMF model. Secondary sulfate was found to be the domi- nant source of PM2.5 in the PRD, at 21 %, followed by vehicle emissions (14 %), industrial emissions (13 %), secondary nitrate (11 %), biomass burning (11 %), SOA (7 %), coal burning (6 %), fugitive dust (5 %), ship emis- sions (3 %) and aged sea salt (2 %). Only aged sea salt and ship emissions did not show obvious seasonal vari- ations. Acknowledgements. This work was supported by the National Natural Science Foundation of China (91744202, 41622304) and the Science and Technology Plan of Shenzhen Municipality (JCYJ20170412150626172, JCYJ20170306164713148). Edited by: James Allan Reviewed by: two anonymous referees Edited by: James Allan Reviewed by: two anonymous referees 3. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Therefore, effective con- trol measures of PM2.5 in the PRD in the future should pay more attention to both local controls and regional joint prevention. Figure 10. The PM2.5 source structures in regional background air and local contributions of the central PRD area under northerly ﬂow. 4 Conclusions Based on the spatial distribution characteristics of PM2.5 sources under typical southerly and northerly air- ﬂow conditions, the central PRD area in between MDS, HS and QA is identiﬁed as a key area for source emis- sions, including SO2, NOx, coal burning, biomass burn- ing, industrial emissions, and vehicle emissions, and thus deserves more attention when implementing local References BIPM, IEC, IFCC, ILAC, ISO, IUPAC, IUPAP, and OIML: Eval- uation of measurement data – Guide to the expression of uncer- tainty in measurement, available at: https://www.bipm.org/utils/ common/documents/jcgm/JCGM_100_2008_E.pdf (last access: 5 August 2018), 2008. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11577 pollution control in the PRD. In addition, ship emissions should be controlled more strictly during summer due to their contribution of approximately 30 % of PM2.5 in the southern coastal area of the PRD under southerly air ﬂow. 0 % 10 % 20 % 30 % 40 % 50 % 60 % 70 % 80 % 90 % 100 % DP QA-DP The contribution of sources to total PM2.5 Ship emiss Industrial e Coal burni Vehicle em Biomass b SV-OOA Secondary LV-OOA Secondary SV-OOA Biomass burning Vehicle emissions Coal burning Industrial emissions Ship emissions Secondary sulfate Regional background (DP) 30.9 % 20.6 % 6.4 % 14.9 % 11.9 % 8.9 % 6.4 % 36.9 % 12.8 % 8.7 % 2.7 % 8.1 % 8.3 % 3.0 % 17.8 % 1.7 % Local contributions of source area (QA-DP) Secondary nitrate LV-OOA Figure 10. The PM2.5 source structures in regional background air and local contributions of the central PRD area under northerly ﬂow. e e 4. Under typical northerly winter ﬂow, the contributions of anthropogenic pollution emissions in the central PRD area contributed 37.7 µgm−3 (45 % of PM2.5) to the regional background air. Secondary sulfate (36.9 %), industrial emissions (17.8 %) and secondary nitrate SV-OOA (12.8 %) were the major PM2.5 sources for the PM2.5 transported in the regional background air mass, while secondary nitrate (30.9 %), biomass burn- ing (20.6 %), vehicle emissions (14.9 %) and coal burn- ing (11.9 %) were the major sources for the PM2.5 pro- duced in the central PRD area. Therefore, effective con- trol measures of PM2.5 in the PRD in the future should pay more attention to both local controls and regional joint prevention. e e 4. Under typical northerly winter ﬂow, the contributions of anthropogenic pollution emissions in the central PRD area contributed 37.7 µgm−3 (45 % of PM2.5) to the regional background air. Secondary sulfate (36.9 %), industrial emissions (17.8 %) and secondary nitrate SV-OOA (12.8 %) were the major PM2.5 sources for the PM2.5 transported in the regional background air mass, while secondary nitrate (30.9 %), biomass burn- ing (20.6 %), vehicle emissions (14.9 %) and coal burn- ing (11.9 %) were the major sources for the PM2.5 pro- duced in the central PRD area. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Under northerly ﬂow conditions, the background DP site, which was barely affected by pol- lution emissions within the PRD, reﬂected regional transport from the north air mass outside the PRD, while the back- ground QA site reﬂected the superposition effect of regional background pollution and the input of the most serious pol- lution area in the PRD. The consistency of the secondary sulfate concentrations at the background QA and DP sites was interpreted to reﬂect almost the same regional back- ground effect during northerly ﬂow; thus, the differences in the six anthropogenic sources between the two background sites, including secondary nitrate (and SV-OOA), biomass burning, industrial emissions, coal burning, vehicle emis- sions and ship emissions, could be used to trace the inter- nal inputs from the most serious pollution area within the PRD to the downwind area. The internal inputs of six anthro- pogenic sources to the corresponding sources of PM2.5 at the background QA site were 66 %, 67 %, 28 %, 76 %, 59 % and 75 %, and the total internal input of 37.7 µgm−3 accounted for 45 % of PM2.5 at the background QA site (83 µgm−3), showing that the local contributions of anthropogenic pollu- www.atmos-chem-phys.net/18/11563/2018/ Atmos. Chem. Phys., 18, 11563–11580, 2018 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Guangzhou Environmental Protection Bureau: The Results of Source apportionment on PM2.5 in Guangzhou in 2016, available at: http://www.gz.gov.cn/gzgov/s5837/201706/ 1dcb25be6dd14dc6ab6506e0a5383745.shtml (last access: 5 August 2018), 2017. Cho, S. H., Kim, P. R., Han, Y. J., Kim, H. W., and Yi, S. M.: Characteristics of Ionic and Carbonaceous Compounds in PM2.5 and High Concentration Events in Chuncheon, Korea, Journal of Korean Society for Atmospheric Environment, 32, 435–447, https://doi.org/10.5572/KOSAE.2016.32.4.435, 2016. Guangzhou Municipal People’s Government: Three-year Action Plan for the Construction of Guangzhou International Shipping Center (2015-2017), available at: http://www.gz.gov.cn/gzgov/ s2811/201509/19601daa69c84e439fe2fb8baea448bb.shtml (last access: 5 August 2018), 2015. Chow, J. C. and Watson, J. G.: Review of PM2.5 and PM10 Ap- portionment for Fossil Fuel Combustion and Other Sources by the Chemical Mass Balance Receptor Model, Energ. Fuel., 16, 222–260, https://doi.org/10.1021/ef0101715, 2002. Hagler, G., Bergin, M., Salmon, L., Yu, J., Wan, E., Zheng, M., Zeng, L., Kiang, C., Zhang, Y., and Lau, A.: Source areas and chemical composition of ﬁne particulate matter in the Pearl River Delta region of China, Atmos. Environ., 40, 3802–3815, https://doi.org/10.1016/j.atmosenv.2006.02.032, 2006. Chow, J. C., Watson, J. G., Pritchett, L. C., Pierson, W. R., Frazier, C. A., and Purcell, R. G.: The DRI thermal/optical reﬂectance carbon analysis system: description, evaluation and applications in U.S. Air quality studies, Atmos. Environ., 27, 1185–1201, https://doi.org/10.1016/0960-1686(93)90245-T, 1993. Hasheminassab, S., Daher, N., Ostro, B. D., and Sioutas, C.: Long- term source apportionment of ambient ﬁne particulate matter (PM2.5) in the Los Angeles Basin: A focus on emissions re- duction from vehicular sources, Environ. Pollut., 193, 54–64, https://doi.org/10.1016/j.envpol.2014.06.012, 2014. Crippa, M., Canonaco, F., Lanz, V. A., Äijälä, M., Allan, J. D., Car- bone, S., Capes, G., Ceburnis, D., Dall’Osto, M., Day, D. A., De- Carlo, P. F., Ehn, M., Eriksson, A., Freney, E., Hildebrandt Ruiz, L., Hillamo, R., Jimenez, J. L., Junninen, H., Kiendler-Scharr, A., Kortelainen, A.-M., Kulmala, M., Laaksonen, A., Mensah, A. A., Mohr, C., Nemitz, E., O’Dowd, C., Ovadnevaite, J., Pan- dis, S. N., Petäjä, T., Poulain, L., Saarikoski, S., Sellegri, K., Swietlicki, E., Tiitta, P., Worsnop, D. R., Baltensperger, U., and Prévôt, A. S. H.: Organic aerosol components derived from 25 AMS data sets across Europe using a consistent ME-2 based source apportionment approach, Atmos. Chem. Phys., 14, 6159– 6176, https://doi.org/10.5194/acp-14-6159-2014, 2014. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11578 http://hbj.jiangmen.gov.cn/thirdData/hbsjzx/hjjc/fs/201712/ t20171218_268676.html (last access: 5 August 2018), 2017. Bressi, M., Sciare, J., Ghersi, V., Bonnaire, N., Nicolas, J. B., Pe- tit, J.-E., Moukhtar, S., Rosso, A., Mihalopoulos, N., and Féron, A.: A one-year comprehensive chemical characterisation of ﬁne aerosol (PM2.5) at urban, suburban and rural background sites in the region of Paris (France), Atmos. Chem. Phys., 13, 7825– 7844, https://doi.org/10.5194/acp-13-7825-2013, 2013. Fröhlich, R., Crenn, V., Setyan, A., Belis, C. A., Canonaco, F., Favez, O., Riffault, V., Slowik, J. G., Aas, W., Aijälä, M., Alastuey, A., Artiñano, B., Bonnaire, N., Bozzetti, C., Bressi, M., Carbone, C., Coz, E., Croteau, P. L., Cubison, M. J., Esser- Gietl, J. K., Green, D. C., Gros, V., Heikkinen, L., Herrmann, H., Jayne, J. T., Lunder, C. R., Minguillón, M. C., Mocnik, G., O’Dowd, C. D., Ovadnevaite, J., Petralia, E., Poulain, L., Priest- man, M., Ripoll, A., Sarda-Estève, R., Wiedensohler, A., Bal- tensperger, U., Sciare, J., and Prévôt, A. S. H.: ACTRIS ACSM intercomparison – Part 2: Intercomparison of ME-2 organic source apportionment results from 15 individual, co-located aerosol mass spectrometers, Atmos. Meas. Tech., 8, 2555–2576, https://doi.org/10.5194/amt-8-2555-2015, 2015. , p g p , Burnett, R. T., Pope, C. A. I., Ezzati, M., Olives, C., Lim, S. S., Mehta, S., Shin, H. H., Singh, G., Hubbell, B., Brauer, M., An- derson, H. R., Smith, K. R., Balmes, J. R., Bruce, N. G., Kan, H., Laden, F., Prüss-Ustün, A., Turner, M. C., Gapstur, S. M., Diver, W. R., and Cohen, A.: An Integrated Risk Function for Estimat- ing the Global Burden of Disease Attributable to Ambient Fine Particulate Matter Exposure, Environ. Health Persp., 122, A235– A235, https://doi.org/10.1289/ehp.122-A235, 2014. Canonaco, F., Crippa, M., Slowik, J. G., Baltensperger, U., and Prévôt, A. S. H.: SoFi, an IGOR-based interface for the efﬁcient use of the generalized multilinear engine (ME- 2) for the source apportionment: ME-2 application to aerosol mass spectrometer data, Atmos. Meas. Tech., 6, 3649–3661, https://doi.org/10.5194/amt-6-3649-2013, 2013. Gao, B., Guo, H., Wang, X., Zhao, X., Ling, Z., Zhang, Z., and Liu, T.: Tracer-based source apportionment of polycyclic aromatic hydrocarbons in PM2.5 in Guangzhou, southern China, using positive matrix factorization (PMF), Environ. Sci. Pollut. R., 20, 2398–2409, https://doi.org/10.1007/s11356-012-1129-0, 2013. Cao, L.-M., Huang, X.-F., Li, Y.-Y., Hu, M., and He, L.-Y.: Volatil- ity measurement of atmospheric submicron aerosols in an urban atmosphere in southern China, Atmos. Chem. Phys., 18, 1729– 1743, https://doi.org/10.5194/acp-18-1729-2018, 2018. www.atmos-chem-phys.net/18/11563/2018/ Atmos. Chem. Phys., 18, 11563–11580, 2018 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD http://hbj.jiangmen.gov.cn/thirdData/hbsjzx/hjjc/fs/201712/ t20171218_268676.html (last access: 5 August 2018), 2017. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD http://hbj.jiangmen.gov.cn/thirdData/hbsjzx/hjjc/fs/201712/ t20171218_268676.html (last access: 5 August 2018), 2017. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11579 tribution to particulate pollution during haze events in China, Na- ture, 514, 218–222, https://doi.org/10.1038/nature13774, 2014. tribution to particulate pollution during haze events in China, Na- ture, 514, 218–222, https://doi.org/10.1038/nature13774, 2014. try (ICP-MS) (HJ 657-2013), available at: http://kjs.mep.gov.cn/ hjbhbz/bzwb/jcffbz/201308/t20130820_257714.shtml (last ac- cess: 5 August 2018), 2013a. Huang, X., Liu, Z., Liu, J., Hu, B., Wen, T., Tang, G., Zhang, J., Wu, F., Ji, D., Wang, L., and Wang, Y.: Chemical characterization and source identiﬁcation of PM2.5 at multiple sites in the Beijing– Tianjin–Hebei region, China, Atmos. Chem. Phys., 17, 12941– 12962, https://doi.org/10.5194/acp-17-12941-2017, 2017. MEE (Ministry of Ecology and Environment): Technical Speci- ﬁcations for gravimetric measurement methods for PM2.5 in ambient air (HJ 656-2013), available at: http://kjs.mep.gov.cn/ hjbhbz/bzwb/jcffbz/201308/t20130802_256857.shtml (last ac- cess: 5 August 2018), 2013b. Huang, X., Yu, J. Z., He, L., and Yuan, Z.: Water-soluble organic carbon and oxalate in aerosols at a coastal ur- ban site in China: Size distribution characteristics, sources, and formation mechanisms, J. Geophys. Res., 111, D22212, https://doi.org/10.1029/2006JD007408, 2006. MEE (Ministry of Ecology and Environment): Ambient Air- Determination of the water soluble anions (F−, Cl−, Br−, NO− 2 , NO− 3 , PO3− 4 , SO2− 3 , SO2− 4 ) from atmospheric particles-Ion chromatography (HJ 799-2016), available at: http://kjs.mep.gov. cn/hjbhbz/bzwb/jcffbz/201605/t20160519_337906.shtml (last access: 5 August 2018), 2016a. Huang, X.-F., He, L.-Y., Hu, M., Canagaratna, M. R., Kroll, J. H., Ng, N. L., Zhang, Y.-H., Lin, Y., Xue, L., Sun, T.- L., Liu, X.-G., Shao, M., Jayne, J. T., and Worsnop, D. R.: Characterization of submicron aerosols at a rural site in Pearl River Delta of China using an Aerodyne High-Resolution Aerosol Mass Spectrometer, Atmos. Chem. Phys., 11, 1865– 1877, https://doi.org/10.5194/acp-11-1865-2011, 2011. MEE (Ministry of Ecology and Environment): Ambient air- Determination of the water soluble cations (Li+, Na+, NH+ 4 , K+, Ca2+, Mg2+) from atmospheric particles-Ion chromatog- raphy (HJ 800-2016), available at: http://kjs.mep.gov.cn/hjbhbz/ bzwb/jcffbz/201605/t20160519_337907.shtml (last access: 5 August 2018), 2016b. Huang, X. F., Hui, Y., Gong, Z. H., Xiang, L., He, L. Y., Zhang, Y. H., and Min, H.: Source apportionment and sec- ondary organic aerosol estimation of PM2.5 in an urban at- mosphere in China, Sci. China Earth Sci., 57, 1352–1362, https://doi.org/10.1007/s11430-013-4686-2, 2014. Ming, L., Jin, L., Li, J., Fu, P., Yang, W., Liu, D., Zhang, G., Wang, Z., and Li, X.: PM2.5 in the Yangtze River Delta, China: Chemical compositions, seasonal variations, and regional pollution events, Environ. Pollut., 223, 200–212, https://doi.org/10.1016/j.envpol.2017.01.013, 2017. Jimenez, J. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD L., Canagaratna, M. R., Donahue, N. M., Prevot, A. S., Zhang, Q., Kroll, J. H., Decarlo, P. F., Allan, J. D., Coe, H., and Ng, N. L.: Evolution of organic aerosols in the atmosphere, Sci- ence, 326, 1525–1529, https://doi.org/10.1126/science.1180353, 2009. Ministry of Environmental Protection: Report on the State of the Environment in China 2015, available at: http://www.zhb.gov.cn/hjzl/zghjzkgb/lnzghjzkgb/201606/ P020160602333160471955.pdf (last access: 5 August 2018), 2016. Kuang, B. Y., Lin, P., Huang, X. H. H., and Yu, J. Z.: Sources of humic-like substances in the Pearl River Delta, China: pos- itive matrix factorization analysis of PM2.5 major compo- nents and source markers, Atmos. Chem. Phys., 15, 1995–2008, https://doi.org/10.5194/acp-15-1995-2015, 2015. Nanfang Daily: PM2.5 level ﬂuctuations down in PRD region in the past decade, available at: http://epaper.southcn.com/nfdaily/ html/2016-01/03/content_7504954.htm (last access: 5 August 2018), 2016. Lanz, V. A., Alfarra, M. R., Baltensperger, U., Buchmann, B., Hueglin, C., and Prévôt, A. S. H.: Source apportionment of sub- micron organic aerosols at an urban site by factor analytical mod- elling of aerosol mass spectra, Atmos. Chem. Phys., 7, 1503– 1522, https://doi.org/10.5194/acp-7-1503-2007, 2007. Norris, G. and Duvall, R.: EPA Positive Matrix Factoriza- tion (PMF) 5.0 Fundamentals and user guide, available at: https://www.epa.gov/sites/production/ﬁles/2015-02/documents/ pmf_5.0_user_guide.pdf (last access: 5 August 2018), 2014. Paatero, P.: The Multilinear Engine – A Table-Driven, Least Squares Program for Solving Multilinear Prob- lems, Including the n-Way Parallel Factor Analy- sis Model, J. Comput. Graph. Stat., 8, 854–888, https://doi.org/10.1080/10618600.1999.10474853, 1999. Leiva, M. A., Araya, M. C., Alvarado, A. M., and Seguel, R. J.: Uncertainty estimation of anions and cations measured by ion chromatography in ﬁne urban ambient particles (PM2.5), Ac- credit. Qual. Assur., 17, 53–63, https://doi.org/10.1007/s00769- 011-0844-4, 2012. Paatero, P. and Tapper, U.: Positive matrix factorization: A non-negative factor model with optimal utilization of er- ror estimates of data values, Environmetrics, 5, 111–126, https://doi.org/10.1002/env.3170050203, 1994. Lelieveld, J., Evans, J. S., Fnais, M., Giannadaki, D., and Pozzer, A.: The contribution of outdoor air pollution sources to pre- mature mortality on a global scale, Nature, 525, 367–371, https://doi.org/10.1038/nature15371, 2015. People’s Government of Guangdong Province: Major Pollutants Emission Reduction implementation plan during the 12th Five- year Plan in Guangdong Province, available at: http://zwgk.gd. gov.cn/006939748/201212/t20121219_359131.html (last access: 5 August 2018), 2012. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Liu, J., Li, J., Zhang, Y., Liu, D., Ding, P., Shen, C., Shen, K., He, Q., Ding, X., and Wang, X.: Source apportionment us- ing radiocarbon and organic tracers for PM2.5 carbonaceous aerosols in Guangzhou, South China: contrasting local- and regional-scale haze events, Environ. Sci. Technol., 48, 12002, https://doi.org/10.1021/es503102w, 2014. People’s Government of Shenzhen Municipality: Air quality im- provement plan in Shenzhen, available at: http://zwgk.gd.gov.cn/ 007543382/201309/t20130930_407564.html (last access: 5 Au- gust 2018), 2013. Mason, B.: Principles of Geochemistry, 4nd edition, John Wiley and Sons, New York, 1982. MEE (Ministry of Ecology and Environment): Ambient air and sta- tionary source emission – Determination of metals in ambient particulate matter – Inductively coupled plasma/mass spectrome- Physick, W. L. and Goudey, R.: Estimating an annual-average RSP concentration for Hong Kong using days characteristic of the X.-F. Huang et al.: Source apportionment of PM2.5 in PRD He, L., Huang, X., Xue, L., Hu, M., Lin, Y., Zheng, J., Zhang, R., and Zhang, Y.: Submicron aerosol analy- sis and organic source apportionment in an urban atmo- sphere in Pearl River Delta of China using high-resolution aerosol mass spectrometry, J. Geophys. Res., 116, D12304, https://doi.org/10.1029/2010JD014566, 2011. Hong Kong Marine Department: Ranking of container ports of the world, https://www.mardep.gov.hk/hk/publication/pdf/portstat_ 2_y_b5c.pdf (last access: 5 August 2018), 2012. Hu, Z. Y.: Studies on the Discharging and Distribution of Heavy Metal Pollution in the Pearl River Delta, Doctoral disserta- tion, Graduate School of the Chinese Academy of Sciences (Guangzhou Institute of Geochemistry), 2004. Elser, M., Huang, R.-J., Wolf, R., Slowik, J. G., Wang, Q., Canonaco, F., Li, G., Bozzetti, C., Daellenbach, K. R., Huang, Y., Zhang, R., Li, Z., Cao, J., Baltensperger, U., El-Haddad, I., and Prévôt, A. S. H.: New insights into PM2.5 chemical composi- tion and sources in two major cities in China during extreme haze events using aerosol mass spectrometry, Atmos. Chem. Phys., 16, 3207–3225, https://doi.org/10.5194/acp-16-3207-2016, 2016. Huang, R., Zhang, Y., Bozzetti, C., Ho, K., Cao, J., Han, Y., Dael- lenbach, K. R., Slowik, J. G., Platt, S. M., Canonaco, F., Zotter, P., Wolf, R., Pieber, S. M., Bruns, E. A., Crippa, M., Ciarelli, G., Piazzalunga, A., Schwikowski, M., Abbaszade, G., Schnelle- Kreis, J., Zimmermann, R., An, Z., Szidat, S., Baltensperger, U., Haddad, I. E., and Prévôt, A. S. H.: High secondary aerosol con- Environmental Protection Agency of Jiangmen City: Key pol- lution sources basic information in Jiangmen, available at: www.atmos-chem-phys.net/18/11563/2018/ Atmos. Chem. Phys., 18, 11563–11580, 2018 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD Yamasoe, M. A., Artaxo, P., Miguel, A. H., and Allen, A. G.: Chemical composition of aerosol particles from direct emis- sions of vegetation ﬁres in the Amazon Basin: water-soluble species and trace elements, Atmos. Environ., 34, 1641–1653, https://doi.org/10.1016/S1352-2310(99)00329-5, 2000. Tan, J., Duan, J., Ma, Y., He, K., Cheng, Y., Deng, S., Huang, Y., and Si-Tu, S.: Long-term trends of chemical charac- teristics and sources of ﬁne particle in Foshan City, Pearl River Delta: 2008–2014, Sci. Total Environ., 565, 519–528, https://doi.org/10.1016/j.scitotenv.2016.05.059, 2016. Yuan, Z., Lau, A., Zhang, H., Yu, J., Louie, P., and Fung, J.: Identiﬁcation and spatiotemporal variations of dominant PM10 sources over Hong Kong, Atmos. Environ., 40, 1803–1815, https://doi.org/10.1016/j.atmosenv.2005.11.030, 2006. Tao, J., Zhang, L., Cao, J., Zhong, L., Chen, D., Yang, Y., Chen, D., Chen, L., Zhang, Z., Wu, Y., Xia, Y., Ye, S., and Zhang, R.: Source apportionment of PM2.5 at urban and suburban ar- eas of the Pearl River Delta region, south China – With em- phasis on ship emissions, Sci. Total Environ., 574, 1559–1570, https://doi.org/10.1016/j.scitotenv.2016.08.175, 2017. Yuan, Z. B., Yu, J. Z., Lau, A. K. H., Louie, P. K. K., and Fung, J. C. H.: Application of positive matrix factorization in estimat- ing aerosol secondary organic carbon in Hong Kong and its rela- tionship with secondary sulfate, Atmos. Chem. Phys., 6, 25–34, https://doi.org/10.5194/acp-6-25-2006, 2006. Zou, B. B., Huang, X. F., Zhang, B., Dai, J., Zeng, L. W., Feng, N., and He, L. Y.: Source apportionment of PM2.5 pollution in an industrial city in southern China, Atmos. Pollut. Res., 8, 1193– 1202, https://doi.org/10.1016/j.apr.2017.05.001, 2017. Taylor, S. R. and Mclennan, S. M.: The geochemical evolu- tion of the continental crust, Rev. Geophys., 33, 293–301, https://doi.org/10.1029/95RG00262, 1995. Ulbrich, I. M., Canagaratna, M. R., Zhang, Q., Worsnop, D. R., and Jimenez, J. L.: Interpretation of organic components from Posi- tive Matrix Factorization of aerosol mass spectrometric data, At- mos. Chem. Phys., 9, 2891–2918, https://doi.org/10.5194/acp-9- 2891-2009, 2009. Villalobos, A. M., Barraza, F., Jorquera, H., and Schauer, J. J.: Chemical speciation and source apportionment of ﬁne particulate matter in Santiago, Chile, 2013, Sci. Total Environ., 512–513, 133–142, https://doi.org/10.1016/j.scitotenv.2015.01.006, 2015. Visser, S., Slowik, J. G., Furger, M., Zotter, P., Bukowiecki, N., Canonaco, F., Flechsig, U., Appel, K., Green, D. C., Tremper, A. H., Young, D. E., Williams, P. I., Allan, J. D., Coe, H., Williams, L. R., Mohr, C., Xu, L., Ng, N. L., Nemitz, E., Barlow, J. F., Halios, C. X.-F. Huang et al.: Source apportionment of PM2.5 in PRD 11580 dominant weather patterns, Atmos. Environ., 35, 2697–2705, https://doi.org/10.1016/S1352-2310(00)00413-1, 2001. dominant weather patterns, Atmos. Environ., 35, 2697–2705, https://doi.org/10.1016/S1352-2310(00)00413-1, 2001. Wang, H., Tian, M., Chen, Y., Shi, G., Liu, Y., Yang, F., Zhang, L., Deng, L., Yu, J., Peng, C., and Cao, X.: Seasonal characteris- tics, formation mechanisms and source origins of PM2.5 in two megacities in Sichuan Basin, China, Atmos. Chem. Phys., 18, 865–881, https://doi.org/10.5194/acp-18-865-2018, 2018. Polissar, A. V., Hopke, P. K., Paatero, P., Malm, W. C., and Sisler, J. F.: Atmospheric aerosol over Alaska: 2. Elemental composition and sources, J. Geophys. Res., 103, 19045–19057, https://doi.org/10.1029/98JD01212, 1998. Wang, J., Ho, S. S. H., Ma, S., Cao, J., Dai, W., Liu, S., Shen, Z., Huang, R., Wang, G., and Han, Y.: Characterization of PM2.5 in Guangzhou, China: uses of organic markers for support- ing source apportionment, Sci. Total Environ., 550, 961–971, https://doi.org/10.1016/j.scitotenv.2016.01.138, 2016. Reyes-Villegas, E., Green, D. C., Priestman, M., Canonaco, F., Coe, H., Prévôt, A. S. H., and Allan, J. D.: Organic aerosol source apportionment in London 2013 with ME-2: exploring the so- lution space with annual and seasonal analysis, Atmos. Chem. Phys., 16, 15545–15559, https://doi.org/10.5194/acp-16-15545- 2016, 2016. Wang, Q. Q., Huang, X. H. H., Zhang, T., Zhang, Q., Feng, Y., Yuan, Z., Wu, D., Lau, A. K. H., and Yu, J. Z.: Organic tracer-based source analysis of PM2.5 organic and elemental carbon: A case study at Dongguan in the Pearl River Delta, China, Atmos. Environ., 118, 164–175, https://doi.org/10.1016/j.atmosenv.2015.07.033, 2015. Rodríguez, S., Van Dingenen, R., Putaud, J.-P., Dell’Acqua, A., Pey, J., Querol, X., Alastuey, A., Chenery, S., Ho, K.-F., Har- rison, R., Tardivo, R., Scarnato, B., and Gemelli, V.: A study on the relationship between mass concentrations, chemistry and number size distribution of urban ﬁne aerosols in Milan, Barcelona and London, Atmos. Chem. Phys., 7, 2217–2232, https://doi.org/10.5194/acp-7-2217-2007, 2007. Wang, Q., Feng, Y., Huang, X. H. H., Grifﬁth, S. M., Zhang, T., Zhang, Q., Wu, D., and Yu, J. Z.: Nonpo- lar organic compounds as PM2.5 source tracers: Investiga- tion of their sources and degradation in the Pearl River Delta, China, J. Geophys. Res.-Atmos., 121, 11862–11879, https://doi.org/10.1002/2016JD025315, 2017. Sarnat, J. A., Marmur, A., Klein, M., Kim, E., Russell, A. G., Sar- nat, S. E., Mulholland, J. A., Hopke, P. K., and Tolbert, P. E.: Fine Particle Sources and Cardiorespiratory Morbidity: An Applica- tion of Chemical Mass Balance and Factor Analytical Source- Apportionment Methods, Environ. Health Persp., 116, 459–66, https://doi.org/10.1289/ehp.10873, 2008. www.atmos-chem-phys.net/18/11563/2018/ Atmos. Chem. Phys., 18, 11563–11580, 2018 X.-F. Huang et al.: Source apportionment of PM2.5 in PRD H., Fleming, Z. L., Baltensperger, U., and Prévôt, A. S. H.: Advanced source apportionment of size-resolved trace el- ements at multiple sites in London during winter, Atmos. Chem. Phys., 15, 11291–11309, https://doi.org/10.5194/acp-15-11291- 2015, 2015. Atmos. Chem. Phys., 18, 11563–11580, 2018
https://openalex.org/W4387773892	https://ecp.ep.liu.se/index.php/sims/article/download/765/670	English	null	Numerical Investigation on Performance of Gas Turbine Blade: Effects of simulation Models and Blade Geometry	Linköping electronic conference proceedings	2,023	cc-by	6,011	Numerical Investigation on Performance of Gas Turbine Blade Effects of Simulation Models and Blade Geometry Heng Hu, Narmin Hushmandi, Magnus Genrup Energy Science department, Lund University, Box 118, 22100 Lund, Sweden heng.hu@energy.lth.se SIMS 64 SIMS 64 SIMS 64 Västerås, Sweden, September 26-27, 2023 Abstract With a significant impact on turbomachinery blade performance, surface curvature distribution becomes one of the essential factors in the design of high-efficiency blades. This study focuses on applying computational fluid dynamics (CFD) to evaluate turbine rotor blade performance. The main aim is to analyze the influence of incidence and geometry shape on the performance of a gas-turbine blade in two dimensions. To achieve this, an investigation was conducted to identify a suitable turbulence model for this case, with two turbulence models combined with two different solvers explored in ANSYS Fluent: Realizable k-ε model in pressure and density based solver; k-ω shear stress transport (SST) model in pressure and density based solver. The blade total pressure loss across different blade exit Mach numbers is the comparison factor, with validation against experimental data. Subsequently, the chosen pressure-based k-ω SST model mode is used to study the performance of various air inflow incidence angles and compare two different blade geometries. In this paper, two geometries, Geometry 1 and Geometry 2, were designed by setting two different exit blade angles, β2=79.5° and β2=70° respectively, while the inlet blade angles have the same value, β1=48.8°. Furthermore, the effect of varying air inflow incidence angles between -48.8° and 10° on the blade performance distribution is also investigated. Within the studied range, the inflow incidence angle of 10° is found to have the best performance in terms of turbine work output. On the other hand, the blade performance of Geometry 2 appears superior to Geometry 1. 1. Introduction This paper involves the 2D round, which is practical The turbine portion in gas turbine systems extracts work from the combusted gases to power the SIMS 64 Västerås, Sweden, September 26-27, 2023 (b) Schematic of blade sections for two geometries. Figure 1: Schematic of the 2D blade. regarding the scope and computational cost. Coupled with the computationally economical RANS models, the approach reflects a common means adopted in industries to offer a quick blade diagnostic tool for trend studies. The selected RANS model is subsequently applied to the turbine blade design to relate the modified parameters to the selected blade performance indicators. In addition, this paper also seeks to demonstrate the key role of CFD in both industry and academic research for turbine blade design. CFD today is an indispensable part of blade design as it provides a performance evaluation for a particular geometry. It can also be used to pick out the blade locations requiring modifications, forming an iterative part of the design process to optimize surface geometry and loadings. ) Schematic of blade sections for two geometries. Figure 1: Schematic of the 2D blade. Schematic of blade sections for two geometr Figure 1: Schematic of the 2D blade. In this research, two blades were designed by setting two different β2: exit blade angle, β2=79.5°, and β2=70° respectively, while the β1: inlet blade angle have the same value, β1=48.8°. The two blade geometries are shown in Figure 1(b). Geometry 1 is a reference blade profile of Atlas (Mee D J et al., 1992). Since Geometry 1 has experimental results, most of the simulations in this paper are based on Geometry 1. In order to study the influence of blade geometric parameters on blade performance, based on Geometry 1, modifications are done to the exit blade angle to obtain Geometry 2. The remaining important blade parameter values of Geometry 1 and Geometry 2 are shown in Table 1. 1. Introduction compressor stages and drive other loads. As such, considerable effort has been poured into turbine blade research to attain maximum extraction of the valuable work output. Additionally, to further push the upper bound of turbine efficiency, much research has been done on limiting flow separation that contributes to decreases in work output (Korakianitis T P. et al., 1989) Unsurprisingly, the surface curvature of the blade determines its loading distribution, forming a crucial factor in controlling flow separation (Nemnem et al., 2014). In further detail, a smooth curvature distribution at the blade leading edge has been found to prevent the formation of separation bubbles, thus suppressing the flow separation (Song Y et al., 2014). The blade geometric profile is designed to determine the efficient aerodynamic performance. Some principal aerodynamic objectives of a turbine blade design are: the blade angles at the inlet and exit must be correctly matched to the fluid flow angles; the throat area determines the flow capacity and must be sized correctly. Besides, Blade surfaces curvature and changes in curvature should be limited, consistent with the necessary turning of the flow in blade passage. So the blade design plays an essential role in the full design process. In turbomachinery, quality blade design is an integral element to efficient aerodynamics (Lebele-Alawa et al., 2008), which can affect the entire blade row's performance, affecting the overall machine efficiency (Fast M et al., 2009). In particular, blade curvature distribution has been shown to influence boundary-layer characteristics, determining blade losses and efficiency (Korakianitis et al., 1993).Even though the field of blade curvature is relatively mature, the potential benefits of sizeable industrial cost-savings and environmental impact from even a tiny efficiency improvement have been sustaining the keen interest in work in this area. This paper considers how blade parameterization affects profile losses and loading diagrams in a numerical approach, including blade angles, incidence, etc. It is worth highlighting that one main objective of the paper is to explore and compare different numerical models against a set of experimental data to identify a suitable model for this application. Designing a turbine blade geometry typically starts from a one-dimensional approach before moving on to two-dimensional (2D) and eventually developing into 3D in the final phase. 2. Numerical Simulation For this research, the fluid flow parameters, e.g., velocity magnitude, pressure, temperature, and Mach number, around the turbine blade will be simulated using numerical methods. Two turbine blade geometries with different exit blade angles were generated to examine the influence of blade geometry. The ANSYS ICEM CFD was used to mesh the geometry. The commercial CFD software, ANSYS Fluent, is used for solving and post- processing. 2.1. Turbine Blade Geometries Table 1: Blade parameters. Parameters Geometry 1 Geometry 2 Chord (m) 0.0474 0.0474 Stagger Angle(°) -37.8 -29.8 Pitch/Chord 0.7593 0.7597 Axial Chord (m) 0.03745 0.0411 The Normalized curvature distribution of the two geometries generated in AxCent showing in Figure 2, which shows that, in the two cases, the blade curvature is continuous and smooth. The blade curvature distribution of the pressure side for the two geometries differs, while the blade curvature distribution on the suction side coincides. (a) Pressure Side Table 1: Blade parameters. Parameters Geometry 1 Geometry 2 Chord (m) 0.0474 0.0474 Stagger Angle(°) -37.8 -29.8 Pitch/Chord 0.7593 0.7597 Axial Chord (m) 0.03745 0.0411 The Normalized curvature distribution of the two geometries generated in AxCent showing in Figure 2, which shows that, in the two cases, the blade curvature is continuous and smooth. The blade curvature distribution of the pressure side for the two geometries differs, while the blade curvature distribution on the suction side coincides. Table 1: Blade parameters. Parameters Geometry 1 Geometry 2 Chord (m) 0.0474 0.0474 Stagger Angle(°) -37.8 -29.8 Pitch/Chord 0.7593 0.7597 Axial Chord (m) 0.03745 0.0411 The Normalized curvature distribution of the two geometries generated in AxCent showing in Figure This paper aims to find the influence of different solvers, incidence angle, and shape of turbine blades. For simplification of simulation cost, 2D geometries are selected for this study. Figure 1(a) shows the schematic diagram of blade section parameters. AxCent of Concepts NREC design tools software provides a good way to generate the geometries by determining the blade section parameters (such as inlet/exit blade angles, stagger angles, gauge angles, wedge angles, chords, and pitch). The Normalized curvature distribution of the two The Normalized curvature distribution of the two geometries generated in AxCent showing in Figure 2, which shows that, in the two cases, the blade curvature is continuous and smooth. The blade curvature distribution of the pressure side for the two geometries differs, while the blade curvature distribution on the suction side coincides. (a) Schematic diagram of blade section parameters (a) Schematic diagram of blade section parameters distribution on the suction side coincides. (a) Pressure Side (a) Pressure Side (a) Pressure Side (a) Schematic diagram of blade section parameters (a) Pressure Side (a) Pressure Side SIMS 64 Västerås, Sweden, September 26-27, 2023 Västerås, Sweden, September 26-27, 2023 (b) Suction Side Figure 2: Curvature distribution of the 2D blade sections for two geometries. 2.1. Turbine Blade Geometries treatment and are expected better to handle the complex turbulent flow around these areas and enhance computational accuracy. The first layer grid near-wall is 0.002 mm, and the y+ value is around 1. Figure 3: Numerical domain and mesh for the analysis of the geometry 1. Figure 3: Numerical domain and mesh for the analysis of the geometry 1. (b) Suction Side Figure 2: Curvature distribution of the 2D blade sections for two geometries. Figure 3: Numerical domain and mesh for the analysis of the geometry 1. 2.2. Mesh Generation and Boundary Conditions A mesh independence analysis was done using various mesh densities to study the effect of grid resolution on the accuracy of numerical results. For this purpose, the grid resolution was increased until the blade total pressure loss had no significant variations. The computational domain of fluid is divided into three parts: the upstream and downstream domains and the blade domain. The computation domain is extended upstream and downstream to achieve a fully developed flow. The inlet of the numerical domain is extended 0.8 times axial chord of the blade upstream of the blade leading edge, and the exit is extended 1.25 times the axial chord downstream of the blade trailing edge. Four types of boundary conditions are presented to solve a blade cascade: wall, periodic, inlet, and outlet (Moshizi S A et al., 2014). Using the periodic boundaries for the blade cascade is common in CFD to reduce the computational domain size and thus decrease the time and memory cost for processing (M. Mahmoudi et al., 2005), so just one blade passage simulated in all simulations. For the inlet and out boundary conditions, pressure-inlet and pressure-outlet are adopted, respectively. No-slip condition is used for the blade wall. The values of the boundary conditions applied to the cases are presented in Table 2. This paper uses the total pressure loss coefficient to characterize the blade total profile loss. The definition of the total pressure loss coefficient: 𝑌𝑝= 𝑝01,𝑖𝑛−𝑝02,𝑜𝑢𝑡 𝑝01,𝑖𝑛−𝑝1𝑠,𝑖𝑛 (1) (1) Where 𝑝01,𝑖𝑛 represents the blade inlet total pressure, 𝑝02,𝑜𝑢𝑡 is the mass-weighted average total pressure at the blade section where extended 0.8 times the axial chord downstream of the blade trailing edge. Nine grid sizes in the range of 5000 to 134 000 structured cells are evaluated. Figure 4 illustrates the variations of blade total pressure loss for the different grids. Due to the importance of computational efficiency, the mesh with 114 400 structured cells was chosen for Geometry 1; Geometry 2 has meshed with the same method, and the number of grids is comparable at 118 650 structured cells. Table 2: Boundary conditions. Parameter Value Inlet/Outlet Total Temperature 1046(K) Inlet Absolute Total Pressure 211325(Pa) Outlet Absolute Static Pressure 126325(Pa) Outlet Mach Number 0.7-1.1 With good quality of the structured mesh, using a higher-order discretization scheme, the solver solution would have a higher convergence rate and precision. 2.5. Model Validation 2.5. Model Validation The experimental results of Atlas, involving the same blade design as Geometry 1, were used to validate the numerical solutions. The blade profile loss against different Mach numbers was profiled in the actual experiment. The inlet airflow direction specification method was set to be normal to the boundary, corresponding to an incidence angle of - 48.8°. Additionally, the specification method for both inlet and outlet boundary conditions was based on turbulence intensity and turbulence viscosity ratios of 5% and 10, respectively. Different exit Mach numbers were obtained by adjusting the outlet pressure value. Figure 5 shows the numerical blade losses across various exit Mach numbers found via the four model-solving cases close to experimental data. The models are pressure-based SST k-ω, density-based SST k-ω, pressure-based Realizable k-ε, and density-based Realizable k-ε. Although the numerical data do not overlap entirely with the experimental ones, the differences are within a reasonable range. The changing trend of the experimental and simulated structures is basically the same and have some slight difference. The plotted loss coefficients are for exit Mach numbers between 0.7 to 1.1. It is seen from the plot that, up to a specific exit Mach number (Ma=0.9), the total pressure losses are low. However, after Ma=0.9, the total pressure loss increases rapidly. The sudden increase is due to the appearance of shocks inside the blade channel. As the exit Mach number continues to grow to about 1.05, the simulation results show that the total pressure loss increases slowly with the exit Mach number, while the experimental results still increase significantly with the increase of the exit Mach number. In Fluent, the pressure-based solver is developed from the original separate solver, which solves the momentum, pressure correction, energy, and other scalar equations in sequence, such as the turbulence equation. Unlike before, the pressure-based solver also adds a coupling algorithm, which can be freely switched between the separation and coupling solutions. The coupling solution is to solve the aforementioned momentum and pressure correction simultaneously and then solve energy, component equations, and other scalar equations, such as Turbulence equations, etc., which have fast convergence speed but require more memory and calculation. 2.4. Solver Settings 1, the density-based simulations took significantly longer to run and converge. The coupled algorithm is used for the pressure-based solver because of its higher accuracy. As the simulation is 2D and the grid resolution is not large, the trade-off in computational time is insignificant. 2.4. Solver Settings The governing equations for viscous compressible fluid are the continuity, Navier-Stokes momentum, and state equations. The equations are discretized with the finite volume method. Firstly, four different RANS models in Fluent - namely the Pressure-based k-epsilon method, Pressure-based SST k-ω method, Density-based Realizable k-ε method, Density- based SST k-ω method were used to solve the discretized equations. For the spatial discretization of pressure-based solver cases, the second-order scheme is used for pressure, and the second-order upwind scheme is used for momentum and turbulent kinetic energy terms. For the spatial discretization of density-based solver cases, the second-order upwind scheme is used for flow and turbulent kinetic energy terms. Besides, ideal gas model has been chosen because the physical fluid is a compressible fluid. Turbulence is chosen to be modeled using the Realizable k-ε and SST k-ω models due to the corresponding theoretical strengths in providing realistic results and superior performance for complex flows (like adverse pressure gradient and separated flows), respectively. The convergence of the solution is monitored by checking the residuals of the numerically solved governing equations, which use the absolute scale of residuals to converge until 1E-6. 2.2. Mesh Generation and Boundary Conditions Therefore, the software ICEM CFD was used to generate the structured grids for calculation domains around the blade surface. Figure 3 shows the meshes of Geometry 1 employed in the computational domain. The whole grid was structured with an O-H type of mesh, using the O- type mesh around the blade and H-type everywhere else. The meshes are refined for the near-wall Table 2: Boundary conditions. Parameter Value Inlet/Outlet Total Temperature 1046(K) Inlet Absolute Total Pressure 211325(Pa) Outlet Absolute Static Pressure 126325(Pa) Outlet Mach Number 0.7-1.1 With good quality of the structured mesh, using a higher-order discretization scheme, the solver solution would have a higher convergence rate and precision. Therefore, the software ICEM CFD was used to generate the structured grids for calculation domains around the blade surface. Figure 3 shows the meshes of Geometry 1 employed in the computational domain. The whole grid was structured with an O-H type of mesh, using the O- type mesh around the blade and H-type everywhere else. The meshes are refined for the near-wall Table 2: Boundary conditions. Parameter Value Inlet/Outlet Total Temperature 1046(K) Inlet Absolute Total Pressure 211325(Pa) Outlet Absolute Static Pressure 126325(Pa) Outlet Mach Number 0.7-1.1 With good quality of the structured mesh, using a higher-order discretization scheme, the solver solution would have a higher convergence rate and precision. Therefore, the software ICEM CFD was used to generate the structured grids for calculation domains around the blade surface. Figure 3 shows Table 2: Boundary conditions. Table 2: Boundary conditions. Parameter Value Inlet/Outlet Total Temperature 1046(K) Inlet Absolute Total Pressure 211325(Pa) Outlet Absolute Static Pressure 126325(Pa) Outlet Mach Number 0.7-1.1 Figure 4: Mesh independence study of Geometry 1 With good quality of the structured mesh, using a higher-order discretization scheme, the solver solution would have a higher convergence rate and precision. Therefore, the software ICEM CFD was used to generate the structured grids for calculation domains around the blade surface. Figure 3 shows the meshes of Geometry 1 employed in the computational domain. The whole grid was structured with an O-H type of mesh, using the O- type mesh around the blade and H-type everywhere else. The meshes are refined for the near-wall Figure 4: Mesh independence study of Geometry 1 Figure 4: Mesh independence study of Geometry 1 Figure 4: Mesh independence study of Geometry 1 SIMS 64 Västerås, Sweden, September 26-27, 2023 2.5. Model Validation The difference between Pressure-based and Density- based: First, the pressure-based solver was mainly used for the solution of low-speed incompressible flow, while the density-based method was mainly designed for high-speed compressible flow, but now both ways have been extended to solve a large flow velocity range method. Second, the density-based solver was developed from the originally coupled solver. It simultaneously solves the continuity, momentum, energy, and component equation, then solves the turbulence and scalar equations. As a result, the density-based solver has a fast convergence speed and requires more memory and calculation time than the pressure-based solver! As a result, the density-based solver is expected to take longer computational time per iteration. According to observation, for the studied Geometry Figure 5: Comparison between numerical and experimental blade profile loss for Geometry 1. As a result, the density-based solver is expected to take longer computational time per iteration. According to observation, for the studied Geometry Figure 5: Comparison between numerical and experimental blade profile loss for Geometry 1. SIMS 64 Västerås, Sweden, September 26-27, 2023 Västerås, Sweden, September 26-27, 2023 blade wall under the same solver, the SST k-ω model shows a better distribution of the reflected oblique shocks and expansion waves near the blade wall. Theoretically, the Realizable k-ε model might face inaccuracies for complex wall-bounded flows, such as predicting the early onset of flow separation or the inability to accurately capture turbulent shock wave- boundary layer interaction. Thus SST k-ω might be more reliable in this respect. The Mach number around the trailing edge of the turbine blade is small, which means the velocity is small, resulting from some adverse flow decrease in kinetic energy. The differences may have come from the following reasons. Firstly, the compressibility of air and the capture of shock waves in the simulation calculations may still differ from the actual problem; some factors, like the turbulent vortex in 3D space and dissipation in the third direction, cannot be captured by the 2D simulations in this research. Secondly, applying constant static pressure at the outlet affects the total pressure loss values. Besides, the empirical data measurement position and statistical approach may differ from the data collection of our numerical approach. Finally, this study is mainly on a transonic turbine blade, with exit Mach number ranging from about 0.7 to 1.1, for which it is notoriously difficult to get a precise solution with RANS models. 2.5. Model Validation Figure 6: The Contour of Mach number of four models with exit Mach number 1.1. Similarly, the differences between the four cases are also slight. The data sets for density-based and pressure-based SST k-ω models are close to each other, while the density-based and pressure-based Realizable k-ε models also have a similar phenomenon. According to the literature (Corriveau D et al., 2007), the profile loss will vary slowly for high Mach numbers, which also can be observed in our current numerical results. When the Mach number is above almost 1.05, the density-based and pressure-based Realizable k-ε model has less variation than the SST k-ω models. So in terms of growth trend, the SST k-ω model is closer to the experimental growth trend than the Realizable k-ε model. Figure 6: The Contour of Mach number of four models with exit Mach number 1.1. Figure 7 and 8 show the contour of static temperature and total pressure of four models, respectively. Besides, in both figures, the ‘inclined strip’ near the outlet part is caused by the blade wakes, and the ‘strips’ are caused by other turbine blades present with the periodic boundary condition selection. 3.1. Comparison across Models 3.1. Comparison across Models Figure 7: The static temperature of four models with exit Mach number 1.1. p Before further research on this turbine blade, some variables, like Mach number, pressure, obtained in the model validation section were identified as key parameters. In addition, comparing the differences caused by different model-solver cases is also critical. Using geometry 1, we have done some simulations to explore the influence of model and solver selection. This study analyzes the conditions of different outlet Mach numbers 0.76 and 1.1, as shown in Figures 6-11. The most apparent difference between these two cases is the observation of shock waves. When the outlet Mach number is 1.1, the shock wave is obvious in all four models, as seen in Figure 6 and 7, while the flow field of four models is subsonic when the outlet Mach number is 0.76, as shown in Figures 9 and 10. Figure 7: The static temperature of four models with exit Mach number 1.1. Figure 8: The Total Pressure of four models with exit Mach number 1.1. Figure 8: The Total Pressure of four models with exit Mach number 1.1. Figure 6 shows the contour of the exit Mach number of 1.1 under the same turbulence models with different solvers. Specifically, the density-based and pressure-based Realizable k-ε models have similar Mach number distributions, including the value range and position of the shock wave, while the density-based and pressure-based SST k-ω models capture a more severe shock. As it is seen from zoom-in figures, near the suction side of the outlet Figure 8: The Total Pressure of four models with exit Mach number 1.1. SIMS 64 SIMS 64 Västerås, Sweden, September 26-27, 2023 In a gas turbine, the turbines would endure some of the harshest operating temperatures. On top of revealing the temperatures the blades will be subject to, the temperature distribution around the blade can potentially allow the designer to measure how design changes might lead to temperature changes in the flow field and uneven distributions, if any. Furthermore, additional design measures such as cooling channels and thermal barrier coating can be incorporated to target the areas with peak temperatures. As the outlet Mach number was decreased to 0.76, the entire flow field becomes subsonic, as shown in Figures 9. The maximum Mach number changed from about 1.31 to 0.85. 3.1. Comparison across Models Besides, the Mach number in the blade's trailing edge is more uniform, and there is no shock wave in the field. Figure 9: The Contour of Mach number of four models with exit Mach number 0.76. Figure 11 shows the total pressure of four models when the outlet Mach number is 0.76. It can be seen that the total pressure drop in Figure 11 is significantly smaller than that in Figure 8. Therefore, the shock wave is one of the significant sources of loss. From this, it can be concluded that the blade curvature distribution should be in a way to minimize the effect of losses with having oblique and expansion waves in the flow. Figure 9: The Contour of Mach number of four models with exit Mach number 0.76. So far, the results of the density-based and the pressure-based are not much different, and the pressure-based costs less computational time. Besides, turbulence models, SST k-ω model compared to Realizable k-ε model has more ability to accurately capture turbulent shock wave- boundary layer interaction. Therefore, the pressure- based SST k-ω model is thought to be more realistic for this study. Therefore, the computations performed and the results presented below are performed entirely with pressure based SST k-ω model. A low Mach number means the kinetic energy loss is slight. Therefore, the static temperature in the trailing edge of the blade increased in a smaller area, and the distribution near the outlet is more even, as shown in Figure 10. The average temperature is higher than the case with a high Mach number. This is because the two cases with the same inlet temperature have higher Mach number with shock waves increasing more loss so that the temperature will be higher. will be higher. Figure 10: The static temperature of four models with exit Mach number 0.76. Figure 11: The Total Pressure of four models with exit Mach number 0.76. The temperature data (Figures 7, 10) also offers information pertinent to the design of turbine blades. g Figure 10: The static temperature of four models with exit Mach number 0.76. 3.2. Influence of Incidence Figure 13 shows the contour of the velocity vector and zoom-in figures. Figure 13(a) compares the contour of different incidence angles on Mach number in Geometry 1. From the zoom-in figure (b), showing some specific details, there is a backflow and vortex near the pressure side boundary region when the incidence angle is -48.8º, which may cause more losses. However, as the incidence angle change to 10º, there is no reverse flow and vortex. ( ) p Figure 14: Influence of incidence on blade loading along the surface of the blade in Geometry 1. Identifiable from Figure 14, the incidence of 10º gives the largest work output (within the studied range). This information could be incorporated either into the design of the trailing edge blade angle of the stator stage upstream of the concerned rotors or the orientation of the rotor blades to obtain the desired incidence angle. Isentropic Mach number and static pressure values along the blade surface normalized with total inlet pressure p_s⁄p_(0,in) are plotted against the axial distance from the leading edge of the blades (x⁄C_axial ), where C_axial is the blade axial chord, is adopted to better evaluate performance due to the small numerical differences for blade profile losses, which shows in Figure 14. The small 'loop' in Figure 14 (a) when the incidence is -48º indicates negative work done by the blade from the flow before the position of 0.25. The net area enclosed by the curve can be related to the work output by the blade so that a larger one will indicate better work output. Besides, we can also know that over-expansion between the throat and trailing edge exits some diffusion, with an increase in loss. Figure 14 (b) compares the influence of different incidences on isentropic Mach number distribution. It can be seen from the Figure 14 (b) that the maximum isentropic Mach number of the four cases does not exceed 1. The blade's performance depends largely on the amount of diffusion and diffusion rate, which is also important when we further optimize the blade performance in the future. On the other hand, gas turbines frequently operate at off-design conditions. The airflow entering each turbine stage can be far from the designed incidences at off-design conditions. 3.2. Influence of Incidence To study the influence of incidence on the flow around the turbine blade, different incidence angles are applied at the inlet with the same geometry 1, setting the same inlet and outlet pressure, which exit Mach number is almost 0.88, and the maximum Mach number is around 1, as shown in Figure 12. Increasing the incidence angle will bring about the translation of the low-speed stagnation regions from the blade leading edge to the pressure side and increase in inlet Mach numbers. Figure 10: The static temperature of four models with exit Mach number 0.76. Figure 11: The Total Pressure of four models with exit Mach number 0.76. Figure 11: The Total Pressure of four models with exit Mach number 0.76. Figure 12: Contour of influence of incidence on Mach number in Geometry 1 Figure 11: The Total Pressure of four models with exit Mach number 0.76. Figure 11: The Total Pressure of four models with exit Mach number 0.76. Figure 12: Contour of influence of incidence on Mach number in Geometry 1 The temperature data (Figures 7, 10) also offers information pertinent to the design of turbine blades. The temperature data (Figures 7, 10) also offers information pertinent to the design of turbine blades. The temperature data (Figures 7, 10) also offers information pertinent to the design of turbine blades. Västerås, Sweden, September 26-27, 2023 SIMS 64 SIMS 64 (a) Contours of velocity vectors (b) Zoom-in Figures of two incidence angle Figure 13: Contours of velocity vectors showing effect of incidence angle on the Geometry 1. (a) Normalized Static Pressure (b) Isentropic Mach Number Figure 14: Influence of incidence on blade loading along the surface of the blade in Geometry 1. (a) Normalized Static Pressure (a) Normalized Static Pressure (a) Normalized Static Pressure (b) Zoom-in Figures of two incidence angle Figure 13: Contours of velocity vectors showing effect of incidence angle on the Geometry 1. Figure 13 shows the contour of the velocity vector and zoom-in figures. Figure 13(a) compares the contour of different incidence angles on Mach number in Geometry 1. From the zoom-in figure (b), showing some specific details, there is a backflow and vortex near the pressure side boundary region when the incidence angle is -48.8º, which may cause more losses. However, as the incidence angle change to 10º, there is no reverse flow and vortex. 3.3. Effects of Exit Blade Angle and Incidence Figure 15 shows the Mach number distribution of the blades in Geometries 1 and 2. The performances of Geometry 1 and Geometry 2 with the incidence of 10º and -10º were compared to observe the influence of the modified exit blade angle on the blade performance. The results are shown in Figures 16. References Lebele-Alawa B T, Hart H I, Ogaji S O T, et al. Rotor-blades’ profile influence on a gas-turbine’s compressor effectiveness[J]. Applied Energy, 2008, 85(6): 494-505. Fast M, Assadi M, De S. Development and multi-utility of an ANN model for an industrial gas turbine[J]. Applied Energy, 2009, 86(1): 9-17. (b) Isentropic Mach Number Figure 16: Influence of incidence on blade loading along the surface of the blade in Geometry 1 and 2. (b) Isentropic Mach Number Korakianitis T. Hierarchical development of three direct-design methods for two-dimensional axial-turbomachinery cascades[J]. 1993. Korakianitis T, Papagiannidis P. Surface-curvature-distribution effects on turbine-cascade performance[C]//Turbo Expo: Power Figure 16(a) shows the normalized static pressure distribution along the blade surface. The enclosed curve area of Geometry 2 for all studied incidence angles is bigger than those of Geometry 1. Figure 16(a) shows the normalized static pressure distribution along the blade surface. The enclosed curve area of Geometry 2 for all studied incidence angles is bigger than those of Geometry 1. From Figure 16(b), Geometry 2 has a smaller maximum isentropic Mach number for the same incidence angle than Geometry 1. Besides, the position of peak velocity on the suction side is earlier, so the diffusion rate is less, with a decrease in loss. for Land, Sea, and Air. American Society of Mechanical Engineers, 1992, 78934: V001T01A044. Korakianitis T P. Design of airfoils and cascades of airfoils[J]. AIAA journal, 1989, 27(4): 455-461. From Figure 16(b), Geometry 2 has a smaller maximum isentropic Mach number for the same incidence angle than Geometry 1. Besides, the position of peak velocity on the suction side is earlier, so the diffusion rate is less, with a decrease in loss. j Nemnem A F, Turner M G, Siddappaji K, et al. A smooth curvature-defined meanline section option for a general turbomachinery geometry generator[C]//Turbo Expo: Power f Land, Sea, and Air. American Society of Mechanical Engineers, 2014, 45615: V02BT39A026. Song Y, Gu C W, Xiao Y B. Numerical and theoretical investigations concerning the continuous-surface-curvature effect in compressor blades[J]. Energies, 2014, 7(12): 8150- 8177. By this measurement, the performance of Geometry 2 is better than Geometry 1, which indicates that decreasing the exit blade angle has led to improving the performance of the current turbine blade. Mee D J, Baines N C, Oldfield M L G, et al. An examination of the contributions to loss on a transonic turbine blade in cascade[J]. 1992. 3.2. Influence of Incidence A transonic or low supersonic flow coupled with a large incidence, possibly leading to a significant flow separation on the turbine blade, would pose a real challenge for turbine designers. The data obtained from incidence angle simulations can be useful for performance analysis of off-design operating conditions. The performances of Geometry 1 and Geometry 2 with the incidence of 10º and -10º were compared to observe the influence of the modified exit blade angle on the blade performance. The results are shown in Figures 16. Västerås, Sweden, September 26-27, 2023 SIMS 64 Figure 15: The Mach number distribution of the blades in Geometries 1 and 2. more accurately, lower computational cost of the pressure-based solver, as well as SST k-ω model’s theoretical superior ability to handle complex flows including those around turbine blades. So this paper finds a cost-effective CFD model that can predict performance trends with reasonable accuracy. It provides a more convenient and reliable method for performance evaluation of 2D turbine blade geometries. g Besides, different incidence angles are studied to see the influence on the blade performance. Within the study range, the inflow incidence angle of 10° is found to have the best performance in terms of turbine work output. Two geometries were designed by setting two different exit blade angles to observe the influence of the modified exit blade angle on the blade performance. The blade performance of Geometry 2 appears superior to Geometry 1. Figure 15: The Mach number distribution of the blades in Geometries 1 and 2. (a) Normalized Static Pressure (b) Isentropic Mach Number Figure 16: Influence of incidence on blade loading along the surface of the blade in Geometry 1 and 2. (a) Normalized Static Pressure Finally, Geometry 2 with an exit blade angle of 70º, coupled with the incidence angle of 10º (among the cases of incidence studied), has been shown to give the largest work output and fewer losses. (a) Normalized Static Pressure (a) Normalized Static Pressure References Moshizi S A, Madadi A, Kermani M J. Comparison of inviscid and viscous transonic flow field in VKI gas turbine blade cascade[J]. Alexandria Engineering Journal, 2014, 53(2): 275- 280. 4. Conclusion Thi i This paper mainly focuses on applying CFD with a suitable turbulence model to evaluate turbine rotor blade performance. The results have shown the adequacy of the four RANS models – pressure and density-based Realizable k-ε and SST k-ω, in simulating the flow field trends for the Geometry blade design to a reasonable accuracy. The pressure- based SST k-ω model has been eventually picked as the model of choice due to the slightly better matching of the experimental data trends, capability to capture shock waves in the performed simulations M. Mahmoudi, M. Ansari, Numerical investigation of turbine blade trailing edge flow ejection effects on mach number distribution of gas turbine blade surface. Using rng.k-e turbulence model[J].2005. Corriveau D, Sjolander S A. Influence of loading distribution on the off-design performance of high-pressure turbine blades[J]. 2007. JNL/27.2.97.
https://openalex.org/W2052972056	https://link.springer.com/content/pdf/10.1007/s40095-014-0133-1.pdf	English	null	Energy potential from the anaerobic digestion of food waste in municipal solid waste stream of urban areas in Vietnam	International journal of energy and environmental engineering	2,014	cc-by	7,226	Int J Energy Environ Eng (2014) 5:365–374 DOI 10.1007/s40095-014-0133-1 Int J Energy Environ Eng (2014) 5:365–374 DOI 10.1007/s40095-014-0133-1 ORIGINAL RESEARCH Energy potential from the anaerobic digestion of food waste in municipal solid waste stream of urban areas in Vietnam Hoa Huu Nguyen • Sonia Heaven • Charles Banks Received: 31 March 2014 / Accepted: 26 June 2014 / Published online: 2 August 2014 The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Anaerobic digestion (AD) was introduced in Vietnam more than 10 years ago, but at a small scale to deal with agricultural wastes, manure, etc. Despite its many advantages, AD does not yet make a signiﬁcant contribu- tion to resolving Vietnams urban waste issues due to a lack of information, data and experience. This paper, using an energy model of food waste digestion, provides a usable source of information regarding energy potential of food waste generated from urban areas in Vietnam in forms of electricity, heat, and upgraded biogas under two different scenarios. Results show that if food waste is separated from the municipal solid waste (MSW) stream and sent to AD plants, total available energy equivalent each day is about 19, 20 and 45 GWh in 2015, 2020, and 2025, respectively. This could contribute between 2.4 and 4.1 % of the elec- tricity demand of Vietnam, as well as double this amount of energy in the form of heat. Alternatively, upgraded biogas could contribute approximately 2.2–4.7 % of fuel consumption for transportation. This suggests AD is a promising method to treat MSW in cities, especially when considering the problematic aspects of other current waste disposal methods such as: landﬁlling, composting and, incineration. H. H. Nguyen (&) S. Heaven C. Banks Water and Environmental Engineering Group, Faculty of Engineering and the Environment, University of Southampton, Southampton, UK e-mail: hhn1g10@gmail.com H. H. Nguyen University of Civil Engineering, Hanoi, Vietnam Introduction Urban waste generation and disposal are a growing global issue, as more people are moving from rural areas into cities. In Vietnam, the generation of MSW is growing rapidly, in parallel with urbanisation. The composition of MSW is mainly food waste which is a highly biodegrad- able material and can be digested with AD to produce biogas. This can then be used to generate heat, electricity, and biofuels. However, in Vietnam food waste is mostly treated with other components in the MSW stream by landﬁlling. The disadvantages of this treatment can be clearly seen: direct landﬁlling of MSW has been known to create lasting detrimental impacts to the environment (emissions to the atmosphere, hydrosphere, risk in landﬁll stability, and scarcity of land). In comparison AD is a better method to treat biogenic wastes from an ecological and economic point of view. It is also receiving increased attention as a promising option for energy recovery to help mitigate against energy shortages. However, applying AD technology has been neglected by the Vietnam government when formulating national strategies to deal with MSW problems due to a lack of information, data, and experi- ence. To date, sources of information about the achievable energy from food waste created in cities in Vietnam using AD technology is unavailable. The purpose of this paper, hence, is to provide estimates of energy potential from food waste in the municipal solid waste stream of urban areas in Vietnam through AD in forms of heat, electricity, and upgraded biogas using an energy model developed at the University of Southampton, UK. Keywords Anaerobic digestion Food waste Aspen Plus Energy Biogas Vietnam Keywords Anaerobic digestion Food waste Aspen Plus Energy Biogas Vietnam Results from estimations can be used as a source of data for evaluation of the AD method as a treatment of food waste in MSW streams from the energy recovery point of view. Furthermore, it can help Vietnam government and H. H. Introduction Nguyen University of Civil Engineering, Hanoi, Vietnam 12 3 3 Int J Energy Environ Eng (2014) 5:365–374 366 Table 1 MSW generation in Vietnam from 2007 to 2010 Contents 2007 2008 2009 2010 Urban population (million) 23.80 27.70 25.50 26.22 % of Vietnam’s population 28.20 28.99 29.74 30.17 Municipal waste generation rate (kg cap-1 day-1) 0.75 0.8 0.95 1.0 Total MSW per day (tonnes day-1) 17,682 20,849 24,225 26,220 Total food waste per daya (tonnes day-1) 10,945 12,905 14,995 16,229 a Estimated from data provided by the MONRE [1] Table 1 MSW generation in Vietnam from 2007 to 2010 industry decision makers to establish more effective and sustainable MSW management strategies. Population growth and increased urbanisation in Vietnam Urbanisation in Vietnam has been increasing rapidly along with the country’s economic growth. In 2000, the number of cities and towns in Vietnam was 649 but this has increased to 715 in 2005 and 755 in 2010. The growth in the number of people moving from rural to urban areas is the main driver in the expansion of Vietnams urban pop- ulation. As of 2009, the urban population was 25.59 mil- lion, accounting for 29.74 % of the total population; this expanded to 26.22 million (30.17 %) a year later. Table 2 Estimation of MSW and food waste generation in Vietnam for the 2015, 2020 and 2025 Contents 2015 2020 2025 Urban population (million)a 35 44 52 % of Vietnam’s population 38 45 50 Municipal waste generation rate (kg cap-1 day-1)b 1.2 1.4 1.6 Total MSW per day (tonnes day-1) 42,000 61,600 83,200 Rate of MSW collected (% of total MSW) 85c 90c 100d Total MSW collected per day (tonnes day-1) 35,700 55,440 83,200 Total food waste collected per day (tonnes day-1)e 21,420 33,264 49,920 a,d Data taken from the GWP [2] b According to the MONRE [1] c According to the GWP [3] e Assumed that food waste fraction in MSW stream is 60 % Table 2 Estimation of MSW and food waste generation in Vietnam for the 2015, 2020 and 2025 According to MONRE [1], it has been estimated that in 2025 the urban population in Vietnam will double to 52 million, accounting for 50 % of Vietnams total population. This rapid urbanisation has put pressure on the government in dealing with environmental problems, including solid waste management in cities; and this is exacerbated by the limited space for treatment of wastes by traditional meth- ods. For Vietnam to develop sustainably more effort is required to solve this challenge. Quantity and composition of MSW in Vietnam According to recent reports, urban areas currently contain 30 % of the country’s population but produce about 42–46 % of total solid wastes. The waste is mainly gen- erated from households, buildings, commercial activities, and other sources similar to households; from commercial enterprises such as ofﬁces, hotels, retail, institutions; and from municipal services such as street cleaning, etc. The municipal waste generated daily in Vietnam from 2007 to 2010 is presented in Table 1 [1]. 123 The problems with landﬁlling One of the most common treatment methods which is applied widely in cities across Vietnam to deal with almost all types of MSW is landﬁlling. It is considered the sim- plest, and in many cases the cheapest, method of disposal. The stabilising solid waste produces leachate and landﬁll gas which can be used for heat and electricity generation. This process has many signiﬁcant economic and environ- mental problems, however, including: limited space for landﬁll sites; cost of waste burial; cost of transporting the waste; the contamination of groundwater with pollutants; and the emission of greenhouse gases to the atmosphere [7]. Due to space limitations landﬁll sites will have to be located further from the cities, considerably increasing transportation costs. The proportion of food waste can be estimated based on the municipal waste generation rate, population growth in urban areas and the organic fraction. Table 2 shows esti- mates for the predicted generation of food waste in cities for 2015, 2020 and 2025. MSW in Vietnams urban areas is mainly composed of food waste, paper, plastic, wood, metal, and glass, with some hazardous household waste such as electric light bulbs, batteries, etc. [1]. Composition of MSW from some of the main cities in Vietnam between 2009 and 2010 is presented in Table 3. The data have been adapted from JICA [4] and MONRE [1]. As can be seen, food waste accounts for a very large proportion of the MSW stream, ranging between 54 and 77 % depending on the city. The problems with landﬁlling Int J Energy Environ Eng (2014) 5:365–374 367 Table 3 Composition of MSW from some of the main cities in Vietnam 2009 - 2010 Components Hanoi (Nam Son) Hanoi (Xuan Son) Haiphong (Trang Cat) Haiphong (Dinh Vu) Hue (Thuy Phuong) Danang (Hoa Khanh) HCM (Da Phuoc) HCM (Phuoc Hiep) Bacninh (Ho) Average Food wastea 53.81 60.79 55.18 57.56 77.1 68.47 64.50 62.83 56.9 61.9 Paper 6.53 5.38 4.54 5.42 1.92 5.07 8.17 6.05 3.73 5.20 Textile 5.82 1.76 4.57 5.12 2.89 1.55 3.88 2.09 1.07 3.19 Wood 2.51 6.63 4.93 3.70 0.59 2.79 4.59 4.18 – 3.74 Plastic 13.57 8.35 14.34 11.28 12.47 11.36 12.42 15.96 9.65 12.15 Leather and rubber 0.15 0.22 1.05 1.90 0.28 0.23 0.44 0.93 0.20 0.60 Metal 0.87 0.25 0.47 0.25 0.40 1.45 0.36 0.59 – 0.58 Glass 1.87 5.07 1.69 1.35 0.39 0.14 0.40 0.86 0.58 1.37 Porcelain 0.39 1.26 1.27 0.44 0.79 0.79 0.24 1.27 – 0.81 Soil and sand 6.29 5.44 3.08 2.96 1.70 6.75 1.39 2.28 27.85 6.42 Cinder 3.10 2.34 5.70 6.06 – 0.00 0.44 0.39 – 2.58 Hazardous waste 0.17 0.82 0.05 0.05 – 0.02 0.12 0.05 0.07 0.17 Sludge 4.34 1.63 2.29 2.75 1.46 1.35 2.92 1.89 – 2.33 Others 0.58 0.05 1.46 1.14 – 0.03 0.14 0.04 – 0.49 Total 100 100 100 100 100 100 100 100 100 a Food waste can be considered as the organic fraction of MSW when it does not contain irrecoverable paper residues [5, 6] Table 3 Composition of MSW from some of the main cities in Vietnam 2009 - 2010 There is no simple solution to the waste issue, but it obviously requires alternative methods to shift the paradigm. glasshouses, and small-scale industry at a community level. A more recent method becoming increasingly popular is to upgrade biogas, resulting in pure methane for gas grid injection, provision of a low-pressure gas supply to the community or for use as a vehicle fuel. Gas upgrading is usually reserved for large installations, but the ability to do this at a smaller community or private enterprise scale would greatly extend the potential applications. Anaerobic digestion technology Anaerobic digestion technology Anaerobic digestion (AD) is a series of natural processes in which microorganisms break down biodegradable material (organic matter) in the absence of oxygen, producing bio- gas and a stabilised digestate. The problems with landﬁlling Compared to landﬁlling and composting there are ecological and economic beneﬁts of using AD to treat biogenic wastes [8]. Compared to land- ﬁlling, anaerobic digesters are fully enclosed systems and allow all the biogas to be collected, reducing gas emissions which can be harmful to the environment. Previous studies [9] indicated that AD has an improved energy balance in comparison with composting. While the AD produces biogas which can be used for energy production, the composting produces mostly carbon dioxide which has no energy value [10, 11]. Food waste as a high energy source for anaerobic digestion Method To estimate energy potential from the anaerobic digestion of food waste in municipal solid waste stream of urban areas in Vietnam, below steps were carried out. • Investigate, collect data from published National Envi- ronment Reports. • Estimate food waste generation based estimation of population in the next milestones and strategies on food waste management of urban area system in Vietnam up to 2025. • Develop a model for simulation of food waste digestion system in terms of mass and energy balance. Biogas production was introduced to Vietnam over 10 years ago. By the end of 2006, more than 18,000 domestic biogas plants had been installed in 10 provinces in Vietnam with support from the Netherlands government. This investment, however, is only to deal with agricultural wastes, manure, etc. at the household or household-group levels [28]. When it comes to MSW, AD has been disre- garded by the government. Food waste as a high energy source for anaerobic digestion Thus, waste-to-energy technologies could not only help to provide a solution to this problem but also meet the national energy consumption policy for sustainable development [1]. solution for waste management and energy recovery in coming decades. Development of anaerobic digestion worldwide and in Vietnam It is widely accepted that the ﬁrst AD plant with biogas collection system to power gas engines was built in Ma- tunga, Bombay, India in 1897 [24]. Since the turn of the 20th century it has become a widely used process and can be found around the world where there are sources of high organic matter from agriculture-by-products. From the 1990s until recently research in AD has developed expo- nentially because of the global shortage in energy and urgent focus for renewable energy. Biogas plants around the world have increased at the rate of 20–30 % each year with the most experienced and well-developed markets being in Germany, Denmark, and Austria [25]. In devel- oping countries AD technology also ﬂourishes as a waste- to-energy solution, but on a smaller scale and in a decen- tralised manner. For example in China, in 2002, there were about 11 million digesters, this has almost doubled to more than 19 million in 2006 [26]. In India, the total number of family size biogas plants installed in 2005 was under 4 million, compared to 12 million in 2010 [27]. 123 Food waste as a high energy source for anaerobic digestion Food waste is a typical type of organic matter containing high potential for energy production through anaerobic degradation. Some characteristics of food wastes that have been reported in the literature are presented in Table 4, showing moisture content of 70–90 %, volatile solids to total solids ratio (VS/TS) of 85–95 %, and carbon to nitrogen ratio (C/N) of 9–36.4. Digestion of food waste produces a highly desirable value of methane potential. Studies showed that the methane yield through anaerobic digestion is among the range of 350–435 mL/g VSadded depending upon hydrolic retention time (HRT), operational conditions, reactor types, and composition of input food waste [18, 21–23]. Because of the beneﬁts in terms of energy saving, environmental espects and waste management, biogas production from food waste, together with other renewable organic sources i.e. agricultural waste has been suggested as an ideal The most important by-product of the AD process is biogas. The biogas produced from the AD process is con- sidered as a renewable energy source because under con- trolled conditions, the biogas produced (consisting mainly of methane and carbon dioxide) can be used for energy production, helping to replace fossil fuels. It can be burnt directly to produce heat for heating and other purposes. Another method is to use the gas in combined heat and power (CHP) plants which again present opportunities for heat use, including supply to public buildings, horticultural 12 3 Int J Energy Environ Eng (2014) 5:365–374 368 Table 4 Characteristics of food wastes listed in literature NA not available Source Characteristics Countries Sources of reference Moisture content (%) VS/TS (%) C/N ratio A dining hall 79.5 95 14.7 Korea [12] University cafeteria 80.03 93.55 NA Korea [13] A dining hall NA 94 18.3 Korea [14] A dining hall 84.1 95.6 NA Korea [15] University’s cafeteria 87.6 89.3 9.2 Korea [16] Segregated domestic food waste 77 92 14 England [17] Separated from MSW of San Francisco 69.1 85.43 14.8 USA [18] University restaurant 81.9 94.47 13.2 Korea [19] Emanating from fruit and vegetable markets, household and juices centres 85 88.5 36.36 India [20] Table 4 Characteristics of food wastes listed in literature NA not available NA not available is necessary to develop waste management systems in which solid wastes are classiﬁed at source, collected, reused, renewed, and treated with progressive technologies to boost technological innovation in waste-to-energy processes [29]. Model boundary streams, process ﬂowsheets can be constructed. Each module in Aspen provides an integrated FORTRAN and Excel environment for calculation or customisation [30]. Model boundary streams, process ﬂowsheets can be constructed. Each module in Aspen provides an integrated FORTRAN and Excel environment for calculation or customisation [30]. To determine how much energy is used in an AD plant and how much is generated, a boundary is required to determine which elements are inputs and which are out- puts from the system. Figure 1 shows the main elements of an AD plant which have been taken into account in this study. The energy model used in this work incorporates an updated investigation on food waste aspects conducted by researchers from the University of Southampton, UK and partners in Austria, Finland and India. Details of compo- nents in system such as mixing, heating, biogas upgrading, etc. can be found in [31]. In this paper, only brief intro- duction has been introduced. There are four main components in our model, including Digester unit, CHP unit, Upgrading unit and Boiler unit. Almost all components of that units modelled by unit operation blocks existed in the Aspen such as reactors, separators, compressors, pumps etc. When the built-in models do not meet calculation purposes, extra steps have been done to involve required subroutines for the simula- tion. In this study, FORTRAN subroutines in calculator blocks, Excel models have been implemented. Biogas production estimation For the energy and mass balance purposes and to limit the complexity, a kinetic free equilibrium model using Aspen plus has been developed. A theoretical stoichiometric method based on Buswell equation is an easy way to estimate products of the anaerobic digestion process (e.g. biogas, digestate). This method has been applied widely in a number of studies [32–37], etc. The energy model of anaerobic digestion of food waste A mass and energy balance model of the anaerobic digestion system based on stoichiometric approach was deliberately built in Aspen Plus ﬂowsheeting software [30]. Aspen is an interactive and ﬂexible process modelling tool for conceptual design, optimisation, process operational improvement, and asset management for chemical indus- tries. The Aspen system has a library of various common industrial operations which are called built-in modules. By interconnecting the modules using material, work and heat The National Environment Report of Vietnam [1] indi- cated that strategies from now to 2025 will focus on methods to recover energy and materials from MSW in cities. Recently, the Vietnam Government again emphasised that it Int J Energy Environ Eng (2014) 5:365–374 369 Fig. 1 System boundaries (inside the dashed line) Assumptions The AD process is very complicated and depends upon a variety of parameters such as the composition and size of feedstock, operational conditions, the trace of tars, and heavy substances, etc. [39]. To make this model simpler, basic assumptions have been made: Symons and Buswell [38] represented an equation for overall process of anaerobic degradation, known as the ‘‘Buswell equation’’: CcHhOoNnSs þ yH2O ! xCH4 þ ðc xÞCO2 þ nNH3 þ sH2S CcHhOoNnSs þ yH2O ! xCH4 þ ðc xÞCO2 þ nNH3 þ sH2S where: x ¼ 1 8 4c þ h 2o 3n 2s ð Þ and y ¼ 1 4 4c þ h ð 2o þ 3n þ 3sÞ 1. The amount of biogas produced can be predicted using Buswell’s Equation [40] which still has been used worldwide, where: x ¼ 1 8 4c þ h 2o 3n 2s ð Þ and y ¼ 1 4 4c þ h ð 2o þ 3n þ 3sÞ where: x ¼ 1 8 4c þ h 2o 3n 2s ð Þ and y ¼ 1 4 4c þ h ð 2o þ 3n þ 3sÞ The validation of this theoretical approach in terms of methane yield and other products for further energy cal- culation can be found in [31]. 2. The digesters work at optimum conditions (adequate temperature, pH, mixing, etc.), BIOGAS BIOGAS ELECTRICITY HEAT HEAT UPGRADED BIOGAS ULT ERLI SAT I ON ELECTRICITY HEAT UPGRADI NG UNI T UPGRADED BIOGAS DI GEST ER UNI T CHP BOI LER FEEDSTOCK DIGESTATE Fig. 1 System boundaries (inside the dashed line) 123 Fig. 1 System boundaries (inside the dashed line) UPGRADI NG UNI T ULT ERLI SAT I ON 123 3 370 Int J Energy Environ Eng (2014) 5:365–374 3. The main impurities are H2S and NH3, other trace gases are minor and can be neglected. As mentioned, biogas can be utilised in the forms of heat, electricity, vehicle fuel, natural gas, fuel cells, etc. Among these, electricity and vehicle fuel are currently the most suitable for use in Vietnam, and therefore these two were used in the scenarios to estimate the possible energy from food waste. These scenarios consider parameters which directly affect the overall energy consumption in operation of the AD plant, such as: ambient temperature, total input food waste, heat loss, etc. Model description Scenario 1 was chosen to obtain the maximum possible usable energy in the form of heat and electricity, by sending all biogas generated from digesters to the CHP unit. The surplus heat and electricity after internal uses (for pumps, mixers, compressors, etc.) is assumed to be sold for commercial purposes. In digester tanks feedstock is fed to a digester where digestion takes place in the absence of oxygen. Gas created from the digestion process (raw biogas) is treated to reduce H2S levels to prevent mechanical corrosion before feeding to the CHP and boiler for the generation of heat and electricity, or to upgrading unit for biofuel production. Both thermal and electrical energy are required to sustain on-site energy requirements, the surplus energy is used for off-site purposes. In some cases, when the demand for heat for internal use exceeds the amount of heat generated by the CHP, a boiler unit is used to make up for this deﬁ- ciency. In the upgrading unit, carbon dioxide and unwanted compounds are separated from the raw biogas to make the puriﬁed biogas which composition meets the requisite standard of vehicle fuel or natural gas. In scenario 2, only a certain amount of raw gas is fed to a CHP unit to produce an adequate amount of electricity for internal uses. The remaining raw gas is then sent to an upgrading unit to enhance the methane content and remove impurities to achieve vehicle fuel standards. The upgraded gas will then be dried and compressed to approximately 200 atm ready for utilisation. When the heat generated by the biogas sent to the CHP unit is less than the internal heat requirement, a boiler unit is added to the system. In this design, the boiler takes cleaned biogas from the upgrading unit. Figure 2 illustrates the modeled AD system. Eight Aspen blocks have been used to simulate the AD system (see Table 5). Physical property method The NRTL global property method which is integrated in Aspen has been used. Gas components i.e. O2, H2S, CO2, and CH4 are assumed to obey Henry’s law for calculating their solubility in the liquid phase [41]. Due to the difﬁ- culties in determining the exact particulates in food waste, for the mass and energy balance purposes, an empirical formula has been used. Themelis and Kim [42] used data conducted by Kayhanian and Tchobanoglous [43] to pro- pose a formula for food waste: C6H9.6O3.5N0.28S0.2 which was used in this study. Assumptions The digester has been assumed to be able to operate at both mesophilic and thermophilic conditions (35 and 42 C, respectively). The required digester volume is also increased by 10 % for gas storage. Other assumptions made in the model are sum- marised in Table 6. Results and discussion The amounts of biogas and digestate derived from the AD of food waste for different years under the two scenarios are shown in Table 7. As can be seen from Table 7, the biogas generated in 2025 is about 9,850 tonnes/day which is two times greater than in 2015 and more than the total biogas generated in 2020. Assumptions and speciﬁcations Assumptions and speciﬁcations Although currently food waste is collected in combination with other components of MSW, the national waste man- agement strategies recently emphasised separation of food waste from MSW for energy recovery [1, 29]. Hence, this paper assumes that all collected food waste is separated from the MSW stream and will be treated at centralised biogas plants. The estimated amount of food waste gen- erated in 2015, 2020, and 2025 as in Table 3 will be used. The seasonal average temperature in Vietnam is taken as 22 C in the winter and 27 C in the summer [44]. Results from running scenario 1 (Table 8) show that energy in the form of heat produced from the CHP unit is about two times greater than the electricity generated. This reﬂects the fact that the efﬁciency of heat from CHP units is about 65 % whereas in electric power it is around 35–43 % [45–47]. From these ﬁgures electricity generated that can be sent to the grid could contribute between 2.4 and 4.1 % of the total electricity demand in Vietnam in 2015 and 2025, respectively. The heat produced can also be 123 Int J Energy Environ Eng (2014) 5:365–374 371 Fig. 2 The Aspen plus modelling of Anaerobic Digestion plant gy g ( ) Fig. 2 The Aspen plus modelling of Anaerobic Digestion plant Table 5 Description of Aspen plus unit operation models Aspen ID Block ID Description RSTOIC DIGESTER Calculations for the digester based on the Buswell equation FLASH SEPARATOR Separates digester output into gas and digestate FLASHTAN Releases almost CH4 and some CO2 in liquid from the absorbe HEATER COOLER1, COOLER2 COOLER3 Cools down the temperature of water streams HRSG Heat Recovery Steam Generator RADFRAC STRIPER1, STRIPER2 Model of Stripers ABSORBER Model of Absorber FSPLIT SPLITER, SPLITER2, SPLITER3 Divides feed based on splits speciﬁed for outlet streams COMPR COMP1, COMP2, COMP3, COMP4 Changes stream pressure to meet the pressure requirement whe energy-related information, such as power requirements FAN1, FAN2 Generates pressure enough for the feeding GASTURB Generates electricity as an isentropic turbine RGIBBS BUNNER1 Equilibrium reactor with Gibbs energy minimization PUMP PUMP1 Changes stream pressure to meet the pressure requirement 123 Fig. Assumptions and speciﬁcations 2 The Aspen plus modelling of Anaerobic Digestion plant Table 5 Description of Aspen plus unit operation models Aspen ID Block ID Description RSTOIC DIGESTER Calculations for the digester based on the Buswell equation FLASH SEPARATOR Separates digester output into gas and digestate FLASHTAN Releases almost CH4 and some CO2 in liquid from the absorber HEATER COOLER1, COOLER2 COOLER3 Cools down the temperature of water streams HRSG Heat Recovery Steam Generator RADFRAC STRIPER1, STRIPER2 Model of Stripers ABSORBER Model of Absorber FSPLIT SPLITER, SPLITER2, SPLITER3 Divides feed based on splits speciﬁed for outlet streams COMPR COMP1, COMP2, COMP3, COMP4 Changes stream pressure to meet the pressure requirement when energy-related information, such as power requirements FAN1, FAN2 Generates pressure enough for the feeding GASTURB Generates electricity as an isentropic turbine RGIBBS BUNNER1 Equilibrium reactor with Gibbs energy minimization PUMP PUMP1 Changes stream pressure to meet the pressure requirement Table 5 Description of Aspen plus unit operation models Calculations for the digester based on the Buswell equation Separates digester output into gas and digestate Releases almost CH4 and some CO2 in liquid from the absorber Cools down the temperature of water streams Heat Recovery Steam Generator Model of Stripers Model of Absorber Divides feed based on splits speciﬁed for outlet streams Changes stream pressure to meet the pressure requirement when energy-related information, such as power requirements Generates pressure enough for the feeding Generates electricity as an isentropic turbine Equilibrium reactor with Gibbs energy minimization Changes stream pressure to meet the pressure requirement Int J Energy Environ Eng (2014) 5:365–374 372 Table 6 AD plant operational parameters Parameters Unit Values Digester temperature C 35/42 Pasteuriser temperature C 70 Ambient temperature C 22/27 Total food waste (wet) 9103 kg day-1 21,420/22,260/49,920 Time in pasteuriser hour 1 Loading rate kg VS m-3day-1 3 TS in food waste % 27.8 VS in food waste % 25 Overall heat transfer coefﬁcient of digesters surroundings Wall W m-2K 0.275 Floor W m-2K 0.823 Roof W m-2K 0.931 Table 6 AD plant operational parameters Parameters Unit Values Digester temperature C 35/42 Pasteuriser temperature C 70 Ambient temperature C 22/27 Total food waste (wet) 9103 kg day-1 21,420/22,260/49,920 Time in pasteuriser hour 1 Loading rate kg VS m-3day-1 3 TS in food waste % 27.8 VS in food waste % 25 Overall heat transfer coefﬁcient of digesters surroundings Wall W m-2K 0.275 Floor W m-2K 0.823 Roof W m-2K 0.931 Table 7 Biogas generated, gas lost and digestate from food waste in different years Contents Unit Years 2015 2020 2025 Food waste tonnes day-1 21,420 22,260 49,920 Gas generated tonnes day-1 5,858 6,088 13,654 fuel requirement for transportation in 2020 will be 23 million tonnes, increasing to around 30 million tonnes in 2025. Assumptions and speciﬁcations Assuming 1 kg fuel (diesel) is equal to 11.5 kWh [52] then upgraded biogas can replace about 2.2 and 4.75 % of anticipated daily fuel use for transportation in 2020 and 2025, respectively. Table 6 AD plant operational parameters Because of the high average ambient temperature, the heat requirements for internal uses such as for heating the digester, pasteurising etc. are small compared to those in cooler climates [53–55]. Therefore, use of the surplus biogas for on-site electricity generation in a CHP plant will produce a large amount of surplus heat. This is potentially available for export. Waste heat can also be used for cooling as well as for industrial purposes and work in these areas is important to effective use of the renewable energy. The embodied energy in the biogas plants was not included in this study, as it is normally quite small relative to the net energy ﬂows in the operation of the plant [56]. Future work could include the embodied energy, speciﬁ- cally for Vietnam, to give a more comprehensive overall energy balance. Table 7 Biogas generated, gas lost and digestate from food waste in different years Contents Unit Years 2015 2020 2025 Food waste tonnes day-1 21,420 22,260 49,920 Gas generated tonnes day-1 5,858 6,088 13,654 Biogas available tonnes day-1 4,221 4,397 9,853 Biogas composition CH4 % 61.6 CO2 % 37.6 H2S ppm 2,055 Digestate tonnes day-1 15,561 16,171 36,265 Table 7 Biogas generated, gas lost and digestate from food waste in different years Conclusion Due to the strong economic growth and urbanisation in recent years, Vietnam faces many environmental chal- lenges. In particular solid waste management in cities has been promoted as the big issue. Solid waste generation in Vietnam is increasing dramatically, mainly generated from households, buildings, commercial activities and other sources whose activities are similar to those of households and commercial enterprises such as wastes from ofﬁces, hotels, supermarkets, shops, institutions, and from muni- cipal services such as street cleaning, etc. The main com- ponent of MSW is food waste which is a source of a very high potential of energy. used for heating/cooling in buildings [48, 49] or other industrial purposes in urban areas. In the second scenario (Table 9), upgraded biogas with a methane content of over 97 % (clean gas) can be used as fuel for trucks, buses, etc. According to Vietnam’s national energy development strategy [51], it is estimated that the Results from running scenarios in an energy balance model based on Aspen Plus show that if the food waste in Results from running scenarios in an energy balance model based on Aspen Plus show that if the food waste in Table 8 Scenario 1: Energy potential in the form of heat and electricity Contents Unit 2015 2020 2025 Digestion temp. M M T T M M T T M M T T Ambient temp. W S W S W S W S W S W S Heat and electricity for external uses Heat GWh 12.6 13.0 12.3 12.6 13.1 13.5 12.7 13.1 28.2 29.0 27.5 28.3 Electricity GWh 6.85 6.85 6.85 6.85 7.10 7.10 7.10 7.10 14.8 14.8 14.8 14.8 Electricity demands of Vietnam p.aa GWh 241 301 360 a Adapted from World Bank [50]; M mesophilic condition (35 C), T thermophilic condition (42 C), S average temperature in summer (27 C), W average temperature in winter (22 C) Table 8 Scenario 1: Energy potential in the form of heat and electricity a Adapted from World Bank [50]; M mesophilic condition (35 C), T thermophilic condition (42 C), S average temperature in summer (27 C), W average temperature in winter (22 C) 123 12 3 373 Int J Energy Environ Eng (2014) 5:365–374 Table 9 Scenario 2: Energy potential in the form of upgraded biogas Contents Unit 2015 2020 2025 Digestion temp. M M T T M M T T M M T T Ambient temp. Conclusion W S W S W S W S W S W S Heat and electricity for external uses Heat GWh 3.06 3.74 2.90 3.58 3.23 3.90 3.03 3.75 7.50 9.09 7.23 8.76 Energy potential of upgraded biogas GWh 16.03 16.11 16.04 16.95 16.85 16.74 16.74 16.95 37.33 37.36 37.65 37.67 M mesophilic condition (35 C), T thermophilic condition (42 C), S average temperature in summer (27 C), W average temperature in winter (22 C) Table 9 Scenario 2: Energy potential in the form of upgraded biogas 3. GWP: Decision No. 798/QD-TTg dated on May 25th, 2011 on ‘‘Approving the investment program for solid waste treatment for the 2011–2020’’. Vietnam, Government Web Portal (2011) the MSW stream from cities in Vietnam could be sepa- rated, it could be a signiﬁcant source of energy in the form of heat, electricity, or biofuel. This can be achieved in the future through changes in people’s behaviour and enforcement of environmental laws. The total surplus exportable energy generated from biogas plants working at standard conditions each day in any form of heat, elec- tricity or puriﬁed gas, after allowing for plant operating demand is about 19, 20 and 45 GWh in 2015, 2020, and 2025, respectively. 4. JICA: Study report on solid waste management in Vietnam (2011) 5. Hartmann, H., Ahring, B.: Anaerobic digestion of the organic fraction of municipal solid waste: inﬂuence of co-digestion with manure. Water Res. 39(8), 1543–1552 (2005) 6. Zhu, H., Parker, W., Basnar, R., Proracki, A., Falletta, P., Beland, M., Seto, P.: Biohydrogen production by anaerobic co-digestion of municipal food waste and sewage sludges. Int. J. Hydr. Ener. 33(14), 3651–3659 (2008) 7. Byrne, K.: Environmental Science. Nelson, Walton-On-Thames (1997) Results from modelling show that when food waste is separated from the MSW stream and sent to AD plants, it could contribute between 2.4 and 4.1 % of the electricity demand of Vietnam, with about two times this energy also in the form of heat. Alternatively upgrading this biogas could contribute approximately 2.2–4.7 % of fuel consumption for transportation. This suggests AD is a promising method to treat MSW in cities, especially when considering the problematic aspects of other waste dis- posal methods such as: landﬁlling, composting, and incineration. 8. Edelmann, W., Schleiss, K., Joss, A.: Ecological, energetic and economic comparison of anaerobic digestion with different competing technologies to treat biogenic wastes. Water Sci. Technol. 41(3), 263–273 (2000) 9. Conclusion Mata-Alvarez, J., Mace, S., Llabres, P.: Review paper: anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Biores. Technol. 74, 3–16 (2000) 10. Komilis, D.P., Ham, R.K.: Carbon dioxide and ammonia emis- sions during composting of mixed paper, yard waste and food waste. Waste Manag 26(1), 62–70 (2006). doi:10.1016/j.wasman. 2004.12.020 11. Martin, D.L., Gershuny, G.: The Rodale Book of Composting: Easy Methods for Every Gardener. Rodale Press, Emmaus (1992) If the organic waste component in the MSW stream of cities in Vietnam could be separated, then AD offers a source of energy generation with many economic beneﬁts. This could provide a contribution towards dealing with the dramatic increases in costs associated with energy supply, waste disposal, space for landﬁlling, and the increasing public concerns with environmental issues. 12. Han, S., Shin, H.: Biohydrogen production by anaerobic fer- mentation of food waste. Int. J. Hydr. Ener. 29(6), 569–577 (2004) 13. Kwon, S., Lee, D.: Evaluation of Korean food waste composting with fed-batch operations I: using water extractable total organic carbon contents (TOCw). Process Biochem. 39(10), 1183–1194 (2004) 14. Shin, H., Youn, J., Kim, S.: Hydrogen production from food waste in anaerobic mesophilic and thermophilic acidogenesis. Int. J. Hydrog. Energy 29(13), 1355–1363 (2004) Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. 15. Kim, S., Han, S., Shin, H.: Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge. Int. J. Hydrog. Energy 29(15), 1607–1616 (2004). doi:10.1016/j.ijhy dene.2004.02.018 16. Kim, J.K., Han, G.H., Oh, B.R., Chun, Y.N., Eom, C.-Y., Kim, S.W.: Volumetric scale-up of a three stage fermentation system for food waste treatment. Bioresour. Technol. 99(10), 4394–4399 (2008). doi:10.1016/j.biortech.2007.08.031 1. MONRE: Vietnam’s 2011 National environment report––solid waste section (2011) 2. GWP: Decision No. 445/QD-TTg dated on April 7th, 2009 on orientations for the development of urban area system in Vietnam up to 2025. Vietnam, Government Web Portal (2009) 2. GWP: Decision No. 445/QD-TTg dated on April 7th, 2009 on orientations for the development of urban area system in Vietnam up to 2025. Vietnam, Government Web Portal (2009) 1. MONRE: Vietnam’s 2011 National environment report––solid waste section (2011) References 1. MONRE: Vietnam’s 2011 National environment report––solid waste section (2011) 17. Banks, C., Chesshire, M., Stringfellow, A.: A pilot-scale com- parison of mesophilic and thermophilic digestion of source seg- regated domestic food waste. Water Sci. Technol. 58(7) (2008) 2. GWP: Decision No. 445/QD-TTg dated on April 7th, 2009 on orientations for the development of urban area system in Vietnam up to 2025. Vietnam, Government Web Portal (2009) 18. Zhang, R., El-Mashad, H.M., Hartman, K., Wang, F., Liu, G., Choate, C., Gamble, P.: Characterization of food waste as 12 12 23 374 Int J Energy Environ Eng (2014) 5:365–374 feedstock for anaerobic digestion. Bioresour. Technol. 98(4), 929–935 (2007). doi:10.1016/j.biortech.2006.02.039 37. Shelton, D., Tiedje, J.: General method for determining anaerobic biodegradation potential. Appl. Environ. Microbiol. 47(4), 850–857 (1984) 19. Zhang, L., Lee, Y.-W., Jahng, D.: Anaerobic co-digestion of food waste and piggery wastewater: focusing on the role of trace elements. Bioresour. Technol. 102(8), 5048–5059 (2011). doi:10. 1016/j.biortech.2011.01.082 38. Symons, G., Buswell, A.: The methane fermentation of carbo- hydrates. J. Am. Chem. Soc. 55(5), 2028–2036 (1933) 39. Gerardi, M.H.: The Microbiology of Anaerobic Digesters. Wiley, New York (2003) 20. Rao, M.S., Singh, S.P.: Bioenergy conversion studies of organic fraction of MSW: kinetic studies and gas yield––organic loading relationships for process optimisation. Bioresour. Technol. 95(2), 173–185 (2004). doi:10.1016/j.biortech.2004.02.013 40. Buswell, A., Neave, S.: Laboratory Studies of Sludge Digestion. Illinois Water Survey Bulletin, vol. 30. Jeffersons Printing & Stationery Co., Springﬁeld (1930) 21. Cho, J., Park, S., Chang, H.: Biochemical methane potential and solid state anaerobic digestion of Korean food wastes. Bioresour. Technol. 52(3), 245–253 (1995). doi:10.1016/0960-8524(95)00 031-9 41. Howe, K.J.: Principles of Water Treatment. Wiley, New Jersey (2012) 42. Themelis, N.J., Kim, Y.H.: Material and energy balances in a large-scale aerobic bioconversion cell. Waste Manag Res 20(3), 234–242 (2002) 22. Banks, C., Chesshire, M., Heaven, S., Arnold, R.: Anaerobic digestion of source-segregated domestic food waste: performance assessment by mass and energy balance. Bioresour. Technol. 102(2), 612–620 (2011). doi:10.1016/j.biortech.2010.08.005 43. Kayhanian, M., Tchobanoglous, G.: Characteristics of humus produced from the anaerobic composting of the biodegradable organic fraction of municipal solid-waste. Environ. Technol. 14(9), 815–829 (1993) 23. Demirel, B., Scherer, P., Yenigun, O., Onay, T.T.: Production of methane and hydrogen from biomass through conventional and high-rate anaerobic digestion processes. Crit Rev Environ Sci Technol 40(2), 116–146 (2010). doi:10.1080/10643380802 013415 44. Usa, I.: Vietnam Ecology and Nature Protection Handbook. International Business Publications, USA (2007) 45. References Verougstraete, A., Nyns, E., Naveau, H., Gasser, J.: Heat recovery from composting and comparison with energy from anaerobic digestion. In: Composting of agricultural and other wastes. Proceedings of seminar organised by Commission of the European Communities, Directorate-General Science, R and D, Environment Res. Prog., Brasenose College, Oxford, March 19–20, (1984). 1985, pp. 135–145. Elsevier Applied Science Publishers, Amsterdam 24. Illinois State Water, S.: Anaerobic fermentations. Urbana, Ill (1939) 25. Curry, N., Pillay, P.: Biogas prediction and design of a food waste to energy system for the urban environment. Renew. Energy 41, 200–209 (2012). doi:10.1016/j.renene.2011.10.019 26. Deublein, D., Steinhauser, A.: Biogas From Waste and Renew- able Resources: An Introduction, 2nd edn. Wiley-VCH, Wein- heim (2011) 46. Weiland, P.: Biogas production: current state and perspectives. Appl. Microbiol. Biotechnol. 85(4), 849–860 (2010) 27. Rao, V., Baral, S., Dey, R., Mutnuri, S.: Biogas generation potential by anaerobic digestion for sustainable energy develop- ment in India. Renew. Sustain. Energy Rev. 14(7), 2086–2094 (2010) 47. Spellman, F.: Water and Wastewater Infrastructure: Energy Efﬁciency and Sustainability. CRC Press, Boca Raton (2013) 48. Deng, J., Wang, R., Han, G.: A review of thermally activated cooling technologies for combined cooling, heating and power systems. Prog. Energy Combust. 37(2), 172–203 (2011) 28. Abbasi, T., Tauseef, S.M., Abbasi, S.A.: Biogas Energy. Springer, New York (2012) 49. Choudhury, B., Saha, B., Chatterjee, P., Sarkar, J.: An overview of developments in adsorption refrigeration systems towards a sustainable way of cooling. Appl. Energy 104, 554–567 (2013) 29. GWP: Decision No. 432/QD-TTg dated on April 12th, 2012 on ‘‘Approving the Vietnam Sustainable Development Strategy for the 2011–2020’’. Vietnam Government Web Portal (2012) 50. World Bank: Electric power consumption (kWh per capita) (2012) 30. AspenTech: Aspen Plus V7.3. Aspen Technology, Inc, Burling- ton (2011) 51. PM: Decision on Vietnam National Energy Development Strat- egy up to 2020 and vision to 2050 (Decision No. 1855/QÐ-TTg) (2007) 31. Nguyen, H.H.: Modelling of Food Waste Digestion Using ADM1 Integrated With Aspen Plus. University of Southampton, South- ampton (2014) 52. Denny, M.: Lights On! The Science of Power Generation. JHU Press, USA (2013) p 32. Sobotka, M., Votruba, J., Havlik, I., Minkevich, I.: The mass- energy balance of anaerobic methane production. Folia Micro- biol. 28(3), 195–204 (1983) 53. Smyth, B., Murphy, J., O’Brien, C.: What is the energy balance of grass biomethane in Ireland and other temperate northern Euro- pean climates? Renew. Sustain. Energy Rev. 13(9), 2349–2360 (2009) 33. References Angelidaki, I., Ahring, B.: Effects of free long-chain fatty acids on thermophilic anaerobic digestion. Appl. Microbiol. Biotech- nol. 37(6), 808–812 (1992) 54. Pertl, A., Mostbauer, P., Obersteiner, G.: Climate balance of biogas upgrading systems. Waste Manag 30(1), 92–99 (2010) ( ) ( ) 34. Hayes, T., Isaacson, H., Pfeffer, J., Liu, Y.: In situ methane enrichment in anaerobic digestion. Biotechnol. Bioeng. 35(1), 73–86 (1990) 55. Deublein, D., Steinhauser, A.: Biogas from Waste and Renewable Resources: An Introduction. Wiley-VCH, Weinheim (2008) 35. Salanitro, J., Diaz, L.: Anaerobic biodegradability testing of surfactants. Chemosphere 30(5), 813–830 (1995) 56. Berglund, M., Borjesson, P.: Assessment of energy performance in the life-cycle of biogas production. Biomass Bioener. 30(3), 254–266 (2006) 36. Sialve, B., Bernet, N., Bernard, O.: Anaerobic digestion of mic- roalgae as a necessary step to make microalgal biodiesel sus- tainable. Biotechnol. Adv. 27(4), 409–416 (2009) 123 123 123
https://openalex.org/W4391434092	http://jurnal.upmk.ac.id/index.php/lensapendas/article/download/3419/1539	Indonesian	null	Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia	Jurnal lensa pendas	2,024	cc-by-sa	5,299	Abstrak Penelitian ini dilatarbelakangi oleh kemampuan pemahaman bacaan siswa yang masih rendah dan masih kurangnya keaktifan siswa dalam kegiatan pembelajaran di kelas. Tujuan penelitian ini adalah untuk mengetahui pengaruh model pembelajaran Cooperative Integrated Reading Composition terhadap kemampuan pemahaman bacaan siswa kelas IV materi cerita. Metode yang digunakan pada penelitian ini adalah quasi eksperimen dengan jumlah sampel sebanyak 30 siswa kelas ekperimen, dengan rancangan penelitian one group pretest postest design. Hasil penelitian ini dilihat dari hasil perhitungan dengan uji t jenis Paired Sampel t-Test dan didapatkan hasil uji t dengan nilai thitung sebesar 25,202 yang dapat diartikan lebih besar dari ttabel 2,042, dengan taraf signifikansi sebesar 0,000 < 0,05 sehingga H0 ditolak dan Ha diterima. Hasil tesebut dapat diartikan bahwa terdapat pengaruh model pembelajaran Cooperative Integrated Reading Composition terhadap kemampuan pemahaman bacaan siswa di kelas IV MIS Al-Hidayah. Dapat disimpulkan bahwa penerapan model pembelajaran CIRC dalam proses pembelajaran memberikan kesempatan siswa untuk lebih aktif, berani bertanya, berdiskusi dan mengemukakan pendapatnya. Hasil penelitian ini diharapkan dapat bermanfaat bagi kepala sekolah, guru, siswa, dan peneliti selanjutnya. Sejarah Artikel: Diterima: 06-10-2023 Direvisi: 28-12-2023 Dipublikasikan: 01-02-2024 Kata Kunci: model pembelajaran CIRC; pemahaman bacaan Keywords: CIRC learning model; reading comprehension Keywords: CIRC learning model; reading comprehension Pengutipan APA: Amelia, L., & Lestari, M. R. D. W. (2024). Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia. Jurnal Lensa Pendas, 9(1). doi: https://doi.org/10.33222/jlp.v9i1.3419 18 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pamahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Lena Amelia1, Mas Roro Diah Wahyu Lestari2 Pendidikan Guru Sekolah Dasar, FIP, Muhammadiyah Jakarta1,2 Jl. K.H. Ahmad Dahlan, Cirendeu, Ciputat, Tangerang Selatan Email: lenaamelia127@gmail.com1, masrorodiah@umj.ac.id2 © 2024 Lena Amelia1, Mas Roro Diah Wahyu Lestari2 Under the license CC BY-SA 4.0 Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pamahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Abstract This research was motivated by students' low reading comprehension abilities and the lack of student activity in classroom learning activities. The aim of this research is to determine the effect of the cooperative integrated reading composition learning model on fourth grade students' reading comprehension ability with story material. The method used in this research was quasi-experimental, with a sample size of 30 experimental class students and a one-group pretest-posttest design. The results of this research can be seen from the results of calculations using the Paired Sample t-Test type t test and the t test results obtained with a t value of 25.202, which can be interpreted as greater than t table 2.042, with a significance level of 0.000 < 0.05, so that H0 is rejected and Ha is accepted. These results can be interpreted as meaning that there is an influence of the cooperative integrated reading composition learning model on students' reading comprehension abilities in class IV of MIS Al-Hidayah. It can be concluded that the application of the CIRC learning model in the learning process provides students with the opportunity to be more active, dare to ask questions, discuss, and express their opinions. It is hoped that the results of this research will be useful for school principals, teachers, students, and future researchers. Pengutipan APA: Amelia, L., & Lestari, M. R. D. W. (2024). Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia. Jurnal Lensa Pendas, 9(1). doi: https://doi.org/10.33222/jlp.v9i1.3419 © 2024 Lena Amelia1, Mas Roro Diah Wahyu Lestari2 Under the license CC BY-SA 4.0 18 18 18 sa Pendas P ISSN: 2541 0199 E ISS 18 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 19 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 JURNAL LENSA PENDAS Volume 9 Nomor 1, Bulan Februari Tahun 2024, Hlm 18-28 Available online at http://jurnal.upmk.ac.id/index.php/lensapendas Alamat Korespondensi : Ciputat - Tangerang Selatan Email : lenaamelia127@gmail.com ISSN 2541-6855 (Online) ISSN 2541-0199 (Cetak) JURNAL LENSA PENDAS Volume 9 Nomor 1, Bulan Februari Tahun 2024, Hlm 18-28 Available online at http://jurnal.upmk.ac.id/index.php/lensapendas ISSN 2541-6855 (Online) ISSN 2541-0199 (Cetak) : Ciputat - Tangerang Selatan : lenaamelia127@gmail.com : Ciputat - Tangerang Selatan : lenaamelia127@gmail.com : Ciputat - Tangerang Selatan : lenaamelia127@gmail.com 19 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia tersebut. Pemahaman akan bacaan sangat diperlukan untuk peningkatan kemampuan siswa baik dalam pendidikan atau pembelajaran karena dalam membaca diperlukan kemampuan untuk mengerti bacaan/teks dan mengetahui informasi yang hendak diketahui dari bacaan/teks. Hal inilah yang disebut dengan pemahaman akan membaca. PENDAHULUAN 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Cooperative Integrated Reading Composition (CIRC) merupakan satu dari sekian model pembelajaran cooperative learning yang dasarnya merujuk kepada pengajaran terpadu dengan saling bekerjasama yang begitu komprehensif dan berkembang serta jelas untuk pembelajaran membaca dan menulis pada siswa kelas tinggi di sekolah dasar (Rahmi dan Marnola, 2020: 665). Menurut Yohana (2022: 17) menjelaskan bahwa pembelajaran Cooperative Integrated Reading Composition adalah bentuk model pembelajaran kooperatif dengan tahapan- tahapan pembelajaran yang disusun untuk mengarahkan peserta didik bekerja sama dan menyelesaikan permasalahan yang ada saat berlangsungnya kegiatan belajar, serta model ini dapat meningkatkan kegiatan pembelajaran dan hasil belajar peserta didik. pengetahuan dan pengalaman sehingga memahami makna dari bacaan tersebut, pemerolehan makna dari bacaan dapat disampaikan penulis baik secara tersurat maupun tersirat. Selain itu, keaktifan siswa sangat berdampak penting dalam mencari informasi dalam bacaan. Pemahaman bacaan sangat dibutuhkan oleh peserta didik karena dengan memahami bacaan siswa tidak hanya membaca teks saja, tetapi melibatkan kemampuan berfikir dan analisisnya. Siswa hendaklah mempunyai tujuan saat membaca dan memperhatikan pesan yang terdapat dalam teks, dengan begitu penguasaan terhadap isi bacaan akan lebih baik jika dibandingkan hanya sekedar membaca. Jika melihat dari pemahaman bacaan siswa saat ini, belum semua siswa mampu melibatkan kemampuan berpikirnya untuk memahami bacaan dengan baik. Cenderung siswa hanya membaca tanpa menganalisis lebih lanjut apa yang sudah dibacanya. Maka dari itu, hal tersebut menjadi penyebab siswa sulit menerima pembelajaran dengan baik. Model pembelajaran CIRC ini salah satu model pembelajaran yang mengikutsertakan siswa agar aktif dan mengembangkan kemampuannya pada saat pembelajaran. Permasalahan yang guru hadapi sekarang adalah bagaimana membuat siswa tidak sekadar hanya mengerti sebuah materi atau konsep saja, akan tetapi siswa bisa menggunakan konsep dan mengembangkannya dalam kegiatan pembelajaran. Selain itu, pembelajaran yang masih satu arah dapat perlahan dikembangkan menjadi pembelajaran yang lebih bermakna, bagaimana siswa aktif dalam mengkonstruksikan pemikiran mereka. Bagaimana siswa terlibat dalam kerjasama dengan para siswa. Bagaimana siswa saling bertukar ide dan pengalaman antar siswa. Serta bagaimana menemukan model yang tepat untuk meningkatkan kualitas pembelajaran di kelas. PENDAHULUAN Pendidikan merupakan upaya seseorang untuk memperoleh pengetahuan. Manusia memperoleh pengetahuan dari sumber-sumber yang tersedia. Membaca adalah salah satu cara dan sumber untuk memperoleh pengetahuan. Membaca merupakan keterampilan dasar yang dibutuhkan manusia untuk memperoleh pengetahuan, dengan membaca pengetahuan seseorang akan bertambah dan berpandangan luas (Wijaya et al., 2021: 2). Dapat disadari bahwa membaca sebagai keterampilan yang harus dimiliki peserta didik, sebab pengetahuan akan bertambah jika pemahaman dalam membaca baik. Dalam setiap tingkat satuan pendidikan hal tersebut akan memudahkan peserta didik dalam setiap pembelajaran. Betapa pentingnya membaca di dalam dunia pendidikan yang akan berpengaruh di kehidupan nyata, lantas sudah selayaknya setiap peserta didik wajib mempunyai keterampilan membaca serta pemahaman dalam membaca. Pemahaman merupakan bagian terpenting dalam kegiatan membaca, pemahaman akan bacaan dapat meningkatkan kemampuan membaca seseorang dan sebagai bagian dalam memperoleh tujuan membaca. Menurut Lestari (2021: 15) dalam membaca tingkat tertinggi yaitu pemahaman bacaan. Ketika mencapai tingkat ini sudah bukan sekadar membaca huruf menjadi kata-kata dan melibatkan gerakan mata saja. Akan tetapi anak sudah pada tahap memahami isi bacaan yang dibacanya. Membaca berdasarkan penjelasan Herlinyanto (2015: 7), membaca adalah proses mengkonstruksi isi atau sebuah makna yang ingin diutarakan penulis dari pesan tersebut melalui lambang-lambang tulisan. Riyanti (2021: 73) pemahaman bacaan adalah kemampuan pemerolehan makna dari bacaan baik itu secara tersurat atau tersirat yang melibatkan pengetahuan dan pengalaman yang dimiliki seseorang agar informasi dari bacaan dapat diterapkan. Membaca dalam keterampilan berbahasa salah satu keterampilan paling dasar di antara empat keterampilan berbahasa yang mencangkup keterampilan menulis, berbicara, membaca dan menyimak. Membaca merupakan keterampilan paling dasar untuk memahami hal lain yang lebih kompleks. Akan tetapi, belum semua siswa mempunyai pemahaman akan bacaan yang telah dibacanya. Pemahaman akan bacaan sangat diperlukan untuk peningkatan kemampuan siswa untuk mengetahui informasi yang hendak diketahui dari bacaan/teks. Bukan hanya sekedar mampu membaca tulisan dengan melafalkan kata atau kalimat saja, namun dalam membaca sangat penting adanya kemampuan memahami akan bacaan/teks Pendapat dari Sutama et al. (2022:73) menjelaskan pemahaman membaca yaitu keterlibatan secara aktif dari seseorang dengan mengaitkan pengetahuan dan pengalaman pembaca yang sudah ada sebelumnya sehingga memperoleh makna dari bacaan serta menghubungkannya dengan isi bacaan tersebut. Oleh karena itu, pemahaman bacaan adalah kemampuan seseorang untuk memaknai bahan bacaan yang melibatkan 20 20 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 21 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 PENDAHULUAN Menyadari begitu sangat pentingnya kemampuan membaca pemahaman siswa, maka upaya yang bisa dilakukan untuk lebih mengembangkan kemampuan membaca siswa harus direncanakan dalam proses pembelajaran agar siswa mempunyai kemampuan pemahaman yang baik. Perencanaan pembelajaran yang bisa dipakai untuk mengembangkan kemampuan tersebut salah satunya adalah dengan diterapkannya model pembelajaran Cooperative Integrated Reading Composition (CIRC) yang harapannya dapat memberi kesempatan kepada peserta didik untuk lebih meningkatkan kemampuan membaca pemahaman. Model pembelajaran 21 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 21 21 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Penerapan model pembelajaran CIRC membantu siswa untuk belajar lebih aktif dan inovatif, dalam pelaksanaannya tidak dipungkiri akan memberikan manfaat bagi siswa dalam meningkatkan pembelajaran. Menurut Rohman (2021: 10) manfaat dari model CIRC adalah sebagai berikut: (1) Penerapan model CIRC diharapkan mampu membantu siswa baik itu dalam meningkatkan kemampuan membaca maupun merangkum. (2) Model pembelajaran CIRC mengembangkan kecakapan siswa dalam memanfaatkan kemajuan ilmu pengetahuan dan teknologi. (3) Pembelajaran CIRC mengembangkan pemikiran siswa dengan mengemukakan pendapatnya secara aktif. (4) Mengembangkan kegiatan belajar serta hasil belajar siswa. (5) Memperdalam informasi yang diterima siswa pada ilmu pengetahuan dan teknologi. Selain itu, model pembelajaran yang digunakan pendidik juga masih konvensional saat berlangsungnya kegiatan belajar mengajar Bahasa Indonesia. Pembelajaran bahasa Indonesia merupakan materi pembelajaran yang sangat penting untuk dipelajari di sekolah dasar. Menurut Hidayah (2015: 193) Pembelajaran bahasa Indonesia untuk SD/MI bisa diartikan bahwa usaha yang dilakukan seorang pendidik untuk memperbaiki sikap siswa dalam berbahasa Indonesia, perbaikan dalam hal ini bisa terlaksana dengan berfokus pada tujuan belajar bahasa Indonesia di SD/MI yang dilaksanakan pendidik saat kegiatan pembelajaran secara terarah. Mata pelajaran bahasa Indonesia dipelajari oleh siswa dengan tujuan dapat berkembangnya kemampuan berbahasa Indonesia dengan baik dan benar. Berdasarkan hal yang sudah disebutkan di atas, maka peneliti ingin melaksanakan penelitian yang berjudul “Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia”. PENDAHULUAN Pembelajaran di kelas seringkali belum begitu memberikan ruang kepada siswa untuk terus mengembangkan konsep dari teks tetapi guru hanya memfokuskan pada penyampaian konsep guru sendiri melalui penjelasan sehingga masih saja ada peserta didik yang belum memiliki kemampuan membaca pemahaman secara optimal dan pemahaman yang baik. diharapkan dengan diterapkannya model CIRC siswa aktif pada saat kegiatan pembelajaran. mengembangkan penelitian. mengembangkan penelitian. sampling. Dikarenakan pengambilan sampel menggunakan teknik purposive sampling yaitu sampel yang dipilih merupakan sampel yang memiliki kriteria tertentu. Adapun kriteria sampel yang berkaitan dengan penelitian ini adalah: menggunakan sampel minimum (sampel kelompok kecil) sebanyak 30 siswa, sampel tersebut termasuk kedalam kelompok yang pemahaman bacaannya masih rendah, sampel tersebut sudah memiliki kemampuan dalam membaca dan kemampuan memahami bacaan dibandingkan dengan kelas di bawahnya, model CIRC yang diterapkan merupakan model yang cocok diterapkan di kelas tinggi sekolah dasar. Prosedur Penelitian Penelitian merupakan kegiatan mengumpulkan data atau informasi secara sistematis yang dapat dibuktikan dan dilakukan berdasarkan tahapan tertentu sehingga menghasilkan pengetahuan baru. Dalam melakukan penelitian ada langkah-langkah yang harus diperhatikan dan dipersiapkan agar penelitian dapat berjalan sesuai dengan tujuan yang akan diperoleh. Penelitian ini memiliki prosedur yang bagiannya melalui 3 tahap yaitu tahap persiapan, tahap pelaksanaan, dan tahap pelaporan hasil atau laporan penelitian. Tabel 1. Desain Penelitian One Group pretest- posttest Kelompok Pretest Perlakuan Posttest Eksperimen Q1 X Q2 Keterangan: Q1 : Pretest (Tes Awal) Q2 : Posttest (Tes Akhir) Waktu dan Tempat Penelitian Tabel 1. Desain Penelitian One Group pretest- posttest Tabel 1. Desain Penelitian One Group pretest- posttest Kelompok Pretest Perlakuan Posttest Eksperimen Q1 X Q2 Keterangan: Q1 : Pretest (Tes Awal) Q2 : Posttest (Tes Akhir) Waktu dan Tempat Penelitian Q1 : Pretest (Tes Awal) Q2 : Posttest (Tes Akhir) Desain Penelitian Desain Quasi Eksperimen yang digunakan oleh peneliti adalah One Group pretest-posttest Design. Pada desain ini, sebelum diberikan perlakuan terlebih dahulu melakukan pretest kemudian diberikan perlakuan dan diakhir pembelajaran diberikan posttest. Di awal pembelajaran siswa akan diberikan uji (pretest) untuk melihat kemampuan awal siswa dalam pembelajaran sebelum diberlakukan perlakuan, kemudian di akhir kegiatan pembelajaran siswa akan diberikan uji (posttest) untuk melihat kemampuan siswa terhadap kegiatan pembelajaran yang telah dilaksanakan dengan penerapan perlakuan. Dalam desain ini hanya menggunakan satu kelompok saja yaitu kelompok eksperimen. rancangan One Group pretest-posttest Design menurut (Seniati et al., 2018: 118) sebagai berikut. Waktu dan Tempat Penelitian Penelitian ini dilakukan di MIS Al- Hidayah Jl. Mawar II No 5, Kel. Pondok Pinang, Kec. Kebayoran Lama, Jakarta Selatan, Kode Pos 12310. 23 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Jenis Penelitian Penelitian ini menggunakan metode Quasi Eksperimen yang bersifat kuantitatif. Menurut Hermawan (2019:16) menjelaskan penelitian kuantitatif ialah sebuah penelitian yang dilakukan secara ilmiah dan terstruktur dengan mengamati beberapa hal mencakup segala aspek yang ada kaitannya dengan objek penelitian, fenomena, dan hubungan yang terkait diantaranya, dalam penelitian kuantitatif ini menggunakan model-model matematis, teori-teori, dan hipotesis dalam Berdasarkan pengamatan peneliti di MIS Al-Hidayah dengan melihat kegiatan belajar mengajar, observasi kemudian wawancara bersama dengan guru kelas IVB menyatakan masih rendahnya kemampuan membaca pemahaman pada peserta didik, penyebab masih rendahnya minat baca dan siswa belum dapat menentukan ide, makna dari bacaan ataupun unsur-unsur yang ada dalam teks yang membutuhkan pemahaman. 22 22 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Teknik Pengumpulan Data Teknik pengumpulan data merupakan salah satu langkah penting dalam penelitian, karena dalam pengumpulan data penelitian harus diamati secara saksama sehingga data yang didapat dari hasil penelitian terjaga tingkat validitas dan reliabilitasnya (Siyoto dan Sodik, 2015:75). Adapun teknik pengumpulan data yang dilakukan oleh peneliti sebagai berikut. (a) Tes Subjek Penelitian Populasi dari penelitian ini adalah semua siswa kelas IV yang terdiri dari 3 rombel dengan total 89 siswa di MIS Al-Hidayah. Sampel yang digunakan dalam penelitian ini yaitu kelas IVB yang berjumlah 30 orang di MIS Al-Hidayah. (a) Tes Tes ialah teknik yang digunakan untuk mengumpulkan informasi atau data dengan cara memberikan sejumlah persoalan atau latihan dan perangkat lainnya kepada subjek yang menjadi tujuannya untuk diperoleh Penelitian ini menggunakan teknik pengambilan sampel non random jenis purposive 23 23 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia dilakukannya uji hipotesis menggunakan uji t. Adapun penjelasan mengenai uji prasyarat analisis dalam penelitian dapat dijelaskan berikut ini. datanya (Nasrudin, 2019: 31). Tes kemampuan pemahaman bacaan yang diujikan kepada siswa berupa tes pilihan ganda dan diujikan sebanyak dua kali, yaitu pretest dan posttest. Pretest dilakukan sebelum dilaksanakannya pembelajaran pada materi cerita. Kemudian diberikan perlakuan berupa model pembelajaran CIRC pada kelas eksperimen. Setelah itu diberikan posttest di kelas eksperimen. Tes ini dilakukan untuk mengetahui adakah peningkatan kemampuan bacaan siswa yang diberikan melalui soal tes tersebut. (b) Dokumentasi Dokumentasi yaitu cara pengumpulan data dengan tujuan untuk mendapatkan data secara langsung di tempat dilakukannya penelitian seperti buku-buku yang relevan, film dokumenter, peraturan-peraturan, foto- foto kegiatan, maupun data lainnya yang berkaitan dengan penelitian (Sudaryono, 2016: 90). Data yang didokumentasikan peneliti pada penelitian ini berupa foto kegiatan belajar mengajar, RPP yang digunakan, serta soal pretest dan posttest. Adapun kriteria pengujian: 𝐻0 diterima apabila sig ≥ α; distribusi sampel normal 𝐻0 ditolak apabila sig < α; distribusi sampel tidak normal Diperoleh nilai signifikansi kemampuan pemahaman bacaan siswa pada pretest sebesar 0,088 > 0,05 serta posttest sebesar 0,062 > 0,05 dengan taraf signifikansi 0,05 atau 5%. Nilai signifikansi baik pada pretest serta posttest mendapat skor lebih besar dari 0,05 maka data berdistribusi normal atau sampel berasal dari populasi berdistribusi normal. Kemampuan Pemahaman Bacaan Kemampuan Pemahaman Bacaan Shapiro-Wilk Statistic df Sig. Pretest .939 30 .088 Posttest .934 30 .062 Adapun kriteria pengujian: 𝐻0 diterima apabila sig ≥ α; distribusi sampel normal 𝐻0 ditolak apabila sig < α; distribusi sampel tidak normal Shapiro-Wilk Statistic df Sig. Pretest .939 30 .088 Posttest .934 30 .062 Adapun kriteria pengujian: 𝐻0 diterima apabila sig ≥ α; distribusi Teknik Analisis Data Sebelum dilakukan penelitian, ada uji coba instrumen yang harus dilakukan dengan tahapan yang terdiri dari uji validitas dan reliabilitas. Uji validitas instrumen pada penelitian ini dilakukan dengan cara pengujian oleh ahli dan pengujian kepada siswa di SDN Cempaka Putih 02. Hasil validitas soal tes diperoleh dari 25 item soal hanya 13 soal yang valid. Hasil reliabilitas menunjukkan variabel Y menghasilkan nilai reliabilitas sebesar 0,810 > 0,6 dengan ini dapat dikatakan instrumen tersebut reliabel. (b) Uji Homogenitas Uji homogenitas dilakukan setelah uji normalitas dan hasilnya normal. Uji homogenitas memakai uji Levene Test. Diperoleh hasil nilai signifikansi Kemampuan Pemahaman Bacaan sebesar 0,485 > 0,05 sebagai taraf signifikansinya. Maka dapat disimpulkan data tersebut bersifat homogen. Sebelum dilakukan uji hipotesis, data yang sebelumnya telah terkumpul selanjutnya diolah serta dianalisis untuk dilakukan uji prasyarat analisis yang terdiri dari uji normalitas dan homogenitas telah memenuhi syarat untuk (a) Uji Normalitas Pada penelitian ini uji normalitas yang digunakan yaitu uji Shapiro-Wilk dengan bantuan software SPSS 25, taraf signifikansi α = 5% (0.05). Tabel 2. Hasil Uji Normalitas Tes Kemampuan Pemahaman Bacaan Tabel 2. Hasil Uji Normalitas Tes Uji t Uji hipotesis yang digunakan dalam penelitian ini uji komparatif atau uji-T. Jenis uji- T yang digunakan adalah Paired Sample t-Test. 24 24 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Berdasarkan data tabel 4.5, maka deskripsi nilai hasil uji kemampuan pemahaman bacaan pada pretest kelas eksperimen dapat dilihat perolehan skor rata-rata 61,2; skor maksimum sebesar 85; skor minimum 31; standar deviasi 12,05; varians 145,40; dan responden 30. Menurut Ramadhani dan Bina (2021: 251) Paired Sample t-Test adalah suatu teknik pengujian hipotesis dengan membandingkan rata-rata dari satu group sampel yang diamati pada dua titik berbeda dalam satu waktu yang sama. Penelitian dengan uji Paired Sample t- Test bertujuan menguji dua sampel yang berpasangan dan membandingkan rata-rata sebelum dan sesudah perlakuan. Maka dari perlakuan tersebut akan didapatkan 2 bentuk data sampel berbeda, yaitu pretest dan posttest. Setelah diberikan pretest, pada kelas eksperimen siswa diberikan perlakuan yaitu dengan menerapkan model pembelajaran Cooperative Integrated Reading Composition. Setelah diberikan perlakuan, selanjutnya siswa diberikan posttest untuk mengetahui apakah terdapat pengaruh dalam menerapkan model pembelajaran tersebut. Hasil perhitungan dari analisis data pada pretest kelas eksperimen bisa dilihat pada tabel dibawah ini: Adapun syarat penggunaannya untuk menentukan hipotesis dengan taraf signifikansi 0,05 yaitu: (a) Jika nilai signifikansi (2 tailed) < 0,05 bahwa ada perbedaan yang signifikan antara pretest dan posttest Tabel 4. Hasil Data Posttest Ukuran Posttest Rata-rata 84,43 Maximum 100 Minimum 62 Standard Deviation 10,34 Sample Variance 107,08 Responden 30 Sumber: Pengolahan Data Ms.Excel 2013 (b) Jika nilai signifikansi (2 tailed) > 0,05 bahwa tidak ada perbedaan yang signifikan antara pretest dan posttest 25 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 25 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 HASIL PENELITIAN DAN PEMBAHASAN Hasil Penelitian Berdasarkan data pada tabel 4.5, maka deskripsi nilai hasil uji kemampuan pemahaman bacaan pada posttest kelas eksperimen dapat dilihat perolehan skor rata-rata 84,43; skor maksimum sebesar 100; skor minimum 62; standar deviasi 10,34; varians 107,08; dan responden 30. Hasil yang diperoleh peneliti dari kelas IVB selaku kelas eksperimen yang berjumlah 30 siswa dengan menggunakan test berupa pretest dan posttest. Sebelum diberikan perlakuan, siswa diberikan pretest terlebih dahulu untuk mengetahui kemampuan awal siswa. Peneliti memberikan tes pilihan ganda kepada siswa. Kemudian didapatkan hasil yang dapat disajikan pada tabel berikut ini: Berdasarkan uraian tersebut, maka peneliti dapat mendeskripsikan data secara keseluruhan baik data hasil pretest dan posttest di kelas eksperimen yang diuraikan dalam bentuk histogram pada gambar berikut: Tabel 3. Hasil Data Pretest Ukuran Pretest Rata-rata 61,2 Maximum 85 Minimum 31 Standard Deviation 12,05 Sample Variance 145,40 Responden 30 Sumber: Pengolahan Data Ms.Excel 2013 25 25 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 a Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Rea position Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indo Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 a Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Read iti T h d K P h B Si S k l h D P d M t P l j B h I d Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesi Gambar 1. Skor Pretest dan Posttest Kemampuan Pemahaman Bacaan 0 20 40 60 80 100 Pretest Posttest Pemahaman Bacaan sebesar 5,04930 dan 0,92187. Tabel 6. Hasil Uji Paired Sample t-Test pada Pretest dan Posttest Paired Differences t df Sig. (2- tailed) 95% Confidence Interval Of the Difference Lower Upper Pair 1 Pretest- Posttest - 25,1187 7 - 21,3478 9 - 25,2 02 29 ,000 Sumber: Pengolahan Data SPSS 25 Gambar 1. HASIL PENELITIAN DAN PEMBAHASAN Hasil Penelitian Skor Pretest dan Posttest Kemampuan Pemahaman Bacaan Histogram nilai rata-rata pretest dan posttest kemampuan pemahaman bacaan dapat dilihat nilai pretest sebesar 61,2 terjadi peningkatan kemampuan pemahaman bacaan siswa setelah diterapkan model CIRC dengan perolehan nilai posttest sebesar 84,43. Hasil output pada tabel 4 uji hipotesis tes kemampuan pemahaman dengan menggunakan Uji Paired Sample T-test diperoleh skor t hitung sebesar 25,202 yang artinya lebih besar dari t tabel 2,042 dengan taraf signifikansi (2-tailed) 0.000 kurang dari 0,05. Selanjutnya dari data yang diperoleh dilakukan uji prasyarat analisis dengan dilakukan uji normalitas dan homogenitas. Berdasarkan uji normalitas bahwa data berdistribusi normal atau sampel berasal dari populasi berdistribusi normal kemudian uji homogenitas dilakukan setelah uji normalitas dan hasilnya normal. Berdasarkan uji homogenitas data tersebut bersifat homogen. Maka didapati uji prasyarat analisis data berdistribusi normal dan bersifat homogen, sehingga dapat melakukan uji perbandingan rata- rata dengan memakai uji Paired Sample t-Test dengan bantuan software SPSS 25 dengan hasil sebagai berikut: 26 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Pembahasan Penelitian ini dilaksanakan untuk mengetahui kemampuan pemahaman bacaan sebelum diberikan perlakuan dengan menerapkan model pembelajaran CIRC dan setelah diterapkan model pembelajaran CIRC. Penelitian ini dilaksanakan di MIS Al-Hidayah pada kelas IVB pada Mata Pelajaran Bahasa Indonesia materi Cerita semester genap tahun pelajaran 2022/2023. Pada penelitian ini dapat dilihat perbedaan kemampuan pemahaman bacaan siswa sebelum dan sesudah penerapan model pembelajaran CIRC dilihat dari nilai pretest dan posttest dengan uji t. Tabel 5. Hasil Statistics Uji Paired Sample t- Test pada Pretest dan Posttest Mean Std Deviation Std. Error Mean Pair 1 Pretest- Posttest - 23,2333 3 5,04930 ,92187 Hasil output pada tabel 3 Uji Paired Sample T-test diperoleh nilai rata-rata (mean) 23,2333. Selain itu didapatkan nilai standard deviation dan standard error mean dari variabel bel 5. Hasil Statistics Uji Paired Sample t- Test pada Pretest dan Posttest Sebelum perlakuan diberikan kepada siswa. Peneliti memberikan pretest pada kelas IVB untuk mengetahui kemampuan awal siswa, dari data pretest ini diketahui nilai kelas IVB memperoleh skor rata-rata 61,2. Dari data pretest dapat diketahui bahwa nilai rata-rata siswa masih rendah. Hal ini disebabkan karena masih kurangnya siswa melatih kemampuan pemahaman bacaan, dan kurang dalam 26 26 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia berbicara menerangkan materi sehingga siswa kurang dalam mengemukakan pendapat. mendiskusikan suatu topik bacaan yang dapat melatih siswa menyampaikan pendapatnya. Selain itu, penggunaan model pembelajaran saat pembelajaran Bahasa Indonesia yang masih kurang, membuat siswa kurang aktif dalam kegiatan pembelajaran. Pembahasan Dengan penerapan model pembelajaran CIRC ini dapat diketahui bahwa dari hasil analisis data menggunakan uji-t dengan desain Paired Sample t-Test, menunjukkan bahwa nilai t hitung sebesar 25,202 yang berarti lebih besar dari t tabel 2,042 dengan taraf signifikansi sebesar 0,000 < 0,05 dapat diartikan bahwa terdapat pengaruh model pembelajaran Cooperatif Integrated Reading Composition terhadap kemampuan pemahaman bacaan siswa sekolah dasar pada mata pelajaran bahasa indonesia. Setelah diketahui nilai rata-rata pretest, kemudian peneliti memberikan perlakuan dengan menerapkan model pembelajaran CIRC dalam proses pembelajaran. Dari penelitian yang telah dilakukan pada kelas eksperimen memberikan hasil yang baik dalam kegiatan pembelajaran, seperti siswa dapat berdiskusi dengan teman, menyampaikan pendapat, bertanya saat kegiatan belajar mengajar berlangsung, dan meningkatkan kemampuan pemahaman bacaan siswa. Setelah diberikan perlakuan diperoleh hasil posttest kelas eksperimen diperoleh skor rata-rata sebesar 84,43. Hasil yang diperoleh dari pretest dan posttest dari kelas eksperimen menunjukkan peningkatan dengan menggunakan model pembelajaran Cooperative Integrated Reading Composition (CIRC). SIMPULAN Jakarta: Kencana. Sutama, I. M., dkk. (2022). PEMBELAJARAN INOVATIF BAHASA dan SASTRA: Ide dan Pengalaman Implementasi dalam Pembelajaran Bahasa Indonesia dan Daerah. Surabaya: Global Aksara Pers. SIMPULAN Berdasarkan penelitian yang dilakukan di MIS Al-Hidayah untuk mengetahui kemampuan pemahaman bacaan siswa dengan menerapkan model pembelajaran Cooperative Integrated Reading Composition pada pelajaran bahasa Indonesia materi cerita kelas IV dapat disimpulkan bahwa model pembelajaran Cooperative Integrated Reading Composition berpengaruh terhadap kemampuan pemahaman bacaan siswa kelas IV pada materi cerita di MIS Al-Hidayah. Hal ini dapat diketahui dari hasil perhitungan uji t dengan menggunakan SPSS tipe 25, dengan perolehan nilai signifikansi 0,000 < 0,05, dapat dijelaskan bahwa terdapat perbedaan yang signifikan antara kemampuan pemahaman bacaan sebelum dan sesudah penggunaan model pembelajaran Cooperative Integrated Reading Composition. Disamping itu berdasarkan hasil pretest dan posttest terjadi peningkatan kemampuan bacaan siswa dengan nilai rata-rata pretest sebesar 61,2 setelah diterapkannya model pembelajaran CIRC diperoleh nilai rata-rata posttest sebesar 84,43. Hal tersebut juga didukung pendapat Pendapat dari Sutama et al. (2022:73) menjelaskan pemahaman membaca yaitu keterlibatan secara aktif dari seseorang dengan mengaitkan pengetahuan dan pengalaman yang telah dimiliki pembaca sehingga memperoleh makna dari bacaan serta menghubungkannya dengan isi bacaan tersebut. Hal ini berbeda dengan yang proses pembelajarannya tidak menerapkan model CIRC melainkan hanya menerapkan pembelajaran konvensional, siswa kurang terlibat aktif dalam proses pembelajaran karena siswa kurang mengemukakan pendapat dan bertanya mengenai materi pembelajaran, selain itu kurangnya tanggung jawab siswa terhadap menyelesaikan tugas yang diberikan. Saat pembelajaran konvensional juga guru yang lebih aktif 27 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 27 Jurnal Lensa Pendas, Volume 9 Nomor 1, Bulan Februari Tahun 2024 Hlm. 18-28 Lena Amelia1, Mas Roro Diah Wahyu Lestari2., Pengaruh Model Pembelajaran Cooperative Integrated Reading Composition Terhadap Kemampuan Pemahaman Bacaan Siswa Sekolah Dasar Pada Mata Pelajaran Bahasa Indonesia Dengan demikian, penerapan model pembelajaran CIRC dalam proses pembelajaran memberikan kesempatan siswa untuk lebih aktif, berani bertanya, berdiskusi dan mengemukakan pendapatnya. Selain itu, melalui diskusi dengan teman siswa akan lebih komunikatif dan interaksi antar teman dapat membuat siswa saling bertukar pemikiran sehingga menghasilkan pengetahuan baru. Serta kemampuan pemahaman bacaan siswa lebih baik karena diterapkan model pembelajaran CIRC sehingga siswa terbiasa untuk bekerjasama untuk menyelesaikan tugas yang telah diberikan. Perhitungan Matematis dan Aplikasi SPSS. Jakarta: Kencana. Perhitungan Matematis dan Aplikasi SPSS. Jakarta: Kencana. Riyanti, A. (2021). KETERAMPILAN MEMBACA. Yogyakarta: Penerbit K- Media. Rohman, S. (2021). Model Pembelajaran, Hasil Belajar dan Respon Peserta Didik. Bogor: Guepedia. Seniati, L., Yulianto, A., & Setiadi, B. N. (2018). Psikologi Eksperimen. Jakarta: Indeks. Siyoto, S., & Sodik, A. (2015). Dasar Metodologi Penelitian. Yogyakarta: Literasi Media Publishing. Sudaryono. (2016). Metodologi Penelitian Pendidikan. DAFTAR PUSTAKA Herlinyanto. (2015). Membaca Pemahaman dengan Strategi KWL Pemahaman dan Minat Membaca. Sleman: Deepublish. Wijaya, P. A., Sutarto, J., & Zulaeha, I. (2021). STATEGI KNOW- WANT TO KNOW- LEARNED DAN STRATEGI DIRECT READING THINGKING ACTIVITY dalam Pembelajaran Pendidikan Dasar. Semarang: CV. Harian Jateng Network. Hermawan, I. (2019). Metodologi Penelitian Pendidikan Kuantitatif, Kualitatif dan Mixed Methode. Kuningan: Hidayatul Quran Kuningan. Hidayah, N. (2015). Penanaman Nilai-Nilai Karakter dalam Pembelajaran Bahasa Indonesia di Sekolah Dasar. Jurnal Pendidikan Dan Pembelajaran Dasar, 2(2), 193. Yohana, S. (2022). KOOPERATIF TIPE INVESTIGATION DAN AKTIVITAS BELAJAR. Lombok Tengah: Penerbit P4I. Lestari, M. R. D. W. (2021). Pengajaran Pemahaman Bacaan Menggunakan Pendekatan Model Pembelajaran di Sekolah Dasar. Tangerang: Media Edukasi Indonesia. Nasrudin, J. (2019). Metodologi Penelitian Pendidikan (buku ajar praktis cara membuat penelitian). Bandung: PT. Panca Terra Firma. Rahmi, Y., & Marnola, I. (2020). Peningkatan Kemampuan Membaca Pemahaman Siswa Melalui Model Pembelajaran Cooperative Integrated Reading and Compotion (Circ). Jurnal Basicedu, 4(3), 662–665. Ramadhani, R., & Bina, N. S. (2021). Statistika Penelitian Pendidikan: Analisis 28 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855 28 28 Jurnal Lensa Pendas, P ISSN: 2541-0199, E ISSN: 2541-6855
https://openalex.org/W2336626347	https://www.siberianlawreview.ru/jour/article/download/604/577	Russian	null	Criminal Law Policy of Counteracting Crimes against Property	Vestnik Omskoj ûridičeskoj akademii	2,016	cc-by	2,082	РАГОЗИНА Ирина Григорьевна – заведующий кафедрой уголовного права и криминологии Омской юридической академии, кандидат юридических наук, доцент (Омск) РАГОЗИНА Ирина Григорьевна – заведующий кафедрой уголовного права и криминологии Омской юридической академии, кандидат юридических наук, доцент (Омск) Ragozina Irina G. – Head of Criminal Law and Criminology Department, Omsk Law Academy, Candi date of Legal Sciences, Associate Professor (Omsk) ragoig@mail.ru ragoig@mail.ru Аннотация. Статья посвящена современному состоянию уголовно-правовой политики противо действия преступлениям против собственности. Анализируются ее основные направления, такие как правотворчество и правоприменение. Автором исследуются вопросы унификации и дифференциации уголовной ответственности за преступления против собственности. Сделан вывод об отсутствии единообразного подхода к конструированию уголовно-правовых запретов, предусматривающих уголов ную ответственность за посягательство на чужую собственность. The article is devoted to the current state of the criminal law policy of counteracting crimes against property. The focus is made on its main directions, such as law-making and law enforcement. The issues of unification and differentiation of criminal liability for crimes against property are under study. The author draws the con clusion that no unified approach is used when producing criminal law prohibitions criminalizing encroachment on someone else's property. Ключевые слова: уголовно-правовая политика, преступления против собственности, уголовно-пра вовой запрет, уголовная ответственность, унификация, дифференциация, правотворчество, правопри менение. i Criminal law policy, crimes against property, penal prohibition, criminal liability, unification, diff lawmaking, law enforcement. Вестник Омской юридической академии. 2016. № 2 (31) Василевский, кроме этого, к одному «из генеральных направлений уголовно- правовой политики любого цивилизованного об щества в сфере борьбы с преступностью» относят дифференциацию ответственности [2, с. 48]. Од ним из оснований дифференциации, по их мне нию, с которым нельзя не согласиться, выступает характер и типовая степень общественной опас ности преступления [2, с. 67]. Вопросам уголовно-правовой охраны соб ственности в теории права уделяется значитель ное внимание. Однако это не свидетельствует о том, что проблем с применением данных за претов становится меньше. К сожалению, стати стика говорит о стабильно высоком количестве хищений имущества, которые составляют около половины всех зарегистрированных преступле ний. По данным Главного информационного аналитического центра МВД России, в 2011 г. зарегистрировано 2404,8 тыс. преступлений, из них 1466,9 тыс. – посягательства на собствен ность, в 2012 г. соответственно – 2302,2 тыс. и 1400,0 тыс., в 2013 г. – 2206,2 тыс. и 1304,6 тыс., в 2014 г. – 2166,4 тыс. и 1238,2 тыс., в 2015 г. – 2388,5 тыс. и 1397,4 тыс. [6] Л. Л. Кругликов и А. В. Василевский, кроме этого, к одному «из генеральных направлений уголовно- правовой политики любого цивилизованного об щества в сфере борьбы с преступностью» относят дифференциацию ответственности [2, с. 48]. Од ним из оснований дифференциации, по их мне нию, с которым нельзя не согласиться, выступает характер и типовая степень общественной опас ности преступления [2, с. 67]. В условиях социальных, экономических, по литических и иных реформ корыстная преступ ность приобретает все более негативные коли чественные и качественные характеристики, организованные формы, элементы криминаль ного профессионализма, что требует адекватных мер борьбы с ней. Сегодня вместо одной статьи, предусматри вающей ответственность за мошенничество, мы имеем семь, одна из которых уже признана не соответствующей нормам Конституции Россий ской Федерации. Из чего исходил законодатель, «клонируя» классическое мошенничество на шесть его раз новидностей, остается загадкой. Если исходить из размера санкций, основанием дифференци ации явилась общественная опасность мошен нических действий в различных сферах обще ственных отношений. Однако почему, например, мошенничество в сфере кредитования или пред принимательской деятельности оказалось «де шевле» на 2 года лишения свободы, чем клас сическое мошенничество, понять достаточно сложно. Из диспозиций новоявленных норм сле дует, что в четырех случаях законодатель провел дифференциацию с учетом специфики тех обще ственных отношений, которые подвергаются криминальному воздействию (мошенничество в сфере кредитования, в сфере предприниматель ской деятельности, в сфере страхования, в сфере Заметим, что составы преступлений против собственности до недавнего времени отличались достаточной стабильностью, что обеспечивало эффективность их применения. Вестник Омской юридической академии. 2016. № 2 (31) будило автора обратиться к проблемам уголовно- правовой охраны собственности. Попытаемся дать оценку этим изменениям исходя из основных направлений уголовно-правовой политики. будило автора обратиться к проблемам уголовно- правовой охраны собственности. Попытаемся дать оценку этим изменениям исходя из основных направлений уголовно-правовой политики. преступлениям против собственности [5, с. 47–49]. б п р у р [ , с. 47–49]. Такой подход законодателя вполне объясним. Собственность является экономической основой существования любого государства независимо от исторического этапа его развития, полити ческого режима и других обстоятельств. Право быть собственником – одно из основных прав, закрепленных во Всеобщей декларации прав че ловека. Обеспечение эффективной защиты соб ственности, в том числе и уголовно-правовыми мерами, является задачей любого государства, и Россия в этом случае не является исключением. Вопросам уголовно-правовой охраны соб ственности в теории права уделяется значитель ное внимание. Однако это не свидетельствует о том, что проблем с применением данных за претов становится меньше. К сожалению, стати стика говорит о стабильно высоком количестве хищений имущества, которые составляют около половины всех зарегистрированных преступле ний. По данным Главного информационного аналитического центра МВД России, в 2011 г. зарегистрировано 2404,8 тыс. преступлений, из них 1466,9 тыс. – посягательства на собствен ность, в 2012 г. соответственно – 2302,2 тыс. и 1400,0 тыс., в 2013 г. – 2206,2 тыс. и 1304,6 тыс., в 2014 г. – 2166,4 тыс. и 1238,2 тыс., в 2015 г. – 2388,5 тыс. и 1397,4 тыс. [6] Такой подход законодателя вполне объясним. Собственность является экономической основой существования любого государства независимо от исторического этапа его развития, полити ческого режима и других обстоятельств. Право быть собственником – одно из основных прав, закрепленных во Всеобщей декларации прав че ловека. Обеспечение эффективной защиты соб ственности, в том числе и уголовно-правовыми мерами, является задачей любого государства, и Россия в этом случае не является исключением. В рамках статьи мы не ставим своей целью рассмотреть все доктринальные подходы к са мому определению уголовной политики. Оста новимся лишь на некоторых из них. По мнению А. И. Коробеева, А. В. Усса и Ю. В. Голика, уго ловная политика в ее традиционном понимании есть генеральная линия, определяющая основные направления, цели и средства воздействия на пре ступность путем формирования уголовного, уго ловно-процессуального, исправительного зако нодательства, практики его применения, а также путем выработки и реализации мер, направлен ных на предупреждение преступлений [1, с. 7]. Как правило, в юридической литературе выделя ют два основных направления уголовно-правовой политики – правотворчество и правоприменение. Л. Л. Кругликов и А. В. DOI: 10.19073/2306-1340-2016-2-35-38 Преступления против собственности с уве ренностью можно отнести к числу классиче ских, или так называемых традиционных, пре ступлений. Это подтверждается тем, что еще в Русской Правде Ярослава Мудрого, состоя щей из 43 статей, 16 из них были посвящены ступлений. Это подтверждается тем, что еще в Русской Правде Ярослава Мудрого, состоя щей из 43 статей, 16 из них были посвящены 35 Вестник Омской юридической академии. 2016. № 2 (31) Вестник Омской юридической академии. 2016. № 2 (31) Изменения, вно симые в статьи, чаще всего были обусловлены об щими изменениями уголовного законодательства, такими как, например, исключение из уголовного закона квалифицирующего признака, предусмат ривающего неоднократность совершения престу плений, критериев оценки размера причиненного ущерба, видов и размеров наказаний. В ноябре 2012 г. законодатель дополнил главу 21 Уголовного кодекса Российской Федерации (далее – УК РФ) сразу шестью составами мошен ничества (ст.ст. 159.1–159.6), появление которых вызвало широкую дискуссию в теории права и по 36 Уголовное право и криминология, уголовно-исполнительное право Уголовное право и криминология, уголовно-исполнительное право компьютерной информации), а в двух – исходя из способа (при получении выплат и с использо ванием платежных карт). Складывается впечат ление, что новые статьи родились в силу того, что увеличилось количество преступных пося гательств в выделенных законодателем сферах. Например, выплаты материнского капитала неиз менно повлекли за собой разработку преступных схем, направленных на неправомерное завладе ние им, что и привело к установлению уголовной ответственности за мошенничество при получе нии выплат. Но в таком случае с учетом послед них событий можно говорить о необходимости дифференциации уголовной ответственности в сфере туристического бизнеса, оказания меди цинских услуг, и этот перечень можно продол жать дальше. Мошенничество – это всего лишь одна из шести форм хищения. Почему же именно она попала в зону дифференциации, а не присво ение или растрата, которые могут иметь место и при производстве выплат, и в сфере предпри нимательской деятельности? ность меньшую, чем предусмотрено ст. 159 УК РФ, а крупный и особо крупный размер при чиненного ущерба, напротив, увеличил по срав нению с классическим мошенничеством, данная норма в этой части признана неконституцион ной [4]. Полагаем, что и оставшиеся нормы ждет такая же участь. Это дело времени. Проведенная дифференциация порождает еще ряд вопросов. Так, из диспозиции ст. 159.6 УК РФ следует, что мошенничеством в сфере компью терной информации признаются действия, со вершаемые путем ввода, удаления, блокирова ния, модификации компьютерной информации либо иного вмешательства в функционирова ние средств хранения, обработки или передачи компьютерной информации или информацион но-телекоммуникационных сетей. Как же быть в данном случае с позицией Верховного Суда Рос сийской Федерации, нашедшей отражение в По становлении от 27 декабря 2007 г. № 51 «О су дебной практике по делам о мошенничестве, присвоении и растрате» [3], относительно того, что использование похищенной платежной кар ты (которая, заметим, является источником ком пьютерной информации) путем снятия наличных средств в банкомате следует признавать кражей? Видимо, необходимо в очередной раз констати ровать, что постановления Пленума Верховного Суда носят лишь рекомендательный характер, и обратиться непосредственно к закону. Библиографический список 1. Коробеев, А. И. Уголовно-правовая политика: тенденции и перспективы / А. И. Коробеев, А. В. Усс, Ю. В. Голик. – Красноярск : Изд-во Краснояр. ун-та, 1991. – 236 с. 2. Кругликов, Л. Л. Дифференциация ответственности в уголовном праве / Л. Л. Кругликов, А. В. Василевский. – СПб. : Юрид. центр Пресс, 2002. – 300 с. р 4. По делу о проверке конституционности положений статьи 159.4 Уголовного кодекса Российской Федерации в связи с запросом Салехардского городского суда Ямало-Ненецкого автономного округа [Электронный ресурс] : постановление Конституц. Суда Рос. Федерации от 11 дек. 2014 г. № 32-П // Справочно-правовая система «КонсультантПлюс». – Режим доступа: локальный. 5. Российское законодательство X–XX веков : в 9 т. / под общ. ред. О. И. Чистякова. – Т. 1. Законодательство Древней Руси. – М. : Юрид. лит., 1984. – 432 с. 3. О судебной практике по делам о мошенничестве, присвоении и растрате : постановление Пленума Верхов. Суда Рос. Федерации от 27 дек. 2007 г. № 51 // Рос. газ. – 2008. – 12 янв. 6. Состояние преступности [Электронный ресурс] // Офиц. сайт М-ва внутр. дел Рос. Федерации. – Режим досту па: https://mvd.ru/folder/101762/4/ (дата обращения: 11.03.2015). Вестник Омской юридической академии. 2016. № 2 (31) Свое отношение к проведенной дифференци ации высказал Конституционный Суд Россий ской Федерации в Постановлении от 11 декабря 2014 г. № 32-П «По делу о проверке конститу ционности положений статьи 159.4 Уголов ного кодекса Российской Федерации в связи с запросом Салехардского городского суда Яма ло-Ненецкого автономного округа». В указанном Постановлении отмечается, что установление мер, направленных на защиту собственности от преступных посягательств, Конституция Рос сийской Федерации возлагает на федерально го законодателя, предоставляя ему достаточно широкую свободу усмотрения, но одновремен но обязывая его руководствоваться имеющими универсальное значение и по своей сути отно сящимися к основам конституционного право порядка общими принципами юридической ответственности, включая принципы юриди ческого равенства и правовой определенности. С учетом того, что законодатель, устанавливая ответственность за мошенничество, сопряжен ное с преднамеренным неисполнением договор ных обязательств в сфере предпринимательской деятельности, установил уголовную ответствен Дифференциация ответственности может за ключаться не только в создании новой нормы, но и в установлении квалифицирующих обсто ятельств. Мы поддерживаем позицию законода теля, который увидел повышенную обществен ную опасность в мошеннических действиях, повлекших лишение права гражданина на жилое помещение, но такого права потерпевший может лишиться и в результате вымогательства, однако этот момент законодатель упустил из виду. На наш взгляд, дифференциация уголовной ответственности – одно из важных направлений уголовной политики, способное с наибольшей полнотой отражать все обстоятельства преступ ного поведения. Только проводиться подобного рода дифференциация должна взвешенно, с уче том всех необходимых оснований. Библиографический список 1. Коробеев, А. И. Уголовно-правовая политика: тенденции и перспективы / А. И. Коробеев, А. В. Усс, Ю. В. Голик. – Красноярск : Изд-во Краснояр. ун-та, 1991. – 236 с. 2. Кругликов, Л. Л. Дифференциация ответственности в уголовном праве / Л. Л. Кругликов, А. В. Василевский. – СПб. : Юрид. центр Пресс, 2002. – 300 с. 37 Вестник Омской юридической академии. 2016. № 2 (31) р 4. По делу о проверке конституционности положений статьи 159.4 Уголовного кодекса Российской Федерации в связи с запросом Салехардского городского суда Ямало-Ненецкого автономного округа [Электронный ресурс] : постановление Конституц. Суда Рос. Федерации от 11 дек. 2014 г. № 32-П // Справочно-правовая система «КонсультантПлюс». – Режим доступа: локальный. у 5. Российское законодательство X–XX веков : в 9 т. / под общ. ред. О. И. Чистякова. – Т. 1. Законодательство Древней Руси. – М. : Юрид. лит., 1984. – 432 с. 6. Состояние преступности [Электронный ресурс] // Офиц. сайт М-ва внутр. дел Рос. Федерации. – Режим досту па: https://mvd.ru/folder/101762/4/ (дата обращения: 11.03.2015). 38
https://openalex.org/W2180424922	https://hal.archives-ouvertes.fr/hal-01417407/document	English	null	Collaborative Supplying Networks: Reducing Materials Management Costs in Healthcare	IFIP advances in information and communication technology	2,015	cc-by	4,911	To cite this version: Lorenzo Tiacci, Chiara Paltriccia. Collaborative Supplying Networks: Reducing Materials Manage- ment Costs in Healthcare. IFIP International Conference on Advances in Production Management Sys- tems (APMS), Sep 2015, Tokyo, Japan. pp.93-101, 10.1007/978-3-319-22756-6_12. hal-01417407 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01417407 https://hal.science/hal-01417407v1 Submitted on 15 Dec 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Collaborative supplying networks: reducing materials management costs in healthcare Lorenzo Tiacci, Chiara Paltriccia Università degli Studi di Perugia - Dipartimento di Ingegneria, Perugia, Italia lorenzo.tiacci@unipg.it chiara.paltriccia@studenti.unipg.it Università degli Studi di Perugia - Dipartimento di Ingegneria, Perugia, Italia lorenzo.tiacci@unipg.it chiara.paltriccia@studenti.unipg.it Abstract. Materials and inventories management is becoming a more and more important subject in the health care sector, because of its impact on efficiency, and quality of services provided. This study is focused on the inventory man- agement and the service of surgical instruments reprocessing related to the op- erating room. The aim is to analyse which benefits may produce the combina- tion of two different factors: the implementation of the RFId technology along the supply chain and the cooperation thorough a long-term collaborative- networked organization of the supplying companies. The first factor, named ‘RFId effect’, allows implementing a Continuous Review policy, instead of the Periodic Review policy normally utilized. The second factor, named ‘Network effect’, gives to supplying companies the possibility to share transportation costs. The model is inspired by a real case study of a long-term collaborative network of supplying companies in the health care sector that operates in cen- tral Italy. A numerical experiment shows that combining the RFId and Network effects may bring to a relevant reduction of expected costs. adfa, p. 1, 2011. © Springer-Verlag Berlin Heidelberg 2011 1 Introduction Providing health services with high quality standards and containing public ex- penditure are increasingly important objectives to be achieved in the context of public healthcare. Among the solutions adopted to contain healthcare spending, one of the most important trend in the public healthcare sector is to entrust some outsourced services, optimizing performance through a contract. In this way, healthcare managers tried to reduce spending without affecting the quality of service offered [1]. The spread of non-core processes outsourcing in healthcare has become very fast because, being the hospital a set of many departments handling a large number of activities, there is the need to entrust outside the no core ones [2]. Cost effectiveness is im- portant even for activities that have greater impact on the hospital core business; among those, there is the surgical instruments reprocessing process, which is consid- ered to be a potential area for important cost savings [3]. van de Klundert, Muls and Schadd [4] studied the logistic activities between the central sterilization service de- partment and the operating theater. These activities affect total costs faced by the operating block. Hospitals that outsource these types of activities are still very few. Inventory management of drugs and medical devices is also become a very im- portant argument for hospital management. Many medical organizations reorganized their logistics in order to contain costs and ensure quality in services provided [5]. This paper investigates a new outsourcing model for the management of the operat- ing room materials, inspired by a real case study of a network of enterprises born in Central Italy, in the Umbria Region. The network is based on the VDO network mod- el [6] and provides the supplying of all the consumables for the operating room and the reprocessing service of surgical instruments. The network business model pro- vides the implementation of the RFId technology for the automatic and continuous monitoring of hospital’s inventories, and for the total traceability of all the materials utilized during surgeries. Through reading tags, stickers on which one can transcribe information on circuits, it is possible to have remote tracking. Inventory tracking be- comes easier than ever and many other probable applications of RFId technologies have been proposed [7]. 1 Introduction As evidenced by Sarac, Absi and Dauzere-Peres [8] this technology may improve the potential benefits along the supply chain reducing inven- tory losses, increasing efficiency and speed of the processes involved and improving information accuracy. The integrated service, provided by the network as a single entity, includes: 1) sur- gical instruments sterilization service; 2) provision of sets of surgical instruments for surgical procedures; 3) provision of disposable non-woven materials, such as surgical drapes, masks, gloves, caps, gowns; 4) provision of disposable surgical devices, such as suture threads, syringes, video-laparoscopic devices; 5) provision, control and maintenance of medical equipment. The specific characteristic of the case study, with respect to traditional models of materials management, consists in the implementation of the RFId technology, and in the long-term strategic cooperation between the supplying companies. Aim of the work is to provide a model to evaluate the effect of these two key factors on total operational costs. Notation i: different products line Qi: order quantity of item i [units] Di: mean annual demand of item i [units/year] A: cost per shipment for Company B without the network effect [€/roundtrip] A’: cost per shipment for Company B with the network effect [€/roundtrip] r: inventory carrying charge [€/€/year] vi: unit variable cost of item i [€/unit] ki: safety factor for item i 2 The model This case study refers to a stable network of companies located in central Italy, in the region of Umbria. The network provides an integrated set of services for the oper- ating room. The network, named Sanitanet, is composed of five companies: four of them are companies already operating as single suppliers in the healthcare sector, while the fifth one is a University Spinoff in the field of collaborative networks man- agement. For the purpose of the present work, we consider in the model just two of the five companies (Company A and Company B). Company A provides a ‘pay per use’ service of sterile surgical instruments kits. These single-use kits are specifically designed for each surgery. This company owns all the surgical instruments composing the kits and reprocesses them in its own plant outside the hospital. At the beginning of each day, the company brings all the surgical kits needed for the daily planned surgery to the hospital. At that time, it withdraws the kits utilized the day before to reprocess them at its plant. Company B provides non-woven surgical protective drapes for patients, operators and furniture (beds, trolleys, etc.). The supplied materials include masks, gloves, caps, gowns and drapes. All these items are packaged as single-use ‘custom pack’, specifi- cally designed for each surgery type. Thus, each custom pack provides all the neces- sary materials for a single surgery. The company periodically delivers its supplies, based on orders placed by the hospital staff every month. As already mentioned, the specific characteristic of the case study consists in: 1) the implementation of the RFId technology; 2) the long-term strategic cooperation between the supplying companies. We will refer to these two key factors with the terms ‘RFId effect’ and ‘Network effect’ respectively. The impact of these two factors on the Expected Total Relevant Costs (ETRC) will be evaluated through an analysis of scenarios. In the next sections we will formalize the ‘Network effect’ and the ‘RFId effect’. 2.1 Network effect The Network effect allows companies to share setup costs, which substantially corre- spond to transportation costs. We assume that cost per shipment is proportional to the Euclidean distance covered for the shipment. Companies A and B may share some part of the way to the hospital. In particular, Company B can take advantage of the daily frequency of the shipment of Company A. In this case, the lower distance that can be covered in a roundtrip is equal to the pe- rimeter of the triangle (see Fig. 1) formed by the Hospital (point H), Company A (point A), and Company B (point B). Thus, when the two companies cooperate as a network, the shipment trip will always start from, and finish at, Company A plant, due to the necessity to bring back the utilized kits from the hospital. The round trip will consist of:  load of sterile kits at Company A plants  trip from Company A to Company B  load of packs from company B  trip from Company B to the Hospital for the delivery of kits and packs  trip from the Hospital to Company A to bring back utilized kits  load of sterile kits at Company A plants  trip from Company A to Company B  load of packs from company B  trip from Company B to the Hospital for the delivery of kits and packs  trip from the Hospital to Company A to bring back utilized kits  load of sterile kits at Company A plants  trip from Company A to Company B  load of packs from company B  trip from Company B to the Hospital for the delivery of kits and packs  trip from the Hospital to Company A to bring back utilized kits Fig. 1. Schematic position of companies and hospital Fig. 1. Schematic position of companies and hospital To estimate the savings achievable through the network effect, the cost per shipment related to the network configuration have to be compared with cost per shipment when companies act individually. The condition for evaluating the convenience of the network collaboration is that ( 2 2 AB AH BH BH AH     ). In this relation, the first term represents the total length of the round trip in a network collaboration. 2.1 Network effect The second term describes the total distance travelled by the two companies, if they work individually. The relation can be simplified in the following way: AB BH AH   . As the sum of two sides of a triangle is always greater than only one side, the last relation is always verified. This implies that working as a network always allows reducing cost per shipment. The total savings SV achievable under the ‘network effect’ will be proportional to 2 2 ( ) SV BH AH AB AH BH      . From a Network perspective, this reduction of shipment cost can be entirely assigned to Company B. Indeed, it is noteworthy that shipment cost does not affect the replenishment policy of Company A, which is obliged to ship the required quantities on a daily basis. On the contrary, a reduction of shipment cost could determine a variation of the optimal quantity shipped by Compa- ny B. In fact, the optimal reorder quantity for each item i is equal to the Economic Order Quantity 2 / EOQ A D v r    . So if the shipment cost A drops to A’ when the network effect is present, the optimal order quantities of each item also decrease. In this view, as the Company B shipment cost without the network is equal to A = 2BH, A’ can be estimated as: A’ = A – SV =2BH – [2BH +2AH – (AB + AH + BH)]=BH + AB – AH. Thus, it is possible to define the ‘Network coefficient’ λ as the ratio be- tween A’ and A, with 0 ≤ λ ≤ 1: 1 2 – 2 A H A H B A A B      (1) (1) λ depends from the mutual positions of Company A, Company B and the Hospital. In summary, when the scenario provides the network effect, the cost per shipment will be equal to λA, and consequently, if a continuous review policy is adopted, reor- der quantity Qi will be calculated as: 2 / i i i Q A D v r     (2) (2) 2.2 RFId effect Among the advantages related to the implementation of RFID technology, we con- sider: 1) to avoid incomplete shipments, misplacements and theft; 2) to possibly im- plement a continuous review policy. The first benefit has been modelled using the study by Ustundag and Tanyas [9]. They considered that RFID technology implemen- tation eliminates item losses caused by theft, incorrect positioning and incomplete shipments. Only damages that can occur during transportation cannot be avoided. This effect can be considered using the following four coefficients: α (incorrect posi- tioning), β (damage), γ (theft) and δ (incomplete shipment). They represent the error rates related to the corresponding causes. Table 1 shows the values, derived from the experimental study of Ustundag and Tanyas [9], that has been assumed in our study. As far as the second benefit is concerned, the conceptual model assumes that without the implementation of the RFId technology a periodic review policy is adopt- ed, while the RFId effect allow implementing a continuous review policy. In the fol- lowing subsections, the description of both policies is carried out. Technology α (%) β (%) γ (%) δ (%) non RFID 2 0.2 0.5 0.3 RFID 0 0.2 0 0 Table 1. Error rates of non-RFId and RFId integrated systems Table 1. Error rates of non-RFId and RFId integrated systems Table 1. Error rates of non-RFId and RFId integrated systems Periodic review – no RFId effect. Continuous review – RFId effect. When RFID technology is implemented, Company B can apply a continuous review policy (s, Q). In this case the optimal order quantity Qi, for each item i, is calculated through the Economic Order Quantity (EOQ): 2 / i i i i Q EOQ AD v r   (5) (5) Note that, if the network effect is simultaneously present, the network coefficient λ has to be considered to calculate order quantities using eq. (2), because A decrease to A’=λA. The reorder-point si and safety stocks SSi are calculated in this case through: si =SSi + Di L; SSi = ki σ(L)I, where σ(L)i is the demand standard deviation during the lead time L. To find the values of the safety factors ki that allows us to reach the fill rate P = 99.9%, it is necessary to calculate the loss function as (see [11]):   ( ) 1- ( ) i i u i L P Q G k   (6) (6) Then the value of ki can be determined using the tabular form of eq(4). Then the value of ki can be determined using the tabular form of eq(4). Periodic review – no RFId effect. Without the RFID effect, Company B actually adopts a periodic review policy (R, S), with a review period R equal to 2 weeks, which is common for all the four items. This type of policy is very common in healthcare [10], and it is often adopted when it is not possible to have an automatic monitoring of the warehouse. Indeed, if the invento- ry has to be checked manually, it is usually preferred to limit the number of times the checking activities have to be performed. Furthermore, if the orders are placed on a periodic basis, different items can be incorporated in the same shipment in order to save on transportation costs. For each item i, the order-up to level Si and the safety stocks SSi are calculated respectively as: Si =SSi + Di (R+L); SSi = ki σ(R+L)I, where σ(R+L)i is the standard deviation of demand during R+L, and ki is the safety factor. The safety factor ki is determined by imposing a specified service level required by the customer. Given the critical function of the supplied items, the required service level is high. We assumed a value of P = 99.9% of demand satisfied immediately from the shelf (‘fill rate’). From this condition, the value of ki can be calculated in the consider- ing the following function [11]:   ( ) 1 ( ) i i u i R L i P D R G k     (3)   ( ) 1 ( ) i i u i R L i P D R G k     (3) where Gu(.) is the loss probability function, equal to: where Gu(.) is the loss probability function, equal to: ( ) ( ) ( )d ( ) (1 ( )) u k G k u k u u k k k          (4) (4) ( ) k  being the unit normal density function, and Φ(k) the corresponding distribu- tion function. From the value of Gu(ki), calculated from (3), it is possible to determine the value of ki using the tabular form of eq. (4). 2.3 Scenario Analysis Four different scenarios will be compared on the basis of the annual Expected To- tal Relevant Costs (ETRC), which is considered composed by four components: re- plenishment costs, holding costs, RFId implementation costs, and costs related to thefts and misplacements. The total annual costs related to RFId implementation has been estimated by summing the periodic amortization payment, related to hardware and software investments, and the annual costs of the ‘one use’ UHF tags, and it is equal to 4434 [€/year] (the detailed costs are omitted due to space limitation). The impact of thefts on the ETRC is calculated considering a cost equal to the value of each item stolen. The cost of items misplaced is considered equal to item value only when item are expired and cannot be used if they are found. In the model it is as- sumed that the 50% of misplaced items expires before the finding, and a cost equal to the value of the item is considered; the other 50% is considered utilizable and for this reason it does not represent a cost. p The four scenarios considered are described in the following. Scenario ‘AS IS’. In the ‘as is’ scenario neither the ‘Network effect’ nor the ‘RFId effect’ are present. Thus, the companies operate individually, Company B utilizes a periodic review policy, with a cost per shipment equal to A, where the products line share the same shipment. ETRC can be estimated through: periodic review policy, with a cost per shipment equal to A, where the products line share the same shipment. ETRC can be estimated through: ( ) 2 2 i i R L i i i i i i D R A ETRC k v r D v R                            (7) (7) In (7), the first term corresponds to replenishment costs, the second one to carrying costs, and the last one approximate costs associated to thefts and misplacements. Scenario 1 - Network effect. Company A and B belong to the same network. Company B still adopts a Periodic Review policy, but it is possible to share the cost per shipment with Company A. Thus, ETRC can be estimated through (7) by substi- tuting the shipment cost A with A’=λA. Scenario 2 - RFID effect. 2.3 Scenario Analysis In this scenario, the companies still operates individual- ly, but Company B implements a continuous review policy, which is enabled by the RFId implementation. The cost per shipment between Company B and the Hospital is still equal to A, because it is not shared. Each shipment contains only one product line, due to the different times each product line reaches the respective reorder point. In this case, there are no costs related to thefts and misplacements, while the annual cost related to RFId implementation is considered: ( ) 2 i i i L i i RFId i i i D EOQ ETRC A k v r C EOQ                (8) (8) Scenario 3 - Network + RFId effects. The Network effect and the RFId effect are simultaneously present. Thanks to RFId technology Company B can implement a continuous review policy. At the same time, each time a product line reaches the re- order point, the corresponding shipment can be shared with Company A, thanks to the network effect. Thus, ETRC can be estimated through eq.(8) by considering a ship- ment cost equal to A’=λA, instead of A. Note that even the optimal quantities EOQi are affected by the decrease of the shipment cost (see eq. (5)). 3 Numerical experiment and discussion In scenario 1, by virtue of the option of cooperating as a network, there is the possibility for Company B to share shipment costs with Compa- ny A, which supplies the hospital on a daily basis. This is of course preferable with respect to the AS IS scenario, because holding costs remain unchanged, and transpor- tation costs are reduced by a factor equal to λ. Results related to scenario 2 show that, even in absence of the network effect, the implementation of the RFId technology makes the adoption of a continuous review policy more convenient. This is due to two reasons: firstly, the RFId annual investment is lower than the annual costs related to thefts and misplacements. Secondly, the parameters of the continuous review policy (s and Q) are optimized for each product line, while in the periodic review policy the review period R is the same for all product lines and it is imposed by the hospital organization (R = 1 month). In scenario 3, the effect obtainable combining the RFId technology with networked cooperation among enterprises is evaluated. to the numerical experiment. In scenario 1, by virtue of the option of cooperating as a network, there is the possibility for Company B to share shipment costs with Compa- ny A, which supplies the hospital on a daily basis. This is of course preferable with respect to the AS IS scenario, because holding costs remain unchanged, and transpor- tation costs are reduced by a factor equal to λ. Results related to scenario 2 show that, even in absence of the network effect, the implementation of the RFId technology makes the adoption of a continuous review policy more convenient. This is due to two reasons: firstly, the RFId annual investment is lower than the annual costs related to thefts and misplacements. Secondly, the parameters of the continuous review policy (s and Q) are optimized for each product line, while in the periodic review policy the review period R is the same for all product lines and it is imposed by the hospital organization (R = 1 month). In scenario 3, the effect obtainable combining the RFId technology with networked cooperation among enterprises is evaluated. The reduction achievable in terms of ETRC is notable, reaching almost the 48% with respect to the AS IS scenario. 3 Numerical experiment and discussion It is noteworthy that the interaction of the two effects brings to a cost reduction higher than the sum of the savings obtainable when the two effects are applied separately. This is due to the fact that the Network Effect, by allowing decreasing the cost per shipment, has a much more great impact on poli- cies with frequent of shipments (s, Q) then on policies with low number of shipments (R, S). Thus, only the simultaneous adoption of the RFId technology and the collabo- rative network model allows maximizing the expected savings. 3 Numerical experiment and discussion The values utilized in the experiments are related to a hospital placed in a city in the south of the Umbria region, in Italy: cost per roundtrip between Company B and the Hospital A = 50 [€], network coefficient λ = 0.1158, inventory carrying charge r = 0.25 [€/€/year], lead time L = 2 days, required service level (demand satisfied directly from the shelf) P = 99.9%. The annual demand data of the four item lines considered are related to the surgeries done in the hospital during the year 2012. Each product line represents a specific surgical discipline custom pack of non-woven drapes. De- mand is assumed to be exponentially distributed, and the annual average values Di and the value vi of each item are reported in Table 2. The value of ki for both the periodic and the continuous review policies have been derived using the procedures described in Section 5. Table 3 shows the results related Product line Description Average Demand [units/year] Item value [€/unit] 1 Surgery 560 208 2 Otorhinolaringologist 20 198 3 Orthopaedics 673 220 4 Obstetrician e genecology 454 247 Table 2. Data related to the 4 product lines Scenarios data Annual Costs Scen. RFId effect Network effect Repl. Policy Unitary shipment cost Transp. Costs [€/y] Holding Costs [€/y] RFId Invest. [€/y] Thefts mispl. [€/y] ETRC [€/y] Saving AS IS NO NO (R, S) A 595 6700 - 4568 11863 / 1 NO YES (R, S) λA 69 6700 - 4568 11337 4.4% 2 YES NO (s, Q) A 2817 3466 4434 - 10717 9.7% 3 YES YES (s, Q) λA 969 1766 4434 - 7170 39.6% Table 3. Results of the numerical experiment Table 2. Data related to the 4 product lines Scenarios data Annual Costs Scen. RFId effect Network effect Repl. Policy Unitary shipment cost Transp. Costs [€/y] Holding Costs [€/y] RFId Invest. [€/y] Thefts mispl. [€/y] ETRC [€/y] Saving AS IS NO NO (R, S) A 595 6700 - 4568 11863 / 1 NO YES (R, S) λA 69 6700 - 4568 11337 4.4% 2 YES NO (s, Q) A 2817 3466 4434 - 10717 9.7% 3 YES YES (s, Q) λA 969 1766 4434 - 7170 39.6% Table 3. Results of the numerical experiment Table 2. Data related to the 4 product lines Table 3. Results of the numerical experiment Table 3. Results of the numerical experiment to the numerical experiment. Conclusions In the paper a methodology to evaluate possible savings of a new business model of collaborative supply network in the healthcare sector is described. The case study show that an integrated supplying service for the operating room can be less expen- sive than traditional models, while maintaining an adequate service level to the pa- tient. In fact, the presence of RFId technology allows the implementation of a contin- uous review policy, which is known to reduce the uncertainty period with respect to periodic review policies. Consequently, relevant savings related to holding costs are achievable. At the same time, the collaborative network of suppliers allows sharing transportation costs. In this way, the costs connected to shipments, which are typically higher in continuous review policy, are limited. It is reasonable to assume that the economic benefits achievable in reality may in- crease as the number of different products handled and the number of companies in- volved increase. In fact, when there are more than four products considered in this work, it is correct to assume that, even by adopting a continuous review policy, the replenishment instant may be the same for more products simultaneously. In the same way, shipment costs can be lowered even in the case where more than two companies are considered, by optimizing the routing from Company A to the Hospital. In order to generalize the results obtainable by the proposed approach, future im- provements consists in performing a sensitivity analysis on the parameters that can have a direct influence on total costs, starting from the network coefficient λ. Finally, a discrete event simulation model could be designed to accurately evaluate the impact of thefts and misplacement on real costs and service level, in order to better quantify the benefits connected to RFId implementation. References 1. Roberts, V.: Managing strategic outsourcing in the healthcare industry. J Healthc Manag 46, 239-249 (2001) 2. Baffo, I., Confessore, G., Liotta, G., Stecca, G.: A Cooperative Model to Improve Hospital Equipments and Drugs Management. Leveraging Knowledge for Innovation in Collaborative Networks 307, 43-50 (2009) 3. Reymondon, F., Pellet, B., Marcon, E.: Optimization of hospital sterilization costs proposing new grouping choices of medical devices into packages. Int J Prod Econ 112, 326-335 (2008) 4. van de Klundert, J., Muls, P., Schadd, M.: Optimizing sterilization logistics in hospitals. Health Care Management Science 11, 23-33 (2008) 5. de Vries, J.: The shaping of inventory systems in health services: A stakeholder analysis. Int J Prod Econ 133, 60-69 (2011) 6. Saetta, S., Tiacci, L., Cagnazzo, L.: The innovative model of the Virtual Development Office for collaborative networked enterprises: the GPT network case study. Int J Comput Integ M 26, 41-54 (2013) 7. Choi, T.M.: Pre-season stocking and pricing decisions for fashion retailers with multiple information updating. Int J Prod Econ 106, 146-170 (2007) 8. Sarac, A., Absi, N., Dauzere-Peres, S.: A literature review on the impact of RFID technologies on supply chain management. Int J Prod Econ 128, 77-95 (2010) 9. Ustundag, A., Tanyas, M.: The impacts of Radio Frequency Identification (RFID) technology on supply chain costs. Transport Res E-Log 45, 29-38 (2009) 10. Nicholson, L., Vakharia, A.J., Erenguc, S.S.: Outsourcing inventory management decisions in healthcare: Models and application. European Journal of Operational Research 154, 271-290 (2004) 11. Silver, E.A., Pyke, D.F., Peterson, R.: Inventory Management and Production Planning and Scheduling. John Wiley & Sons Inc (1998)
https://openalex.org/W4285494687	https://resource-allocation.biomedcentral.com/counter/pdf/10.1186/s12962-022-00366-z	English	null	Costing the outpatient rehabilitation services: time-driven activity-based costing approach	Cost effectiveness and resource allocation	2,022	cc-by	6,637	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Considering the importance of healthcare services costing in resource allocation, the aim of this study is to calculate the cost of rehabilitation services in an outpatient rehabilitation clinic in Tehran, Iran. Methods: The data for this study were categorised as financial data and information about the process of rehabilita- tion services. The first category was extracted from the financial documents and the second was obtained by observa- tion of patient flow and interviews with the clinic staff in 2016. The cost of rehabilitation services has been estimated using the time-driven activity-based costing approach. Results: The findings show that the cost of physical occupational therapy in the Asma rehabilitation center was $18.79 per unit of service. This amount for speech therapy services was $17.23 to $19.40, taking into account the dif- ference in the quality of the service delivered. The cost of mental health occupational therapy service was between $19.46 and $23.57. Comparing the cost of these services with the government’s tariffs makes it clear that there is a huge gap. Conclusion: The limited number of patients referred to the center makes the cost of one unit of rehabilitation services much higher than the official tariffs. This is true for almost all similar institutions and makes the profitability of small rehabilitation institutions extremely unstable. Therefore, proper marketing for rehabilitation services by promot- ing patient referral links with larger healthcare centers and reallocation of resources to the formation of integrated rehabilitation complexes can play a significant role in their profitability. Keywords: Time-driven activity based costing, Rehabilitation services, Costing, Outpatient services organisations face increasing diversity and complexity of services as well as budgetary limits [3]. Awareness of the cost structure through utilisation of an effective costing framework is essential for healthcare providers in order to survive in a competitive economic environment as well as for policy makers for healthcare resources allocation [4–6]. Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 https://doi.org/10.1186/s12962-022-00366-z Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 https://doi.org/10.1186/s12962-022-00366-z Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 https://doi.org/10.1186/s12962-022-00366-z Cost Eﬀectiveness and Resource Allocation Correspondence: Me.basakha@uswr.ac.ir 2 Department of Social Welfare Management, School of Education Sciences and Social Welfare, Social Determinants of Health Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran 1985713834, Iran Full list of author information is available at the end of the article Background One of the main challenges in the sustainability of health- care providers is the development of a cost information system that is suitable for pricing decisions, strategic management and resources allocation [1, 2]. Healthcare managers should be aware of all feasible costing meth- ods to provide affordable and high-quality services as Different costing methods have been used for health- care services, and over time, the accuracy of these meth- ods have been enhanced. The traditional approaches, where there are no solid connections between the activi- ties and the amount of resources used, can be only be employed in cases with limited activities and confined Correspondence: Me.basakha@uswr.ac.ir 2 Department of Social Welfare Management, School of Education Sciences and Social Welfare, Social Determinants of Health Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran 1985713834, Iran Full list of author information is available at the end of the article *Correspondence: Me.basakha@uswr.ac.ir Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 Page 2 of 8 costs [7]. Due to this problem, the Activity-Based Cost- ing (ABC) method has been developed. Nevertheless, setting up an ABC system is time consuming and the sys- tem should be updated regularly, which would increase the cost of accounting considerably [2, 8]. The constraints of this approach have led to a move towards the Time Driven Activity-Based Costing (TDABC) approach. This method was developed by Kaplan and Anderson [9, 10] in order to help healthcare organisations to identify unused capacity and enable them to reduce the cost of services resulting in more efficient service delivery and profitable schemes [11, 12]. system is still underestimated and the government and social insurers have drastically reduced their spend- ing in recent years [25]. This makes access to rehabilita- tion services more difficult for many poorer groups and exacerbates the social cost of disability. Regrettably, most rehabilitation services are not covered by social insur- ances resulting in reduced utilisation of services among these groups of people. Accordingly, direct financing and imposing healthcare expenditure on households has led to the spread of the catastrophic expenditure [26]. Rehabilitation service providers in Iran include pub- lic, private, and charitable organisations. Their tariffs are set annually by the MOHME. The Asma Rehabilita- tion Centre (ARC) is affiliated to the University of Social Welfare and Rehabilitation Sciences and is funded by the MOHME. Therefore, the ARC is an educational thera- peutic center and provides internship opportunities for the students of rehabilitation sciences as well as provid- ing rehabilitative services. The ARC has a range of spe- cialised departments, including speech therapy, physical occupational therapy, mental health occupational ther- apy, psychology, audiology, neuro-feedback, hand occu- pational therapy, and physiotherapy.h Due to the importance of human resources in provid- ing healthcare, personnel costs have a significant share in the costs of these services [13]. Different healthcare costing methods attribute different shares to personnel costs. Some studies have identified personnel costs as the largest part of healthcare costs 58 [14–16], while other studies place a lower proportion of the total cost on per- sonnel [17]. Regardless of these differences, it is agreed that the hidden costs of unused human resources could be one potential reason for unaffordable healthcare ser- vices in developing countries [15, 18]. Iranian healthcare and rehabilitation services profileh Iranian healthcare and rehabilitation services profile The Ministry of Health and Medical Education (MOHME) is responsible for public health in Iran. Healthcare services are accessible through a number of different public and private providers. A significant por- tion of health services in Iran are financed by the Min- istry of Welfare and Social Security through specialised insurer organisations including Social Security Organi- sation, the Army Medical Insurance Organisation and Health Insurance Organisation [19]. The coverage rate of social insurance in Iran is high (more than 90% of Ira- nian people have insurance), but these insurance policies do not provide adequate financial coverage and out-of- pocket (OOP) payments to offset a high proportion of healthcare expenditure [20]. The OOP health payments have the largest contribution in healthcare financing in Iran, accounting for over 31.4% of total healthcare expenditure and 37.6% of rehabilitation services expendi- ture [21]. Therefore, the pre- sent study has tried to calculate the more accurate costs of providing rehabilitation services and to determine the unused capacity of human resources with the aim of pav- ing the way for the reallocation of resources in healthcare sector. The present study used TDABC [11, 27] to calculate the cost of speech therapy services, physical occupational therapy, and mental health occupational therapy ser- vices at the ARC. By calculating the unused capacity of each department, the management of the ARC is able to derive the appropriate decisions for developing a profit- able business. This study intends to calculate the cost of most repeated services based on their contribution to the workload and earnings of the ARC. The implicit purpose of this study is to raise public awareness of the sustain- ability of rehabilitation services. Methodsh There are two main approaches to healthcare cost- ing; highly detailed bottom-up costing and top-down approach. If detailed information is available, the bot- tom-up approach is recommended [28]. However, access to this information is limited in public organisations in developing countries, including Iran. Inescapably, the present study uses top-downapproach in costing the selected rehabilitation services in the ARC. The top- down method is suitable for low-volume, low-cost proce- dures and it is based on average costs [28].h The data needed for this research can be divided into two categories: financial data and information about the process of providing the rehabilitation services. The first category includes personnel costs, depreciation cost for office equipment and medical equipment, rent, and overhead costs. These were collected from the financial documents of the ARC in 2016. The second part of data Despite the challenge of chronic disease in the present century, the increase in the elderly population and the significant relationship between the need for rehabilita- tion services and catastrophic expenditures [22–24], the role of rehabilitation services in the Iranian healthcare Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 Page 3 of 8 was gathered by observing the services processes and by conducting informal interviews with the staff of the ARC after obtaining the necessary permissions. In TDABC method, estimation of two parameters are necessary: cost of a unit of resources, and the time required for each activity [17, 29]. More precisely, the TDABC process has been performed based on the following steps [27]: Subsequently, the practical capacity of each group was considered to be 85% of the theoretical capac- ity (Kaplan and Anderson, 2007) equal to 99,781 min in a year. The practical capacity of medical and non- medical equipment and machinery was also consid- ered in terms of the useful life of the equipment. Subsequently, the practical capacity of each group was considered to be 85% of the theoretical capac- ity (Kaplan and Anderson, 2007) equal to 99,781 min in a year. The practical capacity of medical and non- medical equipment and machinery was also consid- ered in terms of the useful life of the equipment. 4. Calculating the unit cost of each resource group: After calculating the total cost and practical capac- ity for each cost group, the cost of each unit was obtained by dividing these two variables into each other. 1. Methodsh Identifying the various departments: First, a map of the rehabilitation services process was drawn up by observing the process of activities in different departments and talking with the authorities in each department. Figure 1 represents the process map of these three services. Next, the various resource groups were identified. 5. Identifying the time equation: Once a patient has been referred to a unit, the time taken for them receive the service must be calculated. This time was measured by observation and timing for four patients, allowing time equations for rehabilitation services to be determined [2]. i 2. Estimating the total cost of each resource group: The cost groups were identified through interviews with the Finance and Property Departments. Cost groups include buildings, overheads, human resources, and equipment (including medical equipment and office equipment). Through the aggregation of these costs, the total cost of each section was determined. Then, direct and indirect costs were calculated for each department. Costs that could not be catego- rised in the above categories (such as educational and research expenses, printing and purchasing of periodicals, the cost of materials and supplies, and the cost of transport and communications) were included as other overhead costs. The area (squared meter) occupied by each department was considered as basis of cost sharing for the building, and mainte- nance of other assets. i 2. Estimating the total cost of each resource group: The cost groups were identified through interviews with the Finance and Property Departments. Cost groups include buildings, overheads, human resources, and equipment (including medical equipment and office equipment). Through the aggregation of these costs, the total cost of each section was determined. Then, direct and indirect costs were calculated for each department. Costs that could not be catego- rised in the above categories (such as educational and research expenses, printing and purchasing of periodicals, the cost of materials and supplies, and the cost of transport and communications) were included as other overhead costs. The area (squared meter) occupied by each department was considered as basis of cost sharing for the building, and mainte- nance of other assets. 6. Aggregating the costs and calculating the final price of each service: This step was acceded by multiply- ing the unit cost of each resource group in time equa- tions. The cost of rehabilitation servicesh The number of rehabilitation sessions required by each patient varies according to the severity of the disability and the type of service required. Sometimes, due to the financial hardship, the patient does not attend all the ses- sions. Based on the time equations, the total personnel costs of each department were obtained per unit of ser- vice (Table 1). 3. Estimating the practical capacity of resource groups: At this stage, the practical capacity of each group was calculated. Staff hours are from 8:00 am to 2:30 pm from Saturday to Wednesday, and from 8:00 am to 1:00 pm on Thursdays. Therefore, the total work- ing time is 1956.5 h per year (117,390 min per year). For departments where services are provided by a workforce with varying degrees of expertise (includ- ing speech therapy and mental health occupational therapy), rehabilitation activities were divided in terms of quality. High quality (HQ) services were defined as those delivered by rehabilitation specialists with a Ph.D. Figure 1 Outpatient rehabilitation services process map in ARC Figure 1 Outpatient rehabilitation services process map in ARC Figure 1 Outpatient rehabilitation services process map in ARC Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 Page 4 of 8 Table 1 Personnel cost for a regular 10-session rehabilitation Time (Minutes) Cost per minute ($) Cost ($) MQ HQ MQ HQ Reception 1.52 0.08 0.08 0.12 0.12 Counseling 28 0.18 0.18 5.04 5.04 Speech therapy 31.3 0.11 0.18 3.44 5.63 Evaluation 12 0.18 0.18 2.16 2.16 Cashier 1.3 0.08 0.08 0.10 0.10 Total cost of a regular 10-session Speech Therapy 4 6.18 Reception 1.52 0.08 0.08 0.12 0.12 Counseling 28 0.18 0.18 5.04 5.04 Mental health occupational therapy 30 0.08 0.21 2.4 6.3 Evaluation 12 0.18 0.18 2.16 2.16 Cashier 1.3 0.08 0.08 0.10 0.10 Total cost of a regular 10-session Mental health occupa- tional therapy 2.95 7.07 Reception 1.52 0.08 0.08 0.12 0.12 Counseling 28 0.18 0.18 5.04 5.04 Physical occupational therapy 53.6 0.09 - 48.24 - Evaluation 12 0.18 0.18 2.16 2.16 Cashier 1.3 0.08 0.08 0.10 0.10 Total cost of a regular 10-session Physical occupational therapy 5.34 − Table 1 Personnel cost for a regular 10-session rehabilitation per minute for the rehabilitation specialists (HQ) and rehabilitation (MQ) experts in to the time equations, the cost of HQ and MQ services can be calculated. The cost of rehabilitation servicesh In order to estimate the average rehabilitation session for each patient, we have considered a regular 10-session course of treatment for these services. Given that, the counseling and evaluation of the general practitioner are provided only twice, so this item is counted only at the beginning and the end of a regular 10-session rehabilitation.h The non-personnel costs of each department are also admeasured according to the number of visits or the area occupied by the department. For this purpose, the cost of depreciation of equipment, rent of the building, repair and maintenance costs, and administrative costs were calculated for the different departments according to the contribution of each department to the activities of the institute. The cost of rehabilitation services, categorised by personnel and non-personnel costs, of each depart- ment is also presented in Table 2. The highest non- personnel costs in the physical occupational therapy and speech therapy sectors were related to the “other overhead cost”; while in the mental health occupational therapy department, the cost of renting was the high- est and accounted for the highest proportion of non- personnel costs. Given the small scale of the ARC, the cost of repair and maintenance of office equipment has been the lowest non-personnel cost for all three depart- ments. Of the total cost of a speech therapy, mental health occupational therapy, and physical occupational in a relevant field while medium quality (MQ) services were delivered by rehabilitation experts with a bache- lor’s or master’s degree in that field. The cost of rehabilitation servicesh By placing the cost Table 2 Cost of resources for rehabilitation services (US $) The numbers in parentheses represent the share of each cost in the total cost Speech Therapy Mental Health Occupational Therapy Physical occupational Therapy Total non-personnel costs 18,663.57 17,145.43 10,032.19 Rent 8049.95 8017.94 4345.06 Utilities 442.51 440.76 238.84 Medical equipment depreciation 134.76 302.65 98.79 Office equipment depreciation 324.54 214.18 186.85 Outsourcing contract services 3661.70 2413.78 1911.31 Repair and Maintenance of equipment 58.59 38.61 30.58 Repair and maintenance of other assets 5248.75 5227.88 2833.06 Other overhead cost 742.77 489.63 387.70 Non-personnel cost of a rehabilitation session 8.37 10.43 9.56 Personnel cost of a MQ rehabilitation session 4.20 3.15 5.54 Personnel cost of a HQ rehabilitation session 6.37 7.26 - Non-technical personnel cost 4.66 5.88 3.69 Total cost of a MQ rehabilitation session 17.23 19.46 18.79 Total cost of a HQ rehabilitation session 19.40 23.57 - Table 2 Cost of resources for rehabilitation services (US $) Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 Page 5 of 8 therapy session, 8.37 $, 10.43 $ and 9.56 $ were related to non-personnel costs, respectively. of an individual and the number of personnel in each department, the practical capacity of that department is specified.h A closer look at the cost components shows that the shares of employee compensation and benefit in the total cost of speech therapy, physical occupational therapy, and mental health occupational therapy services were 4.20$, 3.15$ and 5.54$, respectively. i The comparison of unused capacity shows that the clinic’s cashier has the highest rate of idle time (Table 4). Total cashier activities in the clinic account for only 14.2% of his practical capacity. This percentage was less than 17% for receptionists. Rehabilitation specialist staffs also have a high rate of unused capacity. Meanwhile, mental health occupational therapists have the highest work less time (83% of total practical capacity) among the different departments. Interestingly, general practitioners in the clinic are the group whose activity time and practi- cal capacity are very similar.h Profitability of the rehabilitation serviceshf The government’s tariffs for a session of speech therapy, physical occupational therapy, and mental health occupa- tional therapy in 2017 are shown in Table 3. The govern- ment’s tariffs did not cover the entire costs of any of these services for the ARC. The comparison of the tariffs for rehabilitation services and their cost shows that it is clear that the ARC has suffered losses for the provision of the services in 2017. The gap in the delivery cost and govern- ment tariffs for some health services has been reported in various studies [30, 31]. The largest gross loss in the clinical activities was due to the high-quality health men- tal occupational therapy. The unused capacity of human resources in the ARC shows that a new combination of work reassignments and better management of human resources could lead to more efficient workforce utilisation and lower average cost of personnel. For example, if the clinic has plans to expand rehabilitation activities, given the unused capac- ity calculated, it only requires more general practitioners to be appointed. Therefore, the development of rehabili- tation activities at the ARC not only does not have high costs, but also reduces per capita indirect costs and ulti- mately reduces the cost of the services. Unused human capital capacityh The practical capacity of personnel was calculated throughout the year. By multiplying the practical capacity Table 3 Profitability of a regular session for rehabilitation services ($) Rehabilitation service Tariffs Cost Gross profit (Lost) MQ HQ MQ HQ Speech therapy 7.01 17.23 19.40 −10.22 −12.39 Mental health occupational therapy 6.17 19.46 23.57 −13.29 −17.40 Physical occupational therapy 7.85 18.79 − −10.94 − Table 3 Profitability of a regular session for rehabilitation services ($) Conclusion In the present study, firstly, costs are calculated using a more accurate method based on the allocated time for each activity. Then, through mapping the process, all stages of the rehabilitation service have been identified for the ARC. This framework facilitates the identification of cost drivers of providing rehabilitation services at the ARC. The findings indicate a significant gap between the cost of services and government tariffs. One of the main reasons for this gap is the existence of unused human resource capacity in the ARC. Thereupon, if the ARC’s staff operate in a situation where their utilised capacity increases, fixed costs per unit of service will be reduced and result in lower prices. One consequence of the high cost of rehabilitation ser- vices at the ARC was the formation of the gap between the cost of service and government tariffs. As a result of this gap, the ARC activities have been financially detri- mental. However, a more accurate costing framework may contribute to lower the costs and increase revenues for rehabilitation activities.h The highest non-personnel costs are due to "other over- head costs”; the most important of which is the build- ing leasing cost. Therefore, avoiding the leasing costs (through resources reallocation like purchasing a build- ing or integration with other rehabilitation institutions) can play an important role in the profitability of the insti- tution’s services. The rent and cost of buildings deprecia- tion in Iran is burdensome [16, 32], and so the provision of rehabilitation services in joint settings, avoiding geo- graphical dispersion, can help to reduce service provid- er’s costs and make their services more profitable. This matter emphasises the importance of resource man- agement and the production scale in the ARC. In other words, due to the low number of patients, overhead costs were divided among a few patients and this contributed significantly to the high cost of rehabilitation services. With that in mind, it is expected that an increase in the number of patients at the ARC could significantly reduce the unit cost of the rehabilitation services and make its activities profitable. This finding can be extended to all rehabilitation clinics that operate on a small scale. This study attempts to apply a more accurate method for costing rehabilitation services, in addition to sensiti- sation about the loss of rehabilitation activities for small institutions. Discussionh The literature lacks case studies of TDABC applica- tion in different rehabilitation services settings [15], which makes the cost of rehabilitation services non- transparent for providers, social insurance organisa- tions and regulatory entities. This lack of transparency challenges the pricing of these services and negatively affects resources productivity and healthcare service utilization. Following the purpose of the study, it was observed that the cost of providing the rehabilitation Table 4 Different capacities for rehabilitation services (min) The numbers in parentheses represent the share of each capacity in practical capacity Practical capacity Used capacity Unused capacity Secretary 117,390 (100%) 19,537 (16.64%) 97,853 (83.36%) General practitioner 224,640 (100%) 161,364 (71.83%) 63,276 (28.17%) Speech therapist 255,060 (100%) 69,799 (27.36%) 185,261 (72.63%) Mental health occupational therapist 272,220 (100%) 44,100 (16.20%) 228,120 (83.80%) Physical occupational therapist 234,780 (100%) 62,390 (26.57%) 172,390 (73.43%) Cashier 117,390 (100%) 16,709 (14.23%) 100,681 (85.77%) Table 4 Different capacities for rehabilitation services (min) Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 Page 6 of 8 Page 6 of 8 Page 6 of 8 services at the ARC is much higher than the govern- ment’s tariffs for these services. Although the ARC is a public institution and its losses are offset by the gov- ernment budget, it is very likely that other rehabilita- tion centers will be in a similar situation [14, 32, 33]. The calculations showed that the cost of high-quality services for speech therapy and mental health occu- pational therapy was 2.76 and 3.82 times of the tariffs of these services, respectively. This ratio was 2.39 for physical occupational therapy services. The highest share of the cost of providing rehabilitation services, especially high-quality services, is related to human resource costs. Among the various types of personnel costs, the compensation of the rehabilitation specialists has the highest share. Indeed, the reason for this high cost should be inquired in the high work less time of rehabilitation specialists. Conclusion Long-term losses for rehabilitation clin- ics will limit the future of such services and will put the welfare community at a greater disadvantage. This study aimed to show that the government tariffs for rehabili- tation services are significantly lower than the costs of providing these services. Second, most of rehabilitation services are not covered by Iranian social insurances, and tariffs are not affordable for families in need of rehabili- tation services. In addition to inspire advocacy of social insurance provision for low-income families, this study sought to suggest an improved costing method for reha- bilitation providers. Evidently, TDABC method may pro- vide a more realistic cost structure information and other valuable information, such as a realistic profit/lost over- view, unused human capacity and a basis for improved resources management for health care providers. All of this may enhance the availability and accessibility of rehabilitation services.h Healthcare is one of the services that has a high diver- sity of human resources with cost variations of more than 10:1[29]. In this regard, the use of the TDABC in this sec- tor could be effective in determining the exact contribu- tion of each type of human resources to the total cost. Studies in Iran have often considered lump sum person- nel costs regardless of the time required to provide health care services [32, 34], that is why the personnel cost makes up a large portion of total costs and makes health care services so expensive. Therefore, use of the TDABC method can be a remedy for controlling costs and price inflation in the health sector. Application of this method could improve human resources management for health- care providers and ensure affordable rehabilitation services. The research has been accompanied by a few limita- tions. One of the limitations of the research was that not all rehabilitation activities cost has been calculated. This selection of activities was based on the number of patients referred to departments. Time and money con- straints made it impossible for calculations to be made for all departments. In addition, it should be stated that all information was provided by the ARC and the accu- racy of this information has not been independently verified. Another issue is the lack of links between the cost and value of rehabilitation services for patients. Mohammadpour et al. Declarations 14. Beyranvand R, Asar FEF, Emamgholipour S, Arab M. Unit-cost calculation of delivered services based on activity based costing method compared with approved tariffs in physiotherapy department of Sina hospital. Hospital. 2016;15(2):49–58 . Consent to publication Not applicable. 17. Kaplan A, Agarwal N, Setlur N, Tan H, Niedzwiecki D, McLaughlin N, et al. Measuring the cost of care in benign prostatic hyperplasia using time- driven activity-based costing (TDABC). Healthcare. 2015. https://doi.org/ 10.1016/j.hjdsi.2014.09.007. Not applicable. Availability of data and materials y The datasets generated and analysed during the current study are not publicly available due to the confidentiality of organizational data; but are available from the corresponding author on reasonable request. 13. Cannavacciuolo L, Illario M, Ippolito A, Ponsiglione C. An activity-based costing approach for detecting inefficiencies of healthcare processes. Bus Process Manag J. 2015;21(1):55–79. Abbreviations ABC A i i b 5. Ensor T, Firdaus H, Dunlop D, Manu A, Mukti AG, ayu Puspandari D, et al. Budgeting based on need: a model to determine sub-national alloca- tion of resources for health services in Indonesia. Cost Eff Resour Alloc. 2012;10(1):1–13. ABC: Activity based costing; ARC: The Asma Rehabilitation Center; TDABC: Time-driver activity based costing; HQ: High quality service; MQ: Medium quality service. Conclusion Cost Effectiveness and Resource Allocation (2022) 20:31 Page 7 of 8 The actual value of the utility that rehabilitation ser- vices given to clients may be more or less than the esti- mated cost. Ultimately, these calculations were related to a small-scale rehabilitation center and the findings cannot be generalised to all active businesses in the rehabilitation services. Funding 11. Rakotondrajoa P, Rakotomamonjy T, Baptiste RJ, Demers L, Kileo P, Anholt M, et al. Achieving self-sustainability of service delivery in an eye care program in Madagascar using time-driven activity based costing. BMC Health Serv Res. 2020;20(1):1–9. This research conducted as a master thesis without any direct financial sup- ports. University of Social Welfare and Rehabilitation Sciences provided us with data and logistic supports. 12. Hoang HT, Pham TL, Vo TT, Nguyen PK, Doran CM, Hill PS. The costs of traumatic brain injury due to motorcycle accidents in Hanoi Vietnam. Cost Eff Resource Allocation. 2008;6(1):1–7. References 1. Neriz L, Núñez A, Ramis F. A cost management model for hospital food and nutrition in a public hospital. BMC Health Serv Res. 2014;14(1):542. 1. Neriz L, Núñez A, Ramis F. A cost management model for hospital food and nutrition in a public hospital. BMC Health Serv Res. 2014;14(1):542. 2. Demeere N, Stouthuysen K, Roodhooft F. Time-driven activity-based costing in an outpatient clinic environment: development, relevance and managerial impact. Health Policy. 2009;92(2–3):296–304. 2. Demeere N, Stouthuysen K, Roodhooft F. Time-driven activity-based costing in an outpatient clinic environment: development, relevance and managerial impact. Health Policy. 2009;92(2–3):296–304. 3. Tibor LC, Schultz SR, Menaker R, Weber BD, Ness J, Smith P, et al. Improv- ing efficiency using time-driven activity-based costing methodology. J Am Coll Radiol. 2017;14(3):353–8. 3. Tibor LC, Schultz SR, Menaker R, Weber BD, Ness J, Smith P, et al. Improv- ing efficiency using time-driven activity-based costing methodology. J Am Coll Radiol. 2017;14(3):353–8. 4. Dejnega O. Method time driven activity based costing. J Appl Econ Sci (JAES). 2011;6(15):9–15. Acknowledgements 6. Jakovljević M, Ranković A, Rančić N, Jovanović M, Ivanović M, Gajović O, et al. Radiology services costs and utilization patterns estimates in South- eastern Europe - a retrospective analysis from Serbia. Value in health regional issues. 2013;2(2):218–25. 6. Jakovljević M, Ranković A, Rančić N, Jovanović M, Ivanović M, Gajović O, et al. Radiology services costs and utilization patterns estimates in South- eastern Europe - a retrospective analysis from Serbia. Value in health regional issues. 2013;2(2):218–25. In this way, the authors appreciate the cooperation of the personnel of the Asma Rehabilitation Centre. Also, the information presented by the Deputy of Treatment and the Department of Finance of the University of Welfare and Rehabilitation Sciences has been very valuable and has contributed to this study. 7. Siguenza-Guzman L, Auquilla A, Van den Abbeele A, Cattrysse D. Using time-driven activity-based costing to identify best practices in academic libraries. J Acad Librariansh. 2016;42(3):232–46. 7. Siguenza-Guzman L, Auquilla A, Van den Abbeele A, Cattrysse D. Using time-driven activity-based costing to identify best practices in academic libraries. J Acad Librariansh. 2016;42(3):232–46. 8. Pernot E, Roodhooft F, Van den Abbeele A. Time-driven activity-based costing for inter-library services: a case study in a university. J Acad Librariansh. 2007;33(5):551–60. 8. Pernot E, Roodhooft F, Van den Abbeele A. Time-driven activity-based costing for inter-library services: a case study in a university. J Acad Librariansh. 2007;33(5):551–60. Author contributions MB and FM conceptualized and designed 338 the study. MB, FM and NA col- lected and analyzed the data. MB and SHMK wrote the manuscript. FM and NA critically reviewed the analysis and provided feedback on the manuscript. All authors read and approved the final manuscript. 9. Kaplan RS, Anderson SR. The innovation of time-driven activity-based costing. J Cost Manag. 2007;21(2):5–15. 9. Kaplan RS, Anderson SR. The innovation of time-driven activity-based costing. J Cost Manag. 2007;21(2):5–15. 10. Kaplan RS, Anderson SR. Time-driven activity-based costing: a simpler and more powerful path to higher profits. Boston: Harvard business press; 2007 10. Kaplan RS, Anderson SR. Time-driven activity-based costing: a simpler and more powerful path to higher profits. Boston: Harvard business press; 2007 Competing interests The authors report no conflict of interest concerning this study or the findings specified in this paper. 18. French KE, Albright HW, Frenzel JC, Incalcaterra JR, Rubio AC, Jones JF, et al. Measuring the value of process improvement initiatives in a preoperative assessment center using time-driven activity-based costing. Healthcare. 2013;1(3):136–42. Ethical approvals and consent to participate Consent from the participants was not required, as this study is an organi- zational study and did not use human or animal data. The financial and organisational data from Asma Rehabilitation Centre provided to researchers and they are required to ensure the data confidentiality. This research has been approved by the Research Ethics Committee IR.USWR.REC.1396.111 at the University of Social Welfare and Rehabilitation Sciences, Tehran, Iran. p 15. Campanale C, Cinquini L, Tenucci A. Time-driven activity-based costing to improve transparency and decision making in healthcare: a case study. Qual Res Account Manage. 2014;11(2):165–86. 15. Campanale C, Cinquini L, Tenucci A. Time-driven activity-based costing to improve transparency and decision making in healthcare: a case study. Qual Res Account Manage. 2014;11(2):165–86.f 16. Javid M, Hadian M, Ghaderi H, Ghaffari S, Salehi M. application of the activity-based costing method for unit-cost calculation in a hospital. Global J Health Sci. 2015;8(1):165–72. Received: 8 September 2021 Accepted: 25 June 2022 Author details 1 Student Research Community, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran. 2 Department of Social Welfare Management, School of Education Sciences and Social Welfare, Social Determinants of Health Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran 1985713834, Iran. 3 Department of Social Welfare Management, School of Education Sciences and Social Welfare, Social Welfare Management Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran. 4 University of Social Welfare and Rehabilitation Sciences, Pediatric Neurorehabilitation Research Center, Tehran, Iran. 19. Ibrahimipour H, Maleki M-R, Brown R, Gohari M, Karimi I, Dehnavieh R. A qualitative study of the difficulties in reaching sustainable universal health insurance coverage in Iran. Health Policy Plan. 2011;26(6):485–95. 19. Ibrahimipour H, Maleki M-R, Brown R, Gohari M, Karimi I, Dehnavieh R. A qualitative study of the difficulties in reaching sustainable universal health insurance coverage in Iran. Health Policy Plan. 2011;26(6):485–95. 20. Davari M, Haycox A, Walley T. The Iranian health insurance system; past experiences, present challenges and future strategies. Iran J Public Health. 2012;41(9):1–9. 20. Davari M, Haycox A, Walley T. The Iranian health insurance system; past experiences, present challenges and future strategies. Iran J Public Health. 2012;41(9):1–9. 21. Statistical Center of Iran. Iranian National Health Accounts. Tehran: 2020. Retrieved from: https://amar.org.ir/Portals/0/Files/abstract/1397/G_ Hesab_Melli_Salamat_97_V2.pdf?ver=EBYR_0680cZSRTzNnoRl2g%3d% 3d. Received: 8 September 2021 Accepted: 25 June 2022 22. Piroozi B, Moradi G, Nouri B, Bolbanabad AM, Safari H. Catastrophic health expenditure after the implementation of health sector evolution plan: a case study in the west of Iran. Int J Health Policy Manag. 2016;5(7):417. 22. Piroozi B, Moradi G, Nouri B, Bolbanabad AM, Safari H. Catastrophic health expenditure after the implementation of health sector evolution plan: a case study in the west of Iran. Int J Health Policy Manag. 2016;5(7):417. Page 8 of 8 Mohammadpour et al. Cost Effectiveness and Resource Allocation (2022) 20:31 23. Soltani S, Hafshejani AM, Salehiniya H. Trend of disability preva- lence in Iran: an evidence to improve disability data. J Res Med Sci. 2015;20(5):531–2. 23. Soltani S, Hafshejani AM, Salehiniya H. Trend of disability preva- lence in Iran: an evidence to improve disability data. J Res Med Sci. 2015;20(5):531–2. 24. Jakovljevic MM, Netz Y, Buttigieg SC, Adany R, Laaser U, Varjacic M. Popu- lation aging and migration–history and UN forecasts in the EU-28 and its east and south near neighborhood–one century perspective 1950–2050. Glob Health. 2018;14(1):1–6. 25. Basakha M. Author details Rehabilitation services in the Iranian health care and eco- nomic system (2002–2017). Archives of Rehabilitation. 2021. https://doi. org/10.32598/RJ.22.3.3131.1. g 26. Arani AA, Atashbar T, Antoun J, Bossert T. Iran’s health reform plan: meas- uring changes in equity indices. Iran J Public Health. 2018;47(3):390. 27. Everaert P, Bruggeman W, Sarens G, Anderson SR, Levant Y. Cost modeling in logistics using time-driven ABC: experiences from a wholesaler. Int J Phys Distrib Logist Manag. 2008;38(3):172–91. 28. Gruen R, Howarth A. Financial management in health services. Berkshire: Open University Press. 2005. 28. Gruen R, Howarth A. Financial management in health services. Berkshire: Open University Press. 2005. 29. Kaplan RS, Witkowski M, Abbott M, Guzman AB, Higgins LD, Meara JG, et al. Using time-driven activity-based costing to identify value improve- ment opportunities in healthcare. J Healthc Manag. 2014;59(6):399–412. 30. Tabibi J, Maleki M, Nourozi T. Computation cost price of clinical laborato- ries services in valiasr hospitals in Tehran in 1387 by using of ABC model. J Hospital. 2010;8(3):5–17. 30. Tabibi J, Maleki M, Nourozi T. Computation cost price of clinical laborato- ries services in valiasr hospitals in Tehran in 1387 by using of ABC model. J Hospital. 2010;8(3):5–17. 31. Markazi Moghaddam N, Goudarzi R, Meshkani Z. Surveying activity based costing of final units (a case study in one of the armed forces hospitals). J Hospital. 2016;15(1):41–50. 32. Janati A, Khosravi MF, Imani A, Javadzadeh A, Gharamaleki MM. Cost analysis of eye surgeries and comparison with approved governmental tariffs. Health Scope. 2017. https://doi.org/10.5812/jhealthscope.39948. f 33. Golmohammad A, EbadifardAzar F, Abutorabi A. The cost of services provided in the physiotherapy department of Shafa Yahyaian Hospital. Health Based Res. 2020;5(4):355–67. 34. Bayati M, Ahari AM, Badakhshan A, Gholipour M, Joulaei H. Cost analysis of MRI services in Iran: an application of activity based costing technique. Iran J Radiol. 2015. https://doi.org/10.5812/iranjradiol.18372v2. 34. Bayati M, Ahari AM, Badakhshan A, Gholipour M, Joulaei H. Cost analysis of MRI services in Iran: an application of activity based costing technique. Iran J Radiol. 2015. https://doi.org/10.5812/iranjradiol.18372v2. 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https://openalex.org/W3213513932	https://www.nature.com/articles/s41598-021-94422-y.pdf	English	null	Semantic segmentation of PolSAR image data using advanced deep learning model	Scientific reports	2,021	cc-by	13,939	Semantic segmentation of PolSAR image data using advanced deep learning model OPEN Rajat Garg1, Anil Kumar1, Nikunj Bansal1, Manish Prateek2 & Shashi Kumar3 Urban area mapping is an important application of remote sensing which aims at both estimation and change in land cover under the urban area. A major challenge being faced while analyzing Synthetic Aperture Radar (SAR) based remote sensing data is that there is a lot of similarity between highly vegetated urban areas and oriented urban targets with that of actual vegetation. This similarity between some urban areas and vegetation leads to misclassification of the urban area into forest cover. The present work is a precursor study for the dual-frequency L and S-band NASA-ISRO Synthetic Aperture Radar (NISAR) mission and aims at minimizing the misclassification of such highly vegetated and oriented urban targets into vegetation class with the help of deep learning. In this study, three machine learning algorithms Random Forest (RF), K-Nearest Neighbour (KNN), and Support Vector Machine (SVM) have been implemented along with a deep learning model DeepLabv3+ for semantic segmentation of Polarimetric SAR (PolSAR) data. It is a general perception that a large dataset is required for the successful implementation of any deep learning model but in the field of SAR based remote sensing, a major issue is the unavailability of a large benchmark labeled dataset for the implementation of deep learning algorithms from scratch. In current work, it has been shown that a pre-trained deep learning model DeepLabv3+ outperforms the machine learning algorithms for land use and land cover (LULC) classification task even with a small dataset using transfer learning. The highest pixel accuracy of 87.78% and overall pixel accuracy of 85.65% have been achieved with DeepLabv3+ and Random Forest performs best among the machine learning algorithms with overall pixel accuracy of 77.91% while SVM and KNN trail with an overall accuracy of 77.01% and 76.47% respectively. The highest precision of 0.9228 is recorded for the urban class for semantic segmentation task with DeepLabv3+ while machine learning algorithms SVM and RF gave comparable results with a precision of 0.8977 and 0.8958 respectively. Synthetic Aperture Radar (SAR) is a type of active sensor that generates its energy which is transmitted in the form of electromagnetic waves and then receives a part of this energy after interaction with the earth’s surface1. In SAR based remote sensing, wavelength plays a crucial role since it determines how the wave interacts with the earth’s surface. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ individual classification algorithms and the overall accuracy obtained is 88%. Another comparative study by Camargo et al.8 using SAR imagery for land-cover classification on Brazilian Savana Forest showed that machine learning algorithms SVM, RF, and Multi Layer Perceptron (MLP) give better results compared to Naïve Bayes and J48 with SVM clocking the highest overall accuracy. On contrary, a similar study done by Lapini et al.9 for the Mediterranean Forest area showed that the RF classifier achieves higher accuracy compared to the SVM classifier reason could be the imbalanced number of samples among classes. individual classification algorithms and the overall accuracy obtained is 88%. Another comparative study by Camargo et al.8 using SAR imagery for land-cover classification on Brazilian Savana Forest showed that machine learning algorithms SVM, RF, and Multi Layer Perceptron (MLP) give better results compared to Naïve Bayes and J48 with SVM clocking the highest overall accuracy. On contrary, a similar study done by Lapini et al.9 for the Mediterranean Forest area showed that the RF classifier achieves higher accuracy compared to the SVM classifier reason could be the imbalanced number of samples among classes. i g One of the core components in the image segmentation task is feature extraction. Texture descriptor matrices measure information about spatial positioning or intensity due to which they are extensively used to extract spatial features of the SAR image10. Texture analysis is generally divided into three categories: transform-domain analysis, statistical analysis, and model-based analysis11. In the transform domain analysis, wavelet transforms are used to analyze SAR images at different orientations. Because of its geometric and stochastic properties the Discrete Fourier Transform (DFT) allows for an efficient multiresolution representation of SAR images12 and the Gabor transform is a variant of Fourier transform, defined as the Fourier transform multiplied by a Gaussian function13. The commonly used statistical analysis methods include Grey-Level Co-occurrence Matrix (GLCM) and Multilevel Local Pattern Histogram (MLPH). The GLCM computes image properties related to second-order statistics like energy, entropy, contrast, homogeneity, and correlation14. The MLPH outlines the size distribu- tions of dark, bright, and homogenous patterns appearing in a moving window at various contrasts15. In the model-based analysis, the Markov Random Field (MRF) is widely used to analyze spatial information between neighboring pixels. To represent the spatial logic relationships, the conditional random field model is integrated with a multiscale region connection calculus model16. www.nature.com/scientificreports/ In the current work, we have used the Gabor filter, Median filter, Gaussian filter, and Canny edge detector to extract the SAR image features. These features combined with the RGB bands of the false-color composite image are used to train the machine learning models for the task of semantic segmentation.i g Optical multispectral remote sensing has successfully demonstrated its potential in feature identification and land use/ land cover classification17–20. The major limitation of optical remote sensing is its dependency on the top surface color of an object. The portion of the electromagnetic spectrum which is used in optical multispec- tral remote sensing is the reflection of light in the visible range (0.4–0.7 μm) and reflected infrared (0.7–3 μm). This range for optical multispectral remote sensing is limited to the top surface color information of the objects and the short wavelength that is easily blocked by the atmospheric cloud. The active imaging microwave remote sensing is carried in the 1 mm to 100 cm wavelength range of the electromagnetic spectrum, and this range is sensitive to the structural and electrical properties of an object. Synthetic aperture Radar remote sensing has the advantages of scattering-based object characterization to identify structural and electrical properties and has several other advantages in data acquisition in the darkened environment and active imaging that can acquire data for SAR-based earth observation at night time too21. Polarimetric Synthetic Aperture Radar (PolSAR) is an advanced technique of SAR remote sensing in which fully polarimetric properties of the transmitted and received electromagnetic waves are used to characterize the scattering behaviour of different objects/scatterers within a SAR resolution cell22. SAR backscatter is a coherent sum of scattering contributed by all the scatterers within a resolution cell, and the information retrieval of different scatterers within a resolution cell is not possible with single or dual-polarised data. All the four possible polarimetric combinations of PolSAR data provide necessary and required scattering information through mathematical modelling of the interaction of electromagnetic waves with different types of objects. Several polarimetric decomposition models have been developed to provide at least three scattering elements within a resolution cell23–25.hh The polarimetric feature is another important feature descriptor for PolSAR image segmentation. The polari- metric features are strongly dependent on scattering indexed values that are captured after a single bounce, double bounce, and volumetric scattering. www.nature.com/scientificreports/ The PolSAR decomposition is used to describe and classify the scattering from man-made, natural targets, and landscapes by splitting the received signal into a sum of different scattering mechanisms representing specific polarimetric signatures23,26. The KNN is one of the popular classifiers used for PolSAR image segmentation. The KNN is highly sensitive to the value of ‘K’ (number of neighbors). It is not efficient for a large volume of training data. A large number of polarimetric features can make it a lazy learner27. In Table 1, it can be observed that its training and inference time both are higher than all the other models. Overall, the KNN model is less sensitive for all the labels are shown in Fig. 1 and Fig. 2 in comparison to other models. The RF model is more robust to handle outliers and noise. The main disadvantage of the RF model is that a large number of trees and selection of other parameters can make the model too slow and may be inef- fective for real-time predictions8,28. In Table 1, it can be observed that its inference time is more than SVM and Deeplabv3+ model. From Fig. 1 and Fig. 2, it can be observed that the RF model has good accuracies for urban and ground classification but less sensitive for water and forest. Support Vector Machine (SVM) algorithm is also a nonparametric classification model, which is insensitive to the distribution of the underlying dataset. The selection of suitable kernel, optimum kernel parameters, and the relatively complex mathematics of the SVM model restricts the effectiveness. The large training data size can also affect the performance of model29. From Fig. 1 and Fig. 2, it can be observed that the SVM model providing better segmentation for the water and urban class labels due to the kernel size and its sensitivity as compared to RF and KNN model. But it is less than the results of the DeepLabv3+ model.h The Deep Neural Network (DNN) models include deep Boltzmann machines30, Convolutional Neural Networks31,32, Deep Belief Networks (DBNs)33, and Stacked Auto Encoders (SAEs)34. It has been proved that DNN models can learn higher and more abstract level of features, which can amplify the discrimination and suppress irrelevant variations in the input data35. Therefore, the framework of DNN improves the state-of-the- art in many computer vision tasks as well as in remote sensing applications36,37. Semantic segmentation of PolSAR image data using advanced deep learning model OPEN The longer is the wavelength, the higher is the penetration depth2. Also, the SAR sensor collects signals in different polarizations. By analyzing these different polarization signals, one can identify the structure of the imaged surface with good accuracy based upon the dominant scattering-type – odd bounce, even bounce, and volume scattering. Target decomposition methods are used for classifying the PolSAR data into different scattering types3. The urban land cover mapping is becoming increasingly important since rapid urbanization has caused steady degradation of urban vegetation4. So, accurate classification of urban land cover is required for better urban planning and to avoid overstressing nature. While the decomposition methods are used to generate a false-color composite image based on the backscatter values, they often misclassify some urban areas as vegeta- tion due to the diffused scattering type from oriented urban targets. This misclassification can be corrected with the help of semantic segmentation tasks using advanced machine learning algorithms. Semantic Segmentation involves assigning a class label to every pixel in the image5,6.fi g g y p g A comparative study by Gupta et al.7 using SAR imagery between different classification algorithms like texture-based, SAR observables based, probability density function (pdf) based, and color based is done and then all of these are fused using the RF classifier. The results of this fusion based RF classifier outperform the 1School of Computer Science, University of Petroleum and Energy Studies, Dehradun, Uttarakhand 248007, India. 2Dev Bhoomi Group of Institutions, Dehradun, Uttarakhand 248007, India. 3Photogrammetry and Remote Sensing Department, Indian Institute of Remote Sensing (IIRS), ISRO, 04 Kalidas Road, Dehradun, Uttarakhand 248001, India. email: anil.kumar@ddn.upes.ac.in \| https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 1. The results of performance evaluation of traditional machine learning algorithms and DeepLabv3+ algorithm. a It is considered only the impact of the training set of size N, however, there are other factors (number of features, dimensionality, etc.) that can also affect the algorithm’s time complexity42,92. b In RF, the variable V = (ntree x mtry), where ‘ntree’ is the number of trees to build and ‘mtry’ represents how many variables need to sample at each node28. In KNN, ‘D’ is the distance between the training set observation and new observation, and ‘K’ represents the number of neighbors27. The variable ‘N’ represents the number of training set size used in SVM29 and other listed algorithms. Parameter RF KNN SVM DeepLabv3+ Pixel accuracyPatch 1 74.74% 78.68% 74.44% 83.51% Pixel accuracyPatch 2 81.09% 74.31% 79.59% 87.78% Overall pixel accuracy 77.92% 76.48% 77.02% 85.65% F1 score 0.7784 0.7762 0.7646 0.8520 Precision (Urban Class) 0.8958 0.7984 0.8977 0.9228 Training time 4204 s 41,091 s 2803 s 13411 s Inference time (per scene) 3.04 s 864.94 s 0.46 s 0.31 s Algorithm complexity O Vx NlogN b O(KND)b O N2 O(N)a Table 1. The results of performance evaluation of traditional machine learning algorithms and DeepLabv3+ algorithm. a It is considered only the impact of the training set of size N, however, there are other factors (number of features, dimensionality, etc.) that can also affect the algorithm’s time complexity42,92. b In RF, the variable V = (ntree x mtry), where ‘ntree’ is the number of trees to build and ‘mtry’ represents how many variables need to sample at each node28. In KNN, ‘D’ is the distance between the training set observation and new observation, and ‘K’ represents the number of neighbors27. The variable ‘N’ represents the number of training set size used in SVM29 and other listed algorithms. Figure 1. Represents the ground truth of Houston City, Texas (patch 1) and respective segmentation results of various classifiers (a) Google Earth image of test patch 1 (b) False color composite image for G4U decomposition on UAVSAR data of a part of Houston City, Texas (USA) and the corresponding image segmentation results for (c) RF classifier, (d) KNN classifier, (e) SVM classifier, and (f) Deeplab V3 respectively. Figure 1. www.nature.com/scientificreports/ Previously, Lv et al.38 develop a DBN for land cover mapping in urban areas using the SAR data. After that, Liu et al.39 designed a Wishart DBN to preliminarily classify and adopt local spatial information to adjust the classified results. Gong et al.40 applied a Scientific Reports \| (2021) 11:15365 \| https://doi.org/10.1038/s41598-021-94422-y www.nature.com/scientificreports/ Represents the ground truth of Houston City, Texas (patch 1) and respective segmentation results of various classifiers (a) Google Earth image of test patch 1 (b) False color composite image for G4U decomposition on UAVSAR data of a part of Houston City, Texas (USA) and the corresponding image segmentation results for (c) RF classifier, (d) KNN classifier, (e) SVM classifier, and (f) Deeplab V3 respectively DNN to produce a change detection map directly from two images, which demonstrates an improvement in the detection performance compared with other traditional algorithms. Chen et al.41 proposed a Deep Convolutional Network (DCN) with sparsely connected layers for target recognition in the SAR data. This study explores some commonly used machine learning techniques along with a comparatively new semantic segmentation model DeepLabv3+42 to resolve the issue of misclassification up to a certain extent. To achieve the task of semantic segmentation, the DeepLabv3+ model uses an encoder-decoder network with Xception as a backbone which consists of convolution, pooling, and filtering layers. In this model, features are extracted from the backbone https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ Figure 2. Represents the ground truth of Houston City, Texas (patch 2) and respective segmentation results of various classifiers (a) Google Earth image of test patch 2 (b) False color composite of UAVSAR image of a part of Houston City, Texas (USA) of test patch 2 and the corresponding image segmentation results for (c) RF classifier, (d) KNN classifier, (e) SVM classifier, and (f) DeepLabv3+ respectively. Figure 2. Represents the ground truth of Houston City, Texas (patch 2) and respective segmentation results of various classifiers (a) Google Earth image of test patch 2 (b) False color composite of UAVSAR image of a part of Houston City, Texas (USA) of test patch 2 and the corresponding image segmentation results for (c) RF classifier, (d) KNN classifier, (e) SVM classifier, and (f) DeepLabv3+ respectively. network Xception and atrous convolution are used in the last stage of the backbone to control the size of the feature map40. Here, Xception has been chosen as the backbone because it performs better compared to other architectures like RESNET-101 for the benchmark datasets like ImageNet43. Overall, the DeepLabv3+ model can retrieve complex textural patterns and polarimetric informative features from PolSAR image data. A detailed comparison of the models is illustrated in Table 1. www.nature.com/scientificreports/ One of the strong limitations of the DeepLabv3+ model is its high dependency on the amount of training data, i.e., ground truth. Although this work solved this limitation to some extent with the support of the transfer learning technique.ti pp g q After the improved performance of deep learning methods in the field of computer vision, researchers from the remote sensing community have made a great effort to implement land use land cover classification using deep learning. Land use land cover maps of an area helps users and remote sensing community to understand the current landscape. It enables the monitoring of temporal dynamics of forest conversions, agricultural eco- system, surface water bodies, biomass estimation, helps to minimize the human and economic loss from floods, tsunamis, and other natural calamities. Since a large amount of data is captured by various satellite sensors on regular basis, it creates a repository of big data. To extract the information from this big data, and classify the data as per requirements, advanced deep learning models are required that must be computationally fast and cost-efficient. The objective of this work is the semantic segmentation of PolSAR data using a deep learning technique, and this work is a part of the NASA-ISRO Synthetic Aperture Radar (NISAR) mission’s L and S-band Airborne SAR Research Announcement (RA) project on Implementation of Evolutionary Computing Algorithm for Polarimetric SAR Data Processing and Classification (TEC-07)44. gi Conclusively, this study aims to create a new dataset for semantic segmentation of PolSAR images captured from Uninhabited Aerial Vehicle Synthetic Aperture Radar (UAVSAR) for Land Use Land Cover applications and effectively implement machine learning and advanced deep learning algorithms. This has been achieved using a small dataset with the help of transfer learning. Another aim of this study is to address the problem of oriented urban targets with the help of advanced machine learning techniques and DeepLabv3+ has addressed this issue quite well. Resultsh Further, area C contains a small forest as clear from the decomposed SAR image, but it has been very sparsely captured by RF and KNN algorithms while it is almost missed by the SVM algorithm as depicted in (Fig. 1c–e). However, the forest has been correctly captured by DeepLabv3+ which is visible in Fig. 1f. It is shown in Fig. 2, area D and area E correspond to oriented urban buildings which are misclassified as vegetation (green color) in the decomposed image. This affects the prediction of traditional machine learning algorithms as well and KNN is worst affected resulting in maximum misclassification of the oriented urban targets as vegetation. However, it is clear from Fig. 2f that the DeepLabv3+ can correctly map the oriented urban targets under the urban class with minimal misclassification. Many performance evaluation matrices are being used to evaluate the image segmentation task like Accu- racy, Kappa coefficient, F1 score, and so on. In this work, a pixel accuracy measure has been used to evaluate the performance of the semantic segmentation task. In addition to pixel accuracy, the F1 score has been reported for the resulting land cover maps. The Precision metric for the urban class has also been reported since one of the aims of this work is to address the issue of oriented urban targets which are generally misclassified as vegetation. Figure 3 is representing the performance measures of the implemented algorithms. It is shown in Fig. 3a for the test patch 1 or scene 1, the highest pixel accuracy of 83.51% has been achieved by the DeepLabv3+ algorithm and the KNN classifier has achieved the highest pixel accuracy of 78.68% among machine learning algorithms with both Random forest and SVM classifiers recording a pixel accuracy of below 75% as reported in Table 1. For test patch 2 or scene 2 as shown in Fig. 2b, the highest pixel accuracy of 87.78% has been achieved by the Many performance evaluation matrices are being used to evaluate the image segmentation task like Accu- racy, Kappa coefficient, F1 score, and so on. In this work, a pixel accuracy measure has been used to evaluate the performance of the semantic segmentation task. In addition to pixel accuracy, the F1 score has been reported for the resulting land cover maps. Resultsh The results of the semantic segmentation task for patch 1 and patch 2 are presented through Figs. 1 and 2 respectively where Figs. 1a and 2a presents the Google Earth images and Figs. 1b and 2b represent the false-color composite images for the selected patches. It can be observed that the DeepLabv3+ generates very smooth and Scientific Reports \| (2021) 11:15365 \| https://doi.org/10.1038/s41598-021-94422-y www.nature.com/scientificreports/ Figure 3. Represents the performance measures. (a) and (b) represents the pixel accuracy for scene 1 and scene 2 respectively; (c) represents overall pixel accuracy for both scenes/patches; (d) represents F1 score for both scenes/patches for RF, KNN, SVM, and DeepLabv3+ algorithms. Figure 3. Represents the performance measures. (a) and (b) represents the pixel accuracy for scene 1 and scene 2 respectively; (c) represents overall pixel accuracy for both scenes/patches; (d) represents F1 score for both scenes/patches for RF, KNN, SVM, and DeepLabv3+ algorithms. accurate land cover maps compared to traditional machine learning algorithms. In Fig. 1, areas A and B cor- responds to a river that falls under the water class but all the traditional algorithms have pointed a small ground patch in these two areas while the DeepLabv3+ has predicted it as water class correctly. Further, area C contains a small forest as clear from the decomposed SAR image, but it has been very sparsely captured by RF and KNN algorithms while it is almost missed by the SVM algorithm as depicted in (Fig. 1c–e). However, the forest has been correctly captured by DeepLabv3+ which is visible in Fig. 1f. It is shown in Fig. 2, area D and area E correspond to oriented urban buildings which are misclassified as vegetation (green color) in the decomposed image. This affects the prediction of traditional machine learning algorithms as well and KNN is worst affected resulting in maximum misclassification of the oriented urban targets as vegetation. However, it is clear from Fig. 2f that the DeepLabv3+ can correctly map the oriented urban targets under the urban class with minimal misclassification. Many performance evaluation matrices are being used to evaluate the image segmentation task like Accu- racy, Kappa coefficient, F1 score, and so on. In this work, a pixel accuracy measure has been used to evaluate the performance of the semantic segmentation task. In addition to pixel accuracy, the F1 score has been reported for the resulting land cover maps. Resultsh The Precision metric for the urban class has also been reported since one of the aims of this work is to address the issue of oriented urban targets which are generally misclassified as vegetation. Figure 3 is representing the performance measures of the implemented algorithms. It is shown in Fig. 3a for the test patch 1 or scene 1, the highest pixel accuracy of 83.51% has been achieved by the DeepLabv3+ algorithm and the KNN classifier has achieved the highest pixel accuracy of 78.68% among machine learning algorithms with both Random forest and SVM classifiers recording a pixel accuracy of below 75% as reported in Table 1. For test patch 2 or scene 2 as shown in Fig. 2b, the highest pixel accuracy of 87.78% has been achieved by the accurate land cover maps compared to traditional machine learning algorithms. In Fig. 1, areas A and B cor- responds to a river that falls under the water class but all the traditional algorithms have pointed a small ground patch in these two areas while the DeepLabv3+ has predicted it as water class correctly. Further, area C contains a small forest as clear from the decomposed SAR image, but it has been very sparsely captured by RF and KNN algorithms while it is almost missed by the SVM algorithm as depicted in (Fig. 1c–e). However, the forest has been correctly captured by DeepLabv3+ which is visible in Fig. 1f. It is shown in Fig. 2, area D and area E correspond to oriented urban buildings which are misclassified as vegetation (green color) in the decomposed image. This affects the prediction of traditional machine learning algorithms as well and KNN is worst affected resulting in maximum misclassification of the oriented urban targets as vegetation. However, it is clear from Fig. 2f that the DeepLabv3+ can correctly map the oriented urban targets under the urban class with minimal misclassification. f l b d l h k l k accurate land cover maps compared to traditional machine learning algorithms. In Fig. 1, areas A and B cor- responds to a river that falls under the water class but all the traditional algorithms have pointed a small ground patch in these two areas while the DeepLabv3+ has predicted it as water class correctly. www.nature.com/scientificreports/ DeepLabv3+ algorithm and the Random Forest classifier has recorded the highest pixel accuracy of 81.09% among machine learning algorithms with SVM and KNN classifiers trailing with a pixel accuracy of 79.59% and 74 31% respectively DeepLabv3+ algorithm and the Random Forest classifier has recorded the highest pixel accuracy of 81.09% among machine learning algorithms with SVM and KNN classifiers trailing with a pixel accuracy of 79.59% and 74.31% respectively.h p y The highest overall pixel accuracy of 85.51% is recorded by the DeepLabv3+ algorithm shown in Fig. 3c while the machine learning algorithms Random forest, KNN, and SVM recorded overall pixel accuracies of 77.92%, 76.48%, and 77.02% respectively. It is observed that the performance of RF is improved in the case of patch 2 that contains more of the urban region as compared to patch 1 that has a good contribution from all the classes because it fails to map the river water accurately in patch 1. Also, the performance of the KNN algorithm drops in the case of patch 2, mainly because it fails to correctly pick up the oriented urban targets and mislabel them as vegetation. In terms of the F1 score, the DeepLabv3+ recorded the highest F1 score of 0.8520 while the machine learning algorithms RF, KNN, and SVM recorded the F1 score of 0.7784, 0.7762, and 0.7646 respectively as illustrated in Fig. 3d. To evaluate the performance of all the algorithms, specifically in the case of urban targets, we also calculated the overall precision for urban class and DeepLabv3+ recorded the highest precision of 0.9228 as shown in Table 1, while all the machine learning algorithms recorded a precision value of less than 0.90 for the urban class for the same region. This shows that the DeepLabv3+ model can correctly classify the oriented urban targets which are misclassified by the decomposition algorithms. Challenges and limitations of SAR data. A radar instrument, especially SAR, has several advantages over the optical multispectral sensor. Since the SAR sensors do not depend on Sun’s illumination for Earth observation, imaging could be done day and night. The longer wavelength is an added advantage that could provide information for land/ocean features in the cloudy environment also. Apart from complements, SAR has various challenges and limitations. The main challenge in SAR data processing for land use/land cover clas- sification and other thematic applications is to ensure distortion-free backscatter image45. www.nature.com/scientificreports/ Mainly three types of distortions affect the SAR data quality: radiometric, geometric, and polarimetric distortions. Appropriate calibration approaches are implemented to minimize the effect of distortions in the SAR imagery46. Radiometric calibration is performed with internal and external calibrators to find the actual return of the radar signals from the objects present on the earth’s surface46–48. The backscatter image represents the radar cross-section obtained after scattering from all the objects within the resolution cell49–51. The concerned space agencies generally pro- vide the radiometric calibration formula to ensure the correct radar return from the different object classes in the SAR data because it is not possible for all the researchers and users to evaluate the radiometric properties with the help of active and passive calibrators. Before implementing any classification approach or modelling for thematic applications, the radiometric calibration is performed to the Single Look Complex (SLC) or intensity/ amplitude data52,53. Geometric distortions in spaceborne SAR occur mainly due to the undulating topography of the imaged surface54. Due to side-looking imaging characteristics of a SAR system, there is the possibility to occur layover, shadow, and foreshortening in SAR image during data acquisition of any hilly or undulating terrain. The geometric distortions mainly affect the SAR image in the range 51direction. The undulating terrain results in geometric distortions and affects the radiometric quality of the SAR data. The precise radiometric calibration of the SAR data requires the angle of incidence information of the incident electromagnetic waves transmitted by the SAR system on the terrain surface. Generally, the angle of incidence information is provided with SAR metadata, but it will be effective only if the topography of the surface is flat. If the terrain is undulating, then the actual angle of incidence will be different from the angle provided with SAR metadata55,56. To find the actual incidence angle for radiometric calibration and to minimize the geometric distortions from the SAR data, high-resolution digital elevation models are required54,57. The side-looking property of the SAR sensor not only gives geometric distortions in the undulating terrain but this also creates slant-range ambiguity50. Due to slant- range ambiguity, the actual shape and size of the ground targets could not be represented in the SAR imagery. The slant to ground range conversion of the data helps in providing actual ground information to the features imaged by the SAR system. Resultsh The Precision metric for the urban class has also been reported since one of the aims of this work is to address the issue of oriented urban targets which are generally misclassified as vegetation. i Figure 3 is representing the performance measures of the implemented algorithms. It is shown in Fig. 3a for the test patch 1 or scene 1, the highest pixel accuracy of 83.51% has been achieved by the DeepLabv3+ algorithm and the KNN classifier has achieved the highest pixel accuracy of 78.68% among machine learning algorithms with both Random forest and SVM classifiers recording a pixel accuracy of below 75% as reported in Table 1. For test patch 2 or scene 2 as shown in Fig. 2b, the highest pixel accuracy of 87.78% has been achieved by the Scientific Reports \| (2021) 11:15365 \| https://doi.org/10.1038/s41598-021-94422-y www.nature.com/scientificreports/ Discussion Th The present study provides an extensive analysis of popular machine learning classifiers Random Forest, K-Near- est Neighbor, and Support Vector Machine and deep learning model DeepLabv3+ for semantic segmentation of Uninhabited Aerial Vehicle Synthetic Aperture Radar (UAVSAR) data captured on a part of Houston City, Texas (USA). This fully polarimetric data with a high resolution of 2 m provides concrete information for the train- ing of machine learning models. Another advantage of using the UAVSAR dataset is that pre-processing steps like calibration and multi-looking are not required to be performed since the data available on the NASA-JPL platform is already radiometrically calibrated and multi-looked. As mentioned earlier that the major hindrance in the large-scale accurate land cover mapping is the unavailability of a common benchmark dataset in a suf- ficient amount that could be used for training the deep learning model. Even if the data is available, the ground truth is unavailable most of the time and it is not possible to validate the results obtained without the availability of proper ground truth learning to the limitation of its usage. Also, labeling a large dataset for training a deep learning model from scratch is a very time-consuming and cumbersome task that can take up to hundreds of man-hours. This issue could be overcome with help of the transfer learning method by using a small dataset as shown in this work. The results obtained by the transfer learning method using a pre-trained DeepLabv3+ model are better than those obtained from the popular traditional machine learning algorithms. p p g g Apart from the smooth overall image segmentation results, the deep learning model is also able to correctly classify the oriented-urban targets while the traditional machine learning algorithms failed to pick them up and misclassified them into vegetation class. This is because deep neural networks can fetch textural features better as compared to traditional algorithms. Upon close observation of the PolSAR data and the false color-composite images, in particular, it can be observed that although the color of oriented urban targets is similar to that of vegetation/forest region, they are texturally different from vegetated areas which are more smooth in terms of image, unlike urban features which have patterns due to the streets running between the urban blocks. www.nature.com/scientificreports/ The precise terrain correction algorithms are implemented with external high-reso- lution DEMs for the minimization of radiometric and geometric distortions from the SAR image58,59. The use of external DEM also helps in creating a simulation-based layover-shadow mask for the orthorectified SAR data of the undulating terrain60,61. The layover-shadow mask provides detailed information for the undulating terrain’s portion affected by the geometric distortions due to layover and shadow effect. The radiometric normalisation and Orthorectification process successfully minimize the radiometric and geometric distortions from the SAR data54. To retrieve all the scattering information contributed by different objects in a small area, fully polarimet- ric quad-pol data are used because retrieval of all the scattering elements within a resolution cell is mathemati- cally and practically rigorous with single or dual polarised SAR data62,63. The remote sensing community has widely used the fully polarimetric quad-pol SAR data to implement model-based decomposition approaches in the retrieval of scattering parameters64–66. The accuracy in polarimetric SAR model-based scattering retrieval is affected by the polarimetric distortion of the SAR data67,68. The polarimetric distortions are cross talk, channel imbalance, and Faraday rotation69,70. Out of these three PolSAR distortions, Faraday rotation is highly effec- tive in low-frequency spaceborne SAR data and this distortion could be removed by implementing appropriate approach71. Several algorithms have been developed to minimize the effect of cross talk and channel imbalance from the PolSAR data72. After minimising radiometric geometric and polarimetric distortions the scattering retrieved from the mathematical modelling and decomposition of the SAR data shows appropriate scattering for different objects46,73,74. The smooth surfaces like barren land and water body could be characterised as sur- face scatterers with a difference in the surface scattering power24. The forest vegetation shows a dominance of volume scattering in the PolSAR decomposition and the urban settlements show a very high amount of double- scattering power26,75. Since, distortion-free polarimetric SAR data helps in identifying the scattering of different features of the imaged area, hence the implementation of classification approaches on these data gives a fruitful https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ result with reliable accuracy. www.nature.com/scientificreports/ This work implemented the land use land cover classification on fully polarimetric quad-pol UAVSAR data after implementing G4U decomposition that targeted the vegetation covers as a forest class and the urban class is representing by the combination of dense and sparse building areas that includes the rural areas, industrial plants and inner-city areas with high rise buildings. The achieved accuracy of urban class using DeepLabv3+ model is 92.28% that is a major significance of this work. Discussion Th The deep learning encoder based on Xception architecture can highlight this dissimilarity much better compared to traditional manual feature extraction methods resulting in the better performance of the deep learning model compared to traditional machine learning algorithms. The transfer learning method has a drawback of nega- tive transfer because of the difference in the datasets used while training the model and the dataset used while transfer learning. It works best only when the two datasets are similar enough. Since the pre-trained model used in this study is trained on an ImageNet dataset that consists of real-life camera images, there could be some negative transfer due to the dissimilarity of the ImageNet dataset from the PolSAR data. But currently, there is no measure to determine the same. In the future, with the availability of a large amount of open-source PolSAR data, the training dataset can be increased to train a deep learning model from scratch and possibly achieve even higher accurate results. Conclusions Land Use Land Cover mapping is important to address the ever-increasing environmental concerns like climate change, soil degradation, deforestation, and water pollution. But this need for highly accurate land cover maps is hindered by the unavailability of a common benchmark labeled dataset used to train the deep learning models. The present work demonstrated and proved that the transfer learning associated with deep learning models can be effectively used to generate accurate land cover maps and their classification even with a small training dataset. The transfer learning algorithms not only address the issue of unavailability of a large amount of labeled dataset in the field of SAR based remote sensing but also takes lesser computational time and resources compared to training a deep learning model from scratch. In both the targeted test patches, DeepLabv3+ clocked the high- est pixel accuracy of 83.51% and 87.78% while among the machine learning algorithms RF classifier achieved the highest overall pixel accuracy of 77.92% for the segmentation task. It has also been validated through these experiments that the KNN classifier is rightly known as the lazy learner since it has taken a maximum training time of around 41,091 s for the overall training dataset and inference time per scene of around 864.94 s. One of the focused areas of this work was to reduce the misclassification of oriented urban targets as vegetation. This could be successfully achieved by using DeepLabv3+, an advanced deep learning model. It can be noted that the current state-of-the-art DeepLabv3+ model works better than the traditional machine learning methods even with small datasets contrary to the popular belief that large datasets are required for deep learning models to achieve better results as compared to traditional machine learning algorithms. In the future, this work can be extended to training and validating the DeepLabv3+ model from scratch subject to the availability of a common benchmark dataset in a sufficient amount. Methods D t T Data. The data used in this research is taken from the open access platform of NASA-JPL76. It’s fully polari- metric L-band data from airborne sensor UAVSAR which has a resolution of 2 m. The target area of Houston City shown in Fig. 4 has been chosen as it provides a variety of land cover features. Houston is a large metropolis city in the state of Texas, USA. Most of its area consists of nearly level, clayey and loamy, prairie soils. The L-band Uninhabited Aerial Vehicle Synthetic Aperture Radar data was used to test the potential of machine learning algorithms for semantic segmentation tasks. A part of the Houston metropolis was imaged by the UAVSAR fully https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ w.nature.com/scientificreports/ Figure 4. Represents the location of the study area compiled using ArcGIS v10.6 (URL: https://desktop.arcgis. com/en/arcmap/) (a) the boundary of Texas state (obtained from URL: https://gis-txdot.opendata.arcgis.com/) (b) the G4U decomposition-based false color composite image of UAVSAR data for part of Houston city, Texas (USA) generated using an open source remote sensing tool PolSARPro v6.0 (open access at URL: https://step. esa.int/main/download/polsarpro-v6-0-biomass-edition-toolbox-download/). Table 2. Details of UAVSAR data and instrument93. SAR system parameters Details Platform Airborne Flight Uninhabited Aerial Vehicle Synthetic Aperture Radar (UAVSAR) Date of acquisition 2 Sep 2017 Acquisition mode PolSAR Frequency 1.257 GHz Wavelength 23.84035 cm Bandwidth 80 MHz Pulse duration 40 µs Transmit power 3.1 kW Look angle range 25°–65° Data type Ground Range Projected (equiangular) and Multi-looked Data (GRD) Polarisation Fully polarimetric quad-pol (HH + HV + VH + VV) Swath width 16 km Range resolution (m) 4.99 m (MLC product) Azimuth resolution (m) 7.2 m (MLC product) Figure 4. Represents the location of the study area compiled using ArcGIS v10.6 (URL: https://desktop.arcgis. com/en/arcmap/) (a) the boundary of Texas state (obtained from URL: https://gis-txdot.opendata.arcgis.com/) (b) the G4U decomposition-based false color composite image of UAVSAR data for part of Houston city, Texas (USA) generated using an open source remote sensing tool PolSARPro v6.0 (open access at URL: https://step. esa.int/main/download/polsarpro-v6-0-biomass-edition-toolbox-download/). SAR system parameters Details Figure 4. Represents the location of the study area compiled using ArcGIS v10.6 (URL: https://desktop.arcgis. com/en/arcmap/) (a) the boundary of Texas state (obtained from URL: https://gis-txdot.opendata.arcgis.com/) (b) the G4U decomposition-based false color composite image of UAVSAR data for part of Houston city, Texas (USA) generated using an open source remote sensing tool PolSARPro v6.0 (open access at URL: https://step. esa.int/main/download/polsarpro-v6-0-biomass-edition-toolbox-download/). www.nature.com/scientificreports/ In the implementation of Canny edge detector81,82, firstly the image undergoes Gaussian filtering and then i , ( ≤, y ≤)i In the implementation of Canny edge detector81,82, firstly the image undergoes Gaussian filtering and then Gradient magnitude and direction is calculated using below Eq. (4) i yi In the implementation of Canny edge detector81,82, firstly the image undergoes Gaussian filtering and then Gradient magnitude and direction is calculated using below Eq. (4) In the implementation of Canny edge detector , , firstly the image undergoes Gaussian filtering and then Gradient magnitude and direction is calculated using below Eq. (4) In the implementation of Canny edge detector , firstly the image undergoes Gauss Gradient magnitude and direction is calculated using below Eq. (4) p y g ,i y Gradient magnitude and direction is calculated using below Eq. (4) (4) G i, j = G2 X i, j + G2 Y i, j , θ i, j = arctanGx x, y Gy x, y (4) where Gx i, j and Gy i, j are the partial-derivative at point i, j in x-direction and y-direction respectively. Machine learning methods. Machine Learning is a process of enabling a computer to take independent, intelligent decisions with the help of supervised or unsupervised learning. The commonly used machine learn- ing algorithms for classification tasks are Random Forest, K Nearest Neighbour, and Support Vector Machine.ii Machine learning methods. Machine Learning is a process of enabling a computer to take independent, intelligent decisions with the help of supervised or unsupervised learning. The commonly used machine learn- ing algorithms for classification tasks are Random Forest, K Nearest Neighbour, and Support Vector Machine. d f l fi fi d b 28 b d d 83 i Random forest classifier was first proposed by Breiman28 in 2001. It is based on Bagging predictors83 pro- posed by Breiman himself in 1996. In random forests, all the trees work independently of each other, and thus, the algorithm can be run in parallel. Random Forest classifier is very robust towards outliers and noise but it requires a large amount of training dataset. Assuming a dataset {r1, r2,…, rn} ∈ RL×n, where L is the number of features and n is the number of samples. ri represents the position of sample i in the space RL×n, while z donates the relationship between ri and rj, where z ∈ Z = {1,-1}. www.nature.com/scientificreports/ polarimetric quad-pol SAR data. Figure 4b shows a false-color composite image of G4U decomposition-based output for the study area. The green color highlights the features responsible for volume scattering. Double- bounce or even-bounce scatters which contribute toward high backscatter are shown in red color and the surface scattering or odd-bounce scattering is represented in blue color. The details about the UAVSAR instrument and the data used are mentioned in Table 2. Methodology. The UAVSAR Quad-Pol Ortho-Rectifying data is provided as the covariance matrix (C3) which is converted to a coherency matrix (T3). Generally, the SAR dataset is influenced by speckle noise which creates problems in object detection and classification. To minimize speckle from the PolSAR data various spe- cialized filters are used which are mainly developed to minimize the speckle effect from polarimetric SAR data. Several filters have been developed for speckle filtering of PolSAR data where the Lee filter is one of the most famous and effective speckle filters which is used in this work for polarimetric speckle filtering of UAVSAR data. After speckle-filtering, PolSAR decomposition is applied to generate a false-color composite image which is used to create a training dataset for our ML models after breaking it into smaller patches. One of the first widely accepted model-based decompositions was proposed by Freeman and Durden63. This model decomposed the SAR backscatter among three different scattering types namely odd-bounce scattering, double-bounce scatter- ing, and volume scattering. Later, an improved model with an additional scattering type, Helix scattering was proposed by Yamaguchi77. Based on Yamaguchi decomposition, Singh et al.78 proposed a general 4-component decomposition method with a unitary transformation that makes use of full polarimetric information in decom- position. In previous models, the T13 component of the Coherency matrix was not being used but this method allowed the usage of the same. It also included an extended volume scattering model which discriminates vol- ume scattering between dihedral and dipole scattering structures caused by the cross-polarized HV component. This lead to an enhanced double-bounce scattering from urban targets compared to the existing model-based decompositions. This is the reason why the G4U decomposition method is used in the present work. ph y p p Four component decomposition is expressed in Eq. (1) h Four component decomposition is expressed in Eq. www.nature.com/scientificreports/ (1) (1) < [T] >= Ps[T]s + Pd[T]d + Pv[T]v + Pc[T]helix (1) < [T] >= Ps[T]s + Pd[T]d + Pv[T]v + Pc[T]helix where Ps, Pd, Pv, and Pc are scattering powers to be determined. [T]s, [T]d, [T]v, and [T]helix are expansion matrices corresponding to surface, double-bounce, and helix scattering respectively.h This false-color composite image is sub-divided into smaller chunks of 500 * 500 pixels and 20 patches are labeled using the Gimp tool to create a dataset for training and testing the machine learning algorithms and the DeepLabv3+ model. From these 20 patches, 18 patches were used for training while 2 patches were kept for testing and validation. After generating the required dataset, manual feature extraction is carried out for the implementation of machine learning models by using Gabor filter, Gaussian filter, Canny edge detector, and Median filter while in the case of DeepLabv3+ feature extraction is automatically carried out by the Xception based encoder. The complete workflow adopted for the task of semantic segmentation is presented in Fig. 5 and the google image, decomposed false-color composite, and the corresponding annotated label for the test patches is shown in Fig. 6. The details about the training and working of different algorithms are discussed ahead separately. Th G b filt i t d b E (2) hi h i i ll i b G b 79 gh g gf g The Gabor filter is represented by Eq. (2) which was originally given by Gabor79. (2) g x, y, T, ∅ = hx(x; T, ∅) · hy y; ∅ = exp x2 ∅ 2σ 2x cos 2πx∅ T · exp −y2 ∅ 2σ 2y (2) where hx—Bandpass filter, hy—Gaussian filter. The Gaussian filter80 is represented by Eq. (3) (3) G x, y = 1 √ 2πσ exp − x2 + y2 /2σ 2 (3) where σ 2 is the variance of Gaussian filter, and l(−l ≤x, y ≤l) is the size of the filter kernel. In the implementation of Canny edge detector81,82, firstly the image undergoes Gaussian filtering and then Gradient magnitude and direction is calculated using below Eq. (4) where σ 2 is the variance of Gaussian filter, and l(−l ≤x, y ≤l) is the size of the filter kernel. Methods D t T Table 2. Details of UAVSAR data and instrument93. SAR system parameters Details Platform Airborne Flight Uninhabited Aerial Vehicle Synthetic Aperture Radar (UAVSAR) Date of acquisition 2 Sep 2017 Acquisition mode PolSAR Frequency 1.257 GHz Wavelength 23.84035 cm Bandwidth 80 MHz Pulse duration 40 µs Transmit power 3.1 kW Look angle range 25°–65° Data type Ground Range Projected (equiangular) and Multi-looked Data (GRD) Polarisation Fully polarimetric quad-pol (HH + HV + VH + VV) Swath width 16 km Range resolution (m) 4.99 m (MLC product) Azimuth resolution (m) 7.2 m (MLC product) Table 2. Details of UAVSAR data and instrument93. Table 2. Details of UAVSAR data and instrument93. https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ If ri and rj belong to the same class, then z = 1, and if ri and rj belong to different classes, then z = −1. For the mth tree, the ensemble is fp(r) = f(r,θm), and a tree f(r,θm) Scientific Reports \| (2021) 11:15365 \| https://doi.org/10.1038/s41598-021-94422-y www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Represents the work flow of the methodology adopted for image segmentation using traditional machine learning algorithms and DeepLabv3+ model. Figure 5. Represents the work flow of the methodology adopted for image segmentation using traditional machine learning algorithms and DeepLabv3+ model. Figure 5. Represents the work flow of the methodology adopted for image segmentation using traditional machine learning algorithms and DeepLabv3+ model. grows along with the training set and the random vector θm, which captures the various stochastic elements of the tree. {θm} are independent and identically distributed84.hi grows along with the training set and the random vector θm, which captures the various stochastic elements of the tree. {θm} are independent and identically distributed84. The estimated probability for predicting class z for a sample is defined in Eq. (5), { m} p y The estimated probability for predicting class z for a sample is defined in Eq. (5), { m} p y The estimated probability for predicting class z for a sample is defin (5) P(z\|r) = 1 M M m=0 Pm(z\|r) (5) where Pm(z\|r) is the estimated density of the class labels of the mth tree and M is the number of trees in the forest. The decision function of the forest is defined in Eq. (6) where Pm(z\|r) is the estimated density of the class labels of the mth tree and M is the number of trees in the forest. The decision function of the forest is defined in Eq. (6) https://doi.org/10.1038/s41598-021-94422-y https://doi.org/10.1038/s41598-021-94422-y https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ Figure 6. Represents the ground truth image, false-color composite image, and labeled image of the patches of Houston City, Texas(USA) (a) Google Earth image, (b) the false-color composite image, and (c) Ground truth label for Patch1, (d) Google Earth image, (e) the false-color composite image of G4U decomposition, and (f) ground truth label for Patch2. Figure 6. C(r) = arg max j∈Z P(j\|r) (6) The margin function for a random forest is defined in Eq. (7) The margin function for a random forest is defined in Eq. (7) (7) ml(r, z) = P(z\|r) −max j∈Z Pm(j\|r) (7) where j ≠ z and if ml(r,z) > 0, we can obtain a correct result. K-Nearest Neighbor has proved to be a powerful nonparametric classifier27. As per the Nearest Neighbor rule, if a set of n pairs (x1,θ1),…., (xn, θn) is considered, where the xi’s take values in a metric space X upon which is defined a metric d, and the θi’s take values in the set {1, 2,…., M}. Every value of the θ set is considered as the index of the category to which ith individual belongs, and every corresponding xi is the outcome of the set of measurements made upon that individual. Given a new pair (x, θ), where x is the measurable quantity and θ needs to be estimated with the help of information available from the set of correctly classified points. Then x’ ∈ {x1, x2,…., xn} is called the nearest neighbor to x if it satisfies the Eq. (8), (8) min d(xi, x) = d(x ′ n, x) i = 1, 2, . . . , n. min d(xi, x) = d(x ′ n, x) i = 1, 2, . . . , n. (8) The Nearest Neighbor rule predicts that x belongs to the category θn′ of its nearest neighbor xn′. A mistake occurs if θn′ ≠ θ. The Nearest Neighbor rule utilizes only the classification of the nearest neighbor. The n-1 remaining classification θi are ignored.ii The Nearest Neighbor rule predicts that x belongs to the category θn′ of its nearest neighbor xn′. A mistake occurs if θn′ ≠ θ. The Nearest Neighbor rule utilizes only the classification of the nearest neighbor. The n-1 remaining classification θi are ignored.ii gi i g Support Vector Machine classifier acts as a parametric classifier when used as a linear SVM while advanced SVMs used to classify complex non-linear data are non-parametric. The complex dataset can be projected as linear in a high-dimensional feature space using kernels like radial basis function (RBF), but the computation is not carried out in that high dimensional feature space. By using kernels, all necessary computations are per- formed directly in the input space29. There are three types of kernels mainly used with non-parametric SVMs – polynomial kernel, RBF kernel, and sigmoid kernel as represented by Eqs. (9), (10), and (11). p y Polynomial Kernel (k), www.nature.com/scientificreports/ Represents the ground truth image, false-color composite image, and labeled image of the patches of Houston City, Texas(USA) (a) Google Earth image, (b) the false-color composite image, and (c) Ground truth label for Patch1, (d) Google Earth image, (e) the false-color composite image of G4U decomposition, and (f) ground truth label for Patch2. The margin function for a random forest is defined in Eq. (7) l l l (k) Polynomial Kernel (k), (9) k (x, y) = (x · y)d k (x, y) = (x · y)d (9) https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ Figure 7. Encoder-Decoder based structure of DeepLabv3+42. Figure 7. Encoder-Decoder based structure of DeepLabv3+42. Figure 7. Encoder-Decoder based structure of DeepLabv3+42. corresponding to a map Φ in the space spanning all products of exactly d dimensions of RN. RBF Kernel (k), corresponding to a map Φ in the space spanning all products of exactly d dimensions of RN. RBF Kernel (k), (10) k x, y = exp − x −y 2/ 2σ2 (10) where 1/2σ2 = γ and γ is the free parameter. Sigmoid Kernel (k), where 1/2σ2 = γ and γ is the free parameter. Sigmoid Kernel (k), (11) k x, y = tanh κ x · y + θ k x, y = tanh κ x · y + θ (11) where κ is the gain and θ is the offset value. where κ is the gain and θ is the offset value. Deep learning methods. In Deep Learning, there are different approaches to implement Semantic Seg- mentation. The most commonly used is based on Fully Convolutional Networks (FCN). Unlike classical CNN, which has fixed fully connected networks, FCN has only convolution and pooling layers that allow it to work with arbitrarily sized inputs85. y p A general Semantic Segmentation architecture consists of an encoder-decoder network. An encoder network can be a classification network like VGG/RESNET/Xception. A decoder network is used to project the low- resolution result of the encoder network to a high-resolution pixel space by upsampling. An encoder-decoder network has been successfully used for the semantic segmentation task86–88. DeepLabv3+ DeepLabv3+ is an extension of the DeepLabv3 model of the DeepLab series. The overall encoder-decoder struc- ture of DeepLabv3+ is represented in Fig. 7. The function of the encoder module is to encode the multi-scale contextual information with help of atrous convolutions, while the decoder module refines the image segmen- tation results along the object boundaries. The DeepLabv3+ uses a modified Xception model as an encoder as shown in Fig. 8. The earlier DCNN’s had a limitation that the repeated use of max-pooling and striding at every step of these networks, significantly reduced the spatial resolution of the output feature maps88. To overcome this problem, atrous convolution, which was originally developed for computing undecimated wavelet transform89, is used. It allows the computation of responses at any desired resolution88. Convolution operation can be defined with the help of three different parameters—kernel size, stride, and padding while an atrous convolution also known as dilated convolution has an additional parameter called dilation rate (r) which is the spacing between Scientific Reports \| (2021) 11:15365 \| https://doi.org/10.1038/s41598-021-94422-y www.nature.com/scientificreports/ Figure 8. The modified Xception architecture used in DeepLabv3+: (1) has higher number of layers, (2) all max pooling layers are replaced by depthwise seperable convolutions with striding and (3) extra batch normalization and ReLU are added after each 3 × 3 depthwise convolution42. Figure 8. The modified Xception architecture used in DeepLabv3+: (1) has higher number of layers, (2) all max pooling layers are replaced by depthwise seperable convolutions with striding and (3) extra batch normalization and ReLU are added after each 3 × 3 depthwise convolution42. the kernel values. If r = 1, this special case of atrous convolution becomes equivalent to normal convolution. For a two dimensional signal, for every location i on the feature map y and the convolution filter w, the atrous convolution is applied over the input feature map x as per the Eq. (12)42 (12) y[i] = k x[i + r.k]w[k] (12) where r = atrous rate, it determines the stride with which the input is sampled. l ll f d fi ld f h l b Atrous convolution allows for a wider field of view at the same computational cost. A comparison between normal convolution and atrous convolution is presented in Fig. References 1. Elachi, C., Bicknell, T., Jordan, R. L. & Wu, C. Spaceborne synthetic-aperture imaging radars: applications, techniques, and tech- nology. Proc. IEEE 70, 1174–1209 (1982). 2. Singh, A., Meena, G. K., Kumar, S. & Gaurav, K. Evaluation of the penetration depth of L- and S-band (NISAR mission) microwave SAR signals into ground. In: 2019 URSI Asia-Pacific Radio Science Conference (AP-RASC) 1 (2019). https://doi.org/10.23919/ URSIAP-RASC.2019.8738217.i 3. van Zyl, J. J. Unsupervised classification of scattering behavior using radar polarimetry data. IEEE Trans. Geosci. Remote Sens. 27 36–45 (1989). 4. Gomez-Chova, L. et al. Urban monitoring using multi-temporal SAR and multi-spectral data. Pattern Recognit. Lett. 27, 234–243 (2006). 5. Khoshboresh-Masouleh, M., Alidoost, F. & Arefi, H. Multiscale building segmentation based on deep learning for remote sensing RGB images from different sensors. J. Appl. Remote Sens. 14, 1–21 (2020).i f 6. Wang, X., Cao, Z., Cui, Z., Liu, N. & Pi, Y. PolSAR image classification based on deep polarimetric feature and contextual informa- tion. J. Appl. Remote Sens. 13, 1–17 (2019).fii 7. Gupta, S., Singh, D., Singh, K. P. & Kumar, S. An efficient use of random forest technique for SAR data classification. In: 2015 IEEE International Geoscience and Remote Sensing Symposium (IGARSS), pp. 3286–3289 (2015). https://doi.org/10.1109/IGARSS. 2015.7326520. 8. Camargo, F. F., Sano, E. E., Almeida, C. M., Mura, J. C. & Almeida, T. A comparative assessment of machine-learning techniques for land use and land cover classification of the brazilian tropical savanna using ALOS-2/PALSAR-2 polarimetric images. Remote Sens. 11, 1600 (2019).i 9. Lapini, A. et al. Comparison of machine learning methods applied to SAR images for forest classification in mediterranean areas Remote Sens. 12, 369 (2020).i ( ) 10. Geng, J., Wang, H., Fan, J. & Ma, X. Deep supervised and contractive neural network for SAR image classification. IEEE Trans. Geosci. Remote Sens. 55, 2442–2459 (2017). 1. He, C., Zhuo, T., Zhao, S., Yin, S. & Chen, D. Particle filter sample texton feature for SAR image classification. IEEE Geosci. Remote Sens. Lett. 12, 1141–1145 (2015). 2. Planins̆ic̆, P., Singh, J. & Gleich, D. SAR Image categorization using parametric and nonparametric approaches within a dual tree CWT. IEEE Geosci. Remote Sens. Lett. 11, 1757–1761 (2014).i 3. Jia, S., Shen, L. & Li, Q. Gabor feature-based collaborative representation for hyperspectral imagery classification. IEEE Trans Geosci. Remote Sens. 53, 1118–1129 (2015). 14. De-yong, H., Xiao-juan, L., Wen-ji, Z. & Hui-li, G. www.nature.com/scientificreports/ It has been reported that by using depthwise separable convolution, the computational complexity in terms of Multiply-Adds is reduced by 33% to 41% while maintaining a similar performance which results in improved speed and accuracy by adapting Xception as a backbone90,91.h y y g The Xception model used as a backbone architecture in the current work has 14 blocks whose details are given in the Appendix. To better understand the working of this deep learning model, a visualization of features maps corresponding to the first hidden layer of each block has been generated. Figure 10 shows the feature maps extracted from Block 1, Block 7, and Block 14 respectively. The features extracted from all 14 blocks are given in the Appendix. It can be noted that the initial layers work on simple and coarser features while the higher layers work on finer details.h i This model has implemented using the python programming language, the supporting libraries like numpy, scipy, sklearn, opencv, pandas, and Tensorflow framework that executed using Anaconda (version 4.9.2) on a workstation with NVIDIA Quadro P4000 GPU and 48 GB RAM. It is easy to implement transfer learning using the TensorFlow in Python with a pre-trained model available on the Github page of DeepLabv3+42. Since the pre-trained model is trained on the ImageNet dataset which has a much higher number of classes compared to our dataset, few changes have been made in the original DeepLabv3+ code for implementing this model using transfer learning on our custom dataset as discussed in the Appendix. Received: 13 February 2021; Accepted: 6 July 2021 Received: 13 February 2021; Accepted: 6 July 2021 References Texture analysis and its application for single-band SAR thematic information extraction. In: IGARSS 2008 - 2008 IEEE International Geoscience and Remote Sensing Symposium, vol. 2, pp. II-935–II-938 (2008).i ( ) 5. Dai, D., Yang, W. & Sun, H. Multilevel local pattern histogram for SAR image classification. IEEE Geosci. Remote Sens. Lett. 8 225–229 (2011).ii 225–229 (2011). 16. Su, X., He, C., Feng, Q., Deng, X. & Sun, H. A Supervised classification method based on conditional random fields with multiscale i ti l l d l f SAR i IEEE G i R t S L tt 8 497 501 (2011) 6. Su, X., He, C., Feng, Q., Deng, X. & Sun, H. A Supervised classification method based on conditional random fields with multiscale region connection calculus model for SAR image. IEEE Geosci. Remote Sens. Lett. 8, 497–501 (2011). g g ( ) 17. Chust, G., Ducrot, D. & Pretus, J. L. L. Land cover discrimination potential of radar multitemporal series and optical multispectral images in a Mediterranean cultural landscape. Int. J. Remote Sens. 25, 3513–3528 (2004). g p ( ) 8. Dibs, H., Hasab, H. A., Al-Rifaie, J. K. & Al-Ansari, N. An optimal approach for land-use/land-cover mapping by integration and fusion of multispectral landsat OLI images: case study in Baghdad. Iraq. Water Air Soil Pollut. 231, 488 (2020). p g y g q 19. Li, X., Ling, F., Foody, G. M. & Du, Y. A superresolution land-cover change detection method using remotely sensed images with different spatial resolutions. IEEE Trans. Geosci. Remote Sens. 54, 3822–3841 (2016).i f p 20. Xu, Y. et al. Advanced multi-sensor optical remote sensing for urban land use and land cover classification: outcome of th IEEE GRSS data fusion contest. IEEE J Sel. Top. Appl. Earth Obs. Remote Sens. 12, 1709–1724 (2019). te Facility. What is SAR? (2021). https://asf.alaska.edu/informatio 22. Kumar, S., Garg, R. D., Govil, H. & Kushwaha, S. P. S. PolSAR-decomposition-based extended water cloud mode aboveground biomass estimation. Remote Sens. 11, 1–27 (2019). g 3. Cloude, S. R. & Pottier, E. A review of target decomposition theorems in radar polarimetry. IEEE Trans. Geosci. Remote Sens. 34 498–518 (1996). 24. M E, B. P. & Kumar, S. PolInSAR decorrelation-based decomposition modelling of spaceborne multifrequency SAR data. Int. J. Remote Sens. 42, 1398–1419 (2021). 25. Yamaguchi, Y., Yajima, Y. & Yamada, H. A four-component decomposition of POLSAR images based on the coherency matrix. IEEE Geosci. DeepLabv3+ 9.f For capturing the contextual information at multiple scales, parallel atrous convolution with different rates which are known as Atrous Spatial Pyramid Pooling (ASPP) was integrated in DeepLabv3. The useful semantic information is present within the last feature map but the information related to object boundaries is missed due Scientific Reports \| (2021) 11:15365 \| https://doi.org/10.1038/s41598-021-94422-y www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 9. Represents the typical and atrous convolution (a) a typical convolution of kernel size = 3, stride = 1 and padding is used to retain the input image size; (b) an atrous convolution which has an additional parameter called dilation rate which deletes every alternate row and column. Figure 9. Represents the typical and atrous convolution (a) a typical convolution of kernel size = 3, stride = 1 and padding is used to retain the input image size; (b) an atrous convolution which has an additional parameter called dilation rate which deletes every alternate row and column. Figure 10. Represents the feature maps extracted from 1st hidden layer of Block1, Block7 and Block14. Feature Maps from all 14 Blocks are presented in Appendix. Figure 10. Represents the feature maps extracted from 1st hidden layer of Block1, Block7 and Block14. Feature Maps from all 14 Blocks are presented in Appendix. Figure 10. Represents the feature maps extracted from 1st hidden layer of Block1, Block7 and Block14. Feature Maps from all 14 Blocks are presented in Appendix. to the convolution operation with striding. This problem can be resolved by applying the atrous convolution to extract denser feature maps but this becomes computationally expensive and inefficient. Whereas in the case of encoder-decoder models no features are dilated in the encoder path and they gradually recover the sharp object boundaries in the decoder path leading to faster computations. To combine the advantages of both methods, DeepLabv3+ extends the DeepLabv3 model by adding a simple but effective decoder module for recovering the sharp object boundaries. https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ Received: 13 February 2021; Accepted: 6 July 2021 References Remote Sens. Lett. 3, 292–296 (2006). 26. Lee, J.-S. & Pottier, E. Polarimetric Radar Imaging From Basics to Applications (CRC Press, 2009). https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ T. & Hart, P. Nearest neighbor pattern classification. IEEE Trans. g pi fh y 28. Breiman, L. Random Forests. Mach. Learn. 45, 5–32 (2001). g pi 28. Breiman, L. Random Forests. Mach. Learn. 45, 5–32 (2001). 28. Breiman, L. Random Forests. Mach. Learn. 45, 5–32 (2001). A S T O l S h lk f S h ll S h A 29. Hearst, M. A., Dumais, S. T., Osuna, E., Platt, J. & Scholkopf, B. Support vector machines. IEEE Intell. Syst. their Appl. 13 (1998). ( ) 30. Salakhutdinov, R. & Statistics, G. H. B. T.-P. of the T. I. C. on A. I. and. Deep Boltzmann Machines. 448–455 (2009). 30. Salakhutdinov, R. & Statistics, G. H. B. T.-P. of the T. I. C. on A. I. and. Deep Boltzmann Machines. 448–455 (2009). 31 L C Y K k l K & F b t C C l ti l t k d li ti i i i I P di f 2010 IEEE I 31. LeCun, Y., Kavukcuoglu, K. & Farabet, C. Convolutional networks and applications in vision. In: Proceedings of 2010 IEEE I national Symposium on Circuits and Systems, pp. 253–256 (2010). https://doi.org/10.1109/ISCAS.2010.5537907. y p y pp p g 32. Chen, X., Xiang, S., Liu, C. & Pan, C. Vehicle detection in satellite images by hybrid deep convolutional neural networks. IEEE Geosci. Remote Sens. Lett. 11, 1797–1801 (2014). . Hinton, G. E., Osindero, S. & Teh, Y.-W. A fast learning algorith 33. Hinton, G. E., Osindero, S. & Teh, Y.-W. A fast learning algorithm for deep belief nets. Neural Comput. 18, 1527–1554 (2006). 34. Ma, X., Geng, J. & Wang, H. Hyperspectral image classification via contextual deep learning. EURASIP J. Image Video Process. 2015, 20 (2015). Bengio, Y. & Hinton, G. Deep learning. Nature 521, 436–444 (201 . LeCun, Y., Bengio, Y. & Hinton, G. Deep learning. Nature 521, 4 l f d l l g p g 36. Liu, C., Yin, J., Yang, J. Application of deep learning to polarimetric SAR classification. In: IET International Radar Conference 2015, pp. 1–4 (2015). https://doi.org/10.1049/cp.2015.1182. 36. Liu, C., Yin, J., Yang, J. Application of deep learning to polarimetric SAR classificatio 2015, pp. 1–4 (2015). https://doi.org/10.1049/cp.2015.1182. pp p g p 7. References Zhang, L., Zhang, L. & Du, B. Deep learning for remote sensing data: a technical tutorial on the state of the art. IEEE Geosci. Remote Sens. Mag. 4, 22–40 (2016).i g 38. Lv, Q. et al. Urban land use and land cover classification using remotely sensed SAR data through deep belief networks. J. Sens. 2015, 538063 (2015).i ( ) 39. Liu, F., Jiao, L., Hou, B. & Yang, S. POL-SAR image classification based on wishart DBN and local spatial information. IEEE Trans. Geosci. Remote Sens. 54, 3292–3308 (2016). 0. Gong, M., Zhao, J., Liu, J., Miao, Q. & Jiao, L. Change detection in synthetic aperture radar images based on deep neural networks IEEE Trans. Neural Netw. Learn. Syst. 27, 125–138 (2016).i y 1. Chen, S., Wang, H., Xu, F. & Jin, Y. Target classification using the deep convolutional networks for SAR images. IEEE Trans. Geosci Remote Sens. 54, 4806–4817 (2016).f 42. Chen, L.-C., Zhu, Y., Papandreou, G., Schroff, F., Adam, H. Encoder–decoder with atrous separable convolution for semanti segmentation. In: Computer Vision and Pattern Recognition (2018). g p g 43. Russakovsky, O. et al. ImageNet large scale visual recognition challenge. Int. J. Comput. Vis. 115, 211–252 (2015). 44. Kumar, A., Garg, R., Prateek, M. & Kumar, S. Implementation of Evolutionary Computing Algorithm for Polarimetric SAR Data Processing and Classification (TEC-07): L&S band Airborne SAR Research Announcement (RA) project under the NASA-ISRO Synthetic Aperture Radar (NISAR) mission. (Space Applications Centre, Indian Space Research Organisation, Ahmedabad, 2021). 44. Kumar, A., Garg, R., Prateek, M. & Kumar, S. Implementation of Evolutionary Computing Algorithm for Polarimetric SAR Processing and Classification (TEC-07): L&S band Airborne SAR Research Announcement (RA) project under the NASA h d ( ) ( l d h h d b d 44. Kumar, A., Garg, R., Prateek, M. & Kumar, S. Implementation of Evolutionary Computing Algorithm for Polarimetric SAR Data Processing and Classification (TEC-07): L&S band Airborne SAR Research Announcement (RA) project under the NASA-ISRO Synthetic Aperture Radar (NISAR) mission (Space Applications Centre Indian Space Research Organisation Ahmedabad 2021) g fi ( ) ( ) p j Synthetic Aperture Radar (NISAR) mission. (Space Applications Centre, Indian Space Research Organisation, Ahmedabad, 2 45. Wood, J. W., White, R. G. & Oliver, C. J. Distortion free SAR imagery and change detection. In: Proceedings of the 1988 IEE National Radar Conference, pp. 95–99 (1988). https://doi.org/10.1109/NRC.1988.10937. g y ar Conference, pp. 95–99 (1988). References & Ottersten, B. Orthorectified polar format algorithm fo SAR imaging with DEM. IEEE Trans. Geosci. Remote Sens. 59, 3999–4007 (2021). i p SAR imaging with DEM. IEEE Trans. Geosci. Remote Sens. 59, 3999–4007 (2021). & Schubert, A. Guide to ASAR Geocoding (University of Zürich, 200 59. Small, D. & Schubert, A. Guide to ASAR Geocoding (Universit , , g ( y , ) 60. Chen, X., Sun, Q. & Hu, J. Generation of complete SAR geometric distortion maps based on DEM and neighbor gradient algorithm. Appl. Sci. 8, 2206 (2018). 61. Cigna, F., Bateson, L. B., Jordan, C. J. & Dashwood, C. Simulating SAR geometric distortions and predicting Persistent Scatterer densities for ERS-1/2 and ENVISAT C-band SAR and InSAR applications: Nationwide feasibility assessment to monitor the landmass of Great Britain with SAR imagery. Remote Sens. Environ. 152, 441–466 (2014). g y 62. Kumar, S. et al. Polarimetric calibration of spaceborne and airborne multifrequency SAR data for scattering-based characteriz of manmade and natural features. Adv. Space Res. https://doi.org/10.1016/j.asr.2021.02.023 (2021). 63. Freeman, A. & Durden, S. L. A three-component scattering model for polarimetric SAR data. IEEE Trans. Geosci. Remote Sens. 36, 963–973 (1998). 64. Shafai, S. S. & Kumar, S. PolInSAR coherence and entropy-based hybrid decomposition mode. Earth Space Sci. 7, 1–17 (202 65 Van Z J & Kim Y Synthetic Aperture Radar Polarimetry (Wile 2011) 64. Shafai, S. S. & Kumar, S. PolInSAR coherence and entropy-based hybrid decomposition mode. Earth S 64. Shafai, S. S. & Kumar, S. PolInSAR coherence and entropy-based hybrid decomposition mode. Earth Space Sci. 7, 1–17 (2020). 65. Van, Z. J. & Kim, Y. Synthetic Aperture Radar Polarimetry (Wiley, 2011). py y p p 65. Van, Z. J. & Kim, Y. Synthetic Aperture Radar Polarimetry (Wiley, 2011). & Kim, Y. Synthetic Aperture Radar Polarimetry (Wiley, 2011). . Van, Z. J. & Kim, Y. Synthetic Aperture Radar Polarimetry (Wile y p y y 6. Yamaguchi, Y., Sato, A., Boerner, W., Sato, R. & Yamada, H. Four-component scattering power decomposition with rotation o coherency matrix. IEEE Trans. Geosci. Remote Sens. 49, 2251–2258 (2011). 67. Sarabandi, K., Pierce, L. E. & Ulaby, F. T. Calibration of a polarimetric imaging SAR. IEEE Trans. Geosci. Remote Sens. 30, 540–549 (1992).fil 68. Sun, G., Huang, L., Chen, K. & Han, C. An efficient polarimetric SAR calibration algorithm using corner reflectors. Can. J. Remote Sens. 43, 286–296 (2017). ( ) 69. Jung, Y. T. References https://doi.org/10.1109/NRC.19 pp p g 46. Freeman, A. SAR calibration: an overview. IEEE Trans. Geosci. Remote Sens. 30, 1107–1121 (1992). 47. Touzi, R., Hawkins, R. K. & Cote, S. High-precision assessment and calibration of polarimetric RADARSAT-2 SAR using tran- sponder measurements. IEEE Trans. Geosci. Remote Sens. 51, 487–503 (2013). 48. Wang, F., Liu, A., Xu, H. & Jiang, T. A Method for estimating and validating polarimetric distortion parameters using co reflectors and its applicability analysis. IEEE J Sel. Top. Appl. Earth Obs. Remote Sens. 12, 5345–5359 (2019). l y y p pp 49. Agrawal, S., Raghavendra, S. & Kumar, S. Geospatial data for the himalayan region: requirements, availability, and challenges. In Remote Sensing of Northwest Himalayan Ecosystems (eds Navalgund, R. R. & Kumar, A. S.) 471–500 (Springer, Singapore, 2018). 50. Richards, J. A. Remote Sensing with Imaging Radar (Springer, 2009). . Richards, J. A. Remote Sensing with Imaging Radar (Springer, 20 g g g p g 1. Woodhouse, I.F. Introduction to Microwave Remote Sensing. (CRC Press, Boca Raton, 2006). https://doi.org/10.1201/9781315272 573. 52. El-Darymli, K., McGuire, P., Gill, E., Power, D. & Moloney, C. Understanding the significance of radiometric calibration for syn- thetic aperture radar imagery. In: 2014 IEEE 27th Canadian Conference on Electrical and Computer Engineering (CCECE), pp. 1–6 (2014). https://doi.org/10.1109/CCECE.2014.6901104.i p g 53. Yang, J., Qiu, X., Ding, C. & Lei, B. Identification of stable backscattering features, suitable for maintaining absolute synthetic aperture radar (SAR) radiometric calibration of sentinel-1. Remote Sens. 10, 1010 (2018). 4. Loew, A. & Mauser, W. Generation of geometrically and radiometrically terrain corrected SAR image products. Remote Sens Environ. 106, 337–349 (2007).i 55. Huang, L., Li, Z. & Tian, B. Local incidence angle referenced classification on polarimetric synthetic aperture radar images in mountain glacier areas. J. Appl. Remote Sens. 10, 1–14 (2016).f 6. Warner, T., Bell, R. & Singhroy, V. Local incidence angle effects on X- and C-band radar backscatter of boreal forest communities Can. J. Remote Sens. 22, 269–279 (1996). 57. Shibayama, T., Yamaguchi, Y. & Yamada, H. Polarimetric scattering properties of landslides in forested areas and the dependence on the local incidence angle. Remote Sens. 7, 15424–15442 (2015).i g 58. Hu, R., Rao, B. S. M. R., Alaee-Kerahroodi, M. & Ottersten, B. Orthorectified polar format algorithm for generalized spot SAR imaging with DEM IEEE Trans Geosci Remote Sens 59, 3999–4007 (2021) g 58. Hu, R., Rao, B. S. M. R., Alaee-Kerahroodi, M. www.nature.com/scientificreports/ A Computational approach to edge detection. IEEE Trans. Pattern Anal. Mach. Intell. 6, 679–698 (1986). 83. Breiman, L. Bagging predictors. Mach. Learn. 24, 123–140 (1996). 83. Breiman, L. Bagging predictors. Mach. Learn. 24, 123–140 (19 , L. Bagging predictors. Mach. Learn. 24, 123–140 (1996). gg g p 84. Dong, Y., Du, B. & Zhang, L. Target detection based on random forest metric learning. IEEE J. Sel. Top. Appl. Earth Obs. Remote Sens. 8, 1830–1838 (2015). 5. Shelhamer, E., Long, J. & Darrell, T. Fully convolutional networks for semantic segmentation. IEEE Trans. Pattern Anal. Mach Intell. 39, 640–651 (2017). 86. Ronneberger, O., Fischer, P. & Brox, T. U-Net: convolutional networks for biomedical image segmentation BT. Medical Image Computing and Computer-Assisted Intervention – MICCAI 2015. in (eds. Navab, N., Hornegger, J., Wells, W. M. & Frangi, A. F.) 234–241 (Springer, 2015). p g 7. Badrinarayanan, V., Kendall, A. & Cipolla, R. SegNet: a deep convolutional encoder-decoder architecture for image segmentation IEEE Trans. Pattern Anal. Mach. Intell. 39, 2481–2495 (2017). 88. Chen, L.-C., Papandreou, G., Kokkinos, I., Murphy, K. & Yuille, A. L. DeepLab: semantic image segmentation with deep convo- lutional nets, atrous convolution, and fully connected CRFs. IEEE Trans. Pattern Anal. Mach. Intell. 40, 834–848 (2018). y 9. Holschneider, M., Kronland-Martinet, R., Morlet, J. & Tchamitchian, P. A Real-Time Algorithm for Signal Analysis with the Help of the Wavelet Transform. (Springer, Berlin, 1990). https://doi.org/10.1007/978-3-642-75988-8_28. ception: deep learning with depthwise separable convolutions. In: 90. Chollet, F. Xception: deep learning with depthwise separable convolutions. In: 2017 IEEE Conference on Computer Vision Pattern Recognition (CVPR), pp. 1800–1807 (2017). https://doi.org/10.1109/CVPR.2017.195.f 1. Chen, L.-C., Papandreou, G., Schroff, F. & Adam, H. Rethinking atrous convolution for semantic image segmentation. in Computer Vision and Pattern Recognition (2017).i g 2. Rebentrost, P., Mohseni, M. & Lloyd, S. Quantum support vector machine for big data classification. Phys. Rev. Lett. 113, 130503 (2014).hi 3. Hensley, S. et al. The UAVSAR instrument: Description and first results. In: 2008 IEEE Radar Conference, pp. 1–6 (2008). https:// doi.org/10.1109/RADAR.2008.4720722. 93. Hensley, S. et al. The UAVSAR instrument: Description and first results. In: 2008 IEEE Radar Conference, pp. 1–6 (2008). https:// doi.org/10.1109/RADAR.2008.4720722. Acknowledgementsh g The authors are grateful to the L&S band Airborne SAR Research Announcement (RA) project on Implementa- tion of Evolutionary Computing Algorithm for Polarimetric SAR Data Processing and Classification (TEC-07) under the NASA-ISRO Synthetic Aperture Radar (NISAR) mission for providing financial support to carry out this study. The authors would like to thank NASA/JPL for providing UAVSAR data and it is open access to all at the URL https://uavsar.jpl.nasa.gov/cgi-bin/data.pl. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 72. Maiti, A., Kumar, S., Tolpekin, V. & Agrawal, S. A computationally efficient hybrid framework for polarimetric calibration of quad-pol SAR data. Earth Space Sci. 8, 1–22 (2021). 3. Li, L., Zhu, Y., Hong, J., Ming, F. & Wang, Y. Design and implementation of a novel polarimetric active radar calibrator for Gaofen-3 SAR. Sensors 18, 2620 (2018). ( ) 4. Shimada, M. Model-based polarimetric SAR calibration method using forest and surface scattering targets. In: 2011 IEEE Inter- ( ) 74. Shimada, M. Model-based polarimetric SAR calibration method using forest and surface scattering target national Geoscience and Remote Sensing Symposium, pp. 3736–3739 (2011). https://doi.org/10.1109/IGA 74. Shimada, M. Model-based polarimetric SAR calibration method using forest and surface scattering targets. In: 2011 IEEE Inter- national Geoscience and Remote Sensing Symposium pp 3736 3739 (2011) https://doi org/10 1109/IGARSS 2011 6050037 national Geoscience and Remote Sensing Symposium, pp. 3736–3739 (2011). https://doi.org/10.1109/IGARSS.2011.605003 g y Polarimetric SAR Imaging Theory and Applications (CRC Press, 202 . Yamaguchi, Y. Polarimetric SAR Imaging Theory and Application g g gh y pp 76. Uninhabited Aerial Vehicle Synthetic Aperture Radar (UAVSAR). (2021). https://uavsar.jpl.nasa.gov/cgi-bin/data.pl. 76. Uninhabited Aerial Vehicle Synthetic Aperture Radar (UAVSAR). (2021). https://uavsar.jpl.nasa.gov/cgi bin/data.pl. 77. Yamaguchi, Y., Moriyama, T., Ishido, M. & Yamada, H. Four-component scattering model for polarimetric SAR image decomposi- 77. Yamaguchi, Y., Moriyama, T., Ishido, M. & Yamada, H. Four-component scattering model for polarimetric SAR image decom tion. IEEE Trans. Geosci. Remote Sens. 43, 1699–1706 (2005). ( ) 78. Singh, G., Yamaguchi, Y. & Park, S. General four-component scattering power decomposition with unitary transformation of coherency matrix. IEEE Trans. Geosci. Remote Sens. 51, 3014–3022 (2013).h y 79. Gabor, D. Theory of communication. Part 1: the analysis of information. J. Inst. Electr. Eng.—Part III Radio Commun. Eng 429–441 (1946). 80. Deng, G. & Cahill, L. W. An adaptive Gaussian filter for noise reduction and edge detection. In: 1993 IEEE Conference Record Nuclear Science Symposium and Medical Imaging Conference, vol. 3, pp. 1615–1619 (1993). https://doi.org/10.1109/NSSMIC. 1993.373563. 81. Dong, Y., Li, M. & Li, J. Image retrieval based on improved Canny edge detection algorithm. In: Proceedings 2013 International Conference on Mechatronic Sciences, Electric Engineering and Computer (MEC), pp. 1453–1457 (2013). https://doi.org/10.1109/ MEC.2013.6885296. 82. Canny, J. A Computational approach to edge detection. IEEE Trans. Pattern Anal. Mach. Intell. 6, 679–698 (1986). 83 B i L B i di M h L 24 123 140 (1996) 82. Canny, J. References & Park, S.-E. Comparative analysis of polarimetric SAR calibration methods. Remote Sens. 10, 2060 (2018). g p y p 70. Villa, A., Iannini, L., Giudici, D., Monti-Guarnieri, A. & Tebaldini, S. Calibration of SAR Polarimetric images by means of a covari- ance matching approach. IEEE Trans. Geosci. Remote Sens. 53, 674–686 (2015). g pp ( ) 1. Kim, J. S., Papathanassiou, K. P., Scheiber, R. & Quegan, S. Correcting distortion of polarimetric SAR data induced by ionospheric scintillation. IEEE Trans. Geosci. Remote Sens. 53, 6319–6335 (2015). https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| Author contributions A.K., R.G., and S.K. carried out the whole experiment and prepared the manuscript with equal contribution. N.B. worked on the annotation of the dataset. A.K., S.K., and M.P. supervised the research work. All the authors contributed to review the manuscript. contributed to review the manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-021-94422-y. Correspondence and requests for materials should be addressed to A.K. 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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps institutional affiliations. https://doi.org/10.1038/s41598-021-94422-y Scientific Reports \| (2021) 11:15365 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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https://openalex.org/W2142006222	https://ccsenet.org/journal/index.php/gjhs/article/download/38626/22539	English	null	Strategic Planning, Implementation, and Evaluation Processes in Hospital Systems: A Survey From Iran	Global journal of health science	2,014	cc-by	5,595	Global Journal of Health Science; Vol. 7, No. 2; 2015 ISSN 1916-9736 E-ISSN 1916-9744 Published by Canadian Center of Science and Education Global Journal of Health Science; Vol. 7, No. 2; 2015 ISSN 1916-9736 E-ISSN 1916-9744 Published by Canadian Center of Science and Education Abstract Aim & Background: Strategic planning has been presented as an important management practice. However, evidence of its deployment in healthcare systems in low-income and middle-income countries (LMICs) is limited. This study investigated the strategic management process in Iranian hospitals. Methods: The present study was accomplished in 24 teaching hospitals in Tehran, Iran from September 2012 to March 2013. The data collection instrument was a questionnaire including 130 items. This questionnaire measured the situation of formulation, implementation, and evaluation of strategic plan as well as the requirements, facilitators, and its benefits in the studied hospitals. Results: All the investigated hospitals had a strategic plan. The obtained percentages for the items “the rate of the compliance to requirements” and “the quantity of planning facilitators” (68.75%), attention to the stakeholder participation in the planning (55.74%), attention to the planning components (62.22%), the status of evaluating strategic plan (59.94%) and the benefits of strategic planning for hospitals (65.15%) were in the medium limit. However, the status of implementation of the strategic plan (53.71%) was found to be weak. Significant statistical correlations were observed between the incentive for developing strategic plan and status of evaluating phase (P=0.04), and between status of implementation phase and having a documented strategic plan (P=0.03). Conclusion: According to the results, it seems that absence of appropriate internal incentive for formulating and implementing strategies led more hospitals to start formulation strategic planning in accordance with the legal requirements of Ministry of Health. Consequently, even though all the investigated hospital had the documented strategic plan, the plan has not been implemented efficiently and valid evaluation of results is yet to be achieved. rds: strategic planning, strategic implementation, strategic evaluation, strategic control, hospital, Iran Keywords: strategic planning, strategic implementation, strategic evaluation, strategic control, hospital, Iran 1. Introduction Strategic Planning, Implementation, and Evaluation Processes in Hospital Systems: A Survey From Iran Jamil Sadeghifar1, Mehdi Jafari1, Shahram Tofighi2, Hamid Ravaghi1,3 & Mohammad Reza Maleki1 1 Department of Health Services Management, School of Health Management and Information Sciences, Iran University of Medical Sciences, Tehran, Iran 2 Health Management Research Centre, Baqiyatallah University of Medical Sciences, Tehran, Iran 3 Health Management and Economics Research Center, Iran University of Medical Sciences, Tehran, Iran Correspondence: Mehdi Jafari, Department of Health Services Management, School of Health Management and Information Sciences, Iran University of Medical Sciences, No 6, Rashid Yasemi st., Vali-e-asr Ave., Tehran 1995614111, Iran. E-mail: mjafari@iums.ac.ir Health Management and Economics Research Center, Iran University of Medical Sciences, Tehran, Iran Correspondence: Mehdi Jafari, Department of Health Services Management, School of Health Management and Information Sciences, Iran University of Medical Sciences, No 6, Rashid Yasemi st., Vali-e-asr Ave., Tehran 1995614111, Iran. E-mail: mjafari@iums.ac.ir Received: July 12, 2014 Accepted: September 1, 2014 Online Published: September 28, 2014 doi:10.5539/gjhs.v7n2p56 URL: http://dx.doi.org/10.5539/gjhs.v7n2p56 1. Introduction In recent years, improving the quality of health services has attracted the attention of many scientific investigations (Arah et al., 2003). The phases that healthcare organizations routinely follow for improvement of services provision quality are setting priorities, establishing sustainable processes and determining an appropriate framework to implement the initiative programs (Glickman, Baggett, Krubert, Peterson, & Schulman, 2007; Rütten, Röger, Abu-Omar, & Frahsa, 2009). On the other hand, increasing global competition has led these organizations to re-engineering of business processes as a means to ensure efficiency, effectiveness and ultimately their success (Jafari, Bastani, Ibrahimipour, & Dehnavieh, 2012). One of the most effective strategies for the success of organizations is running the strategic planning. It is one of the most common managerial practices in healthcare provider organizations, largely in the form of research articles that have not been evaluated. Evidence-based management seems inevitable in these organizations to conduct investigations on such common practice (Kaissi, Begun, & Welson, 2008). Strategic planning is the systematic process whereby an organization creates a document indicating the way it 56 Global Journal of Health Science Vol. 7, No. 2; 2015 www.ccsenet.org/gjhs plans to progress from its current situation to the desired future situation (Perera & Peiró, 2012). According to Swayne, Duncan, and Ginter (2006), strategic planning is a set of processes, which help in identifying the future desired by the organization and to developing guidelines for making the decisions leading to such a future (Swayne, Duncan, & Ginter, 2006). Strategic planning integrates main objectives, policies and associated activities in an individual organization as a whole. A strategy based on the shortcomings and the competence within the enterprise can anticipate changes in the environment, and can be helpful for allocation of enterprise resources in a unique and valuable way (Mintzberg, Quinn, & Ghoshal, 1998). Successful strategic planning necessitates development of the plan in right manner, as well as appropriate implementation, accurate and on-time evaluation of the results, possibly through the Strategic Control System (SCS). The SCS involves tracking strategies to action, identifying the problems or changes in necessary strategies and reforms. Senior manager must constantly ask himself/herself whether the organization is moving in the right direction or not, and if the assumptions about the intended major trends and changes in the environment are correct. Such questions need to set the strategic controls (Pearce & Robinson, 2007). 2.1 Sample The research was conducted on the teaching hospitals affiliated with Tehran University of Medical Sciences (TUMS) and Iran University of Medical Sciences (IUMS) in Tehran (N = 26). There are three large medical universities in Tehran that cover medical services through governmental and teaching hospitals. The TUMS and IUMS have 17 and 10 teaching hospitals, respectively. Three hospitals refused to participate in the study; thus, the study was done on 24 hospitals. From the studied hospitals, a total of 8 hospitals were general and 16 hospitals were specialized. 1. Introduction Evidence of strategic planning in low-income and middle-income countries (LMICs) is very low (El-Jardali, Jamal, Abdallah, & Kassak, 2007). Compared with high-income countries, healthcare environment in LMIC is more complex, dynamic and challenging (Mills, Brugha, Hanson, & McPake, 2002) where resources are generally more limited. Thus, the need for strategic planning in these countries should be understood to a larger degree. Saleh et al. (2013) in research on strategic planning processes and their relation to the financial performance of hospitals in Lebanon examined six dimensions including having a plan, plan development, plan implementation, responsibility of planning activities, governing board involvement, and physicians’ involvement as components of the strategic planning process (Saleh, Kaissi, Semaan, & Natafgi, 2013). In a research study, among hospitals in Texas, Kaissi et al. (2008) demonstrated that 87% of the hospitals had a strategic plan, and concluded that three dimensions including having a strategic plan, assigning the Chief Executive Officer (CEO) for the plan, and involving the board are positively associated with financial performance (Kaissi et al., 2008). Iran as a LMIC is an ancient country located in the Middle East, a region between Asia, Europe, and Africa. The Iranian healthcare system is pluralistic and fragmented because of the public-private mixture in financing and provision of health services. At the national level, Ministry of Health is exercising the governance, policy-making, planning, financing and steering the programs. At the provincial level, the Universities of Medical Sciences and Health Services are responsible for provision of health services, including the environmental health (Mehrdad, 2009). It should be noted that according to the Iranian national accreditation system (effective from 2010); hospitals are required to have a strategic plan that indicates future directions of the hospital (Jafari et al., 2010). Although having a strategic plan is an overarching national health initiative, this study went beyond the requirement of having a strategic plan to assess the actual process and identify the stakeholders involved in the strategic planning within hospitals. The goal of this study was to explore the process of formulation, implementation and evaluation of strategic plans in all the teaching hospitals affiliated of Iran and Tehran Universities of Medical Sciences. 2.2 Data Collection The data gathering tool was a questionnaire developed by the authors based on the literature review (Kaissi et al., 2008; Poister & Streib, 2005) and expert opinion. The questionnaire consisted of 130 questions and its validity was confirmed by seven experts in the field of strategic planning. The questionnaire included the following sections and questions: - Demographic variables (9 items): Specialty of hospital, number of bed, number of employees, hospital age, grade evaluation, field of study and education degree of hospital manager, the number and composition of the strategic planning committee. 57 Global Journal of Health Science Vol. 7, No. 2; 2015 www.ccsenet.org/gjhs - Requirements and Facilitators of Strategic management (7 items): management systems deployed in hospital, using a consultant in strategic planning, having a strategic planning committee, activation of strategic planning committee, having a documented strategic plan, review of the strategic plan, and incentive for developing strategic plan. - The strategic planning phase: extent of stakeholders’ contribution (chief of hospital, hospital manager, assistants and matron, clinical departments officials, officials of the supportive sections, staff at operational levels, external stakeholders, representatives of patient/donor, representative of university) in the strategic planning process in 5 dimensions including participation in meetings, provision of the required information, provision of the initial ideas, provision of reformed and completive suggestions for plan development, presentation of gathering and documentation of plan (45 items), and the extent of attention to various components of the strategic planning (15 items). - The strategic implementation phase: the components of the budget allocation based on strategic priorities (6 items) and taking action based on the strategic plan (7 items). - The strategic implementation phase: the components of the budget allocation based on strategic priorities (6 items) and taking action based on the strategic plan (7 items). The Strategic evaluation phase: status of using indicators for achieving goals (15 items). - The Strategic evaluation phase: status of using indicators for achieving goals (15 items). - The benefits of the strategic planning (25 items) and a question to determine the level of strategic planning usage in hospitals. The scale used for the questions relating to the requirements and facilitator of strategic management was “yes/no”, with a range of weak (0-6), to moderate (7-9) and good (12-10). 2.2 Data Collection For the remaining questions, the 5-degree scale (5: very good to 1: very low) and triple range from weak (0-2.50), to moderate (2.51-4) and good (4.01-5) were used to analyze and interpret the answers. In order to unifying the scorings, the calculated scores in both the requirements and the strategic management process have been changed based on 100% and classified as following, Low: 0 to ≤ 50- Moderate: greater than 50% to ≤ 80 - Good: greater than 80%. Data were collected by the researchers at the studied hospitals and the questionnaire was completed by the investigator in the form of interview. In each hospital, the key person/persons with the greatest involvement and awareness of the hospital's strategic plan were interviewed. Job status of persons that participated in this study in the investigated hospitals included quality improvement officer (13 hospitals), hospital affairs expert (4 hospitals), clinical governance officer (2 hospitals), hospital manager, Research and Development executive (R&D), clinical supervisor, and systems and procedures officer. Objectives of the study were explained to the participants, and to confirm the accuracy of answers, the documents available in the hospitals were also studied if further review required. 2.4 Ethical Issues This study was approved by Ethics Committee of Iran University of Medical Sciences Research. The main ethical issue was respondents’ right to self-determination, anonymity and confidentiality. The questionnaires were distributed among participants with a participant information sheet, explaining the nature of study. The consent was taken verbally from the participants. The questionnaire data were kept confidential and the respondents were assured of their right to withdraw at any time. The respondents’ names were not recorded on the questionnaire; thus, the data were rendered anonymously. 2.3 Data Analysis Data analysis was performed using the Statistical Package for the Social Sciences (SPSS) software, version 18.0. In this study, the strategic management requirements included 10 variables (defined in Table 2) as the independent variables, that all of them have a two-way scale. Furthermore, the strategic management process (including planning, implementation and evaluation) has been considered as the dependent variables. Therefore, in order to compare the mean of the differences between the dimensions of strategic management with against the requirements, we used the t-test (Table 4). 3. Results All the studied hospitals were medical and teaching as national referral hospitals in Tehran providing medical and educational services. More than two-thirds of the hospitals were specialized and most of them had one evaluation degree. Establishment age of most of the hospitals was more than 30, half of them had more than 200 beds and about one-third had more than 500 employees. Most hospital managers had a Master's degree in health services management. 58 Global Journal of Health Science Vol. 7, No. 2; 2015 www.ccsenet.org/gjhs Table 1. Characteristics of hospitals and hospital managers Variables n % Type of hospital General 7 29.2 Specialized 17 70.8 Evaluation degree Excellent degree 6 25 One degree 18 75 Number of beds Less than 200 12 50.0 201 to 500 10 41.7 More than 501 2 8.3 Hospital age Less than 30 2 8.3 31 to 50 9 37.5 More than 51 13 54.2 Number of staff Less than 500 15 62.5 More than 501 9 37.5 Manager's level of education BSc. 2 8.3 MSc. 11 45.8 Professional Doctorate 4 16.7 Ph.D. 7 29.2 Manager's field of education Health Services Management 14 58.3 Others 10 41.7 From all the surveyed hospitals, about one-third had carried out the quality management system i.e. the International Organization for Standardize (ISO 9000) and European Fundamental for Quality Management (EFQM). All the hospitals are currently implementing national programs of accreditation and clinical governance requirements. The Balanced Scorecard method (BSC) was not considered in 15 hospitals (62.5%), while it is running in 9 hospitals (37.5%). More than half of the hospitals had a consultant for strategic planning. The majority of hospitals had a strategic planning committee; that is more than half of them have an active committee with monthly meeting. In more than half of the hospitals, members of strategic planning committee are between 5 to 9 persons. All the hospitals have a documented strategic plan and in the majority of them, plan review was carried out. Most hospitals developed the strategic plan as a legal requirement of the Ministry of Health, while a limited number introduced improvement in performance and future prediction as an incentive for developing strategic plan. 59 Global Journal of Health Science Vol. 7, No. 2; 2015 www.ccsenet.org/gjhs Table 2. 3. Results Situation of strategic management process requirements and facilitators Variables n % Employing consultant Yes 14 58.3 No 10 41.7 Employing ISO 9001 Yes 7 29.2 No 17 70.8 Employing EFQM Yes 8 33.3 No 16 66.7 Having a strategic planning committee Yes 20 83.3 No 4 16.7 Active strategic planning committee Yes 12 60.0 No 8 40.0 Strategic planning committee members 5 to 9 11 55.0 More than 10 9 45.0 Having a documented strategic plan Once 11 45.8 More than once 13 54.2 Having revision in strategic plan Yes 19 79.2 No 5 20.8 Incentive for developing strategic planning Legal driven 15 62.5 Improvement driven 9 37.5 Employing Balanced Scorecard (BSC) Yes 9 37.5 No 15 62.5 In relation to the requirements and facilitators of strategic planning, most hospitals (n=15) have a moderate status. About developing strategic plan, two distinct dimensions were studied. They were stakeholder participation in strategic planning with an average score of 55.7%, and attention to the strategic planning components with an average score of 62.2% (moderate). Implementation of strategic plan consisted of two components. They were budget allocation based on the strategic priorities and action based on the strategic plan. Status of the studied hospitals with an average score of 53.71% was much weaker than other areas and half of the hospitals were weak in this area. Regarding the evaluation of strategic plan, using indicators for objectives achievement was studied and the average score of 59.94% reflected moderate status of the studied hospitals. 60 Global Journal of Health Science Vol. 7, No. 2; 2015 www.ccsenet.org/gjhs Table 3. 3. Results Situation of strategic planning process, requirements, facilitators and benefits Variables n % Mean SD Requirements and facilitators of the strategic planning Weak 4 16.7 68.75 15.59 Moderate 15 62.5 Good 5 20.8 Responsibility for strategic plan Weak 9 37.5 55.74 12.27 Moderate 14 58.3 Good 1 4.2 Documenting the strategic plan Weak 2 8.3 62.22 10.85 Moderate 20 83.3 Good 2 8.3 Implementing the strategic plan Weak 12 50.0 53.71 17.08 Moderate 11 45.8 Good 1 4.2 Evaluating the strategic plan Weak 6 25.0 59.94 17.94 Moderate 14 58.3 Good 4 16.7 Benefits of the strategic planning Weak 3 12.5 65.15 14.20 Moderate 17 70.8 Good 4 16.7 The results of the statistical analysis indicate a significant correlation between the incentive for developing of strategic plan and evaluation of strategic plan (P<0.04); that is the hospitals with incentive such as performance improvement attempted to develop a strategic plan and evaluate strategic plan more effectively. Also, a significant correlation was observed between having a documented strategic plan and implementation of plan (P<0.03). Among other requirements and facilitators, the strategic planning process and planning, implementation and evaluation showed no significant statistical correlation. 61 Global Journal of Health Science Vol. 7, No. 2; 2015 www.ccsenet.org/gjhs Table 4. 3. Results Relationship between strategic planning (SP) requirements and facilitators with planning, implementation, and evaluation phases Variables Planning phase Implementation phase Evaluation phase Totally M±SD F Sig M±SD F Sig M±SD F Sig M±SD F Sig Employing consultant No 60.3±12.5 .97 .33 54.3±17.5 .00 .97 56.6±15.0 1.2 .27 58.7±11.9 .00 .94 Yes 63.5±9.7 53.2±17.4 62.2±19.9 61.8±10.9 Employing ISO 9001 No 60.8±10.3 .23 .63 50.7±15.7 .32 .57 59.8±14.8 2.8 .10 59.1±10.2 1.9 .18 Yes 65.5±12.0 60.8±19.2 60.1±25.3 63.9±13.5 Employing EFQM No 60.1±10.5 .00 .94 51.0±15.4 .33 .56 57.3±16.8 .12 .73 58.2±10.6 .18 .67 Yes 66.4±10.9 59.0±19.9 65.1±20.1 65.1±11.7 Having a SP committee No 59.0±4.1 2.9 .09 41.9±21.1 .16 .68 47.0±14.4 .27 .60 54.4±5.3 2.8 .10 Yes 62.8±11.7 56.0±15.7 62.5±17.7 61.7±11.8 Active SP committee No 56.9±9.0 .05 .81 54.1±19.9 2.4 .13 55.8±18.5 .00 .98 56.3±10.3 .13 .71 Yes 67.5±10.1 53.3±14.5 64.0±17.1 64.8±10.8 Having revision in SP No 57.9±5.2 2.2 .14 47.0±14.9 1.0 .31 53.8±22.7 .86 .36 55.6±8.4 1.1 .29 Yes 63.3±11.7 55.4±17.5 61.5±16.8 61.8±11.7 Incentive for developing of SP No 60.2±9.6 .64 .43 49.4±15.3 .47 .49 54.3±13.5 4.4 .04 57.6±9.1 2.7 .11 Yes 65.5±12.4 60.8±18.3 69.3±21.0 65.5±13.1 Having a documented SP No 58.6±9.2 1.3 .26 47.9±12.2 5.1 .03 58.3±15.4 .99 .32 57.0±7.7 2.9 .10 Yes 65.2±11.5 58.5±19.4 61.3±20.3 63.5±12.5 4. Discussion Making a vision/study to forecast the future, assess the strength and weakness points and check the threats and opportunities have attracted the highest attention. However, assessing the feasibility of proposed strategies, competitive differentiation, understanding the concerns and needs of stakeholders, clarifying the rules and regulations of organizations and understanding customers' demands and community needs was considered the least. The status of strategic plan implementation in the form of budget allocation based on the strategic priorities and doing action according to the strategic plan was poor. Accordingly, in most of the studied hospitals, the university did not review annual budget based on the hospital's strategic objectives. Moreover, hospital managers and authorities did not request budget based on the strategic plan. There was relatively minor attempt to identify and attract new financial resources to achieve the hospital's strategic objectives. This is largely associated with the allocation of governmental budgets to hospitals. Although the administrators and departments authorities in the studied hospitals are responsible for implementation of certain goals associated with the strategic plan, annual evaluation is not practiced based on the achievement of strategic objectives in most of the hospitals, and annual reward in most of the hospitals is not adjusted based on the rate of participation of different wards/individuals in achieving the strategic objectives. In majority of the hospitals, the responsibility for implementing the strategic plan is not assigned to the hospital manager and the annual evaluation is not done based on the achievement of strategic objectives. Unfortunately, hospitals in Iran, only in the formulation of strategic plan efforts to be acted based on existing scientific models and frameworks and in connection with the implementation of the plan are treated merely as traditional. In evaluations of MOH on the hospital's strategic plan, the implementation of the action plan is not considered significantly. In most of the studied hospitals, the rates of goals and projects progress derived from the strategic plan are regularly evaluated, and indicators of goal achievement are reviewed at certain time intervals and when necessary. In some hospitals, cause and effect relationship and balance between the indicators are considered. Analysis of measured values obtained from the indicators is done in limited range. The comparison the measured values with previous values, specified standards, and values of similar hospitals rarely done. 4. Discussion In the present study, status of the formulation, implementation and evaluation of strategic plan in all the teaching hospitals affiliated with two major Medical Universities in Iran was investigated. The results showed that all the studied hospitals had strategic plan as a requirement of the national accreditation system; however, the strategic management was not conceptualized in these centers. Consequently, strategy implementation and particular activities related to the strategic control is not seen systematically. Ideally, the strategic planning process involves all levels of managerial and operational of organization. Undoubtedly, the board of trustees in every organization has the primary responsibility to carry out the strategic planning. Through establishing key policy targets, the board should play a critical role in complex and extensive planning process (Cleverley & Cameron, 2007). In the present study, influential stakeholders, including hospital chief as a representative of physicians’ community, staff of operational levels, external stakeholders, representatives of patient/donor, and the representative of university had very little participation in the formulation of strategic plan. Due to the changing environment of healthcare delivery, physicians not only required to learn strategic planning and be involved in formulating it, but also they should, by engaging actively in the implementation of programs, have an effective role in achieving the organizational objectives (Schwartz, & Pogge, 2000). The study of Khajavi et al. (2009) in Iran showed that physicians’ skills in strategic thinking and planning are in a low level (Khajavi, Vatankhah, Maleki, Barati, Alami, & Heyrani, 2012). Schwartz and Pogge believed that the acquiring such skills requires a systematic and ongoing training (Schwartz & Cohn, 2002). Executive administrator, assistants, matron, supervisors of clinical and supportive departments had appropriate participation in the formulation of strategic plan. It seems essential that key informants at various levels of within and outside of the organization have effective and adequate participation in formulating a strategic plan. In the study by Saleh et al. (2013) at Lebanon hospitals, the extent of participation of doctors and broad in formulating strategic plan was determined as moderate. Also, in the Amer et al. study at Texas hospitals, the board involvement in formulating strategic plan was reported to be medium (Kaissi et al., 2008). 62 Vol. 7, No. 2; 2015 Global Journal of Health Science www.ccsenet.org/gjhs Based on the findings, attention to various elements of strategic planning was at medium and high levels. 4. Discussion Reporting the indicator values to the university, the community and stakeholders as well as design and performing appropriate interventions for improvement of performance are limited. Benchmarking of goal achievement indicators to ensure the effectiveness of strategic projects is limited in other hospitals. In the Mihic et al. study (2012), it is also indicated that healthcare organizations in Serbia attempt to formulate a strategic plan in appropriate manner, however, the monitoring and control of strategies implementation are not based on an scientific method (Mihic, Obradovic, Todorovic, & Petrovic, 2012). The present results indicate that in cases that hospitals have attempted to establish standards of ISO 9001, status of evaluation strategic plan is more appropriate because of regular and systematic processes and activities as well as observance of documentation principles. According to Rusjan and Alic (2010), where ever the BSC model has been established as the strategic management tool, employing the quality management system (ISO 9001 QMS), has a positive impact on the strategic objectives of organization in four perspectives including customer satisfaction, financial results, internal processes, and learning and growth, (Rusjan & Alic, 2010). Also, using of BSC model in combination with EFQM, as an organizational excellence model, can lead to improvement in developing and implementing strategies in the healthcare provider organizations (Groene, Brandt, Schmidt, & Moeller, 2009). Both of these models can be considered as quality management tools, in the same way that it proposed for Spanish hospitals as an integrated management approach (Tejedor, 2009). It is essential to these excellence and quality management systems to be integrated within the organization strategic management. This initiative can be an interesting issue for future studies in the field of strategic management in hospital systems. Finally, most of the studied hospitals something such as focus of hospital instructions on strategic issues, formation of an authentic sense of the hospital missions in community, clear definition of hospital priorities, providing training and development opportunities for all employees and provide high quality services have brought up as most important benefits of strategic management. Stating these benefits is in the condition which the studied hospitals mainly have worked well in the strategic planning, while implementation and evaluation of strategies in these hospitals is seriously impaired. Author’s Contribution Study concept and design: Sadeghifar, Jafari, Tofighi, Ravaghi, and Maleki. Statistical analysis and interpretation of data: Sadeghifar, Tofighi, and Jafari. Drafting of the manuscript: Sadeghifar and Jafari. Critical revision of the manuscript: Tofighi, Ravaghi and Maleki. 6. Limitation This study had some potential limitations that may affect the results. The study was limited to hospitals of two universities in a single city. Therefore, generalizability of the results can be considered as a limitation of the present study. The findings were solely results of a research study and possibly more specialized investigation of these hospitals for strategic planning process will show the most precise results. Another limitation of this study is lack of literature particularly at international level to compare the results. In comparison to other Iranian hospitals, the studied hospitals have a higher proportion of governmental ownership, which limits generalizations of results. 5. Conclusion MOH requires Iranian hospitals have documented strategic plan in order to obtain points of accreditation. Hence, a strategic plan leads to performance improvement and increase in quality of care in hospitals. This is supported 63 Global Journal of Health Science Vol. 7, No. 2; 2015 www.ccsenet.org/gjhs by our finding that the majority of studied hospitals had documented strategic plan. However, levels of development, implementation and evaluation of plans are moderate. The movement toward excellence, success, and favorable results depends on progress in these fields. The inappropriate status of three dimensions of strategic management in the studied hospitals can be attributed to insufficient knowledge about the terminology and process of strategic management, shortage of time to participate, poor beliefs on strategic management, administrative structure characterized by aversion plan, elitism sub-culture, and serious defect in teamwork, distrust in the external environment, and the centralized structure of MOH in notification of the programs. It seems for resolving these problems and acquiring the benefits of strategic management in hospital systems in middle income countries, such as Iran, and low income countries, the authorities should attempt in short and long terms levels. A basic action to reduce many of these problems is creating the culture of program-based strategizing the organization, started in the high levels of the Ministry of Health, and then be flooded within the hospital systems. In the short term, the potentials of the Hospitals’ Accreditation National Program can be employed, and provide sufficient and appropriate training on the concept and on the process of strategic management then provide appropriate financial and non-financial incentives, in order to move hospitals towards centralized planning and strategic thinking. To resolve these problems, the focus of future studies on the identification of infrastructure required for strategizing hospitals and being successful in implementing strategic management system, and on identifying the potential barriers for deployment strategies and evaluating results is recommended. The authors declare no conflict of interest. The authors declare no conflict of interest. Acknowledgement This study was part of a PhD thesis supported by Iran University of Medical Sciences (grant No.: IUMS/SHMIS-2012/527). We would like to extend our gratitude to the participants in the teaching hospitals of IUMS and TUMS, and Sayyed Morteza Hosseini Shokouh for his valuable assistance in data analysis. Jafari, G., et al. (2010). Hospital accreditation standards in Iran. Tehran, Iran: Seda publication. Physician leadership: essential skills in a changing environment. The American journal of surgery, 180(3), 187-192. Swayne, L. E., Duncan, W. J., & Ginter, P. M. (2006). Strategic management of health care organizations. Blackwell Publishing: Malden, MA. Swayne, L. E., Duncan, W. J., & Ginter, P. M. (2006). Strategic management of health care organizations. Blackwell Publishing: Malden, MA. Tejedor, J. P. (2009). On the ultimate goal of management in Spanish hospitals. Gaceta Sanitaria, 23(2), 148-157. http://dx.doi.org/10.1590/S0213-91112009000200014 Tejedor, J. P. (2009). On the ultimate goal of management in Spanish hospitals. Gaceta Sanitaria, 23(2), 148-157. http://dx.doi.org/10.1590/S0213-91112009000200014 Tejedor, J. P. (2009). On the ultimate goal of management in Spanish hospitals. Gaceta Sanitaria, 23(2), 148-157. http://dx.doi.org/10.1590/S0213-91112009000200014 Jafari, G., et al. (2010). Hospital accreditation standards in Iran. Tehran, Iran: Seda publication. Jafari, G., et al. (2010). Hospital accreditation standards in Iran. Tehran, Iran: Seda publication Jafari, M., Bastani, P., Ibrahimipour, H., & Dehnavieh, R. (2012). Attitude of health information system managers and officials of the hospitals regarding the role of information technology in reengineering the business procedures: A qualitative study. Health MED, 6(1), 208-215. Kaissi, A. A., Begun, J. W., & Welson, T. (2008). Strategic planning processes and hospital financial performance. Journal of Healthcare Management, 53(3), 197-208. Khajavi, A., Vatankhah, S., Maleki, M. R., Barati, A., Alami, A., & Heyrani, A. (2012). Conceptual skill in physicians: An overlooked basic competency. HealthMed, 6(10). Mehrdad, R. (2009). Health system in Iran. JMAJ, 52(1), 69-73. Mihic, M. M., Obradovic, V. L., Todorovic, M. L., & Petrovic, D. C. (2012). Analysis of implementation of the strategic management concept in the healthcare system of Serbia. Health Med, 6(10). Mills, A., Brugha, R., Hanson, K., & McPake, B. (2002). What can be done about the private health sector in low-income countries? Bulletin of the World Health Organization, 80(4), 325-330. http://dx.doi.org/10.1590/S0042-96862002000400012 Mintzberg, H., Quinn, J. B., & Ghoshal, S. (1998). The strategy process. Revised European edition, Englewood Cliffs, NJ: Prentice Hall. Pearce, J. A., & Robinson, R. B. (2007). Strategic management: Formulation, implementation, and control. The University of Oklahoma, Irwin. Perera, F. P. R., & Peiró, M. (2012). Strategic Planning in Healthcare Organizations. Revista Española de Cardiología (English Edition), 65(8): 749-754. http://dx.doi.org/10.1016/j.rec.2012.04.004 Poister, T. H., & Streib, G. (2005). Elements of strategic planning and management in municipal government: Status after two decades. Public administration review, 65(1), 45-56. http://dx.doi.org/10.1111/j.1540-6210.2005.00429.x Rusjan, B., & Alic, M. (2010). Capitalising on ISO 9001 benefits for strategic results. International Journal of Quality & Reliability Management, 27(7), 756-778. Rütten, A., Röger, U., Abu-Omar, K., & Frahsa, A. (2009). Assessment of organizational readiness for health promotion policy implementation: test of a theoretical model. Health Promotion International, 24(3), 243-251. http://dx.doi.org/10.1093/heapro/dap016 Saleh, S., Kaissi, A., Semaan, A., & Natafgi, N. M. (2013). Strategic planning processes and financial performance among hospitals in Lebanon. The International Journal of Health Planning and Management, 28, 34-45. http://dx.doi.org/10.1002/hpm.2128 Schwartz, R. W., & Cohn, K. H. (2002). The necessity for physician involvement in strategic planning in healthcare organizations. The American journal of surgery, 184(3), 269-278. Schwartz, R. W., & Pogge, C. (2000). Physician leadership: essential skills in a changing environment. The American journal of surgery, 180(3), 187-192. Schwartz, R. W., & Pogge, C. (2000). References Arah, O. A., Klazinga, N., Delnoij, D., Ten Asbroek, A., & Custers, T. (2003). Conceptual frameworks for health systems performance: a quest for effectiveness, quality, and improvement. International Journal for Quality in Health Care, 15(5), 377-398. http://dx.doi.org/10.1093/intqhc/mzg049 Cleverley, W. O., & Cameron, A. E. (2007). Essentials of Healthcare Finance. Jones & Bartlett Learning: Sudbury, Massachusetts. El-Jardali, F., Jamal, D., Abdallah, A., & Kassak, K. (2007). Human resources for health planning and management in the Eastern Mediterranean region: facts, gaps and forward thinking for research and policy. Human Resources for Health, 5(1), 9. http://dx.doi.org/10.1186/1478-4491-5-9 Glickman, S. W., Baggett, K. A., Krubert, C. G., Peterson, E. D., & Schulman, K. A. (2007). Promoting quality: the health-care organization from a management perspective. International Journal for Quality in Health Care, 19(6), 341-348. http://dx.doi.org/10.1093/intqhc/mzm047 Groene, O., Brandt, E., Schmidt, W., & Moeller, J. (2009). The Balanced Scorecard of acute settings: development process, definition of 20 strategic objectives and implementation. International Journal for Quality in Health Care, 21(4), 259-271. http://dx.doi.org/10.1093/intqhc/mzp024 64 Global Journal of Health Science Vol. 7, No. 2; 2015 www.ccsenet.org/gjhs Copyrights Copyright for this article is retained by the author(s), with first publication rights granted to the journa Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 65
https://openalex.org/W3034645015	https://publikationen.bibliothek.kit.edu/1000122259/83838032	English	null	Eight Weeks Later—The Unprecedented Rise of 3D Printing during the COVID-19 Pandemic—A Case Study, Lessons Learned, and Implications on the Future of Global Decentralized Manufacturing	Applied sciences	2,020	cc-by	7,552	Received: 18 May 2020; Accepted: 14 June 2020; Published: 16 June 2020 Abstract: The eruption of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (corona virus disease, COVID-19) in Wuhan, China, and its global spread has led to an exponentially growing number of infected patients, currently exceeding over 6.6 million and over 390,000 deaths as of the 5th of June 2020. In this pandemic situation, health systems have been put under stress, and the demand for personal protective equipment (PPE) exceeded the delivery capabilities of suppliers. To address this issue, 3D printing was identiﬁed as a possible solution to quickly produce PPE items such as face shields, mask straps, masks, valves, and ear savers. Around the world, companies, universities, research institutions, and private individuals/hobbyists stepped into the void, using their 3D printers to support hospitals, doctors, nursing homes, and even refugee camps by providing them with PPE. In Germany, the makervsvirus movement took up the challenge and connected thousands of end users, makers, companies, and logistic providers for the production and supply of face shields, protective masks, and ear savers. The Karlsruhe Institute of Technology (KIT) also joined the makervsvirus movement and used its facilities to print headbands for face shield assemblies and ear savers. Within this paper, the challenges and lessons learned from the quick ramp up of a research laboratory to a production site for medium-sized batches of PPE, the limitations in material supply, selection criteria for suitable models, quality measures, and future prospects are reported and conclusions drawn. Keywords: COVID-19; pandemic; 3D FFF printing; personal protective equipment (PPE); zero lead-time; customized mass production; quality assessment; makervsvirus applied sciences applied sciences Eight Weeks Later—The Unprecedented Rise of 3D Printing during the COVID-19 Pandemic—A Case Study, Lessons Learned, and Implications on the Future of Global Decentralized Manufacturing Tobias Mueller , Ahmed Elkaseer * , Amal Charles , Janin Fauth, Dominik Rabsch, Amon Scholz, Clarissa Marquardt, Katja Nau and Steﬀen G. Scholz j * Correspondence: ahmed.elkaseer@kit.edu; Tel.: +49-0721-608-25754 1. Introduction The industry for additive manufacturing (AM)/3D printing has grown rapidly in recent years, with increasing numbers of companies adopting various AM technologies for their production and with research in AM growing in output every year. However, researchers in the AM ﬁeld rarely have the opportunity, as they do now, to truly and immediately contribute towards a topic that is genuinely aﬀecting almost everyone in the world at this moment: the 2020 coronavirus virus disease (COVID-19) pandemic. A novel strain of the coronavirus family, SARS-CoV-2, was detected in the city of Wuhan in China in December of 2019. This recent coronavirus strain causes the infectious disease COVID-19, which has A novel strain of the coronavirus family, SARS-CoV-2, was detected in the city of Wuhan in China ecember of 2019. This recent coronavirus strain causes the infectious disease COVID-19, which has Appl. Sci. 2020, 10, 4135; doi:10.3390/app10124135 Appl. Sci. 2020, 10, 4135 Appl. Sci. 2020, 10, 4135 2 of 14 since then spread all over the world, infecting over 6.6 million people as of the 5th of June 2020 and causing the death of over 390,000 people worldwide [1]. Having been identiﬁed in 215 countries and areas, this disease resulted in the shutdown of most travel, global business, and recreational activities. Amidst this, rising demands, panic buying, and disruption of established global supply chains have resulted in massive shortages in some countries of the personal protective equipment (PPE) that is essential for healthcare workers to protect themselves and their patients from the highly infectious virus. PPE includes gloves, medical masks, respirators, goggles, face shields, ear savers, etc., with supplies expected to take months to reach the healthcare professionals that are currently in desperate need of it. The World Health Organization (WHO) estimated that in order to meet the global demand for the current crisis, industries need to increase their current production levels by 40% [2]. However, covering this increased demand is hindered by many factors, with most personnel either working from home or on leave, thereby creating a large gap between the demand and the supply due to the high ramp-up time of traditional manufacturing techniques. This, incidentally, created an opportunity for the AM community to step up and try to fulﬁll local demands. 1. Introduction Diﬀerent movements popped up all over the world with university laboratories, companies, as well as individual hobbyists and makers answering the call to action to supply local hospitals, emergency departments, and those in need with the required PPE. Similar to other European countries such as Belgium, Switzerland, Denmark, and Austria, in Germany, a coalition of enthusiastic makers called makervsvirus [3] was set up as a response to the COVID-19 crisis in order to connect and establish a nationwide network of production hubs ensuring closed connectivity, communication, and mutual fulﬁlment of PPE demands whenever and wherever required. Anyone with access to maker equipment such as 3D printers, either privately or as part of an organization, was able to register as a potential maker and get involved in the movement to immediately start producing PPE as per the requirements given by the hub, see Figure 1. All those in need of personal protection equipment could equally approach the makervsvirus community/hubs (as indicated by a blue symbol in the map below) by signing up on the website to place their request for equipment such as face shields or ear savers. Figure 1. Network of hubs (blue symbols) connected via makervsvirus.org with those requiring personal protective equipment (PPE) [3]. Figure 1. Network of hubs (blue symbols) connected via makervsvirus.org with those requiring personal protective equipment (PPE) [3]. Appl. Sci. 2020, 10, 4135 3 of 14 As a Karlsruhe Institute of Technology (KIT) research group in AM technologies working on the optimization of the fused ﬁlament fabrication (FFF) printing process [4,5], we made the decision to participate in this makersvsvirus movement, and our FFF lab that had been closed due to the COVID-19 crisis re-opened within 24 h with a total of 14 FFF-printers supporting the production of PPE, see Figure 2. After a short ramp-up time required to test the diﬀerent PPE models with the required process parameters, the lab was fully operational to run 24/7 for several weeks, producing the needed PPE in alternating shifts, thereby also minimizing the potential risk to our researchers who were working in the lab. Figure 2. The 3D-printing setup at the Karlsruhe Institute of Technology (KIT) Institute for Automation and Applied Informatics. Figure 2. The 3D-printing setup at the Karlsruhe Institute of Technology (KIT) Institute for Automation and Applied Informatics. Most of the group members committed all of their available working hours to the makersvsvirus project. 1. Introduction The intention of this paper is not to present major scientiﬁc progress in AM as a result of these 3D printing activities, but rather to share opinions and discussions on the lessons learned when converting a research lab into a production line, pumping out PPE parts as quickly as possible, as opposed to our normal practices of scientiﬁc research. The paper, therefore, journeys through the research groups’ experiences of the last 8 weeks of being part of the makersvsvirus movement, concluding with an approach to extrapolate the lessons learned during this time period to provide a vision for the future of decentralized production and its challenges as envisioned by Industry 4.0 principles. 2. Accessibility of 3D Printing for Everyone Along with the European Commission’s call [6] to provide support in the ﬁght against the COVID-19 pandemic, a number of online crowd initiatives and platforms bundling the 3D printing community were set up as a technological strategy to control the pandemic in a cross-border, fast, and eﬀective way. This has resulted in the evolution of a global online network of volunteers and experts from the 3D printing community that works to ensure the optimal exploitation of regional 3D printing capabilities towards a trans-regional response to the primary product shortages and supply chain disruptions; the aim of the network is to supply the right products at the right time in the right place and for the right people. Needs and requests from society are collected in a centralized manner and converted into appropriate measures, which are then communicated via collective online calls for help and can thus be processed by various volunteers and experts from the community. In the case of the COVID-19 crisis, the main requested products for 3D printing were headbands for face shields and straps for surgical masks. This distributed problem-solving format provides an optimal platform to exchange recent information and ﬁndings, make open access ﬁles available, and share relevant Appl. Sci. 2020, 10, 4135 4 of 14 guidelines, data models, and more while reacting to the day-by-day needs and arising challenges to cope with the novelty of required solutions and uncertainty in ﬁghting the pandemic. This recent development is based on a crowd-based organizational model leveraging the collective intelligence of the crowd, while integrating ideas and contributions from outside the traditional organizational boundaries, exploiting technologies like peer-to-peer platforms, and transferring value-creating activities to the crowd [7]. However, this applies not only to the opening of idea and solution generation processes, but also especially to the opening of access to urgently needed goods and services from the crowd. The background idea behind this concept is a shared economy, where access is given to underutilized goods and services [8] in the form of renting, sharing, swapping, borrowing, or other forms of oﬀering goods and services [9]. The concept of a shared economy that is normally used to achieve more sustainable consumption was used here as a means of providing access to highly needed goods and services to be able to implement the support services resulting from the online community. 3. 3D Printing: Capabilities and Challenges The term additive manufacturing encompasses diﬀerent manufacturing processes that allow a layer-wise building of 3D models, which is why a more commonly used term for additive manufacturing is 3D printing. Within the COVID-19 crisis, the capabilities of 3D printing have made it possible to quickly react to the outbreak by producing diﬀerent types of medical or sanitary equipment, of which there were shortages throughout the whole world, by starting production with nearly no ramp-up time. g g y g p y p p One essential aspect of 3D printing compared to conventional manufacturing processes is its opportunity for a short response time [10]. Due to its ability to print a functional part directly from an existing Computer-aided design (CAD) model, there is only a short lead-time compared to traditional manufacturing techniques. The lead-time is mainly needed for preparation of the individual print settings and the setting of the printer itself. The huge variety of available 3D printing materials makes it easy to select the appropriate material for a speciﬁc product application or task, such as sterilization of medical equipment. Printing itself can be done with only a minimum or even no material waste at all, which also makes 3D printing an eco-friendly alternative to subtractive manufacturing methods [11]. Only a small fraction of the used material, such as support structures and brims, is removed in the post-processing of the printed part in cases where such structures are necessary to print the model due to its complex geometry or for increased adhesion on the print bed [12]. The design and relevance of support structures is a complete research ﬁeld on its own due to the fact that the use of supports signiﬁcantly increases print time and energy consumption but also reduces surface quality and is therefore to be avoided whenever possible. p Another advantage of Additive Manufacturing (AM) is its ability to print complex parts directly in one step instead of having to assemble individual parts after manufacturing. Besides that, the part characteristics can be exactly predeﬁned. The deﬁned characteristics such as mechanical strength can be directly inﬂuenced by material choice, inﬁll density, inﬁll pattern, and many additional parameters. As these can be changed after each produced part, optimization routines are also much faster than in traditional manufacturing. With all these properties of 3D printing at hand, some drawbacks also have to be considered. 2. Accessibility of 3D Printing for Everyone An essential part of this concept in the recent crisis was the idea of infrastructure sharing: making infrastructure, such as 3D printers, available for external purposes and use cases to cover the demand for emergency products in the appropriate places. As 3D printing devices are currently available to the public, this infrastructure sharing enables individuals to join in such activities and support their communities in times of critical shortages to cover extraordinary demands. 3. 3D Printing: Capabilities and Challenges Due to the layer-wise building approach and the current technologies available, AM is typically slower than mass-production techniques such as injection molding. The limited adhesion between layers also introduces failures that reduce the mechanical properties of 3D printed parts in comparison to fully dense materials used for subtractive methods or generated by molding or direct extrusion, as those parts exhibit a larger surface area and pores that act as inhibitors for failure or contact points for 5 of 14 Appl. Sci. 2020, 10, 4135 aggressive media [13]. Finally, the printing parameters have a huge impact on the quality of the ﬁnal parts. Small changes in print temperature or printer calibration can lead to totally diﬀerent outcomes and even a total failure of the whole print. Particular interest lies in the inﬂuence of printing parameters on the mechanical properties of printed parts [14,15]. This research shows that the parameters of building direction, layer height, and printing temperature have particular inﬂuence. On the other hand, inﬁll patterns become a less relevant parameter when a certain threshold of inﬁll percentage has been passed. However, the studies indicate that printing parameters have a high inﬂuence on the quality of printed parts and their properties. This indicates that a near 100% quality assurance is necessary for a fast ramp-up of a small-to-medium production, as is the case in a pandemic situation such as the current COVID-19 crisis. Especially in the production of medical equipment, it is crucial to closely monitor the manufacturing processes. The challenge for printing face shields, especially in this crisis, lies in ﬁnding the most eﬃcient means of production, as the printing time of the part increases exponentially with the demand for the highest possible quality of the part. The goal for the selection of optimal printing parameters is to achieve the best possible quality in the shortest possible printing time to eﬃciently cover the time-sensitive high demand for face shields. Therefore, the capabilities of 3D printing such as fast ramp-up, freedom of geometry, and material choice make this technology the ideal tool to respond to a rapidly emerging demand within a very short time without the need for machines or tooling specially developed for this speciﬁc purpose. 4. Design for 3D Printing in a Pandemic Situation A major advantage for 3D printing is the fast way iterations of speciﬁc designs can be brought into a physical form for testing and evaluation. While this is beneﬁcial in the development phase of a product and shortens the time to market, in the case of a pandemic situation, this also can lead to problems. With the outbreak of the COVID-19, diﬀerent makers and companies immediately sprung to action and released CAD designs for diﬀerent medical appliances to overcome shortages in diﬀerent areas. These included surgical face shields [16,17], respiratory masks, and ventilator valves [18] as some examples. As all of these are crucial products for personal protection, the number of diﬀerent CAD designs released posed some challenges for makers willing to help. First of all, most of the CAD designs, particularly those for respiratory masks, were not tested and proven beforehand. This resulted in minor issues such as masks not being comfortable to wear, or more severe issues with the masks not functioning properly or even harming the user (patient) by not providing suﬃcient air ﬂow or lacking viral protection. Some designs also required a large amount of post-production assembly of additional parts, which in itself undermines the use of 3D printing for its ability to create complex parts without the need for additional manufacturing steps. There are some more intricate designs available for respiratory masks [19], but these often have highly individualized parts, making use of 3D scanning techniques to adjust them to the user. Face shields and surgical mask straps are the most commonly produced items in the current pandemic situation. Even for those rather simple products, a variety of diﬀerent designs were published with the original face shield design (Figure 3a) by Josef Prusa [20] being the most commonly used. To satisfy the high demand, print-time optimized models of the face shields were quickly generated and published as well as stacked versions of this model, making use of the whole z-axis of higher volume printers (Figure 3b). Similar eﬀorts were being made for surgical mask straps. A wide variety of CAD designs is available at the moment, ranging from simple, easy, and fast to print types to very intricate ones with a quick release feature (Figure 4 a–c). 6 of 14 Appl. Sci. 2020, 10, 4135 Figure 3. 5.1. Raw Materials The materials used for the printing activities carried out in context of the makervsvirus movement were selected according to the type of item to be printed and the preferences of the individual hubs in Karlsruhe and Ulm with which the KIT was cooperating. As an immediate reaction to the shortage of PPE, polylactic acid (PLA) became the material of choice for the makers due to its general popularity for the fused ﬁlament fabrication process [23]. PLA is a thermoplastic and a so-called biopolymer, because it is produced from renewable resources such as corn starch, tapioca roots, or sugar cane, which makes it environmentally friendlier than classic polymers. It is also biodegradable under certain aerobic conditions such as in industrial composting. It can be printed at relatively low temperatures (190–220 ◦C) and only requires the print bed to be moderately heated to 50–60 ◦C to ensure a secure attachment of the print to the platform. Post-processing is easy, and PLA is perfect for complicated geometries and has excellent coloring options. Therefore, it is favored for many prototyping applications and provides suﬃcient strength in combination with good aesthetic qualities for most applications. In turn, PLA has its drawbacks, such as its relatively low temperature resistance [24]: it begins to soften at around 50 ◦C. It is also not considered food safe [25], mainly because bacteria can build up during the printing process itself or in crevices due to defects in the printed parts, which is always a concern. As a consequence of PLA’s drawbacks, polyethylene terephthalate glycol (PETG) was subsequently selected as the material of choice for printing most of the PPE equipment. PETG, like PLA, belongs to the polyester group of plastics and is a popular 3D printing material for applications requiring high impact resistance and ductility [26]. PETG is a more specialized ﬁlament, with a more sensitive process window compared to PLA. However, for most applications where physical properties of a component are stressed, a material such as PETG is preferred over PLA. Parts made of PETG have a much better temperature resistance than PLA, with some varieties showing stability up to 250 ◦C. In general, most PETG parts remain stable up to a temperature of 70 ◦C, facilitating its use in products intended for temperature-based sterilization processes. PETG is also able to better handle UV-light and other weather-based inﬂuences compared to PLA, favoring its outdoor usage. 4. Design for 3D Printing in a Pandemic Situation Headband designs for protective face shields; (a) simpliﬁed design for faster printing, (b) Prusa design, (c) ﬁnal face shield in use. Figure 3. Headband designs for protective face shields; (a) simpliﬁed design for faster printing, (b) Prusa design, (c) ﬁnal face shield in use. Figure 4. Surgical mask straps; simple, (a) fast printing model, (b) complex quick-release model top, (c) in use. Figure 4. Surgical mask straps; simple, (a) fast printing model, (b) complex quick-release model top, (c) in use. Appl. Sci. 2020, 10, 4135 7 of 14 As most of these PPE designs are being published by private persons on various 3D printing community websites such as thingiverse [21] or myminifactory [22], they did not undergo any of the oﬃcial functional testing, approval, or certiﬁcation processes usually required for PPE products and even more importantly for medical devices. There are a limited number of certiﬁed products available, as the testing and certiﬁcation process is time consuming. Despite the time constraints, Prusa Research had their design and face shield assembly certiﬁed by the Czech Ministry of Health. Often, the makers had their printed parts tested by hospitals or doctors individually for their applicability and sterilization properties. To avoid legal issues, the shields are delivered with a notice attached that these are not certiﬁed medical products and have to be tested for their intended use beforehand. As a consequence, makers have to make an educated decision on which designs to put into production in times of supply shortages. The designs have to fulfil a number of key properties: functionality as intended for the speciﬁc purpose, reliability/durability in use, printability, and short printing time are some of the points to consider when choosing a model. • being sturdy and mechanically sound enough to withstand continuous usage for long amounts of time; 5.2. Quality Control of Final Printed Parts As for any 3D printed component, a number of issues were faced in terms of quality that needed to undergo process optimization in order to establish a standardized process. Some of the issues were typical errors that can occur for other geometries as well, some were speciﬁc to the models used and represent the aforementioned issue of community-based designs often not being tested properly to function as intended. 5.1. Raw Materials PETG is not biodegradable, but can be recycled into new ﬁlament or other products without loss in quality. Finally, it is considered to be food safe, but as every 3D printed part will contain numerous nooks, crannies, cracks, holes, etc. that bacteria can thrive in, a proper cleaning or sterilization before usage is crucial [27]. The application of 3D printing for the production of PPE such as the face shields brings a number of requirements that have to be fulﬁlled: • being sturdy and mechanically sound enough to withstand continuous usage for long amounts of time; Appl. Sci. 2020, 10, 4135 8 of 14 • being ﬂexible enough to be assembled together with elastic components that will stress the part especially because of the head bands; • being ﬂexible enough to be assembled together with elastic components that will stress the part, especially because of the head bands; • being ﬂexible enough to be assembled together with elastic components that will stress the part, especially because of the head bands; • being light weight and not feeling like a burden when being worn; • being light weight and not feeling like a burden when being worn; • being light weight and not feeling like a burden when being worn; having the ability to be sterilized so they can be used under safe conditions in a medical environment • having the ability to be sterilized so they can be used under safe condition Considering these requirements, both PLA and PETG satisfy these criteria and can be used for the production of PPE. However, since PETG is more resistant to elevated temperatures (sterilization) and because of its higher impact resistance, it was the favored material for PPE in the makervsvirus hubs. PLA allows inexperienced makers to quickly produce good printing results due to its ease of use, but is not as mechanically strong and temperature resistant. 5.2. Quality Control of Final Printed Parts 5.2. Quality Control of Final Printed Parts 5.2.1. Issues That Occurred during the Printing Process The following quality issues were observed and will be discussed further in the following paragraphs: insuﬃcient adhesion, stringing, within-stack lack of separation, and breaking of parts. Insuﬃcient adhesion is as result of a cold build platform, a cold preceding layer, or insuﬃcient calibration of the Z-axis. Consequently, the ﬁlament does not attach to the surface of the bed or the previous layer, instead, it detaches from the whole part or the surface due to insuﬃcient bonding between layers. Figure 5 shows an example of mid-print failure due to insuﬃcient layer adhesion. Detachment of the ﬁrst layer can cause the part to be stuck to the heated nozzle, which can result in material build-up at the hot end and in turn fatal damage to the heaters, thermistors, or even the whole printer. Therefore, close monitoring of the ﬁrst layer is crucial for a successful print. Figure 5. Insuﬃcient adhesion between layers on a part made from polylactic acid (PLA). Figure 5. Insuﬃcient adhesion between layers on a part made from polylactic acid (PLA). Stringing is a more common issue with PETG filaments. Due to overheating of the nozzle, small stringy filaments are attached to the actual parts, see Figure 6. These small stringy ﬁlaments are particular 9 of 14 Appl. Sci. 2020, 10, 4135 visible in areas with rapid printhead movements, where the material is stretched out as the retraction of the ﬁlament is not suﬃcient. A solution for this issue is to either print with a lower printing temperature or to tweak the retraction settings. The optimized settings for avoiding major stringing issues with PETG are given in Table 1. Figure 6. Image of 8 head bands that were bonded together and the surface shows stringing of the PETG ﬁlament. Figure 6. Image of 8 head bands that were bonded together and the surface shows stringing of the PETG ﬁlament. Table 1. Optimized setting for 3D printing of polyethylene terephthalate glycol (PETG). Table 1. Optimized setting for 3D printing of polyethylene terephthalate glycol (PETG). Nozzle Temperature (◦C) Print Speed Perimeters (mm/s) Filament Retraction (mm) Retraction Speed (mm/s) Fan Speed (%) 255 50 0.8 35 50 Another major issue was observed during the printing of stacks of the face shields. During the stack printing approach, multiple face shields would unintentionally attach to each other, preventing later separation of the individual parts (Figure 6). 5.2.2. Quality Control of Printed Parts As the printed PPE parts were delivered to system-critical infrastructures, a proper quality testing methodology was developed in order to test the individual components and their suitability for use as PPE. Features to be tested included mechanical strength and integrity, ﬂexibility, as well as defects in the ﬁnal parts. The mechanical strength and integrity of the headbands was tested by using the thumb and index ﬁnger to press in the middle of the headband, the relevant section where the shield is attached and where the band touches the forehead. Pushed together, the band should not snap, crack, or delaminate. A second test was carried out by taking both ends and twisting the headband (pulling one end towards you, the other away from you). The band should not break, crack, or delaminate. Flexibility is particularly crucial for the assembly process of face shields, where the actual shielding foil and the rubber band were attached to the headband. To test the ﬂexibility of the generated part, both ends of the headbands (where the foil is attached to) were pushed forward until the headband was almost a straight line. If this could be done, ﬂexibility was ﬁne. In addition, both ends of the headband were pushed together to see if any failure occurred when the parts are mishandled. All parts were tested with this methodology before tagging and packaging the parts for delivery. Residual stringing ﬁlaments could be removed via heat treatment with a heat gun, and as a ﬁnal step, the inside surface of the headband, which is in contact with a user’s head, was sanded in order to make sure of a smooth surface. 5.2.1. Issues That Occurred during the Printing Process The printing of stacked parts is especially useful, as it maximizes the daily output of the headbands (one hour printing time per headband) and allows for a continuous printing process overnight. However, some prints showed insuﬃcient separation between the individual headbands. This was due to imperfect bridging parameters in the gaps between two parts. After optimizing the printing parameters by increasing the bridging speed and lowering the bridging temperature these issues could be solved, and stacked prints were possible. Breaking of parts during the removal process was another common issue. This issue occurred especially in the initial stages of ramping up the production and was even exacerbated due to issues of increased adhesion to the bed or to other parts (such as the aforementioned bonding in stacks). Establishing a standardized practice for part removal solved this problem, see Figure 7. The issues mentioned in the sections above were mainly speciﬁc to the printing of headbands during the production period. However, as in any production environment, general 3D printing issues were experienced occasionally, such as the clogging of the nozzles or warping of printed parts. 10 of 14 Appl. Sci. 2020, 10, 4135 Figure 7. Part broken while being separated from the stack. Figure 7. Part broken while being separated from the stack. Figure 7. Part broken while being separated from the stack. 6. Lessons Learned In a situation of nearly zero lead-time printing, as occurred in the current COVID-19 crisis, various obstacles had to be overcome that came along with running 3D printers 24 hours per day, 7 days per week. During the process of printing over 1350 headbands for face shields, around 800 surgical mask straps, and over 100 parts for diﬀerent applications such as door opening assistance, we experienced a number of issues. Maintenance: Some of the heat beds showed clear signs of wear during later printing periods, which resulted from printing the same part at the exact same spot repeatedly. This can result in poor bed adhesion, which in turn leads to warping issues and ultimately failing prints. A possible solution for further zero lead-time printing is to process numerous g-codes of the same CAD ﬁle, only diﬀering in model orientation, which consequently will reduce heat bed wear. In addition to this more severe issue, it was necessary to also do some minor maintenance work including cleaning clogged nozzles, lubricating rods and bearings, and removing plastic fragments that accumulated around the printers. Monitoring: We recognized early that monitoring the printing of the ﬁrst layers is very important. Many of the PLA-based prints failed because of insuﬃcient adhesion between the part and the printing Appl. Sci. 2020, 10, 4135 11 of 14 platform. However, once the ﬁrst layers were laid down properly, the printers did not need much surveillance, and there was a very high chance that the print would proceed as expected. Only printer speciﬁc issues occurred, such as ﬁlament loading problems with the Multi-Material-Unit of the Prusa i3 MK3S, but these could usually be solved quickly. This, however, shows that simply starting a print without supervision will only be viable if the setup is proven to run smoothly. In a research lab that is quickly transformed into a production site, this is often not the case, since the printers undergo frequent modiﬁcations, material changes, and parameter changes. Print Quality: We experienced noticeable variation in the quality of printed parts produced by diﬀerent printers, even from those that are similar and came from the same manufacturer. Small diﬀerences such as variations in axis calibration, wear of the print bed, or nozzle wear required printer-speciﬁc solutions. p p Material: There were noticeable diﬀerences in the ﬁlament of the same materials (PETG or PLA) from diﬀerent suppliers or of diﬀerent color. 6. Lessons Learned Printing parameters for achieving a high-quality print were often very diﬀerent depending on the ﬁlament’s brand or even the pigmentation. For example, the recommended printing temperature of some ﬁlaments was not suﬃcient to fuse layers perfectly, which resulted in delamination issues and forced us to raise the temperature by around 40 ◦C (up to 265 ◦C) for this particular kind of PETG. In contrast, other PETG ﬁlament was easily printed using the temperature suggested by the supplier, which was 250 ◦C in this case. Waste: The varying quality also resulted in a high amount of waste, as many face shield headbands either cracked or delaminated during separation of stacked prints or during quality assurance testing. However, the quality improved after we switched to ﬁlament from a single brand. Once an adequate set of printing parameters for this particular PETG was elaborated, the amount of waste dropped noticeably. Consequently, it is highly desirable to only use a single ﬁlament type with well-known printing parameters when it comes to zero lead-time production using FFF printers. Another option would be to increase the stress on manufacturers for more standardized or even certiﬁed ﬁlaments. Quality Control: Finally, since the main products printed in this crisis were medical appliances, a good quality was crucial to make the parts usable and to avoid any harm to the staﬀwearing the PPE. This could only be maintained by setting up 100% quality control for the printed parts. Every printed headband had to be checked for cracks, stringing, material accumulation that could cause hot spots while wearing the face shield, and more. This also included post-processing, such as sanding, to achieve the best possible quality. In comparison to well-dialed mass-production processes, this is certainly a drawback in the 3D printing approach, but one that is also counterweighted by the possibility for a fast set up and ﬂexible manufacturing process chain, able to quickly react to changing demands. 7. Conclusions and Future Prospects This experience has, however, reinforced one of the main benefits of AM technology: its ability to react and adapt to change as quickly as possible, a prerequisite for the enabling of an agile manufacturing environment. This became immediately evident to us as the time taken for the complete deployment of all printers for this eﬀort was only a matter of days from when the decision was made to support the project until full production capabilities were reached with two shifts deployed. This eﬀort proved beneﬁcial as it was an eﬃcient solution that met the requirements for PPEs from the start of the crisis and until others were able to mass-produce the face shields or ear savers using conventional methods. Therefore, 3D printing was validated as an eﬃcient agile production technology, supported by the fact that it is relatively inexpensive to adapt towards changes that may occur in design or parameters, while also enabling the ability for continuous improvement. In contrast, other production processes such as injection molding require signiﬁcant investment for the research, development, and production of tools, after which the design is pretty much locked. However, as 3D printing becomes a more industrially-relevant manufacturing technique, the support of printer manufacturers, reliability of the printers themselves, and the consistency and quality of materials has to evolve as well, providing a much more robust production alternative to classic subtractive or molding techniques. In our case, the FFF process proved to be eﬀective and reliable to provide good quality parts from the moment the need for more PPE arose until mass production systems were able to start fulﬁlling the burden. It is also to be noted that many diﬀerent AM processes do exist, all with diﬀerent production and quality standards; signiﬁcant research as well as policy eﬀorts are needed in all these diﬀerent AM processes in order to be able to react quickly when the next pandemic or crisis situation arrives. Since AM was able to ﬁll the gap this time, next time it might be able to provide mass production capabilities right from the start. As stated beforehand, the ﬂexibility of 3D printing combined with the freedom in geometry allowing very complex shapes and even assembly-free designs is a huge advantage. Three-dimensional FFF has proven itself to be mature enough for 24/7 production and application in the area of Industry 4.0. 7. Conclusions and Future Prospects This paper has reported on our experience as a research group (PIA (Process optimization, Information Management and Applications) at KIT, Karlsruhe, Germany) participating in a German campaign against the COVID-19 crisis by using our 3D FFF printing facilities along with our expertise and being part of a larger manufacturing/supplier hub for the production of medical and sanitary equipment with zero lead time. The fused ﬁlament fabrication technology proved to be the most convenient technology in this situation, as it is relatively easy to quickly set up a manufacturing environment. Due to the low costs of printers and material, the widespread use of FFF also enables a lot of makers to join forces and produce a large number of parts almost without any lead time. Open software applications and platforms also allow for sharing individual models, making it possible for hobbyists with no access to sophisticated CAD or modelling software to print complex designs and support movements such as the makervsvirus coalition. One can conclude that infrastructure sharing has proven itself in the context of a sharing economy as a promising measure for rapid and targeted access to urgently needed infrastructures and in its capacity to cope with crisis situations. Appl. Sci. 2020, 10, 4135 12 of 14 Supplying 3D printed PPE for hospitals, medical staﬀat doctors’ practices, and even in refugee camps with no hands-on or in-depth background in actual mass-production runs has given us the opportunity to experience the chances and challenges of zero lead-time additive mass manufacturing ﬁrsthand. It allows us to give feedback on the requirements that have to be taken into account when adopting AM for manufacturing processes. The experiences described above show that a connected manufacturing environment that includes constant feedback loops and communication between designers, material suppliers, production engineers, and logistics is crucial for AM-based manufacturing approaches. While this might be easy to implement for industries already operating in a just-in-time fashion, such as the automotive industry, it might be more diﬃcult for small enterprises that are facing other challenges such as the implementation of internet-of-things (IoT)-approaches or higher degrees of automation at the same time. The need for stocking spare parts and having an in-depth knowledge of each printers’ individual drawbacks and failure-prone parts can be challenging if only limited staﬀcan be assigned to the adoption of AM. Author Contributions: Conceptualization, S.G.S.; methodology, T.M., A.E., A.C., and S.G.S.; validation, T.M., A.E., A.C., D.R., and A.S.; formal analysis, A.E., T.M., and A.C.; investigation, A.E., T.M., and A.C.; resources, S.G.S.; data curation, A.E., T.M., and A.C.; writing—original draft preparation, T.M., A.C., A.E., J.F., D.R., A.S., and A.E.; writing—review and editing, A.E., C.M., and S.G.S.; visualization, A.E., T.M., and A.C.; project administration, S.G.S. and K.N.; funding acquisition, S.G.S. All authors have read and agreed to the published version of the manuscript. References 1. John Hopkins Corona Virus Resource Center. Available online: https://coronavirus.jhu.edu/map.html (accessed on 5 June 2020). 1. John Hopkins Corona Virus Resource Center. Available online: https://coronavirus.jhu.edu/map.html (accessed on 5 June 2020). 2. World Health Organization. Available online: https://www.who.int/news-room/detail/03-03-2020-shortage- of-personal-protective-equipment-endangering-health-workers-worldwide (accessed on 14 May 2020). 2. World Health Organization. Available online: https://www.who.int/news-room/detail/03-03-2020-shortage- of-personal-protective-equipment-endangering-health-workers-worldwide (accessed on 14 May 2020). 3. MakervsVirus.org. Available online: makervsvirus.org (accessed on 6 June 2020). 4. Elkaseer, A.; Schneider, S.; Scholz, S.G. Experiment-Based Process Modeling and Optimization for High-Quality and Resource-Eﬃcient FFF 3D Printing. Appl. Sci. 2020, 10, 2899. [CrossRef] 4. Elkaseer, A.; Schneider, S.; Scholz, S.G. Experiment-Based Process Modeling and Optimization for High-Quality and Resource-Eﬃcient FFF 3D Printing. Appl. Sci. 2020, 10, 2899. [CrossRef] 5. Elkaseer, A.; Mueller, T.; Charles, A.; Scholz, S. Digital Detection and Correction of Errors in As-built Parts: A Step Towards Automated Quality Control of Additive Manufacturing. In Proceedings of the World Congress on Micro and Nano Manufacturing, Portorož, Slovenia, 18–20 September 2018; Valentincic, J., Ed.; Research Publishing Services: Singapore, 2018; pp. 389–392. 5. Elkaseer, A.; Mueller, T.; Charles, A.; Scholz, S. Digital Detection and Correction of Errors in As-built Parts: A Step Towards Automated Quality Control of Additive Manufacturing. In Proceedings of the World Congress on Micro and Nano Manufacturing, Portorož, Slovenia, 18–20 September 2018; Valentincic, J., Ed.; Research Publishing Services: Singapore, 2018; pp. 389–392. 7. Täuscher, K. Leveraging Collective Intelligence: How to Design and Manage Crowd-Based Business Models. Available online: https://doi.org/10.1016/j.bushor.2016.11.008 (accessed on 8 May 2020). 7. Täuscher, K. Leveraging Collective Intelligence: How to Design and Manage Crowd-Based Business Models. Available online: https://doi.org/10.1016/j.bushor.2016.11.008 (accessed on 8 May 2020). 8. Habibi, M.R.; Davidson, A.; Laroche, M. What managers should know about the sharing economy. Bus. Horiz. 2017, 60, 113–121. [CrossRef] 9. Kurtis, S.K.; Mont, O. Sharing economy business models for sustainability. J. Clean. Prod. 2020. [CrossRef] g y y 10. Pour, M.A.; Zanardini, M.; Bacchetti, A.; Zanoni, S. Additive Manufacturing Impacts on Productions and Logistics Systems. IFAC-Papers 2016, 49, 1679–1684. [CrossRef] 11. Liu, Z.; Jiang, Q.; Zhang, Y.; Li, T.; Zhang, H.C. Sustainability of 3D printing: A critical review and recommendations. In Proceedings of the ASME 2016 International Manufacturing Science and Engineering Conference, Blacksburg, VA, USA, 27 June–1 July 2016. MSEC2016. [CrossRef] g y 12. Jiang, J.; Xu, X.; Stringer, J. Support Structures for Additive Manufacturing: A Review. J. Manuf. Mater. Process. 2018, 2, 64. [CrossRef] 13. Erokhin, K.S.; Gordeev, E.G.; Ananikov, V.P. 7. Conclusions and Future Prospects Coupled with automated handling (e.g., robots removing prints from the print bed) and monitoring (e.g., visual surveillance with automated print parameter adaption) approaches, additive manufacturing has the potential to become the ﬁrst choice as a solution in an ever more ﬂexible production environment and accomplish the goal for mass production of highly customizable products. Appl. Sci. 2020, 10, 4135 13 of 14 13 of 14 Funding: This work was carried out with the support of the Karlsruhe Nano Micro Facility (KNMF, www.knmf. kit.edu), Project ID: 2020-024-029069, a Helmholtz Research Infrastructure at Karlsruhe Institute of Technology (KIT, www.kit.edu) and under the Helmholtz Research Program STN (Science and Technology of Nanosystems) at KIT. The authors acknowledge the support provided by the KIT-Publication Fund of the Karlsruhe Institute of Technology. Acknowledgments: The authors would like to gratefully acknowledge Prof. Dr. Veit Hagenmeyer, Head of the Institute for Automation and Applied Informatics, KIT, for the institutional support to conduct this project. The authors also acknowledge the support of Harald Molle of Newtec GmbH and all the people involved in the makervsvirus movement, in particular the hubs in Karlsruhe and Ulm. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Revealing interactions of layered polymeric materials at solid-liquid interface for building solvent compatibility charts for 3D printing applications. Sci. Rep. 2019, 9, 20177. [CrossRef] 14. Alafaghani, A.; Qattawi, A.; Alrawi, B.; Guzman, A. Experimental Optimization of Fused Deposition Modelling Processing Parameters: A Design-for-Manufacturing Approach. Procedia Manuf. 2017, 10, 791–803. [CrossRef] 15. Popescu, D.; Zapciu, A.; Amza, C.; Baciu, F.; Marinescu, R. FDM process parameters inﬂuence over the mechanical properties of polymer specimens: A review. Polymer Testing 2018, 69, 157–166. [CrossRef] 16. Flanagan, S.T.; Ballard, D.H. 3D Printed Face Shields: A Community Response to the COVID-19 Global P d i A d R di l [C R f] [P bM d] 15. Popescu, D.; Zapciu, A.; Amza, C.; Baciu, F.; Marinescu, R. FDM process parameters inﬂuence over the mechanical properties of polymer specimens: A review. Polymer Testing 2018, 69, 157–166. [CrossRef] 6. Flanagan, S.T.; Ballard, D.H. 3D Printed Face Shields: A Community Response to the COVID-19 Gl Pandemic. Acad. Radiol. 2020. [CrossRef] [PubMed] 17. Sapoval, M.; Gaultier, A.L.; Del Giudice, C.; Pellerin, O.; Kassis-Chikhani, N.; Lemarteleur, V.; Fouquet, V.; Tapie, L.; Morenton, P.; Tavitian, B.; et al. 3D-printed face protective shield in interventional radiology: Evaluation of an immediate solution in the era of COVID-19 pandemic. Diagn. Interv. Imaging 2020. [CrossRef] [PubMed] 18. Ishack, S.; Lipner, S.R. Applications of 3D Printing Technology to Address COVID-19 Related Supply Shortages. Am. J. Med. 2020. [CrossRef] [PubMed] Appl. Sci. 2020, 10, 4135 14 of 14 19. Swennen, G.R.J.; Pottel, L.; Haers, P.E. Custom-made 3D-printed face masks in case of pandemic crisis situations with a lack of commercially available FFP2/3 masks. IJOMS 2020. [CrossRef] [PubMed] 20. Report on Prusas Activities Surrounding the Printing of Face Shields. Available online: https://www.prusa3d. com/covid19/ (accessed on 4 May 2020). 21. Thingiverse – Digital Designs for Physical Objects. Available online: www.thingiverse.com (accessed on 16 May 2020). y 22. MyMiniFactory.com. Available online: www.myminifactory.com (accessed on 16 May 2020). 23. Pan, A.Q.; Huang, Z.F.; Guo, R.J.; Liu, J. Eﬀect of FDM Process on Adhesive Strength of Polylactic Acid(PLA) Filament. Key Eng. Mater. 2015, 667, 181–186. [CrossRef] 24. Macincinova-Benabidillah, K.; Boustta, M.; Coudane, J.; Vert, M. Can the Glass Transition Temperature of PLA Polymers Be Increased. In Polymers from Renewable Resources: Biopolyesters and Biocatalysts; Symposium Series; ACS: Washington, DC, USA, 2000; Volume 764, pp. 200–220. [CrossRef] 25. Is PLA food safe – The Truth. Available online: https://all3dp.com/2/is-pla-food-safe-what-you-really-need- to-know/ (accessed on 2 June 2020). 26. References Szykiedans, K.; Credo, W.; Osi´nski, D. Selected Mechanical Properties of PETG 3D Prints. Procedia Eng. 2017, 177, 455–461. [CrossRef] 27. Oth,O.; Dauchot,C.; Orellana,M.; Glineur,R.HowtoSterilize3DPrintedObjectsforSurgicalUse? AnEvaluation of the Volumetric Deformation of 3D-Printed Genioplasty Guide in PLA and PETG after Sterilization by Low-Temperature Hydrogen Peroxide Gas Plasma. Open Dent. J. 2020, 14, 1874–2106. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4241826065	https://www.qeios.com/read/OJN3V1/pdf	English	null	Ion Channel	Definitions	2,020	cc-by	113	Ion Channel National Cancer Institute National Cancer Institute Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Open Peer Review on Qeios Qeios ID: OJN3V1 · https://doi.org/10.32388/OJN3V1 Source National Cancer Institute. Ion Channel. NCI Thesaurus. Code C16754. A transmembrane pore that presents a hydrophilic channel for ions to cross a lipid bilayer down their electrochemical gradients. Some degree of ion specificity is usually observed and typically a million ions per second may flow. Channels may be permanently open, like the potassium leak channel or they may be voltage gated, like the sodium channel or ligand gated like the acetylcholine receptor. Qeios ID: OJN3V1 · https://doi.org/10.32388/OJN3V1 1/1
https://openalex.org/W4234426773	https://www.qeios.com/read/TSVDQO/pdf	English	null	CD2-Associated Protein	Definitions	2,020	cc-by	67	Qeios · Definition, February 8, 2020 Open Peer Review on Qeios Open Peer Review on Qeios CD2-Associated Protein National Cancer Institute Qeios ID: TSVDQO · https://doi.org/10.32388/TSVDQO Source National Cancer Institute. CD2-Associated Protein. NCI Thesaurus. Code C97249. CD2-associated protein (639 aa, ~71 kDa) is encoded by the human CD2AP gene. This protein plays a role in both cell division and protein-protein binding. Qeios ID: TSVDQO · https://doi.org/10.32388/TSVDQO 1/1
https://openalex.org/W2898798493	https://europepmc.org/articles/pmc6220338?pdf=render	English	null	Increased frequency of systemic pro-inflammatory Vδ1+ γδ T cells in HIV elite controllers correlates with gut viral load	Scientific reports	2,018	cc-by	9,437	Increased frequency of systemic pro-inflammatory Vδ1+ γδ T cells in HIV elite controllers correlates with gut viral load Received: 15 February 2018 Accepted: 17 October 2018 Published: xx xx xxxx Gregory S. Olson 1, Sarah W. Moore1, James M. Richter2, John J. Garber2, Brittany A. Bowman 1, Crystal A. Rawlings1, Meaghan Flagg1, Björn Corleis 1 & Douglas S. Kwon 1 Gregory S. Olson 1, Sarah W. Moore1, James M. Richter2, John J. Garber2, Brittany A. Bowman 1, Crystal A. Rawlings1, Meaghan Flagg1, Björn Corleis 1 & Douglas S. Kwon 1 γδ T cells predominate in the intestinal mucosa and help maintain gut homeostasis and mucosal immunity. Although HIV infection significantly alters these cells, what drives these perturbations is unclear. Growing evidence suggests that impaired intestinal immune function in HIV leads to chronic immune activation and disease progression. This occurs even in HIV controllers – individuals with undetectable HIV viremia without antiretroviral therapy (ART). We show that Vδ1+ cells, a subset of γδ T cells described as being important in intestinal barrier function, increase in frequency in HIV- infected individuals, including HIV controllers. These cells resemble terminally differentiated effector memory cells, producing the pro-inflammatory cytokines IFNγ, TNFα, and MIP-1β upon stimulation. Importantly, pro-inflammatory Vδ1+ cell frequency correlates with levels of HIV RNA in intestinal tissue but not in plasma. This study supports a model in which local viral replication in the gut in HIV controllers disrupts the phenotype and function of Vδ1+ cells, a cell type involved in the maintenance of epithelial barrier integrity, and may thereby contribute to systemic immune activation and HIV disease progression. A small proportion of individuals infected with human immunodeficiency virus type 1 (HIV-1, hereafter HIV) maintain low or undetectable viremia in the absence of antiretroviral therapy (ART). Despite this, these so-called “HIV controllers” still demonstrate increased morbidity and mortality associated with chronic systemic inflam- mation1–5. In addition, they have detectable viral replication in the gut and impaired gut barrier function6. Studies of HIV controllers therefore provide an opportunity to explore the impact of HIV on intestinal immune function in the absence of the confounding effects of ART. f Current models of HIV disease progression suggest that HIV-associated disruption of the gastrointestinal tract results in microbial translocation across a compromised intestinal epithelial barrier and subsequent chronic immune activation, disease progression, and increased mortality in HIV disease7,8. However, the cell types involved with the compromised intestinal barrier and subsequent chronic inflammation are not well understood. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, United States of America. 2Division of Gastroenterology, Massachusetts General Hospital, Boston, Massachusetts, United States of America. Correspondence and requests for materials should be addressed to D.S.K. (email: dkwon@mgh.harvard.edu) Results I Increased frequency of peripheral Vδ1+ cells in HIV controllers. Because the Vδ1+ cell subset is incompletely characterized in HIV controllers, we first used flow cytometry to analyze Vδ1+ cell subsets in PBMCs from HIV-uninfected control subjects and HIV-infected subjects from the following cohorts: HIV con- trollers (further subdivided into elite controllers (EC; HIV viral load (VL) undetectable) and viremic controllers (VC; HIV VL <2000 copies/ml)), ART treated, and ART untreated individuals (Table 1). These cells were defined as CD3+ Vδ1+ Vδ2− (Fig. 1a and see Supplementary Fig. S9). Although Vδ2+ cells represent the majority of circulating γδ T cells in healthy white individuals9,11,23,24, the ratio of Vδ2+ to Vδ1+ cells in healthy individuals is inverted among some self-reported racial groups25,26. Initial analyses were therefore conducted on subsets defined by self-reported race.i We found that the frequency of Vδ1+ cells among total T cells significantly increased in the blood of all white HIV-infected subjects compared to uninfected controls (Fig. 1). This increase was seen even in HIV-infected subjects with undetectable viremia. The Vδ1+ cell frequencies in EC (median 3.25%, range 0.38–9.87%), CT subjects (median 2.60%, range 0.22–11.3%), VC (median 5.78%, range 4.00–9.18%), and CU subjects (median 4.51%, range 2.61–12.3%) were all significantly higher than that in uninfected controls (median 0.85%, range 0.18–3.17%).f Interestingly, African American subjects showed a different pattern, with an increased Vδ1+ cell frequency only observed within the CU group (see Supplementary Fig. S1 and Supplementary Table 1). Due to this observa- tion, the known variations in γδ cell subsets across self-reported racial groups25,26, and limited access to intestinal samples from other self-reported racial groups, further phenotypic analysis of peripheral Vδ1+ cells was only performed in white subjects.iff p j To confirm that the differences in gender composition in our cohorts did not explain the differences in Vδ1+ cell frequency observed, we repeated the analysis after excluding female subjects. Because the Vδ1+ cell fre- quencies in male EC remained significantly increased compared to male uninfected controls (p < 0.05 and see Supplementary Fig. S2), we did not segment by gender in analyses.l Previous reports have shown that the increased frequency of Vδ1+ cells in HIV infection reflects increased absolute counts as well16–18. We used clinical CD4+ T cell counts to approximate absolute numbers of Vδ1+ cells in our HIV-infected cohorts. Increased frequency of systemic pro-inflammatory Vδ1+ γδ T cells in HIV elite controllers correlates with gut viral load CD4+ T cell count and plasma VL not determined for HIV negative cohort. Table 1. Clinical characteristics of white subjects. Values for age, CD4+ T cell count, and days since diagnosis represent mean ± standard deviation. aHCV (#) represents the number of subjects co-infected with Hepatitis C virus. CD4+ T cell count and plasma VL not determined for HIV negative cohort. differently, becoming more likely to produce the pro-inflammatory cytokines IFNγ, TNFα13,19, IL-17A14, and MIP1β15,20. Whether Vδ1+ cells are disturbed in HIV controllers is currently unknown. To better understand HIV-associated alterations in Vδ1+ populations and their potential role in gut dysfunc- tion, we characterized Vδ1+ cell phenotype and function in HIV-infected individuals, including HIV controllers. Since local viral replication in the gut has been implicated in the disruption of resident immune subsets and the impairment of intestinal barrier integrity21,22, we hypothesized that Vδ1+ cells in HIV controllers would resemble those in chronic progressive HIV infection, and that the alterations in Vδ1+ cell frequency and phenotype would be associated with local viral replication within intestinal tissue and not with replication in the blood. Increased frequency of systemic pro-inflammatory Vδ1+ γδ T cells in HIV elite controllers correlates with gut viral load Gamma delta (γδ) T cells are an ‘innate’ T cell type that expresses a semi-invariant T cell receptor (TCR). The differential usage of the Vδ1 or Vδ2 genes in the rearranged TCR differentiate two main subsets of human γδ T cells9. The recognition of both microbial products and stressed host cells allows γδ T cells to play an important role in immune responses against infections in general and viruses in particular10–12. While Vδ2+ cells primar- ily circulate in blood, Vδ1+ cells primarily localize within the mucosa of the gut as intraepithelial lymphocytes (IELs) and help to maintain epithelial function11. Their connection to HIV-associated gut dysfunction remains incompletely characterized. p y Progressive HIV infection drastically changes peripheral γδ T cell subsets13–19, including a depletion of Vδ2+ cells and an expansion of Vδ1+ cells in circulating blood16–18. Controlling viremia with ART does not fully cor- rect the inversion of the normal ratio of peripheral γδ T cell subsets16,17. The expanded Vδ1+ cells also behave 1The Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, United States of America. 2Division of Gastroenterology, Massachusetts General Hospital, Boston, Massachusetts, United States of America. Correspondence and requests for materials should be addressed to D.S.K. (email: dkwon@mgh.harvard.edu) SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 1 www.nature.com/scientificreports/ HIV Negative Elite Controller Chronic Treated Viremic Controller Chronic Untreated # subjects 17 15 13 4 11 Age (years) 43 ± 11 49 ± 9 45 ± 5 53 ± 8 40 ± 8 Male (%) 53 93 69 100 64 Hispanic/Latino (#) 1 0 0 0 2 CD4 + T Cells (/μL) 948 ± 228 679 ± 359 733 ± 172 464 ± 169 Plasma VL (copies/mL) undetectable undetectable 163 ± 53 44,559 ± 36,903 Days since diagnosis 6,386 ± 3,845 4,873 ± 2,549 8,133 ± 891 5,371 ± 2,441 HCV (#)a 0 2 4 0 2 Table 1. Clinical characteristics of white subjects. Values for age, CD4+ T cell count, and days since diagnosis represent mean ± standard deviation. aHCV (#) represents the number of subjects co-infected with Hepatitis C virus. CD4+ T cell count and plasma VL not determined for HIV negative cohort. Table 1. Clinical characteristics of white subjects. Values for age, CD4+ T cell count, and days since diagnosi represent mean ± standard deviation. aHCV (#) represents the number of subjects co-infected with Hepatitis virus. Results I There was a significant correlation between the Vδ1+ cell frequency of CD3+ cells and the absolute count (R2 = 0.6536, p < 0.0001). In addition, the patterns of Vδ1+ cells in EC and other HIV-infected cohorts remained unchanged (see Supplementary Figure S3), suggesting that the increased frequencies of Vδ1+ cells during HIV infection represent an expansion in absolute numbers of these cells. Peripheral Vδ1+ cells display an effector memory phenotype in HIV controllers. To further char- acterize the phenotype of the Vδ1+ cell population, expression of cell surface markers CD45RA and CD27 were used to distinguish four subsets of γδ T cells27–29, that correspond to memory subsets of αβ T cells30. Double positive cells (CD45RA+CD27+) demonstrate characteristics of naïve T cells, CD45RA−CD27+ cells resemble central memory cells, while CD45RA−CD27− cells resemble effector memory cells. CD45RA+CD27− γδ cells are the proposed equivalent of terminally differentiated effector memory cells27,28. p p q yff y We observed shifts in peripheral Vδ1+ cells away from a CD45RA+CD27+ naïve phenotype towards a CD45RA+CD27− terminally differentiated phenotype in all HIV-infected cohorts, including those with con- trolled viremia (Fig. 2a–c). Specifically, the proportion of Vδ1+ cells displaying the CD45RA+CD27+ naïve phenotype was significantly reduced in EC (median 6.03%, range 1.73–25%) and VC (median 2.24%, range SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Vδ1+ cells expanded in HIV-infected white subjects, despite controlled viremia. PBMCs were assessed by flow cytometry and viable CD3+ cells were analyzed for expression of Vδ1 and Vδ2. (a) Representative flow plots from an HIV- uninfected subject (Neg) and an elite controller (EC) are shown. The numbers in the quadrants represent the percentage of total CD3+ cells. (b) Summary data showing the median percentage of viable CD3+ cells that are Vδ1+ for Neg (n = 17), EC (n = 15), chronic treated (CT; n = 13), viremic controller (VC; n = 4), and chronic untreated (CU; n = 11) subjects. The medians of the cohorts were significantly different (p < 0.0001) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between Neg and each HIV- infected group. p < 0.05; p < 0.01; *p < 0.001. Figure 1. Vδ1+ cells expanded in HIV-infected white subjects, despite controlled viremia. PBMCs were assessed by flow cytometry and viable CD3+ cells were analyzed for expression of Vδ1 and Vδ2. (a) Representative flow plots from an HIV- uninfected subject (Neg) and an elite controller (EC) are shown. The numbers in the quadrants represent the percentage of total CD3+ cells. (b) Summary data showing the median percentage of viable CD3+ cells that are Vδ1+ for Neg (n = 17), EC (n = 15), chronic treated (CT; n = 13), viremic controller (VC; n = 4), and chronic untreated (CU; n = 11) subjects. The medians of the cohorts were significantly different (p < 0.0001) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between Neg and each HIV- infected group. p < 0.05; p < 0.01; p < 0.001. Figure 1. Vδ1+ cells expanded in HIV-infected white subjects, despite controlled viremia. PBMCs were assessed by flow cytometry and viable CD3+ cells were analyzed for expression of Vδ1 and Vδ2. (a) Representative flow plots from an HIV- uninfected subject (Neg) and an elite controller (EC) are shown. The numbers in the quadrants represent the percentage of total CD3+ cells. (b) Summary data showing the median percentage of viable CD3+ cells that are Vδ1+ for Neg (n = 17), EC (n = 15), chronic treated (CT; n = 13), viremic controller (VC; n = 4), and chronic untreated (CU; n = 11) subjects. www.nature.com/scientificreports/ The medians of the cohorts were significantly different (p < 0.0001) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between Neg and each HIV- infected group. p < 0.05; p < 0.01; p < 0.001. 1.31–4.89%) compared to uninfected controls (median 38.60%, range 5.73–70%) (Fig. 2a,b). Strikingly, almost all of the Vδ1+ cells displayed a terminally differentiated CD45RA+CD27− phenotype in HIV-infected cohorts. The proportions of Vδ1+ cells that were CD45RA+CD27− in both EC (median 78.50%, range 30.00–96.20%) and VC (median 88.05%, range 76.80–92.90%) were significantly higher than that in uninfected controls (median 23.30%, range 2.17–81.60%) (Fig. 2c).h We next assessed if Vδ1+ cells displayed additional evidence of activation. The percentage of Vδ1+ cells expressing both activation markers CD38 and HLA-DR31–33 increased significantly relative to uninfected controls only in cohorts with detectable viremia (Fig. 2d). Although cohorts with undetectable plasma VL trended towards increased median percentages of CD38+HLA-DR+ Vδ1+ cells, the increase did not reach statistical significance (Fig. 2d). g To determine if direct viral infection could explain the activation, we measured CD4 expression on Vδ1+ cells. Because the median frequency of CD4 on Vδ1+ cells was less than 10% in each cohort (see Supplemental Figure S4,), we did not pursue direct infection as a driver for the population level activation changes observed. Increased frequencies of peripheral Vδ1+ cells producing pro-inflammatory cytokines in HIV controllers. We also characterized whether these Vδ1+ cells had altered patterns of cytokine production in addition to increased markers of activation. Based on recent reports of γδ T cell function13–15,19, we measured the production of the pro-inflammatory cytokines IFNγ, TNFα, MIP-1β, and IL-17A by T cell subsets in response to various stimuli using intracellular cytokine staining. Because IL-17A production in γδ T cells was not detected after any condition—although it was in αβ T cells (see Supplementary Fig. S5)—we restricted subsequent analyses to IFNγ, TNFα, and MIP-1β.if γ, , β Vδ1+ cells from HIV-infected individuals produced significantly different cytokine signatures than those from uninfected controls upon stimulation with PMA/ionomycin, a potent non-specific activator of T cells. Frequencies of Vδ1+ cells producing IFNγ, TNFα, and MIP-1β (IFNγ+TNFα+MIP-1β+ Vδ1+ cells, hereafter SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Vδ1+ cells in HIV controllers display an activated, effector-like phenotype. PBMCs were characterized by flow cytometry and the phenotypes of viable CD3+Vδ1+ cells were assessed. (a) Representative flow plots from an HIV-uninfected subject (Neg) and an elite controller (EC). The numbers in each quadrant represent the percentage of CD3+Vδ1+ cells. (b–d) Summary data of median percentages of Vδ1+ cells that are CD45RA+CD27+ (b), CD45RA+CD27− (c), or HLA-DR+CD38+ (d) for Neg (n = 17), EC (n = 15), CT (n = 13), VC (n = 4), and CU (n = 11) subjects. The medians of the cohorts were significantly different for all three (b-d) summary graphs (p < 0.0001) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between HIV− and HIV+ groups. p < 0.05; p < 0.01; p < 0.001. Figure 2. Vδ1+ cells in HIV controllers display an activated, effector-lik characterized by flow cytometry and the phenotypes of viable CD3+Vδ1 flow plots from an HIV-uninfected subject (Neg) and an elite controller represent the percentage of CD3+Vδ1+ cells. (b–d) Summary data of me CD45RA+CD27+ (b), CD45RA+CD27− (c), or HLA-DR+CD38+ (d) fo VC (n = 4), and CU (n = 11) subjects. The medians of the cohorts were summary graphs (p < 0.0001) as assessed by the Kruskal-Wallis test. Du used to assess differences between HIV− and HIV+ groups. p < 0.05; * Figure 2. Vδ1+ cells in HIV controllers display an activated, effector-like phenotype. PBMCs were characterized by flow cytometry and the phenotypes of viable CD3+Vδ1+ cells were assessed. (a) Representative flow plots from an HIV-uninfected subject (Neg) and an elite controller (EC). The numbers in each quadrant represent the percentage of CD3+Vδ1+ cells. (b–d) Summary data of median percentages of Vδ1+ cells that are CD45RA+CD27+ (b), CD45RA+CD27− (c), or HLA-DR+CD38+ (d) for Neg (n = 17), EC (n = 15), CT (n = 13), VC (n = 4), and CU (n = 11) subjects. The medians of the cohorts were significantly different for all three (b-d) summary graphs (p < 0.0001) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between HIV− and HIV+ groups. p < 0.05; p < 0.01; *p < 0.001. Figure 2. Vδ1+ cells in HIV controllers display an activated, effector-like phenotype. PBMCs were characterized by flow cytometry and the phenotypes of viable CD3+Vδ1+ cells were assessed. www.nature.com/scientificreports/ (a) Representative flow plots from an HIV-uninfected subject (Neg) and an elite controller (EC). The numbers in each quadrant represent the percentage of CD3+Vδ1+ cells. (b–d) Summary data of median percentages of Vδ1+ cells that are CD45RA+CD27+ (b), CD45RA+CD27− (c), or HLA-DR+CD38+ (d) for Neg (n = 17), EC (n = 15), CT (n = 13), VC (n = 4), and CU (n = 11) subjects. The medians of the cohorts were significantly different for all three (b-d) summary graphs (p < 0.0001) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between HIV− and HIV+ groups. p < 0.05; p < 0.01; p < 0.001. “pro-inflammatory Vδ1+ cells”) were increased approximately ten-fold in HIV-infected individuals, even in the absence of detectable viremia (Fig. 3). Specifically, both EC (median 0.84% of CD3+ cells; range 0.19–3.92%) and CT subjects (median 1.18%; range 0.09–3.20%) had significantly higher frequencies of pro-inflammatory Vδ1+ cells than uninfected controls (median 0.10%; range 0.05–0.56%) (Fig. 3a,b). “pro-inflammatory Vδ1+ cells”) were increased approximately ten-fold in HIV-infected individuals, even in the absence of detectable viremia (Fig. 3). Specifically, both EC (median 0.84% of CD3+ cells; range 0.19–3.92%) and CT subjects (median 1.18%; range 0.09–3.20%) had significantly higher frequencies of pro-inflammatory Vδ1+ cells than uninfected controls (median 0.10%; range 0.05–0.56%) (Fig. 3a,b). g g Vδ1+ cells did not consistently produce any of the cytokines measured upon incubation with other stimuli. Stimulation with gag peptide pools resulted in no specific cytokine production in γδ T cells (see Supplementary Fig. S6), although responses were seen in CD8+ T cells in HIV-infected individuals (see Supplementary Fig. S7). Contrary to previous reports14, C. albicans stimulation did not lead to increased cytokine production in Vδ1+ cells (see Supplementary Fig. S6). ( pp y g ) Having shown that pro-inflammatory Vδ1+ cells increased as a percentage of CD3+ cells in HIV infec- tion (Fig. 3b), we investigated the proportion of cells within the Vδ1+ subset that produced pro-inflammatory cytokines. We found that a greater proportion of Vδ1+ cells from HIV-infected individuals produced the SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 4 ificreports/ Figure 3. Vδ1+ cells in HIV controllers produce inflammatory cytokines. PBMCs were stimulated with PMA/ionomycin for 6 hours and cytokine production was measured by intracellular cytokine staining. Viable CD3+Vδ1+ cells were analyzed for production of IFNγ, TNFα, MIP1β, and IL-17A. www.nature.com/scientificreports/ (d) Spearman’s rank correlation between the frequency of pro-inflammatory IFNγ+TNFα+MIP1β+ Vδ1+ cells of total CD3+ cells and the frequency of activated (HLA-DR+CD38+) CD8+ T cells out of total CD8+ T cells; Spearman’s ρ = 0.7107, p < 0.0001; the same subjects as in c, without CT (30 total subjects). Figure 3. Vδ1+ cells in HIV controllers produce inflammatory cytokines. PBMCs were stimulated with PMA/ionomycin for 6 hours and cytokine production was measured by intracellular cytokine staining. Viable CD3+Vδ1+ cells were analyzed for production of IFNγ, TNFα, MIP1β, and IL-17A. (a) Representative flow plots from an HIV-uninfected subject (Neg) and an elite controller (EC) showing percentages of Vδ1+ cells expressing IFNγ and TNFα. (b) Summary data showing the median percentage of viable CD3+ cells that are IFNγ+TNFα+MIP1β+ Vδ1+ for Neg (n = 9), EC (n = 14), CT (n = 11), and CU (n = 7) subjects. The medians were significantly different (p = 0.0008) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between HIV− and HIV+ groups. (c) The median distribution of Vδ1+ cells producing the indicated number of cytokines in each cohort. The distributions in HIV-infected cohorts were significantly different than that of HIV-uninfected subjects as assessed by the permutation analysis in SPICE (n = 10,000 iterations). p < 0.05; p < 0.01; p < 0.001. (d) Spearman’s rank correlation between the frequency of pro-inflammatory IFNγ+TNFα+MIP1β+ Vδ1+ cells of total CD3+ cells and the frequency of activated (HLA-DR+CD38+) CD8+ T cells out of total CD8+ T cells; Spearman’s ρ = 0.7107, p < 0.0001; the same subjects as in c, without CT (30 total subjects). Figure 3. Vδ1+ cells in HIV controllers produce inflammatory cytokines. PBMCs were stimulated with PMA/ionomycin for 6 hours and cytokine production was measured by intracellular cytokine staining. Viable CD3+Vδ1+ cells were analyzed for production of IFNγ, TNFα, MIP1β, and IL-17A. (a) Representative flow plots from an HIV-uninfected subject (Neg) and an elite controller (EC) showing percentages of Vδ1+ cells expressing IFNγ and TNFα. (b) Summary data showing the median percentage of viable CD3+ cells that are IFNγ+TNFα+MIP1β+ Vδ1+ for Neg (n = 9), EC (n = 14), CT (n = 11), and CU (n = 7) subjects. The medians were significantly different (p = 0.0008) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between HIV− and HIV+ groups. www.nature.com/scientificreports/ (a) Representative flow plots from an HIV-uninfected subject (Neg) and an elite controller (EC) showing percentages of Vδ1+ cells expressing IFNγ and TNFα. (b) Summary data showing the median percentage of viable CD3+ cells that are IFNγ+TNFα+MIP1β+ Vδ1+ for Neg (n = 9), EC (n = 14), CT (n = 11), and CU (n = 7) subjects. The medians were significantly different (p = 0.0008) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between HIV− and HIV+ groups. (c) The median distribution of Vδ1+ cells producing the indicated number of cytokines in each cohort. The distributions in HIV-infected cohorts were significantly different than that of HIV-uninfected subjects as assessed by the permutation analysis in SPICE (n = 10,000 iterations). p < 0.05; p < 0.01; p < 0.001. (d) Spearman’s rank correlation between the frequency of pro-inflammatory IFNγ+TNFα+MIP1β+ Vδ1+ cells of total CD3+ cells and the frequency of activated (HLA-DR+CD38+) CD8+ T cells out of total CD8+ T cells; Spearman’s ρ = 0.7107, p < 0.0001; the same subjects as in c, without CT (30 total subjects). www.nature.com/scientificreports/ Figure 3. Vδ1+ cells in HIV controllers produce inflammatory cytokines. PBMCs were stimulated with PMA/ionomycin for 6 hours and cytokine production was measured by intracellular cytokine staining. Viable CD3+Vδ1+ cells were analyzed for production of IFNγ, TNFα, MIP1β, and IL-17A. (a) Representative flow plots from an HIV-uninfected subject (Neg) and an elite controller (EC) showing percentages of Vδ1+ cells expressing IFNγ and TNFα. (b) Summary data showing the median percentage of viable CD3+ cells that are IFNγ+TNFα+MIP1β+ Vδ1+ for Neg (n = 9), EC (n = 14), CT (n = 11), and CU (n = 7) subjects. The medians were significantly different (p = 0.0008) as assessed by the Kruskal-Wallis test. Dunn’s multiple comparison tests were used to assess differences between HIV− and HIV+ groups. (c) The median distribution of Vδ1+ cells producing the indicated number of cytokines in each cohort. The distributions in HIV-infected cohorts were significantly different than that of HIV-uninfected subjects as assessed by the permutation analysis in SPICE (n = 10,000 iterations). p < 0.05; p < 0.01; p < 0.001. www.nature.com/scientificreports/ (c) The median distribution of Vδ1+ cells producing the indicated number of cytokines in each cohort. The distributions in HIV-infected cohorts were significantly different than that of HIV-uninfected subjects as assessed by the permutation analysis in SPICE (n = 10,000 iterations). p < 0.05; p < 0.01; *p < 0.001. (d) Spearman’s rank correlation between the frequency of pro-inflammatory IFNγ+TNFα+MIP1β+ Vδ1+ cells of total CD3+ cells and the frequency of activated (HLA-DR+CD38+) CD8+ T cells out of total CD8+ T cells; Spearman’s ρ = 0.7107, p < 0.0001; the same subjects as in c, without CT (30 total subjects). pro-inflammatory cytokines tested compared with uninfected controls (Fig. 3c). Although Vδ1+ cells not producing any cytokine upon PMA/ionomycin stimulation were the majority in uninfected controls (median 55.80%; range 20.23–74.09%), they were only a small fraction of total Vδ1+ cells in EC (median 15.45%; range 5.71–30.85%), CT subjects (median 11.37%; range 2.12–47.89%), and CU subjects (median 10.98%; range 2.79– 30.56%) (Fig. 3c). pro-inflammatory cytokines tested compared with uninfected controls (Fig. 3c). Although Vδ1+ cells not producing any cytokine upon PMA/ionomycin stimulation were the majority in uninfected controls (median 55.80%; range 20.23–74.09%), they were only a small fraction of total Vδ1+ cells in EC (median 15.45%; range 5.71–30.85%), CT subjects (median 11.37%; range 2.12–47.89%), and CU subjects (median 10.98%; range 2.79– 30.56%) (Fig. 3c).l We next determined whether pro-inflammatory Vδ1+ cells might be associated with chronic immune activa- tion observed during HIV infection31. We found the frequency of peripheral pro-inflammatory Vδ1+ cells sig- nificantly correlated with chronic immune activation as measured by the percentage of CD38+HLA-DR+ among Vδ1−Vδ2−CD8+ T cells) (Spearman’s ρ = 0.7107; p < 0.0001) (Fig. 3d). To avoid the confounding effects intro- duced by the interaction of ART and chronic immune activation, we excluded CT subjects from this analysis. Vδ1+ cells are present in the intestinal mucosa in HIV controllers. Because phenotypic and func- tional perturbations of peripheral Vδ1+ cells are seen even in cohorts with undetectable viremia, we hypothesized that the perturbations might rather be associated with local viral replication in intestinal tissues where Vδ1+ cells reside. To confirm that γδ T cells localize in the intestinal mucosa of HIV controllers, we used IHC to determine the percentage of γδ cells within the colonic epithelial layer. We found that 84% (265/317) of γδ T cells were SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. www.nature.com/scientificreports/ Total populations of mucosal-associated T cells were first identified as CD3+CD103+ cells34 and then the frequency of Vδ1+Vδ2− cells was measured (Fig. 4b). Vδ1+ cells constituted a large percentage of intestinal CD3+CD103+ cells and predominated in the colon of viremic controllers (median ntraepithelial in the colonic mucosa of EC (Fig. 4a), similar to the distribution in uninfected controls (79% 6/122 cells).il We further confirmed that γδ T cells reside in the gut mucosa of HIV controllers by flow cytometry of samples isolated from intestinal pinch biopsies. Total populations of mucosal-associated T cells were first identified as CD3+CD103+ cells34 and then the frequency of Vδ1+Vδ2− cells was measured (Fig. 4b). Vδ1+ cells constituted a large percentage of intestinal CD3+CD103+ cells and predominated in the colon of viremic controllers (median 38.0%; range 27.1–60.10%), elite controllers (median 27.4%; range 0.83–30.0%) and uninfected controls (median 16.10%; range 6.55–28.10%) (Fig. 4c). Frequency of pro-inflammatory Vδ1+ correlate with relative gut VL in HIV controllers. We next addressed whether frequencies of peripheral pro-inflammatory Vδ1+ cells were related to systemic or local gut viral replication in HIV controllers. Relative gut viral load (VL) was quantified from pinch biopsies and Vδ1+ cell functional analysis was performed on 12 EC, 2 VC, and 3 uninfected controls. Similar to a previous report6, we were able to detect HIV viral RNA in multiple gut compartments of the majority of HIV controllers, despite undetectable viral RNA in PBMCs and an undetectable clinical plasma VL (see Supplementary Table S2).il p pp y Significantly more peripheral pro-inflammatory Vδ1+ cells were present in subjects with detectable viral RNA in any gut compartment than in those with no detectable virus (p = 0.0464) (Fig. 5a). To confirm that the expansion of pro-inflammatory Vδ1+ cells was not explained by peripheral viral burden, we repeated the dichotomization using detectable virus measured either by PBMC-associated viral RNA (measured in the same manner as gut-associated virus) or plasma viremia (measured in the clinical setting). No significant difference was found in the frequencies of peripheral pro-inflammatory Vδ1+ cells between individuals dichotomized by PBMC-associated viral RNA (Fig. 5b) or between HIV-infected individuals dichotomized by plasma VL (see Supplementary Figure S8). Furthermore, Spearman’s ranked correlation coefficient showed that the frequency of peripheral pro-inflammatory Vδ1+ cells significantly correlated with the average relative gut VL (Spearman’s ρ = 0.5812, p = 0.0144) (Fig. 5c). www.nature.com/scientificreports/ Thus, the frequency of peripheral pro-inflammatory Vδ1+ cells was closely associated with intestinal but not peripheral levels of HIV. www.nature.com/scientificreports/ A large proportion of CD3+CD103+ mucosal-associated T cells in colon are Vδ1+. (a) A representative 20X image of a colon pinch biopsy stained with a monoclonal antibody against the γδ TCR. Note the intraepithelial location of the γδ+ cells (arrowheads). (b) Cells isolated from fresh intestinal pinch biopsies were analyzed by flow cytometry and viable CD3+ cells were gated for CD103 expression and subsequently analyzed for Vδ1 and Vδ2 expression. (c) Summary data showing the median percentage of CD3+CD103+ cells that are Vδ1+ in the transverse colon, duodenum, and terminal ileum of HIV-uninfected (Neg) subjects (n = 3) or HIV controllers (viremic controllers (n = 3), elite controllers (n = 3)). Figure 4. A large proportion of CD3+CD103+ mucosal-associated T cells in colon are Vδ1+. (a) A Figure 4. A large proportion of CD3+CD103+ mucosal-associated T cells in colon are Vδ1+. (a) A representative 20X image of a colon pinch biopsy stained with a monoclonal antibody against the γδ TCR. Note the intraepithelial location of the γδ+ cells (arrowheads). (b) Cells isolated from fresh intestinal pinch biopsies were analyzed by flow cytometry and viable CD3+ cells were gated for CD103 expression and subsequently analyzed for Vδ1 and Vδ2 expression. (c) Summary data showing the median percentage of CD3+CD103+ cells that are Vδ1+ in the transverse colon, duodenum, and terminal ileum of HIV-uninfected (Neg) subjects (n = 3) or HIV controllers (viremic controllers (n = 3), elite controllers (n = 3)). Figure 4. A large proportion of CD3+CD103+ mucosal-associated T cells in colon are Vδ1+. (a) A representative 20X image of a colon pinch biopsy stained with a monoclonal antibody against the γδ TCR. Note the intraepithelial location of the γδ+ cells (arrowheads). (b) Cells isolated from fresh intestinal pinch biopsies were analyzed by flow cytometry and viable CD3+ cells were gated for CD103 expression and subsequently analyzed for Vδ1 and Vδ2 expression. (c) Summary data showing the median percentage of CD3+CD103+ cells that are Vδ1+ in the transverse colon, duodenum, and terminal ileum of HIV-uninfected (Neg) subjects (n = 3) or HIV controllers (viremic controllers (n = 3), elite controllers (n = 3)). intraepithelial in the colonic mucosa of EC (Fig. 4a), similar to the distribution in uninfected controls (79%, 96/122 cells). We further confirmed that γδ T cells reside in the gut mucosa of HIV controllers by flow cytometry of samples isolated from intestinal pinch biopsies. Discussion The expansion of Vδ1+ cells in EC and VC seen in this study indicates that alterations in Vδ1+ cells occur even with immunologic control of HIV. Interestingly, we did not observe an expansion of Vδ1+ cells in a cohort of African American ECs, emphasizing the need raised by previous reports to account for race in studies on γδ subsets25,26.h alterations and prior studies of chronically HIV-infected cohorts16,17,35. The expansion of Vδ1+ cells in EC and VC seen in this study indicates that alterations in Vδ1+ cells occur even with immunologic control of HIV. Interestingly, we did not observe an expansion of Vδ1+ cells in a cohort of African American ECs, emphasizing the need raised by previous reports to account for race in studies on γδ subsets25,26.h The origin of the expanded Vδ1+ population remains unknown and is a promising area of future investigation. Vδ1+ populations acting as IELs differ from those in the lamina propria in their ontogeny, TCR repertoire, and their propensity to circulate in vasculature at steady state36,37. Deeper characterization of changes in cell localiza- tion during HIV infection and the application of next generation TCR sequencing of Vδ1+ clones in the gut and blood of HIV-infected individuals would help differentiate the possible source of the expanded Vδ1+ population in our cohorts. Direct infection of γδ cells has been reported in the literature38,39, but the rates of infection in these cells are very low and unlikely to fully account for the drastic phenotypic changes seen in the majority of Vδ1+ cells. Although a direct assessment of HIV infection was not conducted in this study, few Vδ1+ cells from each cohort expressed CD4 (see Supplemental Figure S4). Interestingly, HIV-infected individuals did have significantly decreased percentages of Vδ1+ cells that expressed CD4. Future studies to explore the possibility of depletion of CD4+ Vδ1+cells by direct infection might add to the growing appreciation of γδ cells as HIV targets38.htlhi The observed shift of peripheral Vδ1+ cells towards a pro-inflammatory Th1 profile in HIV-infected individ- uals is consistent with prior findings13,14,19, but has never, to our knowledge, been shown in a cohort with viral suppression in the absence of ART. Contrary to previous reports14,40, we did not detect specific IL-17A expression in peripheral γδ T cells stimulated with C. albicans or PMA/ionomycin. Discussion In this study, we found that peripheral Vδ1+ cells are increased in frequency in HIV controllers and produce multiple pro-inflammatory cytokines, similar to ART-treated and -untreated individuals infected with HIV. These perturbations are correlated with VL in the gut, but are independent of peripheral viral burden. We believe the increased frequency of Vδ1+ cells seen in this study represents a true expansion based on the phenotypic 6 SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 www.nature.com/scientificreports/ Figure 5. Levels of pro-inflammatory Vδ1+ cells correlate with gut-associated, but not plasma-associated, relative viral load. HIV gag RNA was quantified in PBMCs and intestinal pinch biopsies by qRT-PCR for HIV-uninfected (Neg) (n = 3), EC (n = 12), and VC (n = 2) subjects. Relative viral load (VL) was determined after normalizing the copies of gag to copies of RPS9. The average gut VL represents the mean VL across three intestinal compartments (transverse colon, duodenum, and terminal ileum). Subjects were divided into groups with detectable or undetectable viral load in any gut compartment or in the blood. (a,b) Summary data showing the percentage of pro-inflammatory (IFNγ+TNFα+MIP1β+) Vδ1+ cells out of total CD3+ cells, grouped by gut VL (a) or PBMC VL (b). Horizontal lines represent the median. A two-tailed Mann-Whitney test was used to compare the medians. (c) Spearman’s rank correlation between the average gut VL and the percentage of CD3+ cells that are pro-inflammatory Vδ1+. Figure 5. Levels of pro-inflammatory Vδ1+ cells correlate with gut-associated, but not plasma-associated, relative viral load. HIV gag RNA was quantified in PBMCs and intestinal pinch biopsies by qRT-PCR for HIV-uninfected (Neg) (n = 3), EC (n = 12), and VC (n = 2) subjects. Relative viral load (VL) was determined after normalizing the copies of gag to copies of RPS9. The average gut VL represents the mean VL across three intestinal compartments (transverse colon, duodenum, and terminal ileum). Subjects were divided into groups with detectable or undetectable viral load in any gut compartment or in the blood. (a,b) Summary data showing the percentage of pro-inflammatory (IFNγ+TNFα+MIP1β+) Vδ1+ cells out of total CD3+ cells, grouped by gut VL (a) or PBMC VL (b). Horizontal lines represent the median. A two-tailed Mann-Whitney test was used to compare the medians. (c) Spearman’s rank correlation between the average gut VL and the percentage of CD3+ cells that are pro-inflammatory Vδ1+. alterations and prior studies of chronically HIV-infected cohorts16,17,35. Methods S d Study participants. The study was approved by the Massachusetts General Hospital Institutional Review Board and was performed in accordance with the approved guidelines. All participants provided written informed consent. HIV elite controllers (EC) were defined by ≥3 undetectable plasma HIV-1 RNA (plasma VL) measurements spanning ≥12 months without ART. Chronic treated (CT) subjects were defined by undetecta- ble plasma VL measurements while on ART for ≥12 months prior to sample date. Viral blips <200 copies/mL were not exclusion criteria in either of these groups if the plasma VL became undetectable within a year. HIV viremic controllers (VC) were defined by detectable low levels of viremia, plasma VL of <2000 copies/mL, for ≥12 months without ART. Chronic untreated (CU) subjects had plasma VL >2000 copies/mL for ≥12 months without ART. Subject characteristics, including ethnicity and sex, were self-reported on intake forms. Flow cytometry analysis. Peripheral blood mononuclear cells (PBMCs) from venous blood collected in acid citrate dextrose tubes were separated by centrifugation on a Histopaque gradient and cryopreserved in liq- uid nitrogen. Cryopreserved PBMCs were thawed and 5 × 106 cells were stained for viability with LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Life Technologies). Surface markers were identified with the following mouse monoclonal antibodies (mAbs): FITC anti-Vδ1 (clone TS8.2, Thermo Fisher Scientific), PE anti-Vδ2 (B6, BD Biosciences), PE-CF594 anti-CD3 (UCHT1, BD Biosciences), Brilliant Violet 605 (BV605) anti-CD4 (RPA-T4, BD Biosciences), v500 anti-CD8 (SK1, BD Biosciences), APC-H7 anti-CD27 (M-T271, BD Biosciences), PE-Cy5 anti-CD45RA (HI100, BD Biosciences), Alexa Fluor 700 (AF700) anti-HLA-DR (G46-6, BD Biosciences), AF647 anti-CD38 (HIT2, Biolegend), PE-Cy7 anti-CD103 (Ber-ACT8, Biolegend), V450 anti-CD19 (HIB19, BD Biosciences), and Pacific Blue anti-CD14 (M5E2, BD Biosciences). Stained cells were fixed with 2% paraform- aldehyde before running on an LSRII flow cytometer (BD Biosciences). Flow data were analyzed with FlowJo (TreeStar). Intracellular cytokine staining analysis. Cryopreserved PBMCs were thawed and rested over- night at 37 °C, 5% CO2 at 2 × 106 cells/mL of R+ media (RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10 mM HEPES buffer, 2mM L-glutamine, 50 IU/mL Penicillin, 50 μg/mL Streptomycin) with 10% (v/v) FBS (Sigma-Aldrich). Cells were resuspended at 4 × 106/mL in R+ with 10% FBS, GolgiPlug (1.0 μg/mL, BD Biosciences), and soluble anti-CD28/CD49d mAbs (BD Biosciences). Stimuli included media only, a pool of gag overlapping peptides (2 μg/mL), C. albicans (106 bodies/mL), or phorbol 12-myristate 13-acetate (PMA; 50 ng/ mL) with ionomycin (1 μg/mL) (PMA/ionomycin, Ebioscience). Discussion This might be explained by the different approaches; our study assayed ex vivo cytokine production using short incubation times (6 hours), rather than following an extended (>1 week) in vitro expansion phase. SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 7 www.nature.com/scientificreports/ Chronic inflammation has been linked to disease progression and increased morbidity in HIV infection in general41,42 and in ECs in particular1,4,5. A disrupted intestinal epithelial barrier is thought to lead to micro- bial translocation from the gut into the periphery and subsequent immune activation, thereby exacerbating the chronic inflammation in HIV infection8. Multiple reports support large perturbations of Vδ1+ cell populations at mucosal surfaces, although the specifics of these changes differ: some groups report they expand in the rectum35 and duodenum43,44 of HIV-infected individuals, while others report they decrease in the duodenum29 and the vaginal mucosa45 during infection. These changes of Vδ1+ cells in the intestinal mucosa, their ability to robustly respond to stressed epithelial cells, and their role in maintaining intestinal barrier integrity support their possible involvement in the proposed immune activation that drives disease progression11,24,44. In fact, a study in rhesus macaques linked levels of microbial translocation in Simian Immunodeficiency Virus (SIV) infection with the expansion of Vδ1+ cells39. Another report connected microbial translocation, Vδ1+ cell expansion, and disease progression in the setting of acute HIV infection28. Our data add an additional component to support a model in which Vδ1+ cells might be involved in chronic inflammation in HIV resulting from loss of the intestinal bar- rier integrity. Specifically, we propose that local viral replication in intestinal tissues rather than systemic viral burden leads to the increased frequency, activated phenotype, and pro-inflammatory function of Vδ1+ cells in HIV-infected individuals. While a perturbed Vδ1+ subset might contribute to impaired gut function and chronic inflammation in EC and other HIV-infected subjects, further work is needed to explore the origins and mechanis- tic role of these cells in this process. Investigating this link will yield insight into the mechanisms of HIV-induced impairment of the intestinal immune system and potentially lead to novel interventions to decrease HIV-related chronic inflammation by targeting Vδ1+ cells. Methods S d After 6 hours at 37 °C, cells were stained with LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Life Technologies) before intracellular cytokine staining with the Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s instructions. i Cells were stained prior to permeabilization with the following mouse mAbs: FITC anti-Vδ1 (clone TS8.2, Thermo Fisher Scientific), PerCP anti-Vδ2 (B6, Biolegend), BV785 anti-PD-1 (EH12.2H7, Biolegend), BV510 anti-CD3 (UCHT1, BD Biosciences), BV605 anti-CD4 (RPA-T4, BD Biosciences), and APC-H7 anti-CD8 (SK1, BD Biosciences). Intracellular antigens were detected using mouse mAbs: AF647 anti-IL-17A (BL168, Biolegend), AF700 anti-TNFα (mAb11, BD Biosciences), PE anti-MIP-1β (D21-1351, BD Biosciences), and PE-Cy7 anti-IFNγ (B27, BD Biosciences).l Data acquisition was performed with an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar). No background subtraction was performed on cytokine-producing subsets. Cytokine expres- sion analysis was performed using SPICE version 5.1, from http://exon.niaid.nih.gov46. Candida albicans preparation. Candida was prepared as previous reported14. Briefly, C. albicans (Ca) was grown in RPMI 1640 medium for 2 days, washed twice in PBS, autoclaved, and used at a final concentration of 106 bodies/mL. 8 SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 www.nature.com/scientificreports/ Mononuclear cell isolation from intestinal tissue. Ten intestinal pinch biopsies from each site were collected by colonoscopy (transverse colon and terminal ileum) and upper endoscopy (duodenum) and placed immediately into 4 °C gut-wash (R+ supplemented with 222 μg/mL piperacillin, 28 μg/mL tazobactam, and 2.5 μg/ mL amphotericin B) and kept on ice <2 hours until processing by an adapted collagenase type II protocol47. Briefly, tissue was mechanically disrupted by passage through a 16-gauge needle before a 20 minute incubation at 37 °C with 0.50 mg/mL collagenase type II (Clostridium histolyticum, Sigma-Aldrich). The cells in the supernatant were filtered through a 70 μm cell strainer and resuspended in gut-wash with 10% FBS. This process was repeated on the remaining tissue and the combined cells from both rounds were kept on ice until further analysis. Immunohistochemical (IHC) staining and quantification of γδ cells in intestinal tissue. Serial sections (4 μm) of formalin-fixed paraffin-embedded pinch biopsies were stained for the TCR γ chain by an adapted protocol48. Briefly, epitope retrieval in 10 mM sodium citrate buffer (pH6.5) for 2 minutes in a Decloaking Chamber (Biocare Medical) preceded staining with anti-TCRγ mouse mAb (clone γ3.20, Thermo Scientific) and the DAB Envision+ system (Dako). References 1. Pereyra, F. et al. Increased coronary atherosclerosis and immune activation in HIV-1 elite controllers. AIDS (London, England) 26 2409–2412, https://doi.org/10.1097/QAD.0b013e32835a9950 (2012). 2. Ling, B. et al. The large intestine as a major reservoir for simian immunodeficiency virus in macaques with long-term, nonprogressing infection. The Journal of infectious diseases 202, 1846–1854, https://doi.org/10.1086/657413 (2010). 2. Ling, B. et al. The large intestine as a major reservoir for simian immunodeficiency virus in macaques with long-term, nonprogressing infection. The Journal of infectious diseases 202, 1846–1854, https://doi.org/10.1086/657413 (2010). p g gh f f p g 3. Hatano, H. et al. Prospective antiretroviral treatment of asymptomatic, HIV-1 infected controllers. PLoS pathogens 9, e1003691, https://doi.org/10.1371/journal.ppat.1003691 (2013). p g gh f f p g 3. Hatano, H. et al. Prospective antiretroviral treatment of asymptomatic, HIV-1 infected controllers. PLoS pathogens 9, e1003691 https://doi.org/10.1371/journal.ppat.1003691 (2013). 4. Hunt, P. W. et al. Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy. The Journal of infectious diseases 197, 126–133, https://doi.org/10.1086/524143 (2008).i 4. Hunt, P. W. et al. Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy. The Journal of infectious diseases 197, 126–133, https://doi.org/10.1086/524143 (2008).i 5. Sanchez, J. L. et al. Lymphoid fibrosis occurs in long-term nonprogressors and persists with antiretroviral therapy but may be reversible with curative interventions. The Journal of infectious diseases 211, 1068–1075, https://doi.org/10.1093/infdis/jiu586 (2015). 5. Sanchez, J. L. et al. Lymphoid fibrosis occurs in long-term nonprogressors and persists with antiretroviral therapy but may be reversible with curative interventions. The Journal of infectious diseases 211, 1068–1075, https://doi.org/10.1093/infdis/jiu586 (2015). 6. Hatano, H. et al. Comparison of HIV DNA and RNA in gut-associated lymphoid tissue of HIV-infected controllers and noncontrollers. AIDS (London, England) 27, 2255–2260, https://doi.org/10.1097/QAD.0b013e328362692f (2013). 7. Dandekar, S. Pathogenesis of HIV in the gastrointestinal tract. Current HIV/AIDS reports 4, 10–15 (2007). 8. Klatt, N. R., Funderburg, N. T. & Brenchley, J. M. Microbial translocation, immune activation, and HIV disease. Trends in microbiology 21, 6–13, https://doi.org/10.1016/j.tim.2012.09.001 (2013). microbiology 21, 6–13, https://doi.org/10.1016/j.tim.2012.09.001 (2013). 9. Pang, D. J., Neves, J. F., Sumaria, N. & Pennington, D. J. Understanding the complexity of gammadelta T-cell subsets in mouse and human. Immunology 136, 283–290, https://doi.org/10.1111/j.1365-2567.2012.03582.x (2012). gy p g j 9. Pang, D. J., Neves, J. F., Sumaria, N. & Pennington, D. J. Methods S d Identification of DAB+ cells in 20X images (TissueFAXS scanning sys- tem; Tissuegnostics) was assisted by HistoQUEST (Tissuegnostics) image analysis software and the percentage of intraepithelial γδ T cells was quantified for 2 slides per biopsy. Detecting and quantifying HIV viral load by qRT-PCR. Pinch biopsies of intestinal tissue were mechanically homogenized using a roto-stator (VWR) and a QIAshredder column. RNA was extracted using the RNeasy kit (Qiagen). Quantitative reverse transcription-PCR (qRT-PCR) was performed on a Roche LightCycler 480 system using the Brilliant II SYBR Green qRT-PCR kit (Agilent Technologies) according to the manufacturer’s instructions with HIV-1 gag SK462 (AGTTGGAGGACATCAAGCAGCCATGCAAAT) and SK431 (TGCTATGTCACTTCCCCTTGGTTCTCT). Relative HIV RNA copy numbers (viral load, VL) were normalized to levels of ribosomal S9 (RPS9) protein as determined by a separate qRT-PCR (forward: AAGGCCGCCCGGGAACTGCTGAC, reverse: ACCACCTGCTTGCGGACCCTGATA). Average relative gut VL was calculated as the mean VL across each measured gut compartment (transverse colon, terminal ileum, and duodenum). The limit of detection (10−7 relative copies) was used for compartments in which no HIV RNA was detected. Subjects were dichotomized into “undetectable” and “detectable” groups separately for PBMCs and for the gut. An individual with HIV viral load below the limit of detection in all measured gut compartments was placed in the “undetectable” group, otherwise they were categorized as “detectable”. Statistical analysis. Nonparametric tests were used to compare medians between groups. The Mann-Whitney test was used for comparisons of 2 groups and the Kruskal-Wallis test followed by Dunn’s post tests for >2 groups. Spearman rank order correlation coefficients were used to assess associations between con- tinuous variables. Differences were considered significant at p < 0.05. Graphpad Prism 5 was used for all analyses except comparison of distributions of cytokine production, which was performed in SPICE using a partial per- mutation test for 10,000 iterations as described46. Data Availability All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). y All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). www.nature.com/scientificreports/ Harris, L. D. et al. Mechanisms underlying gammadelta T-cell subset perturbations in SIV-infected Asian rhesus macaques. Blood 116, 4148–4157, https://doi.org/10.1182/blood-2010-05-283549 (2010). p g 0. Maher, C. O. et al. Candida albicans stimulates IL-23 release by human dendritic cells and downstream IL-17 secretion by Vdelta1 T cells. J Immunol 194, 5953–5960, https://doi.org/10.4049/jimmunol.1403066 (2015).l p g j 1. Deeks, S. G. HIV infection, inflammation, immunosenescence, and aging. Annual review of medicine 62, 141–155, https://doi org/10.1146/annurev-med-042909-093756 (2011).l g 42. Duprez, D. A. et al. Inflammation, coagulation and cardiovascular disease in HIV-infected individuals. PloS one 7, e44454, https:// doi.org/10.1371/journal.pone.0044454 (2012). g j p 3. Nilssen, D. E. et al. Intraepithelial gamma/delta T cells in duodenal mucosa are related to the immune state and survival time in AIDS. J Virol 70, 3545–3550 (1996). ( ) 4. Nilssen, D. E. & Brandtzaeg, P. Intraepithelial gammadelta T cells remain increased in the duodenum of AIDS patients despite antiretroviral treatment. PloS one 7, e29066, https://doi.org/10.1371/journal.pone.0029066 (2012). p g j et al. Loss of Intra-Epithelial Endocervical Gamma Delta (GD) 1 45. Strbo, N. et al. Loss of Intra-Epithelial Endocervical Gamma Delta (GD) 1 T Cells in HIV-Infected Women. American journal of reproductive immunology 75, 134–145, https://doi.org/10.1111/aji.12458 (2016). 46. Roederer, M., Nozzi, J. L. & Nason, M. C. SPICE: exploration and analysis of post-cytometric complex multivariate datasets. Cytometry. Part A: the journal of the International Society for Analytical Cytology 79, 167–174, https://doi.org/10.1002/cyto.a.21015 (2011). ( ) 47. Shacklett, B. L. et al. Optimization of methods to assess human mucosal T-cell responses to HIV infection. Journal of Immunological Methods 279, 17–31, https://doi.org/10.1016/s0022-1759(03)00255-2 (2003).h p g 8. Rodriguez-Pinilla, S. M. et al. TCR-gamma expression in primary cutaneous T-cell lymphomas. The American journal of surgica pathology 37, 375–384, https://doi.org/10.1097/PAS.0b013e318275d1a2 (2013). www.nature.com/scientificreports/ 5. Hudspeth, K. et al. Engagement of NKp30 on Vdelta1 T cells induces the production of CCL3, CCL4, and CCL5 and suppresse HIV-1 replication. Blood 119, 4013–4016, https://doi.org/10.1182/blood-2011-11-390153 (2012). 16. De Paoli, P. et al. A subset of gamma delta lymphocytes is increased during HIV-1 infection. Clinical and experimental immun 83, 187–191 (1991). , ( ) 17. Autran, B. et al. T cell receptor gamma/delta+ lymphocyte subsets during HIV infection. Clinical and experimental immunology 75, 206–210 (1989). 18. Li, H., Chaudhry, S., Poonia, B., Shao, Y. & Pauza, C. D. Depletion and dysfunction of Vgamma2Vdelta2 T cells in HIV disease: mechanisms, impacts and therapeutic implications. Cellular & molecular immunology 10, 42–49, https://doi.org/10.1038/ cmi.2012.50 (2013). 19. Boullier, S., Dadaglio, G., Lafeuillade, A., Debord, T. & Gougeon, M. L. V delta 1 T cells expanded in the blood throughout HIV infection display a cytotoxic activity and are primed for TNF-alpha and IFN-gamma production but are not selected in lymph nodes. J Immunol 159, 3629–3637 (1997). 0. Omi, K. et al. Inhibition of R5-tropic HIV type-1 replication in CD4(+) natural killer T cells by gammadelta T lymphocytes Immunology 141, 596–608, https://doi.org/10.1111/imm.12221 (2014).h gy p g 21. Chun, T. W. et al. Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy. The Journal of infectious diseases 197, 714–720, https://doi.org/10.1086/527324 (2008).f infectious diseases 197, 714–720, https://doi.org/10.1086/527324 f p g 2. Yukl, S. A. et al. Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. The Journal of infectious diseases 202, 1553–1561, https://doi.org/10.1086/656722 (2010). pyh J f f p g ( ) 3. Caccamo, N. et al. Mechanisms underlying lineage commitment and plasticity of human gammadelta T cells. Cellular & molecula i l 10 30 34 htt //d i /10 1038/ i 2012 42 (2013) pyh f f p g ( ) 3. Caccamo, N. et al. Mechanisms underlying lineage commitment and plasticity of human gammadelta T cells. Cellular & molecula immunology 10, 30–34, https://doi.org/10.1038/cmi.2012.42 (2013). pyh f f 23. Caccamo, N. et al. Mechanisms underlying lineage commitment and immunology 10, 30–34, https://doi.org/10.1038/cmi.2012.42 (2013). gy p g 24. Deusch, K. et al. A major fraction of human intraepithelial lymphocytes simultaneously expresses the gamma/delta T cell receptor, the CD8 accessory molecule and preferentially uses the V delta 1 gene segment. European journal of immunology 21, 1053–1059, https://doi.org/10.1002/eji.1830210429 (1991). p g j 5. Cairo, C. et al. www.nature.com/scientificreports/ Impact of age, gender, and race on circulating gammadelta T cells. Human immunology 71, 968–975, https://doi org/10.1016/j.humimm.2010.06.014 (2010). 26. Hviid, L. et al. High frequency of circulating gamma delta T cells with dominance of the v(delta)1 subset in a healthy population. International immunology 12, 797–805 (2000).fflh gy 7. Dieli, F. et al. Differentiation of effector/memory Vdelta2 T cells and migratory routes in lymph nodes or inflammatory sites. The Journal of experimental medicine 198, 391–397, https://doi.org/10.1084/jem.20030235 (2003). f p p g j 8. Li, Z. et al. gammadelta T cells are involved in acute HIV infection and associated with AIDS progression. PloS one 9, e106064 https://doi.org/10.1371/journal.pone.0106064 (2014).ff p g j p 29. Cimini, E. et al. Primary and Chronic HIV Infection Differently Modulates Mucosal Vdelta1 and Vdelta2 T-Cells Differentiation Profile and Effector Functions. PloS one 10, e0129771, https://doi.org/10.1371/journal.pone.0129771 (2015). if p g j p 0. Robinson, H. L. & Amara, R. R. T cell vaccines for microbial infections. Nature medicine 11, S25–32, https://doi.org/10.1038 nm1212 (2005). 1. Paiardini, M. & Muller-Trutwin, M. HIV-associated chronic immune activation. Immunological reviews 254, 78–101, https://doi org/10.1111/imr.12079 (2013). g 32. Jouen-Beades, F. et al. Similarity of expression of activation markers and CD28 on gamma delta and alpha beta-receptor T cells in HIV infection. Clinical immunology and immunopathology 79, 189–193 (1996).i gy p gy 33. Norazmi, M. N., Arifin, H. & Jamaruddin, M. A. Increased level of activated gamma delta lymphocytes correlates with di severity in HIV infection. Immunology and cell biology 73, 245–248, https://doi.org/10.1038/icb.1995.40 (1995). y gy gy p g 34. Shin, H. & Iwasaki, A. Tissue-resident memory T cells. Immunological reviews 255, 165–181, https://doi.org/10.1111/imr.12087 (2013). 35. Poles, M. A. et al. Human Immunodeficiency Virus Type 1 Induces Persistent Changes in Mucosal and Blood T Cells de Suppressive Therapy. Journal of Virology 77, 10456–10467, https://doi.org/10.1128/jvi.77.19.10456-10467.2003 (2003). h 6. Hayday, A. & Tigelaar, R. Immunoregulation in the tissues by gammadelta T cells. Nature reviews. Immunology 3, 233–242, https:/ doi.org/10.1038/nri1030 (2003). g 37. Holderness, J., Hedges, J. F., Ramstead, A. & Jutila, M. A. Comparative biology of gammadelta T cell function in humans, mice, and domestic animals. Annual review of animal biosciences 1, 99–124, https://doi.org/10.1146/annurev-animal-031412-103639 (2013). b l h l d l ll l f f h f p g 8. Soriano-Sarabia, N. et al. Peripheral Vgamma9Vdelta2 T Cells Are a Novel Reservoir of Latent HIV Infection. PLoS pathogens 11 e1005201, https://doi.org/10.1371/journal.ppat.1005201 (2015). p g j pp 39. References Understanding the complexity of gammadelta T-cell subsets in mouse and human. Immunology 136, 283–290, https://doi.org/10.1111/j.1365-2567.2012.03582.x (2012). gy g j 0. Hayday, A. C. Gammadelta T cells and the lymphoid stress-surveillance response. Immunity 31, 184–196, https://doi.org/10.1016/j immuni.2009.08.006 (2009). 1. Vantourout, P. & Hayday, A. Six-of-the-best: unique contributions of gammadelta T cells to immunology. Nature reviews Immunology 13, 88–100, https://doi.org/10.1038/nri3384 (2013). gy p g 2. Poccia, F. et al. Antiviral reactivities of gammadelta T cells. Microbes and infection/Institut Pasteur 7, 518–528, https://doi org/10.1016/j.micinf.2004.12.009 (2005).hhi g j 3. Dobmeyer, T. S. et al. Reciprocal alterations of Th1/Th2 function in gammadelta T-cell subsets of human immunodeficiency virus 1-infected patients. British journal of haematology 118, 282–288 (2002). 13. Dobmeyer, T. S. et al. Reciprocal alterations of Th1/Th2 function in gammadelta T cell subsets of human immunodeficiency virus 1-infected patients. British journal of haematology 118, 282–288 (2002). 14. Fenoglio, D. et al. Vdelta1 T lymphocytes producing IFN-gamma and IL-17 are expanded in HIV-1-infected patients and respond y phh gi y 1-infected patients. British journal of haematology 118, 282–288 (2002). 14. Fenoglio, D. et al. Vdelta1 T lymphocytes producing IFN-gamma and IL-17 are expanded in HIV-1-infected patients and respond to Candida albicans. Blood 113, 6611–6618, https://doi.org/10.1182/blood-2009-01-198028 (2009). p j f gy 4. Fenoglio, D. et al. Vdelta1 T lymphocytes producing IFN-gamma and IL-17 are expanded in HIV-1-infected patients and respond to Candida albicans. Blood 113, 6611–6618, https://doi.org/10.1182/blood-2009-01-198028 (2009). SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 9 www.nature.com/scientificreports/ Author Contributions G.S.O., S.W.M. and D.S.K. designed experiments. G.S.O., S.W.M., B.A.B., C.A.R., M.F. and B.C. conducted and analyzed experiments. J.M.R. and J.J.G. performed all endoscopy procedures. G.S.O. and D.S.K. wrote the manuscript. Acknowledgements g We’d like to thank Cristina Lofton and Shillah Nakulima for technical assistance, the entire clinical core at the Ragon Institute, and the pathology lab at Massachusetts General Hospital. SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 10 www.nature.com/scientificreports/ SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 SCIenTIfIC RepOrTS \| (2018) 8:16471 \| DOI:10.1038/s41598-018-34576-4 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-34576-4 Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W2165513094	https://rjb.revistas.csic.es/index.php/rjb/article/download/100/99/99	Spanish; Castilian	null	On the floristic relationships between Cuba and other Tropical regions based on the distribution of Cuban <i>Asplenium L.</i> species (Aspleniaceae, Pteridophyta)	Anales del Jardín Botánico de Madrid/Anales del Jardín Botánico de Madrid	2,003	cc-by	4,396	Resumen REGALADO GABANCHO, L. & C. SÁNCHEZ VILLA VERDE (2003). Relaciones de afinidad florísti- ca entre Cuba y otras regiones tropicales sobre la base de la distribución de las especies cuba- nas de Asplenium L. (Aspleniaceae, Pteridophyta). Anales Jará. Bot. Madrid 60(2): 395-403. A partir de la distribución mundial de las especies cubanas de Asplenium se confeccionó una matriz de presencia-ausencia de dichas especies en 17 áreas geográficas seleccionadas, sobre la cual se evaluó el índice de similitud de Sorensen y se realizó un Análisis de Parsimonia de Endemismos. Se presentan y discuten las relaciones de afinidad florística entre Cuba y el res- to de las regiones tropicales, obteniendo agrupamientos similares a partir de los dos métodos antes mencionados. Se confirma la estrecha relación existente entre las Antillas Mayores, en cuanto a la composición florística de sus floras pteridológicas. Se corrobora el carácter conti- nental de la pteridoflora de las Antillas Mayores. Palabras claves: Asplenium, Aspleniaceae, Cuba, fitogeografía. RELACIONES DE AFINIDAD FLORÍSTICA ENTRE CUBA Y OTRAS REGIONES TROPICALES SOBRE LA BASE DE LA DISTRIBUCIÓN DE LAS ESPECIES CUBANAS DE ASPLENIUM!.. (ASPLENIACEAE, PTERIDOPHYTA) por LEDIS REGALADO GABANCHO1 & CARLOS SÁNCHEZ VILLAVERDE1 ' Instituto de Ecología y Sistemática. Carretera de Varona, km 3,5. Capdevila, Boyeros. Apartado 8029. C.P. 1800 Ciudad de La Habana (Cuba) <ledisregalado@yahoo.com> 1 Jardín Botánico Nacional. Carretera del Rocío, km 3,5. Calabazar, Boyeros. Ciudad de La Habana (Cuba) <sanchez_villaverde@yahoo.es> por LEDIS REGALADO GABANCHO1 & CARLOS SÁNCHEZ VILLAVERDE1 ' Instituto de Ecología y Sistemática. Carretera de Varona, km 3,5. Capdevila, Boyeros. Apartado 8029. C.P. 1800 Ciudad de La Habana (Cuba) <ledisregalado@yahoo.com> 1 Jardín Botánico Nacional. Carretera del Rocío, km 3,5. Calabazar, Boyeros. Ciudad de La Habana (Cuba) <sanchez_villaverde@yahoo.es> Key words: Asplenium, Aspleniaceae, Cuba, phytogeography. INTRODUCCIÓN América tropical, con 3000 especies de pteridófitos, constituye una de las regiones más ricas del mundo, y son reconocidas cinco áreas importantes en cuanto a número de es- pecies. De acuerdo con TRYON (1972) y TRYON & TRYON (1982), dichas áreas son: las Antillas Mayores, con alrededor de 1200 es- pecies (PRÓCTOR, inéd.), el S de México, con 900; Costa Rica, con alrededor de 700; la re- gión andina, con 1500, y el SE de Brasil, con 600. En cada una de ellas el porcentaje de en- demismos es de alrededor del 40%. De estas regiones, el Caribe y en especial las Antillas han despertado el interés de los biogeógrafos durante tres décadas al compa- rar los patrones de distribución de distintos grupos de animales y plantas con la historia geológica de esta área (ROSEN, 1975; PAGE & LYDEARD, 1994). Asplenium L. es un género cosmopolita, con más de 700 especies descritas (Lovis, 1973; PRÓCTOR, 1985), y se encuentra repre- sentado en todos los continentes, la mayoría de las islas del Atlántico y en las islas del Pa- cífico hasta Hawai. En Cuba, el género Asple- nium ocupa el segundo lugar dentro de los Pteridófitos en cuanto a número de especies con 37 (SÁNCHEZ & REGALADO, 2003), las cuales generalmente se distribuyen en zonas húmedas de montaña a lo largo de todo el país. Este trabajo tiene como objetivo presentar y discutir las relaciones de afinidad florística entre Cuba y el resto de las regiones tropica- les, comparando los resultados obtenidos al aplicar dos métodos de análisis, a partir de la distribución mundial de las especies cubanas de Asplenium. ANALES JARDÍN BOTÁNICO DE MADRID, 60(2) 2003 ANALES JARDÍN BOTÁNICO DE MADRID, 60(2) 2003 396 TABLAI TIPOS COROLÓGICOS DE LAS ESPECIES CUBANAS DEL GÉNERO ASPLENIUM L. [La numeración coincide con la que aparece en la matriz básica (tabla 2) de datos y en el cladograma (fig. 3)] Especies 1. A. abscissum Willd. 2. A. alatum Humb. & Bonpl. ex Willd. 3. A. auriculatum Sw. 4. A. auritum Sw. 5. A. corderoanum Próctor 6. A. cristatum Lam. 7. A. cuneatum Lam. 8. A. delicatulum C. Presl 9. A. delitescens (Maxon) L.D. Gómez 10. A. dentatum L. 11. A. dimidiatum Sw. 12. A. diplosceuum Hieron. 13. A. dissectum Sw. 14. A. erosum L. 15. A. feei Kunze ex Fee 16. A. formosum Willd. 17. A. heterochroum Kunze 18. A. jenmanii Próctor 19. A. juglandifolium Lam. 20. A. laetum Sw. 21. A. auritum x monodon 22. A. monodon Liebm. 23. A. mortonii Duek 24. A. myriophyllum (Sw. ) C. Presl 25. A. praemorsum Sw. 26. A. pteropus Kaulf. 27. A. pumilum Sw. 28. A. radicans L. 29. A. rectangulare Maxon 30. A. rhomboidale Desvaux 31. A. salicifolium L. 32. A. serra Langsd. & Fischer 33. A. serratum L. 34. A. verecundum Champ. ex Fourn. 35. A. radicans x cuneatum 36. A. auriculatum x salicifolium 37. A. serra x erosum Tipo corológico Neotropical Neotropical Neotropical Pan tropical Macroantillano Neotropical Neotropical Neotropical Neotropical Pancaríbeo Surcaríbeo Macroantillano Neotropical Macroantillano Neotropical Pantropical Norcaríbeo Macroantillano Neotropical Pantropical Endemismo Norcaríbeo Endemismo Neotropical Pantropical Neotropical Pantropical Neotropical Macroantillano Macroantillano Neotropical Pantropical Neotropical Norcaríbeo Endemismo Endemismo Endemismo Los híbridos se han identificado sobre la base de caracte- Abstract REGALADO GABANCHO, L. & C. SÁNCHEZ VILLA VERDE (2003). On the floristic relationships between Cuba and other Tropical regions based on the distribution of Cuban Asplenium L. species (Aspleniaceae, Pteridophyta). Anales Jard. Bot. Madrid 60(2): 395-403 (in Spanish). REGALADO GABANCHO, L. & C. SÁNCHEZ VILLA VERDE (2003). On the floristic relationships between Cuba and other Tropical regions based on the distribution of Cuban Asplenium L. species (Aspleniaceae, Pteridophyta). Anales Jard. Bot. Madrid 60(2): 395-403 (in Spanish). Based on the world distribution of the species of Asplenium occurring in Cuba, a presence- absence matrix for 17 selected geographical áreas was constructed. This matrix was separate- ly evaluated with the Similarity Index of Sorensen and Parsimony Analysis of Endemicity (PAE). The floristic relationships between Cuba and the rest of tropical regions are presented and discussed. Similar results were obtained using both methods. The closest relationships among the Greater Antilles islands were confirmed from the composition of their pteridologi- cal floras. The continental affinity of Greater Antilles flora was corroborated. L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM 397 TOR, 1977,1985,1989, respectivamente), flo- ra Mesoamericana (ADAMS, 1995), en las floras de Guatemala, Ecuador y Perú (STOLZE, 1981, 1986, y TRYON & STOLZE, 1993), así como en las floras de Venezuela (MORTON & LELLINGER, 1966), Chiapas (SMITH, 1981) y de Norteamérica (WAGNER & al, 1993), y el trabajo sobre las relaciones florísticas de los pteridófitos entre el Neotrópico y Áfnca-Ma- dagascar (MORAN & SMITH, 2001). Los tipos corológicos fueron definidos a partir de la cla- sificación de BORHIDI (1996) para Cuba y fue- ron utilizados 7 de los 12 tipos, los cuales se relacionan a continuación: TOR, 1977,1985,1989, respectivamente), flo- ra Mesoamericana (ADAMS, 1995), en las floras de Guatemala, Ecuador y Perú (STOLZE, 1981, 1986, y TRYON & STOLZE, 1993), así como en las floras de Venezuela (MORTON & LELLINGER, 1966), Chiapas (SMITH, 1981) y de Norteamérica (WAGNER & al, 1993), y el trabajo sobre las relaciones florísticas de los pteridófitos entre el Neotrópico y Áfnca-Ma- dagascar (MORAN & SMITH, 2001). Los tipos corológicos fueron definidos a partir de la cla- sificación de BORHIDI (1996) para Cuba y fue- ron utilizados 7 de los 12 tipos, los cuales se relacionan a continuación: tropical más austral: el resto del territorio de Brasil, Perú, Bolivia, Paraguay y el NW de Argentina, según DE LA SOTA, 1973), África Tropical, Madagascar, India y Ceilán. Con los datos de distribución de los táxo- nes en dichas regiones se confeccionó una matriz de presencia-ausencia de las especies cubanas por área, donde 1 indica presencia, y 0, ausencia del taxon (tabla 2), a la cual se le aplicó el método Análisis de Parsimonia de Endemismos (PAE) (ROSEN, 1988). Este mé- todo se basa en los principios de la Sistemáti- ca Filogenética y permite inferir las relacio- nes biogeográficas entre diferentes áreas a partir del reconocimiento de patrones de dis- tribución de sus endemismos. No tiene en cuenta la filogenia particular de tales espe- cies, sino que asume como atributos de las áreas la presencia de las especies en éstas. Además, se añadió un área hipotética RA para enraizar el árbol. - Endemismos: Elementos presentes solo en Cuba. - Macroantillanos: Elementos que se en- cuentran distribuidos en las Antillas Ma- yores: Cuba, Jamaica, La Española y Puerto Rico. MATERIALES Y MÉTODOS Para el estudio de las relaciones florísticas entre Cuba y otros territorios a escala mundial se tomaron en cuenta únicamente las especies cubanas de Asplenium (tabla 1) y se utilizó la información recogida en las floras de las An- tillas Menores, Jamaica y Puerto Rico (PROC- Los híbridos se han identificado sobre la base de caracte- res macro- y micromorfológicos intermedios y la presen- cia de esporas marcadamente abortivas. ANALES JARDÍN BOTÁNICO DE MADRID, 60(2) 2003 ANALES JARDÍN BOTÁNICO DE MADRID, 60(2) 2003 398 TABLA 2 MATRIZ DE PRESENCIA/AUSENCIA DE LAS 37 ESPECIES DE ASPLENIUM, EMPLEADA EN EL ANÁLISIS DE PARSIMONIA DE ENDEMISMOS ( P A E ) Y DE SIMILITUD DE SORENSEN, PARA DETERMINAR LAS RELACIONES FLORÍSTICAS ENTRE CUBA Y OTROS TERRITORIOS A ESCALA MUNDIAL RA FLORIDA BERMUDAS BAHAMAS CUBA JAMAICA LA ESPAÑOLA PUERTO RICO ANTILLAS MENORES TRINIDAD-TOBAGO MÉXICO CENTROAMÉRICA N DE AMÉRICA DEL SUR C DE AMÉRICA DEL SUR ÁFRICA TROPICAL MADAGASCAR INDIA CEILÁN Especies 1234567890123456789012345678901234567 0000000000000000000000000000000000000 1000010001000000100001000010000011000 0000000000000000100000010000000000000 0000000001000000000000000000000000000 1111111111111111111111111111111111111 1111011001111111011101011111011110000 1111011001111101111101011111111110000 1011111001000011101100010111011110000 1010011000000001000100000010001100000 1010011000000000001100000100000110000 1011011011000011001101001111001110000 1111011011000011001100011111001110000 1111011011101011001100011111001110000 1111011110001011001000011111001110000 0001000000000001000100001010000100000 0000000000000000000100000000000000000 0000000000000001000000000000000000000 0000000000000001000000000000000000000 Fig. 1 .-Tipos corológicos de las especies cubanas de As- plenium: PT, pantropicales; NT, neotropicales; PC, pan- caríbeos; NC, norcaríbeos; SC, surcaríbeos; MA, macro- antillanos; E, endemismos. L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM - Pancaríbeos: Elementos que se encuen- tran distribuidos en el área del Caribe, desde Colombia y Venezuela, hasta Mé- xico, Florida y las Antillas. El análisis se llevó a cabo utilizando el pro- grama WINCLADA versión 0.9.99Í BETA (NKON, 1999), aplicando métodos heurísticos de búsqueda implícitos en el programa, a partir de un algoritmo de simplicidad (FARRIS, 1970) que se basa en una búsqueda aleatoria de topo- logías y determinación de árboles con el menor número de cambios. La fiabilidad de los resul- tados se determinó mediante los índices de consistencia (CI) (KLUGE & FARRIS, 1969) y de retención (RI) (FARRIS, 1989), implícitos tam- bién en los cálculos del programa. - Norcaríbeos: Elementos que se encuen- tran distribuidos en América Central, México, Bahamas o Florida y en las An- tillas, pero ausentes de Costa Rica, Pana- má, Colombia y Venezuela. - Surcaríbeos: Elementos que se encuen- tran distribuidos en las Antillas y el N de Sudamérica, pero ausentes de América Central, México y Florida. Para sintetizar las topologías obtenidas a partir del PAE, se construyeron árboles de consenso estricto, en los que se mantienen los ciados más consistentes en cuanto a sus rela- ciones florísticas. Este tipo de análisis se utili- za en aquellos casos en los que se obtiene un gran número de árboles con igual cantidad de pasos, lo cual es frecuente en los grupos con una amplia distribución. - Neotropicales: Elementos que se en- cuentran distribuidos en las regiones de América Central, América del Sur y las Antillas. - Pan tropicales: Elementos que se en- cuentran en cualquiera de estas combi- naciones: África-Neotrópico, Asia-Neo- trópico o Pacífico-Neotrópico. Los resultados obtenidos a partir del WIN- CLADA fueron comparados con los origina- dos al calcular el índice de similitud de SOREN- SEN (1948), a la matriz inicial de datos. Con di- cho índice se construye una matriz de simili- tud, cuya expresión gráfica es un dendrograma. Para ello se utilizó el paquete de programas es- tadísticos para el análisis de clusters de COYU- LA(1991). Para el análisis se tuvieron en cuenta 17 unidades geográficas operacionales: Florida, Bermudas, Bahamas, Cuba, Jamaica, La Es- pañola, Puerto Rico, Antillas Menores, Trini- dad-Tobago, México, Centroamérica, N de América del Sur (Colombia, Venezuela, Las Guyanas, cuenca amazónica de Brasil, Perú y Ecuador), centro de América del Sur (franja RESULTADOS Las especies cubanas de Asplenium se cla- sifican, según su tipo corológico, como sigue: seis elementos pantropicales, quince neotro- picales, uno pancaríbeo, tres norcaríbeos y uno surcaríbeo, seis elementos macroantilla- nos, y cinco endemismos cubanos (fig. 1; ta- bla 1). Las relaciones de afinidad florística obtenidas entre las unidades geográficas ope- racionales, a partir de la aplicación del índice de Sorensen a la matriz de presencia-ausencia construida, se reflejan en el dendrograma que se muestra en la figura 2. Como resultado del análisis de parsimonia de endemismos se obtuvieron ocho árboles igualmente parsimoniosos, con 66 pasos y un índice de consistencia CI = 56 y de retención Fig. 1 .-Tipos corológicos de las especies cubanas de As- plenium: PT, pantropicales; NT, neotropicales; PC, pan- caríbeos; NC, norcaríbeos; SC, surcaríbeos; MA, macro- antillanos; E, endemismos. L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM 401 los cuatro híbridos estériles conocidos solo de Cuba (A. auritum X monodon, A. auriculatum x salicifolium, A. serra x erosum y A. radi- cans x cuneatum) se encuentren también en Jamaica, La Española o en los territorios con- tinentales donde coincidan sus posibles espe- cies progenitoras. se relacionan de manera escalonada con el gru- po 1 (fig. 3), y es difícil la delimitación de las afinidades florístícas existentes entre ellos. África Tropical se relaciona con los grupos anteriores por la presencia en su territorio de A. formosum, A. laetum, A. pumilumyA. serra. Bahamas, Bermudas, Madagascar, India-Cei- lán y Florida se separan como grupos indepen- dientes. Las mayores afinidades se establecen entre Cuba, Jamaica y La Española, pues las tres is- las presentan regiones con grandes elevacio- nes (tabla 3), con una gran cantidad de sustra- tos y habitat, dispuestos a manera de un mo- saico ecológico, que ofrecen mayores posibi- lidades a este grupo de plantas. Puerto Rico, con menor afinidad florística, se separa del resto de las Antillas Mayores al poseer meno- res elevaciones y por ser mucho menos com- pleja geológicamente. A esto se debe que el grupo en estudio se encuentre menos repre- sentado en esta isla. El número de especies comunes entre Cuba y los territorios analizados, en orden decre- ciente, se relacionan a continuación: La Espa- ñola, con 27; Jamaica, con 26; Puerto Rico, con 20; N de América del Sur, con 21; Centro- américa y centro de América del Sur, con 19, respectivamente; México, con 18; Antillas Menores y Trinidad-Tobago, con 9, respecti- vamente; Florida, con 8; África Tropical, con 6; Bermudas, con 2, y Bahamas, Madagascar, India y Ceilán, con una especie cada uno. El número de especies de Asplenium en cada área está relacionado con la elevación de las islas y no con su extensión territorial, au- mentando en la medida en que lo hace la altu- ra sobre el nivel del mar (tabla 3). Esta con- clusión se corresponde con la preferencia de este grupo de plantas por los bosques húme- dos de montaña como zonas más favorables para su implantación. L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM 399 Fig. 2.-Dendrograma obtenido al aplicar el índice de Sorensen a la matriz de presencia/ausencia de las especies. 1, Flo- rida; 2, Bermudas; 3, Bahamas; 4, Cuba; 5, Jamaica; 6, La Española; 7, Puerto Rico; 8, Antillas Menores; 9, Trinidad- Tobago; 10, México; 11, Centroamérica; 12, N de América del Sur; 13, C de América del Sur; 14, África Tropical; 15, Madagascar; 16, India; 17, Ceilán. Fig. 2.-Dendrograma obtenido al aplicar el índice de Sorensen a la matriz de presencia/ausencia de las especies. 1, Flo- rida; 2, Bermudas; 3, Bahamas; 4, Cuba; 5, Jamaica; 6, La Española; 7, Puerto Rico; 8, Antillas Menores; 9, Trinidad- Tobago; 10, México; 11, Centroamérica; 12, N de América del Sur; 13, C de América del Sur; 14, África Tropical; 15, Madagascar; 16, India; 17, Ceilán. Estos grupos pueden reconocerse al mismo tiempo en la figura 3, aunque las relaciones que se establecen entre las OGU no son las mismas. El primer grupo obtenido a partir del índice de Sorensen, que relaciona Cuba, Jamaica y La Española, aparece tal cual en el cladograma del PAE. Viene apoyado por la distribución de cuatro especies: A. diplosceuum, A. erosum, AJenmanii, A. monodony A. rhomboidale. El segundo y tercer grupos, obtenidos a partir del índice de Sorensen, formados por los territo- rios continentales y Puerto Rico, y por Antillas Menores y Trinidad-Tobago, respectivamente, RI = 81. La topología de consenso estricto, que resume las ocho topologías anteriores, puede verse en la figura 3. RI = 81. La topología de consenso estricto, que resume las ocho topologías anteriores, puede verse en la figura 3. Al observar la figura 2 se distingue la for- mación de tres grupos coherentes: el primero, formado por Cuba, Jamaica y La Española; el segundo, constituido por los territorios conti- nentales, México y Puerto Rico, y el tercero, integrado por Antillas Menores y Trinidad- Tobago. Los tres grupos se relacionan con África Tropical, con la Florida y, más débil- mente, con Bahamas, Bermudas, Madagas- car, India y Ceilán. ANALES JARDÍN BOTÁNICO DE MADRID, 60(2) 2003 400 ANALES JARDÍN BOTÁNICO DE MADRID, 60(2) 2003 ANALES JARDÍN BOTÁNICO DE MADRID, 60(2) 2003 402 TABLA 3 EXTENSIÓN TERRITORIAL Y ELEVACIÓN DE LAS ANTILLAS MAYORES EN RELACIÓN CON EL NÚMERO DE ESPECIES DE ASPLENIUM PRESENTES EN SUS TERRITORIOS Territorio CUBA JAMAICA LA ESPAÑOLA PUERTO RICO Extensión territorial (km2) 113950 10880 74000 8800 Elevación sobre el nivel del mar (m) 1972 2250 3100 1350 Número total de especies 37 (SÁNCHEZ & REGALADO, 2003) 39 (PRÓCTOR, 1985) 48 (PRÓCTOR, inéd.) 24 (PRÓCTOR, 1989) Número de especies comunes con Cuba — 26 27 20 TABLA 3 TENSIÓN TERRITORIAL Y ELEVACIÓN DE LAS ANTILLAS MAYORES EN RELACIÓN CON EL NÚMERO DE ESPECIES DE ASPLENIUM PRESENTES EN SUS TERRITORIOS viento tendrán un patrón biogeográfico donde las afinidades florísticas estén determinadas por las características ecológicas de las islas más que por las barreras de dispersión exis- tentes entre éstas. migración ocurrió de África a América del Sur. No obstante, teniendo en cuenta los drás- ticos cambios climáticos que afectaron el con- tinente africano durante el Pleistoceno, y que condujeron a numerosas extinciones, es difícil aseverar dicha dirección de migración (MO- RAN & SMITH, 2001). Para explicar las afinidades florísticas del Neotrópico con regiones paleotropicales como África, Madagascar, India y Ceilán, se proponen dos hipótesis, deriva continental y dispersión a larga distancia (MORAN & SMITH, 2001). La primera solo tendría poder explica- tivo en aquellos grupos que se originaron antes de la separación de Gondwana, que ocurrió en el Cretáceo, hace 125-130 millones de años, con la última conexión terrestre direc- ta hace 90 millones de años (RAVEN & AXEL- ROD, 1974). De acuerdo con registros fósiles, los grupos de heléchos leptosporangiados con un anillo vertical interrumpido por el pedicelo, como es el caso de Asplenium, se originaron todos en el Paleoceno, hace 65 millones de años, o en épocas más recientes (COLLINSON, 1996). Es por ello que su distribución paleo- tropical podría explicarse por dispersión a lar- ga distancia, favorecida por el bajo peso y la larga vida de las esporas, las cuales son trasla- dadas a grandes distancias por el viento, de acuerdo con SMITH (1972), TRYON (1970) y WAGNER (1972). La dirección de la migración entre África y América del Sur aún está en dis- cusión, aunque en la zona tropical prevalecen los vientos del NE en el hemisferio Norte y del SE en el hemisferio Sur, lo cual sugiere que la AGRADECIMIENTOS Agradecemos la colaboración de la MSc. Jac- queline Pérez Camacho en la utilización de los mé- todos de estudio biogeográficos; a la Arq. Yanet Pérez Mark, por la confección de los esquemas; a los dos revisores anónimos, por sus comentarios y sugerencias, y al Instituto Sueco (Svenska Institu- tet), por financiar parte de esta investigación. DISCUSIÓN Los resultados obtenidos corroboran las afirmaciones de TRYON (1979) y SÁNCHEZ (1996) de que la flora pteridológica de las An- tillas Mayores presenta un carácter continen- tal, ya que el 54% de las especies cubanas de Asplenium son neotropicales o pantropicales. Esto se debe a la cercanía de las islas entre sí y con las áreas continentales, por lo cual las dis- tancias no representan barreras de aislamiento en relación con las floras pteridológicas conti- nentales. Los territorios continentales presentan gran afinidad entre sí (fig. 2), lo cual se debe, de acuerdo con GÓMEZ (1982), a la migración de plantas que ocurrió una vez formado el ist- mo de Panamá, producto de la interacción de las placas Cocos y Caríbea, al S de Nicaragua y al N de Colombia. Los adversos cambios climáticos ocurridos durante el Pleistoceno influyeron notablemente en las migraciones hacia zonas con mejores condiciones climáti- cas. En la región del istmo y en América Cen- tral en general, prevaleció un régimen de tem- peraturas más cálidas y grandes precipitacio- nes bien distribuidas. Sin embargo, la existencia de seis especies macroantillanas y cinco endemismos cubanos (30% del total) permite inferir que el grupo ha sido y sigue siendo objeto de un proceso de especiación, sobre todo si se tiene en cuenta la gran cantidad de individuos con esporas abor- tivas que aparecen en los estudios palinológi- cos realizados en ejemplares cubanos de As- plenium. De hecho, de las seis especies ma- croantillanas, A. jenmanii, que se distribuye en Cuba, Jamaica y La Española, ha resultado ser un híbrido estéril con esporas abortivas en el total de los ejemplares recolectados en Cuba Central y Oriental (SÁNCHEZ & REGA- LADO, 2003). Por ello, es muy probable que Estos resultados corroboran la predicción de TREJO-TORRES & ACKERMAN (2001) hecha a partir de un análisis biogeográfico de las Antillas, basado en la distribución de especies de orquídeas. Estos autores postulan que cual- quier grupo de plantas con diásporas tan pe- queñas que puedan ser transportadas por el L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM 403 FARRIS, J.S. (1970). Methods of computing Wagner trees. Syst. Zool. 19:83-92. SÁNCHEZ, C. & L. REGALADO (2003). Aspleniaceae. Flo- ra de la República de Cuba 8: 1-65. Koeltz Scientific Books, Licchtenstein. y FARRIS, J.S. (1989). The retentíon index and the rescaled consistency index. Cladistics 5(4): 417-419. p Books, Licchtenstein. SMITH, A.R. (1972). Comparison of fern and flowering plant distribution with some evolutionary interpreta- tions for ferns. Biotropica 4:4-9. y GÓMEZ, L.D. (1982). The origin of the pteridophyte flora of Central America. Ann. Missouri Bot. Gard. 69: 548-556. SMITH, A.R. (1981). Pteridophytes. In: D.E. Breedlove (ed.), Flora ofChiapas 2:35-55. California Academy of Sciences. San Francisco. KLUGE, A.G. & J.S. FARRIS (1969). Quantitative phyloge- netics and the evolution of Anurans. Syst. Zool. 18: 1-32. SORENSEN, T. (1948). A method for establishing groups of equal amplitude in plant sociology based on simila- rity of species content. Biol. Skr. 5(4): 1-34. Lovis, J.D. (1973). A biosystematic approach to phylo- genetic problems and its applications to the Asplenia- ceae. In: A.C. Jermy, J.A. Crabbe & B.A. Thomas (eds.), The Phylogeny and Classification of Ferns. Bot. J. Linn. Soc. 67, Supplement 1:211-229. STOLZE, R.G. (1981). Ferns and ferns allies of Guatema- la. II. Polypodiaceae. Fieldiana Bot. N.S. 6:59-95. STOLZE, R.G. (1986). Polypodiaceae-Asplenioideae. In: G. Harling & G. Sparre (eds.), Flora de Ecuador 23: 1-83. University of Goteberg. MORAN, R.C. & A.R. SMITH (2001). Phytogeographic relationships between Neotropical and African- Madagascan pteridophytes. Brittonia 53(2): 304-351. y g TREIO-TORRES. J.C. & J.D. ACKERMAN (2001). Biogeo- graphy of the Antilles based on a parsimony analysis of orchid distributions. J. Biogeography 28: 775-794. MORTON, C.V. & D.B. LELLJNGER (1966). The Polypo- diaceae subfamily Asplenioideae in Venezuela. Mem. New York Bot. Gard. 15: 1-49. TRYON, R.M. (1970). Development and evolution of ferns floras of oceanic islands. Biotropica 2: 76-84. NIXON, K.C. (1999). WINCLADA ver. 0.9.99 + (BETA). Preliminary Documentaron. p TRYON, R.M. (1972). Endemic áreas and geographic spe- ciation in tropical American ferns. Biotropica 4:121- 131. y PAGE, R.D.M. & CH. LYDEARD (1994). Towards a cladis- tic biogeography of the Caribbean. Cladistics 10: 21-41. TRYON, R.M. (1979). Biogeography of the Antillean Fern Flora. In: D. Bramwell (ed.), Plant and Islands: 55- 68. Academic Press, London, New York, Toronto, Sydney, San Francisco. PRÓCTOR, G.R. (1977). Pteridophytes. In: R.A. Howard (ed.), Flora of the Lesser Antilles, vol. 2. Harvard University, Cambridge, Massachusetts. REFERENCIAS BIBLIOGRÁFICAS ADAMS, C.D. (1995). Asplenium. In: G. Davidse, M. Sousa, S. Knapp. (eds.), Flora Mesoamericana, Psilotaceae a Salviniaceae 1: 234-290. Universidad Nacional Autónoma de México. México D.F. BORHIDI, A. (1996). Phywgeography and Vegetation Ecology ofCuba. Akadémiai Kiadó. Budapest. COLLINSON, M.E. (1996). "What use are fossil ferns?" - 20 years on: with the review of the fossil history of ex- tant pteridophyte families and genera. In: J.M. Ca- mus, M. Gibby & RJ. Johns (eds.), Pteridology in perspective: 349-394. Royal Botanic Gardens, Kew. COYULA, R. (1991). Cluster Analysis Program. Facultad de Biología. Universidad de La Habana. DE LA SOTA, E. (1973). La distribución geográfica de las pteridofitas en el cono Sur de América Meridional. Bol. Soc. Argentina Bot. 15(1): 23-34. L. REGALADO & C. SÁNCHEZ: DISTRIBUCIÓN DE ASPLENIUM Ó y g PRÓCTOR, G.R. (1985). Ferns of Jamaica. British Museum of Natural History. London. y y TRYON, R.M. & G.R. STOLZE (1993). Pteridophyta of Perú. 18. Aspleniaceae-21. Polypodiaceae. Part V. Fieldiana Bot. 32:1-49. PRÓCTOR, G.R. (1989). Ferns of Puerto Rico and the Virgin Islands. Mem. New York Bot. Gard. 53:1-389. TRYON, R.M. & A.F. TRYON, (1982). Ferns and allied plants with special reference to tropical America. Springer Verlag, New York. RAVEN, P.H. & D. AXELROD (1974). Angiosperm biogeo- graphy and past continent movements. Ann. Missouri Bot. Gard. 61:539-673. WAGNER W.H., Jr. (1972). Disjunctions in homosporous vascular plants. Ann. Missouri Bot. Gard. 59: 203- 217. ROSEN, D.E. (1975). A. vicariance model of Caribbean biogeography. Syst. Zool. 24:431-464. WAGNER W.H., Jr., F.S. WAGNER & C. TAYLOR (1993). Aspleniaceae. In: W.H. Wagner, Jr., F.S. Wagner & C. Taylor (eds.), Flora ofNorth America 6: 228-245. Oxford University Press. New York. ROSEN, B.R. (1988). From fossils to earth history: Ap- plied historical biogeography. In: A. Myers & P. Gi- 11er (eds.), Analytical Biogeography: An integrated approach to the study of animal and plant distribu- tion: 437-481. Chapman & Hall, London. SÁNCHEZ, C. (1996). La familia Hymenophyllaceae en Cuba. Tesis en opción al grado científico de Doctor en Ciencias Biológicas. Jardín Botánico Nacional, Uni- versidad de La Habana. Editado por Gonzalo Nieto Feliner Aceptado para publicación: 2-X-2003 Editado por Gonzalo Nieto Feliner Aceptado para publicación: 2-X-2003 Editado por Gonzalo Nieto Feliner Aceptado para publicación: 2-X-2003
W4388497006.txt	https://bop.unibe.ch/BzL/article/download/10373/13364	de		Warum nicht einfach Forschung? Gedanken zur Diskussion um Forschung an den zukünftigen pädagogischen Hochschulen	Beiträge zur Lehrerinnen- und Lehrerbildung	1,999	cc-by	4,584	Zutavern, Michael Warum nicht einfach Forschung? Gedanken zur Diskussion um Forschung an den zukünftigen pädagogischen Hochschulen Beiträge zur Lehrerbildung 17 (1999) 2, S. 211-222 Quellenangabe/ Reference: Zutavern, Michael: Warum nicht einfach Forschung? Gedanken zur Diskussion um Forschung an den zukünftigen pädagogischen Hochschulen - In: Beiträge zur Lehrerbildung 17 (1999) 2, S. 211-222 URN: urn:nbn:de:0111-pedocs-134135 - DOI: 10.25656/01:13413 https://nbn-resolving.org/urn:nbn:de:0111-pedocs-134135 https://doi.org/10.25656/01:13413 in Kooperation mit / in cooperation with: http://www.bzl-online.ch Nutzungsbedingungen Terms of use Gewährt wird ein nicht exklusives, nicht übertragbares, persönliches und beschränktes Recht auf Nutzung dieses Dokuments. Dieses Dokument ist ausschließlich für den persönlichen, nicht-kommerziellen Gebrauch bestimmt. Die Nutzung stellt keine Übertragung des Eigentumsrechts an diesem Dokument dar und gilt vorbehaltlich der folgenden Einschränkungen: Auf sämtlichen Kopien dieses Dokuments müssen alle Urheberrechtshinweise und sonstigen Hinweise auf gesetzlichen Schutz beibehalten werden. 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Kontakt / Contact: peDOCS DIPF \| Leibniz-Institut für Bildungsforschung und Bildungsinformation Informationszentrum (IZ) Bildung E-Mail: pedocs@dipf.de Internet: www.pedocs.de Beiträge zur Lehrerbildung, 17 (2), 1999 http://www.bzl-online.ch 21 1 Warum nicht einfach Forschung? Gedanken zur Diskussion um Forschung an den zukünftigen pädagogischen Hochschulen Michael Zutavern Die Formel "Forschung und Entwicklung" als Aufgabe Pädagogischer Hochschulen beeinhaltet ganz unterschiedliche Funktionen. Der folgende Beitrag analysiert in systematischer Weise die strukturellen Unterschiede zwischen Forschung, Entwicklung und Evaluation und begründet, warum diese unterschiedlichen Funktionen nicht vermischt werden sollten. Einleitung Man kann sich die Diskussion ja einmal umgekehrt denken: Seit Jahrzehnten bewährte, grosse Forschungsinstitute sollen demnächst auch Lehrveranstaltungen anbieten. Entweder muss dazu jedes Mitglied des Forschungsteams einen kleinen Teil Lehrverpflichtungen in sein Pensum integrieren oder es würde eine Dozentin angestellt. Damit sei man so ausgestattet, dass Studierende aufgenommen und durch forschendes Lehren ausgebildet werden. Gleichzeitig könne die neue Steile Lehrmittelentwicklung, Weiterbildung der Forschenden, Beratungsaufgaben und die Qualitätssicherung der Forschungsarbeit übernehmen. Nein, die Etablierung von Forschung in den jetzt entstehenden pädagogischen Hochschulen soll mit diesem Vexierbild keineswegs in Zweifel gezogen werden - irn Gegenteil. Aber man gewinnt zur Zeit den Eindmck, dass dafür nicht Forschung eingeführt, sondern Forschungen erfunden werden, von der angewandten über die kooperative bis zur handelnden. Und man liest von immer neuen Aufträgen, die. die neuen "Forschungs"ste11en einmal übernehmen sollten: Entwicklung, Quaiitätssichemng, Evaluation, Weiterbildung. Die folgenden Überlegungen verstehen sich deshalb als Einwände gegen eine Verwässemng der Forschungsaufgabe und gegen eine Verzettelung der Funktionen der zu schaffenden Forschungsstellen. Es sind im Kern zwei Argumente: Erstens könnten neue Forschungsstellen viel für das Grundlagenwissen über Bildung leisten, wenn ihre Ausrichtung auf empirische Forschung gefördert wird. Dazu muss aber die Anwendungsorientierung, die jetzt als besondere Kennzeichnung herausgestellt wird, zurückgebunden werden auf die Phasen im Forschungsprozess, wo sie hingehört: beim Suchen von Forschungsfragen und beim Verbreiten der Erkenntnisse. Zweitens sollte nicht Forschung, Evaluation, Entwicklung oder Quaiitätssicherung hinter dem Kunstwort "Forschung & Entwicklung" versteckt werden. Vielmehr stehen Entscheidungen an, welche Aufgabe (wo, von wem ) angegangen werden soll jede hat ihre hohen Ansprüche und erfordert für deren Einlösung ausreichende Ressourcen. Die folgenden Gedanken sind aus der Perspektive eines Alltags geschrieben, in dem unser Team seit einigen Jahren den Spagat zwischen Forschung und Lehrerbildung übt. Was im Folgenden behauptet wird, leitet sich also immer aus diesen Erfah- 212 Beiträge zur Lehrerbildung, 17 (Z), 1999 Forschung und Entwicklung in der Lehrerbildung 213 rungen ab, und was in normativen Sätzen formuliert ist, drückt Maximen aus, denen wir zu folgen versuchen. ihren Unterricht erforschen, können spannende Kooperationspartner für Forschungsinstitutionen sein, ersetzen sie aber nicht. 1. Konzentration auf empirische Bildungsforschung Anwendungsorientierung ist ein zwiespältiges Etikett Kein Mangel an Fragen für Forschung Im Bildungswesen und im speziellen im Bereich von Schule und Lehrerinnen- und Lehrerbildung besteht ein Bedarf an empirischer Forschung. Sicher wird manchmal unterschätzt, manchmal gering geschätzt, was die Arbeit von Erziehungswissenschaftlern, Psychologen und Soziologen bereits an Erkenntnissen über Bildungsprozesse zusammengetragen hat. Gegen solche Fehleinschätzungen spricht z.B. die Diskussion über Standards in der Lehrerbildung, die auf wissenschaftlich abgesicherten Erkenntnissen aufbauen (Oser, 1997) Gleichwohl existieren genügend Wissenslücken über Lernen und Sozialisation in der Schule, um viele Forschungsstellen noch lange zu beschäftigen. Gerade über Lehrerhandeln und die wirkungsvollen Prozesse in der Ausbildung von Lehrerinnen und Lehrern weiss man noch recht wenig: "Die kartographische Erfassung der Landschaft des Lehrerwissens hat sich bislang auf einige Ausschnitte beschränkt und Gegenden vernachlässigt, die für die Bewohner des Landes ebenfalls sehr bedeutsam sind" (Bromme, 1992, S. 140). Dieses Manko an abgesichertem Wissen hat ja die Debatte über die Lehrerbildungsreform von Anfang an begleitet. Die Tertiarisierung der Lehrerausbildung ist ein Bekenntnis zur Notwendigkeit einer wissenschaftlich abgesicherten Basis pädagogischen Wissens, das von pädagogischen "Profis" in ihren alltäglichen Entscheidungssituationen "verarbeitet" wird. Gerade wenn man das Erfahrungswissen z.B. von Lehrerinnen und Lehrern ernst nimmt, muss man die Aufgabe fördern, den Kem an wissenschaftlich geprüften Theorien und Erkenntnissen in den zentralen Bereichen des pädagogischen, didaktischen, fachdidaktischen Lehrerwissens zu verbreitern. Speziell als Grundlage für die Fachdidaktiken ist dieser Bedarf gross (z.B. Sitta, 1999). Lehrerbildungsstätten sind der richtige Ort Wie in anderen Berufsfeldem und ihrer Ausbildung sollten auch für den Lehrerberuf die "Wege" zu neuem Wissen besonders für die Novizen sehr kurz sein. Ausbildungsstätten sollten deshalb auch Stätten der Erkenntnisproduktion sein. Forschung soilte an den neuen Pädagogischen Hochschulen ihrer ureigensten Aufgabe nachgehen können, nämlich Wissen zu schaffen. Für diese Aufgabe muss speziell ausgebildetes Personal zur Verfügung stehen. Damit ist gesagt, dass es bei der Schaffung von Forschungsstellen in erster Linie eben nicht um didaktische Anliegen wie das forschende Lernen oder um engere Kooperationen mit reflektierenden Praktikern unter dem Etikett "Lehrerforschung" geht. Beides ist unabhängig von der Forschungsaufgabe zu verwirklichen: Forschendes Lernen ist eine Lemmethode, die durch Forschungsstellen an den Hochschulen zwar angeregt werden kann, sie aber nicht notwendig braucht. Lehrerinnen und Lehrer, die - Erkenntnisgewinn wird es nur durch gute Forschung geben. Anwendungsorientierung kann ein Gütekriterium sein, aber nicht für die Erkenntnis selbst, sondern allenfalls für das Vorgehen bei der Suche nach Forschungsfragen undoder bei der Verbreitung der Erkenntnisse. Gleiches gilt für Kennzeichnungen wie "berufsfeldbezogen" oder "schulbezogen". Ob sich neues Wissen im Schulalltag in veränderten Verhaltensweisen von Lehrerinnen und Lehrern niederschlägt, ist kein Kriterium, das über den Wahrheitsgehalt der Erkenntnis entscheidet. Das Wissen über Kontrollüberzeugungen und ihre Bedeutung für die Leistungsfähigkeit und das psychische Wohlbefinden von Schülerinnen und Schülern z. B. existiert, auch wenn Schulen erst mühsam einen Weg finden müssen, diese KontrollÜberzeugungen & Schülerinnen und Schülern zu stärken. Die Erkenntnisse Piagets über die kognitive Entwicklung haben Jahrzehnte gebraucht, bis sie sich für die Kinder und Jugendlichen als hilfreiche Formen des Lernens und der Unterstützung ihrer Entwicklung in Erziehung und Schulbildung niedergeschlagen haben. In den Studien zum moralischen Urteilen eines Lawrence Kohlberg mit seinen Dilemma-Interviews und ihren aufwändigen Auswertungsverfahren liegt ein sehr "grundlagen-theoretisches" Forschungsprograrnm vor. Und diese Theorie hat sich wie andere intensiv geprüfte Ansätze als immens "praktisch erwiesen. Forschung ist ein von der Gesellschaft zur Verfügung gestellter Freiraum für eine handlungsentlastete Suche nach Wahrheit. Der Elfenbeinturm ist gewollt und hat sich bewährt. In ihm dürfen Dinge auch scheitern. Das Wissen darum, dass etwas nicht der Fall ist, eine Wirkung nicht nachgewiesen werden kann, braucht diesen Schonraum. Und es braucht schonungslose kritische Analysen durch andere Forschungsteams, die ebenfalls die Freiheit haben, neue Erkenntnisse schnell aufzugreifen und kritisch zu prüfen, Gesicherte Erkenntnisse aber, wenn sie bekannt gemacht, wahrund ernst genommen werden, können starke "praktische" Wirkungen entwickeln. Manche Methodenmode wäre Schülerinnen und Schülern erspart geblieben, wenn es selbstverständlicher wäre, nach den gesicherten Grundlagen für ihre Wirkungsbehauptungen zu fragen. Beurteilungs- und Bewertungsvorgänge in Schule könnten heute schon anders aussehen, wenn sich die Wahrnehmung dieser Berufsaufgabe stärker auf Erkenntnisse, die vorliegen, stützen würde. Orientierung am Berufsfeld eijolgt vor und nach der Erkenntnisarbeit Selbstverständlich kann auf der anderen Seite heute kein Elfenbeinturm in einem verschwiegenen Tal Forscherinnen und Forscher von den Realitäten unserer Gesellschaft abschotten. Natürlich unterliegen Forscherinnen und Forscher dem Zwang, ihr Erkentknisinteresse zu begründen. Dies wird schon vor Beginn der Arbeit zum einen durch die Qualitätsaufsicht innerhalb der öffentlichen Institutionen sichergestellt. Zum anderen sind fast keine Forschungsprojekte mehr ohne Drittmittel möglich. Es erfolgt also eine weitere Sicherung durch das Begutachtungsverfahren bei Finanzanträgen 214 Beiträge zur Lehrerbildung, 17 (2), 1999 beispielsweise durch den Nationalfonds. Darüber hinaus wird gerade Bildungsforschung zu Recht kritisch durch die im Bildungswesen arbeitenden Fachleute und durch die "Klienten" wie z.B. Eltern von Schülerinnen und Schülern beobachtet. In der Entdeckungs- und in der Umsetzungsphase ihrer Arbeit können Forscherinnen und Forscher viel dafür tun, dass die Ergebnisse ihrer Arbeit im Bildungsalltag wahrgenommen werden. Sicher garantieren ein ausgewiesener Theoriebezug und der Anschluss an aktuelle Forschungsfragen genauso wie das kreative Entdecken der Lücken und offenen Fragen eines Forschungsfeldes, dass interessante Hypothesen entstehen und spannende Ergebnisse zu erwarten sind. Darüber hinaus jedoch kann in dieser Phase der Entdeckung von Fragen der Bezug zum Berufsfeld und die Kooperation mit Lehrerinnen und Lehrern äusserst fruchtbar sein. Pädagogische Hochschulen, die über die Praktika und den Einbezug von erfahrenen Lehrexinnen und Lehrern in die Ausbildung solche Kontakte pflegen, können hier vorbildhaft wirken. So gerät speziell die Frage nach der Bewährung von Theorien unter komplexeren Rahmenbedingungen in den Blick. Lässt sich beispielsweise die Lernwirksarnkeit der Kooperation von Lernenden auch unter den Alltagsbedingungen einer Schulklasse bestätigen? Doch nur dort, wo dieser Blick auf Fragen der pädagogischen Praxis verbunden wird mit vorhandenem Wissen und seiner theoretischen Ordnung, wird man Forschungsfragen formulieren können, die so überprüfbar sind, dass sie zu gesicherten Erkenntnissen werden können. Hauptkriterium bleibt die Güte der Erkenntnis In der Forschungsphase der Überprüfung von Annahmen kann es nur um Wahrheit gehen. Die bewährten Kriterien von Objektivität, Reliabilität und Validität gelten als Garanten dieses Bemühens um die Begründung von Behauptungen. Gefordert ist die Transparenz der Verfahren. Die Gewinnung, Verdichtung und Interpretation der Daten muss von anderen rekonstruiert werden können. Wie nah oder fern die Hypothesen dem Alltagsgeschehen sind, spielt hier keine Rolle mehr. Und auch die Art des methodischen Zugangs ändert an dieser Maxime nichts. Versuche, die Forschungsaufgaben der neuen Fachhochschulen einseitig auf eine Methodenrichtung festzulegen, sind sicher verfehlt. Empirische Bildungsforschung findet ihre Problemstellungen meist im Schnittbereich von erziehungswissenschaftlichen Theorien mit pädagogischer und entwicklungsorientierter Psychologie und Soziologie. Ihre Hypothesen bedürfen dementsprechend auch Methoden aus diesen Bezugswissenschaften. Und empirische Forschung im Bildungsbereich greift seit längerem sowohl auf das Repertoire erklärender Ansätze mit ihren Quantifizierungen zurück wie auf verschiedene hermeneutische Verfahren. Objektivität Objektivität fordert die Nachvollziehbarkeit- und möglichst oft auch den tatsächlichen Nachvollzug - durch andere Personen. Wie beispielsweise das Verfahren der "objektiven Hermeneutik" (Oevemann, Allert et al., 1979) zeigt, unterwerfen sich gerade auch qualitative Verfahren diesem Kriterium. Hier würden die neuen For- Forschung und Entwicklung in der Lehrerbildung 215 schungseinrichtungen die Chance bieten, dass auch in der Bildungsforschung haufiger kritische Reanalysen entstehen. Reliabilität Reliabilität fordert einen kritischen Blick auf die Verfahren, mit denen Daten gewonnen werden. Methodische Artefakte lassen sich mit den unterschiedlichsten Methoden produzieren und müssen von den Forschenden entdeckt werden. In jedem Methodenrepertoire gibt es die Möglichkeit der falschen Anwendung, des blinden Flecks oder einer unangemessenen Instrumentenwahl. Validität Schliesslich stellt sich in jeder Forschung die Frage, ob ihre Methoden den Bereich erfasst haben, den sie erfassen wollten und wie weit die Veraligemeineningsfiüiigkeit der Daten reicht. Zurecht weisen Kritiker der herkömmlichen Einstellungsforschung darauf hin, dass noch so ausgeklügelte Meinungserhebungen keine validen Aussagen über tatsächliches Verhalten darstellen oder dass eine Fallstudie über eine Junglehrerin wohl Hypothesen mit grösserem Allgemeinheitsgrad erzeugen oder starke Hypothesen falsifizieren, aber kaum allgemeine Aussagen über den Berufseinstieg begründen kann. Güteknterien stehen also für kritische Fragen an die Aussagekraft von Erkenntnissen, nicht aber für irgendwelche Berechnungsverfahren. Zentrales Kriterium bleibt die Transparenz für jeden, der die Erkenntnis prüfen will. Dazu gehören natürlich auch die "Forschungsobjekte". Hier sind Anstrengungen der Forschenden ein Gebot der Fairness. Aber Aussagen über Einstellungen oder Verhaiten können auch gültig sein, wenn ihnen die Befragten oder Beobachteten nicht zustimmen. Die Verabsolutierung kommunikativer Validierung wäre ein Irrweg. Orientierung an der Praxis Keine Rolle kann in dieser Phase eine Orientierung an der Praxis spielen. Erforderlich ist im Gegenteil eine grösstmögliche Unabhängigkeit. Forschung ist die organisierte Entlastung von alltäglichem Entscheidungszwang von Personen, die damit auf der Suche nach neuem Wissen hohe Risiken des Scheiterns eingehen können. Damit ist ein Hauptgrund genannt, weswegen Forschung, auch in den zukünftigen pädagogischen Hochschulen, "freie" Forschung sein sollte. Dies ist auch eine klare Absage an die Handlungsforschung, die Analyse und Entscheidung eng verzahnt und so glaubt, in einer neuen Institution "Forschung und Entwicklung" gleich zwei Aufgaben mit einem Schlag lösen zu können. Der für Erkenntnis notwendige "fremde Blick muss dabei verloren gehen. Dies bedeutet nicht eine unkollegiale Einstellung gegenüber Lehrerinnen und Lehrern, Schülennnen und Schülern, deren Interaktionen beobachtet und analysiert werden. Doch gerade ein fairer Umgang zwischen Forschenden und "Erforschten" und der Respekt vor der Autonomie der Praxis führt zu einer arbeitsteiligen Trennung von Forschung und alltäglicher Bildungsarbeit. Dagegen rückt die Anwendungsonentierung in der Phase der Umsetzung der Ergebnisse wieder stärker ins Zentrum. Gerade in der Bildungsforschung sollte es zu 216 Beiträge zur Lehrerbildung, 17 (2), 1999 den Selbstverpflichtungen von Forscherinnen und Forschern gehören, ihre Erkenntnisse "in der Praxis abzuliefern" und sich verständlich zu machen. Hier kann eine eng mit dem Berufsfeld verbundene Fachhochschule wiederum wertvolle Dienste leisten. Über die Mitarbeit in Lehrerfortbildung, in der berufspraktischen Ausbildung, aber auch durch Kontakte mit interessierten Lehrerinnen und Lehrern, z.B. durch Forschungspraktika,lassen sich neu gewonnene Erkenntnisse schnell verbreiten. Mit den sich daraus ergebenden Anregungen für neue Forschungsfragen schliesst sich der Kreis. 2. Keine Funktionsvermischung Mit der Konzentration auf gute Bildungsforschung wäre also sicher viel gewonnen ob die neuen Institutionen auch Aufträge übernehmen können, die Entwicklung, Evaluation, Qualitätssicherung zum Inhalt haben, muss sich auf Grund ihrer Kapazitäten und der Fachbildung ihrer Mitarbeiterinnen und Mitarbeiter erweisen. Sicher sollte aber nicht in personell dünn ausgestatteten Stellen eine Aufgabenhäufung erzeugt werden. Dies würde auch der Qualität der anderen Funktionen, die jetzt in vielen Planungspapieren auftauchen, nicht gerecht: Entwicklung, Evaluation und Qualitätssicherung folgen einer anderen Logik als die Forschung. Sie werden in andere Abläufe gegliedert und unterliegen anderen Gütekriterien. Forschung in einem Themenbereich braucht Jahre, bis die Kompetenzen und Erkenntnisse so weit sind, dass sie Früchte tragen. Methoden müssen entwickelt werden. Das Personal, das dies leistet, muss im entsprechenden Bereich (z. B. Fachdidaktik oder Entwicklungspsychologie) vorgebildet sein. Fachhochschulen werden kaum für Forschungsnachwuchs sorgen können. Sie werden keine Lizenziate vergeben und kaum Forschungspropädeutik anbieten. Das bedeutet aber auch, dass Forschungsprojekte von denjenigen Experten, die an einer pädagogischen Hochschule arbeiten, aufgrund ihrer spezifischen Kompetenzen und Interessen entworfen werden müssen. Inhaltliche Schwerpunktekönnen nur durch Personalpolitik gebildet werden. Demgegenüber verfolgen Entwicklung und Evaluation andere Ziele und gehen unterschiedlichen Fragen nach. Sie folgen in der Suche nach Antworten unterschiedlichen Leitideen und finden für ihre Ergebnisseje andere Abnehmer. Forschung und Entwicklung in der Lehrerbildung Unterschiedliche Fragestellungen Fnrschuno Evaluation Entwickluna Erkenntnis QualitätsprUfung Problemlßsung Theorie (Praxis) Praxis Praxis Auftrag selbst (frei) fremd selbst I fremd Annahme Gutachten Vertrag I Anordnung Vertrag I Anordnung Ziel Perspektive Quelle Abbildung 1:Unterschiedliche Fragestellungen Forschung Forschung fragt nach der Wahrheit von Behauptungen. Lücken in Theorien sollen geschlossen, Wissen vermehrt werden. Der Theonebezug garantiert die Effizienz von Forschung: Schwach begründete Bausteine verlangen forschenden Einsatz. Falsifizierte Hypothesen brauchen nicht noch einmal aufgegriffen werden. In jedem Forschungsprojekt weiss man arn Ende mehr, aber auch viel genauer, was man noch nicht weiss - solche offenen Fragen von Theorien leiten weitere Forschungsarbeiten. Orientierung an Theorie sichert auch das wechselseitige kritische Überprüfen von Ergebnissen und sorgt für einen systematischen Weiterbau eines Wissenssystems, das als Grundlage für die Analysen von Praxis, die Reflexion praktischer Handlungen und für Planung wichtig wird. Deshalb können Probleme der Bildungspraxis durchaus als Orientierungshilfen für die Auswahl innerhalb eines theoretischen Bereichs dienen. Sie ersetzen aber die Theorieorientierung nicht. Vor zwei möglichen Missverständnissen soll gewarnt werden: Theorieorientierung heisst nicht blindes Vertrauen in eingefahrene Traditionen. Forschungsprojekte müssen gerade auch so angelegt werden, dass Tabubrüche möglich sind und die Macht von Paradigmen in Frage gestellt werden kann. Doch auch "neue" Wahrheiten oder Methoden müssen ihren Geltungsanspruch nach den Regeln der Kunst transparent begründen können. Zum anderen ist mit dem Plädoyer für die Pflege des Elfenbeinturms nicht ein freischwebendes, von jeder gesellschaftlichen Verantwortung losgelöstes Interesse an Erkenntnis gemeint. Die Auswahl von Forschungsfragen, die Durchführung von Forachungsarbeit und die Verwendung von Forschungsergebnissen unterstehen immer auch dem Zwang einer politischen, ökonomischen und vor allem moralischen Legitimation. Theoretische und praktische Interessen müssen transparent gemacht und 218 Beiträge zur Lehrerbildung, 17 (Z), 1999 begründet werden. Öffentlichkeit muss den Zugang zu Forschungsplänen und Forschungsergebnissen haben. Forscherinnen und Forscher müssen auch Übersetzungsarbeit leisten, damit ihr Tun kritisierbar wird. Da dieser Aufwand nötig ist, reicht einer Forschungsstelle "Forschung" als Aufgabe. Dabei sollte auch nicht unterschätzt werden, dass eine kritische Öffentlichkeit durchaus für die Unterstützung der Suche nach Erkenntnis zu gewinnen ist, wenn deren Faszination kommuniziert werden kann - selbst wenn ein unmittelbarer Nutzen nicht erkennbar wird. Warum sollte sich das Interesse arn "Wissenwollen" nur auf den Weltraum oder die Suche nach Atlantis beschränken und sich nicht auch auf die Weltbilder der Kinder oder die kreative Dynamik von Jugendgmppen richten? Wenn daran jedoch Zweifel bestehen, lassen sich statt Forschungsstellen ja Evaluationsoder Entwicklungsstellen schaffen. Deren Aufgaben müssen ebenfalls Standards genügen, die eine Haltung des "so nebenbei" verbieten. Evaluation und Entwicklung Evaluation sucht nicht theoriegeleitet nach neuen Erkenntnissen, sondern prüft und bewertet Produkte, Konzepte, Verfahren. Sie untersucht die Qualität eines Ausschnittes von Bildungswirklichkeit. Entwickierinnen eines Produkts, Verantwortliche für neue Vorschriften oder Anwender eines Systems geben den Auftrag zur Evaluation, um möglichst objektive Rückmeldungen über Brauchbarkeit und Veränderungsbedarf zu erhalten. Ähnlich auftragsgebunden wird Entwicklung initiiert. Probleme in einem Handlungsfeld erfordern meist eine rasche Lösung. Neue Verfahren, Arbeitsmittel oder Konzepte sind gefragt. Sie sollten schnell verfügbar sein, von den Anwendern akzeptiert werden und handhabbar sein. Sie sollen garantieren, dass die Probleme, die die Entwicklung ausgelöst haben, beseitigt, Bedürfnisse befriedigt werden. Für Evaluation und Entwicklung bestehen in aller Regel Aufträge. Für Forschung wären sie Gift. Natürlich braucht es einen allgemeinen Auftrag der Gesellschaft, die Forschung "wollen" muss. Dieser Wille materialisiert sich bislang an den Universitäten. Er sollte auch auf die Fachhochschulen für Lehrerinnen- und Lehrerbildung übertragen werden. Die Forschungsfragen dagegen entstehen in einem thematischen, theorieorientierten Bereich und nicht in engen Auftragsformulierungen. Theorielücken durch Forschung zu schliessen braucht auch kreative Suche nach der weiterführenden Forschungsfrage, ungezieltes brainstonning zur Hypothesenfindung, Aufgreifen von unbeachteten Ideen und Seitensträngen. Und Forschung produziert unangenehme Wahrheiten, Widerlegungen bisheriger Überzeugungen, die einem Auftraggeber vielleicht auch allzu schwer verdaulich wären. Forschende sollten sich also ihre "Aufträge" selbst stellen können. Dazu müssen sie die Chance haben, Mittel für solche Arbeit zu bekommen - qua Institution, die eben nicht nur gebundene ("anwendungsorientierte?") Forschung erlaubt, und / oder durch Institutionen wie dem Nationalfonds, die solche Erkenntnissuche fördern, wenn ihre Vorgehensweisen einem definierten Standard genügen. 219 Forschung und Entwicklung in der Lehrerbildung Leitideen der Antwortsuche Hypothesenprlifung Falsifikation Fragen der Auftraggeber 1 Bewertungskriterien Zeitplan 1 Testphasen I - korrekt genau ,, . . , , . . . ... ...... Prüfung Abbildung 2: Unterschiedliche Leitideen und Vorgangsweisen Auch die Vorgehensweisen bei der Erfüllung der Aufgabe unterscheiden sich zwischen Forschung, Evaluation und Entwicklung stark. Forschung muss den klassischen Weg der Hypothesenprüfung nach verschiedenen Gütekriterien und unter Anwendung eines erprobten Methodenrepertoires gehen. Sie zielt auf verallgemeinerungsfahige Aussagen. Für diese Arbeit ist Kenntnis der Theorien und Erfahrung im Methodeneinsatz notwendig. Die Verfahren müssen transparent sein und von anderen Forscherinnen und Forschern begutachtet werden können. Qualitätskontrolle der Evaluation dagegen orientiert sich an den Fragen der Auftraggeber. Ihre Kriterien liegen in der Regel in der Nützlichkeit und Durchführbarkeit der zu untersuchenden Verfahren und Produkte. Sie muss sich auf die Beantwortung der gestellten Fragen konzentrieren, Blicke nach rechts und links, das Aufgreifen im Prozess entdeckter neuer Fragestellungen lenken nur ab. Sie verwendet häufig aus der Forschung stammende Methoden, um Nutzen und Verwendung des zu untersuchenden Produktes zu testen. Ohne Zweifel ist Evaluation ein naher "Verwandter" von Forschung. Die technologische Theorie, die aus wissenschaftlichen Theorien ableitbar ist, wird als ihr Gebiet betrachtet (Bortz and Döring, 1995). Bleibt im Bildungsbereich nur das ungelöste Problem von Wünsch- und Machbarkeit einer pädagogischen "Technologie". Im Zentrum von Entwicklungsaufgaben steht ein Plan mit verschiedenen Testdurchläufen. Hauptkriterien sind die Wirksamkeit der Lösung für das gesuchte Problem innerhalb einer vertretbaren Zeitspanne. Auch hier werden Methoden aus der Forschung angewendet, vor allem aber gibt es diverse Traditionen des Projektmanagements und der Umsetzung des Prinzips von "Versuch und Irrtum". In Entwicklungsprojekten erweist sich eine Orientiemng an vorhandenen Erkenntnissen aus der Forschung und an Bezugstheorien als sinnvoll, um Irrwege zu vermeiden und vorhandenes Wissen zu nutzen. Neben der theoretischen Orientiemng verlangt Entwick- 220 Beiträge zur Lehrerbildung, 17 (Z), 1999 lungsarbeit Kreativität und Verständnis des Problernfeldes, für das man arbeitet. Die Erfolgsprüfung liegt in den Händen der Auftraggeber und Anwender des entwickelten Produktes. Wem nützt's? Evaluation Medium Entwickluna Wiss. Publikation Bericht an Auftraggeber Vertrieb. Kurse Kurse, (Offentl. Medien) (öffentl. Medien, Kurse) (Medien) Informant Forschende (Journalisten) Evaluateure, Auftraggeber Entwickler, Auftraggeber, Journalisten Reanalysen, Folgeforschung, Praxisnutzen Veränderung, Zufriedenheit Akzeptanz (Verkauf), Probleml6sung ................................. Fs: Neue Fs: Forschungsfragen Eva: uberprüfung der Praxiswirkung Unverstandenes, KenntnislOcken Eva: unterschätzte Elemente ..... .......... Fs: Unverstandenes, KenntnislOcken Eva: Qualiätsprüfung ................ Entw: Umsetzungsideen Entw: Beseitigung von - Enhv: Folgeideen Qualitätsmängeln Abb. 3: Öffentlicher Nutzen und Vermittlung Schliesslich sind auch die Wege der Bekanntmachung von Erkenntnis, Qualitätsergebnis und Produkt unterschiedlich. Forschung muss sich einer wissenschaftlichen Öffentlichkeit stellen. Fachhochschulforschung muss sich - wie universitäre - in der nationalen und internationalen Gemeinschaft der Forschenden bewähren. Forschende sollten aber auch dafür sorgen, dass in möglichen Anwendungsfeldern ihre Ergebnisse bekannt und verstanden werden. Das Feedback kann sowohl in einer erfolgreichen Disseminationsarbeit und damit praktischem Nutzen bestehen, wird sich aber vor allem daran messen lassen, ob die entdeckte Theorielücke geschlossen wurde und ihre Ergebnisse kritischen Reanalysen anderer Forscherinnen und Forscher standhalten. Evaluation schliesst mit Berichten an den Auftraggeber ab, die auch einer breiten Öffentlichkeit zugänglich gemacht werden. Ihre Aussagen betreffen den einen irn Auftrag umschriebenen Gegenstandsbereich und liegen in aller Regel zuerst einmal den Auftraggebern vor, die es in der Hand haben, sie einer weiteren Öffentlichkeit zur Verfügung zu stellen. Evaluation sieht sich dabei oft auch im Dilemma zwischen Interessen des Auftraggebers und möglichen Anwendem des untersuchten Produktes. Evaluation und Entwicklung bekommen Feedback durch den Erfolg der Veränderungen, die sie vorschlagen. Die Zufriedenheit der Benutzer des untersuchten oder entwickelten Produktes oder Verfahrens ist Bestätigung der Arbeit. Manchmal stellen die Verkaufszahlen einen Gradmesser des Erfolges dar. Forschung und Entwicklung in der Lehrerbildung 221 "Un-einheitlichkeit"macht stark Selbstverständlich gibt es viele Nahtstellen zwischen den drei unterschiedlich arbeitenden Funktionen. Sie sind sich gegenseitig von Nutzen. Forschung liefert weiterer Forschung offene Fragen. Aus neuen Erkenntnissen können aber auch Problemlösungen für den Alltag entwickelt und in Folge dann auf ihre Qualität geprüft werden. Aus Evaluationsprojekten können Hinweise für offene Theoriebereicheentstehen, wie sie auch beim Entwickeln von Produkten auftauchen könnten. Aus Evaluation ergeben sich Aufträge an Entwickler, für jene Teile eines Produktes oder Verfahrens, die sich als mangelhaft erwiesen haben, wie umgekehrt das fertige Produkt der Entwickler sich einer Evaluation stellen muss. Auch Evaluation hat Berührungspunkte mit Forschung. Evaluation will einem Auftraggeber Auskunft über die Güte eines Produktes geben. Gerade nach Evaluation besteht zur Zeit ein grosser Bedarf. Sie sollte unabhängig von den Entwicklern des Produktes erfolgen. Schon hier verbietet sich eine zu enge Aufgabenüberschneidung. Evaluation erweist sich als erfolgreich, wenn die Güte getestet und allfällige Verbesserungsvorschläge gemacht wurden. Dies kann aber alles nur auf Grund von vorhandenen Erkenntnissen geschehen. Sicher werden manchmal Fragen auftauchen, die noch nicht beantwortbar sind. Aber die Kriterien, nach denen die Güte eines Produktes beurteilt wird, sind in aller Regel aus einem Forschungsprozess entstanden. Auch bedarf es bei der Evaluation nicht unbedingt eines Theoriebezugs, wohl aber eines klaren Auftrages und Fragen von einem Auftraggeber. Insofern macht es sicher Sinn, wenn Forschungsteams, Evaluationsgruppen und Entwicklungsabteilungen einen Austausch pflegen, zumal wenn sie in einem ähnlichen Fachbereich, z. B. im Berufsfeld "Schule" arbeiten. Ihre Aufgaben und ihre Ansprüche an die jeweilige Personen, die die Aufgaben durchführen sollen, sind jedoch so verschieden und so anspruchsvoll, dass es einen grossen Personalbestand braucht, um wirklich parallel Forschung, Evaluation und Entwicklung zu betreiben. Grosse Zentren könnten dem Anspruch genügen, Projekte der unterschiedlichen Art nebeneinander zu realisieren. Sie sind aber in kleineren pädagogischen Hochschulen, die jetzt geplant werden, kaum zu realisieren. Sind an einer Hochschule Fachleute, die Verfahren, Methoden, Lehrmittel für den Schulbereich entwickeln wollen, dann muss dort eine Entwicklungsabteilung entstehen. Will man sich an der Evaluation von anderswo entwickelten Produkten beteiligen, dann muss man Evaluationsabteilungen schaffen oder die Forschungsabteilungen so ausstatten, dass dort Evaluations- und Forschungsprojekte nebeneinander laufen können. Schlecht wäre nur, wenn man versuchen wollte, diesen Entscheidungszwang zu vertuschen und durch die Benennung der Stellen mit dem Etikett "Forschung" alle möglichen Funktionen zu legitimieren. Ein zeitliches Nacheinander ist auch in kleineren Stellen möglich, aber angesichts des grossen Aufwandes der Einarbeitung, die jedes Feld erfordert, nicht immer effizient. Da für die neuen Fachhochschulen kaum personell und materiell üppig ausgestattete Forschungs-, Evaluations- oder Entwicklungsstellen zu erwarten sind, wird man nicht darum herum kommen zu entscheiden, wo der Schwerpunkt liegen soll und die Kooperation mit anderen Institutionen zu suchen, wenn es die Nahtstellen der eigenen Projekte erfordern. 222 Beiträge zur Lehrerbildung, 17 (2), 1999 Da Forschung von Fachhochschulen gefordert wird, sollten sie sich auch auf Forschung konzentrieren. Dazu müssen ihnen Ressourcen zur Verfügung gestellt werden und Freiräume geschaffen werden, die mit denen an den Universitäten vergleichbar sind. Dann erst können die Vorteile der neuen Institution, besonders ihre Praxisverbundenheit, vielleicht auch ihre Überschaubarkeit und damit Einbindung von Studierenden ausgespielt werden. Selbstverständlich muss eine Qualitätskontroile für die Forschung etabliert werden. Es sollte aber nicht dazu kommen, dass Kriterien schon deshalb nicht erfüllt werden können, weil alle möglichen Aufgaben wie Entwicklung, Evaluation und Qualitätssicherung der Lehre die Forschungsstellen in ihrer Arbeit blockieren. Sollen Forschung und Lehre personell verflochten werden, was für ein schnelles Einfliessen von Erkenntnissen in die Lehre auch von Vorteil wäre, dann muss man entsprechend mehr Personen an der Forschungsarbeit beteiligen. Nicht zu unterschätzen ist der Reibungsverlust, den das Leben in den Parallelwelten von Lehre und Forschung bedeutet. In den Lehrerbildungsstätten wird Lehre immer auch als ein Stück Verpflichtung zum "in der Lehre Vorbild sein" empfunden. Schliesslich sollten die neu entstehenden Institutionen einen grossen Handlungsspielraum in der Übernahme und Ausgestaltung der neuen Aufgaben haben. Koordination braucht es vielleicht zur Verhinderung inhaltlich allzu ähnlicher Ausrichtungen. Zum Schluss: Es besteht jetzt eine einmalige Chance, im Bereich der Bildungsforschung Kapazitäten für die Produktion von Wissen über Erziehung und Bildung zu schaffen, die einer professionellen Weiterentwicklung des Lehrberufs dienen kann und damit auch der Zielgruppe der Profession, nämlich den Kindern, Jugendiichen und erwachsenen Lernerinnen und Lernern. Literatur Bortz, J . & Döring N. (1995). Forschungsmethoden und Evaluation für Sozialwissenschafiler (2., vollst. überarb. und aktualisierte Aufl.). Berlin: Springer. Bromme, R. (1 992). Der Lehrer als Experte. Zur Psychologie des professionellen Wissens. Bem: Huber. Oevermann, U., Allert, T. et al. (1979). Die Methodologie einer 'objektiven Hermeneutik' und ihre allgemeine forschungslogische Bedeutung in den Sozialwissenschaften. In H.-G. Soeffner (Hrsg.), Interpretative Vegahren in den Sozial- und Textwissenschaften (S. 352-434). Stuttgart: Metzler. Oser, F. (1997). Standards in der Lehrerbildung. Teil 1: Berufliche Kompetenzen, die hohen Qualitätsmerkmalen entsprechen. Beiträge zur Lehrerbildung, 15 (I), 26-37. Oser, F. (1997). Standards in der Lehrerbildung. Teil 2: Wie werden Standards in der schweizerischen Lehrerbildung erworben? Erste empirische Ergebnisse. Beiträge zur Lehrerbildung, 15 (2), 210228. Sitta, H. (1999). "Professionalität der Deutschdidaktik." Schweizer Schule, 199916.3-13.
https://openalex.org/W4393130171	https://www.e3s-conferences.org/articles/e3sconf/pdf/2024/33/e3sconf_isac-iccme2023_08002.pdf	English	null	The Characteristic Of Biocomposite Film Of Spirulina Residue As Natural Dyes	E3S web of conferences	2,024	cc-by	4,218	* Corresponding author: siti096@brin.go.id 1Research Center for Agroindustry, National Research and Innovation Agency-Indonesia, Jalan Ra Bogor Km. 46, Cibinong, Jawa Barat, Indonesia 16911 2 Research Center for Biomass and Bioproducts, National Research and Innovation Agency- Indonesia, Jalan Raya Bogor Km. 46, Cibinong, Jawa Barat, Indonesia 16911 Abstract. Spirulina plantesis microalgae contain active ingredients in the form of antioxidants and pigments. Currently, the microalgae Spirulina plantesis is used as an antioxidant in the food, pharmaceutical and cosmetic industries. This manuscript discusses the utilization of Spirulina plantesis microalgae pigment as a natural dye plastic. The need for color plastics for various uses continues to increase. The use of natural dyes for plastics is expected to produce biocomposite films that are safe for the environment or biodegradable and safe to be used. The aim of the study was to determine the effect of adding Spirulina residue as a natural dye on the characteristics of biocomposite films. The variable concentration of Spirulina residue added to the biocomposite film formula was 0.5%,1%, and 1.5%. The film production method used was the solution casting method. In this study, 200ml of distilled water was added with Spirulina residue (SR), then stirred for 30 minutes, and after that successively added 1% Glycerol and stirred for 15 minutes; Carrageenan 1% and stirred for 15 minutes. The solution was heated at 70oC then Polyvinyl Alcohol (PVA) with concentrations of 3%, 4%, and 5%, and Tapioca (3%) were added until complete gelatinization occurred. The solution was poured into a 30 cm x 20 cm mold and dried at 30oC for 48 hours to form a film. Biocomposite films were analyzed for tensile strength and elongation at break based on ASTM D 638, color, and morphology. The results showed that the highest tensile strength and elongation at break of biocomposites were found in biocomposite films with the addition of 0.5% Spirulina residue, namely 96.40±6.04 kgf/m2 and 47.64±7.48%, respectively. Analysis of the color of the biocomposite films showed the highest greenish and yellowish colors in the biocomposite films with the addition of 1.5% Spirulina residue and 5% PVA. Spirulina residue can be utilized as the plastic dyes. E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 https://doi.org/10.1051/e3sconf/202450308002 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). Siti Agustina1,, Aton Yulianto1, Fajriyan1, Ahmad Kamil1, Eddy Sapto Hartanto1, Firda Aulya Syamani2 Siti Agustina1,, Aton Yulianto1, Fajriyan1, Ahmad Kamil1, Eddy Sapto Hartanto1, Firda Aulya Syamani2 1Research Center for Agroindustry, National Research and Innovation Agency-Indonesia, Jalan Raya Bogor Km. 46, Cibinong, Jawa Barat, Indonesia 16911 2 Research Center for Biomass and Bioproducts, National Research and Innovation Agency- Indonesia, Jalan Raya Bogor Km. 46, Cibinong, Jawa Barat, Indonesia 16911 1 INTRODUCTION The need for bioplastics is increasing, especially for the packaging industry, and also for the textile industry, automotive industry, and food industry. In the health sector, plastic is used E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 https://doi.org/10.1051/e3sconf/202450308002 for packaging medical equipment and hospital waste bags. However, after its use, plastic causes environmental problems and creates waste, because it cannot degrade naturally. One way to reduce plastic waste is to use biodegradable plastic (bioplastic). Bioplastics can be produced from cassava starch (1),(2). Then, to improve the performance of starch-based bioplastics, modified starch derived from nuts, glycerol, distilled water, and vinegar is used (3). Apart from that, dragon fruit and turmeric have been used as natural dyes in making bioplastics (4). Additional materials in the form of natural dyes from pine flower waste can be used as bioplastic dyes for food packaging because they do not harm the environment (5). p y p g g y ( ) Other potential materials, such as microalgae are used to make bioplastic because they grow quickly and are not intended as food, which results in their availability (6). Microalgae can be used directly as biomass to produce bioplastics or indirectly using the poly hydroxybutyrate (pHBs) extraction process and also by combining starch with microalgae or it can also be done by producing a mixture of microalgae and polymer through hot molding, melt mixing, solvent casting processes, injection molding or twin screw extrusion (7). Other than starch, bioplastics can be produced from microalgae that are high in protein, such as the Spirulina plantesis microalgae. Spirulina, or blue-green algae, is high in protein (between 50 and 60 percent), antioxidants, vital fatty acids, and other nutrients, it is being used as a nutritional supplement all over the world. Spirulina protein has a higher amino acid content than soy protein, making it one of the best in the plant kingdom (8). The byproduct of Spirulina platensis extraction is a solid residue. The analytical results of Spirulina Residue were for components C (41.36 wt%), H (6.60 wt%), and N (7.17 wt%), O (35.33 wt%), while, for the proximate analysis, the lipids (0.09 wt%) were very low (9). The Spirulina residue has been utilized as bio-oil for renewable energy (9), protein source for Cordyceps militaris culture (10), and so on. Bioplastics produced from mixing polyvinyl alcohol (PVA) and Spirulina can reduce the costs and the use of non-biodegradable materials. 1 INTRODUCTION Bioplastics from Spirulina plantesis have mechanical properties that are almost the same as commercial bioplastics (11). Bioplastic film from Spirulina plantesis has stronger tensile strength than commercial plastic bags, but its elongation at break is low. This bioplastic can be used for food, pharmaceutical, and cosmetic packaging materials (12). Bioplastic can also be produced from spirulina residue by adding polyvinyl alcohol (13). Bioplastic made from 95% Poly Lactic Acid (PLA) and 5% algae shows good mechanical properties, especially for tensile strength (81 MPa) and elongation at break (4%), this bioplastic can be decomposed in 45 days (14). Biodegradation of plastic depends on the physical properties, chemical structure of the polymer bonds, both functional groups and crystallization properties, and also on the environment, such as water content, oxygen, temperature, and pH. One method of using natural materials to make bioplastic is using vegetable waste, which can be converted into bioplastic film with mechanical properties similar to other bioplastics (15). This research aims to determine the effect of adding spirulina residue as a natural dye on the characteristics of biocomposite films based on polyvinyl alcohol, starch, glycerol and carrageenan. Spirulina residue is residue from the spirulina extraction process using CO2 extraction to obtain carotenoids. 2.3 Characterization of biocomposite films The biocomposite films formed were analysis for mechanical properties (tensile strength and elongation at break), colour analysis, and morphology. 2.2 Biocomposite film manufacturing process method In this stage, the process of making a biocomposite film is carried out, with variable concentrations of spirulina residue (0.5%, 1.0% and 1.5%) and variable concentrations of polyvinyl alcohol (3%, 4% and 5%). 200 ml of distilled water was added with spirulina residue (0.5%, 1.0% and 1.5%) stirred until homogeneous for 30 minutes, then 1% glycerol was added, stirred for 15 minutes, then 1% carrageenan was added for 15 minutes, after that the solution was heated at 70oC. added polyvinyl alcohol (3%, 4% and 5%). Then add 3% tapioca and then heat until complete gelatinization occurs. After that, pour the solution into a mould measuring 30 cm x 20 cm. Dry at 30oC for 48 hours. Cool at 27oC, remove the film from the mould and store in a desiccator. The biocomposite film formula made in this research is presented in Table 1. Table 1. Bicomposite films formulation and code. Code PVA Spirulina Residue F1 3% 0.5% F2 4% 0.5% F3 5% 0.5% F4 3% 1% F5 4% 1% F6 5% 1% F7 3% 1.5% F8 4% 1.5% F9 5% 1.5% Table 1. Bicomposite films formulation and code. 2.1 Material This research used materials consisting of spirulina residue (residue from the carotenoid extraction process from Spirulina plantesis using supercritical CO2), tapioca flour (Orang 2 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 https://doi.org/10.1051/e3sconf/202450308002 Tani brand), polyvinyl alcohol (Bratachem), glycerol (Bratachem), Carragenan (Swallow brand). Tani brand), polyvinyl alcohol (Bratachem), glycerol (Bratachem), Carragenan (Swallow brand). 2.3.1 Analysis of mechanical properties and morphology Mechanical properties and morphology structure Parameters observed were tensile strength and elongation, using UTM, based on ASTM. The film sample was measured into 20 mm long and 2 mm wide, given a 500 N load with the speed of 1 mm/minute and range of 10 mm. The analysis was done 5 times. The morphology structure was observed using Scanning Electron Microscope (SEM). 2.3.2 Colour analysis Colour intensity analysis with chromameter based on the Harder’s lab colorimetric system Hunter’s colour notation system is expressed in three values, namely L (lightness), a(redness), and b (yellowness). Lightness value, while = 100, green =0, a* value (+a= 3 3 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 https://doi.org/10.1051/e3sconf/202450308002 red, -a= green), while b* (+b* = clear, -b* = blue). Before use, the tool needs to be calibrated using a while calibration plate. The sample is in the form of 25 gram of powder. red, -a= green), while b (+b* = clear, -b* = blue). Before use, the tool needs to be calibrated using a while calibration plate. The sample is in the form of 25 gram of powder. 3.2 Mechanical Properties of Biocomposite films Analysis of the mechanical properties of biocomposite films is to determine the homogeneity of the mixture of materials used in making biocomposites, consisting of tensile strength and elongation at break. The results of tensile strength analysis show that the biocomposite film uses Spirulina residue 0.5% and PVA 4% is stronger than a biocomposite film with Spirulina residue concentration of 1.0% and 1.5% and PVA concentration of 3% and 5%, as seen in Figure 1. Spirulina residue containing fiber (calculated as carbohydrate) can effect the tensile strength of the biocomposite film. The analysis of elongation at break of biocomposite films, shows that biocomposite films using Spirulina residue with a concentration of 0.5% and PVA with a concentration of 3% produce biocomposite film with higher elongation at break compared to biocomposite films using Spirulina residue 1.0% and 1.5% in PVA 4% and 5% (Figure 2). This shows that the higher the concentration of Spirulina residue will result in a lower elongation at break. It’s interesting to observe that the elongation value for biocomposite film of RS 0.5% in PVA 3% and 4% show similar value. It shows that in lower concentration, Spirulina residue can help the stress tranfer in PVA matrice with concentration of 3% and 4%. It also shows that Spirulina residue can affect the elongation at break properties of biocomposite films. Based on the composition of the residue, Spirulina contains ash, usually in the form of minerals which can affect the flexibility of the biocomposite film. y p To increase the flexibility of the plastic produced from Spirulina plantesis, glycerol was added at different concentrations (15-30%). Higher tensile strengths and lower elongations were observed from this bioplastic with addition of 30% glycerol compared to commercial plastic bags. These results showed that this bioplastic can be used for food packaging in pharmaceutical applications or cosmetics, where high elongation is not needed (9). The use of a compatibilizer also increased the elongation capability of the plastic and enabled smoother surface (8). The microalgae biomass particles which have particles diameter of 5 micrometers did not show efficient strengthening ability. The smaller particles were able to blend with the other materials more efficiently (17). Composite film surface appears to be rough and uneven. In addition, rigidity of the composite film by means of elasticity could affect the tensile strength (18). 3.1 Environmentally Friendly Plastic Formulation In this research, the method used is the solution casting method, where Spirulina residue functions as a natural colour additive. Spirulina residue is obtained from the by-product of the Spirulina plantesis extraction process using a super critical CO2 extractor to obtain carotenoids (16). The composition of Spirulina residue can be seen in Table 2. It shows that Spirulina residue contains quite high total carbohydrate levels (59.94/100g). In the process of making biocomposite films, water is used as a solvent, because spirulina residue can dissolve well in water. It is also easy to get the water, and it is safe as well as environmentally friendly solvent. The spirulina residue used was in concentrations of 0.5%, 1.0% and 1.5%. Stirring was done for 30 minutes, this aimed to obtain Spirulina residue dissolved homogeneously in water. The plasticizer used is glycerol, 1% glycerol is used because glycerol can dissolve well in water. Fiber in biocomposites can function as reinforcement by using 1% carrageenan and 3% tapioca starch. The biocomposite matrix uses polyvinyl alcohol polymer with variables, namely 3%, 4% and 5%, because polyvinyl alcohol is a synthetic polymer that can dissolve in water, so it can mix homogeneously with Spirulina residue and tapioca starch and carrageenan. Polyvinyl alcohol is easily decomposed, so the resulting biocomposite film will decompose easily and is environmentally friendly. Making bicomposites will produce biocomposite films that can be used for packaging. Table 2. Spirulina residue composition. Table 2. Spirulina residue composition. No Parameter Analysis Results 1 Moisture 14.65 g/100g 2 Total ash 12.47 g/100g 3 Protein 3.16 g/100g 4 Total carbohydrate 59.94 g/100g 5 AEAC 14.78 g/100g 6 IC 50 0.32 mg vit C/g sampel 7 Inhibition value 27.25 mg/ml 8 Total fat 2.08 % 9 Vitamin B1 132.49 mg/kg 10 Vitamin B2 - 11 Vitamin B6 80.27 mg/kg 12 Na, Sodium 2840.33 mg/kg 13 K, Potassium 43.93 mg/kg 14 Ca, Calcium 12172.95 mg/kg 15 Mg, Magnesium 4655.96 mg/kg 16 P, Phospor 11624.80 mg/kg 17 Zn, Zinc 55.03 mg/kg 18 Mn, Manganese 3.77 mg/kg 4 https://doi.org/10.1051/e3sconf/202450308002 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 3.2 Mechanical Properties of Biocomposite films Bioplastic film produced from Spirulina plantesis and PVA mixture which has the most similar mechanical properties is bioplastic which uses 6 wt% compatibilizer (8). Fig. 1. Tensile strength of biocomposite film at spirulina residue concentration and poly vinyl alcohol concentration. 96.40 43.52 91.71 0 20 40 60 80 100 120 RS 0.5% RS 1.0% RS 1.5% Tensile strength (kgf/m2) PVA 3% PVA 4% PVA 5% Fig. 1. Tensile strength of biocomposite film at spirulina residue concentration and poly vinyl alcohol concentration. 5 5 https://doi.org/10.1051/e3sconf/202450308002 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 Fig. 2. Elongation at break of biocomposite film at Spirulina residue concentration and poly vinyl alcohol concentration 47.64 25.14 41.79 0.00 10.00 20.00 30.00 40.00 50.00 60.00 RS 0.5% RS 1.0% RS 1.5% Elongation (%) PVA 3% PVA 4% PVA 5% Fig. 2. Elongation at break of biocomposite film at Spirulina residue concentration and poly vinyl alcohol concentration 3.3 Colour Analysis of Biocomposite Films In this research, the use of Spirulina residue did not only affect the mechanical properties of the biocomposite film, but also give colour to the biocomposite film. In general, Spirulina contains bioactive ingredients which have colours; the green from chlorophyll, the blue colour from phycocyanin, and the orange colour from carotenoids. After the extraction using a CO2 supercritical process, in order to obtain carotenoids and chlorophyll, spirulina residue still has colour or pigment. This color or pigment is the color of the biocomposite film. The color of the biocomposites observed with the naked eye does not show any real differences, as presented in Figure 3. Therefore, the color differences between the biocomposites produced were analyzed using the Color Reader tool. Fig. 3. Biocomposite films of PVA filled with Spirulina residue Fig. 3. Biocomposite films of PVA filled with Spirulina residue Based on colour analysis using a Minolta CR 400 Chromameter, it shows that the variable values "L" (Lightness) and "b" (yellowness) and "a" (Greenness). The colour brightness (L) value shows that the highest 'L" value is in the residue concentration 0.5% spirulina and 3% polyvinyl alcohol concentration of 76.18. This shows that the higher the concentration of spirulina residue will produce a lower "L" colour brightness and the higher the poly vinyl alcohol concentration indicates that the "L" colour brightness value is lower., as shown in Figure 4. The greenish colour value "a" shows that the highest greenish colour value is at a spirulina residue concentration of 1.5% and a 5% poly vinyl alcohol concentration of 6.95. This shows the higher the spirulina residue concentration will produce a higher greenness value and the higher the concentration of poly vinyl alcohol will produce a higher greenness value, as seen in Figure 5. In the yellowness value "b" indicates that the highest yellowness value was at a spirulina residue concentration of 1.5% and a poly vinyl alcohol concentration of 5% at 29.97. This shows that the higher the concentration of spirulina residue will produce a higher yellowness value and the higher the poly vinyl alcohol 6 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 https://doi.org/10.1051/e3sconf/202450308002 will produce a higher yellowness value, as can be seen in Figure 6. These results indicate that the level of colour change that occurs is quite significant and relevant to visual observation. 3.3 Colour Analysis of Biocomposite Films There was a colour change when the concentration of spirulina residue is increased and the concentration of poly vinyl alcohol is increased. will produce a higher yellowness value, as can be seen in Figure 6. These results indicate that the level of colour change that occurs is quite significant and relevant to visual observation. There was a colour change when the concentration of spirulina residue is increased and the concentration of poly vinyl alcohol is increased. A comparison was conducted from the same natural dyes –carmine, turmeric, indigo and annatto- with and without encapsulation. Injected into the same PVC matrix (19), the possibility of reducing the UV transparency of two commercial and very common food packages after smart coating with commercial and green materials such as P-Coumatic acid and quinine for PLA matrix (20). Dyeing polyethylene (PE) plastic waste with bioactive photoacid phycocyanobilin (PCB) extracted from the pigment of spirulina blue-green algae the populations of long living photoproduct X*, which are pertinent to deprotonated phyrole species (anionic species) (21). Fig. 4. Colour lightness value "L" of biocomposite film at spirulina residue concentration and polyvinyl alcohol concentration Fig. 5. Greenish colour value "a" of biocomposite film at spirulina residue concentration and polyvinyl alcohol concentration Ϭ ϮϬ ϰϬ ϲϬ ϴϬ ϭϬϬ Z͘^͘Ϭ͘ϱй Z͘^ϭ͘Ϭй Z͘^͘ϭ͘ϱй >ŝŐŚƚŶĞƐƐ͕Η>Η ^ƉŝƌƵůŝŶĂƌĞƐŝĚƵĞĐŽŶĐĞŶƚƌĂƚŝŽŶ Wsϯй Wsϰй Wsϱй Ϭ Ϯ ϰ ϲ ϴ Z͘^͘Ϭ͘ϱй Z͘^͘ϭ͘Ϭй Z͘^͘ϭ͘ϱй 'ƌĞĞŶĞƐƐ͕ΗĂΗ ^ƉŝƌƵůŝŶĂƌĞƐŝĚƵĞĐŽŶĐĞŶƚƌĂƚŝŽŶ Wsϯй Wsϰй Wsϱй Ϭ ϮϬ ϰϬ ϲϬ ϴϬ ϭϬϬ Z͘^͘Ϭ͘ϱй Z͘^ϭ͘Ϭй Z͘^͘ϭ͘ϱй >ŝŐŚƚŶĞƐƐ͕Η>Η ^ƉŝƌƵůŝŶĂƌĞƐŝĚƵĞĐŽŶĐĞŶƚƌĂƚŝŽŶ Wsϯй Wsϰй Wsϱй Fig. 4. Colour lightness value "L" of biocomposite film at spirulina residue concentration an polyvinyl alcohol concentration y y Ϭ Ϯ ϰ ϲ ϴ Z͘^͘Ϭ͘ϱй Z͘^͘ϭ͘Ϭй Z͘^͘ϭ͘ϱй 'ƌĞĞŶĞƐƐ͕ΗĂΗ ^ƉŝƌƵůŝŶĂƌĞƐŝĚƵĞĐŽŶĐĞŶƚƌĂƚŝŽŶ Wsϯй Wsϰй Wsϱй Fig. 5. Greenish colour value "a" of biocomposite film at spirulina residue concentration and polyvinyl alcohol concentration 7 7 https://doi.org/10.1051/e3sconf/202450308002 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 Fig. 6. Yellowness value "b" in spirulina residue concentration and polyvinyl alcohol concentration Ϭ ϱ ϭϬ ϭϱ ϮϬ Ϯϱ ϯϬ ϯϱ Z͘^͘Ϭ͘ϱй Z͘^ϭ͘Ϭй Z͘^ϭ͘ϱй zĞůůŽǁŶĞƐƐΗďΗ ^ƉŝƌƵůŝŶĂƌĞƐŝĚƵĞĐŽŶĐĞŶƚƌĂƚŝŽŶ Wsϯй Wsϰй Wsϱй Fig. 6. Yellowness value "b" in spirulina residue concentration and polyvinyl alcohol concentration 3.4 Morphology residue Spirulina in Polyvinyl alcohol matrix Characteristic of polyvinyl alcohol as a matrix for residue spirulina rheology aimed to observed the form of the polyvinyl alcohol matrix particle. Morphology was analysis by Scanning Electron Microscope (SEM). Figure 7 describes the SEM result with magnification for 500x times and 4000 x times. The particles are isotropic, wavy-round shape, and the particle bond with the matrix. Residue spirulina was very well furthermore, polyvinyl alcohol matrix can protect residue spirulina as natural dyes. The morphology of residue spirulina- polyvinyl alcohol is soft and homogeny. Based on SEM result, the biocomposite film with concentration Spirulina residue of 1.5% in PVA 5%, shows homogeneously shaped and spreading depressively. 3a 3b 3a 3b Fig. 7. SEM Analysis of polyvinyl alcohol 5 % - residue spirulina 1.5 % with magnification 500x (3a) and analysis of polyvinyl alcohol 5 % - residue spirulina 1.5 % with magnification 4000x (3b) 3b 3a Fig. 7. SEM Analysis of polyvinyl alcohol 5 % - residue spirulina 1.5 % with magnification 500x (3a) and analysis of polyvinyl alcohol 5 % - residue spirulina 1.5 % with magnification 4000x (3b) References 1. Gökçe E. Rethinking sustainability: A research on starch based bioplastic. J Sustain Constr Mater Technol. 3(3):249–60. 2018 2. Asrofi M, Sapuan SM, Ilyas RA, Ramesh M. Characteristic of composite bioplastics from tapioca starch and sugarcane bagasse fiber: Effect of time duration of ultrasonication (Bath-Type). Mater Today Proc [Internet]. 46(xxxx):1626–30. Available from: https://doi.org/10.1016/j.matpr.2020.07.254.2020 3. Mojibayo I, Samson AO, Johnson OY, Joshua lagunju O, S.A A. A Preliminary Investigation Of Cassava Starch Potentials As Natural Polymer In Bioplastic Production. Am J Interdiscip Innov Res.02(09):31–9.2020 4. Putra EPD, Thamrin ES, Saputra H. Effect of Dragon Fruit Skin Extract (Hylocereus costaricensis) on Bio-plastic Physical and Mechanical Properties of Cassava Starch and Polyvinyl Alcohol. IOP Conf Ser Earth Environ Sci. 258(1). 2019 5. Nasihin ZD, Masruri M, Warsito W, Srihardyastutie A. Preparation of Nanocellulose Bioplastic with a Gradation Color of Red and Yellow. IOP Conf Ser Mater Sci Eng.833(1).2020 6. Rahman A, Miller CD. Microalgae as a Source of Bioplastics [Internet]. Algal Green Chemistry: Recent Progress in Biotechnology. Elsevier B.V.; 121–138 p. Available from: http://dx.doi.org/10.1016/B978-0-444-63784-0.00006-0. 2017 7. Cinar SO, Chong ZK, Kucuker MA, Wieczorek N, Cengiz U, Kuchta K. Bioplastic production from microalgae: A review. Int J Environ Res Public Health. 17(11):1– 21. 2020 8. Priyanka S, Varsha R, Verma R, Ayenampudi SB. Spirulina: A Spotlight on Its Nutraceutical Properties And Food Processing Applications. J Microbiol Biotech Food Sci / Priyanka et al. 2023:12 (6) e4785. https://doi.org/10.55251/jmbfs.4785 9. Jamilatun S, Rahayu A, Pradana YS, Budhijanto, Rochmadi, Budiman A. Bio-Oil Characterizations of Spirulina platensis Residue (SPR) Pyrolysis Products for Renewable Energy Development. Key Engineering Materials Vol. 849, pp 47-52 10. Iamtham S, Kaewkam A, Chanprame S, Pan-utai W. Effect of Spirulina biomass residue on yield and cordycepin and adenosine production of Cordyceps militaris culture. Bioresource Technology Reports Vol 17, February 2022, 100893. https://doi.org/10.1016/j.biteb.2021.100893 11. Dianursanti, Noviasari C, Windiani L, Gozan M. Effect of compatibilizer addition in Spirulina platensis based bioplastic production. AIP Conf Proc. 2092(April). 2019 11. Dianursanti, Noviasari C, Windiani L, Gozan M. Effect of compatibilizer addition in Spirulina platensis based bioplastic production. AIP Conf Proc. 2092(April). 2019 12 Dianursanti, Gozan M, Noviasari C. The effect of glycerol addition as plasticizer in p p p p ( p ) 12 Dianursanti, Gozan M, Noviasari C. The effect of glycerol addition as plasticizer in Spirulina platensis based bioplastic. E3S Web Conf. 67:11–4. 2018 13. Zhang C, Wang C, Cao G, Wang D, Ho SH. 4 CONCLUSION Spirulina plantesis microalgae residue from the CO2 extraction process can be used as a natural dye in biocomposite films. The method used to make biocomposites is the solution casting method. The results of the research showed that the highest tensile strength showed in biocomposite of 0.5% Spirulina residue and 4% polyvinyl alcohol, which was 96.40 kgf/m2. Meanwhile, the highest elongation at break of the biocomposite made from 0.5% Spirulina residue and3% polyvinyl alcohol was 47.64%. Biocomposite color analysis showed the highest greenish color (6.95) in the biocomposite film of 1.5% spirulina residue dan 5% 8 8 8 https://doi.org/10.1051/e3sconf/202450308002 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 polyvinyl alcohol and the highest yellowish color (29.97) in the biocomposite film of 1.5% spirulina residue dan 5% polyvinyl alcohol. Morphology shows homogeneous dispersion of Spirulina residue in polyvinyl alcohol matrice. polyvinyl alcohol and the highest yellowish color (29.97) in the biocomposite film of 1.5% spirulina residue dan 5% polyvinyl alcohol. Morphology shows homogeneous dispersion of Spirulina residue in polyvinyl alcohol matrice. References A sustainable solution to plastics pollution: An eco-friendly bioplastic film production from high-salt contained Spirulina sp. residues. J Hazard Mater [Internet]. 388(October):121773. Available from: https://doi.org/10.1016/j.jhazmat.2019.121773.2020 14. Simonic M, Zemljic F. Production of bioplastic material from algal biomass. Chem Ind Chem Eng Q. 27(1):79–84. 2021 15 Perotto G, Ceseracciu L, Simonutti R, Paul UC, Guzman-Puyol S, Tran TN, et al. Bioplastics from vegetable waste: Via an eco-friendly water-based process. Green Chem. 20(4):894–902. 2018 16. ( ) 16. Agustina S, Aidha NN, Oktarina E, Haruminda JH. Optimasi Proses Ekstraksi 9 https://doi.org/10.1051/e3sconf/202450308002 E3S Web of Conferences 503, 08002 (2024) ISAC-ICCME 2023 Karoten Dan Klorofil Dari Spirulina Platensis Dengan Teknologi Karbon Dioksida (CO2) Superkritis Menggunakan Metode Permukaan Tanggap. J Kim dan Kemasan. 41(2):95.2019 Karoten Dan Klorofil Dari Spirulina Platensis Dengan Teknologi Karbon Dioksida (CO2) Superkritis Menggunakan Metode Permukaan Tanggap. J Kim dan Kemasan. 41(2):95.2019 17. Ciapponi R, Turri S, Levi M. Mechanical reinforcement by microalgal biofiller in novel thermoplastic biocompounds from plasticized gluten. Materials (Basel). 12(9). 2019 18. Guru Moorthy A, Abdul-Latif NIS, Ong MY, Shamsuddin AH, Nomanbhay S. Enhancement of biodegradability and tensile characteristics of composite plastic film with spirulina algal biomass. IOP Conf Ser Earth Environ Sci. 2020;476(1). 19. Velho SRK, Brum LFW, Petter CO, dos Santos JHZ, Šimunić Š, Kappa WH. Development of structured natural dyes for use into plastics. Dye Pigment. 136:248– 54. 2017 20. Virgili T, Pasini M, Guizzardi M, Tizro N, Bollani M. Natural Dyes Used as Organic Coatings UV Protecting for Food Packages. Coatings.12(3):1–9.2022 21. Alhefeiti M, Chandra F, Gupta RK, Saleh N. Dyeing Non-Recyclable Polyethylene Plastic with Photoacid Phycocyanobilin from Spirulina Algae: Ultrafast Photoluminescence Studies. Polymers (Basel). 14(22). 2022 10
https://openalex.org/W1523018288	https://europepmc.org/articles/pmc4412661?pdf=render	English	null	Experimental assessments of the cross‐reactivity of IgE from patients sensitised with acid‐hydrolysed wheat protein in a cosmetic soap	Clinical and translational allergy	2,015	cc-by	662	1National Institute of Health Sciences, Tokyo, Japan Full list of author information is available at the end of the article Shinobu Sakai1*, Ryosuke Nakamura1, Reiko Adachi1, Yuma Fukutomi2, Yoshiro Saito1, Tomoko Nishimaki-Mogami1, Reiko Teshima1 From Food Allergy and Anaphylaxis Meeting 2014 Dublin, Ireland. 9-11 October 2014 Even after prolonged hydrolysis, acid-HWPs still retained the ability to activate mast cells, with only slightly decreased activity levels. The alkaline and enzymatic hydrolysis samples are currently under investigation to assess cross-reactivity. In conclusion, we showed that aller- genic HWP had specific molecular properties given by hydrolysis. These results showed that it may be possible to reduce the risk of HWP-caused allergy by the regulation of hydrolysis procedures. Hydrolysed wheat protein (HWP), which has high emulsi- fying property and water retentivity, is added to various foods and cosmetics to improve their texture. Recently, several studies showed that a specific HWP formula could induce IgE-mediated hypersensitivity by skin contact and/or food ingestion. In Japan, a number of HWP-caused systemic wheat allergy cases were reported, including wheat-dependent exercise-induced anaphylaxis after sensi- tisation to acid-hydrolysed gluten additive [Glupearl 19S® (Glp19S)] in a cosmetic soap. These allergic manifestations are believed to result from deamidated peptides in high- molecular weight fractions of Glp19S. However, relation- ships between the properties of HWP and its allergenicity remain unknown. Here, we experimentally assessed the cross-reactivity of IgE from Glp19S-sensitised patients’ sera by examining the IgE-binding capacity to various HWPs generated under different conditions and the ability to elicit mast cell activation. We prepared three different HWP types by acidic hydrolysis (0.1M HCl treatment at 100°C), alkaline hydrolysis (1M NaOH treatment at 100°C) and enzymatic hydrolysis (Neutrase® digestion at 50°C) for 0.5, 1, 3, 6, 9, 12 and 24 h, respectively. POSTER PRESENTATION Open Access Sakai et al. Clinical and Translational Allergy 2015, 5(Suppl 3):P16 http://www.ctajournal.com/content/5/S3/P16 Sakai et al. Clinical and Translational Allergy 2015, 5(Suppl 3):P16 http://www.ctajournal.com/content/5/S3/P16 Authors’ details 1 1National Institute of Health Sciences, Tokyo, Japan. 2Sagamihara National Hospital, Sagamihara, Japan. 1National Institute of Health Sciences, Tokyo, Japan. 2Sagamihara National Hospital, Sagamihara, Japan. Published: 30 March 2015 Published: 30 March 2015 doi:10.1186/2045-7022-5-S3-P16 Cite this article as: Sakai et al.: Experimental assessments of the cross- reactivity of IgE from patients sensitised with acid-hydrolysed wheat protein in a cosmetic soap. Clinical and Translational Allergy 2015 5(Suppl 3):P16. We examined the capacity of IgE binding to various acid-HWPs and native gluten using Western blotting. After 0.5-1 h of acid hydrolysis, IgE immunoblotting with a patient’s serum showed smears at around 40-70-kDa proteins. However, after 3 h of acid hydrolysis, IgE binding to 40-70-kDa proteins time-dependently decreased. Regarding mast cell activation by acid-HWPs, a 0.5-h hydrolysis sample showed dramatically increased mast cell activation, whereas native gluten had little effect on it. © 2015 Sakai et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 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https://openalex.org/W1968836000	https://www.scielo.br/j/csp/a/XSPD7SJGbSZ8DVyny8FZvWc/?lang=pt&format=pdf	Portuguese	null	Pesquisa sistemática positiva e relação com conhecimento da população de assentamento e reassentamento de ocupação recente em área de Triatoma sordida (Hemiptera, Reduviidae) no Estado de São Paulo, Brasil	Cadernos de Saúde Pública	2,004	cc-by	7,941	A b s t r a c t Após o controle do Tr i at oma infestans do Esta- do de São Pa u l o, a vigilância entomológica do Pro g rama de Co n t role da Doença de Chagas (PCDCh) foi dirigida às espécies ditas secundá- rias 1, através das atividades de pesquisa siste- mática casa-a-casa, direcionadas às áreas com infestação triatomínica comprovada e do aten- dimento às notificações de triatomíneos en- c aminhadas pelos mora d o re s. A atividade de atendimento às notificações triatomínicas tem sido pri o rizada em relação à pesquisa sistemá- tica. Isso ocorre desde o ano de 1983, quando as atividades educativas do PCDCh incorpora- ram o treinamento das equipes de campo, para que as mesmas pudessem sensibilizar os mo- ra d o res da importância de sua participação na busca de tri a t o m í n e o s, ensinando-os e ori e n- tando-os quanto aos locais mais habituais de e n c o n t ro dos mesmos 2. Desde então, a part i c i- pação da população na detecção de colônias t riatomínicas passou a ser explorada e incenti- vada no Estado, corro b o rando o pre c o n i z a d o por Dias 3 s o b re a participação da população na detecção de colônias triatomínicas e por Vi- nhaes & Si l ve i ra 4 s o b re a sustentabilidade do PCDCh através dessa part i c i p a ç ã o. En t re t a n t o, p a ra que a população participe do PCDCh, é p reciso que a mesma tenha os conhecimentos n e c e s s á rios para suspeitar que um inseto que i n vada seu domicílio seja de espécie ve t o ra da This study aims to analyze the effect of domicil- i a ry infestation by triatomine bugs on the de- g ree to which inhabitants are aware of potential vector transmission of Chagas disease. 1 La b o ratório de Im u n o e p i d e m i o l o g i a , Superintendência de C o n t role de En d e m i a s , São Pa u l o, Bra s i l . 2 Se rviço Regional de Presidente Pr u d e n t e , Superintendência de C o n t role de En d e m i a s , Presidente Pr u d e n t e , Bra s i l . 3 La b o ratório de Pa ra s i t o s e s por Fl a g e l a d o s , Superintendência de Controle de En d e m i a s , Mogi Gu a ç u , Bra s i l . A b s t r a c t Such re- cently constructed dwellings comprise housing g roups classified as settlements and re - s e t t l e- m e n t s , selected re s p e c t i vely in the municipali- ties of Euclides da Cunha Paulista and Pa u l i- c é i a , São Paulo St a t e , Bra z i l . Both municipali- ties are under the jurisdiction of the Pre s i d e n t e Prudente Health Ad m i n i s t ra t i ve Re g i o n . Of the 319 re s i d e n t s , some 100 (76.0% of whom were re-settlement residents) knew about triatomine b u g s . Housing units infested with triatomines w e re inhabited by 93 people. In 79.2% of the in- fested houses, 26.8% of the residents knew about Chagas disease and its ve c t o r s , but in 50.0% of the households, some people did not know what to do in case of triatomine infestation. Po p u l a- tion samples from settlements and re - s e t t l e- m e n t s , re g a rdless of sex , a g e , and the State of o r i g i n ,s h owed no difference in attitudes tow a rd s the pre vention of Chagas disease or know l e d g e of the disease ve c t o r. C o r re s p o n d ê n c i a Rubens Antonio da Si l va La b o ratório de Im u n o e p i d e m i o l o g i a , Superintendência de C o n t role de En d e m i a s . Rua Paula Souza 166, 5o a n d a r, São Pa u l o, S P, 0 1 0 2 7 - 0 0 0 , Bra s i l . r u b e n s @ s u c e n . s p. g ov. Pesquisa sistemática positiva e relação com conhecimento da população de assentamento e reassentamento de ocupação recente em áre a de Triatoma sord i d a (Hemiptera, Reduviidae) no Estado de São Paulo, Brasil Pesquisa sistemática positiva e relação com conhecimento da população de assentamento e reassentamento de ocupação recente em áre a de Triatoma sord i d a (Hemiptera, Reduviidae) no Estado de São Paulo, Brasil Pesquisa sistemática positiva e relação com conhecimento da população de assentamento e reassentamento de ocupação recente em áre a de Triatoma sord i d a (Hemiptera, Reduviidae) no Estado de São Paulo, Brasil Pesquisa sistemática positiva e relação com conhecimento da população de assentamento e reassentamento de ocupação recente em áre a de Triatoma sord i d a (Hemiptera, Reduviidae) no Estado de São Paulo, Brasil Investigation into the effect of household triatomine infestation on awareness among inhabitants of settlements and re-settlements in areas infested with Triatoma sord i d a in São Paulo State, Brazil Rubens Antonio da Si l va 1 Susy Ma ry Perpétuo Sampaio 2 Marisa Poloni 2 Paulo Hi roshi Koyanagui 2 Maria Esther de Ca rvalho 1 Ve ra Lúcia Cortiço Corrêa Rodrigues 3 Rubens Antonio da Si l va 1 Susy Ma ry Perpétuo Sampaio 2 Marisa Poloni 2 Paulo Hi roshi Koyanagui 2 Maria Esther de Ca rvalho 1 Ve ra Lúcia Cortiço Corrêa Rodrigues 3 C o r re s p o n d ê n c i a Rubens Antonio da Si l va La b o ratório de Im u n o e p i d e m i o l o g i a , Superintendência de C o n t role de En d e m i a s . Rua Paula Souza 166, 5o a n d a r, São Pa u l o, S P, 0 1 0 2 7 - 0 0 0 , Bra s i l . r u b e n s @ s u c e n . s p. g ov. b r Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 5 5 5 A RTIGO A RT I C L E 5 5 5 A RTIGO A RT I C L E 5 5 5 A RTIGO A RT I C L E Material e métodos O Município de Euclides da Cunha Pa u l i s t a a p resenta população de 10.214 habitantes, s e n- do 37,3% pertencentes à área ru ral, enquanto o Município de Paulicéia apresenta população de 5.302 habitantes, sendo 25,8% pert e n c e n t e s à área ru ral 7. A seleção destas localidades de- veu-se ao fato de serem ambas de formação re- c e n t e, dois anos para a pri m e i ra localidade e q u a t ro anos para a segunda, e possuírem cara c- terística de assentamento e re a s s e n t a m e n t o, re s p e c t i va m e n t e. As áreas em estudo não apre- sentam histórico de infestação tri a t o m í n i c a , p o rt a n t o, sem atividades de controle re a l i z a d a s. Nestas localidades foi realizada pesquisa i n t e g ral de todas as UD’s, no mês de agosto de 2002, seguindo o normatizado no PCDCh no Estado de São Pa u l o, com direcionamento da pesquisa à fonte de alimentação 8. A atividade de pesquisa foi realizada pelas equipes de cam- po da Su p e rintendência de Co n t role de En d e- m i a s, órgão re s p o n s á vel pelas atividades de c o n t role do pro g rama. Na ocasião da pesquisa t riatomínica no domicílio equipes form a d a s por profissionais da área da saúde foram re s- p o n s á veis pela aplicação de questionários nos residentes de cada UD. Consentimentos fora m obtidos seguindo as normas preconizadas pela De c l a ração de Helsinki. As fichas usadas na in- vestigação continham questões abert a s, per- mitindo ao entre v i s t a d o r, na presença de todos os residentes de uma unidade, buscar inform a- ções sobre os conhecimentos já adquiridos pe- los mesmos em relação aos tra n s m i s s o res da doença de Chagas. Material e métodos doença e notifique o mesmo aos setores com- p e t e n t e s. A Região Ad m i n i s t ra t i va de Saúde de Pre s i d e n- te Prudente situa-se a sudoeste do Estado de São Pa u l o, Brasil, compreendendo 41 municí- p i o s. Nesta região tem-se observado no tra n s- c o r rer dos anos um aumento gra d a t i vo na for- mação de assentamentos e re a s s e n t a m e n t o s ru ra i s. Pa ra o ano de 2002, havia nesta área, 87 assentamentos e 7 reassentamentos abra n g e n- do 17 e 5 municípios, re s p e c t i va m e n t e. As tra n s f o rmações provocadas no ambien- te pelo homem, bem como a necessidade a ter- ra para cultivo, pro m ove ram a formação de c o n g l o m e rados humanos em novas situações, os quais foram denominados de assentamen- tos e re a s s e n t a m e n t o s. O pri m e i ro está re l a c i o- nado ao processo de formação do Mov i m e n t o dos Tra b a l h a d o res Ru rais Sem Te r ra no Estado de São Paulo (MST-SP) que se iniciou nos anos 1979-1984. Nesse período, a articulação das ex- p e riências de luta pela terra que aconteciam, p ri n c i p a l m e n t e, nos Estados do Pa raná, Rio Grande do Sul, São Pa u l o, Mato Grosso do Sul e Santa Ca t a rina levou à fundação do MST, na realização do Pri m e i ro En c o n t ro Nacional, em j a n e i ro de 1984, na cidade de Ca s c a vel, Pa ra n á . Material e métodos Este movimento organizado de luta pelos dire i- tos à pro p riedade dos tra b a l h a d o res sem terra e n vo l ve cerca de 1,5 milhões de pessoas de di- versas regiões bra s i l e i ras 5. Trata-se de uma f o r- ma diferente de reinvidicação social. Já para o segundo caso, com a necessidade do homem ao meio, tra n s f o rmações foram pro m ovidas no a m b i e n t e. Grandes pro p ri e d a d e s, anteri o rm e n- te ocupadas por uma ou duas famílias, fora m divididas em lotes para receber novos mora d o- re s. Um dos motivos desta re d i s t ribuição de t e r ras está baseada na construção de barra g e n s no curso de ri o s, obrigando o re m a n e j a m e n t o de populações inteira s. Essas tra n s f o rm a ç õ e s p ro m ove ram a incorporação de novas Un i d a- des Domiciliares (UD’s) ao meio, entendendo- se por UD o conjunto da casa (intra d o m i c í l i o ) mais seus anexos externos (peridomicílio). Po u- co se conhece quanto às áreas de assentamen- tos e reassentamentos ru rais no Estado de São Pa u l o. Segundo Veiga & Burlandy 6, supostam e n- t e, estes novos grupamentos humanos apre- sentam características especiais que os dife- renciam de outros gru p o s. Pa ra este estudo foram selecionadas as lo- calidades Assentamento Nova Esperança III (41 domicílios e 122 habitantes) e Fa zenda Bu- ritis (81 domicílios e 245 habitantes), pert e n- centes aos municípios de Euclides da Cu n h a Paulista e Paulicéia, re s p e c t i va m e n t e, ambos situados na Região Ad m i n i s t ra t i va de Saúde de Presidente Prudente (Fi g u ra 1). A b s t r a c t b r Chagas Di s e a s e ; Tr i a t o m a ; Communicable Di s- ease Contro l ; Ru ral Se t t l e m e n t s Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 Silva RA et al. 5 5 6 Silva RA et al. 5 5 6 Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 R e s u l t a d o s A pesquisa triatomínica realizada nas localida- des perf ez um total de 107 domicílios, sendo 72 (86,7% dos existentes) pertencentes à Fa ze n d a Bu ritis e 35 (85,3% dos existentes) pert e n c e n t e s ao Assentamento Nova Esperança III. O núme- ro de anexos das duas localidades foi de 572, sendo 72,5% encontrados na Fa zenda Bu ri t i s. Casas habitadas corre s p o n d e ram a 82,3% e o ti- po de construção predominante foi o tijolo, re- bocado ou não, com percentuais de 90,4 e 70,7 p a ra o reassentamento e assentamento, re s p e c- t i va m e n t e. A positividade de pesquisa tri a t o m í- nica foi observada em 24 (22,4%) domicílios, to- dos pertencentes ao re a s s e n t a m e n t o. Fo ra m coletados 136 exemplares de Tr i at oma sord i d a, p rincipalmente no peridomicílio (99,3%), asso- ciados a galinheiros e ninhos de ro e d o r. O exa- me de positividade para ve rificação de infecção por Try p an os oma cruzi re velou resultado nega- t i vo para todos os exemplare s. Quanto à entrevista com os mora d o res da unidade domiciliar foi possível uma cobert u ra de 84,7% da população (319 indivíduos) sendo 62,3% moradores do reassentamento. Sexo m a s- culino (61,4%) predominou sobre o feminino. Na Fi g u ra 2 podemos observar a pro c e d ê n c i a dos entre v i s t a d o s. Ve rificamos que no re a s s e n- tamento há maior número de pessoas nascidas no Estado de São Paulo diferindo do assenta- m e n t o, porém, quando relacionada esta condi- ção ao conhecimento em relação à doença de C h a g a s, não foi encontrada associação (OR = 0,5598; 0,3459 < OR < 0,906, 95% de confiança). radas às entrevistas realizadas no assentamen- to com aquelas do reassentamento, observou-s e baixa associação nas questões re f e rentes ao co- nhecimento do “barbeiro” e no encam i n h amen- to correto do inseto aos setores c o m p e t e n t e s. Material e métodos Ainda procedeu-se ao le- vantamento de características da moradia co- mo tipo de constru ç ã o, situação (habitada ou desabitada) e presença de anexo s. Os grupos populacionais residentes em á re a s de acampamentos, ocupações e assentamen- tos vêm cre s c e n d o, especialmente na última década. Dada a escassez de dados sobre as c o n- dições de vida e saúde dos assentados, re a l i- zou-se o presente estudo que objetiva analisar a relação existente entre pesquisa tri a t o m í n i c a p o s i t i va (UD positiva) e os conhecimentos so- b re ve t o res da doença de Chagas de mora d o re s residentes nestas UD’s formadas re c e n t e m e n- t e, de modo a subsidiar a proposição de ações de controle dirigidas a essas áre a s. Os dados foram re g i s t rados em form u l á ri o s p a d ronizados e tra n s c ritos em bancos de da- A S S E N TAMENTO E REASSENTAMENTO: CONHECIMENTO DA POPULAÇÃO SOBRE DOENÇA DE CHAGAS 5 5 7 Figura 1 d o s. As freqüências foram extraídas através de p ro g rama de análise Epi Info versão 6.0. Di f e- renças de proporções foram analisadas com o teste estatístico odds ratio com intervalo de c o n- fiança de 95%. Região Administrativa de Saúde de Presidente Prudente, Estado de São Paulo. Em destaque os municípios de Paulicéia e Euclides da Cunha Paulista. Paulicéia Euclides da Cunha Paulista • • São Paulo Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 R e s u l t a d o s Figura 2 P e rcentual dos entrevistados no assentamento Nova Esperança III e no reassentamento Fazenda Buritis, 0 10 20 30 40 50 60 70 80 reassentamento assentamento Ignorado Outros Sergipe Mato Grosso Alagoas Minas Gerais Bahia Paraná São Paulo Tabela 1 Conhecimento da população entrevistada sobre doença de Chagas e seus transmissores no assentamento Nova Esperança III e no reassentamento Fazenda Buritis. Região Administrativa de Saúde de Presidente Prudente, Estado de São Paulo, Brasil. R e s u l t a d o s Q u e s t õ e s Assentamento Reassentamento To t a l OR (95% de confiança) (n = 120)* (n = 199)* * S i m % N ã o % S i m % N ã o % S i m % N ã o % Conhece “barbeiro ” 2 4 2 0 , 0 9 6 8 0 , 0 7 7 3 8 , 7 1 2 2 6 1 , 3 1 0 1 3 1 , 7 2 1 8 6 8 , 3 2,525 (1,485 < OR < 4,291) Já foram picados pelo “barbeiro ” 1 0 8 , 3 1 1 0 9 1 , 7 2 4 1 2 , 1 1 7 5 8 7 , 9 3 4 1 0 , 6 2 8 5 8 9 , 4 1,509 (0,695 < OR < 3,276) Já foi encontrado “barbeiro” 0 0 , 0 1 2 0 1 0 0 , 0 8 6 4 3 , 2 1 1 3 5 6 , 8 8 6 2 7 , 9 2 3 3 7 2 , 1 – em sua casa Sabe o que fazer caso encontre 4 1 3 4 , 2 7 9 6 5 , 8 1 0 8 5 4 , 2 9 1 4 5 , 8 1 4 9 4 6 , 7 1 7 0 5 3 , 3 2,287 (1,43 < OR < 3,656) um inseto suspeito Praticam caça 6 6 5 5 , 0 5 4 4 5 , 0 5 4 2 7 , 1 1 4 5 7 2 , 9 1 2 0 3 7 , 6 1 9 9 6 2 , 4 0,305 (0,189 < OR < 0,491) Manipulam caça 6 8 5 6 , 7 5 2 4 3 , 3 7 6 3 8 , 1 1 2 3 6 1 , 9 1 4 4 4 5 , 1 1 7 5 5 4 , 9 0,473 (0,298 < OR < 0,749) * 4 pessoas não responderam as questões. ** 35 pessoas não responderam as questões. cimento da população entrevistada sobre doença de Chagas e seus transmissores no assentamento Nova Esperança III assentamento Fazenda Buritis. Região Administrativa de Saúde de Presidente Prudente, Estado de São Paulo, Brasil. desafiar os poderes constituídos 9. R e s u l t a d o s Quando analisadas as informações sobre o conhecimento de pessoas com idade superi o r a trinta anos nascidas no Estado de São Pa u l o, l e vando-se em consideração a interrupção da t ransmissão natural da doença de Chagas no início da década de 1970, encontramos 69,7% dos entrevistados nesta situação. Estatistica- m e n t e, o fator idade não apresentou relação ao conhecimento adquirido sobre doença de Cha- gas obtendo-se valor de OR de 0,1988 (0,1197 < OR < 0,3301) com intervalo de confiança de 95%. , ; , , , ç ) Em relação aos conhecimentos a respeito d o i n s e t o, 31,7% das famílias conheciam o mesmo (Tabela 1). Re l a t a ram já terem sido picados pe- lo tri a t o m í n e o, 10,6% dos indivíduos. O pro c e- dimento correto a adotar caso encontrem um inseto suspeito em seu domicílio foi observa d o em 46,7% dos entrevistados sendo encontra d o maior percentual no reassentamento se com- p a rado ao assentamento, porém sem re l a ç ã o com o conhecimento sobre a doença de Cha- gas adquirido pela população (OR = 2,7; 0,9352 < OR < 7,7948, 95% de confiança). A prática e manipulação de carne de animais re c o n h e c id o s como re s e rva t ó rios de T. c r u z i a p re s e n t a ra m p e rcentuais mais elevados no assentamento se c o m p a rado ao re a s s e n t a m e n t o. Quando comp a- Os resultados da entrevista para os domicí- lios com pesquisa triatomínica positiva re ve l a- ram que 47,4% ou 93 pessoas estão nestas mo- radias e possuem idade superior a trinta anos (68,0%). Em 79,2% das casas que se coletou t riatomíneo há 26,8% dos indivíduos que apre- sentam conhecimento sobre a doença, mas em 50,0% destas casas há pessoas que não sabem Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 Silva RA 5 5 8 5 5 8 Silva RA et al. 5 5 8 Silva RA et al. R e s u l t a d o s O mov i m e n- to cresceu e se expandiu, não podendo mais ser ignorado pelas autori d a d e s. A opinião pú- blica tem se re velado um elemento import a n t e p a ra as decisões de gove rn o. o que fazer caso encontrem este inseto no seu d o m i c í l i o. Realizam e manipulam caça 45,8% e 58,3% dos mora d o re s, re s p e c t i va m e n t e, nestas re s i d ê n c i a s. Lote de terra foi entregue aos assentados e reassentados sendo disponibilizados para o p ri m e i ro caso recursos financeiros para re a l i- zação de pequenas benfeitorias na pro p ri e d a- d e, como a construção da moradia e, no segun- do caso, a moradia já foi entregue pronta. Qu a n- P e rcentual dos entrevistados no assentamento Nova Esperança III e no reassentamento Fazenda Buritis, segundo local de nascimento. Região Administrativa de Saúde de Presidente Prudente, Estado de São Paulo, Brasil. D i s c u s s ã o Diotaiuti 1 2 e Si l ve i ra et al. 1 3 d e s c re vem que esta espécie apare n t em e n- te não está hoje diretamente associada à tra n s- missão do T. c r u z i ao homem, porém a mesma d e ve ser controlada no peri d o m i c í l i o, para in- t e r rupção do ciclo tão próximo ao ambiente humano e como medida de pre venção contra a f o rmação de eventuais colônias intra d o m i c i- l i a re s. Este estudo demonstrou que a popula- ção trabalhada apresenta conhecimentos so- b re a doença de Chagas, tendo sido observa d o maior percentual no reassentamento (Ta b e l a 1). To rna-se necessári o, então, investir em um p rocesso de continuidade pro m ovendo um c o m p romisso mais efetivo da população com a vigilância entomológica no reassentamento da Fa zenda Bu ritis e uma intervenção educativa no caso do assentamento. O empenho dos téc- nicos em manter um planejamento adequado, c riando uma agenda de compromissos com a p o p u l a ç ã o, que leve em consideração uma m a i- or intensificação das ações educativas deve ser De acordo com Ga rc i a - Zapata & Ma rdsen 1 6 existem fatores de risco como a falta de higie- n e, a desordem intradomiciliar e a presença de animais dentro das habitações, que pare c e m ser re s p o n s á veis pela persistência de focos de t riatomíneos nas áreas ru ra i s. Na população que vive exposta ao risco de contrair a doença de Chagas é de se esperar que ela disponha dos conhecimentos necessários para poder pre ve- nir o contato homem/vetor através de suas ações cotidianas 1 7. Em nosso estudo observa- mos que 69,7% dos entrevistados com idade maior ou igual a trinta anos, principalmente os nascidos no Estado de São Pa u l o, apre s e n t a m conhecimentos sobre a doença de Chagas. Este fato está relacionado a um passado de tra n s- missão ve t o rial ativa no Estado. D i s c u s s ã o No tra n s c o r rer da década de 1990, o MST con- quistou um espaço político importante no q u a- d ro público atual, tendo força suficiente para Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 A S S E N TAMENTO E REASSENTAMENTO: CONHECIMENTO DA POPULAÇÃO SOBRE DOENÇA DE CHAGAS 5 5 9 do ve rificamos os dados re l a t i vos à habitação nestas localidades, observamos uma situação de maior pre c a riedade nas casas do assenta- mento Nova Esperança III, porém, na pesquisa t riatomínica, em todas as moradias não foi en- c o n t rado tri a t o m í n e o. Na Fa zenda Bu ri t i s, per- tencente ao Município de Paulicéia, as casas a p re s e n t a vam-se em melhores condições de c o n s t rução e nesta localidade é que se obser- vou presença de tri a t o m í n e o s. Estes foram co- letados em sua grande maioria no peri d o m i c í- l i o, fato re l e va n t e, pois, na formação deste re a s- s e n t a m e n t o, todo material anteri o rmente en- c o n t rado na moradia foi tra n s p o rtado para es- ta nova área. Os tri a t o m í n e o s, prova ve l m e n t e, podem ter sido tra n s p o rtados passiva m e n t e pelos mora d o re s, uma vez que não há históri c o de infestação nesta área antes de se tornar um re a s s e n t a m e n t o. As populações, do assenta- mento e do re a s s e n t a m e n t o, quando do re c e- bimento do lote de terra para sua nova mora- dia, re c e b e ram junto a este, uma casa constru í- da de tijolo re b o c a d o, refletindo neste fato o e n c o n t ro de percentuais elevados de casas de boa qualidade. D i s c u s s ã o b u s c a d o, para assim podermos manter re s u l t a- dos satisfatóri o s. Pa ra o sucesso de qualquer p ro g rama de controle é fundamental ter a po- pulação informada e participando da vigilân- cia entomológica, como o que se pretende co- mo norma de pro g rama a ser seguido no Esta- do de São Pa u l o. “A participação comunitária é um processo dinâmico em que a população é conscientemente engajada no planejamento, implementação e avaliação de atividades que afetam suas vidas” 1 4 ( p. 128). A maioria das colônias triatomínicas en- c o n t radas no Estado de São Paulo está locali- zada no peri d o m i c í l i o. Em todas as UD’s onde se coletou triatomíneo houve seu encontro no p e ri d o m i c í l i o, sendo os galinheiros (73,5%) re s- p o n s á veis pelo abrigo destes insetos, re f l e t i n d o o comportamento da espécie. Os galinheiro s e ram construídos de madeira originada do p r ó- p rio local da moradia. No peridomicílio sabe- se que a ação do inseticida é reduzida. Po rt a n- t o, caberá à população a vistoria dos anexos lá e n c o n t ra d o s, pro m ovendo a vigilância entom o- lógica peri d o m i c i l i a r. Um pro g rama de contro- le integrado e sustentável no tempo terá êxito com uma participação da comunidade inclusi- ve observando o ambiente peridomiciliar 1 5. Dias 1 0 , 1 1 coloca que, para a adaptação e co- lonização do ambiente doméstico por tri a t o- m í n e o s, entram em jogo pri o ri t a riamente fato- res humanos e sociais, tais como a qualidade e o tipo de habitação, a ação antrópica sobre o ambiente e as migrações humanas, tudo isto sob forte influência de elementos de naturez a política, econômica e cultural, em paralelo c o m as condições ecológicas e ambientais das di- versas micro r regiões da área endêmica. Na p e s- quisa triatomínica realizada, a espécie encon- t rada foi o Tr. s o rd i d a. Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 R e s u m o Pro c u rou-se analisar a relação existente entre Un i d a- de Domiciliar (UD) com triatomíneo e os conhecimen- tos sobre doença de Chagas entre os mora d o res destas U D’s formadas recentemente em conglomerados deno- minados assentamento e re a s s e n t a m e n t o. Fo ram sele- cionadas duas localidades situadas nos municípios de Euclides da Cunha Paulista (assentamento) e Pa u l i- céia (reassentamento) pertencentes à Região Ad m i n i s- t ra t i va de Saúde de Presidente Pr u d e n t e , São Pa u l o, Bra s i l . A entrevista com os mora d o res das UD’s abra n- geu 319 indivíduos. Em relação aos conhecimentos a respeito do inseto, 100 pessoas conheciam o mesmo, sendo 76,0% mora d o ra do re a s s e n t a m e n t o. Qu a n d o analisados os resultados da entrevista para os domicí- lios com pesquisa triatomínica positiva ve r i f i c o u - s e que 93 pessoas (47,4%) estavam nestas mora d i a s . Em 79,2% das casas em que se coletou triatomíneo (Tri a- t oma sord i d a) há 26,8% dos indivíduos que apre s e n- tam conhecimento sobre doença/ve t o r, mas em 50,0% destas casas há pessoas que não sabem o que fazer ca- so encontrem este inseto no seu domicílio. Neste estu- do não foi encontrada relação entre o sexo, a idade, a n a t u ralidade e a adoção de práticas pre ve n t i vas ade- quadas e o conhecimento sobre a doença/vetor na po- pulação entrevistada tanto no assentamento como no re a s s e n t a m e n t o. Todos os autores part i c i p a ram do processo de elabo- ração do art i g o, ficando R. A. Si l va re s p o n s á vel pela e l a b o ração e pré-teste de form u l á ri o s, aplicação de q u e s t i o n á ri o, criação de banco de dados, análise es- tatística e redação do artigo; S. M. P. D i s c u s s ã o Dias & Dias 1 8 o b t i ve ram resultados similares em uma comu- nidade de Minas Ge rais (Brasil) observa n d o que 70,0% dos maiores de vinte anos re c o n h e- ciam o Tr. i n f e s t a n s. É de se esperar que a po- pulação com maior informação sobre a doença m o s t re uma diminuição sobre exposição a fa- t o res de ri s c o. En t re t a n t o, em nosso estudo não foi encontrada relação entre o conhecimento s o b re a doença de Chagas e a adoção de práti- cas pre ve n t i vas adequadas. Ressaltamos que f o i o b s e rvado um comportamento de risco maior na população mora d o ra do assentamento e foi o b s e rvado no re a s s e n t a m e n t o, onde o risco foi menor, um conhecimento em reconhecer o t ri a- Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 Silva RA et al. 5 6 0 Silva RA et al. 5 6 0 Silva RA et al. 5 6 0 tomíneo e encaminhá-lo corretamente aos se- t o res competentes (Tabela 1). de altern a t i vas precoces de interve n ç ã o, pri n- cipalmente no tocante aos assentamentos que por apre s e n t a rem peculiari d a d e s, podem os m o ra d o res vir a ter o mesmo comportamento e conhecimento no que diz respeito à doença de C h a g a s. Si l ve i ra et al. 1 3 colocam em seu tra b a- lho que não se pode pretender um modelo úni- co de ação, ou a universalização de determ i n a- do modelo de opera ç ã o, mesmo porque fatore s como a atividade econômica desenvolvida, o tipo de organização social e hábitos e práticas da população podem favo recer a manutenção da infestação domiciliar. Cassab et al. D i s c u s s ã o 2 0 e m t rabalho realizado na Bolívia sugerem a aplica- ção de diferentes técnicas e meios adequados p a ra intervenção na população levando-se em c o n s i d e ração o grupo a ser trabalhado e as me- tas a serem alcançadas. De acordo com os mes- m o s, visitas semanais junto à comunidade pa- ra prestar assistência técnica e re s o l ver pro b l e- mas que podem surgir devem ser implementa- das pelos técnicos envolvidos nos tra b a l h o s. p No Estado de São Pa u l o, tem-se observa d o, ao longo dos anos, uma preocupação da popu- lação em encaminhar insetos suspeitos aos se- t o res competentes para ave ri g u a ç ã o. Ela o faz quando da invasão deste inseto em seu am- biente domiciliar (intradomicílio) não se pre o- cupando com insetos que possam estar co-ha- bitando com animais no peri d o m i c í l i o. Mo s- t rar à população a importância de atentar-se e cuidar do peridomicílio na busca de insetos ve- t o res são mecanismos que deverão ser busca- dos e incorporados nos pro g ramas de contro l e. Ex i s t e, port a n t o, de acordo com Fe r re i ra et al. 1 9, uma necessidade urgente de que os setore s re s p o n s á veis pela elaboração das políticas pú- blicas realoquem recursos para a interve n ç ã o imediata nas comunidades agrári a s, a fim de s e n s i b i l i z a rem os mora d o res da importância d e p ro m over uma vigilância entomológica cons- tante de seu domicílio. Os resultados encontra- dos em nosso estudo permitem a form u l a ç ã o Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 Doença de Chagas; Tr i a t o m a ; C o n t role de Do e n ç a s Tra n s m i s s í ve i s ; Assentamentos Ru ra i s R e f e r ê n c i a s 1 . Souza AG, Wanderley DMV, Bu ralli GM, Andra d e JCR. Consolidation of the control of Chagas dis- ease vectors in the State of São Pa u l o. Mem In s t Oswaldo Cruz 1984; 79:125-31. 1 1 . Dias JCP. Ecological aspects of the ve c t o rial con- t rol of Chagas’ disease in Brazil. Cad Saúde Públi- ca 1994; 10 Suppl 2:352-9. 1 2 . Diotaiuti L. O risco da domiciliação de novas es- pécies de tri a t o m í n e o s. Rev Soc Bras Med Tro p 2000; 33 Suppl 2:31-5. 2 . Si l va RA, Bonifácio PR, Wanderley DMV. Doença de Chagas no Estado de São Paulo: compara ç ã o e n t re pesquisa ativa de triatomíneos em domicí- lios e notificação de sua presença pela população em área sob vigilância entomológica. Rev So c Bras Med Trop 1999; 32:653-9. 1 3 . Si l ve i ra AC, Arias AR, Se g u ra E, Guillén G, Ru s s o- mando G, Schenone H, et al. El control de la en- f e rmedad de Chagas en los Paises del Cono Su r de America. Ub e raba: Faculdade de Medicina do Triângulo Mi n e i ro; 2002. 3 . Dias JCP. Problemas e possibilidades de part i c i- pação comunitária no controle das grandes en- demias no Brasil. Cad Saúde Pública 1998; 14 Suppl 2:19-37. 1 4 . Dias JCP, Dias RB. Pa rticipação da comunidade no controle da doença de Chagas. Ann Soc Be l g Med Trop 1985; 65 Suppl 1:127-35. 4 . Vinhaes MC, Si l ve i ra AC. The possibilities of maintenance of the natural transmission of Cha- gas endemic disease in Brazil with the elimina- tion of Triatoma infestans. Rev Soc Bras Med Tro p 2001; 34 Suppl 3:61-2. 1 5 . Ce c e re MC, Gu rtler RE, Canale D, Chuit RY, Coh e n JE. El papel del peridomicilio en la eliminación de Triatoma infestans de comunidades ru rales ar- g e n t i n a s. Bol Oficina Sanit Panam 1996; 121:1-8. 1 6 . Ga rc i a - Zapata MTA, Marsden PD. En f e rmedad de Chagas: control y vigilancia con insecticidas y p a rticipación comunitaria en Mambaí, Go i á s, Brasil. R e f e r ê n c i a s Bol Oficina Sanit Panam 1994; 116:97-110. 5 . Movimento dos Tra b a l h a d o res Ru rais Sem Te r ra . A luta do MST e a questão agrária do Estado de São Pa u l o. http://www. m s t . o rg . b r / m s t s p / h i s t . html (acessado em 09/Ma r / 2 0 0 3 ) . 1 7 . Sa n m a rtino M, Crocco L. Conocimentos sobre la e n f e rmedad de Chagas y factores de riesgo en co- munidades epidemiológicamente diferentes de Argentina. Rev Panam Salud Publica 2000; 7:173-8. 6 . Veiga GV, Burlandy L. In d i c a d o res sócio-econô- m i c o s, demográficos e estado nutricional de cri- anças e adolescentes residentes em um assenta- mento ru ral do Rio de Ja n e i ro. Cad Saúde Pública 2001; 17:1465-72. 1 8 . Dias JCP, Dias RB. Las vivendas y la lucha contra los ve c t o res de la enfermedad de Chagas en el h o m b re, en el Estado de Minas Ge ra i s, Brasil. Bo l Oficina Sanit Panam 1982; 93:453-67. 7 . Fundação Instituto Bra s i l e i ro de Ge o g rafia e Est a- tística. Censo demográfico 2000: resultados p re- l i m i n a re s. São Paulo: Fundação Instituto Bra s i l e i- ro de Ge o g rafia e Estatística; 2000. 7 . Fundação Instituto Bra s i l e i ro de Ge o g rafia e Est a- tística. Censo demográfico 2000: resultados p re- l i m i n a re s. São Paulo: Fundação Instituto Bra s i l e i- ro de Ge o g rafia e Estatística; 2000. 1 9 . Fe r re i ra HS, Albuquerque MFM, Ataíde T R , Mo rais MGC, Mendes MCR, Si q u e i ra TC, et al. Es- tado nutricional de crianças menores de dez anos residentes em invasão do “Movimento dos Se m - Te r ra”, Po rto Ca l vo, Alagoas. Cad Saúde Pública 1997; 13:137-9. 8 . Se c re t a ria de Estado da Saúde de São Pa u l o. Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 R e s u m o Sampaio part i c i- pou do pré-teste de form u l á rios e da redação do art i- go; M. Poloni aplicou questionários em campo e re a- l i zou levantamento bibliográfico; P. H. Koyanagui su- p e rvisionou as equipes de campo da SUCEN na pes- quisa triatomínica e controle químico; M. E. Ca rva- lho aplicou questionários e participou da redação do a rtigo; V. L. C. C. Ro d rigues re a l i zou a identificação dos triatomíneos e exame de conteúdo fecal para de- tecção de infecção natural por tri p a n o s s o m a t í d e o s. Doença de Chagas; Tr i a t o m a ; C o n t role de Do e n ç a s Tra n s m i s s í ve i s ; Assentamentos Ru ra i s Doença de Chagas; Tr i a t o m a ; C o n t role de Do e n ç a s Tra n s m i s s í ve i s ; Assentamentos Ru ra i s Cad. Saúde Pública, Rio de Janeiro, 20(2):555-561, mar- a b r, 2004 A S S E N TAMENTO E REASSENTAMENTO: CONHECIMENTO DA POPULAÇÃO SOBRE DOENÇA DE CHAGAS 5 6 1 R e f e r ê n c i a s R e f e r ê n c i a s Re l a- t ó rio final do Grupo de Trabalho do Pro g rama de Co n t role da Doença de Chagas. São Paulo: Su p e- rintendência de Co n t role de En d e m i a s, Se c re t a- ria de Estado da Saúde de São Paulo; 1989. 2 0 . Cassab JRA, No i reau F, Guillén G. La enferm e d a d de Chagas em Bolívia: conocimientos científicos al inicio del Pro g rama de Co n t rol (1998-2002). La Paz: Organización Panamericana de la Salud; 1999. 9 . Co m p a rato BK. A ação política do MST. São Pa u l o em Pe r s p e c t i va 2001; 15:105-18. 9 . Co m p a rato BK. A ação política do MST. São Pa u l o em Pe r s p e c t i va 2001; 15:105-18. 1 0 . Dias JCP. Doença de Chagas, ambiente e part i c i- pação do Estado. Cad Saúde Pública 2001; 17 Suppl 1:165-9. 1 0 . Dias JCP. Doença de Chagas, ambiente e part i c i- pação do Estado. Cad Saúde Pública 2001; 17 Suppl 1:165-9. Recebido em 03/Ma r / 2 0 0 3 Versão final re a p resentada em 19/Se t / 2 0 0 3 Ap rovado em 23/Ou t / 2 0 0 3 Recebido em 03/Ma r / 2 0 0 3 Versão final re a p resentada em 19/Se t / 2 0 0 3 Ap rovado em 23/Ou t / 2 0 0 3
https://openalex.org/W3134073964	https://computingonline.net/computing/article/download/420/390	Ukrainian	null	THE IMPLEMENTATION OF PROLOG INTERPRETER: LEXICAL AND SYNTAX ANALYSES	Computing	2,014	cc-by	6,202	Олександр Цимбал Харківський національний університет радіоелектроніки, м. Харків, проспект Леніна 14, mcdulcimer@ukr.net Резюме: У статті розглядається розробка транслятора мови програмування Prolog у складі лексичного та синтаксичного аналізаторів, блоків керування таблицями та інтерпретації. Надано схеми синтаксичного аналізу та елементи програмного коду транслятора. Програмне забезпечення реалізовано у середовищі розробки Visual C++ 2005 (Beta). Ключові слова: Мова програмування, транслятор, Prolog, інтерпретатор, граматика, лексичний аналіз, синтаксичний аналіз. 1. ВСТУП покоління» [1], в наш час, нею займаються лише спеціалісти в галузі логічного програмування та штучного інтелекту. Натомість Prolog вважається однією з класичних мов програмування для систем штучного інтелекту [2, 3]. Prolog дозволяє розглядати експертні системи не тільки з теоретичної, але і з практичної точки зору [4]. Кожна мова програмування має на меті реалізацію певної моделі подання даних і методів їх обробки. Під час розробки програми відбувається перетворення конструкцій початкових даних задачі у вигляд, відповідний моделі, лексичним та синтаксичним особливостям мови програмування. Вибір мови програмування обумовлюється здатністю у найефективніший спосіб здійснити поставлені перед програмістом завдання. Використання Prolog виглядає особливо доречним з точки зору програмної реалізації моделей подання знань, зокрема продукційної та фреймової [2]. Використання рекурсивних процедур обробки списків також надає широкі можливості організації пошукових функцій інформаційних систем, розв’язання завдань планування, в тому числі у робототехніці [3]. Порівняно з універсальними мовами, мова програмування Prolog (Programming in Logic) виглядає зовсім незвично, реалізація методів обробки даних у ній забезпечується побудовою програми у формі предикатів першого роду. Такі особливості Prolog, проте, не заважають ефективно виконувати складні обчислювальні процедури. Головною особливістю є те, що Prolog з практичної точки зору реалізує логічну модель подання даних та знань [1]. Цікавий спосіб використання Prolog описано у [6], де вирішується зворотне завдання – за допомогою Prolog описуються принципи реалізації інших мов програмування. При цьому визнається максимальна простота та зрозумілість реалізації основних мовних конструкцій, спроектованих саме за допомогою Prolog. Розвиток методів штучного інтелекту та необхідність удосконалення мовних засобів їх практичного втілення ставлять завдання модернізації мови програмування Prolog, тому розробка нового транслятора мови Prolog, адаптованого на розв’язання задач робототехніки, є актуальною та своєчасною. Розгляд можливостей побудови власної реалізації мови Prolog вимагає використання класичних методів розробки, описаних у [4]. Проте, з практичної точки зору, доцільно використовувати програмні конструкції, описані у [7, 8]. Після незначної переробки ці матеріали можна легко використати для створення варіанту власної версії транслятора. computing@tanet.edu.te.ua www.computingonline.net computing@tanet.edu.te.ua www.computingonline.net Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 ISSN 1727-6209 International Journal of Computing computing@tanet.edu.te.ua www.computingonline.net 3. ПОСТАНОВКА ЗАДАЧІ Ідентифікатором у Prolog може бути послідовність літер та цифр, яка починається з літери. Беззнаковим числом є послідовність цифр. З точки зору синтаксису Prolog усі ідентифікатори є однаковими і утворюють лексему IDEN. Якщо розглядати проблематику моделювання робототехнічних систем, можна вказати на існування, на першому рівні, проблем реалізації численних розрахунків у кінематичних моделях, які доцільно реалізувати за допомогою універсальних мов програмування, наприклад мови Cі. Крім того, мова Сі (або мова Асемблер) стає у нагоді під час безпосереднього формування керуючих сигналів системі керування маніпулятора робота. Числа без знаку утворюють лексему NUMB. Числа без знаку утворюють лексему NUMB. Окрім IDEN та NUMB у Prolog визначаються спеціальні дванадцять ідентифікаторів – ключових слів мови. До них належать {"domains", "database", "predicates", "clauses", "goal", "integer", "real", "string", "char", "symbol", ":-", "write"}. Символи пробілу, “\n” та “\t” припустимі у кожному місці програми за винятком ідентифікаторів та беззнакових чисел. Інші службові символи утворюють окремі лексеми мови. Символи пробілу, “\n” та “\t” припустимі у кожному місці програми за винятком ідентифікаторів та беззнакових чисел. Інші службові символи утворюють окремі лексеми мови. На іншому рівні – рівні прийняття рішень, розрахунки стають допоміжним фактором і на передній план виступає необхідність методів логічного програмування. Таким чином загальне завдання моделювання робототехнічних систем можна розглядати сукупністю різнопланових задач, вирішення яких цілком ймовірно потребуватиме залучення різних програмних засобів. З точки зору цілісності проекту такий підхід є небажаним, бо викликатиме проблему взаємодії різних засобів програмування. Інший підхід, має передбачати реалізацію основи проекту базовою (універсальною) мовою програмування та реалізацію додаткових програмних засобів на рівні трансляторів. Prolog у даній реалізації має скалярні дані цілого (integer), дійсного (real), символьного (char та symbol) типів та типу рядок (string). Крім того, послідовність значень визначеного скалярного типу може складати список елементів. Елементи списку відокремлюються комами та розміщуються всередині квадратних дужок, наприклад: [12, 31, -5, 6] або [‘a’, ‘d’, ‘E’]. Довжина списку не визначається. У першому варіанті транслятора реалізовано лише тип integer. Ідентифікатори змінних Prolog-програми записуються з великої, ідентифікатори імен предикатів та констант – з малої літери. Усі змінні є локальними в межах предикату, де вони визначені або до якого передані. Під час розробки системи моделювання та керування інтелектуальним роботом пропонується реалізувати підсистему підтримки та прийняття рішень мовою Prolog. Для забезпечення її функціонування у складі системи виникає завдання розробки інтерпретатора мови програмування Prolog. Вирази Prolog будуються з констант, змінних та імен предикатів (з круглими дужками для позначення списку параметрів або без них). Знаками операцій є “+”, “-“, ““, “/“, “%“. 3. ПОСТАНОВКА ЗАДАЧІ Вирази мають вигляд: v, c, (e1), predicate(e1, e2,…,en ), e1 + e2, e1 – e2, e1 e2, e1 / e2, e1 % e2, + e1, –e1, де v – змінна, с – константа, predicate – ідентифікатор імені предиката, e1, e2,…,en – вирази. Метою статті є опис основних особливостей розробки транслятора мови програмування Prolog. Ставляться завдання побудови граматик мови Prolog, реалізацій лексичного та синтаксичного аналізаторів. Засобом створення транслятора є мова програмування Сі (середовище розробника Visual C++). У Prolog визначаються вбудовані оператори: присвоєння/співвідношення - v=c; та друку - write(v), де v – змінна, e – вираз, c – константа. 2. АНАЛІЗ ЛІТЕРАТУРНИХ ДЖЕРЕЛ Таким чином, якщо задача вимагатиме розробки транслятора мови Prolog, її розв’язання полягатиме у застосуванні відомих методів Хоча свого часу саме мова програмування Prolog визнавалася провідною у проекті «ЕОМ V 15 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 розробки мов програмування. алфавіту складаються лексеми – мінімальні мовні ланцюжки із самостійним змістом. // опис прототипів Останній з них містить прототипи усіх використаних у тексті транслятора функцій (їх вигляд легко зрозуміти з анведених далі у тексті функцій) та визначення структур, що забезпечують режими роботи інтерпретатора: D:\ My Projects\PT04a.exe 1.pro Якщо вхідний Prolog-файл задано, аналіз програми забезпечується викликом функції analys(): typedef struct {int cod,opd;}cmd; typedef struct {char name,owner; int what,val;}odc; typedef struct {char name; int isd,cpt,start;}fnd; typedef struct {int cod,opd;}cmd; typedef struct {int cod,opd;}cmd; typedef struct {int cod,opd;}cmd; typedef struct {char name,owner; int what,val;}odc; typedef struct {char name; int isd,cpt,start;}fnd; void analys (char nf) {err=fopen_s(&PF,nf,"r"); // відкриття Prolog-файлу if(!err) {get (); // введення нової лексеми prog (); // аналіз блоку програми } else printf("Error %s file isn't opened",nf); } void analys (char nf) {err=fopen_s(&PF,nf,"r"); // відкриття Prolog-файлу if(!err) {get (); // введення нової лексеми prog (); // аналіз блоку програми } else printf("Error %s file isn't opened",nf); } Також слід визначити глобальні змінні програми: } else printf("Error %s file isn't opened",nf); } cmd TCD[300], cmd pcmd=TCD; char TNM[400], ptn=TNM, char lstr; static char nch='N'; odc TOB[100]; odc pto=TOB; odc ptol=TOB; fnd TFN[30], fnd ptf=TFN; int adrnm,cpnm,out,lval,lex; int cgv=0,clv=0,tc=0,nst=0; int st[500],sp,t,p; using namespace std; FILE PF; errno_t err; cmd TCD[300], cmd pcmd=TCD; char TNM[400], ptn=TNM, char lstr; static char nch='N'; odc TOB[100]; odc pto=TOB; odc ptol=TOB; fnd TFN[30], fnd ptf=TFN; int adrnm,cpnm,out,lval,lex; int cgv=0,clv=0,tc=0,nst=0; int st[500],sp,t,p; using namespace std; FILE PF; errno_t err; 5. РОЗРОБКА ЛЕКСИЧНОГО ТА СИНТАКСИЧНОГО АНАЛІЗАТОРІВ звичайному стилі консольної програми: звичайному стилі консольної програми: void main(int argc,char argv[]) {printf("\n Prolog translator 01 \n"); if(argc>1 && argv[1]) {analys(argv[1]); cout << "\n Analys is finished \n \n"; if(!error_syntax) {interp(); cout << "\n Interpreting is finished \n \n"; } else cout << "\n Interpreting is impossible \n \n"; } else printf("\n no program file \n"); } звичайному стилі консольної програми: void main(int argc,char argv[]) {printf("\n Prolog translator 01 \n"); if(argc>1 && argv[1]) {analys(argv[1]); cout << "\n Analys is finished \n \n"; if(!error_syntax) {interp(); cout << "\n Interpreting is finished \n \n"; } else cout << "\n Interpreting is impossible \n \n"; } else printf("\n no program file \n"); } 4. ОПИС РЕАЛІЗАЦІЇ МОВИ ПРОГРАМУВАННЯ PROLOG Програма мовою Prolog має логічний та / або обчислювальний зміст. Далі в основному розглядається її обчислювальний зміст. Реалізуємо інтерпретатор мови програмування Prolog, що у своїх синтаксичних конструкціях в основному відповідає Turbo-Prolog, розробленому компанією Borland Inc. За основу побудови транслятора використаємо принципи, застосовані у [7] під час побудови простої мови програмування SPL. Програма на Prolog має чітко визначену структуру і складається з таких розділів: domains визначення імен типів даних, складених типів та списків; database визначення вигляду фактів, що складатимуть вміст бази даних Prolog- програми; predicates визначення прототипів предикатів; clauses реалізація предикатів програми; goal виклик предикатів на виконання або запуск програми. Алфавіт мови Prolog складається з символів латинської мови, десяткових цифр, множини службових символів: {'(', ')', ',', ';', '=', '+', '-', '', '/', '%' ,'[',']', '.', '\|', '>', '<'} та пробілу. З символів 16 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 lval, ідентифікатори із кодом IDEN (входять до таблиці ідентифікаторів TNM), ідентифікатор кінця файла із кодом EOF. lval, ідентифікатори із кодом IDEN (входять до таблиці ідентифікаторів TNM), ідентифікатор кінця файла із кодом EOF. Розділ “goal” Prolog-програми відіграє ту є саму роль, що функція main() програми, написаної мовою Сі. У “goal” відбувається запуск першого з необхідних предикатів, проводиться необхідна ініціалізація параметрів. Додамо, що усі змінні, ініціалізовані у goal є локальними в його межах. Також локальними є змінні, розміщені у записах окремих предикатів Prolog-програми, що цілком відповідає стандарту мови. Інформацію про коди лексем можна зберегти за допомогою перелічення: enum {DOMAINSL=257, DATABASEL, PREDICATESL, CLAUSESL, GOALL, INTL, REALL, STRINGL, CHARL, SYMBOLL, IFL, CONSTL, NUMB, IDEN, WRITEL}; enum {DOMAINSL=257, DATABASEL, PREDICATESL, CLAUSESL, GOALL, INTL, REALL, STRINGL, CHARL, SYMBOLL, IFL, CONSTL, NUMB, IDEN, WRITEL}; Запуск програми транслятора здійснюється у Текст програми, що є реалізацією інтерпретатора мови Prolog розпочинається із директив підключення заголовочних файлів: Текст програми, що є реалізацією інтерпретатора мови Prolog розпочинається із директив підключення заголовочних файлів: ( _ y ) { p() cout << "\n Interpreting is finished \n \n"; } #include<iostream> // за VC++ 8.0 (beta) #include<stdio.h> #include<stdlib.h> #include<math.h> #include<ctype.h> #include<string.h> #include "pt04a.h" // опис прототипів #include<iostream> // за VC++ 8.0 (beta) #include<stdio.h> #include<stdlib.h> #include<math.h> #include<ctype.h> #include<string.h> #include "pt04a.h" // опис прототипів // за VC++ 8.0 (beta) else cout << "\n Interpreting is impossible \n \n"; else printf("\n no program file \n"); } Для виконання Prolog-програми необхідно викликати її з командного рядка операційної системи вказавши у якості параметра ім’я файла з текстом програми, наприклад: За введення лексем відповідає функція get(): Повний синтаксис мови Prolog пропонується у такому вигляді: TYPEL → (intl \| reall \| stringl \| symbol) TYPELS – TYPE ‘’ DOMAINSL → (iden=TYPEL \| iden TYPELS) * DATABASEL → (iden ‘(‘ PARAMSL ‘)’) * PREDICATESL → (iden ‘(‘ PARAMSL ‘)’) * PARAMSL → (TYPEL \| TYPELS) (‘,’ [TYPEL \| TYPELS] )* CLAUSESL → PREDIMP (PREDIMP) * PREDIMP → FACTLS \| RULELS FACTLS → FACT (FACT) * RULELS → RULE (RULE) * FACT → iden (CONSTLS) ‘.’ CONSTLS → ATOM (‘,’ ATOM) * ATOM → iden \| numb \| var RULE → RULEL ‘: - ’ RULER RULEL → iden ‘(’ CONSTLS ‘)’ RULER → STML STML → STAT (‘,’ STAT) * STAT → iden ‘=’ (EXPR \| PREDCALL) PREDCALL → RULEL PROG → (DOMAINSL \| PREDICATESL \| DATABASEL \| CLAUSESL) GOAL eof STML → STAT (‘;’ STAT ) * EXPR → [ ‘+’ \| ‘−’ ] TERM ( ( [ ‘+’ \| ‘−’ ] ) TERM)* TERM → FACT ( ( ‘’ \| ‘/’ \| ‘%’ ) FACT ) TYPEL → (intl \| reall \| stringl \| symbol) TYPELS – TYPE ‘’ DOMAINSL → (iden=TYPEL \| iden TYPELS) DATABASEL → (iden ‘(‘ PARAMSL ‘)’) * PREDICATESL → (iden ‘(‘ PARAMSL ‘)’) * PARAMSL → (TYPEL \| TYPELS) (‘,’ [TYPEL \| TYPELS] )* CLAUSESL → PREDIMP (PREDIMP) * PREDIMP → FACTLS \| RULELS FACTLS → FACT (FACT) * RULELS → RULE (RULE) * FACT → iden (CONSTLS) ‘.’ CONSTLS → ATOM (‘,’ ATOM) * ATOM → iden \| numb \| var RULE → RULEL ‘: - ’ RULER RULEL → iden ‘(’ CONSTLS ‘)’ RULER → STML STML → STAT (‘,’ STAT) * STAT → iden ‘=’ (EXPR \| PREDCALL) PREDCALL → RULEL PROG → (DOMAINSL \| PREDICATESL \| DATABASEL \| CLAUSESL) GOAL eof STML → STAT (‘;’ STAT ) * EXPR → [ ‘+’ \| ‘−’ ] TERM ( ( [ ‘+’ \| ‘−’ ] ) TERM)* TERM → FACT ( ( ‘’ \| ‘/’ \| ‘%’ ) FACT ) TYPEL → (intl \| reall \| stringl \| symbol) TYPELS – TYPE ‘’ DOMAINSL → (iden=TYPEL \| iden TYPELS) DATABASEL → (iden ‘(‘ PARAMSL ‘)’) * PREDICATESL → (iden ‘(‘ PARAMSL ‘)’) * PARAMSL → (TYPEL \| TYPELS) (‘,’ [TYPEL \| TYPELS] )* CLAUSESL → PREDIMP (PREDIMP) * PREDIMP → FACTLS \| RULELS FACTLS → FACT (FACT) * RULELS → RULE (RULE) * FACT → iden (CONSTLS) ‘.’ CONSTLS → ATOM (‘,’ ATOM) * ATOM → iden \| numb \| var RULE → RULEL ‘: - ’ RULER RULEL → iden ‘(’ CONSTLS ‘)’ RULER → STML STML → STAT (‘,’ STAT) * STAT → iden ‘=’ (EXPR \| PREDCALL) PREDCALL → RULEL PROG → (DOMAINSL \| PREDICATESL \| DATABASEL \| CLAUSESL) GOAL eof STML → STAT (‘;’ STAT ) * EXPR → [ ‘+’ \| ‘−’ ] TERM ( ( [ ‘+’ \| ‘−’ ] ) TERM)* TERM → FACT ( ( ‘’ \| ‘/’ \| ‘%’ ) FACT ) За введення лексем відповідає функція get(): odc TOB[100]; void get () {if(nch= ='N')nch=getc(PF); if(nch= =EOF) {lex=EOF;return;} while(isspace(nch)) {if(nch= ='N')nst++;nch=getc(PF);} if(isdigit(nch))number(); else if(islower(nch)) word(); else if(index(nch)) {lex=nch; nch=getc(PF);} else if(nch= =EOF)lex=EOF; else printf("\n error %c illegal simbol",nch); } Під час проведення лексичного аналізу необхідно визначити лексеми, з яких складається програма, зокрема службові слова, якими є "domains", "database", "predicates", "clauses", "goal", "integer", "real", "string", "char", "symbol", ":-", "write". Вказані службові лексеми кодуються відповідними константами DOMAINSL, DATABASEL, PREDICATESL, CLAUSESL, GOALL, INTL, REALL, STRINGL, CHARL, SYMBOLL, IFL, CONSTL, WRITEL. Також до лексем належать знаки операцій та роздільники, беззнакові цілі дані з кодом NUMB та значенням Введення лексем типу “ціле число” та “слово” забезпечується функціями number() та word(): void number () {for(lval=0;isdigit(nch);nch=getc(PF)) lval=lval10+nch-'0'; lex=NUMB; } void word() {static char serv[]={"domains","database","predicates","clauses","goal", "integer","real","string","char","symbol",":-","write"}; у фу void number () {for(lval=0;isdigit(nch);nch=getc(PF)) lval=lval10+nch-'0'; lex=NUMB; } void word() {static char serv[]={"domains","database","predicates","clauses","goal", "integer","real","string","char","symbol",":-","write"}; 17 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 операторів, STAT - оператор, PREDCALL – виклик предиката, GOAL – розділ “goal”, PROG - програма, EXPR – вираз, TERM - доданок, FACT - множник. Повний синтаксис мови Prolog пропонується у такому вигляді: static int cdl[]={DOMAINSL, DATABASEL, PREDICATESL, CLAUSESL, GOALL, INTL, REALL, STRINGL,CHARL,SYMBOLL,IFL,WRITEL}; char tx[40]={""},p; int i; bool keyword=false; if(nch==':'){p=tx;(p++)=nch;nch=getc(PF); if(nch=='-'){(p++)=nch;p=tx;nch='$';} }// окреме визначення лексеми “:-” else for(p=tx;(islower(nch) \|\| isupper(nch) \|\| isdigit(nch) \|\| nch=='_') && !isspace(nch) && !index(nch) && nch!=10;nch=getc(PF)) (p++)=nch; p="\0"; for(i=0;i<12;i++) if(strcmp(serv[i],tx)==0){lex=cdl[i];keyword=true;break;} if(!keyword){lex=IDEN; lstr=add(tx);lval=(int)lstr; }} операторів, STAT - оператор, PREDCALL – виклик предиката, GOAL – розділ “goal”, PROG - програма, EXPR – вираз, TERM - доданок, FACT - множник. Пошук лексем-роздільників забезпечує index(): ) CLAUSESL → PREDIMP (PREDIMP) * PREDIMP → FACTLS \| RULELS bool index(char b) {bool ft=false; char a[16]={'(',')',',',';','=','+','-','','/','%','[',']','.','\|','>','<'}; for(int i=0;i<16;i++) if(a[i]==b){ft=true;break;} return ft; } bool index(char b) {bool ft=false; char a[16]={'(',')',',',';','=','+','-','','/','%','[',']','.','\|','>','<'}; for(int i=0;i<16;i++) if(a[i]==b){ft=true;break;} return ft; } \| FACTLS → FACT (FACT) * RULELS → RULE (RULE) * FACT → iden (CONSTLS) ‘.’ CONSTLS → ATOM (‘,’ ATOM) * } Ідентифікатори, що не знайдені серед списку службових лексем та не введені раніше, мають бути додані до таблиці ідентифікаторів TNM[40], що й забезпечує функція add(): RULER → STML STML → STAT (‘,’ STAT) * char add (char nm) {char p; for(p=TNM;p<ptn;p+=strlen(p)+1) if(strcmp(nm,p)==0)return (p); if(atoi(ptn+=strlen(nm)+1)>atoi(TNM+400)) printf("\n overflow TNM"); strcpy (p,nm); return p; } char add (char * nm) {char p; for(p=TNM;p<ptn;p+=strlen(p)+1) if(strcmp(nm,p)==0)return (p); if(atoi(ptn+=strlen(nm)+1)>atoi(TNM+400)) printf("\n overflow TNM"); strcpy (p,nm); return p; } if(atoi(ptn+=strlen(nm)+1)>atoi(TNM+400)) printf("\n overflow TNM"); strcpy (p,nm); return p; } strcpy (p,nm); Зазначимо, що введення лексем проводиться послідовно, при цьому застосовується непряма рекурсія функцій get(), word(), number(). Конструкція PROG забезпечує обробку тексту файла програми і відповідає функції prog(): Синтаксис Prolog (відповідно [4]) описується розширеними граматиками із використанням символів “[“, “]” в регулярних виразах у правих частинах правил. Обробка розділу “database” ведеться за допомогою функції ddatabase(): Виконання предиката забезпечує pred_exec(): void ddatabase() {do {get(); char nm=(char)lval; newob(nm,"database",0,-1); if(lex=='(') {pred_args(); exam(')'); } }while(lex!=PREDICATESL); } void ddatabase() void ddatabase() void pred_exec(char lstr1) {int st; int cp=0;bool g=false; if(!strcmp(lstr1,"goal"))g=true; odc p; fnd pl; do {get(); switch(lex) {case WRITEL: fact();gen(OPR,2);break; case IDEN: p=findob(lstr); if(!p) newob((char)lval,lstr1, (out?3:4),(out?cgv++:++clv)); p=findob(lstr); exam(IDEN); switch(lex) {case '=': exam('=');expr(); if(p->what==1) printf("\n %s isn't variable",p->name); gen(STI,p->val); break; case '>': stat();break; case '<': stat();break; case '(': pred_pars(lstr); cp=(lex==')' ? 0 : fctl()); exam(')'); pl=eval(lstr1,cp); gen(LIT,cp);cp=gen(CAL,pl->start); if(!pl->isd)pl->start=cp; break; case ',': break; default : ef=true;break; }} if(ef)break; } void pred_exec(char lstr1) int st; int cp=0;bool g=false; if(!strcmp(lstr1,"goal"))g=true; odc p; fnd pl; do {get(); switch(lex) {case WRITEL: fact();gen(OPR,2);break; case IDEN: p=findob(lstr); if(!p) newob((char)lval,lstr1, (out?3:4),(out?cgv++:++clv)); p=findob(lstr); exam(IDEN); switch(lex) {case '=': exam('=');expr(); if(p->what==1) printf("\n %s isn't variable",p->name); gen(STI,p->val); break; case '>': stat();break; case '<': stat();break; case '(': pred_pars(lstr); cp=(lex==')' ? 0 : fctl()); exam(')'); pl=eval(lstr1,cp); gen(LIT,cp);cp=gen(CAL,pl->start); if(!pl->isd)pl->start=cp; break; case ',': break; default : ef=true;break; }} if(ef)break; } Пошук лексем-роздільників забезпечує index(): Нетерміналами є послідовності літер, що позначають синтактичні конструкції програми: case DATABASEL: case PREDICATESL: p=fgoal(); Розгляд розділу “domains” Prolog-програми здійснюється функцією ddomains(): void ddomains() {get(); 18 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 bool noalt=true; do {if(noalt){exam(IDEN); exam('='); } switch(lex) {case INTL: exam(INTL);break; case STRINGL: exam(STRINGL);break; case REALL: exam(REALL);break; case CHARL: exam(CHARL);break; case SYMBOLL: exam(SYMBOLL);break; } lextestmode=true; if(exam(';'))noalt=false; else {noalt=true;exam('');} lextestmode=false; }while(lex!=PREDICATESL && lex!=DATABASEL); } {get(); switch(lex) {case IDEN: exam(IDEN);break; case INTL: exam(INTL);break; case STRINGL: exam(STRINGL);break; case REALL: exam(REALL);break; case CHARL: exam(CHARL);break; case SYMBOLL: exam(SYMBOLL);break; }while(lex==','); return true; }while(lex==','); return true; } lextestmode=true; if(exam(';'))noalt=false; else {noalt=true;exam('');} lextestmode=false; }while(lex!=PREDICATESL && lex!=DATABASEL); } } lextestmode=true; if(exam(';'))noalt=false; else {noalt=true;exam('');} lextestmode=false; }while(lex!=PREDICATESL && lex!=DATABASEL); } Наступний розділ Prolog-програми – “clauses” обробляється функцією dclauses(): void dclauses() {bool bclauses=false; do {get();lstr; if(!bclauses)exam(IDEN); switch(lex) {case '.': break; case IFL: pred_exec(lstr);exam('.');break; case '(': pred_pars(lstr);exam(')'); switch(lex) {case '.': exam('.');break; case IFL: pred_exec(lstr); exam('.');break; }break; } bclauses=true; }while(lex!=GOALL); } void dclauses() {bool bclauses=false; do {get();lstr; if(!bclauses)exam(IDEN); switch(lex) {case '.': break; case IFL: pred_exec(lstr);exam('.');break; case '(': pred_pars(lstr);exam(')'); switch(lex) {case '.': exam('.');break; case IFL: pred_exec(lstr); exam('.');break; }break; } bclauses=true; }while(lex!=GOALL); } Синтаксичний аналіз використовує функцію exam(), яка перевіряє, чи має наступна лексема код lx. Якщо відповідність коду не знайдена, виводиться повідомлення про синтаксичну помилку: bool lextestmode=false; bool exam(int lx) {if(lx!=lex) {if(!lextestmode)printf("\n %d syntax error (exam) %c",nst,lex); return false;} () bool lextestmode=false; bool exam(int lx) {if(lx!=lex) {if(!lextestmode)printf("\n %d syntax error (exam) %c",nst,lex); return false;} () get(); return true; } Обробка розділу “database” ведеться за допомогою функції ddatabase(): Пошук лексем-роздільників забезпечує index(): void prog() {fnd p; lex; while(lex!=EOF) {switch(lex) {case DOMAINSL: ddomains();break; case DATABASEL: ddatabase(); break; case PREDICATESL: dpredicates(); break; case CLAUSESL: dclauses(); break; case GOALL: dgoal(); break; default: printf("\n %d syntax error (prog)",nst); } if(ef){cout<< "\n Predicates sequence error";break;} } p=fgoal(); if(p){adrnm=p->start;cpnm=p->cpt;} } Розгляд розділу “domains” Prolog-програми здійснюється функцією ddomains(): void ddomains() {get(); char nm=(char)lval; newob(nm,"domains",0,0); void prog() {fnd p; lex; while(lex!=EOF) {switch(lex) {case DOMAINSL: ddomains();break; case DATABASEL: ddatabase(); break; case PREDICATESL: dpredicates(); break; case CLAUSESL: dclauses(); break; case GOALL: dgoal(); break; default: printf("\n %d syntax error (prog)",nst); } if(ef){cout<< "\n Predicates sequence error";break;} } p=fgoal(); if(p){adrnm=p->start;cpnm=p->cpt;} } Розгляд розділу “domains” Prolog-програми здійснюється функцією ddomains(): void ddomains() {get(); char nm=(char)lval; newob(nm,"domains",0,0); void prog() {fnd p; lex; while(lex!=EOF) {switch(lex) {case DOMAINSL: ddomains();break; case DATABASEL: ddatabase(); break; case PREDICATESL: dpredicates(); break; case CLAUSESL: dclauses(); break; case GOALL: dgoal(); break; default: printf("\n %d syntax error (prog)",nst); } if(ef){cout<< "\n Predicates sequence error";break;} } p=fgoal(); if(p){adrnm=p->start;cpnm=p->cpt;} } Розгляд розділу “domains” Prolog-програми здійснюється функцією ddomains(): void ddomains() {get(); char nm=(char)lval; newob(nm,"domains",0,0); void prog() {fnd p; lex; while(lex!=EOF) {switch(lex) {case DOMAINSL: ddomains();break; case DATABASEL: ddatabase(); break; case PREDICATESL: dpredicates(); break; case CLAUSESL: dclauses(); break; case GOALL: dgoal(); break; default: printf("\n %d syntax error (prog)",nst); } if(ef){cout<< "\n Predicates sequence error";break;} } p=fgoal(); if(p){adrnm=p->start;cpnm=p->cpt;} } Розгляд розділу “domains” Prolog-програми здійснюється функцією ddomains(): void ddomains() {get(); char nm=(char)lval; newob(nm,"domains",0,0); Якщо r – регулярний вираз, що задає множину R, то [r] – регулярний вираз для множини { } ε ∪ R , деε - порожній ланцюжок. Термі- нальними символами розширеної граматики є лексеми, записані малими літерами. Нетерміналами є послідовності літер, що позначають синтактичні конструкції програми: TYPEL – тип; TYPELS – список типів, DOMAINSL – розділ “domains”, PREDICATESL – розділ “predicates”, PARAMSL – список параметрів, CLAUSEL – розділ “clauses”, PREDIMP – реалізація предиката, FACTLS – список фактів, RULELS - список правил, FACT – опис факту, CONSTLS – список атомів, ATOM - атом, RULE - правило, RULEL – ліва частина правила (консеквент), RULER – права частина правила (антецедент), STML – список Якщо r – регулярний вираз, що задає множину R, то [r] – регулярний вираз для множини { } ε ∪ R , деε - порожній ланцюжок. Термі- нальними символами розширеної граматики є лексеми, записані малими літерами. Обробка предикатів мови Prolog має забезпечуватися викликом dpredicates(), у відповідності до конструкції DPREDICATESL: Обробка предикатів мови Prolog має забезпечуватися викликом dpredicates(), у відповідності до конструкції DPREDICATESL: } }while(lex!=CLAUSESL); } }while(lex!=CLAUSESL); } Обробка розділу database ведеться у аналогічний спосіб. У своєму складі dpredicates() містить послідовні виклики функцій: get() – введення чергової лексеми, pred_args () – обробки параметрів функції: bool pred_args() {do bool pred_args() 19 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 ідентифікатора – iden, числової константи numb, cимволів ‘[‘, ‘(‘ та ‘=’. Обробка параметрів предиката відбувається окремою функцією pred_pars(): void stat() {odc p; int t1,t2; get(); switch(lex) {case IDEN: p=findob((char)lval); get(); if(lex==',' \|\|lex == ')')break; expr(); if(p->what==1)printf("\n %s isn't variable",p->name); gen(STI,p->val); break; case NUMB: gen(LIT,lval); get(); gen(STI,cnl++); if(lex==')')break; if(lex!=','){expr();gen(STI,cnl++); } break; case '[' : test_list("");get();break; case '(' : term(); gen(5,1); break; case '=' : get();expr();break; default: printf("\n %d syntax error (stat)",nst); }} void pred_pars(char lstr1) {odc p; int cp=0; do {get(); switch(lex) {case '[': {test_list(lstr1); exam(']'); break; } case IDEN: if(!findob(lstr)) newob((char)lval,lstr1,3,++clv); exam(IDEN); if(lex=='('){pred_pars(lstr1); exam(')');break;} break; case NUMB: exam(NUMB);break; } } while(lex==','); for(p=ptol;p<pto;p++)p->val-=cp+3; } void pred_pars(char lstr1) {odc p; int cp=0; } while(lex==','); while(lex==','); for(p=ptol;p<pto;p++)p->val-=cp+3; } default: printf("\n %d syntax error (stat)",nst); while(lex , ); for(p=ptol;p<pto;p++)p->val-=cp+3; } Як видно зі схеми конструкції STAT, її складовими є вирази EXPR або списки операторів STML. EXPR є комбінацією операцій додавання, віднімання, множення, ділення виразів та констант і складається з комбінацій конструкцій TERM, та опосередковано – FACT і FCTL. Вказаним вище конструкціям відповідають функції expr(), term(), fact(), fctl(): Обробка списків відіграє значну роль у функціонуванні Prolog-програми, тому для їх обробки призначається окрема функція: void test_list(char lstr) { get(); switch(lex) {case ']': break; case IDEN: get(); switch(lex) {case ']': break; case '\|': pred_pars(lstr);break; case ',': test_list(lstr);break; } break; case NUMB: get(); switch(lex) {case ']': break; case '\|': pred_pars(lstr);break; case ',': test_list(lstr);break; } break; } } void test_list(char lstr) { get(); void expr() {int neg=(lex=='-'); int plusmin=0; if(lex=='+' \|\| lex=='-'){plusmin=1;get();} term(); if(neg)gen(OPR,8); while(lex=='+' \|\| lex =='-') {neg=(lex=='-'?4:3); get();term(); gen(OPR,neg); } if(lex=='.' && plusmin) {neg=(lex=='-'?4:3); gen(OPR,neg); }} void expr() {int neg=(lex=='-'); int plusmin=0; if(lex=='+' \|\| lex=='-'){plusmin=1;get();} term(); if(neg)gen(OPR,8); while(lex=='+' \|\| lex =='-') {neg=(lex=='-'?4:3); get();term(); gen(OPR,neg); } if(lex=='.' && plusmin) {neg=(lex=='-'?4:3); gen(OPR,neg); }} void term() {int op; fact(); while(lex=='' \|\| lex =='/' \|\| lex=='%') {op=(lex=='' ? 5 : (lex=='/' ? 6:7)); get();fact();gen(OPR,op);}} int fctl () {int cf=1; expr(); while(lex = = ',' ) {get();expr();cf++;} return cf; } void fact() {char nm; int cp; odc p;fnd pl; switch(lex) {case IDEN: nm=(char)lval; get(); } break; case NUMB: get(); switch(lex) break; } } Розділ goal у мові Prolog відіграє роль своєрідної функції main() (у розумінні Cі- програми). Тому, фактично у ньому відбувається виклик “предиката” з іменем “goal”: void term() {int op; fact(); while(lex=='' \|\| lex =='/' \|\| lex=='%') {op=(lex=='' ? 5 : (lex=='/' ? ФУНКЦІЙ ІНТЕРПРЕТАТОРА Результатом роботи блоку синтаксичного аналізу є проміжний код, що надалі обробляється функціями інтерпретатора програми. Кінцевою метою розробки транслятора на даному етапі є його доведення до працездатного стану та його інтеграція у Visual С++ проект, що забезпечуватиме моделювання системи підтримки та прийняття рішень інтелектуального робота. Набір функцій інтерпретації формує Prolog- процесор (аналогічний описаному у [7, 8] для мови програмування SPL), який складається: з пам’яті програми, що моделюється масивом TCD та змінними adrnm, cpnm, cgv; стеку st зі значеннями змінних та констант програми. Стек st характеризується покажчиками: вершини стека – t, початку області локальних даних функції – sp (запису активації). Покажчик p – вказує на ідентифікатора – iden, числової константи numb, cимволів ‘[‘, ‘(‘ та ‘=’. 6:7)); get();fact();gen(OPR,op);}} int fctl () {int cf=1; expr(); while(lex = = ',' ) {get();expr();cf++;} return cf; } void fact() {char nm; int cp; odc p;fnd pl; switch(lex) {case IDEN: nm=(char)lval; get(); void dgoal() {defin("goal",0,0); pred_exec("goal"); exam('.'); } void dgoal() {defin("goal",0,0); pred_exec("goal"); exam('.'); } void dgoal() {defin("goal",0,0); pred_exec("goal"); exam('.'); } Синтаксична конструкція STML забезпечує обробку списку операторів, хоча детальну обробку забезпечує найбільш складна конструкція мови – STAT. Цим схемам оброки відповідають дві функції: stml () та stat (). Власне, перша з них здійснює послідовний (в разі потреби) виклик другої – із безпосереднім аналізом поточного 20 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 if(lex=='(') {get(); if(lex==')')fctl(); exam(')'); pl=eval(nm,cp); gen(LIT,cp);cp=gen(CAL,pl->start); if(!pl->isd)pl->start=cp; } else {p=findob(nm); if(!error_syntax && p) gen(p->what==1 ? LIT : (p->what==2 ? LDE:LDI),p->val); } break; case WRITEL: get();fact(); break; case '(': get();expr();exam(')'); break; case NUMB: gen(LIT,lval); get(); break; case '.': break; case '/': break; case '': break; default: printf("\n %d syntax error (fact)",nst); } } void fact() {char nm; int cp; odc p;fnd pl; switch(lex) {case IDEN: nm=(char)lval;get(); if(lex = ='(' ) {get();cp=(lex= =' ) ' ? 0 : fctl()); exam(')');pl=eval(nm,cp); gen(LIT,cp);cp=gen(CAL,pl->start); if(!pl->isd)pl->start=cp; } else { p=findob(nm); gen(p->what==1 ? LIT : (p->what==2 ? LDE:LDI),p->val); } break; case '(': get();expr();exam(')');break; case NUMB: gen(LIT,lval);get();break; default: printf("\n %d syntax error (fact)",nst); }} поточну команду, обрану у STD. поточну команду, обрану у STD. Попереднім, до розробки транслятора мови Prolog, кроком була програмна реалізація інтерпретатора мови програмування SPL, на основі матеріалів, описаних у [7]. Тому процес відлагодження Prolog-транслятора полягав у постійному порівнянні початкових даних, що готуються на етапах лексичного та синтаксичного аналізу обох програм. При цьому, для повторного використання коду, текст блоку інтерпретації практично не змінювався, хоча з точки зору практичної реалізації та розуміння роботи він не є ідеальним, особливо з точки зору мови програмування Prolog. 7. ВИСНОВКИ Таким чином, у запропонованій статті з практичної точки зору розглядається побудова інтерпретатора мови програмування Prolog. Наводиться програмна реалізація блоків лексичного та синтаксичного аналізу. Розв’язання розробки транслятора вимагає великого об’єму копіткої роботи із практичної реалізації розроблених теоретичних моделей, тому значну частину матеріалу займає код функцій інтерпретатора. Звичайно, що подібний опис є лише першим кроком на шляху творення повноцінного транслятора. Перші тестування виявили його здатність коректно виконувати нескладні Prolog- програми. Наступними кроками є відпрацювання схем автоматичного прийняття рішень для задачі функціонування робота спочатку у статичному просторі, потім – додавання засобів адаптації до змін у робочому просторі. Слід одразу зазначити, що у текстах наведених функцій реалізації синтаксичного аналізатора включені додаткові функції контролю таблиць, пов’язані вже з інтерпретацією програми. Наукова цінність роботи полягає у розробці та практичній реалізації граматик мови Prolog. Розроблені граматики можуть використатися для моделювання Prolog за допомогою інших мов програмування. Практична значимість результатів роботи полягає у можливості їх використання для розробки нових і самостійної реалізації власних трансляторів вже існуючих мов програмування. Alexander Tsimbal Kharkiv national university of radio electronics, Kharkiv, Lenin Ave. 14, mcdulcimer@ukr.net Kharkiv national university of radio electronics, Kharkiv, Lenin Ave. 14, mcdulcimer@ukr.net Abstract: The article considers the development of Prolog programming language interpreter with lexical and syntax analyzer, table control and interpreter blocks. There is proposed the schemes of syntax analyzes and the elements of translator’s program code. The software is developed in Visual C++ 2005 (Beta) environment. Keywords: Programming language, interpreter, Prolog, grammar, lexical analysis, syntax analysis. Prolog (Programming in Logic) implements the data processing by program construction in form of predicates. The main option of Prolog is practical implementation of logical knowledge model [1]. various program methods. There is proposed to implement the intellectual robot decision support system on base of Prolog. To supply it’s functionality as system part it’s required to develop the interpreter of Prolog programming language. The development of artificial intelligence methods and the need of their language support set the task of Prolog modernization. The development of new Prolog translator presents an actual problem, aimed to solve the robotics tasks. The article objective is a description of main issues of Prolog translator development. There were set the tasks of Prolog grammar description, the lexical and syntax analyzers implementation. The development tool is C-language. Prolog applications are especially suitable for knowledge representation tasks, namely for production and framework models [2,3]. Also, the recursive list processing procedures give the great possibilities for informational systems organization, for problem solving in various fields including robotics [3, 4]. The interesting way of Prolog application is described in [6], where the reversal task is set – the principles of other language implementation are described in Prolog. Prolog language interpreter has syntax similar to Turbo-Prolog, developed by Borland Inc. There will be used the principles of [7], applied during simple programming language translator (SPL) creation. The alphabet of Prolog consists of Latina characters, decimal values, the service characters: {'(', ')', ',', ';', '=', '+', '-', '', '/', '%' ,'[',']', '.', '\|', '>', '<'} and of space. The alphabet characters construct the lexemes – the minimal language units. The Prolog identity is a sequence of letters and digitals, beginning of character. Unsigned value is a sequence of digitals. From the syntax view point all the identities are the same and correspond lexeme IDEN. Unsigned digitals present the lexeme NUMB. 8. СПИСОК ЛІТЕРАТУРИ [1] Малпасс Дж. Реляционный язык Пролог и его применение. – М.: Наука, 1990. – 464 с. [2] Люгер Дж.Ф. Искусственный интеллект: стратегии и методы решения сложных 21 Олександр Цимбал / Комп’ютинг, 2007, Том 6, Випуск 1, 15-24 проблем. – М.: Изд. дом «Вильямс», 2003. – 894 с. [8] Бєлов Ю.А., Проценко В.С., Чаленко П.Й. Інструментальні засоби програмування. – К.: Либідь, 1993. – 248 с. [3] Гаврилова Т.А., Хорошевский В.Ф. Базы знаний интеллектуальных систем. – СПб.: Питер, 2001. – 384 с. [4] Ахо А., Сети Р., Ульман Дж. - Компиляторы: принципы, технологи, инструменты. – М.: Изд. дом «Вильямс», 2003. – 768 с. Цимбал Олександр Михайлович, канд. техн. наук, докторант, Харківський національний університет радіоелектроніки. Наукові інтереси – мови програмування, системи штучного інтелекту. Адреса: Харків, пр. Леніна 14, ХНУРЕ, каф. ТАВР., тел. (057) 7021486. Цимбал Олександр Михайлович, канд. техн. наук, докторант, Харківський національний університет радіоелектроніки. Наукові інтереси – мови програмування, системи штучного інтелекту. Адреса: Харків, пр. Леніна 14, ХНУРЕ, каф. ТАВР., тел. (057) 7021486. [5] Гордеев А.В., Молчанов А.Ю. Системное программное обеспечение. – СПб.: Питер, 2002. – 736 с. [6] Карпов В.Э. Классическая теория компи- ляторов. – М.: Московский государственный ин-ститут электроники и математики, 2003. – 96 с. [7] Проценко В.С., Чаленко П.Й., Ставровський А.Б. Техніка програмування мовою Сі. – К.: Либідь, 1993. – 224 с. 22 Alexander Tsimbal / Computing, 2007, Vol. 6, Issue 1, 23-24 THE IMPLEMENTATION OF PROLOG INTERPRETER: LEXICAL AND SYNTAX ANALYSES Alexander Tsimbal Alexander Tsimbal It has the defined structure and consists of the following sections: RULELS → RULE (RULE) domains The definition of type names, complex types and lists; database The definition of facts of Prolog database; predicates The declaration of predicate prototypes; clauses The implementation of predicates; goal The call of predicates or the program running. FACT → iden (CONSTLS) ‘.’ During the lexical analyzes, there is defined the lexemes of program, including the keyword "domains", "database", "predicates", "clauses", "goal", "integer", "real", "string", "char", "symbol", ":-", "write". g y The syntax of Prolog (as to [4]) is described by extended grammars with using of characters “[“, “]” in regular expressions. If r is a regular expression which defined the set R, then [r] is a regular expression for set { } ε ∪ R , here ε - is empty chain. The terminal characters of extended grammar are lexemes written by small letters. Non-terminals are letter sequences defining the syntax constructions of program. Every construction is presented by C-language function implementation. The intermediate code, which is a result of syntax analyzes stage, should be processed by interpreter functions of translator. This functions set forms the Prolog processor (analogical to the described in [7, 8] for SPL). There is defined the following non-terminals: TYPEL – the type; TYPELS – the type of list, DOMAINSL – “domains” section, PREDICATESL – “predicates” section, PARAMSL – parameters list, CLAUSEL – “clauses” section, PREDIMP – predicate implementation, FACTLS – the list of facts, RULELS – the list of rules, FACT – the fact description, CONSTLS – the list of atoms, ATOM – the atom, RULE – the rule, RULEL – the left part of the rule (consequent), RULER – the right part of the rule (antecedent), STML – the operators list, STAT – the operator, PREDCALL – the predicate call, GOAL – “goal” section, PROG - program, EXPR – expression, TERM - sum, FACT - multiplication. Therefore, the proposed article practically considers the development of Prolog programming language interpreter. The mentioned description is the first stage on way to full-scale translator. The next steps will include the special settings to implement the robot problem solver, firstly for static and after – for the dynamically changed world. The scientific value of article is in development and practical implementation of Prolog language grammar. The developed grammars can be used for Prolog simulation on other software base. Alexander Tsimbal The review of possibilities to construct new Prolog implementation requires the using of standard methods, described in [4]. But as to practical view point it’s useful to apply the program constructions described in [7, 8]. After the insignificant transformations these materials can be easily used for the translator development. Except IDEN and NUMB Prolog has twelve special keyword - {"domains", "database", "predicates", "clauses", "goal", "integer", "real", "string", "char", "symbol", ":-", "write"}. Robotics requires the decision of several problem classes: from the one hand – the tasks of calculations in cinematic models, which can be decided by universal programming language application, for instance C. Also C-language (or Assembler) is good during the control signals forming and transmission to robot control system. In the current implementation Prolog contains the scalar data of integer, real, symbol and string type. Also the sequence of certain type values can form the list of elements. The elements of list are divided by commas inside the square brackets: [12, 31, -5, 6] or [‘a’, ‘d’, ‘E’]. The identities of Prolog variables are written by capital letter, the constants – by small. All the variables are local inside predicates. From the other hand – on decision support level, the calculation is supplementary aspect, but logical programming is the key issue. Therefore, the common task of robotic systems simulation can be presented as the set of heterogeneous tasks, decisions of which will require the application of The expression of Prolog consists of constants, variables and predicate names (with round brackets for parameter setting or without them). The 23 Alexander Tsimbal / Computing, 2007, Vol. Alexander Tsimbal 6, Issue 1, 23-24 TYPELS – TYPE ‘’ DOMAINSL → (iden=TYPEL \| iden TYPELS) DATABASEL → (iden ‘(‘ PARAMSL ‘)’) * PREDICATESL → (iden ‘(‘ PARAMSL ‘)’) * PARAMSL → (TYPEL \| TYPELS) (‘,’ [TYPEL \| TYPELS] )* CLAUSESL → PREDIMP (PREDIMP) * PREDIMP → FACTLS \| RULELS FACTLS → FACT (FACT) * RULELS → RULE (RULE) * FACT → iden (CONSTLS) ‘.’ CONSTLS → ATOM (‘,’ ATOM) * ATOM → iden \| numb \| var RULE → RULEL ‘: - ’ RULER RULEL → iden ‘(’ CONSTLS ‘)’ RULER → STML STML → STAT (‘,’ STAT) * STAT → iden ‘=’ (EXPR \| PREDCALL) PREDCALL → RULEL PROG → (DOMAINSL \| PREDICATESL \| DATABASEL \| CLAUSESL) GOAL eof STML → STAT (‘;’ STAT ) * EXPR → [ ‘+’ \| ‘−’ ] TERM ( ( [ ‘+’ \| ‘−’ ] ) TERM)* TERM → FACT ( ( ‘’ \| ‘/’ \| ‘%’ ) FACT ) TYPELS – TYPE ‘’ DOMAINSL → (iden=TYPEL \| iden TYPELS) DATABASEL → (iden ‘(‘ PARAMSL ‘)’) * PREDICATESL → (iden ‘(‘ PARAMSL ‘)’) * PARAMSL → (TYPEL \| TYPELS) (‘,’ [TYPEL \| TYPELS] )* CLAUSESL → PREDIMP (PREDIMP) * PREDIMP → FACTLS \| RULELS FACTLS → FACT (FACT) * RULELS → RULE (RULE) * FACT → iden (CONSTLS) ‘.’ CONSTLS → ATOM (‘,’ ATOM) * ATOM → iden \| numb \| var RULE → RULEL ‘: - ’ RULER RULEL → iden ‘(’ CONSTLS ‘)’ RULER → STML STML → STAT (‘,’ STAT) * STAT → iden ‘=’ (EXPR \| PREDCALL) PREDCALL → RULEL PROG → (DOMAINSL \| PREDICATESL \| DATABASEL \| CLAUSESL) GOAL eof STML → STAT (‘;’ STAT ) * EXPR → [ ‘+’ \| ‘−’ ] TERM ( ( [ ‘+’ \| ‘−’ ] ) TERM)* TERM → FACT ( ( ‘’ \| ‘/’ \| ‘%’ ) FACT ) operation signs are “+”, “-“, ““, “/“, “%“. Expressions have view: v, c, (e1), predicate(e1, e2,…,en ), e1 + e2, e1 – e2, e1 e2, e1 / e2, e1 % e2, + e1, –e1, here v – variable, с – constant, predicate – the predicate name, e1, e2,…,en – expressions. p p Prolog has in-build operators of assignment (comparison) v=c; and of print - write(v). Prolog program has logical and / or computational contents. Alexander Tsimbal The practical value of article consists in their applications for new the translator’s development. The general syntax of Prolog language is proposed in the following view: The future objective of translator development is it’s integration to more general project of intellectual robot decision support system. TYPEL → (intl \| reall \| stringl \| symbol) TYPEL → (intl \| reall \| stringl \| symbol) 24
https://openalex.org/W1796121897	https://hal.science/hal-01601452/file/publi17-lipm-012_badet_common_1.pdf	English	null	Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae	Frontiers in plant science	2,015	cc-by	14,929	Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae.f Thomas Badet, Rémi Peyraud, Sylvain Raffaele To cite this version: Thomas Badet, Rémi Peyraud, Sylvain Raffaele. Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae.. Frontiers in Plant Science, 2015, 6, 10.3389/fpls.2015.00776. hal-01601452 Keywords: secretome, Sclerotinia, psychrophily, effector candidates, amino acid usage, intrinsic disorder, antifreeze protein, lytic polysaccharide monooxygenase Edited by: Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its speciﬁc environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are signiﬁcantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identiﬁed through our pipeline as candidate proteins involved in colonization of the environment. Our ﬁndings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases. Reviewed by: Jana Sperschneider, Commonwealth Scientiﬁc and Industrial Research Organisation, Australia Kim Marilyn Plummer, La Trobe University, Australia Correspondence: Sylvain Raffaele, Laboratoire des Interactions Plante Micro-organismes, 24 Chemin de Borde Rouge – Auzeville, 31326 Castanet Tolosan, France sylvain.raffaele@toulouse.inra.fr Specialty section: This article was submitted to Plant Biotic Interactions, a section of the journal Frontiers in Plant Science Received: 23 June 2015 Accepted: 10 September 2015 Published: 24 September 2015 HAL Id: hal-01601452 https://hal.science/hal-01601452v1 Submitted on 27 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. ORIGINAL RESEARCH published: 24 September 2015 doi: 10.3389/fpls.2015.00776 Thomas Badet 1, 2, Rémi Peyraud 1, 2 and Sylvain Raffaele 1, 2 1 Laboratoire des Interactions Plantes-Microorganismes, Institut National de la Recherche Agronomique, UMR441, Castanet-Tolosan, France, 2 Laboratoire des Interactions Plantes-Microorganismes, Centre National de la Recherche Scientiﬁque, UMR2594, Castanet-Tolosan, France Edited by: Delphine Vincent, Department of Environment and Primary Industries, Australia Introduction Genes involved in host-parasite interactions such as pathogen eﬀectors are often subject to strong balancing or directional selection. For example, oomycete eﬀectors commonly evolve rapidly, and natural selection can maintain many diﬀerent alleles in a population (Raﬀaele et al., 2010; Oliva et al., 2015). Therefore, signatures of positive selection are frequent in eﬀector genes and this property has been used to identify novel eﬀector candidates (Wicker et al., 2013; Rech et al., 2014; Sperschneider et al., 2014). However, most of our understanding of the molecular evolution of eﬀector genes and genes involved in colonization of the environment comes from studies of the pairwise co- evolution of a given pathogen with a single host plant. By contrast, fungal pathogens in the Sclerotiniaceae interact with a wide range of hosts in multiple environmental conditions and should therefore be considered as evolving under “diﬀuse” (or “generalized”) interactions (Juenger and Bergelson, 1998). In the Ascomycete genus Metarhizium, signatures of positive selection were observed less frequently in the genome of fungal pathogens FIGURE 1 \| Sclerotinia borealis colonizes different niches than its close relatives S. sclerotiorum and Botrytis cinerea. Number of host plant genera (A) and geographic distribution (B) of the three fungal species according to the USDA Systematic Mycology and Microbiology Laboratory Fungus-Host Database (Farr and Rossman, 2015). under diﬀuse co-evolution compared to Metarhizium acridum evolving under pairwise co-evolution (Hu et al., 2014). It is thus expected that in the Sclerotiniaceae, some genes important for colonization of environment, including fungal eﬀectors involved in diﬀuse interactions, may escape detection by positive selection analysis, and additional detection criteria would be useful. Compared to B. cinerea and S. sclerotiorum, the snow mold pathogen Sclerotinia borealis colonizes a reduced range of environments. Indeed, according to the Fungus-Host database of the U.S. Department of Agriculture (Farr and Rossman, 2015), S. borealis has been reported to infect 14 plant genera only, compared to 332 and 469 for S. sclerotiorum and B. cinerea respectively (Figure 1A). S. borealis host plants include notably Agropyron, Agrostis, Elymus, and Festuca species that have not been reported as hosts for S. sclerotiorum or B. cinerea to date. S. borealis has an economic impact in countries with cold climates, where it causes snow mold on winter cereals and grasses (Schneider and Seaman, 1987). Its geographic range is restricted to the Arctic Circle, including North of Japan, North America, Scandinavia, and Russia, whereas B. cinerea and S. Introduction FIGURE 1 \| Sclerotinia borealis colonizes different niches than its close relatives S. sclerotiorum and Botrytis cinerea. Number of host plant genera (A) and geographic distribution (B) of the three fungal species according to the USDA Systematic Mycology and Microbiology Laboratory Fungus-Host Database (Farr and Rossman, 2015). Fungi from the Sclerotiniaceae family include several devastating plant pathogens with a broad host range. Among those are Botrytis cinerea, the causal agent of gray rot, and Sclerotinia sclerotiorum, causal agent of white and stem rot, each able to infect several hundreds of plant genera and causing multi- billion dollar losses in agriculture every year (Figure 1A) (Bolton et al., 2006; Dean et al., 2012). The geographic distribution of these two fungi is also remarkably broad since they have been reported across ﬁve continents (Figure 1B). Sequencing of the genome of B. cinerea and S. sclerotiorum (Amselem et al., 2011) opened the way to systematic searches for the molecular bases of pathogenicity in these fungi (Guyon et al., 2014; Heard et al., 2015). However, the repertoire of molecules contributing to the ability of plant pathogenic fungi, such as fungi from the Sclerotiniaceae family, to colonize a wide range of hosts and environments remains elusive. Fungal pathogens secrete diverse sets of degrading enzymes and toxins to facilitate colonization of their hosts (Möbius and Hertweck, 2009; Kubicek et al., 2014). In addition, fungal pathogens use molecules designated as eﬀectors to manipulate host cells and achieve successful infection. Their activities include the inactivation of plant defenses, interference with plant hormone balance, or dismantling of the plant cell. However, eﬀectors may also trigger speciﬁc plant defense responses, leading to plant resistance, when recognized directly or indirectly by the plant immune system (Jones and Dangl, 2006). Typical eﬀectors are small secreted proteins, but secondary metabolites and small RNAs can also play the role of eﬀectors (Schardl et al., 2013; Weiberg et al., 2013). Although a subset of bacterial and oomycete protein eﬀectors can be identiﬁed based on conserved N-terminal targeting signals and other sequence signatures (Schornack et al., 2009; McDermott et al., 2011; Meyer et al., 2013), this is not the case in fungi. Eﬀector detection in fungal pathogens relies largely on speciﬁc host responses revealing eﬀector recognition, and bioinformatics approaches based on whole genome sequences and deduced protein repertoires remain challenging (Sperschneider et al., 2015). Citation: Badet T, Peyraud R and Raffaele S (2015) Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae. Front. Plant Sci. 6:776. doi: 10.3389/fpls.2015.00776 September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 1 Protein signatures in Sclerotiniaceae fungi Badet et al. Frontiers in Plant Science \| www.frontiersin.org Introduction sclerotiorum are cosmopolitan fungi found in arctic, temperate and tropical climates (Figure 1B). Consistently, S. borealis is a psychrophile, with an optimal growth temperature about 4–10◦C, whereas optimal growth temperature is ∼23◦C for B. cinerea and S. sclerotiorum (mesophiles) (Wu et al., 2008; Hoshino et al., September 2015 \| Volume 6 \| Article 776 2 Protein signatures in Sclerotiniaceae fungi Badet et al. 2010; Judet-Correia et al., 2010). To successfully thrive in cold environments, psychrophilic pathogens must synthesize enzymes and eﬀectors that perform eﬀectively at low temperatures. Cold-temperature environments present several challenges, in particular reduced reaction rates, increased viscosity, and phase changes of the surrounding medium. A draft genome sequence of S. borealis strain F-4128 has recently been released (Mardanov et al., 2014a,b) providing an opportunity to better understand its adaptation to its ecological niche and particularly to cold environment. The total size of the assembled genome of S. borealis is 39.3 Mb, with a G+C content of 42%, including 10,171 predicted protein coding sequences (Mardanov et al., 2014a). These characteristics are similar for the genomes of S. sclerotiorum 1980 and B. cinerea B05.10 with total sizes of 38.3 Mb and 42.3 Mb respectively, G+C content of 41.8 and 43.1% respectively, and 14,503 and 16,448 predicted protein coding genes respectively (Amselem et al., 2011). the search for signatures of adaptation to S. borealis lifestyle may help revealing proteins essential for host and environment colonization in the Sclerotiniaceae. In this work, we focused our analysis on adaptations to S. borealis environment that lead to alterations in core functions (genes and proteins) conserved in S. sclerotiorum and B. cinerea. We analyzed a set of 5531 groups of core orthologous proteins for amino acid usage and intrinsic protein disorder patterns speciﬁcally associated with S. borealis proteins. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identiﬁed through our pipeline as candidate proteins involved in colonization of the environment. Our ﬁndings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases and for the bio-conversion of plant biomass. Frontiers in Plant Science \| www.frontiersin.org Introduction Cellular adaptations to low temperatures and the underlying molecular mechanisms are not fully understood but include membrane ﬂuidity, the production of cold-acclimation and antifreeze proteins and maintenance of enzyme-catalyzed reactions and protein-protein interactions involved in essential cellular processes (Feller, 2003; Casanueva et al., 2010). Attempts to correlate protein thermal adaptation with sequence and structure derived features have accumulated with the multiplication of genomic sequencing programs. For instance, analysis of the complete predicted proteome of the psychrophilic bacterium Colwellia psychrerythraea supported the view that psychrophilic lifestyle probably involves speciﬁc sets of genes in addition to changes in the overall genome content and amino acid composition (Methé et al., 2005). Because microorganisms are at complete thermal equilibrium with their environment, it is indeed conceivable that adaptation to low temperature lead to global alterations of proteomes in psychrophiles. Comparative genomic and metagenomic analyses in prokaryotes demonstrated that the summed frequency of amino acids Ile, Val, Tyr, Trp, Arg, Glu, Leu (IVYWREL) correlates with optimal growth temperature (Zeldovich et al., 2007). In another study on bacteria, Ala, Asp, Ser, and Thr were found preferred in the genome of psychrophiles compared to mesophiles, whereas Glu and Leu are less frequent (Metpally and Reddy, 2009). The analysis of amino acid usage in thermophilic fungi showed that these species indeed have a higher total frequency of IVYWREL amino acids than their mesophilic relatives, but show also signiﬁcant depletion in Gly and enrichment in Arg and Ala (Van Noort et al., 2013). At the structural level, cold environments seem to release selective pressure for stable proteins, and increase selection for highly active heat-labile enzymes, relying on improved intrinsic disorder to maintain optimal conformation dynamics (Feller, 2003, 2007). Besides these seemingly general principles and given the existence of psychrophiles in lineages across the tree of life, multiple mechanisms contributing to cold adaptation may exist. For a fungal pathogen such as S. borealis, completion of its A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments g p g g Several studies reported speciﬁc amino acid usage patterns and intrinsic disorder frequency in proteins from psychrophilic bacteria as compared to related mesophilic bacteria (Methé et al., 2005; Metpally and Reddy, 2009). To test whether S. borealis proteins have a distinctive pattern of amino acid usage and disorder compared to S. sclerotiorum and B. cinerea proteins, we designed a bioinformatics pipeline to process complete proteomes deduced from the whole genome sequences of these three fungal pathogens (Figure 2) (Amselem et al., 2011; Mardanov et al., 2014a). To exclude patterns that may be due to factors unrelated to adaptation in S. borealis proteins, we focused our analysis on core groups of orthologous proteins with one member from each species. A total of 6717 core orthologous groups (COGs) were identiﬁed using two pairwise InParanoid proteome comparisons (Ostlund et al., 2010) as explained in material and methods section and presented in Figure 2, covering between ∼42% (B. cinerea) to ∼66% (S. borealis) of complete predicted proteomes. We used multiple alignments of the three proteins in each COG to select S. sclerotiorum protein regions conserved in S. borealis and B. cinerea. To retrieve core protein regions conserved in all three members of COGs, we ran another round of InParanoid pairwise comparisons using conserved regions of S. sclerotiorum proteins as input. Short alignments can artiﬁcially cause strong variations in amino acid proportions. To reduce this confounding eﬀect, we excluded alignments producing a consensus sequence shorter than 200 amino acids. We obtained a total of 5531 COG alignments matching these criteria that were processed for amino acid frequency and intrinsic protein disorder analysis. S. borealis Proteins Show Speciﬁc Intrinsic Disorder and Amino Acid Usage Patterns Compared to Their Sclerotiniaceae Orthologs To document intrinsic protein disorder and amino acid usage in Sclerotiniaceae COGs, we calculated frequencies of each of the 20 amino acids in the aligned protein regions as well S. borealis Proteins Show Speciﬁc Intrinsic Disorder and Amino Acid Usage Patterns Compared to Their Sclerotiniaceae Orthologs For a fungal pathogen such as S. borealis, completion of its life cycle requires successful plant colonization, and a subset of secreted virulence factors is likely involved in essential cellular processes. Results A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments Besides, secreted proteins in both yeasts and mammals were shown to evolve slightly faster than intracellular proteins (Julenius and Pedersen, 2006; Liao et al., 2010), suggesting that p g To document intrinsic protein disorder and amino acid usage in Sclerotiniaceae COGs, we calculated frequencies of each of the 20 amino acids in the aligned protein regions as well September 2015 \| Volume 6 \| Article 776 3 Badet et al. Protein signatures in Sclerotiniaceae fungi FIGURE 2 \| Bioinformatics pipeline for the identiﬁcation of S. borealis protein sequence signatures in multiple ortholog alignments. Our pipeline uses complete predicted proteomes of S. borealis, S. sclerotiorum, and B. cinerea as inputs. It identiﬁes orthologous protein pairs in S. borealis and S. sclerotiorum; and in S. borealis and B. cinerea using Inparanoid. Using S. sclerotiorum proteins as a reference, it identiﬁes non-redundant core ortholog groups (COG) and overlapping regions (1). A second Inparanoid run is then used to deﬁne the longest aligned region in all three genomes (“consensus”) for each COG (2). Next, protein sequence metrics (disorder probability and amino acid frequencies) are calculated for consensus regions of all proteins (3). Finally, Wilcoxon sum rank tests are performed to identify metrics signiﬁcantly different in S. borealis proteins. FIGURE 2 \| Bioinformatics pipeline for the identiﬁcation of S. borealis protein sequence signatures in multiple ortholog alignments. Our pipeline uses complete predicted proteomes of S. borealis, S. sclerotiorum, and B. cinerea as inputs. It identiﬁes orthologous protein pairs in S. borealis and S. sclerotiorum; and in S. borealis and B. cinerea using Inparanoid. Using S. sclerotiorum proteins as a reference, it identiﬁes non-redundant core ortholog groups (COG) and overlapping regions (1). A second Inparanoid run is then used to deﬁne the longest aligned region in all three genomes (“consensus”) for each COG (2). Next, protein sequence metrics (disorder probability and amino acid frequencies) are calculated for consensus regions of all proteins (3). Finally, Wilcoxon sum rank tests are performed to identify metrics signiﬁcantly different in S. borealis proteins. when Wilcoxon sum rank tests were signiﬁcant (p < 0.05) for S. borealis—S. sclerotiorum and S. borealis—B. cinerea comparisons but not (p > 0.05) for S. sclerotiorum—B. cinerea comparison. The “hot loops” frequencies measure of intrinsic protein disorder was found signiﬁcantly associated with S. borealis COG aligned regions, whereas “Coils” and “Remark465” were not (Figure 3A). A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments borealis and the other fungi (p < 0.05) but not between S. sclerotiorum and B. cinerea (p > 0.05). These properties were considered as associated with S. borealis lifestyle. FIGURE 3 \| Adaptation to S. borealis lifestyle associates with speciﬁc amino acid usage and protein disorder patterns. Distribution of the p-values of Wilcoxon sum rank tests performed to identify intrinsic disorder probabilities (A) and amino acid frequencies (B) that are signiﬁcantly different in S. borealis core orthologs. For each amino acid frequency and intrinsic disorder probability, three pairwise tests were performed to compare (i) values in B. cinerea and S. sclerotiorum orthologs (p-values shown along the X-axis), (ii) values in S. borealis and B. cinerea orthologs (p-values shown along the Y-axis in green), and (iii) values in S. borealis and S. sclerotiorum orthologs (p-values shown along the Y-axis in red). Amino acid frequencies and intrinsic disorder probabilities that fell in the shaded areas were considered signiﬁcantly different between S. borealis and the other fungi (p < 0.05) but not between S. sclerotiorum and B. cinerea (p > 0.05). These properties were considered as associated with S. borealis lifestyle. and 5.91% in B. cinerea proteins. Lys and Glu were signiﬁcantly depleted in S. borealis. Lysine represented 5.26% of amino acids in S. borealis instead of 5.41% in S. sclerotiorum and B. cinerea proteins; Glu represented 6.43% of amino acids in S. borealis instead of 6.54% in S. sclerotiorum and 6.57% in B. cinerea proteins (Figure 3B). These ﬁndings are consistent with the view that cold adaptation includes the directional adaptation of pre- existing protein functions (intrinsic protein structure and amino acid composition) in addition to speciﬁc sets of genes conferring a psychrophilic lifestyle, such as previously reported in bacteria (Methé et al., 2005). proteomes of S. borealis, S. sclerotiorum, and B. cinerea. First, we compared the distribution of sTEKhot values in COGs by plotting all values in a ternary plot (Figure 4A). This revealed that sTEKhot values are distributed along an axis pointing toward S. borealis angle, clearly showing that sTEKhot values of S. borealis orthologs are major contributors to the structure of the dataset. There was 692 COGs in which S. borealis sTEKhot value accounted for >40% of the total sTEKhot for the COG, but only 388 and 345 in which S. sclerotiorum and B. A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments “Hot loops,” corresponding to protein regions predicted not to adopt helix or strand secondary structure and having a high degree of ﬂexibility, were found signiﬁcantly depleted in S. borealis COG aligned regions. S. borealis proteins had a median hot loop frequency of 3.43% in COG aligned regions, vs. 3.67% in S. sclerotiorum and 3.71% in B. cinerea proteins. Regarding frequency of amino acids, three were found signiﬁcantly associated with S. borealis aligned COG regions. Thr frequency was signiﬁcantly enriched, representing 6.00% of amino acids in S. borealis instead of 5.93% in S. sclerotiorum as their disorder frequencies. Determination of the disorder frequencies were obtained by assigning to each amino acid of the aligned protein regions their disorder probability obtain by submitting the full length protein to disEMBL analyses (Linding et al., 2003). The disEMBL output contained three measures of intrinsic protein disorder designated as “Coils,” “Hot loops,” and “Remark465” corresponding to their probability to be involved in disorder region. To test whether any of these 20 amino acid frequencies plus 3 disorder metrics frequencies showed a signiﬁcantly diﬀerent distribution in S. borealis COG aligned regions compared to S. sclerotiorum and B. cinerea, we performed pairwise Wilcoxon sum rank tests to compare distributions of each of the 23 properties in S. borealis and S. sclerotiorum, in S. borealis and B. cinerea, and in S. sclerotiorum and B. cinerea (Table S1). We considered that a protein property was signiﬁcantly associated with S. borealis COG aligned regions September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 4 Protein signatures in Sclerotiniaceae fungi Badet et al. FIGURE 3 \| Adaptation to S. borealis lifestyle associates with speciﬁc amino acid usage and protein disorder patterns. Distribution of the p-values of Wilcoxon sum rank tests performed to identify intrinsic disorder probabilities (A) and amino acid frequencies (B) that are signiﬁcantly different in S. borealis core orthologs. For each amino acid frequency and intrinsic disorder probability, three pairwise tests were performed to compare (i) values in B. cinerea and S. sclerotiorum orthologs (p-values shown along the X-axis), (ii) values in S. borealis and B. cinerea orthologs (p-values shown along the Y-axis in green), and (iii) values in S. borealis and S. sclerotiorum orthologs (p-values shown along the Y-axis in red). Amino acid frequencies and intrinsic disorder probabilities that fell in the shaded areas were considered signiﬁcantly different between S. A Pipeline to Reveal S. borealis Protein Sequence Signatures in Multiple Ortholog Alignments cinerea sTEKhot values respectively accounted for >40% of the total sKTEHhot for the COG (Figure 4A). Consistently, S. borealis has the highest sTEKhot value in 42.7% of COGS (2761), whereas S. sclerotiorum and B. cinerea have the highest sTEKhot value in 28.3% (1845) and 28.8% (1865) of the COGs respectively (Figure 4B). The Distribution of sTEKhot Index Is Biased in S. borealis Orthologous Proteins and Complete Predicted Proteome Therefore, orthologs that have similar sTEKhot values in all three species appear at the center of the plot, while COGs appear near the corner of the species harboring the ortholog with the highest sTEKhot otherwise. The size of bubbles is proportional to the sTEKhot value of S. borealis orthologs. Data points are frequent above the 40% line for S. borealis sTEKhot, and less so for S. sclerotiorum and B. cinerea sTEKhot indicating frequent higher sTEKhot values in S. borealis orthologs. (B) Species distribution of orthologs having the highest sTEKhot value in COGs. (C) Distribution of the sTEKhot index in the whole predicted proteome of the three fungi. FIGURE 4 \| The sTEKhot index discriminates S. borealis proteins in core ortholog groups and whole predicted proteomes. (A) Overall distribution of sTEKhot values from the three fungal species within COGs. Each bubble represents a COG positioned according to the contribution of each ortholog (sTEKhot%) to the total sTEKhot of the COG. Therefore, orthologs that have similar sTEKhot values in all three species appear at the center of the plot, while COGs appear near the corner of the species harboring the ortholog with the highest sTEKhot otherwise. The size of bubbles is proportional to the sTEKhot value of S. borealis orthologs. Data points are frequent above the 40% line for S. borealis sTEKhot, and less so for S. sclerotiorum and B. cinerea sTEKhot indicating frequent higher sTEKhot values in S. borealis orthologs. (B) Species distribution of orthologs having the highest sTEKhot value in COGs. (C) Distribution of the sTEKhot index in the whole predicted proteome of the three fungi. simulations indicate that sTEKhot clearly departs from random in describing speciﬁc intrinsic protein disorder and amino acid usage patterns in S. borealis proteins. measures to discriminate the proteome of S. borealis from that of S. sclerotiorum and B. cinerea, we randomly shuﬄed the 23 measures for intrinsic protein disorder and amino acid usage in equation (1) and calculated the proteome median value for shuﬄed indices in S. borealis, S. sclerotiorum, and B. cinerea (Table S3). In 300 shuﬄing iterations, the p-value for diﬀerence between the distribution of shuﬄed index in S. borealis and S. sclerotiorum or B. cinerea was < 5.1.e−104 (highest observed p- value) in only 6 instances. The median shuﬄed index value for S. borealis proteome was higher than the observed sTEKhot median in only 2 instances over 300 (0.6%). The Distribution of sTEKhot Index Is Biased in S. borealis Orthologous Proteins and Complete Predicted Proteome At the whole proteome level, sTEKhot median was 0.366 in S. borealis, but only 0.314 in S. sclerotiorum and 0.313 in B. cinerea (Figure 4C, Table S2). The overall sTEKhot distributions were signiﬁcantly diﬀerent when comparing S. borealis to the two other species (p < 5.1.e−104) but not when comparing S. sclerotiorum to B. cinerea (p = 0.84). However, a subset of S. sclerotiorum and B. cinerea proteins appeared to have high sTEKhot values. Indeed, as mentioned previously, S. sclerotiorum and B. cinerea each account for the highest sTEKhot in ∼30% of the COGs. Furthermore, the proportion of proteins with a sTEKhot > 1 was 6.2% in S. borealis, 4.6% in S. sclerotiorum and 5.0% in B. cinerea. This suggests that the general pattern of intrinsic protein disorder and amino acid usage observed in S. borealis protein may be shared by a subset of S. sclerotiorum and B. cinerea predicted proteome. Several studies reported biases in amino acid usage in the proteome of extremophiles and proposed indices able to discriminate proteins from extremophilic and related mesophilic organisms (Suhre and Claverie, 2003; Zeldovich et al., 2007; Wang and Lercher, 2010). To analyze the degree to which intrinsic protein disorder and amino acid usage of individual proteins matches with speciﬁc patterns identiﬁed in S. borealis predicted proteome, we designed the S. borealis T (Thr), E (Glu), K (Lys), hot (hot loops) index as follows: sTEKhot = T E + K + hot (1) (1) where “T,” “E,” and “K” are the normalized frequencies of Thr, Glu and Lys respectively in a given protein sequence, and “hot” is the normalized frequency of hot loops in this sequence. We computed the sTEKhot index for each protein in the predicted To verify that the sTEKhot index was an optimized combination of intrinsic protein disorder and amino acid usage September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org Badet et al. Protein signatures in Sclerotiniaceae fungi FIGURE 4 \| The sTEKhot index discriminates S. borealis proteins in core ortholog groups and whole predicted proteomes. (A) Overall distribution of sTEKhot values from the three fungal species within COGs. Each bubble represents a COG positioned according to the contribution of each ortholog (sTEKhot%) to the total sTEKhot of the COG. The Distribution of sTEKhot Index Is Biased in S. borealis Orthologous Proteins and Complete Predicted Proteome Wilcoxon ranking tests comparing random medians distribution to real sTEKhot median showed p < 4.72e−33 in the three species. The result of these Secreted Enzymes are Enriched among S. borealis Proteins with High sTEKhot To identify protein functions important for adaptation to S. borealis environment, we analyzed annotations of proteins with a sTEKhot value higher than 1 in S. borealis proteome. Overall, 4794 (47%) S. borealis proteins had no Gene Ontology (GO) annotation assigned. There were 635 proteins with sTEKhot > 1, among which 349 (55%) had no GO annotation. We looked for GO term enrichment in the 635 S. borealis with sTEKhot > 1 September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 6 Protein signatures in Sclerotiniaceae fungi Badet et al. and cell wall compartments. Consistently, enriched “biological processes” and “molecular functions” related to secreted enzymes involved in cell wall modiﬁcation (glycosyl hydrolases and carboxylic ester hydrolases, among which are pectinesterases and cutinases) and binding to cellulose. Cellulose is a major compared to all annotated proteins. Forty two GO terms appeared signiﬁcantly enriched (p < 0.05) among proteins with sTEKhot > 1, including 16 leaves (GO with no child term) of the GO network (Figure 5). GO terms for “cellular component” enriched in proteins with sTEKhot > 1 included extracellular FIGURE 5 \| Network representation of gene ontologies (GOs) of proteins with sTEKhot >1 in S. borealis proteome. Nodes correspond to GOs are sized according to the number of proteins with sTEKhot >1. They are colored from yellow to orange according to the p-value of a hypergeometric test for enrichment in proteins with sTEKhot >1 compared to whole proteomes. White nodes are GOs not signiﬁcantly enriched among proteins with sTEKhot > 1 (p > 0.05). GOs labeled in bold font correspond to functions possibly associated with host interaction. FIGURE 5 \| Network representation of gene ontologies (GOs) of proteins with sTEKhot >1 in S. borealis proteome. Nodes correspond to GOs are sized according to the number of proteins with sTEKhot >1. They are colored from yellow to orange according to the p-value of a hypergeometric test for enrichment in proteins with sTEKhot >1 compared to whole proteomes. White nodes are GOs not signiﬁcantly enriched among proteins with sTEKhot > 1 (p > 0.05). GOs labeled in bold font correspond to functions possibly associated with host interaction. September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 7 Protein signatures in Sclerotiniaceae fungi Badet et al. component of plant cell walls that fungal pathogens are able to detect and bind. Secreted Proteins Have Higher sTEKhot Than Non-secreted Proteins in the Three Sclerotiniaceae Species The enrichment of extracellular proteins among proteins with sTEKhot > 1 prompted us to compare the distribution of sTEKhot for secreted and non-secreted protein in the Sclerotiniaceae. We considered as predicted secreted proteins those identiﬁed as secreted with SignalP 4.0 no-TM network and as extracellular by WoLF PSORT. This produced lists of 667, 661, and 748 predicted secreted proteins (secretome) for S. borealis, S. sclerotiorum, and B. cinerea respectively. In all three fungal species, secreted proteins had signiﬁcantly higher sTEKhot values than non-secreted proteins, with median sTEKhot values for secreted proteins of 1.13 in S. borealis, 1.06 in S. sclerotiorum and 1.08 in B. cinerea (Figure 6A). The distribution of sTEKhot in secreted proteins was found signiﬁcantly higher than its distribution in non-secreted proteins with p-value of 8.8e−239 in S. borealis, 9.1e−265 in S. sclerotiorum and 4.1e−275 in B. cinerea respectively. To evaluate the likelihood of obtaining such distributions with other intrinsic protein disorder and amino acid usage parameters, we randomly shuﬄed the 23 measures for intrinsic protein disorder and amino acid usage in Equation (1), and calculated shuﬄed indices for each protein in the predicted secretome in the three species. In 300 rounds of shuﬄing, the median secretome index was found higher than the observed median secretome sTEKhot in 3, 1 and 1 instance for S. borealis, S. sclerotiorum and B. cinerea respectively (Table S3). FIGURE 6 \| Predicted secreted proteins have high sTEKhot values. (A) Distribution of sTEKhot values in the proteome and the secretome of (Continued) FIGURE 6 \| Predicted secreted proteins have high sTEKhot values. (A) Distribution of sTEKhot values in the proteome and the secretome of (Continued) Remarkably, although secreted proteins account for 6.5% of total proteome in S. borealis, 4.5% in S. sclerotiorum and 4.5% in B. cinerea, the proportion of secreted proteins among those with sTEKhot > 1.5 raised to 76.9% (206 out of 268) in S. borealis, 68.2% (182 out of 267) in S. sclerotiorum and 65.0% (206 out of 317) in B. cinerea, representing ∼13.6 fold enrichment in secreted proteins (Figure 6B). These results suggest that intrinsic protein disorder and amino acid usage patterns associated with S. borealis lifestyle and secretion are largely overlapping in the Sclerotiniaceae. Secreted Enzymes are Enriched among S. borealis Proteins with High sTEKhot Also plants aerial parts are protected by a cuticle composed by cutin. Fungal pathogens are able to hydrolyze cutin through cutinases, thus facilitating host colonization. In addition, proteins involved in carbohydrate metabolism were enriched among proteins with sTEKhot > 1. These functions are associated with colonization of the environment, especially plant- associated environment. Similar enrichments where observed when looking at GO annotations for S. sclerotiorum and B. cinerea proteins harboring a sTEKhot > 1 (Figures S1, S2). In addition, copper ion binding GO was found to be enriched in S. sclerotiorum and B. cinerea. FIGURE 6 \| Predicted secreted proteins have high sTEKhot values. (A) Distribution of sTEKhot values in the proteome and the secretome of (Continued) Frontiers in Plant Science \| www.frontiersin.org FIGURE 6 \| Continued S. borealis, S. sclerotiorum and B. cinerea. (B) Proportion of predicted secreted proteins according to sTEKhot cutoff values. In complete proteomes (sTEKhot ≥0), the proportion of secreted proteins is ∼5% in all three fungal proteomes, whereas among proteins with sTEKhot ≥1.5 (dotted line) it reaches an average ∼70%. (C) Proportion of whole proteomes and proteins with sTEKhot > 1.5 that are secreted, contain GPI-anchors, are N-glycosylated or contain transmembrane (TM) domains. Enrich., enrichment fold among sTEKhot > 1.5 as compared to whole proteomes. To test whether proteins with high sTEKhot could be enriched in other types of motifs, we predicted glycosylphosphatidylinositol (GPI) anchors, transmembrane (TM) domains and N-glycosylation sites in the proteome of S. borealis, S. sclerotiorum and B. cinerea. We found an average of 5.0% of proteins with GPI-anchors, 9.9% proteins with TM domains and 3.8% of proteins with >10 predicted N-glycosylation sites in the Sclerotiniaceae species (Table S6, Figure 6C). As compared to whole proteomes, the list of proteins with sTEKhot >1.5 showed an average 7.1-fold enrichment in proteins with GPI-anchors, 2.1-fold enrichment in proteins with >10 predicted N-glycosylation sites and no enrichment in proteins with TM domains (Figure 6C). Secreted proteins showed the strongest enrichment among proteins with sTEKhot >1.5. Overall these analyses suggest that a signiﬁcant overlap exists between the constraints imposed on protein sequence by adaptation to S. borealis lifestyle and to secretion in the Sclerotiniaceae. non-secreted proteins) could be rejected with p < 0.05 for all three fungal species. Among the 23 measures for protein disorder and amino acid usage, 21 could be signiﬁcantly associated with fungal secretomes, supporting the view that function outside the cell imposes speciﬁc constraints on amino acids usage in secreted proteins, such as evolution toward reduced synthetic cost of proteins (Smith and Chapman, 2010). Similar to patterns associated with S. borealis lifestyle, we found that enrichment in Thr, depletion in Glu and reduced frequency of hot loops disorder are among the properties most signiﬁcantly associated with secretion (p-values ranging from 7.62e−3 to 2.67e−194) (Table S4). We considered several hypotheses to explain the observed common signatures for S. borealis lifestyle and secretion. First, we envisaged that prevalence of secreted proteins in COGs may have biased signatures of S. borealis lifestyle toward properties associated with secretion. However, ratios of secreted proteins in COG sets were similar to those observed for total proteomes (7% in S. borealis, 6.7% in S. Secreted Proteins Have Higher sTEKhot Than Non-secreted Proteins in the Three Sclerotiniaceae Species To independently validate this observation, we compared the distribution of all amino acid frequencies and the distribution of the three intrinsic protein disorder measures used previously in secreted and non-secreted proteins from the three fungal species. We considered that a protein property is associated with secretion when the null hypothesis of the Wilcoxon sum-rank test (distribution of property no diﬀerent between secreted and FIGURE 6 \| Predicted secreted proteins have high sTEKhot values. (A) Distribution of sTEKhot values in the proteome and the secretome of (Continued) September 2015 \| Volume 6 \| Article 776 8 Protein signatures in Sclerotiniaceae fungi Badet et al. with their closest homolog in S. sclerotiorum, this average score was 521.6 for S. borealis secreted proteins (Figure S4). This indicates that globally, S. borealis secretome is more divergent from S. sclerotiorum proteome than S. borealis non-secreted proteins. A similar tendency was observed when comparing S. borealis and B. cinerea proteomes. The high sTEKhot average observed in Sclerotiniaceae secretomes is therefore not due to higher similarity in secretomes compared to non-secreted proteins. FIGURE 6 \| Continued sclerotiorum and 6.4% in B. cinerea proteins from the set of 5531 COGs). Furthermore, we excluded COGs that comprised secreted proteins and tested whether amino acid usage patterns associated with S. borealis proteins as previously. Amino acids enriched in S. borealis proteins included Thr and amino acids depleted in S. borealis included Glu and Lys (p < 0.05), similar to what we found in our initial analysis taking all COGs into account. In addition, we also found His enriched in S. borealis sequences and Asn depleted (p < 0.05). We conclude that the detection of a bias in the usage of these amino acids in S. borealis proteins was not due to the abundance of secreted proteins in COGs (Table S5). Second, we hypothesized that intrinsic protein disorder and amino acid usage in secreted proteins might be due to signal peptide regions. To test this, we analyzed protein properties associated with mature secreted proteins (signal peptide region removed). We found that mature secreted proteins had signiﬁcantly higher sTEKhot than the rest of the proteome (p < 2.4.e−232), similar to what we found with full length secreted proteins (Figure S3). Therefore high sTEKhot in secretomes is not due to signal peptide sequence. Third, we considered that high sTEKhot in secretomes could arise if secretomes were be less divergent than the rest of the proteomes, leading to S. borealis signature being more conserved in secreted proteins of S. sclerotiorum and B. cinerea. To test this, we analyzed the distribution of similarity between S. borealis proteins and their closest homologs in S. sclerotiorum and B. cinerea. Whereas the average BLASTP score was 630.9 for S. borealis non-secreted proteins aligned S. Sclerotiorum Genes Encoding Proteins with High sTEKhot are Enriched in Genes Induced in planta Frontiers in Plant Science \| www.frontiersin.org S. Sclerotiorum Genes Encoding Proteins with High sTEKhot are Enriched in Genes Induced in planta p To further support the association between high sTEKhot index and colonization of the environment, and particularly host plants, we analyzed the distribution of sTEKhot values in S. sclerotiorum genes diﬀerentially regulated in planta. For this, we took advantage of S. sclerotiorum microarray gene expression data generated by Amselem et al. from infected sunﬂower cotyledons (Amselem et al., 2011). In this dataset, out of 14 503 predicted protein coding genes, 615 were induced at least two-fold during infection of sunﬂower (4.31%) and 458 genes down-regulated at least two-fold (3.21%). The proportion of genes induced in planta reached 27.1% of S. sclerotiorum genes encoding proteins with sTEKhot ≥2, representing ∼6.3-fold enrichment (Figure 7). The proportion of genes down-regulated in planta reached 12.1% of S. sclerotiorum genes encoding proteins with sTEKhot ≥2, representing ∼3.8-fold enrichment. S. sclerotiorum proteins with sTEKhot > 1 include six proteins with CFEM domain, a Cys-rich domain with proposed role in fungal pathogenesis, two proteins with a cerato-platanin domain, one of which being the ortholog of B. cinerea pathogen associated molecular pattern BcSpl1 (Frías et al., 2011), 27 proteins with a pectin lyase fold found in Aspergillus virulence factors (Mayans et al., 1997), and 29 out of 78 eﬀector candidates proposed by Guyon et al. (2014). These ﬁndings are consistent with important role in the colonization of the host plant for some proteins with high sTEKhot values. September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 9 Protein signatures in Sclerotiniaceae fungi Badet et al. may stabilize folding like, although these residues were not predicted to form disulﬁde bonds by Disulﬁnd (Ceroni et al., 2006). Antifreeze proteins have been reporting that rely on disulﬁde bonds for folding (Basu et al., 2015) whereas others do not (Kondo et al., 2012; Sun et al., 2014). Like other known fungal antifreeze proteins (Kondo et al., 2012), but unlike Maxi, SBOR_9046 and its orthologs are predicted to be secreted. A unique feature of Maxi among antifreeze proteins is the presence of ice-binding residues buried within the four-helix bundle instead of exposed on their surface (Sun et al., 2014). A prediction of SS1G_10836 dimer structure supports the existence of rather hydrophilic pockets buried within the four-helix bundle, suggesting that the mechanism of ice binding of Maxi could be conserved in SS1G_10836 and its orthologs (Figure 8C). S. Sclerotiorum Genes Encoding Proteins with High sTEKhot are Enriched in Genes Induced in planta To get insights into SS1G_10836 function, we analyzed the expression of the corresponding gene in mycelium grown in Potato Dextrose Broth (PDB), during the colonization of Arabidopsis plants and in sclerotia by quantitative RT-PCR. This revealed a 3.3-fold induction (log2 = 1.7) speciﬁc to sclerotia (Figure 8F). Since sclerotia overwinter in the soil, putative antifreeze proteins may contribute to survival of these structures both in arctic and temperate climates. FIGURE 7 \| Proportion of S. sclerotiorum proteins encoded by genes differentially expressed in planta according to sTEKhot cutoff values. In S. sclerotiorum complete genome (sTEKhot ≥0), the proportion of genes induced ≥2-fold in planta is ∼4.31%, whereas it reaches ∼27.1%.among proteins with sTEKhot ≥2 (dotted line). FIGURE 7 \| Proportion of S. sclerotiorum proteins encoded by genes differentially expressed in planta according to sTEKhot cutoff values. In S. sclerotiorum complete genome (sTEKhot ≥0), the proportion of genes induced ≥2-fold in planta is ∼4.31%, whereas it reaches ∼27.1%.among proteins with sTEKhot ≥2 (dotted line). p The COG including SS1G_03146, BC1G_07573, and SBOR_1255 is remarkable for including three proteins with high (>1) but with very variable sTEKhot, ranging from 1.58 (SS1G_03146) to 7.07 (BC1G_07573). No interproscan domain or GO terms were associated with these proteins of 223 amino acids in average, but all three were predicted to include a N-terminal signal peptide for secretion. To get insights into their putative function, we performed protein structure modeling and fold recognition using the I-TASSER server (Zhang, 2008). The closest structural analog was Aspergillus oryzae AA11 (AoAA11) Lytic Polysaccharide Monooxygenase (LPMO) (Hemsworth et al., 2014). Sequence similarity with AoAA11 was limited (from 9.6% identity for SBOR_1255 to 10.9% identity for SS1G_03146), superimposition of SS1G_03146 predicted structure with AoAA11 structure showed a Root Mean Square Deviation < 3.1Å and a TM-score of 0.677, indicating structural similarity deviating signiﬁcantly from random (Figures 8D,E). Similar to the Sclerotiniaceae proteins, full length AoAA11 (accession number XM_001822611) harbors a N-terminal signal peptide. AoAA11, SBOR_1255, and BC1G_07573 feature two conserved predicted disulﬁde bonds, SS1G_03146 is predicted to contain only one (Figure 8D). The catalytic triad of AoAA11 appears nicely conserved in the Sclerotiniaceae proteins, with the exception of the catalytic Tyr replaced by a Ser in SS1G_03146 (Figure 8D). LPMOs are enzymes oxidizing recalcitrant polysaccharides such as cellulose, starch and chitin. They present excellent potential for use in biomass conversion and the production of biofuels. S. Sclerotiorum Genes Encoding Proteins with High sTEKhot are Enriched in Genes Induced in planta Aspergillus oryzae AA11 represents a new class of LPMOs that include a putative chitin-binding domain (Hemsworth et al., 2014). We analyzed the expression of the SS1G_03146 gene in mycelium grown in PDB, during the colonization of Arabidopsis plants and in sclerotia by quantitative RT-PCR. This revealed up to 9.5-fold induction (log2 = 3.25) during plant infection (Figure 8F). This suggests that SS1G_03146 may be involved in colonization of the Frontiers in Plant Science \| www.frontiersin.org High sTEKhot Index and Secretion Signal Reveal Candidate Proteins Associated with Colonization of the Environment To illustrate the value of the sTEKhot index for the exploration of the proteome of fungi from the Sclerotiniaceae, we analyzed in detail the sequence of two proteins with high sTEKhot but with no assigned function. Over the three proteomes analyzed, S. borealis SBOR_9046 had the highest sTEKhot (10.01). In S. sclerotiorum, its ortholog is SS1G_10836 which ranked as the 5th highest sTEKhot in S. sclerotiorum (7.34). In B. cinerea, its ortholog is BC1G_03854 which ranked as the 23rd highest sTEKhot in B. cinerea (4.29). No interproscan domain or GO terms were associated with these proteins of 171 amino acids (except SS1G_10836 which is 173 amino acids long). To get insights into their putative function, we performed protein structure modeling and fold recognition using the I-TASSER server (Zhang, 2008). The closest structural analog was the antifreeze protein Maxi from winter ﬂounder (Pseudopleuronectes americanus) (Sun et al., 2014). Although sequence similarity with Maxi was limited (from 15.2% identity for SBOR_9046 to 16.2% identity for SS1G_10836), superimposition of SS1G_10836 predicted structure with Maxi structure showed a Root Mean Square Deviation < 2.3Å and a TM-score of 0.875, indicating structural similarity deviating signiﬁcantly from random (Figures 8A,B). Analysis of SBOR_9046, SS1G_10836 and BC1G_03854 sequence by TargetFreeze (He et al., 2015) supported the prediction as antifreeze proteins. The Sclerotiniaceae proteins contain four Cys residues located in the kink of predicted structures that Frontiers in Plant Science \| www.frontiersin.org September 2015 \| Volume 6 \| Article 776 10 Badet et al. Protein signatures in Sclerotiniaceae fungi FIGURE 8 \| Candidate proteins associated with colonization of the environment identiﬁed based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). High sTEKhot Index and Secretion Signal Reveal Candidate Proteins Associated with Colonization of the Environment (F) SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates. FIGURE 8 \| Candidate proteins associated with colonization of the environment identiﬁed based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). (F) SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates. FIGURE 8 \| Candidate proteins associated with colonization of the environment identiﬁed based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). (F) SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates. Discussion Understanding how fungal plant pathogens colonize their environment, including their host plants, is critical for food security and the sustainable management of ecosystems (Roux et al., 2014). In particular B. cinerea and S. sclerotiorum are threatening hundreds of plant species and important crop species in the majority of regions of the globe. Fungi also represent a remarkable reservoir of enzymes with very diverse catalytic abilities that are employed in industrial processes. We have conducted a comparative analysis of the proteome and secretome of fungal species from the Sclerotiniaceae revealing common principles of sequence optimization for secreted proteins. g y Enrichment analyses revealed that signatures associated with S. borealis lifestyle are frequent in plant cell wall degrading enzymes, carbohydrate binding domain containing proteins and ion binding proteins. More generally, secreted proteins showed high sTEKhot values in S. borealis, S. sclerotiorum and B. cinerea. The proportion of predicted secreted proteins reaches over 75% of S. borealis proteins with sTEKhot > 1.5 and the proportion of proteins encoded by in-planta induced genes reaches over 27% of S. sclerotiorum proteins with sTEKhot > 2, suggesting that sTEKhot may be a useful criterion to identify proteins associated with environmental adaptation or potential virulence factors. More speciﬁcally, there were 117 proteins predicted to be secreted and harboring a sTEKhot > 1.5 with no annotation in S. sclerotiorum that could include uncharacterized virulence factors. Although some classes of protein eﬀectors from bacteria and oomycete pathogens can be identiﬁed relatively easily thanks to conserved N-terminal sequence signals, this strategy has proven limited for fungal pathogens. Alternative bioinformatics approaches have been developed exploiting known eﬀector properties for searching eﬀector candidates in the secretome of fungal pathogens (Saunders et al., 2012; Guyon et al., 2014). Typical eﬀector properties include the presence of a N-terminal secretion signal, small protein size, high Cys content, the absence of characterized protein domains, high rate of non-synonymous over synonymous substitutions (Hacquard et al., 2012; Saunders et al., 2012; Persoons et al., 2014; Sperschneider et al., 2014). However, validated virulence factors do not all comply with these properties, such as Verticillium dahlia isochorismatase VdIsc1 harboring an isochorismatase domain but no conventional secretion signal (Liu et al., 2014) or Melampsora lini AvrM that lacks any Cys (Catanzariti et al., 2006). High sTEKhot Index and Secretion Signal Reveal Candidate Proteins Associated with Colonization of the Environment September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 11 Protein signatures in Sclerotiniaceae fungi Badet et al. plant, but functional analysis will be required to determine its actual role. are therefore consistent with some previous observations made for psychrophilic proteins. Nevertheless, our approach does not allow dissociating psychrophily and other speciﬁc life traits of S. borealis (speciﬁc host range, geographic habitat) as drivers of the observed protein signatures. We observed a reduction in the frequency of intrinsic disorder in hot loops in S. borealis proteins. By contrast, cold adapted enzymes were often reported to harbor low conformational stability to maintain high reaction rates at low temperature (Feller, 2007; Casanueva et al., 2010) and intrinsically disordered proteins were shown to be more resistant to cold than globular proteins (Tantos et al., 2009). A global study of intrinsic protein disorder in 332 prokaryotes showed however that psychrophilic bacteria have a lower level of intrinsic disorder than mesophiles, although this was proposed to be due to the loss of cellular functions relying on intrinsically disordered proteins (Burra et al., 2010). This analysis also supports the view that adaptations to S. borealis lifestyle include directional changes in the sequence of conserved proteins, in addition to possible gene gains and losses that have not been analyzed in this work. Based on these predicted functions, we propose that SS1G_10836 and SS1G_03146 have important functions in the colonization of the environment, the identiﬁcation of which was facilitated by the implementation of the sTEKhot index. Functional studies will be required to test predicted functions of these proteins. Furthermore, these two proteins have predicted properties that may be exploited for biotechnology purposes. Frontiers in Plant Science \| www.frontiersin.org Discussion In Pseudomonas syringae, amino acid biases and patterns at the N-terminus were used to identify type III September 2015 \| Volume 6 \| Article 776 12 Protein signatures in Sclerotiniaceae fungi Badet et al. eﬀector candidates. Enrichment in Thr and depletion in Leu is a characteristic of bacterial type III proteins secreted into animal and plant cells, although high sequence variability and high tolerance of mutations make these properties diﬃcult to generalize (Arnold et al., 2009; McDermott et al., 2011; Schechter et al., 2012). To identify novel eﬀectors in Fusarium sp., Stagonospora nodorum, and Puccinia graminis f.sp. tritici fungi, Sperschneider et al. performed unsupervised clustering based on 35 sequence-derived features, including amino acid composition (Sperschneider et al., 2013, 2014). Several clusters were characterized by strong biases in amino acid usage, such as the cluster including the three S. nodorum eﬀectors SnToxA, SnTox1 and SnTox3 enriched in small and non-polar amino acids and the cluster including F. oxysporum f. sp. lycopersici SIX3 featuring high average positive protein charge and a signiﬁcantly higher percentage of Pro, Ser and Thr (Sperschneider et al., 2013). Similarly, secreted eﬀectors of fungi from the Sclerotiniaceae family could be enriched in Thr and depleted in Glu and Lys compared to the rest of the proteome. This suggests that amino acid usage bias is a property that may be shared by sets of secreted proteins with unrelated function and from distant pathogen lineages. Consistent with Glu and Lys being disorder-promoting amino acids, we found that secreted proteins of Sclerotiniaceae species show lower disorder frequency in hot loops that the rest of the proteome. Eﬀectors of bacterial pathogens were shown to be highly enriched in long disordered regions, presumably to facilitate eﬀector translocation into the host cell, host function mimicry and evasion of the host immune system (Marín et al., 2013). Intrinsic protein disorder was shown to promote high speciﬁcity and low aﬃnity protein-ligand interactions (Zhou, 2012; Chu and Wang, 2014). While these properties could be advantageous for host-speciﬁc eﬀectors of biotrophic pathogens, for which avoiding detection by the host is critical, opposite requirements may shape the evolution of eﬀectors from broad- range necrotrophic pathogens. Indeed, a relatively low speciﬁcity may allow eﬀectors to function during colonization of diverse host species. It is also believed that detection by the host would not be detrimental, and could even be beneﬁcial, to some necrotrophic plant pathogens (Govrin and Levine, 2000). Genome Sources We retrieved three predicted proteomes (Sclerotinia sclerotiorum v1.0, Botrytis cinerea v1.0 and Sclerotinia borealis F-4157) from the Joint Genome Institute (http://jgi.doe.gov/) and NCBI (http://www.ncbi.nlm.nih.gov/) in fasta format. As a cautionary note: the proteome sequences that form the basis of our analyses had originally been predicted by various techniques and may thus be of varying quality and completeness. S. sclerotiorum gene expression data was obtained from http://urgi.versailles.inra.fr/ Data/. Discussion Consistent with Glu and Lys being disorder-promoting amino acids, we found that secreted proteins of Sclerotiniaceae species show lower disorder frequency in hot loops that the rest of the proteome. Eﬀectors of bacterial pathogens were shown to be highly enriched in long disordered regions, presumably to facilitate eﬀector translocation into the host cell, host function mimicry and evasion of the host immune system (Marín et al., 2013). Intrinsic protein disorder was shown to promote high speciﬁcity and low aﬃnity protein-ligand interactions (Zhou, 2012; Chu and Wang, 2014). While these properties could be advantageous for host-speciﬁc eﬀectors of biotrophic pathogens, for which avoiding detection by the host is critical, opposite requirements may shape the evolution of eﬀectors from broad- range necrotrophic pathogens. Indeed, a relatively low speciﬁcity may allow eﬀectors to function during colonization of diverse host species. It is also believed that detection by the host would not be detrimental, and could even be beneﬁcial, to some necrotrophic plant pathogens (Govrin and Levine, 2000). In that case, eﬀectors with high aﬃnity for their targets would not be counter-selected by the host immune system, and would instead favor Sclerotiniaceae fungi in the competition with other microbes for plant-derived resources. Discussion In the present study we designed a bioinformatics pipeline aiming at identifying species-speciﬁc patterns of amino acid usage and intrinsic protein disorder in the proteome of closely related species. We applied this pipeline to agriculturally important fungal pathogens from Sclerotiniaceae family to reveal speciﬁc signatures associated with S. borealis lifestyle. Compared to S. sclerotiorum and B. cinerea orthologs, we observed in S. borealis proteins a signiﬁcant increase in Thr usage and a signiﬁcant decrease in Glu and Lys usage. To minimize the impact of phylogenetic distance on the deﬁnition of S. borealis sequence signature, we have restricted our analysis to species from the Sclerotiniaceae family and we discarded any sequence signature diﬀering signiﬁcantly between S. sclerotiorum and B. cinerea. It is also worth noting that S. borealis, S. sclerotiorum and B. cinerea have a very similar G+C content, so that G+C bias is not expected to have an impact on the diﬀerential usage of amino acids. Speciﬁc trends in amino acid composition have been reported to associate with protein stability at extreme temperatures. Given the diversity of ecological groups including psychrophiles, it has been challenging to identify universal trends in amino acids usage associated with cold adaptation (Casanueva et al., 2010). Enrichment in Thr has been reported in solvent-accessible areas of proteins from two cold-adapted Archaea (Goodchild et al., 2004) and in proteins from several psychrophilic bacteria (Metpally and Reddy, 2009). This was proposed to reduce surface charge while minimizing risk of aggregation (Goodchild et al., 2004). Frequent substitutions of Glutamate were observed in exposed sites of selected psychrophilic enzymes (Gianese et al., 2001) and more generally in the proteome of the psychrophilic Archea Halorubrum lacusprofundi (Dassarma et al., 2013). Glu is also part of a set of amino acids shown to correlate signiﬁcantly with optimal growth temperature of prokaryotes (Zeldovich et al., 2007). Speciﬁc signatures of amino acid usage we found in S. borealis Amino acid composition is a feature used to predict candidate bacterial eﬀectors. Positive charge, richness in alkaline (H, R, K) amino acids and Glu in the 30 C-terminal amino acids is for instance a property often found in type IV secreted eﬀectors (Meyer et al., 2013; Zou et al., 2013; Wang et al., 2014). Gene Ontology Annotation and Enrichment Analysis The Gene Ontology was collected from the Gene Ontology Consortium website (http://geneontology.org/) in obo format. Assignment of the Gene Ontology annotation of the three sets of protein sequences was performed using InterProScan (Jones et al., 2014). GO enrichments analysis was performed using the Biological Networks Gene Ontology plug-in (Maere et al., 2005) in Cytoscape 3.2.1 with the following parameters: a hypergeometric test for statistical analysis with a Bonferroni Family-Wise Error rate correction and a signiﬁcance level of 0.05. Cross species comparative analysis has been successfully applied to the identiﬁcation of novel and specialized virulence mechanisms on the one hand, and to the identiﬁcation of optimization principles governing the evolution of proteins under given constraints on the other hand. In nature, S. borealis proteins have undergone optimization under speciﬁc environmental constraints, including cold, over an irreproducible time at the scale of human life. Comparative genomics approaches therefore have the potential to reveal protein specialization and optimization principles that are not easily accessible through experimental evolution experiments. Indeed, selecting optimized enzyme variants, especially for thermostability, through random mutagenesis often requires exploring a large library of mutants or experimental setups Frontiers in Plant Science \| www.frontiersin.org Discussion In that case, eﬀectors with high aﬃnity for their targets would not be counter-selected by the host immune system, and would instead favor Sclerotiniaceae fungi in the competition with other microbes for plant-derived resources. maintaining an appropriate pressure of selection to collect the optimized variants (Kuchner and Arnold, 1997; Lebbink et al., 2000). Comparative genomics can accelerate discoveries usually relying on time consuming screens (Xiao et al., 2008). The biochemical properties of cold-active proteins make them attractive in biochemical, bioremediation, and industrial processes for food, biofuels and pharmaceutical production notably (Cavicchioli et al., 2011). Plant pathogenic fungi in particular present a vast reservoir of biopolymer degrading enzymes adapted to a wide range of temperatures and environments. Functional analyses will be required to test whether the activity of candidates highlighted in this work have applied potential. In the long term, the analysis of optimization principles governing the evolution of secreted proteins from important fungal pathogens may prove useful in improving plant health with the design of crops resistant to broad host range pathogens and to cold stress, and to develop novel strategies for the production of renewable energy relying on the bio-conversion of plant biomass. eﬀector candidates. Enrichment in Thr and depletion in Leu is a characteristic of bacterial type III proteins secreted into animal and plant cells, although high sequence variability and high tolerance of mutations make these properties diﬃcult to generalize (Arnold et al., 2009; McDermott et al., 2011; Schechter et al., 2012). To identify novel eﬀectors in Fusarium sp., Stagonospora nodorum, and Puccinia graminis f.sp. tritici fungi, Sperschneider et al. performed unsupervised clustering based on 35 sequence-derived features, including amino acid composition (Sperschneider et al., 2013, 2014). Several clusters were characterized by strong biases in amino acid usage, such as the cluster including the three S. nodorum eﬀectors SnToxA, SnTox1 and SnTox3 enriched in small and non-polar amino acids and the cluster including F. oxysporum f. sp. lycopersici SIX3 featuring high average positive protein charge and a signiﬁcantly higher percentage of Pro, Ser and Thr (Sperschneider et al., 2013). Similarly, secreted eﬀectors of fungi from the Sclerotiniaceae family could be enriched in Thr and depleted in Glu and Lys compared to the rest of the proteome. This suggests that amino acid usage bias is a property that may be shared by sets of secreted proteins with unrelated function and from distant pathogen lineages. Ortholog Prediction Ortholog prediction was performed with standalone InParanoid 4.0 (Ostlund et al., 2010) using all vs. all Basic Local Alignment Search Tool (BLAST) algorithms and the following parameters: the BLOSUM62 matrix, a score cut-oﬀof 50 bits and a minimal sequence overlap area of 0.5 (Altschul et al., 1990; Remm et al., 2001). Two pairwise InParanoid comparisons (S. borealis vs. S. sclerotiorum and S. borealis vs. B. cinerea) were ran ﬁrst on complete proteomes, leading to the identiﬁcation 6717 COGs, then using only conserved regions of S. sclerotiorum proteins (“overlapping regions”) as input (Figure 2). Finally alignments September 2015 \| Volume 6 \| Article 776 13 Protein signatures in Sclerotiniaceae fungi Badet et al. producing a consensus sequence shorter than 200 amino acids were excluded leading to 5531 COGs. producing a consensus sequence shorter than 200 amino acids were excluded leading to 5531 COGs. enriched or depleted amino acids and disorder frequency in S. borealis common set of core ortholog groups’ alignments compared to S. sclerotiorum and B. cinerea core ortholog groups alignments, but found to be not signiﬁcantly diﬀerent between S. sclerotiorum and B. cinerea, were further used for computing the environmental condition adaptation index (sTEKhot). Thr frequency (Tf) found to be over represented in S. borealis were added to the numerator of the index, whereas Lys (Kf), Glu (Ef) and hot loops (HotLOOPf) frequencies found to be under represented were added to the denominator. Each metrics were normalized by their own median (Xmf, where X is the considered metric) through the all set of proteome used in the analysis (S. borealis plus S. sclerotiorum plus B. cinerea). This normalization assures similar contribution of each metrics to the index. Gene Expression Analysis One-centimeter long leaves were collected and grinded twice for 30 s at maximum frequency in a Retsch MM40 mixer. Total RNA extraction was performed with Macherey-Nagel Nucleospin RNA extraction kit following the manufacturer’s instructions. One µg of total RNA was used for cDNA synthesis in a 20-µL reaction according to Roche Transcriptor Reverse Transcriptase protocol, using 0.5 µL of SuperScript II reverse transcriptase Random Shufﬂing of sTEKhot Protein amino acid usage was assessed by calculating the frequency of each of 20 amino acids in protein sequences. Prediction of disorder probability of protein amino acid was performed with DisEMBL vs. 1.4 computational tool (Linding et al., 2003) on the full length proteins. In case of analysis of a protein sequence subset, like for the core ortholog groups alignments (see previous section), the disorder probability of each amino acid in the subset were taken from the disorder probability of this amino acid in the full length protein. This was done to avoid miss attribution of disorder probability in a subset of a sequence since surrounding of amino acid in the sequence are of importance to calculate its own disorder probability. Random sTEKhot indexes were calculated by shuﬄing amino acid and hotloop frequencies in Equation (2) with any of the observed amino acid and hotloop frequencies for a given organism. The random index is therefore deﬁned by Equation (3) in which W, X, Y, and Z are randomly selected observed frequencies. RANDOMindex = Xf Xmf Yf Ymf + Zf Zmf + Wf Wmf (3) (3) Indexes were calculated separately for the three proteomes and secretomes. Random sTEKhot medians and Wilcoxon ranking test p-values were extracted from 300 independent runs. Protein Structure Modeling and Analysis g y Protein structure modeling was performed with the I-TASSER server (Zhang, 2008) using SS1G_10836 and SS1G_03146 full length sequences as queries. SS1G_10836 best model C-score was -3.22; best TM score was 0.875 (RMSD 2.27Å) with model 4KE2. SS1G_03146 best model C-score was -2.28; best TM score was 0.677 (RMSD 3.07Å) with model 4MAH. Pipeline for Collecting Multiple Ortholog Alignments g First, ortholog predictions were performed as described in previous section between one organism, called reference organism in the following (S. sclerotiorum), and each other organism included in the analysis (B. cinerea and S. borealis). Only core groups of orthologous proteins harboring one member from each species were retained. Then, the common overlapping sequences in the reference organism to the others organisms were selected according to BLAST begin and end alignment positions. The maximal begin and the minimal end were used to deﬁned the overlapping sequences. Overlapping sequences with lower than 200 amino acids length were excluded. The obtained overlapping sequences in the reference organism were used to run a new round of ortholog prediction with each other organisms. The consensus sequences, or core ortholog groups alignments, in each organisms were selected accordingly to BLAST begin and end alignment positions using the minimal begin and the maximal end obtained through the all orthologs predicted. The consensus sequences with lower than 200 amino acids length were excluded. First, ortholog predictions were performed as described in previous section between one organism, called reference organism in the following (S. sclerotiorum), and each other organism included in the analysis (B. cinerea and S. borealis). Only core groups of orthologous proteins harboring one member from each species were retained. Then, the common overlapping sequences in the reference organism to the others organisms were selected according to BLAST begin and end alignment positions. h l b d h l d d d ﬁ d h sTEKhot = Tf Tmf Kf Kmf + Ef Emf + HotLOOPf HotLOOPmf (2) (2) sTEKhot value was calculated for every protein of the three proteomes according to (2). The list of proteins with the top 635 sTEKhot (>1) corresponded exactly to proteins with the top Tf-(Ef+Kf+HotLOOPf) values supporting the robustness of the arithmetic design of the sTEKhot index in this dataset. Secretome Prediction and Protein Motif Annotation Analysis by SignalP4.1 was performed at http://www.cbs.dtu.dk using default parameters. Protein localization was predicted with PSORT II software using the WoLF PSORT extension (Horton et al., 2007) for organism type “fungi.” Proteins were deﬁned as part of the secretome when containing both signal peptide and extracellular predicted localization and were excluded if they possess a trans-membrane region predicted by TMHMM (Sonnhammer et al., 1998). Glycosylphosphatidylinositol anchored proteins were identiﬁed using Fraganchor (Poisson et al., 2007); N-glycosylation sites were predicted using GlycoEP (Chauhan et al., 2013). References Chauhan, J. S., Rao, A., and Raghava, G. P. S. (2013). In silico platform for prediction of N-, O- and C-glycosites in eukaryotic protein sequences. PLoS ONE 8:e67008. doi: 10.1371/journal.pone.0067008 Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. doi: 10.1016/S0022- 2836(05)80360-2 Chu, X., and Wang, J. (2014). Speciﬁcity and aﬃnity quantiﬁcation of ﬂexible recognition from underlying energy landscape topography. PLoS Comput. Biol. 10:e1003782. doi: 10.1371/journal.pcbi.1003782 Amselem, J., Cuomo, C. A., van Kan, J. A. L., Viaud, M., Benito, E. P., Couloux, A., et al. (2011). Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet. 7:e1002230. doi: 10.1371/journal.pgen.1002230 Dassarma, S., Capes, M. D., Karan, R., and Dassarma, P. (2013). Amino acid substitutions in cold-adapted proteins from halorubrum lacusprofundi, an extremely halophilic microbe from antarctica. PLoS ONE 8:e58587. doi: 10.1371/journal.pone.0058587 Arnold, R., Brandmaier, S., Kleine, F., Tischler, P., Heinz, E., Behrens, S., et al. (2009). Sequence-based prediction of type III secreted proteins. PLoS Pathog. 5:e1000376. doi: 10.1371/journal.ppat.1000376 Dean, R., Van Kan, J. A. L., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., et al. (2012). The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414–430. doi: 10.1111/j.1364- 3703.2011.00783.x Basu, K., Graham, L. A., Campbell, R. L., and Davies, P. L. (2015). Flies expand the repertoire of protein structures that bind ice. Proc. Natl. Acad. Sci. U.S.A. 112, 737–742. doi: 10.1073/pnas.1422272112 Farr, D. F., and Rossman, A. Y. (2015). Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Available online at: http://nt.ars- grin.gov/fungaldatabases/ (Retrieved March 31, 2015). Bolton, M. D., Thomma, B. P. H. J., and Nelson, B. D. (2006). Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Mol. Plant Pathol. 7, 1–16. doi: 10.1111/j.1364-3703.2005.00316.x grin.gov/fungaldatabases/ (Retrieved March 31, 2015). Feller, G. (2003). Molecular adaptations to cold in psychrophilic enzymes. Cell. Mol. Life Sci. 60, 648–662. doi: 10.1007/s00018-003-2155-3 Burra, P. V., Kalmar, L., and Tompa, P. (2010). Reduction in structural disorder and functional complexity in the thermal adaptation of prokaryotes. PLoS ONE 5:e12069. doi: 10.1371/journal.pone.0012069 Feller, G. (2007). Life at low temperatures: is disorder the driving force? Extremophiles 11, 211–216. doi: 10.1007/s00792-006-0050-1 Casanueva, A., Tuﬃn, M., Cary, C., and Cowan, D. A. (2010). Molecular adaptations to psychrophily: the impact of “omic” technologies. Trends Microbiol. 18, 374–381. Author Contributions TB, RP, and SR designed and performed analyses. SR conceived the study. TB, RP, and SR wrote the manuscript. Figure S3 \| Distribution of sTEKhot values for non-secreted proteins and mature secreted proteins (signal peptide removed) in S. borealis, S. sclerotiorum and B. cinerea. Statistical Analysis and sTEKhot Index Determination All statistical tests were computed with R.Studio software. Wilcoxon test was used for signiﬁcance analysis. Diﬀerence was considered signiﬁcant for p-values inferior to 0.05. Signiﬁcantly September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 14 Protein signatures in Sclerotiniaceae fungi Badet et al. BBRIC computational facilities for providing bioinformatics tools. BBRIC computational facilities for providing bioinformatics tools. (Invitrogen), 1 µg of oligo(dT), and 10 nmol of dNTP. cDNAs (diluted 1:10) were used as templates in the quantitative RT- PCR analysis. Quantitative RT-PCR was performed using gene- speciﬁc primers (Table S6) with LightCycler 480 apparatus (Roche Diagnostics). Quantitative PCR reaction was performed using the SYBR GREEN I protocol (5 pmol of each primer and 5 µL of RT reaction product in a 7 µL ﬁnal reaction volume). The PCR conditions were 9 min at 95◦C, followed by 45 cycles of 5 s at 95◦C, 10 s at 65◦C, and 20 s at 72◦C. Expression values of SS1G_10836 and SS1G_03146 were normalized based on expression of SS1G_04652 and SS1G_12196 housekeeping genes. Values from two biological replicates are shown, error bars show standard error of the mean. (Invitrogen), 1 µg of oligo(dT), and 10 nmol of dNTP. cDNAs (diluted 1:10) were used as templates in the quantitative RT- PCR analysis. Quantitative RT-PCR was performed using gene- speciﬁc primers (Table S6) with LightCycler 480 apparatus (Roche Diagnostics). Quantitative PCR reaction was performed using the SYBR GREEN I protocol (5 pmol of each primer and 5 µL of RT reaction product in a 7 µL ﬁnal reaction volume). The PCR conditions were 9 min at 95◦C, followed by 45 cycles of 5 s at 95◦C, 10 s at 65◦C, and 20 s at 72◦C. Expression values of SS1G_10836 and SS1G_03146 were normalized based on expression of SS1G_04652 and SS1G_12196 housekeeping genes. Values from two biological replicates are shown, error bars show standard error of the mean. Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015. 00776 Figure S1 \| Network representation of gene ontologies (GOs) of proteins with sTEKhot >1 in S. sclerotiorum proteome. Nodes correspond to GOs are sized according to the number of proteins with sTEKhot >1. They are colored from yellow to orange according to the p-value of a hypergeometric test for enrichment in proteins with sTEKhot >1 compared to whole proteomes. Figure S2 \| Network representation of gene ontologies (GOs) of proteins with sTEKhot >1 in B. cinerea proteome. Nodes correspond to GOs are sized according to the number of proteins with sTEKhot >1. They are colored from yellow to orange according to the p-value of a hypergeometric test for enrichment in proteins with sTEKhot >1 compared to whole proteomes. Acknowledgments Figure S4 \| Distribution of best BlastP bit scores (log-scaled scores) using S. borealis non-secreted proteins and secreted proteins as queries against S. sclerotiorum or B. cinerea proteomes. Lower scores for searches using S. borealis secretome as query indicate that S. borealis secreted proteins are less conserved than non-secreted proteins. P-values of a Student t-test for differences between non-secreted and secreted proteins are indicated. Figure S4 \| Distribution of best BlastP bit scores (log-scaled scores) using S. borealis non-secreted proteins and secreted proteins as queries This work was supported by a Starting Grant of the European Research Council (ERC-StG 336808 project VariWhim) and a Marie Curie grant (MC-CIG 334036 project SEPAraTE) to SR and the French Laboratory of Excellence project TULIP (ANR-10-LABX-41; ANR-11-IDEX-0002-02). We thank the against S. sclerotiorum or B. cinerea proteomes. Lower scores for searches using S. borealis secretome as query indicate that S. borealis secreted proteins are less conserved than non-secreted proteins. P-values of a Student t-test for differences between non-secreted and secreted proteins are indicated. References doi: 10.1105/tpc.113.116319 He, X., Han, K., Hu, J., Yan, H., Yang, J.-Y., Shen, H.-B., et al. (2015). TargetFreeze: identifying antifreeze proteins via a combination of weights using sequence evolutionary information and pseudo amino acid composition. J. Membr. Biol. doi: 10.1007/s00232-015-9811-z. [Epub ahead of print]. Mayans, O., Scott, M., Connerton, I., Gravesen, T., Benen, J., Visser, J., et al. (1997). Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases. Structure 5, 677–689. doi: 10.1016/S0969- 2126(97)00222-0 Heard, S., Brown, N. A., and Hammond-Kosack, K. (2015). An interspecies comparative analysis of the predicted secretomes of the necrotrophic plant pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS One 10:e0130534. doi: 10.1371/journal.pone.0130534 Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014). Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol. 10, 122–126. doi: 10.1038/nchembio.1417 McDermott, J. E., Corrigan, A., Peterson, E., Oehmen, C., Niemann, G., Cambronne, E. D., et al. (2011). Computational prediction of type III and IV secreted eﬀectors in gram-negative bacteria. Infect. Immun. 79, 23–32. doi: 10.1128/IAI.00537-10 Horton, P., Park, K.-J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C. J., et al. (2007). WoLF PSORT: protein localization predictor. Nucleic Acids Res. 35, W585–W587. doi: 10.1093/nar/gkm259 Methé, B. A., Nelson, K. E., Deming, J. W., Momen, B., Melamud, E., Zhang, X., et al. (2005). The psychrophilic lifestyle as revealed by the genome sequence of Colwellia psychrerythraea 34H through genomic and proteomic analyses. Proc. Natl. Acad. Sci. U.S.A. 102, 10913–10918. doi: 10.1073/pnas.0504766102 Hoshino, T., Terami, F., Tkachenko, O. B., Tojo, M., and Matsumoto, N. (2010). Mycelial growth of the snow mold fungus, Sclerotinia borealis, improved at low water potentials: an adaption to frozen environment. Mycoscience 51, 98–103. doi: 10.1007/S10267-009-0013-3 l. Acad. Sci. U.S.A. 102, 10913–10918. doi: 10.1073/pnas.05047661 Metpally, R. P. R., and Reddy, B. V. B. (2009). Comparative proteome analysis of psychrophilic versus mesophilic bacterial species: insights into the molecular basis of cold adaptation of proteins. BMC Genomics 10:11. doi: 10.1186/1471- 2164-10-11 Hu, X., Xiao, G., Zheng, P., Shang, Y., Su, Y., Zhang, X., et al. (2014). Trajectory and genomic determinants of fungal-pathogen speciation and host adaptation. Proc. Natl. Acad. Sci. U.S.A. 111, 16796–16801. doi: 10.1073/pnas.1412662111 Meyer, D. F., Noroy, C., Moumène, A., Raﬀaele, S., Albina, E., and Vachiéry, N. (2013). References doi: 10.1016/j.tim.2010.05.002 Frías, M., González, C., and Brito, N. (2011). BcSpl1, a cerato-platanin family protein, contributes to Botrytis cinerea virulence and elicits the hypersensitive response in the host. New Phytol. 192, 483–495. doi: 10.1111/j.1469- 8137.2011.03802.x Catanzariti, A.-M., Dodds, P. N., Lawrence, G. J., Ayliﬀe, M. A., and Ellis, J. G. (2006). Haustorially expressed secreted proteins from ﬂax rust are highly enriched for avirulence elicitors. Plant Cell 18, 243–256. doi: 10.1105/tpc.105.035980 Gianese, G., Argos, P., and Pascarella, S. (2001). Structural adaptation of enzymes to low temperatures. Protein Eng. 14, 141–148. doi: 10.1093/protein/14.3.141 Goodchild, A., Saunders, N. F. W., Ertan, H., Raftery, M., Guilhaus, M., Curmi, P. M. G., et al. (2004). A proteomic determination of cold adaptation in the Antarctic archaeon, Methanococcoides burtonii. Mol. Microbiol. 53, 309–321. doi: 10.1111/j.1365-2958.2004.04130.x Cavicchioli, R., Amils, R., Wagner, D., and McGenity, T. (2011). Life and applications of extremophiles. Environ. Microbiol. 13, 1903–1907. doi: 10.1111/j.1462-2920.2011.02512.x Govrin, E. M., and Levine, A. (2000). The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr. Biol. 10, 751–757. Ceroni, A., Passerini, A., Vullo, A., and Frasconi, P. (2006). DISULFIND: a disulﬁde bonding state and cysteine connectivity prediction server. Nucleic Acids Res. 34, W177–W181. doi: 10.1093/nar/gkl266 September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 15 Protein signatures in Sclerotiniaceae fungi Badet et al. Guyon, K., Balagué, C., Roby, D., and Raﬀaele, S. (2014). Secretome analysis reveals eﬀector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum. BMC Genomics 15:336. doi: 10.1186/1471-2164-15-336 Mardanov, A. V., Beletsky, A. V., Kadnikov, V. V., Ignatov, A. N., and Ravin, N. V. (2014a). Draft genome sequence of Sclerotinia borealis, a psychrophilic plant pathogenic fungus. Genome Announc. 2:e01175-13. doi: 10.1128/genomea.01175-13 Hacquard, S., Joly, D. L., Lin, Y.-C., Tisserant, E., Feau, N., Delaruelle, C., et al. (2012). A comprehensive analysis of genes encoding small secreted proteins identiﬁes candidate eﬀectors in Melampsora larici-populina (poplar leaf rust). Mol. Plant. Microbe. Interact. 25, 279–293. doi: 10.1094/MPMI-09-11-0238 Mardanov, A. V., Beletsky, A. V., Kadnikov, V. V., Ignatov, A. N., and Ravin, N. V. (2014b). The 203 kbp mitochondrial genome of the phytopathogenic fungus Sclerotinia borealis reveals multiple invasions of introns and genomic duplications. PLoS ONE 9:e107536. doi: 10.1371/journal.pone.0107536 Marín, M., Uversky, V. N., and Ott, T. (2013). Intrinsic disorder in pathogen eﬀectors: protein ﬂexibility as an evolutionary hallmark in a molecular arms race. Plant Cell 25, 3153–3157. References Searching algorithm for type IV secretion system eﬀectors 1.0: a tool for predicting type IV eﬀectors and exploring their genomic context. Nucleic Acids Res. 41, 9218–9229. doi: 10.1093/nar/gkt718 Jones, J. D. G., and Dangl, J. L. (2006). The plant immune system. Nature 444, 323–329. doi: 10.1038/nature05286 Jones, P., Binns, D., Chang, H.-Y., Fraser, M., Li, W., McAnulla, C., et al. (2014). InterProScan 5: genome-scale protein function classiﬁcation. Bioinformatics 30, 1236–1240. doi: 10.1093/bioinformatics/btu031 Möbius, N., and Hertweck, C. (2009). Fungal phytotoxins as mediators of virulence. Curr. Opin. Plant Biol. 12, 390–398. doi: 10.1016/j.pbi.2009.06.004 Oliva, R. F., Cano, L. M., Raﬀaele, S., Win, J., Bozkurt, T. O., Belhaj, K., et al. (2015). A recent expansion of the RXLR eﬀector gene Avrblb2 is maintained in global populations of Phytophthora infestans indicating diﬀerent contributions to virulence. Mol. Plant. Microbe. Interact. 28, 901–912. doi: 10.1094/MPMI-12- 14-0393-R Judet-Correia, D., Bollaert, S., Duquenne, A., Charpentier, C., Bensoussan, M., and Dantigny, P. (2010). Validation of a predictive model for the growth of Botrytis cinerea and Penicillium expansum on grape berries. Int. J. Food Microbiol. 142, 106–113. doi: 10.1016/j.ijfoodmicro.2010.06.009 Juenger, T., and Bergelson, J. (1998). Pairwise versus diﬀuse natural selection and the multiple herbivores of scarlet gilia, Ipomopsis aggregata. Evolution 52, 1583–1592. doi: 10.2307/2411332 Ostlund, G., Schmitt, T., Forslund, K., Köstler, T., Messina, D. N., Roopra, S., et al. (2010). InParanoid 7: new algorithms and tools for eukaryotic orthology analysis. Nucleic Acids Res. 38, D196–D203. doi: 10.1093/nar/gkp931 Julenius, K., and Pedersen, A. G. (2006). Protein evolution is faster outside the cell. Mol. Biol. Evol. 23, 2039–2048. doi: 10.1093/molbev/msl081 Persoons, A., Morin, E., Delaruelle, C., Payen, T., Halkett, F., Frey, P., et al. (2014). Patterns of genomic variation in the poplar rust fungus Melampsora larici-populina identify pathogenesis-related factors. Front. Plant Sci. 5:450. doi: 10.3389/fpls.2014.00450 Kondo, H., Hanada, Y., Sugimoto, H., Hoshino, T., Garnham, C. P., Davies, P. L., et al. (2012). Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation. Proc. Natl. Acad. Sci. U.S.A. 109, 9360–9365. doi: 10.1073/pnas.1121607109 Poisson, G., Chauve, C., Chen, X., and Bergeron, A. (2007). FragAnchor: a large-scale predictor of glycosylphosphatidylinositol anchors in eukaryote protein sequences by qualitative scoring. Genomics Proteomics Bioinformatics 5, 121–130. doi: 10.1016/S1672-0229(07)60022-9 Kubicek, C. P., Starr, T. L., and Glass, N. L. (2014). Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Annu. Rev. Phytopathol. 52, 427–451. doi: 10.1146/annurev-phyto-102313-045831 Kuchner, O., and Arnold, F. H. References doi: 10.1371/journal.pgen.1003323 Wang, G.-Z., and Lercher, M. J. (2010). Amino acid composition in endothermic vertebrates is biased in the same direction as in thermophilic prokaryotes. BMC Evol. Biol. 10:263. doi: 10.1186/1471-2148-10-263 Wang, Y., Wei, X., Bao, H., and Liu, S.-L. (2014). Prediction of bacterial type IV secreted eﬀectors by C-terminal features. BMC Genomics 15:50. doi: 10.1186/1471-2164-15-50 Schechter, L. M., Valenta, J. C., Schneider, D. J., Collmer, A., and Sakk, E. (2012). Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae. PLoS ONE 7:e36038. doi: 10.1371/journal.pone.0036038 Weiberg, A., Wang, M., Lin, F.-M., Zhao, H., Zhang, Z., Kaloshian, I., et al. (2013). Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science 342, 118–123. doi: 10.1126/science. 1239705 Schneider, E. F., and Seaman, W. L. (1987). Snow mold diseases and their distribution on winter wheat in Ontario in 1982-84. Can. Plant Dis. Surv. 67, 35–39. Wicker, T., Oberhaensli, S., Parlange, F., Buchmann, J. P., Shatalina, M., Roﬄer, S., et al. (2013). The wheat powdery mildew genome shows the unique evolution of an obligate biotroph. Nat. Genet. 45, 1092–1096. doi: 10.1038/ng.2704 Schornack, S., Huitema, E., Cano, L. M., Bozkurt, T. O., Oliva, R., Van Damme, M., et al. (2009). Ten things to know about oomycete eﬀectors. Mol. Plant Pathol. 10, 795–803. doi: 10.1111/j.1364-3703.2009.00593.x Wu, B. M., Subbarao, K. V., and Qin, Q. M. (2008). Nonlinear colony extension of Sclerotinia minor and S. sclerotiorum. Mycologia 100, 902–910. doi: 10.3852/ 08-021 Smith, D. R., and Chapman, M. R. (2010). Economical evolution: microbes reduce the synthetic cost of extracellular proteins. MBio 1, 28–32. doi: 10.1128/mBio.00131-10 Xiao, Z., Bergeron, H., Grosse, S., Beauchemin, M., Garron, M.-L., Shaya, D., et al. (2008). Improvement of the thermostability and activity of a pectate lyase by single amino acid substitutions, using a strategy based on melting-temperature- guided sequence alignment. Appl. Environ. Microbiol. 74, 1183–1189. doi: 10.1128/AEM.02220-07 Sonnhammer, E. L., von Heijne, G., and Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol. 6, 175–182. Sperschneider, J., Dodds, P. N., Gardiner, D. M., Manners, J. M., Singh, K. B., and Taylor, J. M. (2015). Advances and challenges in computational prediction of eﬀectors from plant pathogenic fungi. PLoS Pathog. 11:e1004806. doi: 10.1371/journal.ppat.1004806 Zeldovich, K. B., Berezovsky, I. N., and Shakhnovich, E. I. (2007). References (1997). Directed evolution of enzyme catalysts. Trends Biotechnol. 15, 523–530. doi: 10.1016/S0167-7799(97)01138-4 Raﬀaele, S., Farrer, R. A., Cano, L. M., Studholme, D. J., MacLean, D., Thines, M., et al. (2010). Genome evolution following host jumps in the Irish potato famine pathogen lineage. Science 330, 1540–1543. doi: 10.1126/science.1193070 Lebbink, J. H., Kaper, T., Bron, P., van der Oost, J., and de Vos, W. M. (2000). Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution. Biochemistry 39, 3656–3665. doi: 10.1021/bi991483q Rech, G. E., Sanz-Martín, J. M., Anisimova, M., Sukno, S. A., and Thon, M. R. (2014). Natural selection on coding and noncoding DNA sequences is associated with virulence genes in a plant pathogenic fungus. Genome Biol. Evol. 6, 2368–2379. doi: 10.1093/gbe/evu192 Liao, B.-Y., Weng, M.-P., and Zhang, J. (2010). Impact of extracellularity on the evolutionary rate of mammalian proteins. Genome Biol. Evol. 2, 39–43. doi: 10.1093/gbe/evp058 Remm, M., Storm, C. E., and Sonnhammer, E. L. (2001). Automatic clustering of orthologs and in-paralogs from pairwise species comparisons. J. Mol. Biol. 314, 1041–1052. doi: 10.1006/jmbi.2000.5197 Linding, R., Jensen, L. J., Diella, F., Bork, P., Gibson, T. J., and Russell, R. B. (2003). Protein disorder prediction: implications for structural proteomics. Structure 11, 1453–1459. doi: 10.1016/j.str.2003.10.002 Roux, F., Voisin, D., Badet, T., Balagué, C., Barlet, X., Huard-Chauveau, C., et al. (2014). Resistance to phytopathogens e tutti quanti: placing plant quantitative disease resistance on the map. Mol. Plant Pathol. 15, 427–432. doi: 10.1111/mpp.12138 Liu, T., Song, T., Zhang, X., Yuan, H., Su, L., Li, W., et al. (2014). Unconventionally secreted eﬀectors of two ﬁlamentous pathogens target plant salicylate biosynthesis. Nat. Commun. 5, 4686. doi: 10.1038/ncomms5686 Saunders, D. G. O., Win, J., Cano, L. M., Szabo, L. J., Kamoun, S., and Raﬀaele, S. (2012). Using hierarchical clustering of secreted protein families to classify and rank candidate eﬀectors of rust fungi. PLoS ONE 7:e29847. doi: 10.1371/journal.pone.0029847 Maere, S., Heymans, K., and Kuiper, M. (2005). BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21, 3448–3449. doi: 10.1093/bioinformatics/bti551 September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 16 Badet et al. Protein signatures in Sclerotiniaceae fungi Schardl, C. L., Young, C. A., Hesse, U., Amyotte, S. G., Andreeva, K., Calie, P. J., et al. (2013). Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci. PLoS Genet. 9:e1003323. September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org References Protein and DNA sequence determinants of thermophilic adaptation. PLoS Comput. Biol. 3:e5. doi: 10.1371/journal.pcbi.0030005 Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40. doi: 10.1186/1471-2105-9-40 Sperschneider, J., Gardiner, D. M., Taylor, J. M., Hane, J. K., Singh, K. B., and Manners, J. M. (2013). A comparative hidden Markov model analysis pipeline identiﬁes proteins characteristic of cereal-infecting fungi. BMC Genomics 14:807. doi: 10.1186/1471-2164-14-807 Zhou, H.-X. (2012). Intrinsic disorder: signaling via highly speciﬁc but short-lived association. Trends Biochem. Sci. 37, 43–48. doi: 10.1016/j.tibs.2011.11.002 Sperschneider, J., Ying, H., Dodds, P. N., Gardiner, D. M., Upadhyaya, N. M., Singh, K. B., et al. (2014). Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate eﬀectors. Front. Plant Sci. 5:372. doi: 10.3389/fpls.2014.00372 Zou, L., Nan, C., and Hu, F. (2013). Accurate prediction of bacterial type IV secreted eﬀectors using amino acid composition and PSSM proﬁles. Bioinformatics 29, 3135–3142. doi: 10.1093/bioinformatics/btt554 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Suhre, K., and Claverie, J.-M. (2003). Genomic correlates of hyperthermostability, an update. J. Biol. Chem. 278, 17198–17202. doi: 10.1074/jbc.M301327200 Sun, T., Lin, F.-H., Campbell, R. L., Allingham, J. S., and Davies, P. L. (2014). An antifreeze protein folds with an interior network of more than 400 semi- clathrate waters. Science 343, 795–798. doi: 10.1126/science.1247407 Copyright © 2015 Badet, Peyraud and Raﬀaele. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Tantos, A., Friedrich, P., and Tompa, P. (2009). Cold stability of intrinsically disordered proteins. FEBS Lett. 583, 465–469. doi: 10.1016/j.febslet.2008.12.054 Van Noort, V., Bradatsch, B., Arumugam, M., Amlacher, S., Bange, G., Creevey, C., et al. (2013). Consistent mutational paths predict eukaryotic thermostability. BMC Evol. Biol. 13:7. doi: 10.1186/1471-2148-13-7 September 2015 \| Volume 6 \| Article 776 Frontiers in Plant Science \| www.frontiersin.org 17
https://openalex.org/W4214696292	https://www.research-collection.ethz.ch/bitstream/20.500.11850/592039/3/s41095-022-0268-6.pdf	English	null	Specificity-preserving RGB-D Saliency Detection	2021 IEEE/CVF International Conference on Computer Vision (ICCV)	2,021	cc-by	15,498	ETH Library ETH Library Journal Article Author(s): Zhou, Tao; Fan, Deng-Ping; Chen, Geng; Zhou, Yi; Fu, Huazhu Speciﬁcity-preserving RGB-D saliency detection Tao Zhou1,2, Deng-Ping Fan3 (), Geng Chen4, Yi Zhou5, and Huazhu Fu6 c⃝The Author(s) 2022. c⃝The Author(s) 2022. c⃝The Author(s) 2022. Abstract Salient object detection (SOD) in RGB and depth images has attracted increasing research interest. Existing RGB-D SOD models usually adopt fusion strategies to learn a shared representation from RGB and depth modalities, while few methods explicitly consider how to preserve modality-speciﬁc characteristics. In this study, we propose a novel framework, the speciﬁcity-preserving network (SPNet), which improves SOD performance by exploring both the shared information and modality-speciﬁc properties. Speciﬁcally, we use two modality-speciﬁc networks and a shared learning network to generate individual and shared saliency prediction maps. To eﬀectively fuse cross-modal features in the shared learning network, we propose a cross-enhanced integration module (CIM) and propagate the fused feature to the next layer to integrate cross-level information. Moreover, to capture rich complementary multi-modal information to boost SOD performance, we use a multi-modal feature aggregation (MFA) module to integrate the modality- speciﬁc features from each individual decoder into the shared decoder. By using skip connections between encoder and decoder layers, hierarchical features can be fully combined. Extensive experiments demonstrate that our SPNet outperforms cutting-edge approaches on six popular RGB-D SOD and three camouﬂaged object detection benchmarks. The project is publicly available at https://github.com/taozh2017/SPNet. Keywords salient object detection (SOD); RGB-D; cross-enhanced integration module (CIM); multi-modal feature aggregation (MFA) 1 School of Computer Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094, China. E-mail: taozhou.ai@gmail.com. 2 Key Laboratory of System Control and Information Processing, Ministry of Education, Shanghai, China. 3 Computer Vision Lab, ETH Z¨urich, Z¨urich, Switzerland. E-mail: dengpfan@gmail.com, dengpingfan@mail.nankai.edu.cn (). 4 School of Computer Science and Engineering, North- western Polytechnical University, Xi’an, China. E-mail: geng.chen.cs@gmail.com. 5 School of Computer Science and Engineering, Southeast University, Nanjing, China. E-mail: yizhou.szcn@gmail.com. 6 Inception Institute of Artiﬁcial Intelligence, Abu Dhabi, United Arab Emirates. E-mail: hzfu@ieee.org. Manuscript received: 2021-10-19; accepted: 2022-01-01 5 School of Computer Science and Engineering, Southeast University, Nanjing, China. E-mail: yizhou.szcn@gmail.com. 6 Inception Institute of Artiﬁcial Intelligence, Abu Dhabi, United Arab Emirates. E-mail: hzfu@ieee.org. Manuscript received: 2021-10-19; accepted: 2022-01-01 1 School of Computer Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094, China. E-mail: taozhou.ai@gmail.com. Originally published in: Originally published in: Computational Visual Media 9(2), https://doi.org/10.1007/s41095-022-0268-6 Computational Visual Media 9(2), https://doi.org/10.1007/s41095-022-0268-6 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Computational Visual Media https://doi.org/10.1007/s41095-022-0268-6 Vol. 9, No. 2, June 2023, 297–317 Research Article Research Article 4 School of Computer Science and Engineering, North- western Polytechnical University, Xi’an, China. E-mail: geng.chen.cs@gmail.com. 2 Key Laboratory of System Control and Information Processing, Ministry of Education, Shanghai, China. 3 Computer Vision Lab, ETH Z¨urich, Z¨urich, Switzerland. E-mail: dengpfan@gmail.com, dengpingfan@mail.nankai.edu.cn (). 1 Introduction Salient object detection (SOD, also called saliency detection) aims to emulate the mechanisms of human visual attention and locate the most visually distinctive object(s) in a given scene [1]. SOD has been widely applied in various vision-related tasks, such as image understanding [2], action recognition [3, 4], video/semantic segmentation [4, 5], and person re-identiﬁcation [6]. Although signiﬁcant progress has been made, it is still challenging to accurately locate salient objects in many challenging scenarios, such as images with cluttered backgrounds, low-contrast lighting conditions, and salient object(s) having a similar appearance to the background. Recently, with the ready availability of depth sensors in smart devices, depth maps have been introduced to provide geometric and spatial information to improve SOD performance. Consequently, fusing RGB and depth images has gained increasing interest in the SOD community [7–15], and it is a challenging task to adaptively fuse RGB and depth modalities. Over past years, various RGB-D SOD methods have been proposed; they often focus on how to eﬀectively fuse RGB and depth images. Existing fusion strategies can be divided into categories using early fusion, late fusion, and intermediate fusion. The early fusion strategy often adopts a simple concatenation to integrate the two modalities. For example, methods in Refs. [1, 16–18] directly integrate RGB and depth images to form four- T. Zhou, D.-P. Fan, G. Chen, et al. 298 channel input. However, this type of fusion does not consider the distribution gap between the two modalities, which could result in an inaccurate feature fusion. The late fusion strategy uses two parallel network streams to generate independent saliency maps for RGB and depth data, which are fused to obtain a ﬁnal prediction map [19–21]. However, it is still challenging to capture the complex interactions between the two modalities. methods focus on learning shared representations by fusing them and then use a decoder to generate the ﬁnal saliency map. Furthermore, there is no supervised decoder to guide the depth-based feature learning [30, 31], which may prevent optimal depth features from being obtained. From a multi-modal learning perspective, several works [34–37] have shown that exploring both the shared information and modality-speciﬁc characteristics can improve model performance. However, in the RGB- D SOD community, few methods explicitly exploit modality-speciﬁc characteristics. 1 Introduction Recent research mainly focuses on intermediate fusion, which utilizes two independent networks to learn intermediate features of the two modalities separately, and then the fused features are fed into a subsequent network or decoder (see Fig. 1(a)). Other methods carry out cross-modal fusion at multiple scales [22–28]. As a result, complex correlations can be eﬀectively exploited from the two modalities. Further methods utilize depth infor- mation to enhance RGB features via an a auxiliary subnetwork [29–31] (see Fig. 1(b)). For example, Zhao et al. [30] introduced a contrast prior into a CNN-based architecture to enhance the depth information, which was then integrated with RGB features using a ﬂuid pyramid integration module. Zhu et al. [31] utilized an independent subnetwork to extract depth-based features, which were then incorporated into the RGB network. The above Thus, in this paper, we propose a novel RGB-D SOD framework, the speciﬁcity-preserving network (SPNet), which can eﬀectively explore the shared information as well as capture modality-speciﬁc characteristics to improve the SOD performance. Two encoder subnetworks are used to extract multi-scale features for the two modalities (i.e., RGB and depth), and a cross-enhanced integration module (CIM) is proposed to integrate cross-modal features in diﬀerent feature layers. Then, we use a simple U-Net [38] structure to construct a modality-speciﬁc decoder, in which skip connections between the encoder and decoder layers are used to combine hierarchical features. In this way, we can learn powerful modality- speciﬁc features in each independent decoder, which Fig. 1 Comparison of two existing RGB-D salient object detection frameworks and our proposed model. (a) RGB and depth images are fed into two independent network streams, and then fused high-level features are fed into a decoder to predict saliency maps (e.g., Refs. [22–25]). (b) Depth features are integrated into the RGB network using an auxiliary subnetwork (e.g., Refs. [29–33]). (c) Our method adopts two modality-speciﬁc networks and a shared learning network to explicitly explore modality-speciﬁc characteristics and shared information. Features learned from the modality-speciﬁc decoders are integrated into the shared decoder to boost SOD performance. Fig. 1 Comparison of two existing RGB-D salient object detection frameworks and our proposed model. (a) RGB and depth images are fed into two independent network streams, and then fused high-level features are fed into a decoder to predict saliency maps (e.g., Refs. [22–25]). (b) Depth features are integrated into the RGB network using an auxiliary subnetwork (e.g., Refs. [29–33]). 2.1 RGB salient object detection Early salient object detection methods were based on hand-crafted features and various saliency priors, such as a background prior [40], color contrast [41], a compactness prior [42], and a center prior [43]. However, the generalizability and eﬀectiveness of these traditional methods are limited. With the breakthrough of deep learning in the ﬁeld of computer vision, various deep learning-based salient object detection methods have been developed with promising results. For example, Hou et al. [44] proposed a novel salient object detection method by introducing short connections to the skip-layer structures within the holistically-nested edge detector architecture. Wang et al. [45] proposed a recurrent fully convolutional network framework for salient object detection with promising results. Liu et al. [46] proposed to hierarchically embed global and local context modules into the top–down pathway, which can generate attention over context regions for each pixel. Deng et al. [47] proposed a recurrent residual reﬁnement network with residual reﬁnement blocks to accurately detect salient objects. Further methods can be found in a survey [48]. Scale variation is a key challenge in the SOD task, so several methods have been proposed to integrate multi-level or scale features [49–52] to improve SOD results. In our method, we consider how to eﬀectively combine cross- modal (RGB and depth) features, and how multi-level information can be exploited via a cross-enhanced integration module. • A cross-enhanced integration module (CIM) to integrate cross-modal features and learn shared representations for the two modalities. The output of each CIM is propagated to the next layer to explore rich cross-level information. • An eﬀective multi-modal feature aggregation (MFA) module to integrate learned modality- speciﬁc features. It allows our model to make full use of the features learned in the modality-speciﬁc decoder to improve salient object detection. • Extensive experiments on six public RGB-D SOD and three camouﬂaged object detection (COD) datasets demonstrate the superiority of our model over other cutting-edge methods. Moreover, we carry out an attribute-based evaluation on various state-of-the-art RGB-D SOD methods under varying conditions (e.g., number of salient objects, indoors or outdoors, lighting, and object scale), which has not been done previously. This paper signiﬁcantly extends our previous work in Ref. [39], as follows: • We discuss diﬀerences between (i) our proposed CIM and existing fusion strategies, and (ii) the proposed CIM and MFA. 1 Introduction (c) Our method adopts two modality-speciﬁc networks and a shared learning network to explicitly explore modality-speciﬁc characteristics and shared information. Features learned from the modality-speciﬁc decoders are integrated into the shared decoder to boost SOD performance. Fig. 1 Comparison of two existing RGB-D salient object detection frameworks and our proposed model. (a) RGB and depth images are fed into two independent network streams, and then fused high-level features are fed into a decoder to predict saliency maps (e.g., Refs. [22–25]). (b) Depth features are integrated into the RGB network using an auxiliary subnetwork (e.g., Refs. [29–33]). (c) Our method adopts two modality-speciﬁc networks and a shared learning network to explicitly explore modality-speciﬁc characteristics and shared information. Features learned from the modality-speciﬁc decoders are integrated into the shared decoder to boost SOD performance. Fig. 1 Comparison of two existing RGB-D salient object detection frameworks and our proposed model. (a) RGB and depth images are fed into two independent network streams, and then fused high-level features are fed into a decoder to predict saliency maps (e.g., Refs. [22–25]). (b) Depth features are integrated into the RGB network using an auxiliary subnetwork (e.g., Refs. [29–33]). (c) Our method adopts two modality-speciﬁc networks and a shared learning network to explicitly explore modality-speciﬁc characteristics and shared information. Features learned from the modality-speciﬁc decoders are integrated into the shared decoder to boost SOD performance. Speciﬁcity-preserving RGB-D saliency detection 299 eﬀectiveness of the shared decoder, and to examine the eﬀects of diﬀerent numbers of CIMs. We also show that our model can eﬀectively handle variations in object scale. also captures modality-speciﬁc characteristics to provide cross-modal complementarity. Further, we construct a shared decoder to combine hierarchical features from outputs of the previous CIM via a skip connection. To make full use of the modality- speciﬁc features, a multi-modal feature aggregation module (MFA) is proposed to integrate them into the shared decoder. Finally, we formulate a uniﬁed and end-to-end trainable framework where shared and modality-speciﬁc information are simultaneously exploited to boost SOD performance. • We apply SPNet to a new RGB-D task: COD, and demonstrate its superiority over existing methods. • We apply SPNet to a new RGB-D task: COD, and demonstrate its superiority over existing methods. 2 Related work In this section, we review three types of works most related to the proposed model, i.e., RGB salient object detection, RGB-D salient object detection, and multi- modal learning. The main contributions of our paper in summary are: • A novel RGB-D salient object detection frame- work, the speciﬁcity-preserving network (SPNet), which explores shared information from RGB and depth images as well as preserving modality- speciﬁc characteristics. 2.1 RGB salient object detection • We provide further details, including (i) a review of existing RGB SOD methods, (ii) a discussion of the importance of integrating multi-level/scale features, and (iii) better characterisation of our evaluation metrics. 2.2 RGB-D salient object detection Early RGB-D based SOD methods often extracted hand-crafted features from the input RGB-D data. For example, Lang et al. [53] in the ﬁrst RGB-D SOD • We provide an additional ablation study and attribute-based evaluation, to validate the T. Zhou, D.-P. Fan, G. Chen, et al. 300 work utilized Gaussian mixture models to model the distribution of depth-induced saliency. Subsequently, several methods explored diﬀerent principles, such as center-surround diﬀerence [19, 54], contrast [1, 16, 55], a center/boundary prior [56, 57], and background enclosure [58]. However, these methods typically provide poor results due to the limited expressivity of handcrafted features. Beneﬁting from the rapid development of deep convolutional neural networks (CNNs), several deep learning-based works [7, 12, 30, 59, 60] have recently obtained promising results. For example, Qu et al. [59] used a CNN model to fuse saliency cues from diﬀerent low levels into hierarchical features to boost SOD abilities. Chen and Li [22] proposed a complementarity-aware fusion module to eﬀectively integrate cross-modal and cross-level features for RGB and depth modalities. Piao et al. [60] proposed a depth-induced multi- scale recurrent attention network to enhance cross- modality feature fusion. Fan et al. [7] designed a depth puriﬁcation unit to remove some low-quality depth maps. Most other models [23–26, 61, 62] employ cross-modal fusion at multiple scales using diﬀerent integration strategies. methods have been developed to explicitly fuse the complementary information from diﬀerent modalities to improve model results. These popular methods can be divided into three types: (i) co-training [63, 64] tries to minimize the disagreement between diﬀerent modalities, (ii) multiple kernel learning [65] utilizes a predeﬁned set of kernels for multiple modalities and integrates these modalities using the learned kernel weights, and (iii) subspace learning [66, 67] assumes that a latent subspace exists shared by diﬀerent modalities, with one underlying latent representation. To eﬀectively fuse multi-modal data, several deep learning-based models have also been explored. For example, Ngiam et al. [68] proposed to learn a shared representation from audio and video inputs. Eitel et al. [69] adopted two separate CNN streams for RGB and depth, combining them using a late fusion network for RGB-D object recognition. Hu et al. [34] presented a shared and individual multi-view learning algorithm to explore further properties of multi-modal data. Lu et al. [35] presented a shared-speciﬁc feature transfer framework to perform a cross-modal person ReID task. 2.3 Multi-modal learning In this section, we ﬁrst present the overall SPNet. Then we describe the two key components in our model, the modality-speciﬁc learning network and shared learning network, and ﬁnally provide the overall loss function. Recently, multi-modal (or multi-view) learning has attracted increasing attention: much data can be collected from multiple sources or represented using diﬀerent types of features. One traditional strategy directly concatenates feature vectors from such multi- modal data into a feature vector. However, this may fail to exploit the complex correlations within multi-modal data. Thus, several multi-modal learning 3.1 Overview Figure 2 shows the framework of our proposed speciﬁcity-preserving network for RGB-D SOD. First, Fig. 2 Architecture of SPNet, consisting of two modality-speciﬁc learning networks and a shared learning network. The former preserve individual properties for RGB or depth, while the shared network fuses cross-modal features and explores complementary information. Skip connections combine hierarchical features between encoder and decoder layers. Learned features from the modality-speciﬁc decoder are integrated into the shared decoder to provide rich multi-modal complementary information, boosting saliency detection. C denotes feature concatenation. Fig. 2 Architecture of SPNet, consisting of two modality-speciﬁc learning networks and a shared learning network. The former preserve individual properties for RGB or depth, while the shared network fuses cross-modal features and explores complementary information. Skip connections combine hierarchical features between encoder and decoder layers. Learned features from the modality-speciﬁc decoder are integrated into the shared decoder to provide rich multi-modal complementary information, boosting saliency detection. C denotes feature concatenation. Speciﬁcity-preserving RGB-D saliency detection 301 RGB and depth images are fed into two stream modality-speciﬁc learning networks to obtain their multi-level feature representations, and a CIM learns their shared feature representation. Secondly, the individual and shared decoder subnetworks are each utilized to generate saliency prediction maps. The original features from the encoder networks are integrated into the decoder via skip connections. Finally, to make full use of the features learned by using the modality-speciﬁc decoder, an MFA module integrates these features into the shared decoder. We detail each key part below. ﬁnal saliency map. We again adopt skip connections between the encoder and decoder layers to combine hierarchical features. Moreover, to make full use of the features learned by the modality-speciﬁc decoder, we integrate them into the shared decoder to improve saliency detection. 3.3.2 Cross-enhanced integration module Our CIM is used to eﬀectively fuse cross-modal features. Let the width, height, and number of channels for the m-th layer be denoted Wm, Hm, and Cm, respectively. Taking f R m ∈RWm×Hm×Cm and f D m ∈RWm×Hm×Cm as an example, we use a 1 × 1 convolutional layer to reduce the number of channels to Cm/2 for speed. The CIM has two parts, for cross-modal feature enhancement and adaptive feature fusion. First, we use a cross-enhanced strategy to exploit correlations between the two modalities by learning their enhanced features. Speciﬁcally, as shown in Fig. 3.1 Overview 3, the two features are fed into a 3 × 3 convolutional layer with a sigmoid activation function to obtain the normalized feature maps, wR m = σ(Conv3(f R m)) ∈[0, 1] and wD m = σ(Conv3(f R m)) ∈ [0, 1], where σ is the logistic sigmoid activation function. To exploit correlations between the two modalities, the normalized feature maps can be regarded as feature-level attention maps to adaptively enhance the feature representation. In this way, the feature map from one modality can be used to enhance the other modality. To preserve the original information of each modality, a residual connection is used to combine the enhanced features with the original features. Thus, the cross-enhanced feature representations for the two modalities are as Eq. (1): ′ 3.2 Modality-speciﬁc learning network As Fig. 2 shows, the modality-speciﬁc subnetwork is built upon Res2Net-50 [70], pretrained on the ImageNet [71] dataset. Thus, there are ﬁve multi- level features, i.e., F R = {f R m, m = 1, · · ·, 5]} and F D = {f D m, m = 1, · · ·, 5}, in the modality-speciﬁc encoder subnetworks for RGB and depth, respectively. The input resolution of the modality-speciﬁc encoder subnetwork is W × H. Thus, we have a feature resolution of (H/8) × (W/8) for the ﬁrst layer, and a general resolution of (H/2m) × (W/2m) (for m > 1). The number of channel features in the m-th layer is denoted Cm, where Cm = [64, 256, 512, 1024, 2048]. After obtaining the high-level features f R 5 and f D 5 , they are then fed into the modality-speciﬁc decoder subnetworks to generate individual saliency maps. We further utilize a U-Net [38] structure to construct the modality-speciﬁc decoder, where the skip connections between encoder and decoder layers are used to combine hierarchical features. Moreover, the concatenated features (only f R 5 and f D 5 in the ﬁrst stage of the decoder subnetwork) are fed to the receptive ﬁeld block (RFB) [72] to capture global context information. This modality-speciﬁc learning network enables us to learn eﬀective and powerful individual features for each modality by retaining its speciﬁc properties. These features are then integrated into the shared decoder subnetwork to improve saliency detection. ( f R′ m = f R m + f R m ⊗wD m f D′ m = f D m + f D m ⊗wR m (1) (1) where ⊗denotes element-wise multiplication. where ⊗denotes element-wise multiplication. Fig. 3 Cross-enhanced integration module (CIM). C, +, ×, and M denote feature concatenation, element-wise addition, multiplication, and maximization, respectively. 3.3.1 Structure As Fig. 2 shows, in the shared learning network, we fuse the cross-modal features from the RGB and depth modalities to learn their shared representation, which is fed into the shared decoder to generate the Fig. 3 Cross-enhanced integration module (CIM). C, +, ×, and M denote feature concatenation, element-wise addition, multiplication, and maximization, respectively. T. Zhou, D.-P. Fan, G. Chen, et al. 302 3.3.3 Multi-modal feature aggregation To make full use of the features learned in the modality-speciﬁc decoder, we propose a simple but eﬀective MFA module to integrate them into the shared decoder. Speciﬁcally, in the m-th layer of the shared decoder, we have the shared representation gS m, and the learned features gR m and gD m in the modality- speciﬁc decoder. As Fig. 4 shows, two features gR m and gD m are multiplied by the shared features of the current layer: gRS m = gS m ⊗gR m and gDS m = gS m ⊗gD m. The two features are further concatenated ([gDR m , gDS m ]) and then fed into a BConv(·) operation to obtain gSc m . Finally, we obtain the output of the MFA module to combine the convolutional feature gSc m with the original feature gS m via an addition operation. 3.3.3 Multi-modal feature aggregation Having obtained the cross-enhanced feature representations f R′ m and f D′ m , the critical task is to eﬀectively fuse them. Various strategies can be used to fuse features from diﬀerent modalities, including element-wise multiplication and maximiza- tion. However, it is unclear which is best for speciﬁc tasks. In order to beneﬁt from the advantages of diﬀerent strategies, we apply element-wise multiplication and maximization, and concatenate the results. Speciﬁcally, the two features f R′ m and f D′ m are ﬁrst fed into a 3 × 3 convolutional layer to obtain smooth representations, and then we carry out element-wise multiplication and maximization, giving ( pmul = BConv3(f R′ m ) ⊗BConv3(f D′ m ) ( pmul = BConv3(fm ) ⊗BConv3(fm ) pmax = max(BConv3(f R′ m ), BConv3(f D′ m )) (2) (2) pmax = max(BConv3(f R′ m ), BConv3(f D′ m )) In the MFA, the learned modality-speciﬁc features are used to enhance the shared features and provide rich and complementary cross-modal information. Speciﬁcally, we use the two modality-speciﬁc features gR m and gD m to enhance gS m. 3.3.1 Structure More importantly, the modality-speciﬁc decoder is given a supervision signal to guide feature learning for modality-speciﬁc property preservation, which beneﬁts the ﬁnal prediction results when integrating them in the shared decoder. We also note the diﬀerences between the CIM and the MFA: the CIM is used to learn the fused multi-modal (RGB and depth) feature representation, while the MFA utilizes the learned modality-speciﬁc features to form an aggregate feature representation in the shared decoder. where BConv(·) is a sequential operation that applies 3 × 3 convolution followed by batch normalization, then a ReLU function. Then, we concatenate the results as pcat = [pmul, pmax] ∈RWm×Hm×Cm, and obtain p1 cat = BConv3(pcat) through a BConv3 operation to adaptively weigh the two parts. Further, the output p1 cat is concatenated with the previous output f S m−1 of the (m −1)-th CIM, and fed into the second BConv3 operation. Finally, we obtain the output f S m of the m-th CIM. Note that, when m = 1, we do not need to use a 1 × 1 convolutional layer to reduce the number of channels. Furthermore, there is no previous output f S m−1 when m = 1, so we only feed the concatenated features into a BConv3 operation. 3.4 Loss function We note that our CIM can eﬀectively exploit correlations between the two modalities via cross-enhanced feature learning, and fuse them by adaptively weighting the diﬀerent feature representations. The fused feature representation f S m is propagated to the next layer to capture and integrate cross-level information. Some works [1, 17, 18] directly integrate RGB images and depth maps to form four-channel input (cascaded operation), and other methods carry out cross-modal fusion strategies, e.g., using attention-based fusion modules [24, 26], fusion-reﬁnement modules (e.g., using summation) [23], etc. Unlike these methods, our proposed CIM mainly exploits the correlation between RGB and depth images, and then adaptively integrates enhanced cross-modal features to obtain a fused feature representation. We may now formulate a uniﬁed, end-to-end trainable framework. The overall loss function has two parts, Lsp and Lsh, for the modality-speciﬁc and decoders, respectively. For convenience, SR and SD denote the prediction maps for RGB and depth images, respectively, Ssh denotes the prediction map using their shared representation, and G denotes the ground Fig. 4 Multi-modal feature aggregation (MFA) module. C, +, and × denote feature concatenation, element-wise addition, and element-wise multiplication respectively. Fig. 4 Multi-modal feature aggregation (MFA) module. C, +, and × denote feature concatenation, element-wise addition, and element-wise multiplication respectively. Speciﬁcity-preserving RGB-D saliency detection 303 it to a binary map M, and then compute the Precision and Recall [41] using truth map. Therefore, the overall loss function can be formulated as Eq. (3): ( ) Ltotal = Lsh(Ssh, G) + Lsp(SR, G) + Lsp(SD, G) (3) Ltotal = Lsh(Ssh, G) + Lsp(SR, G) + Lsp(SD, G) ( ) Precision = \|M ∩G\| \|M\| , Recall = \|M ∩G\| \|G\| (5) Precision = \|M ∩G\| \|M\| , Recall = \|M ∩G\| \|G\| (5) where G denotes the ground truth. A popular strategy is to partition S using a set of thresholds varying from 0 to 255. For each threshold, we calculate a pair of recall and precision scores, and then combine all scores to obtain a PR curve. Precision = \|M ∩G\| \|M\| , Recall = \|M ∩G\| \|G\| (5) where G denotes the ground truth. A popular strategy is to partition S using a set of thresholds varying from 0 to 255. For each threshold, we calculate a pair of recall and precision scores, and then combine all scores to obtain a PR curve. 3.4 Loss function (5) (3) ( ) Here, we utilize the pixel position-aware loss [73] for Lsp and Lsh, which can pay diﬀerent attention to hard and easy pixels to improve results. 4.1.2 Evaluation metrics 4.1.2 Evaluation metrics We adopt four widely used metrics to evaluate the eﬀectiveness of the proposed model. Their deﬁnitions are as follows. 4 Experimental results and analysis The F-measure Fβ [41] combines both precision and recall, via a weighted harmonic mean: In this section, we first give the experimental setup, including datasets, evaluation metrics, and implementation details. Then we carry out a quan-titatively and qualitatively evaluation, as well as conducting ablation studies to validate the effectiveness of each key component. Finally, we conduct an attribute-based evaluation to show the eﬀectiveness of our model in dealing with diﬀerent challenges. Fβ = 1 + β2 Precision × Recall β2Precision + Recall (6) (6) where β2 is set to 0.3 to emphasize precision [41]. We use diﬀerent ﬁxed [0, 255] thresholds to compute the F-measure. This yields a set of F-measure values; we report the maximum Fβ values from our experiments. Enhanced-alignment Measure. Eφ [78] is used to capture image-level statistics and local pixel matching information. It is deﬁned as 4.1.1 Datasets To validate the eﬀectiveness of the proposed model, we have evaluated it on six public RGB-D SOD datasets: NJU2K [54], NLPR [1], DES [74], SSD [75], STERE [76], and SIP [7]. Details of each dataset can be found at https://github.com/taozh2017/ RGBD-SODsurvey. Eφ = 1 WH W X i=1 H X i=1 φF M (i, j) (7) (7) where φF M denotes the enhanced-alignment matrix [78]. Mean Absolute Error (M). It is adopted to evaluate the average pixel-level relative error between the ground truth (i.e., G) and normalized prediction (i.e., S), which is deﬁned by For a fair comparison, we utilized the same protocol to form the training and test sets as introduced in Refs. [7, 60]. The training set includes 2195 samples in total, with 1485 samples from NJU2K [54] and 700 samples from NLPR [1]. The remaining samples from NJU2K (500) and NLPR (300), and the entire DES (135), SSD (80), STERE (1000), and SIP (929) datasets were used for testing. M = 1 W ∗H W X i=1 H X i=1 \|S (i, j) −G (i, j)\| (8) (8) where W and H denote the width and height of the map, respectively. M estimates the similarity between the saliency map and the ground-truth map, and normalizes it to [0, 1]. 4.1.3 Implementation details Our proposed model was implemented with the PyTorch library, and trained on an nVidia Tesla V100 GPU with 32 GB memory. Res2Net-50 [70], pre- trained on ImageNet [71], was used as the backbone network. Since RGB and depth images have diﬀerent numbers of channels, the input channel for the depth encoder was modiﬁed to 1. We utilized the Adam algorithm to optimize the proposed model. The initial learning rate was set to 10−4 and divided by 10 every 60 epochs. The input RGB and depth images were • Structure Measure. The S-measure Sα [77] assesses the structural similarity between regional perception (Sr) and object perception (So), and is deﬁned as Sα = αSo + (1 −α) Sr (4) (4) where α ∈[0, 1] is a trade-oﬀparameter, set to 0.5 by default [77]. • F-measure. Given a saliency map S, we convert 304 T. Zhou, D.-P. Fan, G. Chen, et al. [84], A2dele [85], JLDCF [11], S2MA [86], UCNet [12], SSF [87], HDFNet [88], Cas-GNN [89], CMMS [61], D3Net [7], CoNet [90], DANet [91], and PGAR [92]. See also the survey in Ref. [10]. resized to 352×352. To enhance the generalizability of the proposed learning algorithm, we adopted multiple data augmented strategies: random ﬂipping, rotation, and border clipping. The batch size was set to 20 and the model was trained over 200 epochs. 4.2.2 Quantitative evaluation For testing, the RGB and depth images were ﬁrst resized to 352 × 352 and then fed into the model to obtain the predicted saliency map. The predicted saliency map was then resized back to the original size of the input images. The output of the shared decoder is regarded as the ﬁnal prediction of our model. As Table 1 shows, our method is superior to the eight traditional methods LHM [1], ACSD [54], LBE [58], DCMC [80], SE [19], MDSF [17], CDCP [56], and DTM [81] by a large margin, on all six datasets. Our method furthermore outperforms all compared state- of-the-art methods and obtains the best performance in terms of the four evaluation metrics on NJU2K, DES, and SIP datasets. It is worth noting that our model obtains better results on STERE and NLPR than most compared RGB-D saliency detection methods. Our model is also comparable with CoNet on the STERE dataset, and JLDCF and PGAR on the NLPR dataset. 4.1.3 Implementation details Overall, our proposed SPNet obtains promising results in locating salient object(s) in a given scene. We further show PR curves in Fig. 5 and F-measure curves in Fig. 6, giving results for 4.2.1 Models compared We compared our proposed SPNet with 31 RGB-D saliency detection methods, including 8 handcrafted traditional models: LHM [1], ACSD [54], LBE [58], DCMC [80], SE [19], MDSF [17], CDCP [56], and DTM [81], and 23 deep models: DF [59], CTMF [25], PCF [22], AFNet [20], CPFP [30], MMCI [29], TANet [24], DMRA [60], cmSalGAN [82], ASIFNet [83], ICNet Table 1 Benchmarking results using 8 representative traditional models and 23 deep models on six public RGB-D saliency detection datasets using four widely used evaluation metrics: Sα [77], max Eφ [78], max Fβ [41], and M [79]). ↑, ↓indicate that larger or smaller is better. The subscript for each model denotes the publication year. esults using 8 representative traditional models and 23 deep models on six public RGB-D saliency detection datasets luation metrics: Sα [77], max Eφ [78], max Fβ [41], and M [79]). ↑, ↓indicate that larger or smaller is better. The enotes the publication year. Best results are highlighted in bold Table 1 Benchmarking results using 8 representative traditional models and 23 deep models on six public RGB-D saliency detection datasets using four widely used evaluation metrics: Sα [77], max Eφ [78], max Fβ [41], and M [79]). ↑, ↓indicate that larger or smaller is better. The subscript for each model denotes the publication year. Best results are highlighted in bold 4.2.1 Models compared Best results are highlighted in bold Model NJU2K [54] STERE [76] DES [74] NLPR [1] SSD [75] SIP [7] Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑Fβ ↑Eφ ↑M ↓ Sα ↑Fβ ↑Eφ ↑M ↓ Sα ↑Fβ ↑Eφ ↑M ↓ LHM14 [1] 0.514 0.632 0.724 0.205 0.562 0.683 0.771 0.172 0.562 0.511 0.653 0.114 0.630 0.622 0.766 0.108 0.566 0.568 0.717 0.195 0.511 0.574 0.716 0.184 ACSD14 [54] 0.699 0.711 0.803 0.202 0.692 0.669 0.806 0.200 0.728 0.756 0.850 0.169 0.673 0.607 0.780 0.179 0.675 0.682 0.785 0.203 0.732 0.763 0.838 0.172 LBE16 [58] 0.695 0.748 0.803 0.153 0.660 0.633 0.787 0.250 0.703 0.788 0.890 0.208 0.762 0.745 0.855 0.081 0.621 0.619 0.736 0.278 0.727 0.751 0.853 0.200 DCMC16 [80] 0.686 0.715 0.799 0.172 0.731 0.740 0.819 0.148 0.707 0.666 0.773 0.111 0.724 0.648 0.793 0.117 0.704 0.711 0.786 0.169 0.683 0.618 0.743 0.186 SE16 [19] 0.664 0.748 0.813 0.169 0.708 0.755 0.846 0.143 0.741 0.741 0.856 0.090 0.756 0.713 0.847 0.091 0.675 0.710 0.800 0.165 0.628 0.661 0.771 0.164 MDSF17 [17] 0.748 0.775 0.838 0.157 0.728 0.719 0.809 0.176 0.741 0.746 0.851 0.122 0.805 0.793 0.885 0.095 0.673 0.703 0.779 0.192 0.717 0.698 0.798 0.167 CDCP17 [56] 0.669 0.621 0.741 0.180 0.713 0.664 0.786 0.149 0.709 0.631 0.811 0.115 0.669 0.621 0.741 0.180 0.603 0.535 0.700 0.214 0.595 0.505 0.721 0.224 DTM20 [81] 0.706 0.716 0.799 0.190 0.747 0.743 0.837 0.168 0.752 0.697 0.858 0.123 0.733 0.677 0.833 0.145 0.677 0.651 0.773 0.199 0.690 0.659 0.778 0.203 DF17 [59] 0.763 0.804 0.864 0.141 0.757 0.757 0.847 0.141 0.752 0.766 0.870 0.093 0.802 0.778 0.880 0.085 0.747 0.735 0.828 0.142 0.653 0.657 0.759 0.185 CTMF18 [25] 0.849 0.845 0.913 0.085 0.848 0.831 0.912 0.086 0.863 0.844 0.932 0.055 0.860 0.825 0.929 0.056 0.776 0.729 0.865 0.099 0.716 0.694 0.829 0.139 PCF18 [22] 0.877 0.872 0.924 0.059 0.875 0.860 0.925 0.064 0.842 0.804 0.893 0.049 0.874 0.841 0.925 0.044 0.841 0.807 0.894 0.062 0.842 0.838 0.901 0.071 AFNet19 [20] 0.772 0.775 0.853 0.100 0.825 0.823 0.887 0.075 0.770 0.729 0.881 0.068 0.799 0.771 0.879 0.058 0.714 0.687 0.807 0.118 0.720 0.712 0.819 0.118 CPFP19 [30] 0.878 0.877 0.923 0.053 0.879 0.874 0.925 0.051 0.872 0.846 0.923 0.038 0.888 0.867 0.932 0.036 0.807 0.766 0.852 0.082 0.850 0.851 0.903 0.064 MMCI19 [29] 0.859 0.853 0.915 0.079 0.873 0.863 0.927 0.068 0.848 0.822 0.928 0.065 0.856 0.815 0.913 0.059 0.813 0.781 0.882 0.082 0.833 0.818 0.897 0.086 TANet19 [24] 0.878 0.874 0.925 0.060 0.871 0.861 0.923 0.060 0.858 0.827 0.910 0.046 0.886 0.863 0.941 0.041 0.839 0.810 0.897 0.063 0.835 0.830 0.895 0.075 DMRA19 [60] 0.886 0.886 0.927 0.051 0.886 0.886 0.938 0.047 0.900 0.888 0.943 0.030 0.899 0.879 0.947 0.031 0.857 0.844 0.906 0.058 0.806 0.821 0.875 0.085 cmSalGAN20 [82] 0.903 0.896 0.940 0.046 0.900 0.894 0.936 0.050 0.913 0.899 0.943 0.028 0.922 0.907 0.957 0.027 0.791 0.735 0.867 0.086 0.865 0.864 0.906 0.064 ASIFNet20 [83] 0.889 0.888 0.927 0.047 0.878 0.878 0.927 0.049 0.934 0.935 0.974 0.019 0.906 0.888 0.944 0.030 0.857 0.834 0.884 0.056 0.857 0.859 0.896 0.061 ICNet20 [84] 0.894 0.891 0.926 0.052 0.903 0.898 0.942 0.045 0.920 0.913 0.960 0.027 0.923 0.908 0.952 0.028 0.848 0.841 0.902 0.064 0.854 0.857 0.903 0.069 A2dele20 [85] 0.871 0.874 0.916 0.051 0.878 0.879 0.928 0.044 0.886 0.872 0.920 0.029 0.898 0.882 0.944 0.029 0.802 0.776 0.861 0.070 0.828 0.833 0.889 0.070 JLDCF20 [11] 0.903 0.903 0.944 0.043 0.905 0.901 0.946 0.042 0.929 0.919 0.968 0.022 0.925 0.916 0.962 0.022 0.830 0.795 0.885 0.068 0.879 0.885 0.923 0.051 S2MA20 [86] 0.894 0.889 0.930 0.053 0.890 0.882 0.932 0.051 0.941 0.935 0.973 0.021 0.915 0.902 0.953 0.030 0.868 0.848 0.909 0.052 0.872 0.877 0.919 0.057 UCNet20 [12] 0.897 0.895 0.936 0.043 0.903 0.899 0.944 0.039 0.933 0.930 0.976 0.018 0.920 0.903 0.956 0.025 0.865 0.854 0.907 0.049 0.875 0.879 0.919 0.051 SSF20 [87] 0.899 0.896 0.935 0.043 0.893 0.890 0.936 0.044 0.904 0.884 0.941 0.026 0.914 0.896 0.953 0.026 0.845 0.824 0.897 0.058 0.876 0.882 0.922 0.052 HDFNet20 [88] 0.908 0.911 0.944 0.038 0.900 0.900 0.943 0.041 0.926 0.921 0.970 0.021 0.923 0.917 0.9630.023 0.8790.870 0.9250.045 0.886 0.894 0.930 0.047 Cas-GNN20 [89] 0.911 0.903 0.933 0.035 0.899 0.901 0.930 0.039 0.905 0.906 0.947 0.028 0.919 0.904 0.947 0.028 0.872 0.862 0.915 0.047 0.875 0.879 0.919 0.051 CMMS20 [61] 0.900 0.897 0.936 0.044 0.895 0.893 0.939 0.043 0.937 0.930 0.976 0.018 0.915 0.896 0.949 0.027 0.874 0.864 0.922 0.046 0.872 0.877 0.911 0.058 CoNet20 [90] 0.895 0.893 0.937 0.046 0.908 0.905 0.949 0.040 0.909 0.896 0.945 0.028 0.908 0.887 0.945 0.031 0.853 0.840 0.915 0.059 0.858 0.867 0.913 0.063 DANet20 [91] 0.899 0.910 0.935 0.045 0.901 0.892 0.937 0.043 0.924 0.928 0.968 0.023 0.915 0.916 0.953 0.028 0.864 0.866 0.914 0.050 0.875 0.892 0.918 0.054 PGAR20 [92] 0.909 0.907 0.940 0.042 0.907 0.898 0.939 0.041 0.913 0.902 0.945 0.026 0.9300.916 0.961 0.024 0.865 0.838 0.898 0.057 0.876 0.876 0.915 0.055 D3Net21 [7] 0.900 0.900 0.950 0.041 0.899 0.891 0.938 0.046 0.898 0.885 0.946 0.031 0.912 0.897 0.953 0.030 0.857 0.834 0.910 0.058 0.860 0.861 0.909 0.063 SPNet (ours) 0.925 0.935 0.954 0.028 0.907 0.915 0.944 0.037 0.945 0.950 0.980 0.014 0.927 0.9250.959 0.0210.871 0.8830.915 0.0440.8940.9160.9300.043 Model NJU2K [54] STERE [76] DES [74] NLPR [1] SSD [75] SIP [7] Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑Fβ ↑Eφ ↑M ↓ Sα ↑Fβ ↑Eφ ↑M ↓ Sα ↑Fβ ↑Eφ ↑M ↓ LHM14 [1] 0.514 0.632 0.724 0.205 0.562 0.683 0.771 0.172 0.562 0.511 0.653 0.114 0.630 0.622 0.766 0.108 0.566 0.568 0.717 0.195 0.511 0.574 0.716 0.184 ACSD14 [54] 0.699 0.711 0.803 0.202 0.692 0.669 0.806 0.200 0.728 0.756 0.850 0.169 0.673 0.607 0.780 0.179 0.675 0.682 0.785 0.203 0.732 0.763 0.838 0.172 LBE16 [58] 0.695 0.748 0.803 0.153 0.660 0.633 0.787 0.250 0.703 0.788 0.890 0.208 0.762 0.745 0.855 0.081 0.621 0.619 0.736 0.278 0.727 0.751 0.853 0.200 DCMC16 [80] 0.686 0.715 0.799 0.172 0.731 0.740 0.819 0.148 0.707 0.666 0.773 0.111 0.724 0.648 0.793 0.117 0.704 0.711 0.786 0.169 0.683 0.618 0.743 0.186 SE16 [19] 0.664 0.748 0.813 0.169 0.708 0.755 0.846 0.143 0.741 0.741 0.856 0.090 0.756 0.713 0.847 0.091 0.675 0.710 0.800 0.165 0.628 0.661 0.771 0.164 MDSF17 [17] 0.748 0.775 0.838 0.157 0.728 0.719 0.809 0.176 0.741 0.746 0.851 0.122 0.805 0.793 0.885 0.095 0.673 0.703 0.779 0.192 0.717 0.698 0.798 0.167 CDCP17 [56] 0.669 0.621 0.741 0.180 0.713 0.664 0.786 0.149 0.709 0.631 0.811 0.115 0.669 0.621 0.741 0.180 0.603 0.535 0.700 0.214 0.595 0.505 0.721 0.224 DTM20 [81] 0.706 0.716 0.799 0.190 0.747 0.743 0.837 0.168 0.752 0.697 0.858 0.123 0.733 0.677 0.833 0.145 0.677 0.651 0.773 0.199 0.690 0.659 0.778 0.203 DF17 [59] 0.763 0.804 0.864 0.141 0.757 0.757 0.847 0.141 0.752 0.766 0.870 0.093 0.802 0.778 0.880 0.085 0.747 0.735 0.828 0.142 0.653 0.657 0.759 0.185 CTMF18 [25] 0.849 0.845 0.913 0.085 0.848 0.831 0.912 0.086 0.863 0.844 0.932 0.055 0.860 0.825 0.929 0.056 0.776 0.729 0.865 0.099 0.716 0.694 0.829 0.139 PCF18 [22] 0.877 0.872 0.924 0.059 0.875 0.860 0.925 0.064 0.842 0.804 0.893 0.049 0.874 0.841 0.925 0.044 0.841 0.807 0.894 0.062 0.842 0.838 0.901 0.071 AFNet19 [20] 0.772 0.775 0.853 0.100 0.825 0.823 0.887 0.075 0.770 0.729 0.881 0.068 0.799 0.771 0.879 0.058 0.714 0.687 0.807 0.118 0.720 0.712 0.819 0.118 CPFP19 [30] 0.878 0.877 0.923 0.053 0.879 0.874 0.925 0.051 0.872 0.846 0.923 0.038 0.888 0.867 0.932 0.036 0.807 0.766 0.852 0.082 0.850 0.851 0.903 0.064 MMCI19 [29] 0.859 0.853 0.915 0.079 0.873 0.863 0.927 0.068 0.848 0.822 0.928 0.065 0.856 0.815 0.913 0.059 0.813 0.781 0.882 0.082 0.833 0.818 0.897 0.086 TANet19 [24] 0.878 0.874 0.925 0.060 0.871 0.861 0.923 0.060 0.858 0.827 0.910 0.046 0.886 0.863 0.941 0.041 0.839 0.810 0.897 0.063 0.835 0.830 0.895 0.075 DMRA19 [60] 0.886 0.886 0.927 0.051 0.886 0.886 0.938 0.047 0.900 0.888 0.943 0.030 0.899 0.879 0.947 0.031 0.857 0.844 0.906 0.058 0.806 0.821 0.875 0.085 cmSalGAN20 [82] 0.903 0.896 0.940 0.046 0.900 0.894 0.936 0.050 0.913 0.899 0.943 0.028 0.922 0.907 0.957 0.027 0.791 0.735 0.867 0.086 0.865 0.864 0.906 0.064 ASIFNet20 [83] 0.889 0.888 0.927 0.047 0.878 0.878 0.927 0.049 0.934 0.935 0.974 0.019 0.906 0.888 0.944 0.030 0.857 0.834 0.884 0.056 0.857 0.859 0.896 0.061 ICNet20 [84] 0.894 0.891 0.926 0.052 0.903 0.898 0.942 0.045 0.920 0.913 0.960 0.027 0.923 0.908 0.952 0.028 0.848 0.841 0.902 0.064 0.854 0.857 0.903 0.069 A2dele20 [85] 0.871 0.874 0.916 0.051 0.878 0.879 0.928 0.044 0.886 0.872 0.920 0.029 0.898 0.882 0.944 0.029 0.802 0.776 0.861 0.070 0.828 0.833 0.889 0.070 JLDCF20 [11] 0.903 0.903 0.944 0.043 0.905 0.901 0.946 0.042 0.929 0.919 0.968 0.022 0.925 0.916 0.962 0.022 0.830 0.795 0.885 0.068 0.879 0.885 0.923 0.051 S2MA20 [86] 0.894 0.889 0.930 0.053 0.890 0.882 0.932 0.051 0.941 0.935 0.973 0.021 0.915 0.902 0.953 0.030 0.868 0.848 0.909 0.052 0.872 0.877 0.919 0.057 UCNet20 [12] 0.897 0.895 0.936 0.043 0.903 0.899 0.944 0.039 0.933 0.930 0.976 0.018 0.920 0.903 0.956 0.025 0.865 0.854 0.907 0.049 0.875 0.879 0.919 0.051 SSF20 [87] 0.899 0.896 0.935 0.043 0.893 0.890 0.936 0.044 0.904 0.884 0.941 0.026 0.914 0.896 0.953 0.026 0.845 0.824 0.897 0.058 0.876 0.882 0.922 0.052 HDFNet20 [88] 0.908 0.911 0.944 0.038 0.900 0.900 0.943 0.041 0.926 0.921 0.970 0.021 0.923 0.917 0.9630.023 0.8790.870 0.9250.045 0.886 0.894 0.930 0.047 Cas-GNN20 [89] 0.911 0.903 0.933 0.035 0.899 0.901 0.930 0.039 0.905 0.906 0.947 0.028 0.919 0.904 0.947 0.028 0.872 0.862 0.915 0.047 0.875 0.879 0.919 0.051 CMMS20 [61] 0.900 0.897 0.936 0.044 0.895 0.893 0.939 0.043 0.937 0.930 0.976 0.018 0.915 0.896 0.949 0.027 0.874 0.864 0.922 0.046 0.872 0.877 0.911 0.058 CoNet20 [90] 0.895 0.893 0.937 0.046 0.908 0.905 0.949 0.040 0.909 0.896 0.945 0.028 0.908 0.887 0.945 0.031 0.853 0.840 0.915 0.059 0.858 0.867 0.913 0.063 DANet20 [91] 0.899 0.910 0.935 0.045 0.901 0.892 0.937 0.043 0.924 0.928 0.968 0.023 0.915 0.916 0.953 0.028 0.864 0.866 0.914 0.050 0.875 0.892 0.918 0.054 PGAR20 [92] 0.909 0.907 0.940 0.042 0.907 0.898 0.939 0.041 0.913 0.902 0.945 0.026 0.9300.916 0.961 0.024 0.865 0.838 0.898 0.057 0.876 0.876 0.915 0.055 D3Net21 [7] 0.900 0.900 0.950 0.041 0.899 0.891 0.938 0.046 0.898 0.885 0.946 0.031 0.912 0.897 0.953 0.030 0.857 0.834 0.910 0.058 0.860 0.861 0.909 0.063 SPNet (ours) 0.925 0.935 0.954 0.028 0.907 0.915 0.944 0.037 0.945 0.950 0.980 0.014 0.927 0.9250.959 0.0210.871 0.8830.915 0.0440.8940.9160.9300.043 Speciﬁcity-preserving RGB-D saliency detection 305 Fig. 4.2.1 Models compared 5 PR curves for six datasets: NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], and SIP [7]. Fig. 6 F-measure curves for diﬀerent thresholds, for NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], and SIP [7]. 29 RGB-D saliency detection methods, including 28 state-of-the-art models with complete saliency maps. The superiority of our model is clearly visible on these datasets. Speciﬁcity-preserving RGB-D saliency detection 305 Fig. 5 PR curves for six datasets: NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], and SIP [7]. Fig. 5 PR curves for six datasets: NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], and SIP [7]. Fi PR f d NJU K [ 4] STERE [7 ] DES [74] NLPR [ ] SSD [7 ] d SIP [7] Fig. 5 PR curves for six datasets: NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], and SIP [7]. Fig. 5 PR curves for six datasets: NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], and SIP [7]. Fig. 6 F-measure curves for diﬀerent thresholds, for NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], and SIP [7]. Fig. 6 F-measure curves for diﬀerent thresholds, for NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], and SIP [7]. Fig. 6 F-measure curves for diﬀerent thresholds, for NJU2K [54], STERE [76], DES [74], NLPR [1], SSD [75], an The superiority of our model is clearly visible on these datasets. The superiority of our model is clearly visible on these datasets. 29 RGB-D saliency detection methods, including 28 state-of-the-art models with complete saliency maps. 29 RGB-D saliency detection methods, including 28 state-of-the-art models with complete saliency maps. T. Zhou, D.-P. Fan, G. Chen, et al. 306 model using ResNet-50 as backbone still performs better than other methods (see Table 1). 4.2.3 Qualitative evaluation In addition, we compared our SPNet to 13 recent state-of-the-art models on the ReDWeb-S dataset. Results for the other methods are from https:// github.com/nnizhang/SMAC, while results for our method were obtained by testing the model (trained using NJU2K [54] and NLPR [1]) on the ReDWeb-S dataset. The comparison is shown in Table 2. Our method works better than most compared methods, and is comparable to UCNet and JLDCF on the ReDWeb-S dataset. Figure 7 shows several representative samples of results comparing our model with those from eight state-of-the-art methods. 4.2.1 Models compared The ﬁrst row shows a scene with a small object. Our method, A2dele, PGAR, and D3Net accurately detect the salient object, while JLDCF, S2MA, SSF, and UCNet predict some non-object regions. Rows 2 and 3 show two examples of scenes with complex backgrounds. Our method and S2MA produce reliable results, while other RGB- D saliency detection models fail to locate the object or confuse the background with a salient object. In row We further compared using diﬀerent backbone networks in the proposed model, with the results shown in Table 3. The proposed model works better when using Res2Net-50 as the backbone, yet the Table 2 Results from our model and 13 state-of-the art methods: CTMFF [25], PCF [22], AFNet [20], MMCI [29], CPFP [30], DMRA [60], TANet [24], A2dele [85], UCNet [12], JLDCF [11], S2MA [86], SSF [87], and D3Net [7]) on the ReDWeb-S dataset Model CTMF PCF AFNet MMCI CPFP DMRA TANet A2dele UCNet JLDCF S2MA SSF D3Net Ours Sα ↑ 0.641 0.655 0.546 0.660 0.685 0.592 0.656 0.641 0.713 0.734 0.711 0.595 0.689 0.710 Fβ ↑ 0.607 0.627 0.549 0.641 0.645 0.579 0.623 0.603 0.710 0.727 0.696 0.558 0.673 0.715 Eφ ↑ 0.739 0.743 0.693 0.754 0.744 0.721 0.741 0.672 0.794 0.805 0.781 0.710 0.768 0.800 M ↓ 0.204 0.166 0.213 0.176 0.142 0.188 0.165 0.160 0.130 0.128 0.139 0.189 0.149 0.129 Table 3 Results from our model using diﬀerent backbone networks Backbone NJU2K [54] STERE [76] DES [74] NLPR [1] SSD [75] SIP [7] Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑M ↓ Sα ↑ Fβ ↑ Eφ ↑M ↓ Sα ↑ Fβ ↑ Eφ ↑M ↓ ResNet-50 0.922 0.934 0.952 0.030 0.904 0.914 0.942 0.037 0.936 0.944 0.974 0.016 0.930 0.931 0.965 0.020 0.869 0.876 0.906 0.044 0.896 0.916 0.934 0.041 Res2Net-50 0.925 0.935 0.954 0.028 0.907 0.915 0.944 0.037 0.945 0.950 0.980 0.014 0.927 0.925 0.959 0.021 0.871 0.883 0.915 0.044 0.894 0.916 0.930 0.043 Fig. 7 Visual comparison of results from our method and eight state-of-the-art methods: A2dele [85], JLDCF [11], S2MA [86], UCNet [12], SSF [87], D3Net [7], DANet [91], and PGAR [92]. 4.2.1 Models compared Table 2 Results from our model and 13 state-of-the art methods: CTMFF [25], PCF [22], AFNet [20], MMCI [29], CPFP [30], DMRA [60], TANet [24], A2dele [85], UCNet [12], JLDCF [11], S2MA [86], SSF [87], and D3Net [7]) on the ReDWeb-S dataset Model CTMF PCF AFNet MMCI CPFP DMRA TANet A2dele UCNet JLDCF S2MA SSF D3Net Ours Sα ↑ 0.641 0.655 0.546 0.660 0.685 0.592 0.656 0.641 0.713 0.734 0.711 0.595 0.689 0.710 Fβ ↑ 0.607 0.627 0.549 0.641 0.645 0.579 0.623 0.603 0.710 0.727 0.696 0.558 0.673 0.715 Eφ ↑ 0.739 0.743 0.693 0.754 0.744 0.721 0.741 0.672 0.794 0.805 0.781 0.710 0.768 0.800 M ↓ 0.204 0.166 0.213 0.176 0.142 0.188 0.165 0.160 0.130 0.128 0.139 0.189 0.149 0.129 Table 3 Results from our model using diﬀerent backbone networks Backbone NJU2K [54] STERE [76] DES [74] NLPR [1] SSD [75] SIP [7] Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑ M ↓ Sα ↑ Fβ ↑ Eφ ↑M ↓ Sα ↑ Fβ ↑ Eφ ↑M ↓ Sα ↑ Fβ ↑ Eφ ↑M ↓ ResNet-50 0.922 0.934 0.952 0.030 0.904 0.914 0.942 0.037 0.936 0.944 0.974 0.016 0.930 0.931 0.965 0.020 0.869 0.876 0.906 0.044 0.896 0.916 0.934 0.041 Res2Net-50 0.925 0.935 0.954 0.028 0.907 0.915 0.944 0.037 0.945 0.950 0.980 0.014 0.927 0.925 0.959 0.021 0.871 0.883 0.915 0.044 0.894 0.916 0.930 0.043 Fig. 7 Visual comparison of results from our method and eight state-of-the-art methods: A2dele [85], JLDCF [11], S2MA [86], UCNet [12], SSF [87], D3Net [7], DANet [91], and PGAR [92]. Fig. 7 Visual comparison of results from our method and eight state-of-the-art methods: A2dele [85], JLDCF [11], S2MA [86], UCNet [12] SSF [87], D3Net [7], DANet [91], and PGAR [92]. Fig. 7 Visual comparison of results from our method and eight state-of-the-art methods: A2dele [85], JLDCF [11], S2MA [86], UCNet [12], SSF [87], D3Net [7], DANet [91], and PGAR [92]. Speciﬁcity-preserving RGB-D saliency detection 307 This also indicates the contribution of the CIM in improving the saliency detection results. Going further, there are two parts to CIM: cross-modal feature enhancement and adaptive feature fusion. Thus, to evaluate the contribution of each part, we modiﬁed CIM to have only cross-modal feature enhancement or adaptive feature fusion, with results denoted A2 and A3, respectively. 4.3.2 Eﬀectiveness of MFA In the proposed framework, the MFA is proposed to make full use of the features learned in the modality-speciﬁc decoder, which are then integrated into the shared decoder to provide more multi- modal complementary information. To validate its eﬀectiveness, we deleted this module in an approach denoted B1. We also considered two other feature fusion strategies: see Fig. 8. One provides cross- modal feature enhancement fusion; the other is a simple concatenation strategy. Results for the two strategies are denoted B2 and B3. Table 5 demonstrates, by comparing results of B1 and our full model, the eﬀectiveness of integrating the features learned into the shared decoder. Comparing results of B2 and B3 with our full model, we can see that the MFA module outperforms both other fusion strategies. 4.3.3 Eﬀectiveness of modality-speciﬁc decoders 4.3 Ablation studies To verify the relative importance of diﬀerent key components of our model, we conducted ablation studies by removing or replacing them. 4.2.1 Models compared When comparing them to the full version of CIM, we can see the eﬀectiveness of the proposed CIM. Moreover, in CIM, the features of the last layer are propagated to the next layer to capture cross-level correlations. To validate the eﬀectiveness of the propagation strategy, we removed this propagation in the CIM, with results denoted A4, showing that the propagation strategy does improve saliency detection results. 4 3 2 Eﬀectiveness of MFA 4, the compared methods other than D3Net locate a non-salient and small object. In row 5, we show an example with multiple salient objects, where it is challenging to accurately locate them all. Our method locates all salient objects and segments them more accurately, generating sharper edges than other approaches. We show an example under low-light conditions in the last row. While some approaches fail to detect the entire extent of the salient object, our model suppresses background distractors and gives good saliency detection results. 4.2.4 Inference time and model size We tested the inference time for different methods on an NVIDIA TESLA P40 GPU with 24 GB memory. The inference time and model size of different methods, including our SPNet, JLDCF [11], S2MA [86], UCNet [12], SSF [87], and HDFNet [88], are shown in Table 4. Because our model adopts two modality- specific networks and a shared learning network to generate individual and shared saliency prediction maps, it has a relatively large model size and takes more inference time for saliency prediction than other methods. We thus hope to design light-weight networks to improve the efficiency of SPNet in future work. 4.4.2 Indoor vs. outdoor 4.3.4 Eﬀects of varying numbers of CIMs We evaluated the results of diﬀerent RGB-D SOD models on indoor and outdoor scenes. As DES [74] and NLPR [1] include indoor and outdoor scenes, we constructed a hybrid dataset collected from the two datasets. Results are shown in Fig. 9(b). As can be observed, many models ﬁnd it harder to detect salient objects in indoor scenes than outdoor scenes, while JLDCF, S2MA, UCNet, ICNet, SSF, DANet, and our model work a little better on outdoor scenes. 4.4.3 Lighting conditions To investigate the eﬀects of changing the numbers of CIMs, we compare our full model using ﬁve CIMs with two degraded versions, CIM1, which only applies a CIM to the features from the last layer in the encoder network, and CIM3, using CIMs on the features from each of the last three layers in the encoder network. Table 6 shows the results; our model with ﬁve CIMs works better for most datasets. 4.3.1 Eﬀectiveness of CIM 308 Table 5 Quantitative evaluation for ablation studies NJU2K [54] STERE [76] DES [74] NLPR [1] SSD [75] SIP [7] Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ Ours 0.925 0.028 0.907 0.037 0.945 0.014 0.927 0.021 0.871 0.044 0.894 0.043 A1 0.916 0.034 0.898 0.042 0.939 0.016 0.926 0.022 0.869 0.047 0.892 0.044 A2 0.921 0.031 0.895 0.042 0.938 0.016 0.925 0.022 0.865 0.051 0.896 0.042 A3 0.919 0.032 0.895 0.043 0.938 0.016 0.929 0.020 0.864 0.049 0.887 0.048 A4 0.924 0.029 0.903 0.038 0.930 0.019 0.927 0.023 0.867 0.049 0.888 0.046 B1 0.918 0.034 0.901 0.041 0.939 0.017 0.922 0.024 0.858 0.050 0.885 0.048 B2 0.924 0.029 0.900 0.041 0.941 0.015 0.926 0.022 0.864 0.049 0.893 0.044 B3 0.921 0.031 0.903 0.039 0.938 0.016 0.925 0.022 0.863 0.050 0.891 0.045 C1 0.913 0.037 0.900 0.047 0.935 0.019 0.922 0.025 0.861 0.055 0.880 0.051 C2 0.916 0.034 0.906 0.040 0.923 0.021 0.924 0.022 0.866 0.049 0.882 0.051 and weaknesses of state-of-the-art models in handling these challenges. To further evaluate the eﬀectiveness of the combination of the two modality-speciﬁc decoders, we added an experiment to compare the SOD results when using the output from the shared decoder and the combination of the two modality-speciﬁc decoders. Results are shown in C2 of Table 5. We can see that the shared decoder outperforms the combination of the two modality-speciﬁc decoders, indicating that the shared decoder can combine multi-modal shared information and modality-speciﬁc characteristics to improve SOD results. 4.4.1 Single vs. multiple objects 4.4.1 Single vs. multiple objects In this evaluation, we constructed a hybrid dataset with 1229 images from the NLPR [1] and SIP [7] datasets. Results using Sα are shown in Fig. 9(a). As can be observed, it is easier to detect a single salient object than several. Our model outperforms other state-of-the-art methods in locating single and multiple objects. 4.3.1 Eﬀectiveness of CIM 4.3.1 Eﬀectiveness of CIM Since the proposed CIM is used to fuse cross-modal features and learn their shared representation, we compared it to an alternative of a direct concatenation strategy. Speciﬁcally, the two features f R m and f D m (see Fig. 3) are directly concatenated and then fed into a 3 × 3 convolutional layer to obtain the fused representation in each layer. We denote this approach as A1 in Table 5, which shows that our model performs better when using the proposed CIM than using a simple feature concatenation strategy. We deleted the two modality-speciﬁc decoders, with results shown in C1 in Table 5. Performance degrades when not using the two parts. This indicates the eﬀectiveness of the modality-speciﬁc decoders, which provide supervision signals to ensure that modality- speciﬁc properties can be learned. Fig. 8 Comparison of MFA module with other fusion strategies. Table 4 Comparisons of inference time and model size for diﬀerent methods Method Ours JLDCF S2MA Model size (MB) 175.3 124.5 82.7 Inference time (ms) 91.7 21.8 22.1 Method UCNet SSF HDFNet Model size (MB) 31.3 32.9 153.2 Inference time (ms) 31.8 45.7 57.1 Table 4 Comparisons of inference time and model size for diﬀerent methods Fig. 8 Comparison of MFA module with other fusion strategies. T. Zhou, D.-P. Fan, G. Chen, et al. 4.4 Attribute-based evaluation There are several challenging factors that may aﬀect results from RGB-D saliency detection models, such as the number of salient objects, indoor versus outdoor environments, lighting conditions, and so on. Thus, we evaluated saliency detection results under diﬀerent conditions, to show the strengths We carried out this evaluation on the SIP dataset [7], with examples grouped into two categories, sunny and low-light. Results are shown in Fig. 9(c). All models struggle to detect salient objects in low- light conditions, conﬁrming that low-light negatively impacts SOD performance. Table 6 Results for diﬀerent numbers of CIMs NJU2K STERE DES NLPR SSD SIP Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ CIM1 0.918 0.034 0.908 0.039 0.929 0.019 0.928 0.022 0.865 0.047 0.889 0.046 CIM3 0.920 0.032 0.900 0.041 0.935 0.017 0.928 0.021 0.857 0.049 0.891 0.045 Ours 0.925 0.028 0.907 0.037 0.945 0.014 0.927 0.021 0.871 0.044 0.894 0.043 Speciﬁcity-preserving RGB-D saliency detection 309 Fig. 9 Attribute-based evaluation with respect to (a) number of salient objects (one or multiple), (b) indoor versus outdoor environments, and (c) lighting conditions (low-light versus sunny). Fig. 9 Attribute-based evaluation with respect to (a) number of salient objects (one or multiple), (b) indoor versus outdoor environments, and (c) lighting conditions (low-light versus sunny). Fig. 9 Attribute-based evaluation with respect to (a) number of salient objects (one or multiple), (b) indoor ver and (c) lighting conditions (low-light versus sunny). evaluation with respect to (a) number of salient objects (one or multiple), (b) indoor versus outdoor environments, (low-light versus sunny). 4.5 Failures and discussion Our proposed SPNet shows good RGB-D saliency detection in most cases. However, it fails to detect salient objects in some challenging scenes such as those with complex backgrounds and low-quality depth data. Some failures of our model are shown in Fig. 11. In the ﬁrst row, the depth data quality is very poor, so our model can only roughly locate the boat without ﬁne details. This suggests that it is helpful to enhance or ﬁlter depth maps to improve saliency detection results. In the second row, the annotated salient object has a similar appearance to other objects in the scene, so it is challenging to accurately detect the salient object. In the third row, the object has ﬁne details, but our model only locates the main regions without the ﬁne details. There is still considerable room to improve our model to handle such scenes with ﬁne structures. We compare our method to other existing COD models, including FPN [93], MaskRCNN [94], PSPNet [95], PiCANet [46], BASNet [96], PFANet [97], CPD [98], EGNet [99], and SINet [105] (results are from Ref. [105]). Since there are few works for RGB-D camouﬂaged object detection, we also compared two recent RGB-D salient object detection methods, DANet [91] and HDFNet [88], in this experiment. We re-trained the two RGB-D SOD models and our model using RGB and depth images. Table 7 shows quantitative results for three public datasets. Our model performs better than the other COD methods. Our model and the two RGB-D COD methods use depth cues, and work better than other methods which do not, indicating that depth cues can provide spatial information to improve COD results. Figure 12 shows qualitative results for diﬀerent COD We compare our method to other existing COD models, including FPN [93], MaskRCNN [94], PSPNet [95], PiCANet [46], BASNet [96], PFANet [97], CPD [98], EGNet [99], and SINet [105] (results are from Ref. [105]). Since there are few works for RGB-D camouﬂaged object detection, we also compared two recent RGB-D salient object detection methods, DANet [91] and HDFNet [88], in this experiment. We re-trained the two RGB-D SOD models and our model using RGB and depth images. 4.4.4 Object scale scale variation, we constructed a hybrid dataset with 2444 images from STERE [76], NLPR [1], SSD [75], DES [74], and SIP [7]. Figure 10 shows results of this attribute-based evaluation with respect to the scales of the salient objects. All methods work better at detecting small salient objects and relatively at To characterize the scale of a salient object, we compute the ratio r of the size of the salient region to the whole image, and deﬁne three object scales: small, when r < 0.1, large, when r > 0.4, and medium otherwise. To evaluate how diﬀerent methods handle Fig. 10 Attribute-based evaluation with respect to scale of the salient object. Fig. 10 Attribute-based evaluation with respect to scale of the salient object. T. Zhou, D.-P. Fan, G. Chen, et al. 310 detecting large salient objects. The most recent models, JLDCF, DANet, PGAR, and our model, obtain the promising results. research [103] suggests that depth can provide useful spatial information to improve COD results. Thus, we extended SPNet to the RGB-D COD task. We conducted this experiment on three public benchmark datasets for camouﬂaged object detection: (i) CHAMELEON [100], consisting of 76 camouﬂaged images, (ii) CAMO [104], with 1250 images (1000 for training, 250 for testing) in 8 categories, and (iii) COD10K [100], with 5066 camouﬂaged images (3040 for training, 2026 for testing) in 5 super-classes and 69 sub-classes. Following the same setting in Ref. [105], we divided the training and testing sets and then trained our model on the training set. Table 7 Results for camouﬂaged object detection models on benchmark datasets using evaluation metrics Sα [77] and M [79]. ↑, ↓indicate that larger or smaller is better 4.6 Application to RGB-D camouflaged object detection Table 7 shows quantitative results for three public datasets. Our model performs better than the other COD methods. Our model and the two RGB-D COD methods use depth cues, and work better than other methods which do not, indicating that depth cues can provide spatial information to improve COD results. Figure 12 shows qualitative results for diﬀerent COD SPNet was originally designed for the RGB-D SOD task, which can be easily extended to other related RGB-D tasks, e.g., RGB-D based camouﬂaged object detection (COD). The aim of COD is to identify objects that are “seamlessly” embedded in their background surroundings. This is a very challenging task due to the high intrinsic similarities between the target object and the background [100–102]. Recent Table 7 Results for camouﬂaged object detection models on benchmark datasets using evaluation metrics Sα [77] and M [79]. ↑, ↓indicate that larger or smaller is better Model CHAMELEON CAMO COD10K Sα ↑ M ↓ Sα ↑ M ↓ Sα ↑ M ↓ FPN [93] 0.794 0.075 0.684 0.131 0.697 0.075 MaskRCNN [94] 0.643 0.099 0.574 0.151 0.613 0.080 PSPNet [95] 0.773 0.085 0.663 0.139 0.678 0.080 PiCANet [46] 0.769 0.085 0.609 0.156 0.649 0.090 BASNet [96] 0.687 0.118 0.618 0.159 0.634 0.105 PFANet [97] 0.679 0.144 0.659 0.172 0.636 0.128 CPD [98] 0.853 0.052 0.726 0.115 0.747 0.059 EGNet [99] 0.848 0.050 0.732 0.104 0.737 0.056 SINet [100] 0.869 0.044 0.751 0.100 0.771 0.051 DANet [91] 0.874 0.043 0.752 0.100 0.765 0.051 HDFNet [88] 0.875 0.032 0.778 0.085 0.779 0.045 SPNet (ours) 0.895 0.027 0.795 0.082 0.797 0.042 Fig. 11 Cases in which our model fails. Fig. 11 Cases in which our model fails. Fig. 11 Cases in which our model fails. Fig. 11 Cases in which our model fails. Speciﬁcity-preserving RGB-D saliency detection 311 Fig. 12 COD results of our SPNet and three state-of-the-art COD methods: SINet [105], DANet [91], and HDFNet [88]. Fig. 12 COD results of our SPNet and three state-of-the-art COD methods: SINet [105], DANet [91], and HDFNet [88]. Fig. 12 COD results of our SPNet and three state-of-the-art COD methods: SINet [105], DANet [91], and HDFNet [88]. Fig. 12 COD results of our SPNet and three state-of-the-art COD methods: SINet [105], DANet [91], and HDFNet [88]. Fig. 12 COD results of our SPNet and three state-of-the-art COD methods: SINet [105], DANe methods. Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article. 4.6 Application to RGB-D camouflaged object detection Compared to other COD models, our SPNet achieves better results by detecting more accurate boundaries of camouﬂaged objects. to study the ability of many cutting-edge RGB- D SOD approaches to meet diﬀerent challenges. Finally, we extended SPNet to the recently proposed RGB-D camouﬂaged object detection task, and its eﬀectiveness was veriﬁed. Acknowledgements In this paper, we have presented a novel RGB-D salient object detection framework, SPNet. Unlike most existing RGB-D SOD methods, which focus on learning shared representations, SPNet not only explores shared cross-modal information but also uses modality-speciﬁc characteristics to improve SOD results. To learn the shared representations for the two modalities, we introduce a cross-enhanced integration module (CIM) to fuse the cross-modal features, and the output of each CIM is propagated to the next layer to explore rich cross-level information. We further adopt a multi-modal feature aggregation (MFA) module to integrate the learned modality- speciﬁc features to enhance the complementary multi- modal information. Extensive results on benchmark datasets show the eﬀectiveness of our model in comparison to other state-of-the-art RGB-D SOD methods. Moreover, we have thoroughly validated the eﬀectiveness of key components of our framework, and an attribute-based evaluation was conducted This work was supported in part by the National Natural Science Foundation of China under Grant No. 62172228, in part by an Open Project of the Key Laboratory of System Control and Information Processing, Ministry of Education (Shanghai Jiao Tong University, No. Scip202102). References [1] Peng, H.; Li, B.; Xiong, W.; Hu, W.; Ji, R. RGBD salient object detection: A benchmark and algorithms. In: Computer Vision – ECCV 2014. Lecture Notes in Computer Science, Vol. 8691. Fleet, D.; Pajdla, T.; Schiele, B.; Tuytelaars, T. Eds. Springer Cham, 92–109, 2014. 312 T. Zhou, D.-P. Fan, G. Chen, et al. [2] Zhu, J.-Y.; Wu, J.-J.; Xu, Y.; Chang, E.; Tu, Z. W. Unsupervised object class discovery via saliency- guided multiple class learning. IEEE Transactions on Pattern Analysis and Machine Intelligence Vol. 37, No. 4, 862–875, 2015. Pattern Analysis and Machine Intelligence doi: 10.1109/TPAMI.2021.3073564, 2021. [13] Chen, H.; Li, Y. F.; Deng, Y. J.; Lin, G. S. CNN- based RGB-D salient object detection: Learn, select, and fuse. International Journal of Computer Vision Vol. 129, No. 7, 2076–2096, 2021. [3] Rapantzikos, K.; Avrithis, Y.; Kollias, S. Dense saliency-based spatiotemporal feature points for action recognition. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 1454–1461, 2009. [14] Li, G. Y.; Liu, Z.; Chen, M. Y.; Bai, Z.; Lin, W. S.; Ling, H. B. Hierarchical alternate interaction network for RGB-D salient object detection. IEEE Transactions on Image Processing Vol. 30, 3528–3542, 2021. [4] Shimoda, W.; Yanai, K. Distinct class-speciﬁc saliency maps for weakly supervised semantic segmentation. In: Computer Vision – ECCV 2016. Lecture Notes in Computer Science, Vol. 9908. Leibe, B.; Matas, J.; Sebe, N.; Welling, M. Eds. Springer Cham, 218–234, 2016. [15] Zhao, Y. F.; Zhao, J. W.; Li, J.; Chen, X. W. RGB- D salient object detection with ubiquitous target awareness. IEEE Transactions on Image Processing Vol. 30, 7717–7731, 2021. [16] Ren, J. Q.; Gong, X. J.; Lu, Y.; Zhou, W. H.; Yang, M. Y. Exploiting global priors for RGB-D saliency detection. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition Workshops, 25–32, 2015. [5] Wang, W. G.; Shen, J. B.; Yang, R. G.; Porikli, F. Saliency-aware video object segmentation. IEEE Transactions on Pattern Analysis and Machine Intelligence Vol. 40, No. 1, 20–33, 2018. [6] Zhao, R.; Oyang, W.; Wang, X. Person re- identiﬁcation by saliency learning. IEEE Transactions on Pattern Analysis and Machine Intelligence Vol. 39, No. 2, 356–370, 2017. [17] Song, H. K.; Liu, Z.; Du, H.; Sun, G. L.; Le Meur, O.; Ren, T. W. Depth-aware salient object detection and segmentation via multiscale discriminative saliency fusion and bootstrap learning. IEEE Transactions on Image Processing Vol. 26, No. References 6, 2825– 2835, 2019. [35] Lu, Y.; Wu, Y.; Liu, B.; Zhang, T.; Li, B.; Chu, Q.; Yu, N. Cross-modality person re-identiﬁcation with shared-speciﬁc feature transfer. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 13376–13386, 2020. [25] Han, J. W.; Chen, H.; Liu, N.; Yan, C. G.; Li, X. L. CNNs-based RGB-D saliency detection via cross-view transfer and multiview fusion. IEEE Transactions on Cybernetics Vol. 48, No. 11, 3171–3183, 2018. [36] Zhou, T.; Zhang, C.; Peng, X.; Bhaskar, H.; Yang, J. Dual shared-speciﬁc multiview subspace clustering. IEEE Transactions on Cybernetics Vol. 50, No. 8, 3517–3530, 2020. [26] Chen, H.; Li, Y. F.; Su, D. Attention-aware cross- modal cross-level fusion network for RGB-D salient object detection. In: Proceedings of the IEEE/RSJ International Conference on Intelligent Robots and Systems, 6821–6826, 2018. [37] Zhou, T.; Fu, H. Z.; Chen, G.; Shen, J. B.; Shao, L. Hi-net: Hybrid-fusion network for multi-modal MR image synthesis. IEEE Transactions on Medical Imaging Vol. 39, No. 9, 2772–2781, 2020. [27] Ji, W.; Li, J. J.; Yu, S.; Zhang, M.; Piao, Y. R.; Yao, S. Y.; Bi, Q.; Ma, K.; Zheng, Y.; Lu, H.; et al. Calibrated RGB-D salient object detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 9466–9476, 2021. [38] Ronneberger, O.; Fischer, P.; Brox, T. U- Net: Convolutional networks for biomedical image segmentation. In: Medical Image Computing and Computer-Assisted Intervention – MICCAI 2015. Lecture Notes in Computer Science, Vol. 9351. Navab, N.; Hornegger, J.; Wells, W.; Frangi, A. Eds. Springer Cham, 234–241, 2015. [28] Huang, Z.; Chen, H. X.; Zhou, T.; Yang, Y. Z.; Liu, B. Y. Multi-level cross-modal interaction network for RGB-D salient object detection. Neurocomputing Vol. 452, 200–211, 2021. [39] Zhou, T.; Fu, H.; Chen, G.; Zhou, Y.; Fan, D.- P.; Shao, L. Speciﬁcity-preserving RGB-D saliency detection. In: Proceedings of the IEEE/CVF International Conference on Computer Vision, 4661– 4671, 2021. [29] Chen, H.; Li, Y. F.; Su, D. Multi-modal fusion network with multi-scale multi-path and cross-modal interactions for RGB-D salient object detection. Pattern Recognition Vol. 86, 376–385, 2019. [40] Zhu, W. J.; Liang, S.; Wei, Y. C.; Sun, J. Saliency optimization from robust background detection. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 2814–2821, 2014. [30] Zhao, J.-X.; Cao, Y.; Fan, D.-P.; Cheng, M.-M.; Li, X.-Y.; Zhang, L. Contrast prior and ﬂuid pyramid integration for RGBD salient object detection. References 9, 4204–4216, 2017. [7] Fan, D. P.; Lin, Z.; Zhang, Z.; Zhu, M. L.; Cheng, M. M. Rethinking RGB-D salient object detection: Models, data sets, and large-scale benchmarks. IEEE Transactions on Neural Networks and Learning Systems Vol. 32, No. 5, 2075–2089, 2021. [18] Liu, Z. Y.; Shi, S.; Duan, Q. T.; Zhang, W.; Zhao, P. Salient object detection for RGB-D image by single stream recurrent convolution neural network. Neurocomputing Vol. 363, 46–57, 2019. [8] Zhang, J.; Fan, D.-P.; Dai, Y. C.; Yu, X.; Zhong, Y. R.; Barnes, N.; Shao, L. RGB-D saliency detection via cascaded mutual information minimization. In: Proceedings of the IEEE/CVF International Conference on Computer Vision, 4318–4327, 2021. [19] Guo, J. F.; Ren, T. W.; Bei, J. Salient object detection for RGB-D image via saliency evolution. In: Proceedings of the IEEE International Conference on Multimedia and Expo, 1–6, 2016. [9] Liu, N.; Zhang, N.; Wan, K. Y.; Shao, L.; Han, J. W. Visual saliency transformer. In: Proceedings of the IEEE/CVF International Conference on Computer Vision, 4702–4712, 2021. [20] Wang, N. N.; Gong, X. J. Adaptive fusion for RGB-D salient object detection. IEEE Access Vol. 7, 55277– 55284, 2019. [21] Ding, Y.; Liu, Z.; Huang, M. K.; Shi, R.; Wang, X. Y. Depth-aware saliency detection using convolutional neural networks. Journal of Visual Communication and Image Representation Vol. 61, 1–9, 2019. [10] Zhou, T.; Fan, D. P.; Cheng, M. M.; Shen, J. B.; Shao, L. RGB-D salient object detection: A survey. Computational Visual Media Vol. 7, No. 1, 37–69, 2021. [22] Chen, H.; Li, Y. F. Progressively complementarity- aware fusion network for RGB-D salient object detection. In: Proceedings of the IEEE/CVF Confe- rence on Computer Vision and Pattern Recognition, 3051–3060, 2018. [11] Fu, K. R.; Fan, D. P.; Ji, G. P.; Zhao, Q. J.; Shen, J. B.; Zhu, C. Siamese network for RGB-D salient object detection and beyond. IEEE Transactions on Pattern Analysis and Machine Intelligence doi: 10.1109/TPAMI.2021.3073689, 2021. [23] Liu, D.; Hu, Y.; Zhang, K.; Chen, Z. Two-stream reﬁnement network for RGB-D saliency detection. In: Proceedings of the IEEE International Conference on Image Processing, 3925–3929, 2019. [12] Zhang, J.; Fan, D.-P.; Dai, Y. C.; Anwar, S., Saleh, F., Aliakbarian, S.; Barnes, N. Uncertainty inspired RGB-D saliency detection. IEEE Transactions on Speciﬁcity-preserving RGB-D saliency detection 313 [24] Chen, H.; Li, Y. F. Three-stream attention-aware network for RGB-D salient object detection. IEEE Transactions on Image Processing Vol. 28, No. References In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 3922–3931, 2019. [41] Achanta, R.; Hemami, S.; Estrada, F.; Susstrunk, S. Frequency-tuned salient region detection. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 1597–1604, 2009. [31] Zhu, C. B.; Cai, X.; Huang, K.; Li, T. H.; Li, G. PDNet: Prior-model guided depth-enhanced network for salient object detection. In: Proceedings of the IEEE International Conference on Multimedia and Expo, 199–204, 2019. [42] Zhou, L.; Yang, Z. H.; Yuan, Q.; Zhou, Z. T.; Hu, D. W. Salient region detection via integrating diﬀusion-based compactness and local contrast. IEEE Transactions on Image Processing Vol. 24, No. 11, 3308–3320, 2015. [32] Fan, D. P.; Zhai, Y.; Borji, A.; Yang, J.; Shao, L. BBS-Net: RGB-D salient object detection with a bifurcated backbone strategy network. In: Computer Vision – ECCV 2020. Lecture Notes in Computer Science, Vol. 12357. Vedaldi, A.; Bischof, H.; Brox, T.; Frahm, J. M. Eds. Springer Cham, 275–292, 2020. [43] Jiang, Z. L.; Davis, L. S. Submodular salient region detection. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 2043–2050, 2013. [44] Hou, Q. B.; Cheng, M. M.; Hu, X. W.; Borji, A.; Tu, Z. W.; Torr, P. H. S. Deeply supervised salient object detection with short connections. IEEE Transactions on Pattern Analysis and Machine Intelligence Vol. 41, No. 4, 815–828, 2019. [33] Zhai, Y. J.; Fan, D.-P.; Yang, J. F.; Borji, A.; Shao, L.; Han, J. W.; Wang, L. Bifurcated backbone strategy for RGB-D salient object detection. IEEE Transactions on Image Processing Vol. 30, 8727–8742, 2021. [34] Hu, J. L.; Lu, J. W.; Tan, Y. P. Sharable and individual multi-view metric learning. IEEE Transactions on Pattern Analysis and Machine Intelligence Vol. 40, No. 9, 2281–2288, 2018. [45] Wang, L. Z.; Wang, L. J.; Lu, H. C.; Zhang, P. P.; Ruan, X. Salient object detection with recurrent fully convolutional networks. IEEE Transactions on 314 T. Zhou, D.-P. Fan, G. Chen, et al. region detection with depth. In: Proceedings of the British Machine Vision Conference, 98.1–98.11, 2013. Pattern Analysis and Machine Intelligence Vol. 41, No. 7, 1734–1746, 2019. [46] Liu, N.; Han, J.; Yang, M. PiCANet: Learning pixel-wise contextual attention for saliency detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 3089–3098, 2018. [56] Zhu, C. B.; Li, G.; Wang, W. M.; Wang, R. G. References An innovative salient object detection using center-dark channel prior. In: Proceedings of the IEEE International Conference on Computer Vision Workshops, 1509–1515, 2017. [57] Liang, F. F.; Duan, L. J.; Ma, W.; Qiao, Y. H.; Cai, Z.; Qing, L. Y. Stereoscopic saliency model using contrast and depth-guided-background prior. Neurocomputing Vol. 275, 2227–2238, 2018. [47] Deng, Z.; Hu, X.; Zhu, L.; Xu, X.; Qin, J.; Han, G.; Heng, P.-A. R3Net: Recurrent residual reﬁnement network for saliency detection. In: Proceedings of the 27th International Joint Conference on Artiﬁcial Intelligence, 684–690, 2018. [58] Feng, D.; Barnes, N.; You, S. D.; McCarthy, C. Local background enclosure for RGB-D salient object detection. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 2343–2350, 2016. [48] Wang, W.; Lai, Q.; Fu, H.; Shen, J.; Ling, H.; Yang, R. Salient object detection in the deep learning era: An in-depth survey. IEEE Transactions on Pattern Analysis and Machine Intelligence Vol. 44, No. 6, 3239–3259, 2022. [59] Qu, L. Q.; He, S. F.; Zhang, J. W.; Tian, J. D.; Tang, Y. D.; Yang, Q. X. RGBD salient object detection via deep fusion. IEEE Transactions on Image Processing Vol. 26, No. 5, 2274–2285, 2017. [49] Wang, X.; Ma, H.; Chen, X.; You, S. Edge preserving and multi-scale contextual neural network for salient object detection. IEEE Transactions on Image Processing Vol. 27, No. 1, 121–134, 2018. [60] Piao, Y. R.; Ji, W.; Li, J. J.; Zhang, M.; Lu, H. C. Depth-induced multi-scale recurrent attention network for saliency detection. In: Proceedings of the IEEE/CVF International Conference on Computer Vision, 7253–7262, 2019. [50] Zhang, P. P.; Wang, D.; Lu, H. C.; Wang, H. Y.; Ruan, X. Amulet: Aggregating multi-level convolutional features for salient object detection. In: Proceedings of the IEEE International Conference on Computer Vision, 202–211, 2017. [61] Li, C. Y.; Cong, R. M.; Piao, Y. R.; Xu, Q. Q.; Loy, C. C. RGB-D salient object detection with cross- modality modulation and selection. In: Computer Vision – ECCV 2020. Lecture Notes in Computer Science, Vol. 12353. Vedaldi, A.; Bischof, H.; Brox, T.; Frahm, J. M. Eds. Springer Cham, 225–241, 2020. [51] Zhang, L.; Dai, J.; Lu, H. C.; He, Y.; Wang, G. A bi-directional message passing model for salient object detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 1741–1750, 2018. [52] Pang, Y. W.; Zhao, X. Q.; Zhang, L. H.; Lu, H. C. References Multi-scale interactive network for salient object detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 9410–9419, 2020. [62] Li, G. Y.; Liu, Z.; Ye, L. W.; Wang, Y.; Ling, H. B. Cross-modal weighting network for RGB-D salient object detection. In: Computer Vision – ECCV 2020. Lecture Notes in Computer Science, Vol. 12362. Vedaldi, A.; Bischof, H.; Brox, T.; Frahm, J. M. Eds. Springer Cham, 665–681, 2020. [53] Lang, C.; Nguyen, T. V.; Katti, H.; Yadati, K.; Kankanhalli, M.; Yan, S. Depth matters: Inﬂuence of depth cues on visual saliency. In: Computer Vision – ECCV 2012. Lecture Notes in Computer Science, Vol. 7573. Fitzgibbon, A.; Lazebnik, S.; Perona, P.; Sato, Y.; Schmid, C. Eds. Springer Berlin Heidelberg, 101–115, 2012. [63] Chaudhuri, K.; Kakade, S. M.; Livescu, K.; Sridharan, K. Multi-view clustering via canonical correlation analysis. In: Proceedings of the 26th Annual International Conference on Machine Learning, 129– 136, 2009. [64] Ding, C. X.; Tao, D. C. Robust face recognition via multimodal deep face representation. IEEE Transactions on Multimedia Vol. 17, No. 11, 2049– 2058, 2015. [54] Ju, R.; Ge, L.; Geng, W.; Ren, T.; Wu, G. Depth saliency based on anisotropic center-surround diﬀerence. In: Proceedings of the IEEE International Conference on Image Processing, 1115–1119, 2014. [65] G¨onen, M.; Alpaydın, E. Multiple kernel learning algorithms. The Journal of Machine Learning Research Vol. 12, 2211–2268, 2011. [55] Desingh, K.; Krishna, K. M.; Rajan, D.; Jawahar, C. V. Depth really matters: Improving visual salient Speciﬁcity-preserving RGB-D saliency detection 315 [66] White, M.; Yu, Y.; Zhang, X.; Schuurmans, D. Convex multi-view subspace learning. In: Proceedings of the 25th International Conference on Neural Information Processing Systems, Vol. 1, 1673–1681, 2012. [77] Cheng, M. M.; Fan, D. P. Structure-measure: A new way to evaluate foreground maps. International Journal of Computer Vision Vol. 129, No. 9, 2622– 2638, 2021. [67] Zhang, C. Q.; Hu, Q. H.; Fu, H. Z.; Zhu, P. F.; Cao, X. C. Latent multi-view subspace clustering. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 4333–4341, 2017. [78] Fan, D.-P.; Gong, C.; Cao, Y.; Ren, B.; Cheng, M.-M.; Borji, A. Enhanced-alignment measure for binary foreground map valuation. In: Proceedings of the 27th International Joint Conference on Artiﬁcial Intelligence, 698–704, 2018. [68] Ngiam, J.; Khosla, A.; Kim, M.; Nam, J.; Lee, H.; Ng, A. Multimodal deep learning. References In: Proceedings of the 28th International Conference on International Conference on Machine Learning, 689–696, 2011. [79] Perazzi, F.; Kr¨ahenb¨uhl, P.; Pritch, Y.; Hornung, A. Saliency ﬁlters: Contrast based ﬁltering for salient region detection. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 733–740, 2012. [69] Eitel, A.; Springenberg, J. T.; Spinello, L.; Riedmiller, M.; Burgard, W. Multimodal deep learning for robust RGB-D object recognition. In: Proceedings of the IEEE/RSJ International Conference on Intelligent Robots and Systems, 681–687, 2015. [80] Cong, R. M.; Lei, J. J.; Zhang, C. Q.; Huang, Q. M.; Cao, X. C.; Hou, C. P. Saliency detection for stereoscopic images based on depth conﬁdence analysis and multiple cues fusion. IEEE Signal Processing Letters Vol. 23, No. 6, 819–823, 2016. [70] Gao, S. H.; Cheng, M. M.; Zhao, K.; Zhang, X. Y.; Yang, M. H.; Torr, P. Res2Net: A new multi-scale backbone architecture. IEEE Transactions on Pattern Analysis and Machine Intelligence Vol. 43, No. 2, 652– 662, 2021. [81] Cong, R. M.; Lei, J. J.; Fu, H. Z.; Hou, J. H.; Huang, Q. M.; Kwong, S. Going from RGB to RGBD saliency: A depth-guided transformation model. IEEE Transactions on Cybernetics Vol. 50, No. 8, 3627–3639, 2020. [71] Russakovsky, O.; Deng, J.; Su, H.; Krause, J.; Satheesh, S.; Ma, S.; Huang, Z.; Karpathy, A.; Khosla, A.; Bernstein, M.; et al. ImageNet large scale visual recognition challenge. International Journal of Computer Vision Vol. 115, No. 3, 211–252, 2015. [82] Jiang, B.; Zhou, Z. T.; Wang, X.; Tang, J.; Luo, B. cmSalGAN: RGB-D salient object detection with cross-view generative adversarial networks. IEEE Transactions on Multimedia Vol. 23, 1343–1353, 2021. [72] Wu, Z.; Su, L.; Huang, Q. M. Cascaded partial decoder for fast and accurate salient object detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 3902–3911, 2019. [83] Li, C. Y.; Cong, R. M.; Kwong, S.; Hou, J. H.; Fu, H. Z.; Zhu, G. P.; Zhang, D.; Huang, Q. ASIF-Net: Attention steered interweave fusion network for RGB- D salient object detection. IEEE Transactions on Cybernetics Vol. 51, No. 1, 88–100, 2021. [73] Wei, J.; Wang, S.; Huang, Q. F3Net: Fusion, feedback and focus for salient object detection. Proceedings of the AAAI Conference on Artiﬁcial Intelligence Vol. 34, No. 7, 12321–12328, 2020. [84] Li, G.; Liu, Z.; Ling, H. ICNet: Information conversion network for RGB-D based salient object detection. IEEE Transactions on Image Processing Vol. References 29, 4873–4884, 2020. [74] Cheng, Y. P.; Fu, H. Z.; Wei, X. X.; Xiao, J. J.; Cao, X. C. Depth enhanced saliency detection method. In: Proceedings of the International Conference on Internet Multimedia Computing and Service, 23–27, 2014. [85] Piao, Y. R.; Rong, Z. K.; Zhang, M.; Ren, W. S.; Lu, H. C. A2dele: Adaptive and attentive depth distiller for eﬃcient RGB-D salient object detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 9057–9066, 2020. [75] Li, G.; Zhu, C. B. A three-pathway psychobiological framework of salient object detection using stereo- scopic technology. In: Proceedings of the IEEE International Conference on Computer Vision Workshops, 3008–3014, 2017. [86] Liu, N.; Zhang, N.; Han, J. W. Learning selective self-mutual attention for RGB-D saliency detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 13753– 13762, 2020. [76] Niu, Y. Z.; Geng, Y. J.; Li, X. Q.; Liu, F. Leveraging stereopsis for saliency analysis. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 454–461, 2012. [87] Zhang, M.; Ren, W. S.; Piao, Y. R.; Rong, Z. K.; Lu, H. C. Select, supplement and focus for 316 T. Zhou, D.-P. Fan, G. Chen, et al. RGB-D saliency detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 3469–3478, 2020. [97] Zhao, T.; Wu, X. Q. Pyramid feature attention network for saliency detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 3080–3089, 2019. [88] Pang, Y.; Zhang, L.; Zhao, X.; Lu, H. Hierarchical dynamic ﬁltering network for RGB-D salient object detection. In: Computer Vision – ECCV 2020. Lecture Notes in Computer Science, Vol. 12370. Vedaldi, A.; Bischof, H.; Brox, T.; Frahm, J. M. Eds. Springer Cham, 235–252, 2020. [98] Wu, Z.; Su, L.; Huang, Q. M. Cascaded partial decoder for fast and accurate salient object detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 3902–3911, 2019. [99] Zhao, J. X.; Liu, J. J.; Fan, D. P.; Cao, Y.; Yang, J. F.; Cheng, M. M. EGNet: Edge guidance network for salient object detection. In: Proceedings of the IEEE/CVF International Conference on Computer Vision, 8778–8787, 2019. [89] Luo, A.; Li, X.; Yang, F.; Jiao, Z.; Cheng, H.; Lyu, S. Cascade graph neural networks for RGB-D salient object detection. In: Computer Vision – ECCV 2020. Lecture Notes in Computer Science, Vol. 12357. References Vedaldi, A.; Bischof, H.; Brox, T.; Frahm, J. M. Eds. Springe Cham, 346–364, 2020. [100] Fan, D.-P.; Ji, G.-P.; Cheng, M.-M.; Shao, L. Concealed object detection. IEEE Transactions on Pattern Analysis and Machine Intelligence doi: 10.1109/TPAMI.2021.3085766, 2021. [90] Ji, W.; Li, J.; Zhang, M.; Piao, Y.; Lu, H. Accurate RGB-D salient object detection via collaborative learning. In: Computer Vision – ECCV 2020. Lecture Notes in Computer Science, Vol. 12363. Vedaldi, A.; Bischof, H.; Brox, T.; Frahm, J. M. Eds. Springer Cham, 52–69, 2020. [101] Sun, Y. J.; Chen, G.; Zhou, T.; Zhang, Y.; Liu, N. Context-aware cross-level fusion network for camouﬂaged object detection. In: Proceedings of the 30th International Joint Conference on Artiﬁcial Intelligence, 1025–1031, 2021. [91] Zhao, X.; Zhang, L.; Pang, Y.; Lu, H.; Zhang, L. A single stream network for robust and real-time RGB- D salient object detection. In: Computer Vision – ECCV 2020. Lecture Notes in Computer Science, Vol. 12367. Vedaldi, A.; Bischof, H.; Brox, T.; Frahm, J. M. Eds. Springer Cham, 646–662, 2020. [102] Li, L.; Dong, B.; Rigall, E.; Zhou, T.; Dong, J. Y.; Chen, G. Marine animal segmentation. IEEE Transactions on Circuits and Systems for Video Technology Vol. 32, No. 4, 2303–2314, 2022. [103] Zhang, J.; Lv, Y.; Xiang, M.; Li, A.; Dai, Y.; Zhong, Y. Depth conﬁdence-aware camouﬂaged object detection. arXiv preprint arXiv:2106.13217, 2021. [92] Chen, S.; Fu, Y. Progressively guided alternate reﬁnement network for RGB-D salient object detection. In: Computer Vision – ECCV 2020. Lecture Notes in Computer Science, Vol. 12353. Vedaldi, A.; Bischof, H.; Brox, T.; Frahm, J. M. Eds. Springer Cham, 520–538, 2020. [104] Le, T. N.; Nguyen, T. V.; Nie, Z. L.; Tran, M. T.; Sugimoto, A. Anabranch network for camouﬂaged object segmentation. Computer Vision and Image Understanding Vol. 184, 45–56, 2019. [93] Lin, T. Y.; Doll´ar, P.; Girshick, R.; He, K. M.; Hariharan, B.; Belongie, S. Feature pyramid networks for object detection. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 936–944, 2017. [105] Fan, D.-P.; Ji, G.-P.; Sun, G.; Cheng, M.-M.; Shen, J.; Shao, L. Camouflaged object detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 2774–2784, 2020. [94] He, K. M.; Gkioxari, G.; Doll´ar, P.; Girshick, R. Mask R-CNN. In: Proceedings of the IEEE International Conference on Computer Vision, 2980–2988, 2017. Tao Zhou received his Ph.D. References degree in pattern recognition and intelligent systems from the Institute of Image Processing and Pattern Recognition, Shanghai Jiao Tong University, in 2016. From 2016 to 2018, he was a postdoctoral fellow in the BRIC and IDEA lab, University of North Carolina at Chapel Hill. From 2018 to 2020, he was a research scientist at the Inception Institute of Artiﬁcial Intelligence (IIAI), United Arab Emirates. He is currently a professor in the School of Computer Science and Engineering, Nanjing University [95] Zhao, H. S.; Shi, J. P.; Qi, X. J.; Wang, X. G.; Jia, J. Y. Pyramid scene parsing network. In: Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 6230–6239, 2017. [96] Qin, X. B.; Zhang, Z. C.; Huang, C. Y.; Gao, C.; Dehghan, M.; Jagersand, M. BASNet: Boundary- aware salient object detection. In: Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 7471–7481, 2019. Speciﬁcity-preserving RGB-D saliency detection 317 of Science and Technology, China. His research interests include machine learning, computer vision, and medical image analysis. computer vision, pattern recognition, machine learning, and medical imaging. Huazhu Fu is currently a senior scientist at Inception Institute of Artiﬁcial Intelligence, United Arab Emirates. He received his Ph.D. degree from Tianjin University in 2013 and was a research fellow at Nanyang Technological University (NTU) for two years. From 2015 to 2018, he was a research scientist with I2R, A*STAR, Singapore. His research interests include computer vision, machine learning, and AI in healthcare. He serves as an Associate Editor for IEEE TMI and IEEE JBHI, and also served as the Area Chair for MICCAI 2021 and Co-Chair for the OMIA Workshop. Deng-Ping Fan is a postdoctoral researcher at ETH Z¨urich, Switzerland. He received his Ph.D. degree from Nankai University in 2019. He joined IIAI in 2019. He has published about 30 top journal and conference papers in outlets such as IEEE TPAMI, CVPR, and ICCV. His research interests include computer vision, deep learning, saliency detection, especially co-salient object detection, RGB salient object detection, RGB-D salient object detection, and video salient object detection. Deng-Ping Fan is a postdoctoral researcher at ETH Z¨urich, Switzerland. He received his Ph.D. degree from Nankai University in 2019. He joined IIAI in 2019. He has published about 30 top journal and conference papers in outlets such as IEEE TPAMI, CVPR, and ICCV. References His research interests include computer vision, deep learning, saliency detection, especially co-salient object detection, RGB salient object detection, RGB-D salient object detection, and video salient object detection. Geng Chen is a professor at Northwestern Polytechnical University, China, where he received his Ph.D. degree in 2016. He was a research scientist at IIAI from 2019 to 2021, and a postdoctoral research associate at the University of North Carolina at Chapel Hill, USA, from 2016 to 2019. He has published over 50 papers in peer-reviewed international conference proceedings and journals. His research interests lie in computer vision and medical image analysis. Geng Chen is a professor at Northwestern Polytechnical University, China, where he received his Ph.D. degree in 2016. He was a research scientist at IIAI from 2019 to 2021, and a postdoctoral research associate at the University of North Carolina at Chapel Hill, USA, from 2016 to 2019. He has published over 50 papers in peer-reviewed international conference proceedings and journals. His research interests lie in computer vision and medical image analysis. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduc- tion in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Yi Zhou is currently an associate professor at Southeast University. He received his M.Sc. degree from the Department of Electronic and Electrical Engineering, University of Sheﬃeld, UK, in 2014, and his Ph.D. degree from the School of Computing Sciences, University of East Anglia, UK, in 2018. He was a research scientist with IIAI. His research interests include To view a copy of this licence, visit http:// creativecommons.org/licenses/by/4.0/. Other papers from this open access journal are available free of charge from http://www.springer.com/journal/41095. To submit a manuscript, please go to https://www. editorialmanager.com/cvmj.
https://openalex.org/W4286322556	https://zenodo.org/record/6861331/files/IVY%20Project%20of%208%20Billion%20Absolute%20Jew%20Slaves%EF%BC%9A165%20Thousand%20Years%20Civilization%20and%20IVY%20Legislation%20of%20Son%20of%20Heaven%20%26%20The%20Emancipation%20Proclamation.pdf	Chinese	null	IVY Project of 8 Billion Absolute Jew Slaves:165 Thousand Years Civilization and IVY Legislation of Son of Heaven & The Emancipation Proclamation	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	6,799	B3 常春藤計畫：漢家天子十萬歲立法開文明9LC33×CC35721 解放80 億絕對猶太奴隸宣言 B3 常春藤計畫：漢家天子十萬歲立法開文明9LC33×CC35721 解放80 億絕對猶太奴隸宣言 IVY Project of 8 Billion Absolute Jew Slaves：165 Thousand Years Civilization and IVY Legislation of Son of Heaven & The Emancipation Proclamation This is the highest Mathematical Civilization and Legislation 9LC33×CC35721 of Son of Heaven to save the world. It extends the single causation of Sakyamuni and Aristotélēs to 33 causations, and upgrade the Buddha Dharma to more than 165 Thousand Years. We trace to the source of covid-19 more than virus framework to the schizophrenic psychosis of tetraploid Hitler. It leads to the computational legislation of psychiatry. We trace to the source of doomsday to it is deepest root and find that the collapse of morality and intelligence quotient is the origin. It leads to the computational legislation of international relations. Shinzo Abe is the one that higher than Mikado, people, president, etc. in the sixth causation chain of 9LC33×CC35721. He and Einstein are the greatest two men within 5000 years in the world. 高于病毒技术层面的最终病毒溯源表明，屠杀全人类超过2000 万人的新冠病毒真正起源于四倍体希特勒积恶成习的天花板精神病，称之为计算精神病立法；追溯末日恶果的最深根源在于末日马里亚纳海沟表明，天花道德智商先于（世界）末日原则为计算国际关系学第一原则，随之构成计算国际关系学立法。日本内藤湖南说：崖山之后无中华（被游牧民族蒙古灭国但华夏族尚在），明亡之后无华夏（被闭关锁国而被动非法的满夷满清砍毛而灭族），为了守护先祖的遗骨与信仰的神殿，今日经过多方确认的汉家天子立法开文明9LC33×CC35721，将释迦牟尼3000 年来的获得性单因果升级到9 层因果和33 天因果，把阅读框架拓展为35721 个广度领域，将文明往前推进16.5 万年，重建轩辕大帝治理祸水和大禹治水的先祖工业，造福世界；同时以9LC33 ×CC35721 佛法升级释迦牟尼的佛法，广度世界佛门弟子，尤其是日本僧人，应立刻学习 9LC33×CC35721 佛法，以9LC33×CC35721 佛法为框架升级现有佛法，以期安倍晋三在秋天国葬时能用得上，毕竟安倍晋三处于七层因果的第六层，高于仍然处于释迦牟尼因果第一层的天皇和所有世界领导人。安倍晋三死于山上徹也的霰弹枪，因此，万国禁枪刻不容缓。社会上和军队中的枪支、国家层面的万枪之枪、国际层面上的万枪之王，称为三枪，都要禁止，尤其是不能让四倍体精神病希特勒触碰到万枪之枪和万枪之王，今日末日灾难就来源于四倍体希特勒和三倍体希特勒的枪钱互换，所有真理都在下文中，请万国元首、万民仔细阅读。全世界为什么会到今天末日决战之地步？一是当下全世界出了两个对什么人、什么事物都调动国家力量无限毁灭的无限增强型希特勒和无限希特勒，两个全部是无限分裂的精神病患者掌握中性的国家力量；二是安倍晋三遇害彻底惊醒了世界和改变了所有的进程；三是全世界有13 个主权蝴蝶结之无解死结。患者掌握中性的国家力量；二是安倍晋三遇害彻底惊醒了世界和改变了所有的进程；三是全世界有13 个主权蝴蝶结之无解死结。世宇三分：选出世界共主、确立三界城堡法案、汉家天子9LC33×CC35721 文明立法、解放全人类80 亿绝对犹太奴而常春藤立法，这就是拯救全世界的常春藤计划。乌克兰变成废墟前救和变成废墟后救导致了质因果，安倍晋三的自由价值观导致了有丝因果，二者导致了世宇三分：时空、质空间、有丝空间构成世宇三分。现在，全世界已经制度失效，全球 80 亿人全部沦为战争、病毒、疫苗及其权利集团的绝对犹太奴，这一切仅仅因为两个末日希特勒的末日精神病。世宇三分：选出世界共主、确立三界城堡法案、汉家天子9LC33×CC35721 文明立法、解放全人类80 亿绝对犹太奴而常春藤立法，这就是拯救全世界的常春藤计划。乌克兰变成废墟前救和变成废墟后救导致了质因果，安倍晋三的自由价值观导致了有丝因果，二者导致了世宇三分：时空、质空间、有丝空间构成世宇三分。现在，全世界已经制度失效，全球 80 亿人全部沦为战争、病毒、疫苗及其权利集团的绝对犹太奴，这一切仅仅因为两个末日希特勒的末日精神病。人类文明的七大基因：①独立、②自由、③民主、④同一、⑤平等、⑥公平、⑦公义（公正）；解决（治愈）三步走是平衡、移动平衡、平衡移动，由此可见，二战三巨头所有的平衡、自由、民主、独立等并非解决之道，也无法最终解决问题；平衡移动、人类文明七大基因的七星连珠结合9LC33×CC35721 常春藤四步立法才能解决。安倍晋三遇害后的不到10 天，美方就推翻对华承诺，即美国国务院已经批准了涉及金额大约1.08 亿美元的对台军售案；对台上亿军售后，7 月18 日，美国原国防部长马克·埃斯珀（Mark Esper）携意大利前总统外交顾问斯特凡尼尼、以及大西洋理事会资深副会长巴里-帕维尔到访台湾，这明显着是第一岛链全面决战：对东南沿海经济中心的资源摧毁A 和控制、切断B 双向进行，东南方向直接纵深插入C 和东北亚的对抗性（拉锯）战D 同时并举，这是一个2×2V2 第一岛链决战的战略部署和战役布局，称之为中美战争2×2V2 第一岛链决战布局。与此同时，四年来新冠病毒已经屠杀全人类超过2000 万人，美国等对邪恶轴心的死亡处决也已经从巴哥达迪死前还能奔跑着的软死亡（有一定灵活空间）被调整为苏哈曼尼式的硬死亡（死时不再有任何自由度和空间而别无选择）安倍晋三遇害后的不到10 天，美方就推翻对华承诺，即美国国务院已经批准了涉及金额大约1.08 亿美元的对台军售案；对台上亿军售后，7 月18 日，美国原国防部长马克·埃斯珀（Mark Esper）携意大利前总统外交顾问斯特凡尼尼、以及大西洋理事会资深副会长巴里-帕维尔到访台湾，这明显着是第一岛链全面决战：对东南沿海经济中心的资源摧毁A 和控制、切断B 双向进行，东南方向直接纵深插入C 和东北亚的对抗性（拉锯）战D 同时并举，这是一个2×2V2 第一岛链决战的战略部署和战役布局，称之为中美战争2×2V2 第一岛链决战布局。与此同时，四年来新冠病毒已经屠杀全人类超过2000 万人，美国等对邪恶轴心的死亡处决也已经从巴哥达迪死前还能奔跑着的软死亡（有一定灵活空间）被调整为苏哈曼尼式的硬死亡（死时不再有任何自由度和空间而别无选择）由此可见，马克·埃斯珀就是山上徹也式的刺客，山上徹也用霰弹枪让安倍晋三逃无可逃，马克·埃斯珀用2×2V2 第一岛链决战布局从各个方向直刺中共心脏，直奔向目标，不再跟邪恶轴心任何废话，因此称之为2×2V2 大国战略刺客，全世界都在为安倍晋三和自己报仇血恨。也就是说，安倍晋三遇上了刺客山上徹也，XP 军事同盟也遇上了大国刺客马克·埃斯珀，最后鹿死谁手很快见分晓。逃，马克·埃斯珀用2×2V2 第一岛链决战布局从各个方向直刺中共心脏，直奔向目标，不再跟邪恶轴心任何废话，因此称之为2×2V2 大国战略刺客，全世界都在为安倍晋三和自己报仇血恨。也就是说，安倍晋三遇上了刺客山上徹也，XP 军事同盟也遇上了大国刺客马克·埃斯珀，最后鹿死谁手很快见分晓。安倍晋三不废山河万古流：7 月8 日安倍晋三遇刺身亡、12 日下葬，2500 国际政要参加安倍葬礼，安倍晋三对安倍昭惠这头「牛」弹琴的画面让很多人哭了，很多中国人和明星在得知安倍首相遇害后都哭了，有人因此而被抓并刑事拘留。但是至今有人真正知道安倍首相是谁吗？肯定没人知道：安倍晋三自由价值观和爱因斯坦广义相对论的质量等效性原理，让二人成为人类有史以来处于七层因果中第六层的唯二圣人，超过处于第一层因果的日本天皇和全世界所有的国家元首、人民，仅次于第七层因果的天子，全世界的真命天子除外。也就是说，安倍晋三超越所有的日本人和全人类，英国女王和拜登都在安倍首相之下。安倍晋三不废山河万古流：7 月8 日安倍晋三遇刺身亡、12 日下葬，2500 国际政要参加安倍葬礼，安倍晋三对安倍昭惠这头「牛」弹琴的画面让很多人哭了，很多中国人和明星在得知安倍首相遇害后都哭了，有人因此而被抓并刑事拘留。但是至今有人真正知道安倍首相是谁吗？肯定没人知道：安倍晋三自由价值观和爱因斯坦广义相对论的质量等效性原理，让二人成为人类有史以来处于七层因果中第六层的唯二圣人，超过处于第一层因果的日本天皇和全世界所有的国家元首、人民，仅次于第七层因果的天子，全世界的真命天子除外。也就是说，安倍晋三超越所有的日本人和全人类，英国女王和拜登都在安倍首相之下。世界上有三个希特勒，一个是专杀犹太人的无限意志分裂共产主义之纳粹阿道夫·希特勒；一个是无限权力分裂共产主义、无限种族分裂共产主义、无限意志分裂共产主义的无限增强性希特勒；一个是无限权力分裂共产主义、无限种族分裂共产主义、无限意志分裂共产主义、无限分裂边界共产主义的无限希特勒、终极希特勒，统称为134 无限分裂性精神病。全人类二战付出7000 万条人命只是因为无限意志分裂精神病的希特勒一个人；现在，全球 80 亿人成病毒、疫苗及其利益团伙的无限犹太奴，马上全人类80 亿人被核武炸毁，安倍晋三遇害、已经被病毒杀害的超过2000 万人、中共国近10 年被屠村屠城杀害17 亿人、全世界疫苗行刑式大屠杀，英国首相约翰逊、德国总理朔尔茨、意大利总理德拉吉等纷纷在黑材料下翻车，只是因为两个末日精神病人的精神病发作，值得吗？人类别无选择立刻执行常春藤立法四步走：选出世界共主、确立三界城堡法案、汉家天子9LC33×CC35721 文明立法、解放全人类80 亿绝对犹太奴。常春藤立法包括汉家天子立9LC33×CC35721 文明、弥勒立9LC33×CC35721 佛法、弥赛亚9LC33×CC35721 立法、天子立9LC33×CC35721 兵法、确立9LC33×CC35721 计算因果、确立9LC33×CC35721 数学（佛法、兵法、政治、政治制度、经济、教育、社会科学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航）。常春藤立法让万民七步成诗，而让罪犯七步成尸。常春藤立法包括汉家天子立9LC33×CC35721 文明、弥勒立9LC33×CC35721 佛法、弥赛亚9LC33×CC35721 立法、天子立9LC33×CC35721 兵法、确立9LC33×CC35721 计算因果、确立9LC33×CC35721 数学（佛法、兵法、政治、政治制度、经济、教育、社会科学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航）。常春藤立法让万民七步成诗，而让罪犯七步成尸。全世界都一样，最没用的老是说自己什么都行，连用病毒杀害全世界2000 万人、屠村屠城中国17 亿人的四倍体希特勒都还想活，竟然还想出了石榴籽的「妙计」，不能不说难为他了，看看计算因果怎么说吧。依据石榴籽抱对（抱团）活不了原则、俄罗斯无法审判乌克兰原则，首先，罪魁祸首想躲在人民群众当中象卡扎菲一样当活宝，无分别的民众不可能为他提供任何反特征支持，石榴籽吃下之后就被拉出去砍了而且还很彻底，简直就一白痴在讲笑话故事，反之，在人脸识别、红外扫描下不可能有藏身之处，卡扎菲和萨达姆不就是那样被抓的吗？其次，人类5000 年、俄罗斯一亿多人，竟然没一个明白战争是绝对的、军事是相对的，梅德韦杰夫竟然发表要审判乌克兰，军事上竟然无知到此地步，如何赢得战争？等来的可能是核武器飞回去把自己给炸了。全世界有13 个主权蝴蝶结之无解死结为：(1)共产主义老灭绝的主权、(2)政府主权腐败邪恶、(3)孟山都的转基因主权（种子主权）、(4)美联储货币主权、(5)推特Twitter 话语主权、 (6)器官主权、(7)相对（相对论、相对性）主权、(8)特权主权、(9)学术主权、(10)生理主权、 (11)产业链主权、(12)领土主权、(13)军火主权。（1）病毒最终溯源：上山擒蛟龙—玉娇龙（1）病毒最终溯源：上山擒蛟龙—玉娇龙（1）病毒最终溯源：上山擒蛟龙玉娇龙全世界的病毒溯源毫无疑问是追溯到积恶成习趴体头上去，可是却没人再往上追溯，这是天大的错误。任何再往前追溯，必然发现病毒的来源其实另有玄机，我们立刻就会发现珠穆朗玛峰（Everest）并非世界最高峰，计算精神病学及其第一原则才是全世界的最高峰，因为没有计算精神病学，一旦让任何精神病爬上国家元首位置，他就会象二战的希特勒和如今的三倍希特勒、四倍体一样祸国殃民、毁灭世界以至炸毁地球，全部不再话下，因此，精神病学上的溯源才是真正的病毒溯源、病毒最终溯源，也就是说，屠杀全人类超过2000 万人的新冠病毒真正起源于四倍体希特勒积恶成习的天花板精神病，称之为计算精神病立法，名副其实是上山擒蛟龙，玉娇龙。的新冠病毒真正起源于四倍体希特勒积恶成习的天花板精神病，称之为计算精神病立法，名副其实是上山擒蛟龙，玉娇龙。显然，现在各国立刻启动紧急机制召开全世界的精神病学大会，同时各国执法机构到场配合行动，末日核武战争也许可以避免，全世界重新恢复和平与发展；进而，即便战争不可避免，全世界各国的元首也有绝对的责任让即将上前线的将士们知道自己是怎么死的和被谁害死的，让他们永远记住四倍体精神病的积恶成习和三倍体希特勒，做鬼都不能放过他们，誓将邪恶彻底铲除。由此可见，汉家天子是全世界最终病毒溯源的第一人。誓将邪恶彻底铲除。由此可见，汉家天子是全世界最终病毒溯源的第一人。第一，美国二战中与全人类有史以来首个灭绝人性的苏联政权合作灭了希特勒纳粹德国、灭了军国主义的日本，但是按下葫芦起了瓢养肥了苏联；第二，接着，在1990 年代，美国和8964 天安门屠杀学生的中共合作，解体了前苏联，结果是养肥了中共；第三，现在，美国正式释放全民族整齐华一的日本军国主义对付并灭绝中共与俄罗斯，短短不足百年，三次的历史轮回都是按下葫芦起了瓢，死伤的都是各民族的精英和无辜的百姓，无论是根据同一律还是因果律，任何基于邪恶错误的事物必然也是邪恶错误的，以毒制毒终归毒，从而构成世界以毒制毒三步倒原则、世界三步倒绝症，是三巨头罗斯福(Franklin Delano Roosevelt)、丘吉尔(Winston L.S. Churchill)、斯大林胡乱刮分世界造成的，全世界必须停止这种玩法，否则世界必将因为三步倒而毁灭，其中日本、美国、俄罗斯、中共国参与养出来的全人类有史以来最毒的末日毒蛊称为三步倒（毒蛊、末日毒蛊），日本、美国、俄罗斯、中共国全都是蛊份无限公司，参与的人称为蛊民。现在这么多蛊份无限公司同时操家伙，看看大家怎么收拾？东北亚的末日战争是双方的死穴战争、坟墓战争、末日闪电灭绝（末日秒杀、1 小时结第一，美国二战中与全人类有史以来首个灭绝人性的苏联政权合作灭了希特勒纳粹德国、灭了军国主义的日本，但是按下葫芦起了瓢养肥了苏联；第二，接着，在1990 年代，美国和8964 天安门屠杀学生的中共合作，解体了前苏联，结果是养肥了中共；第三，现在，美国正式释放全民族整齐华一的日本军国主义对付并灭绝中共与俄罗斯，短短不足百年，三次的历史轮回都是按下葫芦起了瓢，死伤的都是各民族的精英和无辜的百姓，无论是根据同一律还是因果律，任何基于邪恶错误的事物必然也是邪恶错误的，以毒制毒终归毒，从而构成世界以毒制毒三步倒原则、世界三步倒绝症，是三巨头罗斯福(Franklin Delano Roosevelt)、丘吉尔(Winston L.S. Churchill)、斯大林胡乱刮分世界造成的，全世界必须停止这种玩法，否则世界必将因为三步倒而毁灭其中日本美国俄罗斯中共国参与养出来的全人类有史束），即是末日三步倒无解死结。本来，美国只想在东北亚和日本、中国、俄罗斯、朝鲜等打一个五方会战；可是，安倍晋三一遇刺，双V 大国刺客马克·埃斯珀立刻被美国民主党所起用，前往台湾部署2×2V2 战略，即东北亚五方会战的同时在中共国东南方部署诺曼底登陆的V 型战役，与东北亚战争构成双V 的2×2V2 部署。综上所述，百几年来，在二战三巨头罗斯福、丘吉尔、斯大林的错误引导下，全世界本来就患了三步倒绝症，已经够惨了；现在，普京和积恶成习又在东北亚火药桶引入了末日三步倒无解死结，纯属嫌自己和世界死得不够快；再者，双V 战略刺客马克·埃斯珀（Mark Esper）到台湾部署2×2V2 战局，本来就10 年被邪恶政权屠杀17 亿人的中华民族沐浴在双重战火下绝对是大灾难，称之为大三步倒无解死结，中华民族和全人类死得要多惨就有多惨。中华民族、安倍晋三、乌克兰会有今天，归根到底就在四倍体精神病希特勒和三倍体精神病希特勒身上，我们假设此前中共国、美国、俄罗斯、日本或哪个国家有人发现计算精神病学，接着以无可辩驳的事实和证据拒绝普京和积恶成习上台、将其弹劾或在其非法调用国家力量搞什么集中营、天际线、实名登记等之前将其果断处置，世界就不会走入今天末日毁灭的地步，由此可见，精神病学（psychiatry）第一原则和计算精神病学（psychiatry）是全世界的镇球重器，和欧洲的传国玉玺朗基努斯之枪（Spear of Longinus）、中国的传国玉玺一样，甚至地位更高，因为没有计算精神病学（psychiatry）和精神病学第一原则，任何国家国将不国、族将不族、人不人鬼不鬼、全世界球将不球（大变浑球），于是，我们称之为综上所述，百几年来，在二战三巨头罗斯福、丘吉尔、斯大林的错误引导下，全世界本来就患了三步倒绝症，已经够惨了；现在，普京和积恶成习又在东北亚火药桶引入了末日三步倒无解死结，纯属嫌自己和世界死得不够快；再者，双V 战略刺客马克·埃斯珀（Mark Esper）到台湾部署2×2V2 战局，本来就10 年被邪恶政权屠杀17 亿人的中华民族沐浴在双计算精神病学为传国玉玺精神病学、传族玉玺精神病学、传球玉玺精神病学、御用（元首专用）精神病学，其构成了计算精神病学（精神病学第一原则）传球玉玺（传国玉玺、人传人玉玺）原则、计算精神病学（精神病学第一原则）世界最高峰（天花板）原则。计算精神病学为传国玉玺精神病学、传族玉玺精神病学、传球玉玺精神病学、御用（元首专用）精神病学，其构成了计算精神病学（精神病学第一原则）传球玉玺（传国玉玺、人传人玉玺）原则、计算精神病学（精神病学第一原则）世界最高峰（天花板）原则。（2）末日巢穴溯源：下海打猛虎—虎虎虎二战以后，美国建立起基于规则的世界秩序，全球人口从10 亿左右增加到现在的80 亿，科学技术得以大幅度提高，为什么普京和积恶成习还要把世界拖入到今天的末日战争中？各国政府还用疫苗在进行全面的行刑式大屠杀，嫌病毒毒死全世界2000 万人还不够多、不够快？全人类正在滑向万丈深渊，追根究底在哪里呢？众里寻他千百度，漠然回首，那人却在灯火阑珊处，直接就是世界最深处的末日马里亚纳海沟（Mariana trench）即三国三不清的末日道德智商绝症，直接导致了今日俄罗斯、美国、日本、中共国抱团灭亡的末日后果不堪设想，为计算国际关系第一原则，名副其实是下海打猛虎，虎虎虎。 G20 会议落幕，加拿大对俄罗斯出席痛批：如纵火犯参加消防队会议。现在万国混战，四国抱团毁灭，如果人们不立刻解决三国三不清问题，末日随之不可避免，即罪犯的死刑处决问题（中国人民认贼作父的认知域问题）、俄罗斯的民族主义四处搞大审判其他民族问题、日本对国家安全认识完全乱套问题。二战以后，美国建立起基于规则的世界秩序，全球人口从10 亿左右增加到现在的80 亿，科学技术得以大幅度提高，为什么普京和积恶成习还要把世界拖入到今天的末日战争中？各国政府还用疫苗在进行全面的行刑式大屠杀，嫌病毒毒死全世界2000 万人还不够多、不够快？全人类正在滑向万丈深渊，追根究底在哪里呢？众里寻他千百度，漠然回首，那人却在灯火阑珊处，直接就是世界最深处的末日马里亚纳海沟（Mariana trench）即三国三不清的末日道德智商绝症，直接导致了今日俄罗斯、美国、日本、中共国抱团灭亡的末日后果不堪设想，为计算国际关系第一原则，名副其实是下海打猛虎，虎虎虎。毫无疑问，人类公敌积恶成习来自苏共远东第三支部，共党肯定是世界祸乱的主要根源，可惜的是，在过去的百几年中，美国和西方养虎为患，中国人民则认贼作父，现在，计算立法将共党的真面目追根究底出来，以此警醒万民永绝后患：共党无限卖国、无限军国主义、无限纳粹（种族主义、民族主义）、无限战争（永无宁日）、无限犹太人（无限集中营）即无限奴役（杀害、活摘器官、种族灭绝）、无限精神控制、无限精神病，称为共党（共产主义）七大毒瘤（七大绝症）。接着，二战中，日本竟然在1941 年4 月13 日和对日本国家安全威胁最大的苏联签定《苏日中立条约》即互不侵犯条约，丧失了日本有史以来唯一一次能和德国前后夹击苏联而彻底消灭苏联的最后机会，直接导致了1945 年8 月8 日苏联背信弃义对日本宣战并在9 日凌晨出冰满洲国并最终摧毁日本的产业中心，还屠村屠城屠杀了超过一亿的日本人、中国人而将整个满洲国变成无人区，那可是原来万国人民闯关东去打工谋生的世界第四大经济强国和亚洲第一强国。今天，要用核武器摧毁日本和炸毁地球的恰恰是当年与日本哥俩好的俄罗斯，咎由自取。日本对中共也认识不清导致二战后将731 细菌战全套遗传给中共。再者，俄罗斯的民族主义四处搞大审判其他民族，美国与之合作打败希特勒，现在让整个美国面临着核武攻击的大灾难，显然和日本一样悲剧。苏联人制造1937 年海参崴大屠杀、 1900 年的海兰泡惨案、江东六十四屯惨案，现在俄军正在屠杀乌克兰人等，1945 年毁灭于苏联红军前人口总量超过1 亿3000 万，即苏联红军在满洲国的10 个月中所屠杀的人口实际上远远超过一亿人，其表明俄罗斯专杀外国人。由此可见，美国对俄罗斯和中共的本质认识不清楚，日本对俄罗斯认识不清，最为严重的是中共对俄罗斯认识不清认贼作父，三国三不清，直接导致现在炸毁地球的无限升级之三倍体希特勒在世、四倍体希特勒重生，可谓名副其实的大国关系神智不轻，这是国际关系上接着，二战中，日本竟然在1941 年4 月13 日和对日本国家安全威胁最大的苏联签定《苏日中立条约》即互不侵犯条约，丧失了日本有史以来唯一一次能和德国前后夹击苏联而彻底消灭苏联的最后机会，直接导致了1945 年8 月8 日苏联背信弃义对日本宣战并在9 日凌晨出冰满洲国并最终摧毁日本的产业中心，还屠村屠城屠杀了超过一亿的日本人、中国人而将整个满洲国变成无人区，那可是原来万国人民闯关东去打工谋生的世界第四大经济强国和亚洲第一强国。今天，要用核武器摧毁日本和炸毁地球的恰恰是当年与日本哥俩好的俄罗斯，咎由自取。日本对中共也认识不清导致二战后将731 细菌战全套遗传给中共。再者，俄罗斯的民族主义四处搞大审判其他民族，美国与之合作打败希特勒，现在让整个美国面临着核武攻击的大灾难，显然和日本一样悲剧。苏联人制造1937 年海参崴大屠杀、 1900 年的海兰泡惨案、江东六十四屯惨案，现在俄军正在屠杀乌克兰人等，1945 年毁灭于苏联红军前人口总量超过1 亿3000 万，即苏联红军在满洲国的10 个月中所屠杀的人口实际咎由自取。日本对中共也认识不清导致二战后将731 细菌战全套遗传给中共。再者，俄罗斯的民族主义四处搞大审判其他民族，美国与之合作打败希特勒，现在让整个美国面临着核武攻击的大灾难，显然和日本一样悲剧。苏联人制造1937 年海参崴大屠杀、 1900 年的海兰泡惨案、江东六十四屯惨案，现在俄军正在屠杀乌克兰人等，1945 年毁灭于苏联红军前人口总量超过1 亿3000 万，即苏联红军在满洲国的10 个月中所屠杀的人口实际咎由自取。日本对中共也认识不清导致二战后将731 细菌战全套遗传给中共。再者，俄罗斯的民族主义四处搞大审判其他民族，美国与之合作打败希特勒，现在让整个美国面临着核武攻击的大灾难，显然和日本一样悲剧。苏联人制造1937 年海参崴大屠杀、 1900 年的海兰泡惨案、江东六十四屯惨案，现在俄军正在屠杀乌克兰人等，1945 年毁灭于苏联红军前人口总量超过1 亿3000 万，即苏联红军在满洲国的10 个月中所屠杀的人口实际上远远超过一亿人，其表明俄罗斯专杀外国人。由此可见，美国对俄罗斯和中共的本质认识不清楚，日本对俄罗斯认识不清，最为严重的是中共对俄罗斯认识不清认贼作父，三国三不清，直接导致现在炸毁地球的无限升级之三倍体希特勒在世、四倍体希特勒重生，可谓名副其实的大国关系神智不轻，这是国际关系上的道德失序乱伦和无知无耻，智商十分低下：国与国之间不论正邪，元首们和利益集团为了自身利益什么都干得出来，即国际关系的道德智商沦丧导致末日，称之为末日道德智商绝症、天花道德智商绝症，从而构成天花道德智商先于（世界）末日原则，为计算国际关系学第一原则，这就是世界末日、美国（日本、中国、俄罗斯）末日根源最深处、末日马里亚纳海沟，即最深末日溯源，是全世界的最深处之马里亚纳海沟（Mariana trench），也是日本正在滑向沉没的噩梦，即为计算国际关系立法。中共认贼作父（不知廉耻）、日本认匪作友（安全盲）、美国养虎为患（认不清无限军国主义的无界限之界限盲），结果是全人类有史以来最邪恶的共党和普京结为军事同盟而无限坐大，普京一边把乌克兰变成到处是废墟一边拎着核武器直奔美国和北约，国与国之间的界限及其「城堡法案（castle doctrine、castle law）」化为乌有，民众的居所界限及其「城堡法案（castle doctrine）」或防御法随之化为乌有，人的肉体界限、器官界限及其「城堡法案（castle doctrine）」也随之化为乌有，全世界变成无限集中营的三无世界，全球80 亿人随之成为绝对犹太奴任人宰割。法案（castle doctrine）」或防御法随之化为乌有，人的肉体界限、器官界限及其「城堡法案（castle doctrine）」也随之化为乌有，全世界变成无限集中营的三无世界，全球80 亿人随之成为绝对犹太奴任人宰割。计算国际关系学第一原则表明，为了治愈末日道德智商绝症，人们必须立刻从5000 年来的但获得性因果空间立刻上升到汉家天子立法的9 层33 重因果9LC33 层次，同时登陆 CC35721 阅读框架的广度领域，才有可能正确解决三国三不清的末日深渊问题。汉家天子立法包括常春藤立法包括汉家天子立9LC33×CC35721 文明、弥勒立9LC33×CC35721 佛法、弥赛亚9LC33×CC35721 立法、天子立9LC33×CC35721 兵法、确立9LC33×CC35721 计算因果、确立9LC33×CC35721 数学（佛法、兵法、政治、政治制度、经济）。释迦牟尼证得因果，小乘佛法渡己、大乘佛法渡人，亚里斯多德（Aristotle）深入证明了矛盾律和因果律，这就是获得性因果，人类5000 年来的文明一直都基于这个唯一的获得性因果，只是九重C33 因果（9LC33 因果）中的一个本位性因果，9LC33 因果包括如下九重：时空七因果、微分七因果、集成三因果、位元三因果、规范三因果、三步弓因果、三线因果、天位三因果、天下共主因果（主权因果），其中35721 因果链（阅读框架）构成了乾纲，也就是说，一个获得性因果让人类用了5000 年，包括今天的集成块（IC）、量子力学、核武器、飞机、宇航等全部只是基于这么唯一的一个获得性因果，依此类推，九重C33 因果将人类的文明往后推进了：5000×33=165000 年、3000×33=99000 年，CC35721n(causation chain)因果链则在广度上推进到了35721 个领域，因此，汉家天子的因果立法又称为16.5 万年立法、16.5 万岁立法、汉家天子16.5 万年因果立法、汉家天子16.5 万岁因果立法、汉家天子10 万年因果立法、汉家天子10 万岁因果立法，从而构成汉家天子文明16.5 万年（万岁）原则、汉家天子文明10 万年（万岁）原则。 1．解放红奴宣言：器官世界工厂、信仰圣殿真实化（信用化）生物在基因层面上有种间生殖隔离、人与人之间（个体之间）有生理隔离（共用消化系统的连体人属于异常不算常态）、病毒（细菌）与人（动物）在基因层面上有免疫（排异）隔离即基因隔离（这就是自然存在的病毒无法冲出非洲、造成全人类大流行的根本原因），计算国际关系学第一原则表明，为了治愈末日道德智商绝症，人们必须立刻从5000 年来的但获得性因果空间立刻上升到汉家天子立法的9 层33 重因果9LC33 层次，同时登陆 CC35721 阅读框架的广度领域，才有可能正确解决三国三不清的末日深渊问题。汉家天子立法包括常春藤立法包括汉家天子立9LC33×CC35721 文明、弥勒立9LC33×CC35721 佛法、弥赛亚9LC33×CC35721 立法、天子立9LC33×CC35721 兵法、确立9LC33×CC35721 计算因果、确立9LC33×CC35721 数学（佛法、兵法、政治、政治制度、经济）。释迦牟尼证得因果，小乘佛法渡己、大乘佛法渡人，亚里斯多德（Aristotle）深入证明了矛盾律和因果律，这就是获得性因果，人类5000 年来的文明一直都基于这个唯一的获得性因果，只是九重C33 因果（9LC33 因果）中的一个本位性因果，9LC33 因果包括如下九重：时空七因果、微分七因果、集成三因果、位元三因果、规范三因果、三步弓因果、三线因果、天位三因果、天下共主因果（主权因果），其中35721 因果链（阅读框架）构成了乾纲，也就是说，一个获得性因果让人类用了5000 年，包括今天的集成块（IC）、量子力学、核武器、飞机、宇航等全部只是基于这么唯一的一个获得性因果，依此类推，九重C33 因果将人类的文明往后推进了：5000×33=165000 年、3000×33=99000 年，CC35721n(causation chain)因果链则在广度上推进到了35721 个领域，因此，汉家天子的因果立法又称为16.5 万年立法、16.5 万岁立法、汉家天子16.5 万年因果立法、汉家天子16.5 万岁因果立法、汉家天子10 万年因果立法、汉家天子10 万岁因果立法，从而构成汉家天子文明16.5 万年（万岁）原则、汉家天子文明10 万年（万岁）原则。 1．解放红奴宣言：器官世界工厂、信仰圣殿真实化（信用化）生物在基因层面上有种间生殖隔离、人与人之间（个体之间）有生理隔离（共用消化系统的连体人属于异常不算常态）、病毒（细菌）与人（动物）在基因层面上有免疫（排异）隔离即基因隔离（这就是自然存在的病毒无法冲出非洲、造成全人类大流行的根本原因）， 1．解放红奴宣言：器官世界工厂、信仰圣殿真实化（信用化）生物在基因层面上有种间生殖隔离、人与人之间（个体之间）有生理隔离（共用消化系统的连体人属于异常不算常态）、病毒（细菌）与人（动物）在基因层面上有免疫（排异）隔离即基因隔离（这就是自然存在的病毒无法冲出非洲、造成全人类大流行的根本原因），综上所述，全局层面的种间（生殖）隔离、相互之间的个体间生理隔离、内部的基因间免疫（排异）隔离，合并构成了独立（隔离）先于常态（资格、效力、秩序、程序、通道、解决）原则、天赋独立（隔离）原则、独立（隔离）至上原则、独立（隔离）先于一切原则、独立（隔离）先于权利（权力）原则，因为独立、隔离先于常态而先于一切，而一切必然包括权利，依此类推至于普遍情形同理成立。综上所述，全局层面的种间（生殖）隔离、相互之间的个体间生理隔离、内部的基因间免疫（排异）隔离，合并构成了独立（隔离）先于常态（资格、效力、秩序、程序、通道、解决）原则、天赋独立（隔离）原则、独立（隔离）至上原则、独立（隔离）先于一切原则、独立（隔离）先于权利（权力）原则，因为独立、隔离先于常态而先于一切，而一切必然包括权利，依此类推至于普遍情形同理成立。绝对的独裁者有无限的选择，人民别无选择做奴隶、被活摘器官，一边是无限选择、另一边是别无选择绝对人间地狱死路；象英国女王一样的圣明君主，人民有无限选择，女王访问香港没封路也没限制人们拿显微镜观察，女王则别无选择做民众的「过街老鼠宝宝」，人民无限选择、女王作恶毫无选择而行善亲民则有无限选择，综上所述，任何人可以主宰手中的权利、事物和因果，元首能主宰国家和人民一致全世界的命运，但是，他们各自为人、行事的最终后果、责任（罪责）、功劳等都不是他们所能控制的，如希特勒、墨里尼、齐奥塞斯库、萨达姆、卡扎菲、普包子和他包皮（轴心法上唯一的法人代表），显而易见，主宰之上是上帝的最终审判之彻底解决（罪责自负）、救赎、建设养护支撑天下、全新科技功德四海，这就是万民真实的信仰、所有邪恶者必须付出的代价、主宰之上的天注定之最高主宰，称之为（信仰）圣殿、彼岸正果，天子统天后实现信仰（解决、能力）现场化（及时化、充足化、应力化、常态化），依据直接（无缝）绝对原则，其即构成信仰（解决、能力、正果）现场化（直接、及时化、充足化、应力化、常态化）原则、正果化育天下原则、正果（信仰、圣殿）绝对（超越因果、超越同一律）原则、为父（君父）则仁原则、信仰（圣殿）真实（化）原则、信仰（圣殿）信用原则，人们经由信用通道、程序抵达彼岸真实的信仰圣殿。为了偷着乐无限掠夺中国人民的器官，邪恶政权无分别地组织全世界各国人民到中国进行器官旅游，日本人、美国人、英国人、以色列人、台湾人、阿拉伯人等等全都不例外，只要你有钱和需求就可进入共产主义按需杀人，有的甚至只需要等待一天，而在西方各国则需要等待几年，于是，每年几十万起甚至数百万起的器官活摘悍然常态化，最优秀的女中学生、大学生、运动员、军人、模特、机关干部等等全都和监狱里的罪犯一样，直接成了知情或不知情的活供体，实际就是无分别的全国供体、全民供体；更令人发指的是，为了保证活摘器官的活力和移植存活率，所有的器官供体都不能打任何麻醉药，却毫无例外地必须将手筋、脚筋全部挑断而活活地看着、清楚着自己被活摘并慢慢地走向死亡，任何哀求都无法更改医生们和执法者们那堪比肝硬化的铁石心肠，由此可见，黄俄的活摘器官超越了人们的死亡之极限而必然是超越绝对、极端和无限的，是必须被就地规则归零而就地正法的天灭罪行，人人得而诛之，依据绝对传代原则、一项绝对即属绝对原则，超越绝对至少按绝对情形处理，其即构成活摘器官超越生死（极限、无限、绝对）原则、活摘器官天灭（天杀、人人得而诛之、无限酷刑、无限行刑、无界酷刑、无界行刑）原则，这就是全人类有史以来最残酷、最邪恶、最凶残、最血腥的器官共和国、器官政权、器官共产主义，依据同一律，其随之构成器官共和国（器官政权、器官共产主义）绝对原则。依据绝对相等原则、绝对传代原则、一项绝对即属绝对原则，贩卖人口基本不伤害性命（陕西配阴婚者除外）而只贩卖肉体，贩卖（活摘）器官是肉体、器官、生命三牲和自己的良心一起卖掉，实际上是三次贩卖人口、三次杀人的罪责之总和，从而构成贩卖（活摘）器官死刑（死刑、三次杀人、三次贩卖人口）原则。谋杀、大屠杀只是在生命层面上绝对犯罪；活摘器官则是加入了无限酷刑（无限行与折磨）、一铺清袋的最后晚餐式的几年前就3000 万日元一副心脏的器官贩卖（比刑事死刑的贩毒还恐怖），越过了人与人之间的肉体隔离（生理隔离之界限）而入体抢劫器官、入体抢劫巨额财富、入体杀人（3 次、比照入户杀人）、入体贩卖人口（3 次），随之违背了造物主亲自制定的独立（隔离）先于一切（权利）的天规，还要以珍爱生命、红十字会、国家等为名，名副其实不得好死，从而构成活摘器官（三次）入体杀人（入体贩卖人口）原则、活摘器官入体抢劫器官（入体抢劫）原则，显然，美国的城堡法案必须推进到每个人的肉体层面，文明才算基本到位。全世界在过去五千多年的发展中，全世界法律最先进的英美法系也只是关注居所的安全，对于进一步将城堡法案推进到人体、器官层面则一片荒芜，这到导致近七十年来全体中国人民沦为共党、全世界政要和特权阶级的供体，世界各国为富不仁或有情报、话语价值的人们全部到中国进行器官旅游，移植供体从头到尾不打任何麻醉药而存活率最高的高质量活摘器官，归根结底，中国就是名副其实的器官世界工厂，全中国人民沦陷于全世界最残酷的器官监狱，人间地狱的中国版、真实版，从而构成中国器官世界工厂原则，也就是说，中国不仅是全世界消费品的世界工厂，更是全人类器官移植的器官世界工厂，无分别的供体天下之人间民族惨案，在世界的发展史上前所未有。 1862 年9 月22 日林肯发表《解放黑奴宣言（The Emancipation Proclamation: Negro 谋杀、大屠杀只是在生命层面上绝对犯罪；活摘器官则是加入了无限酷刑（无限行与折磨）、一铺清袋的最后晚餐式的几年前就3000 万日元一副心脏的器官贩卖（比刑事死刑的贩毒还恐怖），越过了人与人之间的肉体隔离（生理隔离之界限）而入体抢劫器官、入体抢劫巨额财富、入体杀人（3 次、比照入户杀人）、入体贩卖人口（3 次），随之违背了造物主亲自制定的独立（隔离）先于一切（权利）的天规，还要以珍爱生命、红十字会、国家等为名，名副其实不得好死，从而构成活摘器官（三次）入体杀人（入体贩卖人口）原则、活摘器官入体抢劫器官（入体抢劫）原则，显然，美国的城堡法案必须推进到每个人的肉体层面，文明才算基本到位。全世界在过去五千多年的发展中，全世界法律最先进的英美法系也只是关注居所的安全，对于进一步将城堡法案推进到人体、器官层面则一片荒芜，这到导致近七十年来全体中国人民沦为共党、全世界政要和特权阶级的供体，世界各国为富不仁或有情报、话语价值的人们全部到中国进行器官旅游，移植供体从头到尾不打任何麻醉药而存活率最高的高质量活摘器官，归根结底，中国就是名副其实的器官世界工厂，全中国人民沦陷于全世界最残酷的器官监狱，人间地狱的中国版、真实版，从而构成中国器官世界工厂原则，也就是说，中国不仅是全世界消费品的世界工厂，更是全人类器官移植的器官世界工厂，无分别的供体天下之人间民族惨案，在世界的发展史上前所未有。全世界在过去五千多年的发展中，全世界法律最先进的英美法系也只是关注居所的安全，对于进一步将城堡法案推进到人体、器官层面则一片荒芜，这到导致近七十年来全体中国人民沦为共党、全世界政要和特权阶级的供体，世界各国为富不仁或有情报、话语价值的人们全部到中国进行器官旅游，移植供体从头到尾不打任何麻醉药而存活率最高的高质量活摘器官，归根结底，中国就是名副其实的器官世界工厂，全中国人民沦陷于全世界最残酷的器官监狱，人间地狱的中国版、真实版，从而构成中国器官世界工厂原则，也就是说，中国不仅是全世界消费品的世界工厂，更是全人类器官移植的器官世界工厂，无分别的供体天下之人间民族惨案，在世界的发展史上前所未有。之人间民族惨案，在世界的发展史上前所未有。 1862 年9 月22 日，林肯发表《解放黑奴宣言（The Emancipation Proclamation: Negro Slaves）》解放黑奴而造福美国；今天，我们必须立刻解放全中国器官供体奴隶，即红色邪教下的全民红奴、血奴，这是全人类有史以来最血腥的罪行，超越了生死极端的命运层面之绝对犯罪，这就是全人类的良心解放红奴（血奴、赤奴、器官奴）宣言：《解放红奴宣言（The Emancipation Proclamation: Emancipate Red Slaves）》。器官罪犯属于超越生死极限的罪行，全人类人人得而诛之将其处决、斩首都是有天大功劳的。城堡法案不仅要进家门，更要送瘟神到个人，人人城堡法案。在人类文明已经进入宇航文明60 年后的今天，全人类有史以来最血腥的全民器官奴隶制度竟然存在于早已AI 现代化之科技、法治的光天化日之下，而且象传销一样公然行销全世界几十年无法禁止，或者屡禁不止，全中国原来的20 亿人到现在被屠村屠城剩下的5.6 亿人全部沦为全世界的器官奴隶，这是全世界法律的耻辱、文明的悲剧：技术越发达、中国人被活摘器官的规模和速度越快，到了现在一年被活摘几十万副甚至几百万副器官的规模。全中国人民的心脏在滴血，苍天在流泪，说邪恶政权是畜牲绝对侮辱了全世界的所有畜牲。现在，普包子和他的包皮兄弟给全世界带来了超越任何人生死的核武末日，全人类不出地狱即被毁灭，任何救援都刻不容缓，全人类才有和平的机会和生的希望，把死亡留给恶魔吧。在人类文明已经进入宇航文明60 年后的今天，全人类有史以来最血腥的全民器官奴隶制度竟然存在于早已AI 现代化之科技、法治的光天化日之下，而且象传销一样公然行销全世界几十年无法禁止，或者屡禁不止，全中国原来的20 亿人到现在被屠村屠城剩下的5.6 亿人全部沦为全世界的器官奴隶，这是全世界法律的耻辱、文明的悲剧：技术越发达、中国人被活摘器官的规模和速度越快，到了现在一年被活摘几十万副甚至几百万副器官的规模。全中国人民的心脏在滴血，苍天在流泪，说邪恶政权是畜牲绝对侮辱了全世界的所有畜牲。现在，普包子和他的包皮兄弟给全世界带来了超越任何人生死的核武末日，全人类不出地狱即被毁灭，任何救援都刻不容缓，全人类才有和平的机会和生的希望，把死亡留给恶魔吧。在活摘器官的罪行中，医生、警察Wj(j∈N)负责提供供体和杀人，器官移植者Q 出钱，医生和警察强夺、活摘受害者的器官去换取器官移植者或其保险公司的金钱，器官移植者Q 扮演着销赃的角色，显然，假设其没有器官移植的需求并支付如3000 万日元作为代价，供体U 的器官就不会因为器官移植者Q 而被摘除，此时，如果器官移植者（和其保险公司） Q 不是医生、警察Wj 的同谋、共犯，那么，其主动因素必然是予以排除的，记为结论F；然而，器官移植者（和其保险公司）Q 的付款是主动的、到中共国起换器官也是主动的，案件中的主动因素并未被排除的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 否定肯定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得器官移植者（相关保险公司等）非同谋（共犯）否定原则和器官移植者（相关保险公司等）同谋（共犯）原则、器官移植者（相关保险公司等）绝对罪行（死刑、终身监禁）原则，即器官移植者（相关保险公司等）、器官媒介等都是绝对犯罪，不是死刑即是终身监禁。如今，疫情的全民强制核酸检测而建立起来的全国以至全世界之大数据基因库和HLA 配型数据库，必然令全人类成为供体、器官传销组织中的非必要移植者（被忽悠的）和必要移植者，器官绑架全球政要家族、富豪家族等随之不可阻挡，器官恐怖主义随之席卷全球，全人类的灾难因为民众不断加深失去话语权、知情权而越来越无法自拔，最终全部堕落邪恶组织的地狱中任人宰割。扮演着销赃的角色，显然，假设其没有器官移植的需求并支付如3000 万日元作为代价，供体U 的器官就不会因为器官移植者Q 而被摘除，此时，如果器官移植者（和其保险公司） Q 不是医生、警察Wj 的同谋、共犯，那么，其主动因素必然是予以排除的，记为结论F；然而，器官移植者（和其保险公司）Q 的付款是主动的、到中共国起换器官也是主动的，案件中的主动因素并未被排除的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 否定肯定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得器官移植者（相关保险公司等）非同谋（共犯）否定原则和器官移植者（相关保险公司等）同谋（共犯）原则、器官移植者（相关保险公司等）绝对罪行（死刑、终身监禁）原则，即器官移植者（相关保险公司等）、器官媒介等都是绝对犯罪，不是死刑即是终身监禁。如今，疫情的全民强制核酸检测而建立起来的全国以至全世界之大数据基因库和HLA 配型数据库，必然令全人类成为供体、器官传销组织中的非必要移植者（被忽悠的）和必要移植者，器官绑架全球政要家族、富豪家族等随之不可阻挡，器官恐怖主义随之席卷全球，全人类的灾难因为民众不断加深失去话语权、知情权而越来越无法自拔，最终全部堕落邪恶组织的地狱中任人宰割。扮演着销赃的角色，显然，假设其没有器官移植的需求并支付如3000 万日元作为代价，供体U 的器官就不会因为器官移植者Q 而被摘除，此时，如果器官移植者（和其保险公司） Q 不是医生、警察Wj 的同谋、共犯，那么，其主动因素必然是予以排除的，记为结论F；然而，器官移植者（和其保险公司）Q 的付款是主动的、到中共国起换器官也是主动的，案件中的主动因素并未被排除的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 否定肯定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得器官移植者（相关保险公司等）非同谋（共犯）否定原则和器官移植者（相关保险公司等）同谋（共犯）原则、器官移植者（相关保险公司等）绝对罪行（死刑、终身监禁）原则，即器官移植者（相关保险公司等）、器官媒介等都是绝对犯罪，不是死刑即是终身监禁。如今，疫情的全民强制核酸检测而建立起来的全国以至全世界之大数据基因库和HLA 配型数据库，必然令全人类成为供体、器官传销组织中的非必要移植者（被忽悠的）和必要移植者，器官绑架全球政要家族、富豪家族等随之不可阻挡，器官恐怖主义随之席卷全球，全人类的灾难因为民众不断加深失去话语权、知情权而越来越无法自拔，最终全部堕落邪恶组织的地狱中任人宰割。 5000 年来，全人类只关心、关注常态性的居所安全，却没有解决自身的切身安全，从而为罪犯留下了漏洞，亡羊补牢为时未晚，现在立刻采取行动补救别无选择。 5000 年来，全人类只关心、关注常态性的居所安全，却没有解决自身的切身安全，从而为罪犯留下了漏洞，亡羊补牢为时未晚，现在立刻采取行动补救别无选择。 2．（XP 军事同盟、枪指挥党、无能党指挥枪）活摘真理绝对（归零）原 2．（XP 军事同盟、枪指挥党、无能党指挥枪）活摘真理绝对（归零）原则普包子和他包皮（轴心法上唯一的法人代表），一个上浮向台前（如直接国有化萨哈林 2 号天然气项目）、一个下沉到幕后（不再亲自指挥亲自部署），无论怎么折腾，他们都是要推翻雅尔塔体系的秩序，建立起无限行刑的绝对丛林秩序，即以武力解决一切的专制秩序取代以金钱、法律解决一切的雅尔塔秩序，可是这是违背并活摘真理的而永远也不可能成功的。全人类为什么要跟这两个连常识都不理解的人倒退回原始社会之前的大灾难洪荒社会呢？普京指挥包子是枪指挥党，犯了中共所有党员的忌讳，因为普京有枪无弹；现在普京伪造了一种党指挥枪的假象，假装让毫无能力的文盲加神经病持枪指挥，实际上犯了太阿倒持、授人以柄的致命错误，而且是千古经典。任何人想象一下，一个神经病拿着子弹上膛的枪四处挥舞，威胁恐吓全世界，不要说让子弹飞伤到他人要赔偿医药费，就是全世界的愤怒都会把你普京和俄罗斯直接销毁。况且，就是在持枪最自由到泛滥成灾的美国，神经病持枪也是不可能被允许的，普京的权力果然大过美帝，兜底并加持了神经病持枪。中国10 年来已经有超过17 亿人死于屠村屠城和活摘器官、杀着玩等，显然都是普包子的功劳。 2022 年2 月4 日开始的共俄军事同盟第一阶段，普包子枪指挥党犯中共灭党的太平间忌讳；2022 年6 月15 日的共俄军事同盟第二阶段，普包子让神经病持枪而假装党指挥枪，跨国有组织太阿倒持、授人以柄全出来了，不惜一切代价打造了两人的末日，包子和包子馅不挂绝对没天理。这就是说，包子和包子馅无论秩序怎么排列组合（这是数学语言他们肯定听不懂的），结果都一样：飞流直下三千尺，疑是「银核」落九天，中国人民无偿进贡给俄罗斯打仗的血汗钱、器官钱（银核的银）全没了，炸毁地球的核武器（银核的核）马上下来了。普京很坦白也够直接，单刀直入地指明了李白的老婆叫赵紫烟、女儿叫李香炉：日照香炉生紫烟。由此可见，普京与包子的二人转，无论是男上女下还是女上男下，反正都是天然不归路。听不懂的），结果都一样：飞流直下三千尺，疑是「银核」落九天，中国人民无偿进贡给俄罗斯打仗的血汗钱、器官钱（银核的银）全没了，炸毁地球的核武器（银核的核）马上下来了。普京很坦白也够直接，单刀直入地指明了李白的老婆叫赵紫烟、女儿叫李香炉：日照香炉生紫烟。由此可见，普京与包子的二人转，无论是男上女下还是女上男下，反正都是天然不归路。在XP 的共俄军事同盟第一阶段，俄罗斯的GDP 就大约中国广州一个市的水平即经济很不好，不从中国拿钱就无法支持俄乌战争，因此俄罗斯普京P 的实力显然是有限的；共党历来伪称自己的是永远光荣、正确、伟大、全人类的最高理想，其党魁X 更是宇宙之王、万教圣主、红黄蓝圣殿主任、罂粟花圣化教材主编，显然是无限大，于是，普京P 枪指挥党X 是：P>X→∞，lim(P/X)=0>lim(X/X)=1，即0>1，XP（普京的包皮和包子馅普京两货）两个人完全活摘了真理，必被规则（真理）彻底归零而天打五雷劈遭天杀，自己从不想想自己是几斤几两，在全世界的众目睽睽之下，悍然直接把上帝、玉皇大帝挤出了灵霄宝殿，然后自己一屁股坐上了宇宙之王的宝座，在人间大地上就地上演了当年一出生就贩毒的罂粟猴（泼猴泼皮poppy 猴）齐天大圣孙悟空大闹天宫的闹剧，从而构成了（XP 军事同盟、枪指挥党）活摘真理绝对（归零）原则、枪指挥党绝对（归零）原则，依此类推至于XP 军事同盟第二阶段的（XP 军事同盟、无能者党指挥枪）活摘真理绝对（归零）原则、无能（者）党指挥枪绝对（归零）原则同理成立，因为，文盲、智力有缺陷者是被动的而无法达到主动层面，从而不可能胜任主动的指挥任务。在XP 的共俄军事同盟第一阶段，俄罗斯的GDP 就大约中国广州一个市的水平即经济很不好，不从中国拿钱就无法支持俄乌战争，因此俄罗斯普京P 的实力显然是有限的；共党历来伪称自己的是永远光荣、正确、伟大、全人类的最高理想，其党魁X 更是宇宙之王、万教圣主、红黄蓝圣殿主任、罂粟花圣化教材主编，显然是无限大，于是，普京P 枪指挥党X 是：P>X→∞，lim(P/X)=0>lim(X/X)=1，即0>1，XP（普京的包皮和包子馅普京两货）两个人完全活摘了真理，必被规则（真理）彻底归零而天打五雷劈遭天杀，自己从不想想自己是几斤几两，在全世界的众目睽睽之下，悍然直接把上帝、玉皇大帝挤出了灵霄宝殿，然后自己一屁股坐上了宇宙之王的宝座，在人间大地上就地上演了当年一出生就贩毒的罂粟猴（泼猴泼皮poppy 猴）齐天大圣孙悟空大闹天宫的闹剧，从而构成了（XP 军事同盟、枪指挥党）活摘真理绝对（归零）原则、枪指挥党绝对（归零）原则，依此类推至于XP 军事同盟第二阶段的（XP 军事同盟、无能者党指挥枪）活摘真理绝对（归零）原则、无能（者）党指挥枪绝对（归零）原则同理成立，因为，文盲、智力有缺陷者是被动的而无法达到主动层面，从而不可能胜任主动的指挥任务。由此可见，俄乌战争不仅终结了二战以来的雅尔塔体系，更终结了上帝的真理并取而代之用武力解决一切：普京和他的包皮企图以别无选择而无限行刑的专制秩序取代二战后以金钱利益和法律解决问题的文治和平体系，后者也有问题需要改革，但取代金钱解决国际争端的秩序绝对不是XP 两个人武力解决一切的独裁体系（如香港反送中大屠杀就是邪恶暴力践踏和平），这是一场独裁与民主之战，更是一场屠杀如吃顿犯的恶魔与上帝之战，只是一言不合就天翻地覆造反、但还有点可爱的猴子孙悟空变成了两只一出生就贩毒的罂粟猴（毒猴） XP 二人转。在XP 的共俄军事同盟第一阶段，俄罗斯的GDP 就大约中国广州一个市的水平即经济很不好，不从中国拿钱就无法支持俄乌战争，因此俄罗斯普京P 的实力显然是有限的；共党历来伪称自己的是永远光荣、正确、伟大、全人类的最高理想，其党魁X 更是宇宙之王、万教圣主、红黄蓝圣殿主任、罂粟花圣化教材主编，显然是无限大，于是，普京P 枪指挥党X 是：P>X→∞，lim(P/X)=0>lim(X/X)=1，即0>1，XP（普京的包皮和包子馅普京两货）两个人完全活摘了真理，必被规则（真理）彻底归零而天打五雷劈遭天杀，自己从不想想自己是几斤几两，在全世界的众目睽睽之下，悍然直接把上帝、玉皇大帝挤出了灵霄宝殿，然后自己一屁股坐上了宇宙之王的宝座，在人间大地上就地上演了当年一出生就贩毒的罂粟猴（泼猴泼皮poppy 猴）齐天大圣孙悟空大闹天宫的闹剧，从而构成了（XP 军事同盟、枪指挥党）活摘真理绝对（归零）原则、枪指挥党绝对（归零）原则，依此类推至于XP 军事同盟第二阶段的（XP 军事同盟、无能者党指挥枪）活摘真理绝对（归零）原则、无能（者）党指挥枪绝对（归零）原则同理成立，因为，文盲、智力有缺陷者是被动的而无法达到主动层面，从而不可能胜任主动的指挥任务。由此可见，俄乌战争不仅终结了二战以来的雅尔塔体系，更终结了上帝的真理并取而代之用武力解决一切：普京和他的包皮企图以别无选择而无限行刑的专制秩序取代二战后以金钱利益和法律解决问题的文治和平体系，后者也有问题需要改革，但取代金钱解决国际争端的秩序绝对不是XP 两个人武力解决一切的独裁体系（如香港反送中大屠杀就是邪恶暴力践踏和平），这是一场独裁与民主之战，更是一场屠杀如吃顿犯的恶魔与上帝之战，只是一言不合就天翻地覆造反、但还有点可爱的猴子孙悟空变成了两只一出生就贩毒的罂粟猴（毒猴） XP 二人转。由此可见，俄乌战争不仅终结了二战以来的雅尔塔体系，更终结了上帝的真理并取而代之用武力解决一切：普京和他的包皮企图以别无选择而无限行刑的专制秩序取代二战后以金钱利益和法律解决问题的文治和平体系，后者也有问题需要改革，但取代金钱解决国际争端的秩序绝对不是XP 两个人武力解决一切的独裁体系（如香港反送中大屠杀就是邪恶暴力践踏和平），这是一场独裁与民主之战，更是一场屠杀如吃顿犯的恶魔与上帝之战，只是一言不合就天翻地覆造反、但还有点可爱的猴子孙悟空变成了两只一出生就贩毒的罂粟猴（毒猴） XP 二人转。 3．海洛因（Heroin）以药物救人为名贩毒绝对归零原则 3．海洛因（Heroin）以药物救人为名贩毒绝对归零原则 1806 年，德国药剂师泽尔蒂纳（F.W.A.Serturner）首次从阿片中提取出含氮植物碱，即吗啡。1874 年，英国伦敦圣玛莉医院的化学家莱特（R.Wright），在吗啡中加入醋酸酐等物质，首次提炼出镇痛效果更佳的半合成化衍生物，二乙酸吗啡，这就是最早合成的海洛因。 1897 年，德国拜耳（Bayer）药厂化学家霍夫曼（FelixHoffmann）将海洛因制成药物，其止痛效力远高于吗啡，至少提高了4-8 倍，可明显抑制肺痨病人的剧咳、久喘和胸痛，促进患者情绪安定，且无明显不良反应。1898 年拜耳药厂开始规模化生产该药，并正式注册商品名为「海洛因」（Heroin），源自德文heroisch 一词，英语意即女英雄。 1898 年，该药以不会上瘾的吗啡之名上市，其后更曾用作儿童止咳药。拜耳公司很快就发现海洛因并不只是能治咳嗽，公司后来建议在治疗疼痛、抑郁、支气管炎、哮喘甚至胃癌时都可以使用海洛因，以至于在当时人们了解的疾病中，只有很少几种不在海洛因的适用 3．海洛因（Heroin）以药物救人为名贩毒绝对归零原则 1806 年，德国药剂师泽尔蒂纳（F.W.A.Serturner）首次从阿片中提取出含氮植物碱，即吗啡。1874 年，英国伦敦圣玛莉医院的化学家莱特（R.Wright），在吗啡中加入醋酸酐等物质，首次提炼出镇痛效果更佳的半合成化衍生物，二乙酸吗啡，这就是最早合成的海洛因。 1897 年，德国拜耳（Bayer）药厂化学家霍夫曼（FelixHoffmann）将海洛因制成药物，其止痛效力远高于吗啡，至少提高了4-8 倍，可明显抑制肺痨病人的剧咳、久喘和胸痛，促进患者情绪安定，且无明显不良反应。1898 年拜耳药厂开始规模化生产该药，并正式注册商品名为「海洛因」（Heroin），源自德文heroisch 一词，英语意即女英雄。 1898 年，该药以不会上瘾的吗啡之名上市，其后更曾用作儿童止咳药。拜耳公司很快就发现海洛因并不只是能治咳嗽，公司后来建议在治疗疼痛、抑郁、支气管炎、哮喘甚至胃癌时都可以使用海洛因，以至于在当时人们了解的疾病中，只有很少几种不在海洛因的适用范围之内。甚至包括疯人院：那不勒斯精神病院的大夫给病人们开出海洛因，记录说有「持久的镇定作用」、「甚至有几个痊愈的病例」。俄国精神病医生用海洛因驱散「灵魂的痛苦」，甚至登山俱乐部都建议俱乐部成员在登山前服用此物，因为它能使呼吸更为顺畅，能让他们登得更高。海洛因作为商品出售后获得了巨大利润，1902 年海洛因的利润占整个药品行业的5%，这在一定程度上得益于拜耳公司的营销手段。公司给全世界的医生们免费发放海洛因试用品，委托一些专家做带有宣传海洛因神奇疗效的研究。在这些研究人员的记录里，海洛因仅仅具有昏沉、晕眩和便秘这些微不足道的副作用。拜耳甚至在《德国医生报》的广告中公开要求医生们用「公认的出色的」海洛因医治吗啡成瘾，称海洛因是吗啡的下一代产品，并且不会让人上瘾，但却事与愿违，人们很快就发现海洛因比吗啡的水溶性更大，吸收亦更快，脂溶性也较大，更容易通过血脑屏障进神经中枢发挥作用，更为严重的是，它的成瘾性更强烈对个人和社会所导致的危害后果已远远地超过了其医用价值烈。对个人和社会所导致的危害后果，已远远地超过了其医用价值。 1910 年起各国取消了海洛因在临床上的应用。1912 年在荷兰海牙召开的鸦片问题国际会议上，到会代表一致赞成管制鸦片、吗啡和海洛因的贩运。1924 年，美国参众两院立法禁止进口、制造和销售海洛因。1953 年，首先发明了海洛因生产工艺的英国政府也将海洛因从《英国药典》中删去。烈。对个人和社会所导致的危害后果，已远远地超过了其医用价值。 1910 年起各国取消了海洛因在临床上的应用。1912 年在荷兰海牙召开的鸦片问题国际会议上，到会代表一致赞成管制鸦片、吗啡和海洛因的贩运。1924 年，美国参众两院立法禁止进口、制造和销售海洛因。1953 年，首先发明了海洛因生产工艺的英国政府也将海洛因从《英国药典》中删去。这就是海洛因（Heroin）以药物B 救人为名贩毒A 的绝对罪行：-\|A\|=\|B\| ⇒ A=B=0； {A}∩{B}=Ø ⇒ A∈Ø、B∈Ø 而A、B 不存在而归零，其中A、B 为任意实数，即属绝对归零的，从而构成海洛因以药物救人为名贩毒绝对归零原则、海洛因以药物救人为名贩毒绝对（罪行）原则。这就是海洛因（Heroin）以药物B 救人为名贩毒A 的绝对罪行：-\|A\|=\|B\| ⇒ A=B=0； {A}∩{B}=Ø ⇒ A∈Ø、B∈Ø 而A、B 不存在而归零，其中A、B 为任意实数，即属绝对归零的，从而构成海洛因以药物救人为名贩毒绝对归零原则、海洛因以药物救人为名贩毒绝对（罪行）原则。就是海洛因以药物救人为名贩毒的对罪行 \| \| \| \| ； {A}∩{B}=Ø ⇒ A∈Ø、B∈Ø 而A、B 不存在而归零，其中A、B 为任意实数，即属绝对归零的，从而构成海洛因以药物救人为名贩毒绝对归零原则、海洛因以药物救人为名贩毒绝对（罪行）原则。 4．海洛因（Heroin）蝴蝶结原则、蝴蝶结（挂钩、挂狗）绝对归零原则、十三（13）蝴蝶结（挂钩）绝对归零原则国际上，毒品叫hard drug 即硬药品，与硬通货的黄金、美元一样；国际禁毒日叫the International Day against Drug Abuse and Illicit Traffickin、吸毒的瘾君子叫drug addict、毒贩子叫drug dealer、缉毒叫capture drug smuggler、涉毒或制毒的叫drug criminal，等等，归根结底就是，全世界最大的毒品海洛因（Heroin）最早是德国拜耳（Bayer）以常用药的咳嗽药上市的，海洛因作为商品出售后获得了巨大利润，1902 年海洛因的利润占整个药品行业的5%，总之，世界最大量的毒品海洛因以治病救人的常用药物为名上市毒死人、杀害人，即以救人圣药之名杀人、祸国殃民、毁灭世界而且历经26 年（1898~1924）才被美国国会首次禁止、历经55 年（1898~1953）才被首先发明海洛因生产工艺的英国政府从《英国药典》中删去，恶魔伪装的时间跨越了至少两到三代人才被揭穿真面目。于是，现代的任何与毒品相关的事务都直接与被海洛因彻底污名化的药物（drug）对等、挂钩，就象美国在大喊脱钩一样之前的挂钩，实际上就是挂狗的蝴蝶结，真正被卡住了的人或政府才知道个中滋味，为了海洛因将整个药物行业污名化的无法忘却之纪念，人们必须称之为海洛因（Heroin）蝴蝶结、挂钩（挂狗）蝴蝶结、海洛因（Heroin）效应（模式）、拜耳（Bayer）效应（模式），从而构成全世界到处都是的通用公司的海洛因（Heroin）蝴蝶结原则、挂钩（挂狗）蝴蝶结原则、海洛因（Heroin）挂钩（挂狗）原则。从而构成全世界到处都是的通用公司的海洛因（Heroin）蝴蝶结原则、挂钩（挂狗）蝴蝶结原则、海洛因（Heroin）挂钩（挂狗）原则。从而构成全世界到处都是的通用公司的海洛因（Heroin）蝴蝶结原则、挂钩（挂狗）蝴蝶结原则、海洛因（Heroin）挂钩（挂狗）原则。人家是挂羊头卖狗肉，德国拜耳（Bayer）是挂狗（挂钩）贩毒，利用了人们的善意、良心和无知吃人血馒头、祸国殃民、毁灭世界，全人类的教训不可谓不惨痛，历史经验历历在目却无法警示世人和无法阻止人们前仆后继、风起云涌的飞蛾扑火式自取灭亡，心痛有什么用？行动最重要，认知真理和真相最有效。人家是挂羊头卖狗肉，德国拜耳（Bayer）是挂狗（挂钩）贩毒，利用了人们的善意、良心和无知吃人血馒头、祸国殃民、毁灭世界，全人类的教训不可谓不惨痛，历史经验历历在目却无法警示世人和无法阻止人们前仆后继、风起云涌的飞蛾扑火式自取灭亡，心痛有什么用？行动最重要，认知真理和真相最有效。么用？行动最重要，认知真理和真相最有效。依据海洛因以药物救人为名贩毒绝对归零原则、海洛因（Heroin）蝴蝶结原则、挂钩（挂狗）蝴蝶结原则，任何蝴蝶结、挂钩（挂狗）都是进退不能的，即进B 和退A 都是一样不能的：-\|A\|=\|B\| ⇒ A=B=0；{A}∩{B}=Ø ⇒ A∈Ø、B∈Ø 而A、B 不存在而归零，其中A、B 为任意实数，即属绝对归零的，从而构成蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则，依此类推至于如下的13 个蝴蝶结同理成立而构成十三（13）蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则，包括但不仅限于： (1)共产主义蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(2)政府主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(3)孟山都的转基因主权（种子主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(4)美联储货币主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(5)推特Twitter 话语主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(6)器官主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(7)相对（相对论、相对性）主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(8)特权主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(9)学术主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(10)生理主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(11)产业链主权、(12)领土主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(13)军火主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则。全世界的13 个蝴蝶结为：(1)共产主义老灭绝的主权、(2)政府主权腐败邪恶、(3)孟山都的转基因主权（种子主权）、(4)美联储货币主权、(5)推特Twitter 话语主权、(6)器官主权、 (7)相对（相对论、相对性）主权、(8)特权主权、(9)学术主权、(10)生理主权、(11)产业链主权、(12)领土主权、(13)军火主权。么用？行动最重要，认知真理和真相最有效。依据海洛因以药物救人为名贩毒绝对归零原则、海洛因（Heroin）蝴蝶结原则、挂钩（挂狗）蝴蝶结原则，任何蝴蝶结、挂钩（挂狗）都是进退不能的，即进B 和退A 都是一样不能的：-\|A\|=\|B\| ⇒ A=B=0；{A}∩{B}=Ø ⇒ A∈Ø、B∈Ø 而A、B 不存在而归零，其中A、B 为任意实数，即属绝对归零的，从而构成蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则，依此类推至于如下的13 个蝴蝶结同理成立而构成十三（13）蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则，包括但不仅限于： (1)共产主义蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(2)政府主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(3)孟山都的转基因主权（种子主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(4)美联储货币主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(5)推特Twitter 话语主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(6)器官主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(7)相对（相对论、相对性）主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(8)特权主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(9)学术主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(10)生理主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(11)产业链主权、(12)领土主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(13)军火主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则。全世界的13 个蝴蝶结为：(1)共产主义老灭绝的主权、(2)政府主权腐败邪恶、(3)孟山都的转基因主权（种子主权）、(4)美联储货币主权、(5)推特Twitter 话语主权、(6)器官主权、 (7)相对（相对论、相对性）主权、(8)特权主权、(9)学术主权、(10)生理主权、(11)产业链主权、(12)领土主权、(13)军火主权。依据海洛因以药物救人为名贩毒绝对归零原则、海洛因（Heroin）蝴蝶结原则、挂钩（挂狗）蝴蝶结原则，任何蝴蝶结、挂钩（挂狗）都是进退不能的，即进B 和退A 都是一样不能的：-\|A\|=\|B\| ⇒ A=B=0；{A}∩{B}=Ø ⇒ A∈Ø、B∈Ø 而A、B 不存在而归零，其中A、B 为任意实数，即属绝对归零的，从而构成蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则，依此类推至于如下的13 个蝴蝶结同理成立而构成十三（13）蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则，包括但不仅限于：钩挂狗海洛因拜耳绝对归零原则包括但不仅限于 (1)共产主义蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(2)政府主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(3)孟山都的转基因主权（种子主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(4)美联储货币主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(5)推特Twitter 话语主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(6)器官主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(7)相对（相对论、相对性）主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(8)特权主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(9)学术主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(10)生理主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(11)产业链主权、(12)领土主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则、(13)军火主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）绝对（归零）原则。狗、海洛因、拜耳）绝对（归零）原则。全世界的13 个蝴蝶结为：(1)共产主义老灭绝的主权、(2)政府主权腐败邪恶、(3)孟山都的转基因主权（种子主权）、(4)美联储货币主权、(5)推特Twitter 话语主权、(6)器官主权、 (7)相对（相对论、相对性）主权、(8)特权主权、(9)学术主权、(10)生理主权、(11)产业链主权、(12)领土主权、(13)军火主权。狗、海洛因、拜耳）绝对（归零）原则。全世界的13 个蝴蝶结为：(1)共产主义老灭绝的主权、(2)政府主权腐败邪恶、(3)孟山都的转基因主权（种子主权）、(4)美联储货币主权、(5)推特Twitter 话语主权、(6)器官主权、 (7)相对（相对论、相对性）主权、(8)特权主权、(9)学术主权、(10)生理主权、(11)产业链主权、(12)领土主权、(13)军火主权。 5．十三（13）蝴蝶结 5．十三（13）蝴蝶结（1）全人类有史以来最血腥的共产主义，至今屠杀全人类几十亿人，如文革饿死一亿人、斗死几千万人，近来短短几年内大肆屠村屠城而屠杀了中国17 亿人，加上种族灭绝、宗教灭绝罪行、计划生育等，单在中国就有远远超过20 亿人被杀害，马克思、列宁、老毛等全都是以全人类最高理想欺骗全人类近200 年、吞噬几十亿条人命，其中大都是觉醒或可觉醒的精英，由此可见，共产主义不仅危害、摧毁中华民族的主权，如今更危害、摧毁世界各国的主权和具备支持俄罗斯用核武器炸毁地球而危害地球的主权，而且全都以全人类最高理想、为人民服务之名行灭绝之实，共产主义国家的这种权力Q 决定世界各国主权的生死存亡而必然是高于世界各国主权的，称为共产主义主权，显然是海洛因（Heroin）模式、拜耳（Bayer）效应，马克思、霍夫曼（Felix Hoffmann）、拜耳（Bayer）等都是德国人，可见，德国人在杀人方面有无以伦比的天分和犹太血统的优势，从而构成共产主义（主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则，以解放之名行无分别大屠杀之实。（2）如2021 年美国军费8010 亿美元、俄罗斯军费659 亿美元，两国相差12 倍，只要两国或俄罗斯和北约一开战，俄罗斯直接就进坑里去了。可是，现在是中共和华尔街相互深度勾结，不断无条件地给俄罗斯输送美元和弹药，原来有枪无弹的普京和俄罗斯民族立刻如猛虎下山扑向乌克兰、日本、北约和美国，重新变成了可与美国抗衡的超级大国；另一方面，给了钱交保护费的中共政权更加有恃无恐，更加变本加厉地四处用病毒去威胁恐吓各国和大捞特捞，完全不顾廉耻和自身死活，临时也要拉个垫背的，由此可见，俄罗斯政权给了共党政权无限作恶的加持而直接危害各国安全，中共政权支撑了俄罗斯用核武器毁灭任何一个民主国家主权的斯拉夫民族之传统战斗力，即华尔街联手中共政府向俄罗斯输送资金导致俄罗斯能危害、摧毁世界任何各国的主权，这种一个政府向另一个政府输送金钱的权力Q 称为政府（相对、增量）主权，显然，现在全世界各国都被卡住了，全人类随时会被双方交战的核武器所灭绝，地球随之被炸毁，从而构成政府（相对、增量）主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（3）美国的孟山都公司，是转基因种子市场的垄断巨头，在玉米、大豆、棉花等多种重要作物的转基因种子市场上，占据70%至100%的份额。全世界超过90%的转基因种子，都使用它的专利，Monsanto 犹太人家族Olga Mendez Monsanto 的姓氏，一代目是德国的Issac Rodriguez Monsanto；约翰·奎恩伊(John Francis Queeny)是吃软饭的创始人。2016 年9 月，拜耳和孟山都宣布双方签署最终并购协议。2018 年6 月7 日起，德国拜耳成为孟山都公司的唯一股东，孟山都公司股票于纽约证券交易所退市，孟山都的股东获得每股128 美元的现金，孟山都公司被拜耳公司正式收购。孟山都的橙剂、PCBs 等从生物、基因、遗传上毁灭越南人、令美国国鸟白头海雕濒临灭绝、毁灭全世界等已经举世皆知，普京等到俄乌战争开战后才发现俄罗斯的粮食主权已经被孟山都攻陷，现在，随着转基因对人类生育和健康的毁灭性影响和打击（如喂转基因的小白鼠三代而绝育），全人类公认孟山都妄图毒杀世界，是世界上最邪恶的公司孟山都，致癌农药只是冰山一角，孟山都的转基因已经形成世界性的种子主权、转基因主权、粮食主权，从而构成孟山都（转基因、粮食主权、种子主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（3）美国的孟山都公司，是转基因种子市场的垄断巨头，在玉米、大豆、棉花等多种重要作物的转基因种子市场上，占据70%至100%的份额。全世界超过90%的转基因种子，都使用它的专利，Monsanto 犹太人家族Olga Mendez Monsanto 的姓氏，一代目是德国的Issac Rodriguez Monsanto；约翰·奎恩伊(John Francis Queeny)是吃软饭的创始人。2016 年9 月，拜耳和孟山都宣布双方签署最终并购协议。2018 年6 月7 日起，德国拜耳成为孟山都公司的唯一股东，孟山都公司股票于纽约证券交易所退市，孟山都的股东获得每股128 美元的现金，孟山都公司被拜耳公司正式收购。孟山都的橙剂、PCBs 等从生物、基因、遗传上毁灭越南人、令美国国鸟白头海雕濒临灭绝、毁灭全世界等已经举世皆知，普京等到俄乌战争开战后才发现俄罗斯的粮食主权已经被孟山都攻陷，现在，随着转基因对人类生育和健康的毁灭性影响和打击（如喂转基因的小白鼠三代而绝育），全人类公认孟山都妄图毒杀世界，是世界上最邪恶的公司孟山都，致癌农药只是冰山一角，孟山都的转基因已经形成世界性的种子主权、转基因主权、粮食主权，从而构成孟山都（转基因、粮食主权、种子主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（4）美国联邦储备系统（The Federal Reserve System）和华尔街利用货币主权扶植希特勒上台招致了对犹太人的大屠杀和二战的开始；美联储和华尔街以世界货币主权对共产主义、共党的无底线扶持，由此导致了如今的俄乌战争和普京准备拿核武器炸毁地球，导致了全球最大监控系统和基因数据库的建立并用来活摘器官、种族灭绝和无限行刑中国人民，导致了美国政要几乎全部被共党绑定，导致了香港反送中大屠杀和全世界毒品的泛滥成灾，更导致，等等，可见，美联储货币主权已经形成，货币主权和它所带来的利益是把双刃剑，可以救人也可以灭世，从而构成美联储（货币主权、利益主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（5）推特(Twitter)等能在2020 年美国总统中通过封杀、审核等手段彻底扭转全世界最高权力的选举制度，可见，话语（宣传）主权的时代已经到来，用得好的话拯救世界、用得不好的话就象今天普京炸毁地球（如拼命支持俄乌战争的共党抹黑乌克兰），埃隆·马斯克 (Elon Musk)收购推特肯定就看出了这一点，从而构成推特Twitter（话语权、话语主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（6）共党有史以来一直让中国成为世界器官工厂和全人类器官的世界工厂，还无分别地组织全世界各国人民到中国进行器官旅游，日本人、美国人、英国人、以色列人、台湾人、阿拉伯人等等全都不例外，只要你有钱和需求就可进入共产主义按需杀人，有的甚至只需要等待一天，而在西方各国则需要等待几年，于是，每年几十万起甚至数百万起的器官活摘悍然常态化，最优秀的女中学生、大学生、运动员、军人、模特、机关干部等等全都和监狱里的罪犯一样，直接成了知情或不知情的活供体，实际就是无分别的全国供体、全民供体；更令人发指的是，为了保证活摘器官的活力和移植存活率，所有的器官供体都不能打任何麻醉药，却毫无例外地必须将手筋、脚筋全部挑断而活活地看着、清楚着自己被活摘并慢慢地走向死亡，任何哀求都无法更改医生们和执法者们那堪比肝硬化的铁石心肠……全人类用器官移植延长生命和治疗绝症的最高生命主权就掌握在中共手上，称之为器官主权，现在邪恶政权用病毒屠杀全人类超过2000 万人却连全世界情报能力最厉害的美国CIA 都「查不出来」、「无法确定」等，可见，跨国有组织犯罪集团用活摘中国人民的器官对全世界总统、政要、官员、权贵等进行渗透、绑架是何等之深，从而构成活摘器官（生命主权、最高生命主权、器官主权、器官移植）蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则、器官移植（世界）最高生命主权原则。因为，金钱、性贿赂、赌博等对全世界总统、政要、官员、权贵来说全部并不希奇，但无须等待的不打麻醉药的最高质量健康器官只有共党能提供，世界百年老店仅此一家别无分店，不想死的话全世界都得闭口，现在，共党做到了，美国做到了，全世界也做到了。（6）共党有史以来一直让中国成为世界器官工厂和全人类器官的世界工厂，还无分别地组织全世界各国人民到中国进行器官旅游，日本人、美国人、英国人、以色列人、台湾人、阿拉伯人等等全都不例外，只要你有钱和需求就可进入共产主义按需杀人，有的甚至只需要等待一天，而在西方各国则需要等待几年，于是，每年几十万起甚至数百万起的器官活摘悍然常态化，最优秀的女中学生、大学生、运动员、军人、模特、机关干部等等全都和监狱里的罪犯一样，直接成了知情或不知情的活供体，实际就是无分别的全国供体、全民供体；更令人发指的是，为了保证活摘器官的活力和移植存活率，所有的器官供体都不能打任何麻醉药，却毫无例外地必须将手筋、脚筋全部挑断而活活地看着、清楚着自己被活摘并慢慢地走向死亡，任何哀求都无法更改医生们和执法者们那堪比肝硬化的铁石心肠……全人类用器官移植延长生命和治疗绝症的最高生命主权就掌握在中共手上，称之为器官主权，现在邪恶政权用病毒屠杀全人类超过2000 万人却连全世界情报能力最厉害的美国CIA 都「查不出来」、「无法确定」等，可见，跨国有组织犯罪集团用活摘中国人民的器官对全世界总统、政要、官员、权贵等进行渗透、绑架是何等之深，从而构成活摘器官（生命主权、最高生命主权、器官主权、器官移植）蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则、器官移植（世界）最高生命主权原则。因为，金钱、性贿赂、赌博等对全世界总统、政要、官员、权贵来说全部并不希奇，但无须等待的不打麻醉药的最高质量健康器官只有共党能提供，世界百年老店仅此一家别无分店，不想死的话全世界都得闭口，现在，共党做到了，美国做到了，全世界也做到了。（7）邪恶政权释放新冠病毒屠杀全人类超过2000 万人（Bill Gates 证实的），全人类不得不封城，上海清零、全中国清零，全世界人人被政府、卫生组织等当成犯人、传染源来看待而进入绝对零度冻结状态，不断有人跳楼、自杀等，这就是说，疫情人为地制造了一个抗疫的极端制高点和相应的极端权力Q，所有人都不能违背，称之为相对性（生命）主权、相对（相对论、相对性）主权，依据极端绝对原则、绝对归一原则、绝对主权原则、一项绝对即属绝对原则，极端权力Q 又称为主权范畴的病毒（疫苗）生命主权、相对性（相对论、相对）绝对主权，背后的官商勾结、国际内外勾结的核酸检测利益集团、疫苗集团为所欲为，一切以个人的和利益为转移，从而构成基于利益、政府权力的病毒（疫苗）生命主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（8）在病毒来源上，邪恶政权一会说是穿山甲、一会说是野生动物、一会说是美国实验室，反正一开始不久吹哨人李文亮就被害死了，其与从2002 年11 月至2003 年7 月间在 17 个国家造成774 例死亡SARS 同族，爆发地就在武汉，等等，其掌握了政权就掌握了颠倒黑白、指鹿为马的特权Q，称之为基于政府权利（包括金钱收编）的特权主权，如他们把杀害抗日县长的刘胡兰说成是生的伟大、死的光荣的民族英雄（那抗日的县长是什么呢），把违背常识而自己人害死自己子弟兵的长津湖惨败说成是民族英雄冰雕连，它们所有的英雄几乎全部是加工出来的，真正的民族英雄岳飞却被挖坟毁骨，这些只是冰山一角，从而构成特权主权（特别主权）蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则、特权主权（特别主权）横跨正反原则、共党以反共之名义灭反共原则，反正都是党、权力说了算，最为邪恶的是以反共之名义灭反共。（9）从2019 年开始，当病毒大流行开始时，《柳叶刀》、《自然》、《科学》等高影响因子的杂志就一直为病毒来源于自然站台，并且成功掩盖真相、掩护邪恶政权至今未被追责，依此类推至于其他各种与邪恶政权勾兑的学术平台，这就是国际上的学术主权，新冠病毒杀害全人类超过2000 万人（Bill Gates 证实的）的刑事犯罪被拖到今天，从而构成学术主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（9）从2019 年开始，当病毒大流行开始时，《柳叶刀》、《自然》、《科学》等高影响因子的杂志就一直为病毒来源于自然站台，并且成功掩盖真相、掩护邪恶政权至今未被追责，依此类推至于其他各种与邪恶政权勾兑的学术平台，这就是国际上的学术主权，新冠病毒杀害全人类超过2000 万人（Bill Gates 证实的）的刑事犯罪被拖到今天，从而构成学术主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（10）任何吸毒者成瘾后都会不顾一切后果地铤而走险（daredevil），杀人越货、贩卖毒品、卖国求荣等，什么都干得出来，然后别有用心的团伙、政府就满足其金钱、毒品等需求的方式来控制他，如同现在的黄俄政权控制全世界的黑帮、贩毒组织一样，依此类推至于其他成瘾性的生理需求同理成立，这种基于成瘾性生理的群体性以至世界范围的控制权力Q 称为生理主权，很多贩毒团伙和吸毒群体如同中了邪恶政权的蝴蝶结而无法自拔，南美的芬太尼群体就是例子，从而构成生理主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（11）在疫情之后，邪恶政权趁着美国连口罩都制造不了情形下断开所有供应链，企图以供应链崩溃美国经济打败美国，由此可见，全世界的供应链主权、产业链主权形成的，我们称之为供应链主权，从而构成供应链（产业链）主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（10）任何吸毒者成瘾后都会不顾一切后果地铤而走险（daredevil），杀人越货、贩卖毒品、卖国求荣等，什么都干得出来，然后别有用心的团伙、政府就满足其金钱、毒品等需求的方式来控制他，如同现在的黄俄政权控制全世界的黑帮、贩毒组织一样，依此类推至于其他成瘾性的生理需求同理成立，这种基于成瘾性生理的群体性以至世界范围的控制权力Q 称为生理主权，很多贩毒团伙和吸毒群体如同中了邪恶政权的蝴蝶结而无法自拔，南美的芬太尼群体就是例子，从而构成生理主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（11）在疫情之后，邪恶政权趁着美国连口罩都制造不了情形下断开所有供应链，企图以供应链崩溃美国经济打败美国，由此可见，全世界的供应链主权、产业链主权形成的，我们称之为供应链主权，从而构成供应链（产业链）主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。（12）二战后，三巨头罗斯福(Franklin Delano Roosevelt)、丘吉尔(Winston L.S. Churchill)、斯大林胡乱刮分世界，如将库页岛南部交给俄罗斯、将日本北方四岛交给苏联、将钓鱼岛交给日本、将琉球群岛（Ryukyu）Wj(j∈N)交给日本，等等，现在，俄罗斯等国利用这些领土四处挑起世界三战的争端，涉及其中的各国轻一点象乌克兰被炸成废墟、重一点整个地球被炸毁，由此可见，这些领土Wj 争端已经危急相关民族、国家的生死存亡和主权，也就是说，争端领土Wj 的归属权Qj 已经上升到正常国家的主权高度，称之为领土主权，从而构成领土主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则，你进也不是退也不是，象日本进被灭国、退则辱国。（13）这次的俄乌战争为三战的第一阶段（XP 两货示范统治世界阶段）；接下来，俄罗斯携邪恶轴心和北约、美国开战，如果双方都不动用核武器，战争打得月强烈、残酷，军火商的生意就会越红火，正如疫情下世界各国的棺材商都发大财产象发大水一样没法形容，因此，世界各国的军火商为了能发财肯定拼命挑拨是非、推动战争，为自己的钱包添砖加瓦，惟恐天下不乱，这种行为直接危害到各国主权的生死存亡，如现在的乌克兰变成废墟、二战中法国和欧洲各国迅速被灭等，因此，军火商如转基因的孟山都的转基因主权一样形成了能决定各国主权生死而高于各国主权的军火主权，从而构成军火主权蝴蝶结（挂钩、挂狗、海洛因、拜耳）原则。 6．五位四因果律、三十字架秩序同样是5000 年来，全人类只关注因果关系中导致结果R 的前提、条件C，却没有注意到不良和邪恶的结果R 会导致毁灭性的后果、效果E，因此没有建立起因果关系对应正义、真理J 之间的机制，即面向规则（真理、正义）的选择机制，包括E~J（因果与后果）之间约定、道路、程序等的信用连线和面向彼岸灯塔的真实有效性对应（瞄准），所谓的看前不看后，这才导致如今的真理不设防、正义裸奔，由此而来，我们必须将因果律上升为因果标律、因果目标律、因果效果律、因果果律（Cause-Result-Effect--Discrimination CRED Law 律），也就是说，我们要将原因、结果和真理（正义）的灯塔性目标对应起来，同时形成判断以决定事物的正确与否和要不要继续（及时止损）真理J 之间的机制，即面向规则（真理、正义）的选择机制，包括E~J（因果与后果）之间约定、道路、程序等的信用连线和面向彼岸灯塔的真实有效性对应（瞄准），所谓的看前不看后，这才导致如今的真理不设防、正义裸奔，由此而来，我们必须将因果律上升为因果标律、因果目标律、因果效果律、因果果律（Cause-Result-Effect--Discrimination CRED Law 律），也就是说，我们要将原因、结果和真理（正义）的灯塔性目标对应起来，同时形成判断以决定事物的正确与否和要不要继续（及时止损）在中共国，大量的最健康的男女中学生、小学生W 只因为通过常规体检而HLA 被配型，就被强行绑架而被活摘器官去满足那些将死之人U 的器官需求Q 和绑架者X 的金钱需求Y，邪恶政权拿一条甚至几条W 最健康、最有前途的学生的生命去救另一条行将就木者的生命，中学生、小学生W 无辜（因C）却被活摘器官而被无限行刑之结果R，也就是说，任何无辜者进入中共国都可能因为健康和HLA 配型信息而被活摘器官而无锡行刑，无因出死果R，这个结果称为去毛（去皮）结果、校验结果、初始化结果、第零结果、本位因果，也是现场主体（事物）的实时结果、穿梭结果之初始化结果，比照商场里的电子秤的去毛、去皮，而其因果称为去毛（去皮）因果、系统初始化的初始因果（律、关系）、净因果（律、关系）、校验因果（律、关系）、第零因果（律、关系），也是现场主体（事物）的实时因果、穿梭因果之初始化因果，从而构成第零因果律，如上述最优秀的男女中小学生无辜成为被活摘器官的无限行刑死刑犯，这种政权无辜行刑、犯罪有钱收、受害者要被灭门而灭口，如同香港陈彦霖一样，人类历经5000 年，今天去毛（去皮）因果才被提出来，这是怎么地悲剧？全人类的罪恶一切必须到此为止。一个人A 到泰国谋杀杀人B，如浙江那个老公谋杀自己孕妇妻子的罪犯，人B 的受伤害或死亡和A 的谋杀C 构成了一对直接因果，称为直接因果或第一因果（律、关系）、纵向因果（律、关系），其结果称为第一结果、直接（因果）结果；罪犯（嫌犯）杀人后必然要受到法律L 的制裁K，制裁K 即第二结果、法律结果，是社会系统罪责自负的清算结果、结算结果、横向拦截的横向结果，这就是第二因果（律、关系）、横向因果（律、关系）；如果罪犯A 象普京一样有着足够的权利按下核按钮发射核武器谋杀他人B，如几年前俄罗斯军事推演发射核导弹攻击川普的海湖庄园谋杀美国总统，地球顺便被炸毁了，罪犯（嫌犯） A 可谓万死莫赎，他被处死也无法赔偿和匹配他炸毁地球、害死所有无辜者的罪行H，我们称之为第三结果、垂直结果，从而构成第三因果（律、关系）、垂直因果（律、关系）。第零因果、第一因果、第二因果、第三因果构成了（五位、五元）四因果（律、关系），成为通向文明法线上的殿堂天之道、大道、正道，从而构成了（五位、五元）四因果神之道（大道、正道）原则，其中五元、五位是起因A、第一结果B、第二结果C、第三结果D 和事物本人、本位E，构成因果五位（五元）原则。第零因果（对象因果）、第一因果（过程因果、程序因果、道路因果）、第二因果（社会因果、系统因果、面向对象因果、目标因果）、第三因果（文明因果、出因果）构成了从点到线性到平面到立体的点线面体结构，从而构成点线面体因果（律、关系）。一个人A 到泰国谋杀杀人B，如浙江那个老公谋杀自己孕妇妻子的罪犯，人B 的受伤害或死亡和A 的谋杀C 构成了一对直接因果，称为直接因果或第一因果（律、关系）、纵向因果（律、关系），其结果称为第一结果、直接（因果）结果；罪犯（嫌犯）杀人后必然要受到法律L 的制裁K，制裁K 即第二结果、法律结果，是社会系统罪责自负的清算结果、结算结果、横向拦截的横向结果，这就是第二因果（律、关系）、横向因果（律、关系）；如果罪犯A 象普京一样有着足够的权利按下核按钮发射核武器谋杀他人B，如几年前俄罗斯军事推演发射核导弹攻击川普的海湖庄园谋杀美国总统，地球顺便被炸毁了，罪犯（嫌犯） A 可谓万死莫赎，他被处死也无法赔偿和匹配他炸毁地球、害死所有无辜者的罪行H，我们称之为第三结果、垂直结果，从而构成第三因果（律、关系）、垂直因果（律、关系）。第零因果、第一因果、第二因果、第三因果构成了（五位、五元）四因果（律、关系），成为通向文明法线上的殿堂天之道、大道、正道，从而构成了（五位、五元）四因果神之道（大道、正道）原则，其中五元、五位是起因A、第一结果B、第二结果C、第三结果D 和事物本人、本位E，构成因果五位（五元）原则。第零因果（对象因果）、第一因果（过程因果、程序因果、道路因果）、第二因果（社会因果、系统因果、面向对象因果、目标因果）、第三因果（文明因果、出因果）构成了从点到线性到平面到立体的点线面体结构，从而构成点线面体因果（律、关系）。第零因果（对象因果）、第一因果（过程因果、程序因果、道路因果）、第二因果（社会因果、系统因果、面向对象因果、目标因果）、第三因果（文明因果、出因果）构成了从点到线性到平面到立体的点线面体结构，从而构成点线面体因果（律、关系）。第零因果和第一因果构成第一（因果）十字架，第一因果和第二因果构成第二（因果）十字架，第二因果和第三因果构成第三（因果）十字架，称为三十字架因果（结果）、三因果十字架，从而构成（四）因果三十字架因果（律、关系、原则）、三因果十字架原则。在第一（因果）十字架A 中，嫌犯Q 如果能及时终止犯罪就能免于刑事惩罚（对个人有利），如执迷不悟继续犯罪就必然被法律所惩处而进监牢、下地狱（对个人不利）；一个人如果做好事有功于民族就能被嘉奖而个人得益（个人有功），因果的过程在十字架的纵轴上而结果在十字架的竖轴上，一念天堂在上、一念地狱在下万丈深渊，称为第一（十字、十字架）秩序、是非（黑白）分明秩序、门户秩序。在第二（因果）十字架B 中，嫌犯Q 犯了罪肯定要受到法律的制裁而进监牢、下地狱（罪犯与团伙遭清算）；个人如果做好事有功于民族就会被嘉奖（个人领功得尊敬），就象真正的好官得万民伞受人尊敬，因果的过程在十字架的纵轴上而结果在十字架的横轴上，在社会法律体系中一念天堂在上、一念地狱在下万丈深渊，称为第二（十字、十字架）秩序、后果秩序、台面秩序。在第三（因果）十字架B 中，嫌犯Q 到了炸毁地球、放病毒杀害全球2000 万人的层次肯定是万死莫赎、遗臭万年，进入文明层面的十字架的鄙视链之最底端；如果是华盛顿建立自由、民主美国一样的英雄，肯定是上总统山接受万民万世景仰，犹如长江之水滔滔不绝，因果的过程在十字架的横轴上而结果在十字架的垂直轴上，在全局体系、文明体系中一念天堂在上、一念地狱在下万丈深渊，称为第三（十字、十字架）秩序、全局（文明、永恒）秩序、总秩序。第一（十字）秩序、第二（十字）秩序、第三（十字）秩序统称为三（十字架）秩序，线性是因果、正义（殿堂、信仰、天堂、光明）在上、地狱（黑暗、罪恶、毁灭）在下，善恶辨、功罪分，世界见承平，从而构成三（十字架）秩序分善恶（定功罪）原则。第一（十字）秩序、第二（十字）秩序、第三（十字）秩序统称为三（十字架）秩序，线性是因果、正义（殿堂、信仰、天堂、光明）在上、地狱（黑暗、罪恶、毁灭）在下，善恶辨、功罪分，世界见承平，从而构成三（十字架）秩序分善恶（定功罪）原则。 7．天子七和弦（七因果）立法、天子方程（ĤU=λU）先于一切正果原则如同新冠疫情中一位西班牙老奶奶说的：好好的日子不过，为什么要放病毒来杀人啊？如今，全世界已经超过2000 万人死于新冠病毒，任何人不是有罪被处死，而全部是无辜被毒死，即全世界的去毛（去皮）结果（责任）是无分别地无辜者死刑；另一方面，放病毒的团伙帮你做核酸检测是救你有功、行刑毒针的疫苗不仅要你命还要你钱（疫苗开打的前几个月全世界就有最健康的几万飞行员和空乘一上天就疫苗猝死），这就是病毒（疫苗）末日罪行。 7．天子七和弦（七因果）立法、天子方程（ĤU=λU）先于一切正果原则如同新冠疫情中一位西班牙老奶奶说的：好好的日子不过，为什么要放病毒来杀人啊？如今，全世界已经超过2000 万人死于新冠病毒，任何人不是有罪被处死，而全部是无辜被毒死，即全世界的去毛（去皮）结果（责任）是无分别地无辜者死刑；另一方面，放病毒的团伙帮你做核酸检测是救你有功、行刑毒针的疫苗不仅要你命还要你钱（疫苗开打的前几个月全世界就有最健康的几万飞行员和空乘一上天就疫苗猝死），这就是病毒（疫苗）末日罪行。全世界都热衷到中共国进行器官旅游，用几天甚至一天时间就能移植一副供体无打麻醉药物的活摘器官，一年几十万甚至几百万副器官被活摘，大量中小学男女学生和有体检纪律的公务员、军人一样被无分别的无辜活摘而杀害，然后全家被灭门，象香港陈彦霖一样，只是因为自己的HLA 配型掌握在邪恶政权手上，这就是活摘器官末日罪行。任何人在乌克兰做任何事情，首先要面临着普京的导弹袭击甚至荒谬的饱和打击，如同坐上了2014 年7 月17 日被俄罗斯山毛榉导弹（BUK）的马航MH17（波音777）一样，绝对不是任何机上的人能用自行火炮控制的，这就是战争末日罪行。病毒（疫苗）末日罪行、活摘器官末日罪行、战争末日罪行、共俄军事同盟已经进入第二阶段准备发动三战炸毁地球，等等，名副其实的末日天灾人祸A→-∞，此时，我们令 ĤU=λU，称为天子方程，Ĥ 为代表至上天廷的算符或算符式的矩阵：Ĥ→∞，依据同一律， λ 为天子B 的本征根必然是无限大的：λ→+∞，天子在天而令天子的本征函数（特征向量） U 必然也是无限大的：U→+∞，天灾人祸A 要吞噬天子B 时必然吞噬其无限大的特征向量即权力U：-\|A\|=\|U\| ⇒ A=U=0，这就是天子剑方程（天子剑函数），则依据ĤU=λU 有： λ=lim(ĤU/U)=Ĥ→∞，即天子及其本征根是固有的而并不随着特征向量U 被归零而归零；然而，天灾人祸A 在吞噬天子的特征向量U 时已经被伴随着特征向量U 的绝对归零原则T 所归零，自然无法再进一步去吞噬天子的本征根或者天子本身，此时，伴随着天子特征向量的绝对归零原则T 就是诛杀一切恶魔与邪恶的至上宝剑而称为无限天子剑，从而构成天灾人祸天灭原则、天子无害归零天灾人祸原则，也就是说，天子一出生、一出现，所有的天灾人祸和恶魔就自取灭亡而到头了，天子与生俱来的特征向量（函数）包括跟随的文武众星，将无限攻击恶魔及其天灾人祸直到彻底消灭，这就是天子无限围剿恶魔（天灾人祸）原则、天子生灾难灭（人祸绝）原则、天子生妖魔死（灭、灭亡）原则、天子妖魔不相容原则、天子计划生育恶魔（天灾人祸）原则、天子登临天位（就职）妖魔鬼怪灭绝原则、天子登临（天位、就职）安天下原则、天子登临（天位、就职）天下太平原则、天子（圣子）定乾坤原则、天子一剑定乾坤原则、天子剑绝对（无限）原则、天子剑（无限）无害（无损）灭绝（剿灭）妖魔原则、天子剑与妖魔鬼怪水火不相容原则，犹如长江之水滔滔不绝。任何人在乌克兰做任何事情，首先要面临着普京的导弹袭击甚至荒谬的饱和打击，如同坐上了2014 年7 月17 日被俄罗斯山毛榉导弹（BUK）的马航MH17（波音777）一样，绝对不是任何机上的人能用自行火炮控制的，这就是战争末日罪行。病毒（疫苗）末日罪行、活摘器官末日罪行、战争末日罪行、共俄军事同盟已经进入第二阶段准备发动三战炸毁地球，等等，名副其实的末日天灾人祸A→-∞，如果其没有天子B 登临世界（国家、社会）最高位立法并行使最高权力进行全局部署：B→+∞，任何人、组织无法单凭自己的力量改变这些来自世界全局、国家和社会全局的制度性大灾难、丧尽天良的灭绝性人祸，因为，当末日天灾人祸A 吞噬任何人Q 时，非天子者并非在天而必然不是绝对的：-A>Q 即必然无力对抗而被摧毁的，包括任何假冒伪劣产品通通是能力受限的而不可能是主动的，自然无法主动解决并了断这些去皮后仍然是无限大祸害的末日罪行；另一方面，依据无分别绝对原则，任何全局性的事物、灾难、人祸、战争都是无分别而绝对的，此时，依据天子无害归零天灾人祸原则，能对抗这种绝对性无限大力量者只有上帝、圣子即真命天子本人才有可能，这就是非（真命）天子无法阻止全局灾难（人祸、天灾人祸）原则、非（真命）天子阻止全局灾难（人祸、天灾人祸）否定原则、唯（真命）天子阻止全局灾难（人祸、天灾人祸）原则、天子宙斯盾(aegis of Zeus)原则、天子（圣子）安天下原则。天子（圣子）定乾坤原则和天子（圣子）安天下原则统称为天子（圣子）定乾坤（安天天子（圣子）定乾坤原则和天子（圣子）安天下原则统称为天子（圣子）定乾坤（安天下）原则、天子（圣子）生乾坤定（天下安）原则。第零因果律是获得性因果，第一因果律是分布性因果，第二因果律是输出性因果即门票自由，第三因果律是承受性因果即饭票自由，在这四大因果中，唯一一个非全局解决全部死（炸毁地球）就是承受性因果：除非全局解决，否则任何人无法独善其身而必死或必死恐怖威胁无法排除，任何独立、自由、民主（选择、判断）、非全局的解决治愈都失效，即华盛顿总统等先贤未知奋斗一生的自由宪政全打水漂，今天普京和他包皮联手用核武器炸毁地球、用病毒毒死全世界的问题无人能解就是证据，比照泰坦尼克号沉穿或水鬼凿船，船被凿沉而船上的人无人能幸免于难，因此，承受性因果又称为主宰因果、决定（瓶颈、生死、阎罗王、末日）因果、全局因果、本征因果、凿船因果、沉船因果，从而构成承受性因果决定（主宰）一切原则、承受性因果先于一切原则、承受性因果凿船（沉船）原则、同一先于本征因果原则、本征因果同一原则。上述的天子方程（ĤU=λU）（Ĥ 为代表至上天廷的算符或算符式的矩阵、λ 是天子本征根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞），是解决所有的末日因果、生死因果、阎罗王因果的唯一方案、钥匙，同时解决了制度性以至天地的独立、自由、民主（选择、判断）和解决而最终实现标准化、全局性治愈，由此而来，天子方程（ĤU=λU）解决末日因果而得出至上的天正果，我们称天子方程为天子因果、圣子因果、天因果、正果因果、正因果，也就是说，全世界以至天地间所有的正果都必须无分别地、别无选择地经过天子完成，依据别无选择绝对原则、同一律，天子因果必然是绝对的而必然是最后的正果、最初的因果，正如同耶稣所说的：我是最初的、我是最后的，从而构成天子方程（ĤU=λU）先于（一切、任何）正果原则、天子先于（一切、任何）正果原则、天子天地正果原则、天子正果原则，归根结底，天子比吃一口就能长生不老的唐僧肉更厉害。根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞），是解决所有的末日因果、生死因果、阎罗王因果的唯一方案、钥匙，同时解决了制度性以至天地的独立、自由、民主（选择、判断）和解决而最终实现标准化、全局性治愈，由此而来，天子方程（ĤU=λU）解决末日因果而得出至上的天正果，我们称天子方程为天子因果、圣子因果、天因果、正果因果、正因果，也就是说，全世界以至天地间所有的正果都必须无分别地、别无选择地经过天子完成，依据别无选择绝对原则、同一律，天子因果必然是绝对的而必然是最后的正果、最初的因果，正如同耶稣所说的：我是最初的、我是最后的，从而构成天子方程（ĤU=λU）先于（一切、任何）正果原则、天子先于（一切、任何）正果原则、天子天地正果原则、天子正果原则，归根结底，天子比吃一口就能长生不老的唐僧肉更厉害。天子方程（ĤU=λU）中，天子通过天子函数U、天子本征根与万物、众神、众生Pj(j ∈N)在全局的本征层面上开放相互沟通、相互帮助、相互救济： (Ĥ-λ")U"=0……（Eq.TZ01）其中Ĥ 为代表至上天廷的算符或算符式的矩阵、λ 是天子本征根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞，λ"为λ 合并万物、众神、众生Pj(j∈N)后的本征根、U"为合并万物、众神、众生Pj(j∈N)后的特征向量，由此而来，久期方程(Ĥ-λ")U"=0 称为天子权杖方程（函数）、天子久期方程，永恒的天子久期方程则称为永恒的天子权杖，天子通过永恒的天子久期方程保守万物众生、为他们诛杀任何邪恶（恶魔）而提供足够的天赋安全（救赎、拯救）、支撑（支持）万物众生苍天大地竞自由地发展和发挥自己的一切潜力，即三保非不可救药的万物众生，这就是唯有天子降生（登临）才能提供的全局性永恒之久期大爱，称之为天子（降生、登临）先于一切（万物、众生）的永恒之三保久期大爱（博爱）、天子（久期、永恒、绝对、三保）大爱（博爱），也是万物（众生）的永恒之天子三宝（即天子的降生为众生与万物带来了永恒的三宝保守天下苍生），从而构成天子（圣子）安天下（安万物）原则、天子一杖（天子一根、天子权杖、天子本征根）安天下（安万物）原则、天子杖（天子权杖、天子本征根）永恒（无限、绝对）原则、天子杖（天子权杖、天子本征根）先于天下安定（乾坤安定、和平）原则、天子杖（天子权杖、天子本征根）先于万物（众生）原则、天子（降生、登临）先于一切（万物、众生）原则、天子保守（三保）天下苍生（铸永恒）原则、天子三宝大爱（博爱）永恒（无限、绝对）原则。任何）正果原则、天子先于（一切、任何）正果原则、天子天地正果原则、天子正果原则，归根结底，天子比吃一口就能长生不老的唐僧肉更厉害。天子方程（ĤU=λU）中，天子通过天子函数U、天子本征根与万物、众神、众生Pj(j ∈N)在全局的本征层面上开放相互沟通、相互帮助、相互救济： (Ĥ-λ")U"=0……（Eq.TZ01）其中Ĥ 为代表至上天廷的算符或算符式的矩阵、λ 是天子本征根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞，λ"为λ 合并万物、众神、众生Pj(j∈N)后的本征根、U"为合并万物、众神、众生Pj(j∈N)后的特征向量，由此而来，久期方程(Ĥ-λ")U"=0 称为天子权杖方程（函数）、天子久期方程，永恒的天子久期方程则称为永恒的天子权杖，天子通过永恒的天子久期方程保守万物众生、为他们诛杀任何邪恶（恶魔）而提供足够的天赋安全（救赎、拯救）、支撑（支持）万物众生苍天大地竞自由地发展和发挥自己的一切潜力，即三保非不可救药的万物众生，这就是唯有天子降生（登临）才能提供的全局性永恒之久期大爱，称之为天子（降生、登临）先于一切（万物、众生）的永恒之三保久期大爱（博爱）、天子（久期、永恒、绝对、三保）大爱（博爱），也是万物（众生）的永恒之天子三宝（即天子的降生为众生与万物带来了永恒的三宝保守天下苍生），从而构成天子（圣子）安天下（安万物）原则、天子一杖（天子一根、天子权杖、天子本征根）安天下（安万物）原则、天子杖（天子权杖、天子本征根）永恒（无限、绝对）原则、天子杖（天子权杖、天子本征根）先于天下安定（乾坤安定、和平）原则、天子杖（天子权杖、天子本征根）先于万物（众生）原则、归根结底，天子比吃口就能长生不老的唐僧肉更厉害。天子方程（ĤU=λU）中，天子通过天子函数U、天子本征根与万物、众神、众生Pj(j ∈N)在全局的本征层面上开放相互沟通、相互帮助、相互救济： (Ĥ-λ")U"=0……（Eq.TZ01）其中Ĥ 为代表至上天廷的算符或算符式的矩阵、λ 是天子本征根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞，λ"为λ 合并万物、众神、众生Pj(j∈N)后的本征根、U"为合并万物、众神、众生Pj(j∈N)后的特征向量，由此而来，久期方程(Ĥ-λ")U"=0 称为天子权杖方程（函数）、天子久期方程，永恒的天子久期方程则称为永恒的天子权杖，天子通过永恒的天天子方程（ĤU=λU）中，天子通过天子函数U、天子本征根与万物、众神、众生Pj(j ∈N)在全局的本征层面上开放相互沟通、相互帮助、相互救济： (Ĥ-λ")U"=0……（Eq.TZ01）其中Ĥ 为代表至上天廷的算符或算符式的矩阵λ 是天子本征根U 是天子本征函数或 ( ) q 其中Ĥ 为代表至上天廷的算符或算符式的矩阵、λ 是天子本征根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞，λ"为λ 合并万物、众神、众生Pj(j∈N)后的本征根、U"为合并万物、众神、众生Pj(j∈N)后的特征向量，由此而来，久期方程(Ĥ-λ")U"=0 称为天子权杖方程（函数）、天子久期方程，永恒的天子久期方程则称为永恒的天子权杖，天子通过永恒的天子久期方程保守万物众生、为他们诛杀任何邪恶（恶魔）而提供足够的天赋安全（救赎、拯救）、支撑（支持）万物众生苍天大地竞自由地发展和发挥自己的一切潜力，即三保非不可救药的万物众生，这就是唯有天子降生（登临）才能提供的全局性永恒之久期大爱，称之为天子（降生、登临）先于一切（万物、众生）的永恒之三保久期大爱（博爱）、天子（久期、永恒、绝对、三保）大爱（博爱），也是万物（众生）的永恒之天子三宝（即天子的降生为众生与万物带来了永恒的三宝保守天下苍生），从而构成天子（圣子）安天下（安万物）原则、天子一杖（天子一根、天子权杖、天子本征根）安天下（安万物）原则、天子杖（天子权杖、天子本征根）永恒（无限、绝对）原则、天子杖（天子权杖、天子本征根）先于天下安定（乾坤安定、和平）原则、天子杖（天子权杖、天子本征根）先于万物（众生）原则、天子（降生、登临）先于一切（万物、众生）原则、天子保守（三保）天下苍生（铸永恒）原则、天子三宝大爱（博爱）永恒（无限、绝对）原则。依据天子先于（一切、任何）正果原则，天子剑、天子权杖、天子影响力之天子函数三位一体于天子在位，天子剑是规则归零的规则因果，天子权杖是天下万物的上天层面的电动势化育万物而为造化分布因果，天子影响力之天子函数诛杀任何邪恶（恶魔）而提供足够的天赋安全（救赎、拯救）即为造化安全因果，称之为天位三因果，从而构成天位因果三分原则。如下的广义相对论方程表明，能量动量张量Tαβ 与代表相对性协变参数化的度规gαβ 和偏离差异性的里奇(Ricci)张量Rαβ 之间具有不可排除的参数化同步性（包括可移动的copy、 paste 模块和不可移动的静态同步性），我们称之为（参数化）同步论、（参数化）同步性论、（参数化）同步化论。显然，爱因斯坦白将量子力学和时空的同步性拆家复杂化后没有参数化回去，这就导致他没有最终得出量子力学的本征同步性，也是量子力学以线性、线性相对化来解决基于本征同步性而一直无法得以最终完善的原因，今天，基于本征同步性的天子方程（ĤU=λU）的矩阵动力学将最终升级广义相对论和量子力学，从而登陆广义相对同步论和参数化同步性本征量子力学。 Gαβ=Rαβ-gαβR/2=χTαβ……（Eq.TZ02） Gαβ=Rαβ-gαβR/2=χTαβ……（Eq.TZ02）其中gαβ 是度规或距离张量、Rαβ 是黎曼曲率缩并后的里奇(Ricci)张量、R 代表时空曲率、 Tαβ 为能量动量张量、Gαβ 为爱因斯坦张量、χ=8πG/c4、G 为引力常数、c 光光速。全人类的文明从丛林混沌已经上升到线性的独立、自由、民主、博爱平等层面，但尚未实现相对与常态（固有）的隔离即相对性立法（法律相对论），相对性立法（法律相对论）之上的比照广义相对论的参数化同步论则完全未入门，线性（板性、体级等）、线性（板性、体级等）相对论、相对参数同步论经由天子本征根三位一体于天（自然）的三位一体解决，称为本征三位一体：本位（参数化）P（plant 植物性植物和立足）、相对性（参数化）R、同步性（参数化）S，即PSR 三位一体。此时，天子方程、天子剑方程和天子权杖方程带来了PSR 参数化的全面解决即治愈因果，因此，同步因果包括本征因果和参数化同步性因果（如广义相对论）：parametric synchronism。同步性涉及正反两方面的承载平台，从而构成同步正反（承载平台）原则；相对涉及本身和外部之差异，从而构成相对差异原则。伽利略（Galileo）证明不同的物体在同一个引力场是无分别地以同一个加速度（重力加速度）运动的，即重力场加速度产生自由落体加速度：f=mg=Ma，其中f 为物体所受的重力、 m 为引力质量、M 为惯性质量、g 为重力加速度、a 为自由落体加速度：a=g，则其必然有 M=m，依据无分别绝对原则，M=m 的质量等效即属绝对的，从而构成质量等效（动力学效应）绝对原则、（与时间无关的）时间自由之万物平等绝对原则；爱因斯坦在广义相对论中将任何物体在同一个引力场的引力质量与惯性质量等同，其必然也是绝对的，这就是广义相对论中的质量等效性（动力学效应）原理、质量等效性（动力学效应）绝对原理（如果不成立则伽利略的质量等效原则即不是绝对的与事实矛盾）、（与时间无关的）时间自由之万物解决因果包括本征因果⑤、参数化同步性因果⑥、治愈因果⑦。平等绝对原则，同时广义相对论也已经被诸多事实证明成立，依此类推至于所有效应中同理成立。平等绝对原则，同时广义相对论也已经被诸多事实证明成立，依此类推至于所有效应中同理成立。平等绝对原则，同时广义相对论也已经被诸多事实证明成立，依此类推至于所有效应中同理成立。（与时间无关的）时间自由之万物平等绝对原则表明，万物的本征方程（ĤU=λU）从万物之间延伸到万物之外的平等之统一平台，即宇宙万物产生之后即处于一个统一的绝对平等的平台W（即质量等效平台）上，平台W 称之为大本征平台、（参数化）同步性平台，这就万物登陆殿堂的真实彼岸、天堂彼岸，从而构成（绝对、无限、无分别）平等先于（天堂）彼岸原则、（绝对、无限、无分别）平等先于同步性（同步、大本征）因果原则、同步性（同步、大本征）因果平等原则、同步性（同步、大本征）绝对平等原则、彼岸（天堂彼岸）绝对平等原则。广义相对论方程（Gαβ=Rαβ-gαβR/2=χTαβ）表明，一个质量为M 的物体W 会引起时空扭曲，从而将时空作为通解方程y=f(x,t)=0 而将物体W 作为特解方程g0(x,t)而构成时空扭曲平衡Q 的方程：y=f(x,t)=g0(x,t)，x 为空间变量、t 为时间变量，时空扭曲平衡Q 成为凌驾与本征和大本征之上的公共平等，称为跨越时空、事物以至一切（包括动力所在的内空间）的全局框架K 之公平，跨越时空、事物以至一切（包括动力所在的内空间）的全局框架K 之最高本征则称为朝廷（枢纽）及其乾纲，从而构成公平公共平等原则、公平公共本征原则、公平先于平等（同一、本征、大本征）原则。平先于平等（同、本征、大本征）原则。进而，上述的天子方程（ĤU=λU），系数矩阵的秩小于n 元未知数：rank(A)<n(2<n∈ N)，因为至少是天子和一个非天子的事物存在，天子方程（ĤU=λU）以无限解无缝无限接轨本征因果之同一性、张量多元时间性的参数化同步性因果之平等律、超越事物本征和大本征平台的全局框架（朝廷）之公平，从而解决、治愈万物、天下而实现公正、正义之公义，从而构成天子（方程）公平原则、上天公义（公正、正义）原则、天子先于朝廷（公平）原则、上天公义（公正、正义）先于天子（公平）原则、天子万解之解原则、天子治愈（解决）原则，其中，Ĥ 为代表至上天廷的算符或算符式的矩阵、λ 是天子本征根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞。在天子的全局框架的朝廷公平中，天子方程将本征因果的同一性和大本征因果的绝对平等性（质量等效性）推广到算符的跨越时空、事物以至一切（包括动力和万物起源）的通解、特解之全局框架上，全国以至全世界、全人类的动力无缝充盈自由、水位充盈（产业、技术、资金、人才）、安全无分别足够有效、法律无缝对接正义，任何人如鱼得水、如入无人之境、犹如长江之水滔滔不绝，道路（解决、程序）多元化、人才济济、群策群力，同时实现动力公平。平先于平等（同、本征、大本征）原则。进而，上述的天子方程（ĤU=λU），系数矩阵的秩小于n 元未知数：rank(A)<n(2<n∈ N)，因为至少是天子和一个非天子的事物存在，天子方程（ĤU=λU）以无限解无缝无限接轨本征因果之同一性、张量多元时间性的参数化同步性因果之平等律、超越事物本征和大本征平台的全局框架（朝廷）之公平，从而解决、治愈万物、天下而实现公正、正义之公义，从而构成天子（方程）公平原则、上天公义（公正、正义）原则、天子先于朝廷（公平）原则、上天公义（公正、正义）先于天子（公平）原则、天子万解之解原则、天子治愈（解决）原则，其中，Ĥ 为代表至上天廷的算符或算符式的矩阵、λ 是天子本征根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞。在天子的全局框架的朝廷公平中，天子方程将本征因果的同一性和大本征因果的绝对平等性（质量等效性）推广到算符的跨越时空、事物以至一切（包括动力和万物起源）的通解、特解之全局框架上，全国以至全世界、全人类的动力无缝充盈自由、水位充盈（产业、技术、资金、人才）、安全无分别足够有效、法律无缝对接正义，任何人如鱼得水、如入无人之境、犹如长江之水滔滔不绝，道路（解决、程序）多元化、人才济济、群策群力，同时实现动力公平。进而，上述的天子方程（ĤU=λU），系数矩阵的秩小于n 元未知数：rank(A)<n(2<n∈ N)，因为至少是天子和一个非天子的事物存在，天子方程（ĤU=λU）以无限解无缝无限接轨本征因果之同一性、张量多元时间性的参数化同步性因果之平等律、超越事物本征和大本征平台的全局框架（朝廷）之公平，从而解决、治愈万物、天下而实现公正、正义之公义，从而构成天子（方程）公平原则、上天公义（公正、正义）原则、天子先于朝廷（公平）原则、上天公义（公正、正义）先于天子（公平）原则、天子万解之解原则、天子治愈（解决）原则，其中，Ĥ 为代表至上天廷的算符或算符式的矩阵、λ 是天子本征根、U 是天子本征函数或特征向量：Ĥ、U、λ→+∞。象泰坦尼克号的甲板沉没了，任何乘客还能到美国纽约吗？任何人乘坐的飞机失事了还能活吗？败类用兵核武器把地球炸毁了，人类还能生存吗？全世界濒临三战的问题不解决，人类还有路可走吗？依此类推至于普遍情形同理成立，这就是本征（全局）因果先于相对因果原则、同步性（因果）先于相对（因果）原则。广义相对论表明，矩阵是张量的退化，物体在不同的引力环境中具有不同的本征矩阵，引力环境如同航空母舰的甲板决定甲板上的舰载机一样，从而构成参数化同步性因果先于本征因果原则。征因果原则。任何人如果无路可走，肯定象上海疫情清零政策中被活活饿死、饿到吃大便、纷纷跳楼、不让看病当场身亡的人们一样，象乌克兰人出门被枪打死一样，象武汉人在家被病毒毒死一样等，这就是道路（程序、过程、秩序）先于存在原则、相对先于状态（本源、存在）原则、相对因果先于植物因果（固定因果、常态因果）原则。任何时候，如果世界没有天子、天子本征根，任何人无法单独抵挡邪恶组织放病毒毒死全世界超过2000 万的人为大灾难，也无法抵挡象在乌克兰的人出门被俄罗斯军队用枪打死一样的战争，更加无法抵挡整天把核武器挂在嘴上的普京炸毁地球，等等；同时，如果全世界没有天子和天子剑，任何人、组织没有办法单独去将放病毒毒死全人类超过2000 万人的罪魁祸首和犯罪组织解决掉，也没办法将整天用核武器炸毁地球的XP 军事同盟解体，更加没办法将用疫苗毒死全世界的投资者进行法办，等等，综上所述，没有天子、天子剑、天子权杖（本征根），全世界的任何全局性问题如瘟疫、气候、环保等就无法解决，这就是天子（天子剑、天子权杖、天子本征根、天子特征向量、天子因果）先于同步因果（本征因果、参数化同步性因果）原则。任何人如果无路可走，肯定象上海疫情清零政策中被活活饿死、饿到吃大便、纷纷跳楼、不让看病当场身亡的人们一样，象乌克兰人出门被枪打死一样，象武汉人在家被病毒毒死一样等，这就是道路（程序、过程、秩序）先于存在原则、相对先于状态（本源、存在）原则、相对因果先于植物因果（固定因果、常态因果）原则。任何时候，如果世界没有天子、天子本征根，任何人无法单独抵挡邪恶组织放病毒毒死全世界超过2000 万的人为大灾难，也无法抵挡象在乌克兰的人出门被俄罗斯军队用枪打死一样的战争，更加无法抵挡整天把核武器挂在嘴上的普京炸毁地球，等等；同时，如果全世界没有天子和天子剑，任何人、组织没有办法单独去将放病毒毒死全人类超过2000 万人的罪魁祸首和犯罪组织解决掉，也没办法将整天用核武器炸毁地球的XP 军事同盟解体，更加没办法将用疫苗毒死全世界的投资者进行法办，等等，综上所述，没有天子、天子剑、天子权杖（本征根），全世界的任何全局性问题如瘟疫、气候、环保等就无法解决，这就是天子（天子剑、天子权杖、天子本征根、天子特征向量、天子因果）先于同步因果（本征因果、参数化同步性因果）原则。存在（植物性存在、状态）的常态因果（植物因果、承载性因果、立足因果①）A、相对因果（开天地、独立因果②、自由因果③、民主选择判断因果④）B、同步因果（本征因果⑤、张量多元时间性的参数化同步性因果⑥）C、天子因果（定乾坤、治愈因果⑦）D，构成了治愈四因果、积体（点线面体）四因果、点线面体四因果。对于万物而言，承受性因果包括本征因果⑤、张量多元时间性的参数化同步性因果⑥、天子因果⑦，从而构成承受三因果原则，这三个因果全部是框架性、平台性、社会性的因果，是非全局改变无法解决的一因果，从而又构成承受三因果非全局改变无法解决原则。治愈四因果包含有7 个独立因果称为七层文明（文明七层协议）、七步因果、七因果、七弦琴因果、七步成诗因果、七和弦、天子七步因果（统天、登天）、（天子）七因正果、天子七和弦（乐章）、天子七律（乐章）、天子（七和弦、七因果、七律、七乐章）立法：存在（植物性存在、状态）的常态因果（植物因果①）A、相对因果（独立因果即获得性因果②、自由因果③、民主选择判断因果④）B、同步因果（本征因果⑤同一、张量多元时间性的参数化同步性因果⑥平等）C、天子因果（治愈因果⑦博爱公平）D 而公义（公正、正义之公义）在天，从而构成（天子）七步因果统天（登天）原则、天子（七和弦、七因果、七律、七乐章）立法原则、七因果（七步因果、七弦琴因果、七步成诗因果、七和弦）原则，其中①独立、②自由、③民主、④同一、⑤平等、⑥公平、⑦公义（公正），称为人类社会（天地、文明）七（大）基因。存在（植物性存在、状态）的常态因果（植物因果、承载性因果、立足因果①）A、相对因果（开天地、独立因果②、自由因果③、民主选择判断因果④）B、同步因果（本征因果⑤、张量多元时间性的参数化同步性因果⑥）C、天子因果（定乾坤、治愈因果⑦）D，构成了治愈四因果、积体（点线面体）四因果、点线面体四因果。解决因果包括本征因果⑤、参数化同步性因果⑥、治愈因果⑦。五位四因果为：第零因果（独立因果或获得性因果）①、分布因果（程序道路自由因果） ②、输出性因果（民主选择判断因果如罪犯被处决）③、承受性因果（解决治愈因果、乌克兰飞机失事）④。上海疫情清零导致人们纷纷跳楼（所谓校园民谣中的畜牲一回头全校师生齐跳楼）、被活活饿死、饿到吃大便、不让病人看病当场身亡，等等，无辜的上海人突然在清零中纷纷折五位四因果为：第零因果（独立因果或获得性因果）①、分布因果（程序道路自由因果） ②、输出性因果（民主选择判断因果如罪犯被处决）③、承受性因果（解决治愈因果、乌克兰飞机失事）④。 8．天子七乐章统天、邪恶（罪犯）七步成尸、天子四喜、邪恶四悲独立让事物、主体脱离束缚而获得相对论式的相对性自主，这就是万物包容而仁慈平行并列，从而构成独立相对原则、独立事物（万物、主体）相对原则、独立包容（兼容并蓄、并置仁行）原则并置、仁行）原则。自由让船行使在江河湖海的航道W 上，令航道与河床之道得以独立，依此类推至于普自由让船行使在江河湖海的航道W 上，令航道与河床之道得以独立，依此类推至于普遍情形同理成立，从而构成自由道路（过程、程序）相对（独立、平行、兼容并蓄）原则。正确的选择判断（即民主）令人们、事物避免陷入万劫不复之清算性地狱、法律惩处或制裁，同时令人们、事物有机会抓住机遇全速发展到世界先进水平而功在当代、利在千秋；反之，象普京和他包皮是一床新被褥、两个过来人（共产主义窝里斗的老手），祸国殃民、放病毒毒死全世界、用核武器炸毁地球，每一宗罪行都足够让这一类的罪犯亲自下地狱如入无人之境，从而构成选择（判断、民主）罪责自由（独立、平行、不粘锅）原则、选择（判断、民主）目标（彼岸、灯塔）自由（独立、平行、不粘锅）原则、选择（判断、民主）先于功罪（罪责、彼岸门票）原则、门票自由（独立）先于彼岸原则、民主先于门票自由（独立）原则、民主（门票、自由、独立）先于彼岸原则。任何人在乌克兰做任何事情，首先要面临着普京的导弹袭击甚至荒谬的饱和打击，如同坐上了2014 年7 月17 日被俄罗斯山毛榉导弹（BUK）的马航MH17（波音777）一样，绝对不是任何机上的人能用自行火炮控制的；反之，任何人在英美国家生活、学习，没有山毛榉导弹导致正义不举之忧，相对有一定的安全保障，这种由制度提供的安全保障、奋斗机会的正义称为全局正义、本征正义（对应并比照久期方程）、保守正义，只有无分别绝对归一的全局管理者、主宰者才能支撑、保证并实现，与真理、秩序（程序、过程）、效力、良心等息息相关，因此，非上帝（圣子、真命天子）亲自降临并就职、立法无法实现，即真命天子（圣子）降临（立法、就职）先于制度（保守）自由（独立、自主、安全），而制度自由（独立、自主、安全）先于人们对制度、环境、全局的安全、自由、独立而成家立业、建功立业，这就是（真命）天子（立法、就职、降临）先于制度（保守）安全（自由、独立、自主）原则、真命天子（圣子、上帝）先于制度安全（自由、独立）原则、真命天子（圣子、上帝）先于（制度、无分别）保守原则。上帝（圣子、真命天子）亲自降临并就职、立法，从而实现制度自由（独立、自主、安全）和全局保守，这就是文明法线上信仰的圣殿和光明殿堂，解决、治愈由此实现，由此可见，万民、万物、人们无法凭借自己获得有效的门票，真命天子、圣子、上帝才是全局面向制度（规则）而面向对象、面对人们的保守、安全、独立、自由之渊源性长期饭票，尤其是真命天子的能力、智慧、知识、经验和判断，从而构成真命天子（圣子、上帝）先于制度安全（自由、独立）饭票原则、真命天子（圣子、上帝）能力（智慧、知识、经验、判断）先上帝（圣子、真命天子）亲自降临并就职、立法，从而实现制度自由（独立、自主、安全）和全局保守，这就是文明法线上信仰的圣殿和光明殿堂，解决、治愈由此实现，由此可见，万民、万物、人们无法凭借自己获得有效的门票，真命天子、圣子、上帝才是全局面向制度（规则）而面向对象、面对人们的保守、安全、独立、自由之渊源性长期饭票，尤其是真命天子的能力、智慧、知识、经验和判断，从而构成真命天子（圣子、上帝）先于制度安全（自由、独立）饭票原则、真命天子（圣子、上帝）能力（智慧、知识、经验、判断）先于制度安全（自由、独立）饭票原则、解决（治愈）先于制度安全（自由、独立）原则、解决（治愈）先于制度饭票原则。我们假设英美如销毁乌克兰核武器时所承诺而在乌克兰被打成一片废墟之前出兵拯救，让乌克兰免于被打成废墟或免于战争，这是文明十字架上的正果、天堂正义、义薄云天、治愈、解决、圣殿；可是，事实是英美并未履行承诺而导致乌克兰现在到处是无药可救的废墟，人间地狱一片，也就是说，英美、北约以至全世界在乌克兰上面的解决方法绝对有问题，解决方案决定了全局绩效的效力和高低。于制度安全（自由、独立）饭票原则、解决（治愈）先于制度安全（自由、独立）原则、解决（治愈）先于制度饭票原则。我们假设英美如销毁乌克兰核武器时所承诺而在乌克兰被打成一片废墟之前出兵拯救，让乌克兰免于被打成废墟或免于战争，这是文明十字架上的正果、天堂正义、义薄云天、治愈、解决、圣殿；可是，事实是英美并未履行承诺而导致乌克兰现在到处是无药可救的废墟，人间地狱一片，也就是说，英美、北约以至全世界在乌克兰上面的解决方法绝对有问题，解决方案决定了全局绩效的效力和高低。承受性因果包括本征因果⑤、张量多元时间性的参数化同步性因果⑥、天子因果⑦，其中天子因果属于最高因果，从而构成天子最高因果原则；而天子所实现的公义、正义为最高结果，其随之构成公义（正义）最高结果原则。第零因果律是获得性解决（解放），第一因果律是分布性（期望性、过程性、程序性、面向过程、面向程序、面向道路）解决（解放），第二因果律是输出性解决（解放）即门票自由，第三因果律是承受性解决（解放）即饭票自由，四大解决合称为四仁解放（解决），包括解放主体、道路（程序、过程、秩序）、真理门票、文明（正义）饭票，称之为四仁开天地（解放），从而构成解放（独立、自由、民主、解决）四仁（四仁爱）原则、行四仁开天地原则、行仁开天地原则、四仁开天地原则。承受性因果包括本征因果⑤、张量多元时间性的参数化同步性因果⑥、天子因果⑦，其中天子因果属于最高因果，从而构成天子最高因果原则；而天子所实现的公义、正义为最高结果，其随之构成公义（正义）最高结果原则。第零因果律是获得性解决（解放），第一因果律是分布性（期望性、过程性、程序性、面向过程、面向程序、面向道路）解决（解放），第二因果律是输出性解决（解放）即门票自由，第三因果律是承受性解决（解放）即饭票自由，四大解决合称为四仁解放（解决），包括解放主体、道路（程序、过程、秩序）、真理门票、文明（正义）饭票，称之为四仁开天地（解放），从而构成解放（独立、自由、民主、解决）四仁（四仁爱）原则、行四仁开天地原则、行仁开天地原则、四仁开天地原则。独立、自由、民主（选择、判断）、解决治愈而解放万物，万物从植物状态脱离而获得相对性和新生，这就是四仁神之道，从而构成（四仁）神之道相对（降生、创生）原则、行仁开天地（创生）原则，万物今天正式降生。独立、自由、民主（选择、判断）、解决治愈而解放万物，万物从植物状态脱离而获得相对性和新生，这就是四仁神之道，从而构成（四仁）神之道相对（降生、创生）原则、行仁开天地（创生）原则，万物今天正式降生。天子站在最高点向下拯救万民万物，众官和常态下的万民站在正高点同心协力救赎；天子携众官、万民站在国家力量、世界力量的立足点，向上推进科技、产业、教育、就业、医疗、健康保障、灾难应急、战争防范措施等发展；天子携众官、万民维持常态性文明社会，建设道路、道路防护栏等各种维持机制，由此形成覆盖正反两方面的救赎、常态维持、向上推进发展，三足鼎立，统称为三义，即天子携众生万物立三义而立乾坤，比照人类和小孩直立行走，称为天子七步统天、天子七乐章（七弦琴、七步）统天、万物（众生）七步成诗，从而构成三义立乾坤原则、行义立乾坤原则、天子七步（七乐章、七弦琴）原则、万物（众生）七步成诗原则、天子七乐章（七弦琴、七步）统天原则。章依据天子剑（无限）无害（无损）灭绝（剿灭）妖魔原则、行仁开天地创生原则、行义立乾坤原则，解放万物（开放世界）、治愈全局（女娲补天）、普度众生而统天（开放殿堂、佛教的证得正果），邪恶被灭绝、罪犯伏法，而不是象今天邪恶多如狗、罪犯满地走而灭绝万物，邪恶（罪犯）七步成尸随之形成，这就是四仁三义七步走的邪恶（罪犯）七步成尸原则、罪魁祸首（罪大恶极）七步成尸原则。依据天子剑（无限）无害（无损）灭绝（剿灭）妖魔原则、行仁开天地创生原则、行义立乾坤原则，解放万物（开放世界）、治愈全局（女娲补天）、普度众生而统天（开放殿堂、佛教的证得正果），邪恶被灭绝、罪犯伏法，而不是象今天邪恶多如狗、罪犯满地走而灭绝万物，邪恶（罪犯）七步成尸随之形成，这就是四仁三义七步走的邪恶（罪犯）七步成尸原则、罪魁祸首（罪大恶极）七步成尸原则。上海疫情清零导致人们纷纷跳楼（所谓校园民谣中的畜牲一回头全校师生齐跳楼）、被活活饿死、饿到吃大便、不让病人看病当场身亡，等等，无辜的上海人突然在清零中纷纷折上海疫情清零导致人们纷纷跳楼（所谓校园民谣中的畜牲一回头全校师生齐跳楼）、被活活饿死、饿到吃大便、不让病人看病当场身亡，等等，无辜的上海人突然在清零中纷纷折磨至死，绝大多数的上海人的权利、安全被清零到一铺清袋、扑街趴体加入永远伟大光荣正确的共产党万衰，于是，大地在清零中露出了邪恶的面目吞噬人们的生命、财富、安全和健康，显然是天地在清零的净因果为绝对邪恶、绝对刑事犯罪之常态因果（植物因果）△W→-∞，依此类推至于合饭班主任毒死2000 万全世界的无辜者同理成立，地球已经不是厚德载物的大地而是山崩地裂的黑山老妖之巢穴穷山恶水、红楼、血污海，依此类推至于杭州清零、北京清零、全国清零等同理成立，邪恶政权直接摧毁上海以至全中国的产业链、拆毁世界各国产业链，比资本主义大萧条更彻底的直接探底的共产主义经济大解体瞬间来到，从而构成大地黑山老妖（吞噬一切）原则、大地黑洞原则、大地绝对罪行原则、大地因果黑洞（负无限大、吞噬一切）原则。磨至死，绝大多数的上海人的权利、安全被清零到一铺清袋、扑街趴体加入永远伟大光荣正确的共产党万衰，于是，大地在清零中露出了邪恶的面目吞噬人们的生命、财富、安全和健康，显然是天地在清零的净因果为绝对邪恶、绝对刑事犯罪之常态因果（植物因果）△W→-∞，依此类推至于合饭班主任毒死2000 万全世界的无辜者同理成立，地球已经不是厚德载物的大地而是山崩地裂的黑山老妖之巢穴穷山恶水、红楼、血污海，依此类推至于杭州清零、北京清零、全国清零等同理成立，邪恶政权直接摧毁上海以至全中国的产业链、拆毁世界各国产业链，比资本主义大萧条更彻底的直接探底的共产主义经济大解体瞬间来到，从而构成大地黑山老妖（吞噬一切）原则、大地黑洞原则、大地绝对罪行原则、大地因果黑洞（负无限大、吞噬一切）原则。大、吞噬切）原则。依据大地因果黑洞（负无限大、吞噬一切）原则、依据并比照天子剑（无限）无害（无损）灭绝（剿灭）妖魔原则，天子必须以无限天子剑诛杀吞噬一切的黑山老妖，人类才能免于无辜受害的新冠已经之人祸、工业化活摘器官之入体抢劫器官（入体抢劫）刑事犯罪、杀人有功的行刑疫苗犯罪等，这就是天子征恶地、天子安天下（平四海、主大地、守土）、天子剑征恶地、天子剑安天下（平四海、主大地、守土）、天子修理地球（厚德载物），从而构成天子（诛邪）安天下（平四海、主大地、征恶地、守土）原则、天子剑（诛邪）安天下（平四海、主大地、征恶地、守土）原则、天子剑（无限）无害（无损）诛邪安天下（平四海、主大地、征恶地、守土）原则、天子修理地球（厚德载物）原则。由此而来，天子行仁开天地、行义立乾坤（直立行走、义薄云天）、治愈补天定乾坤、诛邪守土安天下（修理地球、厚德载物），构成了天子四喜、人间（万物、万民、众生）四喜：修理地球（厚德载物）、开天、立乾坤、补天定乾坤。天子剑无限诛邪安天下定山河（修理地球）、天子剑（无限）无害（无损）灭绝（剿灭）妖魔、天子审判断生死不废山河四海流、天子权杖震慑四方妖魔鬼怪和不安定因素随时随地灰飞烟灭，称为邪恶（罪犯、盒饭班主任）四悲。依据大地因果黑洞（负无限大、吞噬一切）原则、依据并比照天子剑（无限）无害（无损）灭绝（剿灭）妖魔原则，天子必须以无限天子剑诛杀吞噬一切的黑山老妖，人类才能免于无辜受害的新冠已经之人祸、工业化活摘器官之入体抢劫器官（入体抢劫）刑事犯罪、杀人有功的行刑疫苗犯罪等，这就是天子征恶地、天子安天下（平四海、主大地、守土）、天子剑征恶地、天子剑安天下（平四海、主大地、守土）、天子修理地球（厚德载物），从而构成天子（诛邪）安天下（平四海、主大地、征恶地、守土）原则、天子剑（诛邪）安天下（平四海、主大地、征恶地、守土）原则、天子剑（无限）无害（无损）诛邪安天下（平四海、主大地、征恶地、守土）原则、天子修理地球（厚德载物）原则。由此而来，天子行仁开天地、行义立乾坤（直立行走、义薄云天）、治愈补天定乾坤、诛邪守土安天下（修理地球、厚德载物），构成了天子四喜、人间（万物、万民、众生）四喜：修理地球（厚德载物）、开天、立乾坤、补天定乾坤。天子剑无限诛邪安天下定山河（修理地球）、天子剑（无限）无害（无损）灭绝（剿灭）妖魔、天子审判断生死不废山河四海流、天子权杖震慑四方妖魔鬼怪和不安定因素随时随地灰飞烟灭，称为邪恶（罪犯、盒饭班主任）四悲。造物主造物。天子通过七因果令万界限万物经由解决而治愈：相对因果让万物、法律、制度真正独立而出生和出生而独立、获得生命力，参数化同步性因果让万物直立行走，天子方程让万物顶天立地归于上帝和正义、让罪恶七步成尸，此时，万物独立出生、直立行走、顶天立地而治愈，称之为造物后之升级造化，天子的出生是造物主的升级而令宇宙天地的保守成为可能，事物从固定的植物状态走向独立、自由、民主、同一、平等、博爱（公平）而公义。造物主造物。天子通过七因果令万界限万物经由解决而治愈：相对因果让万物、法律、制度真正独立而出生和出生而独立、获得生命力，参数化同步性因果让万物直立行走，天子方程让万物顶天立地归于上帝和正义、让罪恶七步成尸，此时，万物独立出生、直立行走、顶天立地而治愈，称之为造物后之升级造化，天子的出生是造物主的升级而令宇宙天地的保守成为可能，事物从固定的植物状态走向独立、自由、民主、同一、平等、博爱（公平）而公义。独立（宪法）面向对象让万物起源和生命出生；自由立法面向过程（程序）让过程（道独立（宪法）面向对象让万物起源和生命出生；自由立法面向过程（程序）让过程（道路、程序）相对而独立；选择（判断）性立法面向规则（法律）让真理相对而独立；解决（信独宪法面向对象万物起源和命出；自法面向程程序程路、程序）相对而独立；选择（判断）性立法面向规则（法律）让真理相对而独立；解决（信路、程序）相对而独立；选择（判断）性立法面向规则（法律）让真理相对而独立；解决（信仰）立法面向正义让文明相对而独立。解决（信仰）立法面向正义，选择性立法面向规则（法律），自由立法面向过程（程序），独立（宪法）面向对象。仰）立法面向正义让文明相对而独立。解决（信仰）立法面向正义，选择性立法面向规则（法律），自由立法面向过程（程序），独立（宪法）面向对象。 9．天子三生有幸原则、信仰（公义）在天原则天子以相对性因果开天而让生命在真正意义上得以开始，比照计划生育游击队多子多福，从而构成天子创生原则；天子以天子剑诛杀邪恶而保护众生，从而构成天子保生原则；天子；天子以天子权杖支持众生存在、发展而养生、资生，从而构成天子养生（资生）原则，综上所述三方面，其即构成天子三生（有幸、超生游击队队长）原则。造物主造物，天子创生、三生有幸而比照计划生育超生游击队。天子方程、天子剑方程（定乾坤）、天子久期方程（安天下）化育万物而创生，从而构成天子造化（化育万物）原则。天子以相对性因果开天而让生命在真正意义上得以开始，比照计划生育游击队多子多福，从而构成天子创生原则；天子以天子剑诛杀邪恶而保护众生，从而构成天子保生原则；天子；天子以天子权杖支持众生存在、发展而养生、资生，从而构成天子养生（资生）原则，综上所述三方面，其即构成天子三生（有幸、超生游击队队长）原则。造物主造物，天子创生、三生有幸而比照计划生育超生游击队。天子方程天子剑方程（定乾坤）天子久期方程（安天下）化育万物而创生从而构造物主造物，天子创生、三生有幸而比照计划生育超生游击队。天子方程、天子剑方程（定乾坤）、天子久期方程（安天下）化育万物而创生，从而构成天子造化（化育万物）原则。信仰是什么？天子所实现的公义、正义为最高结果，其随之构成公义（正义）最高结果原则，七级因果之上的公义在天（公正、正义真来）即是信仰、信仰的圣殿之所在，从而构成信仰（公义）在天（公正、正义真来）原则。 10．无产业阶级大革命俄罗斯土地至上而称为死性不改的土匪国；中共四处送国土卖国而虚荣心、名声至上、利益至上，还四处搞器官传销捆绑世界各国权贵而捆绑各国政府，不拿人民的命当命是死性不改的人匪国、民匪国，和从肉体上解决问题、解决一切的肉匪；美国是改邪归正的海盗（Pirates of the Caribbean）国、海匪国；日本是失败时群体性神风特攻队同归于尽和传统性切腹自杀，雷击一锅端，是比照眼镜王蛇的王匪、对人对自己都是亡命之徒的匪中之匪；象朝鲜一样的失败国家、非洲那些伸手大将军的流氓国家，它们依靠自己无法存在几天，靠吸血为生又都把自己吹成宇宙牛皮魔王，无分别残杀自己的百姓、兄弟，称之为丐匪。土匪坐地吸土，民匪共匪共产共妻杀人为乐，海盗是海底捞月（国家政要权贵到处海底捞拣了芝麻丢了西瓜而渗透颠覆自己国家送货上门），亡命之徒的王匪是自杀性核武战争，丐匪是杀自己人来吓阻对方和威胁全世界，从而构成五胡乱华原则、五胡乱世原则、五胡灭世（末日、三战）原则，五胡指王匪、肉匪、海盗海匪、王匪（神风特攻队的神匪）、流氓加瘪三的丐匪。 90%以上的全国化工厂被炸毁、80%以上的兵工厂也被炸毁、99%的外资企业逃离中国、 80%以上的民营生产企业倒闭或逃离，时至今日整个国家经济大解体，整个中国正在进行无产业大革命；几亿工人群体性失业而回乡再创业、全部特权阶级垄断了无产业和产业绝对收敛下的所有的财富（连核酸检测、行刑毒疫苗都不放过），从而构成了双向的无产业阶级大革命，包括失业人群的无产阶级A 无产业大革命和特权阶级B 无产业大革命，失业人群的无产阶级A 和特权阶级B 一起走向并变成无产业阶级，也就是说，共党从无产阶级大革命到无产业阶级大革命，直奔地狱的倒行逆施开倒车已经不可逆转而必然是绝对性地终结一切，这不仅是产业和产业链的末日，也是全人类核武战争的末日。无产阶级大革命掠夺一切财产，杀害绝大多数地主和资产阶级、没收所有产业国有化；无产业阶级大革命和无产业大革命是掠夺一切财产加摧毁一切产业，绝大多数情形连没收产业进行国有化都不允许而必须予以摧毁、炸毁（如南京化工厂在被炸毁前曾由地方官员求移交给利益集团而别炸毁都不获准许），JD 老板的亲妹妹被一尸体两命或三命地杀害只因为哥哥有钱，尤其是病毒清零政策对所有生命、人性的践踏和灭绝，更是前所未有、前所未闻，综上所述，无产业阶级大革命是对产业、财产、就业机会（人生尊严）和人命的绝对性摧毁之三光政策，依据无分别绝对原则，无产业阶级大革命即属无分别毁灭而必然是绝对收敛的，从而构成无产业（产业链）大革命三光（政策）原则、无产业阶级大革命三光（政策）原则、无产业（产业链）大革命绝对原则、无产业阶级大革命绝对原则。显然，无产业阶级大革命比无产阶级大革命更彻底、更变态也更凶残，二者都是共产党强加给人民的，让所有人为他去陪葬。如果无产业（产业链）大革命、无产业阶级大革命是可以悬崖勒马、危地马拉的，那么，它们就不是绝对的，记为结论F；然而，依据无产业（产业链）大革命绝对原则、无产业阶级大革命绝对原则，无产业（产业链）大革命、无产业阶级大革命都是绝对的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得无产业（产业链）大革命（无产业阶级大革命、末日灭绝）悬崖勒马（危地马拉、刹车）否定原则和无产业（产业链）大革命（无产业阶级大革命、末日灭绝）末路狂奔（一条路走到黑）原则、无产业（产业链）大革命（无产业阶级大革命、末日灭绝）不可能悬崖勒马（危地马拉、刹车）原则，一切为时已晚、不可救药，全人类惟有团结一致奋斗到底才有可能夺取最后的生机。依据无产业（产业链）大革命（无产业阶级大革命末日灭绝）不可能悬崖勒马（危地如果无产业（产业链）大革命、无产业阶级大革命是可以悬崖勒马、危地马拉的，那么，它们就不是绝对的，记为结论F；然而，依据无产业（产业链）大革命绝对原则、无产业阶级大革命绝对原则，无产业（产业链）大革命、无产业阶级大革命都是绝对的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得无产业（产业链）大革命（无产业阶级大革命、末日灭绝）悬崖勒马（危地马拉、刹车）否定原则和无产业（产业链）大革命（无产业阶级大革命、末日灭绝）末路狂奔（一条路走到黑）原则、无产业（产业链）大革命（无产业阶级大革命、末日灭绝）不可能悬崖勒马（危地马拉、刹车）原则，一切为时已晚、不可救药，全人类惟有团结一致奋斗到底才有可能夺取最后的生机。依据无产业（产业链）大革命（无产业阶级大革命、末日灭绝）不可能悬崖勒马（危地马拉、刹车）原则，任何人要停止无产业阶级大革命的末日灭绝，可谓是：与虎谋皮难，与盒饭班主任谋盒子皮更是难上加难。邪恶政权用病毒杀害全世界超过2000 万人，乌克兰战争已经令全国到处一片废墟，安倍晋三已经用自己的生命向全人类控诉这个世界的邪恶，全世界对这两个罪犯的任何处罚远远小于其对世界的危害，永远也不可能弥补其对世界所犯下的罪行，善恶有报在预防机制上出现了天大的漏洞。马拉、刹车）原则，任何人要停止无产业阶级大革命的末日灭绝，可谓是：与虎谋皮难，与盒饭班主任谋盒子皮更是难上加难。邪恶政权用病毒杀害全世界超过2000 万人，乌克兰战争已经令全国到处一片废墟，安倍晋三已经用自己的生命向全人类控诉这个世界的邪恶，全世界对这两个罪犯的任何处罚远远小于其对世界的危害永远也不可能弥补其对世界所犯下的罪行善恶有报在预防机制上马拉、刹车）原则，任何人要停止无产业阶级大革命的末日灭绝，可谓是：与虎谋皮难，与盒饭班主任谋盒子皮更是难上加难。邪恶政权用病毒杀害全世界超过2000 万人，乌克兰战争已经令全国到处一片废墟，安倍晋三已经用自己的生命向全人类控诉这个世界的邪恶，全世界对这两个罪犯的任何处罚远远小于其对世界的危害，永远也不可能弥补其对世界所犯下的罪行，善恶有报在预防机制上出现了天大的漏洞。 11．天门秩序取代二战以来的雅尔塔规则体系：修理地球、治愈补天而安定天下，安全第一、文明升级依据（唯一）确定绝对原则，主体或事物的真实性、资格、效力都是立足确定性的，从而构成确定性先于真实（效力、资格、实力、能力）原则、真实（效力、资格、实力、能力）确定（绝对、锁定）原则。轨道、程序、过程是分布性的、动态的、相对的，从而构成相对（分布性、动态）先于程序（过程、轨道）原则、程序（过程、轨道）相对（分布性、动态、非绝对）原则。如果基于规则的秩序W 是适合作为世界秩序的，那么，依据程序（过程、轨道）相对（分布性、动态、非绝对）原则，秩序W 必然不是绝对的，记为结论F；然而，依据真实（效力、资格、实力、能力）确定（绝对、锁定）原则、同一律，世界万物的真实性（效力、 11．天门秩序取代二战以来的雅尔塔规则体系：修理地球、治愈补天而安定天下，安全第一、文明升级 11．天门秩序取代二战以来的雅尔塔规则体系：修理地球、治愈补天而安定天下，安全第一、文明升级依据（唯一）确定绝对原则，主体或事物的真实性、资格、效力都是立足确定性的，从而构成确定性先于真实（效力、资格、实力、能力）原则、真实（效力、资格、实力、能力）确定（绝对、锁定）原则。轨道、程序、过程是分布性的、动态的、相对的，从而构成相对（分布性、动态）先于程序（过程、轨道）原则、程序（过程、轨道）相对（分布性、动态、非绝对）原则。如果基于规则的秩序W 是适合作为世界秩序的，那么，依据程序（过程、轨道）相对（分布性、动态、非绝对）原则，秩序W 必然不是绝对的，记为结论F；然而，依据真实（效力、资格、实力、能力）确定（绝对、锁定）原则、同一律，世界万物的真实性（效力、依据（唯一）确定绝对原则，主体或事物的真实性、资格、效力都是立足确定性的，从而构成确定性先于真实（效力、资格、实力、能力）原则、真实（效力、资格、实力、能力）确定（绝对、锁定）原则。轨道、程序、过程是分布性的、动态的、相对的，从而构成相对（分布性、动态）先于程序（过程、轨道）原则、程序（过程、轨道）相对（分布性、动态、非绝对）原则。如果基于规则的秩序W 是适合作为世界秩序的，那么，依据程序（过程、轨道）相对（分布性、动态、非绝对）原则，秩序W 必然不是绝对的，记为结论F；然而，依据真实（效力、资格、实力、能力）确定（绝对、锁定）原则、同一律，世界万物的真实性（效力、程序（过程、轨道）原则、程序（过程、轨道）相对（分布性、动态、非绝对）原则。如果基于规则的秩序W 是适合作为世界秩序的，那么，依据程序（过程、轨道）相对（分布性、动态、非绝对）原则，秩序W 必然不是绝对的，记为结论F；然而，依据真实（效力、资格、实力、能力）确定（绝对、锁定）原则、同一律，世界万物的真实性（效力、程序（过程、轨道）原则、程序（过程、轨道）相对（分布性、动态、非绝对）原则。如果基于规则的秩序W 是适合作为世界秩序的，那么，依据程序（过程、轨道）相对（分布性、动态、非绝对）原则，秩序W 必然不是绝对的，记为结论F；然而，依据真实（效力、资格、实力、能力）确定（绝对、锁定）原则、同一律，世界万物的真实性（效力、基于规则的秩序如同过筛子一样，符合尺寸的沙子和米粒一样都能通过筛子，开放的体系给邪恶势力留下了直接近身渗透、颠覆以至谋杀的后门漏洞Q，此时，我们要补上一台选沙机W 来确定通过筛子前后的必须都是粮食，这台选沙机就是程序前后对事物的真实性进行检验并确认其效力：一方面，程序官在程序先于权利之前确立真实先于程序（轨道），以此确保程序（轨道）面向对象的有效性；另一方面，在程序（轨道）完成之后，人们必须确认抵达终点的是否被调包、是否变质等，以此确定承受性因果层面上结果的有效性，综上所述两方面，其即构成真实性确认A、信用性确保程序（轨道、过程）B、效力检验C 三元并立，同时确立真实先于程序（轨道、过程）原则（AB）、程序（轨道、过程）先于权利原则（BC）、效力先于结果（确定）原则即效力确认原则和结果效力原则（CE），其中，AB 为从实力出发的真实原则、BC 为程序先于权利的诚信（信用）原则、CE 为效力（先于结果）原则，效力先于因果（程序）的秩序由此确立，这就是效力先于因果程序，依此类推至于普遍情形同理成立。如果真实是不可确认的，那么，其即是测不准的，即属分布或离散的而必然是排除确认性的，记为结论F；然而，任何真实都是确认不可排除的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得真实不可确认否定原则和真实确认原则。如果程序不具有真实性，其即是无效的，这就是真实先于程序（轨道、过程）原则（AB）。如果权利不经由程序，其即是无程序之无条件的，即属绝对之无限大而为变量的，也就是无法被确认的，违背了真实先于程序的可被确认之真实性原则，这就是程序（轨道、过程）先于权利原则（BC）。如果效力不先于因果、程序（轨道、过程），因果、程序（轨道、过程）即是不排除后门、漏洞与虚假的，随之违背真实先于程序的可被确认之真实性原则，这就是效力先于结果原则（CE）和效力先于因果（程序）原则（CC）。真实先于程序（轨道、过程）原则（AB）、程序（轨道、过程）先于权利原则（BC）、效力先于结果原则（CE）和效力先于因果（程序）原则（CC），共同建立起真实（从实力出发）a、信用（程序）b、效力（结果、因果）c 三元并立的秩序W，称为基于效力的秩序即效力秩序，包括真实立足（从实力出发）a（承载因果）、信用（程序）b（相对因果、规则因果）、效力（结果、因果）c（承受性因果），也就是说，效力秩序覆盖基于实力的秩序（实力秩序）、基于规则的秩序（规则秩序），由此而来，实力秩序、规则秩序、效力秩确，我们于是获得真实不可确认否定原则和真实确认原则。如果程序不具有真实性，其即是无效的，这就是真实先于程序（轨道、过程）原则（AB）。如果权利不经由程序，其即是无程序之无条件的，即属绝对之无限大而为变量的，也就是无法被确认的，违背了真实先于程序的可被确认之真实性原则，这就是程序（轨道、过程）先于权利原则（BC）。如果效力不先于因果、程序（轨道、过程），因果、程序（轨道、过程）即是不排除后原则（CE）和效力先于因果（程序）原则（CC）。真实先于程序（轨道、过程）原则（AB）、程序（轨道、过程）先于权利原则（BC）、效力先于结果原则（CE）和效力先于因果（程序）原则（CC），共同建立起真实（从实力出发）a、信用（程序）b、效力（结果、因果）c 三元并立的秩序W，称为基于效力的秩序即效力秩序，包括真实立足（从实力出发）a（承载因果）、信用（程序）b（相对因果、规则因果）、效力（结果、因果）c（承受性因果），也就是说，效力秩序覆盖基于实力的秩序（实力秩序）、基于规则的秩序（规则秩序），由此而来，实力秩序、规则秩序、效力秩序依次三位一体于公正秩序（三元秩序、三星秩序），即信仰的圣殿之秩序，从而构成公正秩序三位一体（三星）原则、公正秩序实力规则效力原则。也就是说，公正秩序经由承载因果（从实力出发因果）、相对因果（规则因果）、（比照承受性因果的）效力因果并抵达信仰圣殿而建立，因此必须遵循文明七层协议、天子四喜、邪恶（罪犯）四悲，这就是公正秩序的三大框架，人类通往光明之路R 被打通、光明之门D 向世界敞开，从而构成公正秩序顶天立地（光明）原则，其中，通往光明之路R 也就是神之道、神圣之道，光明之门D 即天门、天堂之门、天门开。来算法由此而来，公正秩序又称为光明秩序、天门秩序、天门算法，随之构成光明秩序天门开（大道通）原则。由此而来，公正秩序又称为光明秩序、天门秩序、天门算法，随之构成光明秩序天门开（大道通）原则。一战及一战以前的秩序是狭路相逢勇者胜的丛林实力秩序，武力解决一切；二战以来的秩序是以规则为前提的秩序，文斗之谈判、外交、法律解决一切，但存在着开放体系都有的后门、漏洞问题，民主国家被渗透、颠覆而防不胜防是常有的事，现在直接导致了两个独裁者用病毒杀人、用核武器炸毁地球的末日大灾难到来，三战随时降临地球。邪恶轴心要推翻的二战以来的雅尔塔体系以规则为基础，传承的是英国的程序先于权利，用过程来检验其资格、效力，此时，为了不让三战爆发或者让三战的破坏程度降到最低，全世界必须立刻以涵盖实力秩序、规则秩序、效力秩序的光明秩序来取代二战以来的雅尔塔规则体系，以此修理地球而加固大地、治愈补天而安定天下，让所有人免于恐惧而安天下，安全第一、文明升级。一战及一战以前的秩序是狭路相逢勇者胜的丛林实力秩序，武力解决一切；二战以来的秩序是以规则为前提的秩序，文斗之谈判、外交、法律解决一切，但存在着开放体系都有的后门、漏洞问题，民主国家被渗透、颠覆而防不胜防是常有的事，现在直接导致了两个独裁者用病毒杀人、用核武器炸毁地球的末日大灾难到来，三战随时降临地球。邪恶轴心要推翻的二战以来的雅尔塔体系以规则为基础，传承的是英国的程序先于权利，用过程来检验其资格、效力，此时，为了不让三战爆发或者让三战的破坏程度降到最低，全世界必须立刻以涵盖实力秩序、规则秩序、效力秩序的光明秩序来取代二战以来的雅尔塔规则体系，以此修理地球而加固大地、治愈补天而安定天下，让所有人免于恐惧而安天下，安全第一、文明升级。两个事物、主体或国家A、B 以各自的实力（武力）F1、F2（之差△F=F1-F2）为前提决定获得性结果，如美国打赢日本而建立二战后的日本政府R，称之为差异性因果、从实力出发因果、丛林因果，依此类推至于普遍情形同理成立。雅尔塔体系的联合国以规则对朝鲜等国家进行考察，违反规则W 即启动制裁R，，这就是规则性因果、规则因果，属于获得性因果，依此类推至于普遍情形同理成立。北约对于想要申请加入北约的任何国家进行资格、效力上的全面考察，符合即接纳，否则即不接纳，这就是效力性因果，属于获得性因果，依此类推至于普遍情形同理成立。从实力出发因果、规则因果、效力性因果构成元（位元、前提、条件）三因果、元（位元、前提、条件）因果、以元（位元、前提、条件）达到的公义三因果（位元秩序）。 12．共党七大终极秘密武器、末日三步倒三死结、三步倒末日毒蛊历史性轮回（1）共党的七大终极秘密武器：无限卖国、无限军国主义、无限纳粹军国主义（militarism）就是面粉厂，如日本在侵化战争中掠夺来的财富就象面粉厂里的面粉，但二战中日本的下场和如今继承日本731 而继承日本军国主义的中共放病毒的下场一样惨烈，还有跟共党军国主义穿同一条裤子而在行动上上纲上线的普京，从而构成军国主义面粉厂（大爆炸）原则。 12．共党七大终极秘密武器、末日三步倒三死结、三步倒末日毒蛊历史性轮回（1）共党的七大终极秘密武器：无限卖国、无限军国主义、无限纳粹军国主义（militarism）就是面粉厂，如日本在侵化战争中掠夺来的财富就象面粉厂里的面粉，但二战中日本的下场和如今继承日本731 而继承日本军国主义的中共放病毒的下场一样惨烈，还有跟共党军国主义穿同一条裤子而在行动上上纲上线的普京，从而构成军国主义面粉厂（大爆炸）原则。明石元二郎在俄罗斯的极端民族主义、种族主义中植入了日本的军国主义，最后捣蛊成祸害全人类的列宁主义，同时传染给了中共如今大爆发；日本在二战战败后将731 等全部保留给中共就是证据。日本的军国主义是自己有无限的意志去入侵他国和杀害别国人民绝不留情，即自己有无限意志去剥夺他人全部意志、生命、意志、财产，就象731 对中国人民进行惨无人道的活摘器官等实验，但是有自己人的概念，是集体对外的群体性行动，即日本人对中国人的大屠杀，从而构成日本军国主义群体性犯罪原则、日本军国主义群体内无限意志（对外剥夺一切意志、肉体、生命、财产）原则、日本军国主义自己人（国家化）原则。中共继承日本的军国主义后，将国家界限取消，取而代之的是党（特权阶级）和人民的界限，甚至以阶级斗争为名推进到党的任何角落而无死角；进而，依据无分别绝对原则，党（特权阶级）无限意志（想怎么做就怎么做），剥夺人民一切意志、肉体、生命、财产，由此可见，共党就是无界限、无底线的无限（绝对）军国主义，称之为无限（无界限、无分别、绝对）军国主义、无限（绝对）杀戮（杀人）、无分别的人民（人类）灭绝罪，从而构成共党军国主义无上限（无限、无界限、无分别、绝对）原则、共党无限（绝对、无底线）军国主义原则。在共党的无限军国主义中，任何人都会死（如陈毅的小儿子陈小鲁、国家主席刘少奇、国家副主席林彪、彭德怀等），文革饿死一亿人、斗死几千万人，杀人不偿命、罪责不负；现在，邪恶政权放病毒毒死全人类2000 万人，与俄罗斯搞无上限无限卖国的军事同盟要炸毁地球，连美国都控制不住，彻底展现共党的无限军国主义是没有界限和底线的，从而构成无限军国主义不可控制（不可抗力）原则、无限军国主义四海无内外原则。纳粹的种族主义鼓吹日尔曼民族最优秀至高无上，对犹太人无限杀戮无责有功；共党则取消了种族间的界限，执行特权阶级至高无上而人民是畜牲都不如的植物人仅供割韭菜，挥之则来，来之红黄蓝幼儿园圣殿（教材），红黄蓝全家灭门；同时，共党还以阶级斗争为名将种族主义的界限推进到党内而无死角，任何人都会被共产共妻而红黄蓝，任何人都会死（如陈毅的小儿子陈小鲁、国家主席刘少奇、国家副主席林彪、彭德怀等），称之为无限（绝对）种族主义、无限（绝对）纳粹，从而构成共党无限（绝对）纳粹原则、共党无限（绝对）种族主义（民族主义）原则、共产主义人民无限（绝对）犹太人（无限集中营、无限毒气室）原则、共党无限（绝对）犹太人（无限集中营、无限毒气室、无限大监狱、无限监控、无限破门消毒、无限清零、无限核酸检测）原则、共党无限（绝对）奴役（杀害、活摘器官、种族灭绝）。战场上，将士们都知道自己面临着敌人的枪林弹雨，因此必然将警戒提到最高，自己人和督战队背后捅刀子也是常有的事但不是主流；共产党讲究阶级斗争为纲归根结底就是，任何地方、时间都是无限战场，国内、国际、单位里、食堂、会议、监狱、家里、床上等都是战场不受任何限制，这才有两任总书记枪毙自己的父亲，文革中丈夫出卖妻子、儿子出卖母亲等等都是常有的事情，从而构成共党无限（绝对）战争原则、共党超限战原则、共党侮辱阶级斗争（侮辱性不大至少看不出来、伤害性极强、伤害性极大、无限伤害、无限祸害）原则，说阶级斗争那绝对是侮辱了阶级斗争。依据无限大绝对原则，2022 年2 月初日，共党和普京签定合作无上限的军事挑衅，并且向全世界宣告，那就是无限出卖国家利益、民族利益的无限卖国，从而构成邪恶政权无限卖国原则、共党无限腐败原则，掏别人腰包就象掏自己的一样，如共产JD 集团还杀害老板的妹妹一尸三命。依据无偿（无条件）绝对原则，共产主义讲究的是无产阶级国家之间的无偿援助、无条件割让国土，如白头山一半无缘无故就给了朝鲜等，可见，共产主义的无偿援助、无条件割让国土直接就是无条件卖国、无限卖国，从而构成共党无限（绝对）卖国原则。依据无限绝对原则，日本讲究的是精神控制，共党则是无限洗脑、无限忠诚于党，从而构成共党无限（绝对）精神控制原则。共产主义认为你的就是他的、你的即不是你的，名义上他的也是你的但实际上他的永远也不可能是你的，归根结底：{A}∩{B}={A}∩{┓A}=Ø(B=┓A) ⇒ A∈Ø、B∈Ø 而A、B 不存在；-\|A\|=\|B\| ⇒ A=B=0，其中A、B 为任意实数，即属绝对归零的连坐即是绝对归零的，也就是说，共产主义是绝对分裂的，从而构成共产主义（共党）绝对（无限）精神病原则。（1）共党无限（绝对、无底线）军国主义原则、（2）共党无限（绝对、无底线）纳粹（种族主义、民族主义）原则、（3）共党无限（绝对）犹太人（无限集中营、无限毒气室、无限大监狱、无限监控、无限破门消毒、无限清零、无限核酸检测）原则、（4）共党无限（绝对）战争原则（共党超限战原则）、（5）共党无限卖国（无限腐败、塌方）原则、（6）共党无限（绝对）精神控制原则、（7）共党无限（绝对）精神病原则，合称为共党七条军归（铁七条）原则。共党无限卖国、无限军国主义、无限纳粹（种族主义、民族主义）、无限战争（永无宁日）、无限犹太人（无限集中营）即无限奴役（杀害、活摘器官、种族灭绝）、无限精神控制、无限精神病，称为共党（共产主义）七大毒瘤（七大绝症）。中国人在文革中饿死一亿人、斗死几千万人，国库全空，后来邓小平找到安倍晋三的父亲安倍晋太郎帮助恢复经济，大量的日资企业因此进入中国，欧美企业随之跟进，中国的经济这才发展到2012 年的巅峰，随之被人不断L 型摧毁至今天：90%以上的全国化工厂被炸毁、80%以上的兵工厂也被炸毁、99%的外资企业逃离中国、80%以上的民营生产企业倒闭或逃离，时至今日整个国家经济大解体，整个中国正在进行无产业大革命；几亿工人群体性失业而回乡再创业、全部特权阶级垄断了无产业和产业绝对收敛下的所有的财富（连核酸检中国人在文革中饿死一亿人、斗死几千万人，国库全空，后来邓小平找到安倍晋三的父亲安倍晋太郎帮助恢复经济，大量的日资企业因此进入中国，欧美企业随之跟进，中国的经济这才发展到2012 年的巅峰，随之被人不断L 型摧毁至今天：90%以上的全国化工厂被炸毁、80%以上的兵工厂也被炸毁、99%的外资企业逃离中国、80%以上的民营生产企业倒闭或逃离，时至今日整个国家经济大解体，整个中国正在进行无产业大革命；几亿工人群体性失业而回乡再创业、全部特权阶级垄断了无产业和产业绝对收敛下的所有的财富（连核酸检测、行刑毒疫苗都不放过），从而构成了双向的无产业阶级大革命，也就是说，安倍晋三的父亲安倍晋太郎让中国人在饿死1 亿多的情形下吃上了咸鸭蛋（台湾人则造谣大陆人吃不起咸鸭蛋），恩同再造，安倍晋三则延续父亲的政策，从而构成安倍晋三咸鸭蛋（恩同再造）原则。日本、美国、英法、欧洲等就象养蛊一样将中共在人类文明和法治的反向上越养越大，时至今日从天津大蛊养成了末日世界最大蛊，终于酿成了今天之炸毁地球的滔天大祸，日本、美国、英法、欧洲等就象成立了一家蛊份无限公司（中共国）的蛊东一样，共党放病毒毒死全世界2000 万人、联合普京炸毁地球，蛊东们人人有责而难辞其咎，从而构成日本英美养虎为患（捣蛊末日、养肥中共）原则，原因就在于他们没认清共党的真实本质：无限卖国、无限军国主义、无限纳粹（种族主义、民族主义）、无限战争（永无宁日）、无限犹太人（无限集中营）即无限奴役（杀害、活摘器官、种族灭绝）、无限精神控制、无限精神病。（2）三步倒末日毒蛊历史性轮回原则、天子三宝救世三步曲ABC、轩辕核按钮第一，美国二战中与全人类有史以来首个灭绝人性的苏联政权合作灭了希特勒纳粹德国、灭了军国主义的日本，但是按下葫芦起了瓢养肥了苏联并且尾巴大不掉，出现了三巨大头；第二，接着，在1990 年代，美国和8964 天安门屠杀学生的全人类有史以来最邪恶的中共合作，解体了尾大不掉的前苏联，结果是养肥了中共并且尾大不掉；第三，现在，美国正式释放全民族整齐华一的日本军国主义对付并灭绝中共与俄罗斯，短短不足百年，三次的历史轮回都是按下葫芦起了瓢，死伤的都是各民族的精英和无辜的百姓，全世界死于核武战争的三光即将到来，无论是根据同一律还是因果律，任何基于邪恶错误的事物必然也是邪恶错误的，以毒制毒终归毒，从而构成世界以毒制毒三步倒原则、世界三步倒绝症，是三巨头罗斯福(Franklin Delano Roosevelt)、丘吉尔(Winston L.S. Churchill)、斯大林胡乱刮分世界造成的，全世界必须停止这种玩法，否则世界必将因为三步倒而毁灭，其中日本、美国、俄罗斯、中共国参与养出来的全人类有史以来最毒的末日毒蛊称为三步倒（毒蛊、末日毒蛊）、三步倒末日，日本、美国、俄罗斯、中共国全都是蛊份无限公司，参与的人称为蛊民。依据丛林历史轮回原则、世界以毒制毒三步倒原则，三步倒毒蛊、蛊民、蛊份无限公司，全都是近百年来全人类误入歧途的历史性轮回，从而构成三步倒（毒蛊、末日毒蛊）历史性轮回原则、蛊民（蛊份无限公司）历史轮回原则。刺杀肯尼迪总统的杀手从现场逃掉了，没有留下足够的线索，这是谋杀无线索（逃脱）原则，符合人的生命只有一次原则；本·拉登的人体炸弹几乎都命丧当场，符合背后的老大保护自己贪生怕死的原则，从而构成人体炸弹断线索（同归于尽）原则。众所周知，恐怖主义使用同归于尽的人体炸弹就是为了获取目标对象不设防的通道和机会。山上徹也杀害安倍晋三是不杀伤别人也不逃跑，而是留下来宣示线索和背后恐怖主义后台的主权，违背了自己要命和背后邪恶组织要命的常理，属于双违规现象，显然其目的只有一个就是制造寒蝉效应，从而构成刺杀安倍寒蝉效应原则、刺杀安倍恐怖主义三倍体原则，因此，安倍昭雪至关重要，日本警方不能放弃查明真相的任何机会。英国首相约翰逊、德国总理朔尔茨、意大利总理德拉吉等纷纷在黑材料下翻车，显然是一种常态性的寒蝉效应。安倍晋三遇害的第二天，普京立刻宣布将1 亿吨当量的末日核鱼雷波塞冬、萨尔马特核导弹列装，要到陆地上去打鱼、掠过海洋去炸毁地球，人祸的末日洪水已经令全世界退无可退，此时，依据天下共主（万王之王、万主之主）先于末日解决原则，全世界民主世界重中之重是刻不容缓地选举出一位世界共主，末日洪水才能得以启动携带着33 层因果、35721n 阅读框架因果链的轩辕治水方案解决，也就是说，B.全世界将以33 天（35721、C33）透天秩序取代雅尔塔体系（体系更迭），C.全人类将以33 天（35721n、C33）透天因果乾纲建立第三圣殿和处置万物（制度更迭），轩辕治水才能在世界范围内得以成功；可是，A.天下共主的选举（主权升级）先于上述A、B 两步。33 天（35721n、C33）透天因果乾纲简称为透天乾纲、33 天外天乾纲。安倍晋三遇害邪恶轴心变本加厉可谓丧尽天良主权升级秩序更迭制度更迭安倍晋三遇害，邪恶轴心变本加厉可谓丧尽天良，主权升级A、秩序更迭B、制度更迭 C 是轩辕治水的核心与世界永久性和平的反转核按钮，称为轩辕救世三步曲（ABC）。 C 是轩辕治水的核心与世界永久性和平的反转核按钮，称为轩辕救世三步曲（ABC）。拜登、超常待机现在必须立刻触发启动的英国伊丽莎白女王、日本德仁天皇、泽连斯基、马克龙、英国首相约翰逊以及有志于拯救世界的其他人等，都必须立刻参选，以确立天下共主，这就是轩辕治水方案启动的核按钮，称之为轩辕核按钮，从而构成天下共主（万王之王、万主之主）轩辕核按钮原则。为今之计，全世界必须立刻选举天下共主（万王之王、万主之主）按下轩辕核按钮，之后，当33 天（35721n、C33）透天乾纲与天下共主的权杖合一、《天子兵法》与天子剑合一时，即真命天子一手大棒一手胡萝卜，33 天外天的光明才能照在大地上和乾坤中，人们才能朝着文明圣殿的方向前进，邪恶才能最终被涤荡干净。天下共主轩辕核按钮、《天子兵法》与天子剑、33 天（35721n、C33）透天乾纲与天子为今之计，全世界必须立刻选举天下共主（万王之王、万主之主）按下轩辕核按钮，之后，当33 天（35721n、C33）透天乾纲与天下共主的权杖合一、《天子兵法》与天子剑合一时，即真命天子一手大棒一手胡萝卜，33 天外天的光明才能照在大地上和乾坤中，人们才能朝着文明圣殿的方向前进邪恶才能最终被涤荡干净才能朝着文明圣殿的方向前进，邪恶才能最终被涤荡干净。天下共主轩辕核按钮、《天子兵法》与天子剑、33 天（35721n、C33）透天乾纲与天子才能朝着文明圣殿的方向前进，邪恶才能最终被涤荡干净。天下共主轩辕核按钮、《天子兵法》与天子剑、33 天（35721n、C33）透天乾纲与天子权杖，合称为天子三宝、末日三宝、浑天三宝。普京是整个俄罗斯民族要为他去陪葬，这是煎烤全人类的烈火；普京兜底的二哈是要全部中国人为他去陪葬，同时将中国变成战败国，这是淹没全世界的祸水。本来，所谓水火不相容是真理；现在，冰火两重天军事同盟挑战全世界，全人类陷于水深火热之中，病毒屠杀全人类超过2000 万、邪恶轴心把乌克兰变成到处是废墟后还要炸毁地球。明朝太监刘瑾白天被凌迟割千刀，晚上回到牢房，还能连喝两大碗稀饭，第二天才失血而死，依此类推，任何手术都一样，速度越快病人的输血量和失血量越小，时间长了往往有性命之忧甚至后果不堪设想，从而构成速度先于手术（生死）原则。（4）养恶兵不可能成功（胜利）原则、邪恶轴心军事同盟不可能取得胜利（成功）原则、卖国绝对归零（天绝地灭）原则（3）末日三步倒三死结、东北亚1 小时战争地球末日结束（3）末日三步倒三死结、东北亚1 小时战争地球末日结束美国原国防部长马克·埃斯珀（Mark Esper）近日在布鲁金斯学会（Brookings Institution）说，东北亚（日本、朝鲜、俄罗斯、中国）是经济和军事交织区而成为全社会世界最不稳定的地区，成为日本当今世界最大的火药桶、成为面向北京的诺曼底登陆（Operation Overlord）基地；双方是生死存亡的最终决战：中共经由无上限绝对卖国输送支撑俄罗斯普京发动战争的动力，解决北京即解决俄罗斯，而直接端掉共党的巢穴北京则是最佳选择，就象奥斯卡金像奖一样迷人；再者，俄中如果解决日本必然导致美国退出西太平洋，俄中因此聚焦第一岛链核心的日本，由此可见，北京和日本是本次战争的两个死穴性制高点，因此，东北亚的战争成为死穴战争，双方一剑定输赢，即战略已经清晰，聚焦式的预置战争打击靶点一定是一场两败俱伤的战争，等于是三光玉碎必然发生，任何一方连逃都无法逃。中共经由无上限绝对卖国输送支撑俄罗斯普京发动战争的动力，俄罗斯反过来包庇纵容共党，也就是说，俄罗斯为中共接入了军事人工心肺ECMO、中共为俄罗斯接入了经济人工心肺ECMO，俄罗斯的战斗部（battle disposition）与自己的经济实力无关、中共的战斗部与自己的军事实力无关，双方都在各自卖国的情形下军事最大化，这就是链式军国主义，全世界的第一个外挂式（尿袋式）互补作战链由此产生，二者相互体外循环，从而构成链式军国主义互补体外循环（ECMO）原则。他们的实质是俄罗斯和中共国什么都不顾了，只要军事征服全世界，显然只能是军国主义的复活，日本二战开的头、干的好事、吃饱了没事找事遗传给共党。普京和二哈两者必然都很清楚这种链式军国主义的脆弱性，他们必然采取比独立的日本军国主义更家凶残的模式来对抗各自被击破而影响到对方生存、生命的风险和弥补其弱点，显然，中共北京是英美日等各国聚焦饱和打击的第一死穴（七寸），日本则成为俄中聚焦饱和打击的第一死穴（七寸），因此这场战争必然是两败俱伤的死穴战争，从而构成死穴战争（三战）原则、战争（三战）互为死穴原则、战争（三战）死穴原则；同时，北京是普京的外挂心脏，普京必然在心脏被毁灭后的几个小时甚至是一个小时内对日本、美国等实施同归于尽的饱和核打，对等地，美日等必然礼尚往来，由此可见，地球将在几个小时甚至是一个小时内被炸毁（各方的各种预案早已演练得滚瓜烂熟），人类末日降临，称之为临终战争、死亡战争、挖坟（掘墓、坟墓）战争、1 小时（炸毁地球）战争、冥王（Pluto）战争、反噬（回旋镖）战争、断头蛇飞（起咬）死人的同归于尽战争、再没有黎明的暗夜战争，从而构成东北亚1 小时（最终解决）战争原则、东北亚战争临终（挖坟、掘墓、坟墓、炸毁地球、冥王之战、同归于尽、反噬、回旋镖、断头蛇咬死人、尿袋、外挂人工心肺、链式军国主义、末日、再没有黎明的暗夜）原则、东北亚末日战争原则。依据经济先于国家原则，日本的军国主义只想解决生存空间，却无视也没有生存空间的安全隐患，如二战中，日本竟然在1941 年4 月13 日和对日本国家安全威胁最大的苏联签定《苏日中立条约》即互不侵犯条约，丧失了日本有史以来唯一一次能和德国前后夹击苏联而彻底消灭苏联的最后机会，直接导致了1945 年8 月8 日苏联背信弃义对日本宣战并在9 日凌晨出冰满洲国并最终摧毁日本的产业中心，还屠村屠城屠杀了超过一亿的日本人、中国人而将整个满洲国变成无人区，那可是原来万国人民闯关东去打工谋生的世界第四大经济强国和亚洲第一强国；今日，俄罗斯携带着自相残杀而废物的中共要最终解决日本，难道不悲哀吗？由此可见，日本整个民族短视俄罗斯、有眼无珠而无视国家安全到了必死的鄙视链的最末端，从而构成日本（民族）短视俄罗斯（有眼无珠无视国家安全）必死原则、日本（民族）无知（不知道）谁是敌人原则、日本群体性（民族性）国家安全盲（绝症）原则，超越天皇的安倍晋三也没看出这一点。日本好战但又存在着绝对的国家安全盲区、安全民族绝症，导致今日俄罗斯要彻底灭亡日本、抢劫日本，不能不说是整个大和民族的悲剧。如果二战中日本不是与苏联合作，而是与希特勒前后夹击苏联彻底解决真正毁灭大和民族的恶魔悬剑俄罗斯，今天，日本不会面临着再次被核打击的灭国之命运，地球也不会面临着被彻底炸毁的末日，证据如下：安倍晋三遇害的第二天，普京立刻宣布将1 亿吨当量的末日核鱼雷波塞冬、萨尔马特核导弹列装，要到陆地上去打鱼、掠过海洋去炸毁日本和地球。显然，俄罗斯是世界最强国美国的头号敌人，灭亡日本绝对不在话下，说灭就灭，日本什么军费开支天花板不天花板的，天花都没用，从而构成俄罗斯日本头号敌人原则、俄罗斯日本国家安全头号元凶（天敌、无解）原则，因为俄罗斯整个民族特性就是不靠自己发展靠掠夺。日本的军国主义只顾往前冲，只想掠夺、殖民，如同蜜蜂采蜜却没有留下警戒蜂群；俄罗斯就象中共公安系统对人民标注的菜农、粮农之蜂农，专门在日本的老巢热烈地割蜂蜜，从而构成日本军国主义工蜂原则、俄罗斯对日本蜂农（老巢割蜂蜜、非洲平头哥、蜜獾、头号杀手）原则，那可是号称世界上最无畏的动物，小日本怎么能承受得了？日本的宗主国美国是全世界唯一一个能与之抗衡的国家。现在日本是盲人骑瞎马，方向不对直本死路，任何无限军国主义都没用，天皇也一样，唯一之路是赶紧全民明白这个道理而制定可行的应对国策，安倍晋三才不会白死，从而构成日本盲人骑瞎马（死路、无解、任何军国主义都无效、任何军国主义都无用）原则。依据不限制绝对原则，日本政府已经宣布2023 年日本的军事开支没有天花板，全人类依据不限制绝对原则，日本政府已经宣布2023 年日本的军事开支没有天花板，全人类马上得天花，这就是无限而绝对的军国主义复活，而且是最高绝对的军国主义、雷电军国主依据不限制绝对原则，日本政府已经宣布2023 年日本的军事开支没有天花板，全人类马上得天花，这就是无限而绝对的军国主义复活，而且是最高绝对的军国主义、雷电军国主马上得天花，这就是无限而绝对的军国主义复活，而且是最高绝对的军国主义、雷电军国主义，从而构成日本军事开支无天花板绝对（最高、无限）军国主义原则。产业全世界最发达的东北亚经济、军事高强度相互交织成为世界最高度不稳定的死结，由此成为兵家和利益集团的必争之地，东北亚的人民和民族也成为世界各国最争相控制的最佳奴役民族，因为他们最勤劳勇敢也最富于工匠精神，比日尔曼民族还机器化和智能化，从而构成东北亚世界产业核心（民族核心、劳动核心、人民中心、人民金融中心、消费金融中心）原则、东北亚人民（民族）世界天花板原则，反之，相比之下的俄罗斯民族就成为世界的天花了，随之构成俄罗斯世界天花原则，天女散花、异常美丽，欢迎光临、下次优惠。 2019 年，罪魁祸首释放新冠病毒无分别毒死全人类超过2000 万人；2022 年2 月4 日，罪魁祸首与普京结为军事同盟开启俄罗斯入侵乌克兰之战争和世界三战；十年来，罪魁祸首努力并以刺杀安倍晋三事件最终解禁二战投降时还有700 万部队的日本军事力量，日本政府已经宣布2023 年日本的军事开支没有天花板，全人类马上得天花，其实等同于已经向俄罗斯、中共国宣战，这三宗罪行让世界70 年来的和平与科技的高速发展戛然而止，称之为三罪灭世、灭世（末日）三罪（三宗罪），从而构成邪恶轴心三罪（三宗罪）灭世（末日）原则、邪恶轴心三罪（三宗罪）直达（直奔）末日原则。明朝太监刘瑾白天被凌迟割千刀，晚上回到牢房，还能连喝两大碗稀饭，第二天才失血而死，依此类推，任何手术都一样，速度越快病人的输血量和失血量越小，时间长了往往有性命之忧甚至后果不堪设想，从而构成速度先于手术（生死）原则。美国解决北京和俄罗斯解决日本一样，速度越快损失越小，方案早已制定完毕而随时待命；况且，美国一旦解决了北京和三宗罪直奔末日的罪魁祸首，世界三战必然就此结束，俄罗斯的经济体量无法支撑持续性战争，由此而来，依据并比照速度先于手术（生死）原则、同一律，末日战争如同马克·埃斯珀（Mark Esper）所说是双方的生死存亡之战，即活摘俄中联盟链式军国主义外挂心脏之战，邪恶轴心的凶残程度一定提到极限和最高而那就是核武大战，当然美国和日本都不是省事的主，于是，美俄对抗70 年来，相互之间的摸底早已经全部完成只等付诸实施的最后一不，俄中和美日同归于尽的核武战争将在一小时内完成，地球因此被炸毁、人类随之被灭亡，称之为末日无限闪电战、无限闪电死、无限闪电灭绝（灭世）、无限闪电末日（在虚假的美好光明中碉堡而毁灭）、末日闪电灭绝（用秒来计算的核武大屠杀之末日秒杀、末日闪电死、末日闪电死局），从而构成美俄日中（全球）末日闪电死（闪电灭亡、秒杀、闪电死）原则、无限闪电末日原则、无限闪电灭绝（灭世）原则，这是希特勒的绝对复活并无限延伸，随之构成末日无限希特勒（纳粹）复活原则。由此可见，东北亚的末日战争是双方的死穴战争、坟墓战争、末日闪电灭绝（末日秒杀、 1 小时结束），称之为末日双坟闪电死局（死路）灭绝三步曲、（俄罗斯、日本、中国、美国、全世界）末日三步倒（死局、死路、灭绝）、末日三步倒（俄罗斯、日本、中国、美国、全世界）死局（死路、灭绝）、末日三步倒（秒少杀、1 小时灭绝）、三步倒末日，从而构成东北亚（战争）末日三步倒死局（死路、灭绝）原则，俄罗斯人及其军方、日本有史以来的任何人、美国、一心要跟随普京全球治理的中共国所有人和有史以来的所有中国人都没认识到此次末日是全人类、俄罗斯、日本、中国、美国等无分别三步倒，无人能幸免。美国解决北京和俄罗斯解决日本一样，速度越快损失越小，方案早已制定完毕而随时待命；况且，美国一旦解决了北京和三宗罪直奔末日的罪魁祸首，世界三战必然就此结束，俄罗斯的经济体量无法支撑持续性战争，由此而来，依据并比照速度先于手术（生死）原则、同一律，末日战争如同马克·埃斯珀（Mark Esper）所说是双方的生死存亡之战，即活摘俄中联盟链式军国主义外挂心脏之战，邪恶轴心的凶残程度一定提到极限和最高而那就是核武大战，当然美国和日本都不是省事的主，于是，美俄对抗70 年来，相互之间的摸底早已经全部完成只等付诸实施的最后一不，俄中和美日同归于尽的核武战争将在一小时内完成，地球因此被炸毁、人类随之被灭亡，称之为末日无限闪电战、无限闪电死、无限闪电灭绝（灭世）、无限闪电末日（在虚假的美好光明中碉堡而毁灭）、末日闪电灭绝（用秒来计算的核武大屠杀之末日秒杀、末日闪电死、末日闪电死局），从而构成美俄日中（全球）末日闪电死（闪电灭亡、秒杀、闪电死）原则、无限闪电末日原则、无限闪电灭绝（灭世）原则，这是希特勒的绝对复活并无限延伸，随之构成末日无限希特勒（纳粹）复活原则。由此可见，东北亚的末日战争是双方的死穴战争、坟墓战争、末日闪电灭绝（末日秒杀、 1 小时结束），称之为末日双坟闪电死局（死路）灭绝三步曲、（俄罗斯、日本、中国、美国、全世界）末日三步倒（死局、死路、灭绝）、末日三步倒（俄罗斯、日本、中国、美国、全世界）死局（死路、灭绝）、末日三步倒（秒少杀、1 小时灭绝）、三步倒末日，从而构成东北亚（战争）末日三步倒死局（死路、灭绝）原则，俄罗斯人及其军方、日本有史以来的任何人、美国、一心要跟随普京全球治理的中共国所有人和有史以来的所有中国人都没认识到此次末日是全人类、俄罗斯、日本、中国、美国等无分别三步倒，无人能幸免。苏联人制造1937 年海参崴大屠杀、1900 年的海兰泡惨案、江东六十四屯惨案，现在俄军正在屠杀乌克兰人等，1945 年毁灭于苏联红军前人口总量超过1 亿3000 万，即苏联红军在满洲国的10 个月中所屠杀的人口实际上远远超过一亿人，其表明俄罗斯专杀外国人，从而构成俄中或共俄（无分别）大屠杀平民原则、俄罗斯民族绝对（无限）排他（无限杀戮、绝对杀戮、杀光最后一个中国人、杀光最后一个日本人）原则、俄罗斯民族绝对（无限）种族主义（民族主义）原则。在世界末日三步倒1 小时灭绝中，日本从来没意识到俄罗斯不仅是美国的头号敌人更是日本的最高敌人，因为俄罗斯民族是具有绝对的无限排他性的，这是日本国家安全错误的死结；美国没意识到俄罗斯民族绝对（无限）排他性死结即使无限杀戮、绝对杀戮之种族灭绝死结；中国人民和全世界都没认清中共的无限（绝对）人民（人类）灭绝罪死结，2019 年放病毒毒死全世界2000 万人后就是邪恶轴心的死刑和共党的解体问题，也就是说，俄罗斯的基于种族灭绝的无限野心死结、日本的安全错误死结、中共国的人民（人类）灭绝死结，称为末日三步倒三死结，从而构成末日三步倒三死结原则。（4）养恶兵不可能成功（胜利）原则、邪恶轴心军事同盟不可能取得胜利（成功）原则、卖国绝对归零（天绝地灭）原则（4）养恶兵不可能成功（胜利）原则、邪恶轴心军事同盟不可能取得胜利（成功）原则、卖国绝对归零（天绝地灭）原则共党用病毒清零、不断封城折腾、断粮、断房等养蛊养恶兵Wj(j∈N)，也就是说，这些共党用病毒清零、不断封城折腾、断粮、断房等养蛊养恶兵Wj(j∈N)，也就是说，这些蛊兵Wj 都是被逼迫的而必然是被动的，从而构成蛊兵（恶兵）被动原则。共党用病毒清零、不断封城折腾、断粮、断房等养蛊养恶兵Wj(j∈N)，也就是说，这些蛊兵Wj 都是被逼迫的而必然是被动的，从而构成蛊兵（恶兵）被动原则。蛊兵（恶兵）为逃脱邪恶政权或督战队的刑罚而奋勇向前，回头即被射杀，即属无退路、共党用病毒清零、不断封城折腾、断粮、断房等养蛊养恶兵Wj(j∈N)，也就是说，这些蛊兵Wj 都是被逼迫的而必然是被动的，从而构成蛊兵（恶兵）被动原则。蛊兵（恶兵）为逃脱邪恶政权或督战队的刑罚而奋勇向前，回头即被射杀，即属无退路、别无选择的，从而构成蛊兵（恶兵）无退路（别无选择、无回头）原则。任何胜利都是胜利者有无限选择的，想怎么做就怎么做，从而构成胜利者主动（非被动、任何胜利都是胜利者有无限选择的，想怎么做就怎么做，从而构成胜利者主动（非被动、无限选择、无压力）原则。无限选择、无压力）原则。蛊兵（恶兵）的新生必须建立在与敌人进行拼杀并赢得机会的前提下，否则回头逃跑一定被处决，前路的战场撕杀是唯一的出路，显然是有条件的，从而构成蛊兵（恶兵）命不由无限选择、无压力）原则。蛊兵（恶兵）的新生必须建立在与敌人进行拼杀并赢得机会的前提下，否则回头逃跑一定被处决，前路的战场撕杀是唯一的出路，显然是有条件的，从而构成蛊兵（恶兵）命不由己（有条件）原则。蛊兵（恶兵）的新生必须建立在与敌人进行拼杀并赢得机会的前提下，否则回头逃跑定被处决，前路的战场撕杀是唯一的出路，显然是有条件的，从而构成蛊兵（恶兵）命不由己（有条件）原则。依据无分别绝对原则，战场上刀枪无眼、是无分别面向对象的，从而构成战场撕杀绝对原则。如果蛊兵（恶兵）W 能取得胜利，那么，依据蛊兵（恶兵）命不由己（有条件）原则，蛊兵（恶兵）W 必然是有限的，记为结论F；然而，依据战场撕杀绝对原则，战场撕杀U 是无限的，命题表明有限的蛊兵（恶兵）W 是大于无限的战场撕杀U：∞>W>U→∞，即∞>∞，这是不可能成立的命题，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得蛊兵（恶兵）胜利否定（错误）原则、邪恶政权临时抱佛脚胜利否定原则和蛊兵（恶兵）不可能胜利（取胜）原则、邪恶政权临时抱佛脚没用（无效）原则、蛊兵（恶兵）人民灭绝（罪）原则、养恶兵不可能成功（胜利）原则，邪恶政权白白牺牲人民生命，属于人民灭绝罪。邪恶轴心是在2019年放病毒毒死全人类2000万人后得知美国要灭它们的情形下才去哀求普京搞军事同盟的，纯属不得已之被动的，从而构成二哈求普京军事同盟迫不得已（被动、无回头路）原则。无回头路）原则。普京的经济ECMO 是中共给插的、中共的军事ECMO 是普京给插的，整个XP 军事同盟都是有条件的、相对的，从而构成邪恶轴心军事同盟有条件（有限、相对）原则。如果邪恶轴心军事同盟W 能取得胜利，那么，依据邪战场撕杀绝对原则、同一律，邪恶轴心军事同盟W 能取得胜利必然是经过了无限撕杀并且超越战场撕杀U 而获得的，即邪恶轴心军事同盟W 是大雨无限大之战场撕杀的：W>U→∞，记为结论F；依据二哈求普京军事同盟迫不得已（被动、无回头路）原则、邪恶轴心军事同盟有条件（有限、相对）原则、前提决定结果原则，W 必然是有限的而小于无限大的：W< U→∞，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得邪恶轴心军事同盟胜利否定原则和邪恶轴心军事同盟不可能取得胜利（成功）原则、邪恶轴心军事同盟必灭原则。如二哈对普京成承诺是你想拿什么就拿什么，合作无上限就是无限（绝对）卖国，从而构成二哈无限（绝对）卖国原则如二哈对普京成承诺是你想拿什么就拿什么，合作无上限就是无限（绝对）卖国，从而构成二哈无限（绝对）卖国原则。如二哈对普京成承诺是你想拿什么就拿什么，合作无上限就是无限（绝对）卖国，从而构成二哈无限（绝对）卖国原则。依据无条件绝对原则，任何人U 卖国中出卖的都不是自己的权利、金钱、武器W 等，权利、金钱、武器W 等必然是无条件的，从而构成卖国无条件（绝对）原则。任何个人的承担都是属于自己的，不属于外部的，也就必然不是外部无分别之绝对的，从而构成个人承担非绝对原则。构成二哈无限（绝对）卖国原则。依据无条件绝对原则，任何人U 卖国中出卖的都不是自己的权利、金钱、武器W 等，构成二哈无限（绝对）卖国原则。依据无条件绝对原则，任何人U 卖国中出卖的都不是自己的权利、金钱、武器W 等，权利、金钱、武器W 等必然是无条件的，从而构成卖国无条件（绝对）原则。任何个人的承担都是属于自己的不属于外部的也就必然不是外部无分别之绝对的权利、金钱、武器W 等必然是无条件的，从而构成卖国无条件（绝对）原则。任何个人的承担都是属于自己的，不属于外部的，也就必然不是外部无分别之绝对的，权利、金钱、武器W 等必然是无条件的，从而构成卖国无条件（绝对）原则。任何个人的承担都是属于自己的，不属于外部的，也就必然不是外部无分别之绝对的，从而构成个人承担非绝对原则任何个人的承担都是属于自己的，不属于外部的，也就必然不是外部无分别之绝对的，从而构成个人承担非绝对原则。从而构成个人承担非绝对原则。如果任何一个人W 卖国U 是无报应的，那么，依据个人承担非绝对原则，其必然都是自己能承担的而必然不是绝对的，记为结论F；然而，依据卖国无条件（绝对）原则，卖国 U 必然都是绝对的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得卖国无报应否定原则和卖国报应原则。如果卖国不是有命拿没命花的，那么，一个人W 卖国U 是他一个人W 必须承担的责任，即卖国是他一个人的事情P，记为结论F；然而，依据卖国无条件（绝对）原则、个人承担非绝对原则，任何人卖国是无条件的、绝对的之无限大的，显然不是自己能承担的，即卖国超过了他一个人能承担的Q，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 否定肯定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得卖国非有命拿没命花否定原则和卖国有命拿没命花（不得好死、永不赦免）原则。一个人卖国P 得肯定是遗臭万年的而必然是负面的：\|P\|=-P；然而，任何人的卖国P 都是主动的即是大于零的：P>0，P=\|P\|，综上所述，其必然有\|P\|=-\|P\|则P=0，即属绝对归零的，从而构成卖国绝对归零原则。一个人卖国P 得肯定是遗臭万年的而必然是负面的：\|P\|=-P；然而，任何人的卖国P 都是主动的即是大于零的：P>0，P=\|P\|，综上所述，其必然有\|P\|=-\|P\|则P=0，即属绝对归零的，从而构成卖国绝对归零原则。象老毛卖国竟然得好死，表面上那样；但是，依据卖国绝对归零原则，老毛等任何卖国的狗毛的灵魂必然被宇宙制定的规则彻底摧毁而魂飞魄散，从而构成（老毛）卖国魂飞魄散（齑粉、绝对归零）原则、（老毛）卖国生无立足之地（死无葬身之处）原则、卖国天绝地灭原则。象老毛卖国竟然得好死，表面上那样；但是，依据卖国绝对归零原则，老毛等任何卖国的狗毛的灵魂必然被宇宙制定的规则彻底摧毁而魂飞魄散，从而构成（老毛）卖国魂飞魄散（齑粉、绝对归零）原则、（老毛）卖国生无立足之地（死无葬身之处）原则、卖国天绝地灭原则。养恶兵不可能成功（胜利）原则、邪恶轴心军事同盟不可能取得胜利（成功）原则、卖国绝对归零（天绝地灭）原则结合后续的9LC33×CC35721 计算因果，构成了9LC33× CC35721 兵法（计算兵法、计算因果兵法、因果兵法），依此类推至于其他情形同理成立。由此可见，只要人们具有足够强大的量子计算机，决胜千里之外的兵法是可以被精确地计算出来的，而不只是被沙盘推演出来的，因此，我们称之为天子兵法（二）。 14．国家元首（总统）禁枪、汉家天子立法（1）主动（全面、进攻）安保取代被动（传统）安保原则、山上徹也四重高度模糊化狙击原则、山上徹也自带拆墙功能（高度隐形）杀手（狙击手）原则枪支、大炮、火箭都是瞄准的、有方向的，瞄准目标的而必然是相对的，扫射的枪和霰弹枪（canister~shotgun）也是有一定瞄准的和具有从枪手到受害者之间的距离差别的，从构成枪支（大炮、火箭）相对（有限、非绝对）原则、扫射的枪（霰弹枪）相对（有限、非绝对）原则。装成相机Q 的枪G 从外观看来已经不是枪支，同时以按动相机快门的伪动作掩饰扣动扳机动作（激发钨丝熔断点燃黑火药发射霰弹），白天即便是枪口冒烟外人看来也会误认为是附近导致的或现代电子产品的意外性自我爆炸甚至目标对象也是受害者，综上所述三点，谋杀安倍晋三的霰弹枪（喷子）具有三重伪装，在三个层面上三度排除了人们的注意力量，从而构成谋杀安倍晋三霰弹枪（喷子）三重伪装（三度排除注意力）原则。在安倍被谋杀过程中，山上徹也象个大隐隐于市的狙击手（sniper）：相机的外表按快门而非扣动扳机的常态性操作、目标对象可能也是受害者（目标模糊化）摆在那里，更搞笑的是山上徹也穿得象人畜无害的平民粉丝，四重伪装四个现代化全面实现，要多无辜就有多无辜，你让安保人员如何下得了手？其结果就是让所有安保人权坐视安倍晋三被击倒后才能作出正确判断，白白送给他杀手两枪三秒与死神赛跑的时间，从而构成杀手（山上徹也）四个现代化狙击手化原则、杀手（山上徹也）四重高度模糊化狙击原则。众所周知，狙击手的生存和成功前提就是高度模糊化自己，否则肯定容易出师未捷身先死和被发现而被打成筛子，从而构成（高度）模糊化先于狙击手原则、狙击手（高度）模糊化（最大限度模糊化）原则、狙击手第一（生存、成功）原则。在疑犯开第一枪或确定之前，以防卫为首的安保人员和基于判断的军队一样基于判断；在疑犯开第一枪或确定之前，以防卫为首的安保人员和基于判断的军队一样基于判断；安保人员在确认嫌犯后才进入基于保护对象为主的进攻状态中，也是基于判断；同时，安保人员以保护对象为主即是反应第一、进攻第二，从而构成安保（人员）基于判断原则（安保第一原则、安保人员第一原则）、安保（人员）判断先于反应原则（安保第一原则、安保人员第一原则、安保（人员）反应第一进攻第二原则（反应先于进攻原则、安保行动第一原则）、安保（人员）防卫为首（进攻次之）原则。安保（人员）防卫为首（进攻次之）原则。面对这么一个高度模糊化的狙击手山上徹也，其完全消除了安保基于判断的第一安保原则和反应先于进攻原则的安保行动第一原则，纯属自带拆墙功能的高度甚至全隐形杀手、狙击手，堪称安保死神，从而构成山上徹也自带拆墙功能（高度隐形、全隐形）杀手（狙击手）原则、山上徹也死神（经典）原则、山上徹也黑马（防不住）原则，单就本点而言，孤狼怎么做得到？其肯定要普京这个级别的KGB 高手才能训练得出来。对于山上徹也，安保人员一旦误杀或滥杀无辜必然毁灭自己的职业生涯，同时也过不了自己心理上滥杀无辜的这一关，除非其人本来就是精神变态、心理扭曲，从而构成应对山上徹也双重无解原则。由此可见，山上徹也杀害安倍晋三事件本身就展现了安保上的绝对盲区和黑洞、漏洞，山上徹也本身则就是安保无解的克星和天敌，这是任何反应速度快和缩短行动时间、提高效率等无法解决的，从而构成山上徹也安保无解（克星、天敌）原则、山上徹也杀害安倍晋三事件安保绝对盲区（黑洞、漏洞、无效）原则、山上徹也标志安保失败原则、安保人员防为首攻次则面对这么一个高度模糊化的狙击手山上徹也，其完全消除了安保基于判断的第一安保原则和反应先于进攻原则的安保行动第一原则，纯属自带拆墙功能的高度甚至全隐形杀手、狙击手，堪称安保死神，从而构成山上徹也自带拆墙功能（高度隐形、全隐形）杀手（狙击手）原则、山上徹也死神（经典）原则、山上徹也黑马（防不住）原则，单就本点而言，孤狼怎么做得到？其肯定要普京这个级别的KGB 高手才能训练得出来。么做得到？其肯定要普京这个级别的KGB 高手才能训练得出来。对于山上徹也，安保人员一旦误杀或滥杀无辜必然毁灭自己的职业生涯，同时也过不了自己心理上滥杀无辜的这一关，除非其人本来就是精神变态、心理扭曲，从而构成应对山上徹也双重无解原则。由此可见，山上徹也杀害安倍晋三事件本身就展现了安保上的绝对盲区和黑洞、漏洞，山上徹也本身则就是安保无解的克星和天敌，这是任何反应速度快和缩短行动时间、提高效率等无法解决的，从而构成山上徹也安保无解（克星、天敌）原则、山上徹也杀害安倍晋三事件安保绝对盲区（黑洞、漏洞、无效）原则、山上徹也标志安保失败原则、山上徹也式刺杀与安保提高反应速度快（缩短行动时间、提高效率）无关原则、山上徹也式刺杀安保提高反应速度快（缩短行动时间、提高效率）无效原则。如果人们要解决山上徹也杀害安倍晋三事件本身展现出来的安保绝对盲区和黑洞、漏洞，即解决安保上的标志性失败，那么，此时，人们要确保安全的话，有罪推定的主动安保、全面安保、进攻性安保成为不二选择，即安保必须基于如下两个原则：进攻先于防守（进攻先于反应）之主动（全面、进攻）安保行动第一原则、面对（反应）先于判断之主动（全面、进攻）安保第一原则，这是不得不进行的安保革命，即主动（全面、进攻）安保取代被动（传统）安保原则。山上徹也式刺杀与安保提高反应速度快（缩短行动时间、提高效率）无关原则、山上徹也式刺杀安保提高反应速度快（缩短行动时间、提高效率）无效原则。己所无法控制的而必然是绝对的，从而构成外部风险绝对原则。如果枪支W 可以防范独裁和自卫，那么，依据外部风险绝对原则、独裁（专制）绝对原则、同一律，与绝对的独裁和外部风险保持同一的枪支W 必然是绝对的，记为结论F；然而，依据自卫相对（有限）原则、枪支（大炮、火箭）相对（有限、非绝对）原则、扫射的枪（霰弹枪）相对（有限、非绝对）原则，任何枪支、防范、自卫都是相对的，记为结论 G，此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得枪支防范独裁（防范专制、防范暴政、防范、自卫）否定原则和枪支无法（不可能、不能、不）防范独裁（防范专制、防范暴政、防范、自卫）原则。原则如果不禁止枪支，象美国民间组织有2.9 亿支枪支，因为任何主动的人都可能卷入是非而手委屈和冲动无限大，等于是释放了冲动是魔鬼的人人是魔鬼之悬剑，依据常态绝对原则，你拿枪来防卫杀害居所入侵者毕竟不是常态，但人人拥枪而成为冲动中的魔鬼则是常态而必然是绝对的，美国即成为枪林弹雨、人人被打成筛子（不可排除）的战场，依此类推至于其他国家同理成立，从而构成不禁枪战场（枪林弹雨、人人被打成筛子）绝对原则、不禁枪战场（枪林弹雨、人人被打成筛子）原则、不禁枪战场（枪林弹雨、人人被打成筛子、人人自危）不可排除绝对原则、不禁枪战场（枪林弹雨、人人被打成筛子、人人自危）不可排除原则，由此可见，世界各国禁枪别无选择。香港、新加坡、日本等禁止枪支的国家、地区，象香港在反送中被大屠杀前的犯罪率也就是十万分之10 左右，美国一年三万人死于枪击的死亡率就已经等于或高于香港的十万分之10 犯罪率了，另外必然再有十万分之10 的持枪罪犯需要法办，则一年至少有十万分之 20 与枪击死亡有关的犯罪，你美国法律再公证都是马后炮，有什么用？况且，在美国还有超过枪击犯罪很多的其他犯罪，与香港、新加坡、日本等禁止枪支的国家、地区根本无法相如果不禁止枪支，象美国民间组织有2.9 亿支枪支，因为任何主动的人都可能卷入是非而手委屈和冲动无限大，等于是释放了冲动是魔鬼的人人是魔鬼之悬剑，依据常态绝对原则，你拿枪来防卫杀害居所入侵者毕竟不是常态，但人人拥枪而成为冲动中的魔鬼则是常态而必然是绝对的，美国即成为枪林弹雨、人人被打成筛子（不可排除）的战场，依此类推至于其他国家同理成立，从而构成不禁枪战场（枪林弹雨、人人被打成筛子）绝对原则、不禁枪战场（枪林弹雨、人人被打成筛子）原则、不禁枪战场（枪林弹雨、人人被打成筛子、人人自危）不可排除绝对原则、不禁枪战场（枪林弹雨、人人被打成筛子、人人自危）不可排除原则，由此可见，世界各国禁枪别无选择。则，由此可见，世界各国禁枪别无选择。香港、新加坡、日本等禁止枪支的国家、地区，象香港在反送中被大屠杀前的犯罪率也就是十万分之10 左右，美国一年三万人死于枪击的死亡率就已经等于或高于香港的十万分之10 犯罪率了，另外必然再有十万分之10 的持枪罪犯需要法办，则一年至少有十万分之 20 与枪击死亡有关的犯罪，你美国法律再公证都是马后炮，有什么用？况且，在美国还有超过枪击犯罪很多的其他犯罪，与香港、新加坡、日本等禁止枪支的国家、地区根本无法相香港、新加坡、日本等禁止枪支的国家、地区，象香港在反送中被大屠杀前的犯罪率也就是十万分之10 左右，美国一年三万人死于枪击的死亡率就已经等于或高于香港的十万分之10 犯罪率了，另外必然再有十万分之10 的持枪罪犯需要法办，则一年至少有十万分之 20 与枪击死亡有关的犯罪，你美国法律再公证都是马后炮，有什么用？况且，在美国还有超过枪击犯罪很多的其他犯罪，与香港、新加坡、日本等禁止枪支的国家、地区根本无法相比，由此可见，法律解决比枪支暴力解决更有效，依此类推至于其他情形同理成立，从而构成法律先于枪支原则、法律先于枪支解决原则。美国、巴西等放着好好的法律不用，非要用枪支去解决问题，合理吗？有必要吗？比，由此可见，法律解决比枪支暴力解决更有效，依此类推至于其他情形同理成立，从而构成法律先于枪支原则、法律先于枪支解决原则。美国、巴西等放着好好的法律不用，非要用枪支去解决问题，合理吗？有必要吗？人死不能复生，依据不可逆转绝对原则即属绝对的，从而构成人死不能复生绝对原则。任何人都要为生活而奔波、不以生活为前提，同时生命是以新陈代谢为前提的，即不可能是绝对的，从而构成生命相对原则。如果不禁止枪支，依据不禁枪战场（枪林弹雨、人人被打成筛子、人人自危）绝对原则、一项绝对即属绝对原则、同一律，人人都是极度不安全的，人们要生活就必须在高度上与泛滥成灾的枪支保持同一而必然是绝对之无限的，记为结论F；然而，任何人都要为生活而奔波、不以生活为前提，即不可能是绝对的，记为结论G，此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得不禁枪否定原则和禁枪（绝对）原则、禁止枪支原则。依据不禁枪战场（枪林弹雨、人人被打成筛子、人人自危）绝对原则、生命相对原则，如果不禁枪，任何人的生命都不可能与绝对的不禁枪的枪林弹雨相抗衡，从而构成不禁枪生命不设防原则。（3）一石二鸟计划和一鸟二石计划：邪恶轴心非无限卖国否定原则、邪恶政权网络（通信）封锁（审查）延续政权（继续统治、获得支持、继续活摘器官）不可能（乌龙）原则本来放病毒毒杀全人类超过2000 万人的罪犯是钉在了死刑的法场上而不可赦免的。美国拜登向普京伸出橄榄枝，普京可以跟美国联手灭共而刮分半个中共国，同时站在正义的一边A→+∞；可是，普京最终拒绝了美国拜登的橄榄枝，在2022 年2 月4 日与元首签定了军事同盟协议，转而向欧美全面开战，彻底站在了用病毒屠杀全人类2000 万的最特大罪行的邪恶一边B→-∞，A=-B ⇒ C=A-B=2∞，也就是说，如果其在中间无人双向卖国C 最大化（两倍无限大而把正无限大和负无限大都卖给了普京即把整个中国的主权、经济、军事、人民等全部卖给了普京和俄罗斯），普京是不可能在A 与B 中间选择最差的：B=-∞，普京身边有一大群利益集团做参照而显然不可能任普京胡来，称之为普京甘当炮灰（炮灰、弃明投暗、弃善投恶）原则，记为结论F；然而，事实上普京的确选择了与恶魔站在一起临终关怀的一边，记为结论G，此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得邪恶轴心非无限卖国否定原则和邪恶轴心无限（绝对）卖国原则、邪恶轴心军事同盟无限（绝对）卖国原则。普京如果和美国合作，无法拿回前苏联的地缘版图，最终还是得与美国进行最后一战，在干掉中共的过程中还可能被美国背后一刀而一石二鸟，可谓赔了老命又折兵，这是A 计划；于是，普京选择和中共合作，既得了中共的全部家当又一次性干掉美国阵营，然后再灭掉中共实现大欧亚主义，拿回前苏联的地盘版图，这是B 计划也是普京的如意算盘。在上述B 计划中，普京采取联弱打强的作战方案，免除来掉臀部的后顾之忧和无情的伤害，这叫保守疗法；在上述A 计划中，普京联强打弱，即联合核武器势均力敌的竞争对手干掉卑鄙无耻、猥琐下流的低端产品，随时会被黑吃黑丢了老命，这叫叶利钦玩过一次没死成的休克服疗法，这就是一石二鸟方案，综上所述两方面，军事上联弱打强的保守疗法是最安全的，联强打弱最终必然是被一石二鸟黑吃黑而挂掉休克，这也是当年蒙古帝国联宋灭金再灭宋的翻版，可见普京相当地有战略眼光，既然不会被一石二鸟那就洗心革面好好做鸟，称之为一鸟二石方案，普京既吃掉了美国又吃光了中国，要死也可拉个神经病当垫背的，做个CCTV 的新冠病毒害人精也确实没什么大不了（普京胸部就那么点想大也大不起来啊），可谓老婆挖坑老公埋，从而构成害人精作战方法。此时，人们只见普京一只鸟抓起了两块石头精卫填海，象当年的汪精卫直接当汉奸去了，绝对不只是鸟人，从而构成鸟人填海（新景象）原则，反正做鸟不做人不重要，重要的是没有生命危险，安全第一、面子第二。可见，普京比当年叶利钦强多了，一下直奔目标，惨了美国、正中包子的下部，一旦美国释放了全民整体性神经的大日本帝国后，普京可能要一尸三命了。怀才就象怀孕，普京那是想藏都藏不住了。划；于是，普京选择和中共合作，既得了中共的全部家当又一次性干掉美国阵营，然后再灭掉中共实现大欧亚主义，拿回前苏联的地盘版图，这是B 计划也是普京的如意算盘。在上述B 计划中，普京采取联弱打强的作战方案，免除来掉臀部的后顾之忧和无情的伤害，这叫保守疗法；在上述A 计划中，普京联强打弱，即联合核武器势均力敌的竞争对手干掉卑鄙无耻、猥琐下流的低端产品，随时会被黑吃黑丢了老命，这叫叶利钦玩过一次没死成的休克服疗法，这就是一石二鸟方案，综上所述两方面，军事上联弱打强的保守疗法是最安全的，联强打弱最终必然是被一石二鸟黑吃黑而挂掉休克，这也是当年蒙古帝国联宋灭金再灭宋的翻版，可见普京相当地有战略眼光，既然不会被一石二鸟那就洗心革面好好做鸟，称之为一鸟二石方案，普京既吃掉了美国又吃光了中国，要死也可拉个神经病当垫背的，做个CCTV 的新冠病毒害人精也确实没什么大不了（普京胸部就那么点想大也大不起来啊），可谓老婆挖坑老公埋，从而构成害人精作战方法。此时，人们只见普京一只鸟抓起了两块石头精卫填海，象当年的汪精卫直接当汉奸去了，绝对不只是鸟人，从而构成鸟人填海（新景象）原则，反正做鸟不做人不重要，重要的是没有生命危险，安全第一、面子第二。可见，普京比当年叶利钦强多了，一下直奔目标，惨了美国、正中包子的下部，一旦美国释放了全民整体性神经的大日本帝国后，普京可能要一尸三命了。怀才就象怀孕，普京那是想藏都藏不住了。划；于是，普京选择和中共合作，既得了中共的全部家当又一次性干掉美国阵营，然后再灭掉中共实现大欧亚主义，拿回前苏联的地盘版图，这是B 计划也是普京的如意算盘。在上述B 计划中，普京采取联弱打强的作战方案，免除来掉臀部的后顾之忧和无情的伤害，这叫保守疗法；在上述A 计划中，普京联强打弱，即联合核武器势均力敌的竞争对手干掉卑鄙无耻、猥琐下流的低端产品，随时会被黑吃黑丢了老命，这叫叶利钦玩过一次没死成的休克服疗法，这就是一石二鸟方案，综上所述两方面，军事上联弱打强的保守疗法是最安全的，联强打弱最终必然是被一石二鸟黑吃黑而挂掉休克，这也是当年蒙古帝国联宋灭金再灭宋的翻版，可见普京相当地有战略眼光，既然不会被一石二鸟那就洗心革面好好做鸟，称之为一鸟二石方案，普京既吃掉了美国又吃光了中国，要死也可拉个神经病当垫背的，做个CCTV 的新冠病毒害人精也确实没什么大不了（普京胸部就那么点想大也大不起来啊），可谓老婆挖坑老公埋，从而构成害人精作战方法。此时，人们只见普京一只鸟抓起了两块石头精卫填海，象当年的汪精卫直接当汉奸去了，绝对不只是鸟人，从而构成鸟人填海（新景象）原则，反正做鸟不做人不重要，重要的是没有生命危险，安全第一、面子第二。可见，普京比当年叶利钦强多了，一下直奔目标，惨了美国、正中包子的下部，一旦美国释放了全民整体性神经的大日本帝国后，普京可能要一尸三命了。怀才就象怀孕，普京那是想藏都藏不住了。在上述的一石二鸟计划和一鸟二石计划中，希特勒选择墨索里尼做伴侣是不怕狼一样的对手就怕猪一样的队友；普京选择小普京做队友，那是热爱猪一样的队友藐视狼一样的对手，至少精神上是先胜了一局，肉体上争取再胜第二局，得寸进尺、吃着碗里的看着锅里的，可谓是精神肉体双丰收。普京爱猪如命可谓具有大无畏的无产阶级革命家超大无畏的牺牲精神，人为财死、鸟为食亡，普京为地奋斗一生、战斗一生、快活一生，俄罗斯民族却完了：遭全人类所唾弃，所谓人在做、天在看，人生在世不够短短几十年，何必呢？察必活着绝对不允许这么干，人活着，民族却死了，普京你这是察哪里了？由此可见，猪一样的队友、狼一样的对手都不重要，重要的是自己不能误判。邪恶轴心以建设为名行互联网建设防火墙、严厉审查之实封锁互联网，已经到了走火入魔、登峰造极的最后一步，现在，整个中共国，通过正常网络无法访问google 等外网，全民被禁止访问外网、翻墙即刑事犯罪并刑事拘留和罚款等，即属被动的，依据一项绝对即属绝对原则，中共的网络即属绝对的，从而构成（共党、邪恶政权体系下）全民（全国、人人）被动（绝对）原则、全民（全国、人人）绝对被动原则、（共党、邪恶政权）网络（通信）封锁（审查）绝对原则。邪恶政权意图用绝对封锁、审查网络、通信等让五毛、粪青、人民Wj(j∈N)等继续在正面Q 上主动支持、服从它们Ti(i∈N)割韭菜、统治、活摘器官等，至少在中性层面上J 不拆穿它们，如果这是可能的，那么，上述情形中五毛、粪青、人民Wj(j∈N)即不是被动的，记为结论F；然而，依据（共党、邪恶政权体系下）全民（全国、人人）绝对被动原则，五毛、粪青、人民Wj(j∈N)是绝对被动的而不可能有主动的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明邪恶轴心以建设为名行互联网建设防火墙、严厉审查之实封锁互联网，已经到了走火入魔、登峰造极的最后一步，现在，整个中共国，通过正常网络无法访问google 等外网，全民被禁止访问外网、翻墙即刑事犯罪并刑事拘留和罚款等，即属被动的，依据一项绝对即属绝对原则，中共的网络即属绝对的，从而构成（共党、邪恶政权体系下）全民（全国、人人）被动（绝对）原则、全民（全国、人人）绝对被动原则、（共党、邪恶政权）网络（通信）封锁（审查）绝对原则。邪恶政权意图用绝对封锁、审查网络、通信等让五毛、粪青、人民Wj(j∈N)等继续在正面Q 上主动支持、服从它们Ti(i∈N)割韭菜、统治、活摘器官等，至少在中性层面上J 不拆穿它们，如果这是可能的，那么，上述情形中五毛、粪青、人民Wj(j∈N)即不是被动的，记为结论F；然而，依据（共党、邪恶政权体系下）全民（全国、人人）绝对被动原则，五毛、粪青、人民Wj(j∈N)是绝对被动的而不可能有主动的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明邪恶轴心以建设为名行互联网建设防火墙、严厉审查之实封锁互联网，已经到了走火入魔、登峰造极的最后一步，现在，整个中共国，通过正常网络无法访问google 等外网，全民被禁止访问外网、翻墙即刑事犯罪并刑事拘留和罚款等，即属被动的，依据一项绝对即属绝对原则，中共的网络即属绝对的，从而构成（共党、邪恶政权体系下）全民（全国、人人）被动（绝对）原则、全民（全国、人人）绝对被动原则、（共党、邪恶政权）网络（通信）封锁（审查）绝对原则。邪恶政权意图用绝对封锁、审查网络、通信等让五毛、粪青、人民Wj(j∈N)等继续在正面Q 上主动支持、服从它们Ti(i∈N)割韭菜、统治、活摘器官等，至少在中性层面上J 不拆穿它们，如果这是可能的，那么，上述情形中五毛、粪青、人民Wj(j∈N)即不是被动的，记为结论F；然而，依据（共党、邪恶政权体系下）全民（全国、人人）绝对被动原则，五毛、粪青、人民Wj(j∈N)是绝对被动的而不可能有主动的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论 G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得（共党、邪恶政权）网络（通信）封锁（审查）延续政权（继续统治、获得支持、继续活摘器官）否定（不可能）原则和（共党、邪恶政权）网络（通信）封锁（审查）延续政权（继续统治、获得支持、继续活摘器官）不可能（乌龙、终结、搬起石头砸自己的脚、（共党、邪恶政权）网络（通信）封锁（审查）延续政权、（共党、邪恶政权）网络（通信）封锁回旋镖（自找死路）原则、共党（邪恶政权）终结（必灭、灭绝）原则、（共党、邪恶政权）网络（通信）封锁（审查）绝对归零原则。因此，邪恶政权再怎么样封锁网络、通信也不可能挽救、延续其血腥统治，只能被规则无条件归零。依据勒夏特列原理（又称平衡移动原理、Le Chatelier's principle），我们观察一下氨的反应：N2+3H2⇌2NH3，当平衡后压力突然增加，反应会朝向气体系数和气体体积较小的方（4）两木一针解决灭世三步倒之毒一战之前，世界上解决问题的方法是用战争进行博弈即武力解决一切，和丛林里的动物武斗是一样的，于是，邪恶的一方和胜利的一方都可能在这种自由搏击中胜出，出问题的筛选机制问题没解决，还会继续制造问题而导致周而复始、循环往复的历史轮回，这就是战争（弱肉强食、自由博弈）无法解决问题原则、（武斗、武力）胜负不解决原则、（武斗、武力）胜负（武力自由竞争）历史轮回原则、丛林历史轮回原则，自然界的繁殖周期脑震荡就这么折腾出来的，何况人类？依此类推至于肉体胜负（共产党的从肉体上解决问题）历史轮回原则、规则胜负历史轮回原则等同理成立，从而构成问题解决问题三弄（弄巧成拙）原则。这也是资本主义危机、金融风暴周而复始、循环往复的根源。在一个面粉仓库，火焰的燃烧没有得到控制，很快就会发生大灾难性的蘑菇云大爆炸，子弹、炮弹、火箭、原子弹都是这样的原理，这就是非控制大灾难（大祸临头）原则。于是，上帝发明并制定了水火不相容的方程性平衡来解决问题，将任何事物及其行为、品性等限制在一定范围内，如柴火在灶堂里烧但不能超过爆炸的限度，一旦发生过限灾难即用水来解决，因此，水火不相容的平衡将事物限制在安全的许可范围内，称之为弓弦平衡、位弓、构造弓（弦）、固有（平衡）弓（弦），这就是上帝的平衡弓（平衡弦、方程、方程组）解决原则、平衡弓（平衡弦、方程、方程组）限度原则、限度先于平衡弓（平衡弦、方程、方程组）原则、弓弦平衡解决原则，方程解决的不仅是万物本身，更包括是万物的安全界限，这是有史以来人们一直不注意到的，也就是说，方程（代数）、平衡是万物、乾坤宇宙、上帝的第一解决方案。平衡不仅有水火不相容，还有吸收和非必要不生效的预备役。在上帝的平衡（方程）第一方案中，自由（竞争）是根深蒂固的，平衡（方程）是建立在自由（竞争）之上的，因此，平衡（方程）、弓弦平衡又称为上帝的第一平衡木，从而构成自由（竞争）先于平衡（方程）原则。在上帝的平衡（方程）第一方案中，自由（竞争）是根深蒂固的，平衡（方程）是建立在自由（竞争）之上的，因此，平衡（方程）、弓弦平衡又称为上帝的第一平衡木，从而构成自由（竞争）先于平衡（方程）原则。依据勒夏特列原理（又称平衡移动原理、Le Chatelier's principle），我们观察一下氨的反应：N2+3H2⇌2NH3，当平衡后压力突然增加，反应会朝向气体系数和气体体积较小的方向进行，在此例中也就是朝向增加NH3 的方向进行；反之，如果平衡后压力突然减小，反应会朝向气体系数和气体体积较大的方向进行，故每两分子NH3 将会分解成一分子N2 和三分子H2，显然，如果上述平衡不移动，上述反应U 被无限增压下去而发生化工厂蘑菇云大爆炸的时间就会在最短的时间内发生，因此，勒夏特列原理下平衡移动W 最大限度地延迟了这个爆炸过程，为解决方案争取最大的时间容忍度，平衡移动W 如同天平横梁（crossbeam）上的游标（I beam），也就是说，反应U 如果没有平衡移动W 就不可能有任何的足够的稳定和安全，称之为弓举弦张平衡、开弓平衡，反之则为弓恢复常态，从而构成平衡移动（横梁、游标）先于平衡原则、上帝的平衡弓移动（平衡弦移动、方程特解、方程组特解阵列）解决原则、上帝的平衡弓屈移（平衡弦屈移）解决原则、上帝的平衡张弛（平衡弦张弛）解决原则、弓举弦张（开弓、张弛弓）平衡解决原则、弓举弦张（开弓、张弛弓）解决原则，其中方程y=f(x,t)进行特解函数g(x,t)规范如下：y=f(x,t)=g(x,t)，x 为空间变量、t 为时间变量，依此类推至于普遍情形同理成立。勒夏特列原理（又称平衡移动原理、Le Chatelier's principle）是一个定性预测化学平衡点的原理，主要内容为：在一个已经达到平衡的反应中，如果改变影响平衡的条件之一（如温度、压强以及参加反应的化学物质的浓度），平衡将向着能够减弱这种改变的方向移动，即：Every system in stable chemical equilibrium submitted to the influence of an exterior force which tends to cause variation either in it s temperature or condensation (pressure, concentration, number of molecules in the unit of volume), in its totality or only in some of it s parts, can undergo only those interior modifications change of temperature, or of condensation, of a sign contrary to that resulting from the exterior force. 如果一个天平W 的水准泡不在正中央，那么，天平尤其是指针天平的基准就不是归零的，天平上任何游标的移动都是不准的，称之为水准泡（指针、基准、基准平衡）、水准泡（指针、基准、基准平衡）校验，由此而来，天平将随着底座的移动而不同位置称重，从而无限扩大天平的应用范围和地点分布，如同我们将一张比照自行火炮的弓W 不断地望靠近敌人阵地的方向移动而不断射箭，因此称为（无限）分布弓（弦），从而构成水准泡（指针、基准、基准平衡、分布弓）先于平衡移动原则、水准泡（指针、基准、基准平衡、分布弓）校验先于平衡移动原则、上帝的基准平衡（水准泡、指针、基准、分布弓）解决原则、分布弓移动（分布弓时间阵列、方程特解阵列）解决原则、弓（弦）移动（阵列、时间阵列、分布）解决原则。固有（平衡）弓（弦）是奥运会体操项目的平衡木（beam）；拉开分弓是天平的横梁（crossbeam）和游标（I beam）；布弓是在移动平衡之上的水准泡（指南针compass、指针 index、基准benchmark）移动层面上之（无限）分布性扩展，又比照建筑上的水平桶（barrel 枪管），从而构成一弓3B（两木一针、定海神针）原则、一弓3B（两木、两木定海神针）原则、一弓3B 解决原则、解决一弓3B（两木、两木定海神针）原则。其中，一弓又叫一张弓、三步弓：立弓、开弓、移动分布（移形换位），平衡P、移动平衡Q、平衡移动R 而又构成三平衡原则。上述的平衡P 属于弓弦因果、移动平衡Q 属于张弛弓（堆栈）因果、平衡移动R 属于分布因果，统称为一弓3B（三步、平衡）因果、三步弓因果，构成天地一张弓（三分、三步）原则、天地张弛弓（三分、三步）原则、天地平衡弓（三分、三步）原则。上述的平衡P 属于弓弦因果、移动平衡Q 属于张弛弓（堆栈）因果、平衡移动R 属于分布因果，统称为一弓3B（三步、平衡）因果、三步弓因果，构成天地一张弓（三分、三步）原则、天地张弛弓（三分、三步）原则、天地平衡弓（三分、三步）原则。上述的平衡P 属于弓弦因果、移动平衡Q 属于张弛弓（堆栈）因果、平衡移动R 属于分布因果，统称为一弓3B（三步、平衡）因果、三步弓因果，构成天地一张弓（三分、三步）原则、天地张弛弓（三分、三步）原则、天地平衡弓（三分、三步）原则。华夏5000 文明起源于亚当伏羲与夏娃女娲，共494 帝王，但主角只有七位：（1）轩辕大帝（天下万族的万主之主、千古第一君王）、（2）周文王（千古文治第一君王、确立文官治国）、（3）秦始皇（定局中国2000 年的千古万族第一人主、千古万族第一君父即伏羲与女祸亲生、灭六国而不灭其族与人民：人民领袖）、（4）汉武大帝刘彻（汉家第一天子）、（5）唐太宗李世民（天下万族之主、通天教主）、（6）宋仁宗赵祯（千古第一仁君）、（7）明成祖朱棣（世界之王、万王之王、联合国第一任大统领），满清闭关锁国令人民无分别为被动而被排除在外，康熙、乾隆一样处置，称之为九州七君子（七君王），这就是中华七君（君主、君王、皇帝、帝王）原则。秦时明月汉时关，大唐广宋明大统：万主之主的轩辕大帝、千古文治第一君王周文王（君子动口不动手）、千古第一人主秦始皇、汉家第一天子汉武大帝刘彻、万族之主通天教主唐太宗李世民、千古第一仁君宋仁宗赵祯、（7）万国来朝圣殿的世界之王明成祖朱棣，他们七位从如下七方面解决了国家的七个属性：主权、偃武修文文官治国、人权、民族主权、民主、治愈（上帝情怀的人人平等）、真正世界之王的全球万国主权（第一次确立），从而构成国家七要素原则。依据中华七君原则、国家七要素原则，中华君主全部完美地解决了自己所面临的主要问题并作为案例法得以被继承下去，依据中华七君原则、国家七要素原则，中华君主全部完美地解决了自己所面临的主要问题并作为案例法得以被继承下去，依据中华七君原则、国家七要素原则，中华君主全部完美地解决了自己所面临的主要问题并作为案例法得以被继承下去，天下的灾难感同身受，苍生的幸福即是君王的成就，拥有足够的权力解决邪恶与罪犯，具有足够的能力与资源普惠万民，君民同一、家国无差、诛邪化育从实力出发，德才配位与实力原则是天下大治和问题解决的两大基本前提，其位、其职、其力之心国合一的博爱国家化（实力化）、圣心国家化（实力化），从而构成心国合一（德才位合一、德才位三位一体、德才配位）先于解决（国家治理、治愈）原则、博爱国家化（博爱实力化、圣心国家化、圣心实力化）先于解决（国家治理、治愈）原则、博爱国家化（博爱实力化、圣心国家化、圣天下的灾难感同身受，苍生的幸福即是君王的成就，拥有足够的权力解决邪恶与罪犯，具有足够的能力与资源普惠万民，君民同一、家国无差、诛邪化育从实力出发，德才配位与实力原则是天下大治和问题解决的两大基本前提，其位、其职、其力之心国合一的博爱国家化（实力化）、圣心国家化（实力化），从而构成心国合一（德才位合一、德才位三位一体、德才配位）先于解决（国家治理、治愈）原则、博爱国家化（博爱实力化、圣心国家化、圣心实力化）先于解决（国家治理、治愈）原则、博爱国家化（博爱实力化、圣心国家化、圣心实力化）先于解决（国家治理、治愈）原则，依此类推至于如下原则同理成立：心实力化）先于解决（国家治理、治愈）原则，依此类推至于如下原则同理成立：博爱民族化（博爱实力化圣心民族化圣心实力化）先于解决（国家治理治愈）原博爱民族化（博爱实力化、圣心民族化、圣心实力化）先于解决（国家治理、治愈）原则、博爱民族化（博爱实力化、圣心民族化、圣心实力化）先于解决（国家治理、治愈）原则，博爱民族化（博爱实力化、圣心民族化、圣心实力化）先于解决（国家治理、治愈）原则、博爱民族化（博爱实力化、圣心民族化、圣心实力化）先于解决（国家治理、治愈）原则、博爱民族化（博爱实力化、圣心民族化、圣心实力化）先于解决（国家治理、治愈）原则；博爱国际化（博爱实力化、圣心国际化、圣心实力化）先于解决（国家治理、治愈）原则、博爱国际化（博爱实力化、圣心国际化、圣心实力化）先于解决（国家治理、治愈）原则，博爱民族化（博爱实力化、圣心国际化、圣心实力化）先于解决（国家治理、治愈）原则、博爱民族化（博爱实力化、圣心国际化、圣心实力化）先于解决（国家治理、治愈）原由此可见，解决（国家治理、治愈）必须排除分裂的力不从心（有心无力）、德不配位（有力无心），从而构成解决（国家治理、治愈）排除分裂（力不从心、有心无力、德不配位、有力无心）原则。在经济全球化的时代，人们单单解决国内问题而不解决国际问题、民族问题都无效，象邪恶政权就是在国际层面把病毒播散到各国而毒蛇全人类超过2000 万人的；进而，人们解决国内问题而不解决国际问题、民族问题也没有，作为优等防疫生的台湾就那样不断被病毒从国际外部传染进去而几度崩溃的，综上所述就是解决（国家治理、治愈）四海无内外（无分别）原则。由此可见，解决（国家治理、治愈）必须排除分裂的力不从心（有心无力）、德不配位（有力无心），从而构成解决（国家治理、治愈）排除分裂（力不从心、有心无力、德不配位、有力无心）原则。在经济全球化的时代，人们单单解决国内问题而不解决国际问题、民族问题都无效，象邪恶政权就是在国际层面把病毒播散到各国而毒蛇全人类超过2000 万人的；进而，人们解决国内问题而不解决国际问题、民族问题也没有，作为优等防疫生的台湾就那样不断被病毒从国际外部传染进去而几度崩溃的，综上所述就是解决（国家治理、治愈）四海无内外（无分别）原则。博爱（圣心）国家（民族、国际）化先于解决（国家治理、治愈）原则属于心国合一、顶天立地的正向因果A、解决（国家治理、治愈）四海无内外（无分别）原则属于常态性因果B、解决（国家治理、治愈）排除分裂（力不从心、有心无力、德不配位、有力无心）原则C 属于负面因果，构成天子解决（国家治理、治愈）因果三线原则、天子解决（国家治理、治愈）三线原则、解决（国家治理、治愈）三线因果原则、（天子）三线因果，其中A、 B、C 三个因果称为天子三线因果、三线因果。现在，邪恶政权用病毒屠杀全人类超过2000 万人，乌克兰从世界粮仓变成到处是废墟，新冠疫苗行刑毒死毒害全人类，邪恶轴心核武器准备炸毁地球，等等，这些都是资不抵债、刑不抵责的滔天大祸难，就是你把罪犯杀死2000 万次也无法赔偿其所造成的损失，称之为无限死莫赎、无限资不抵债、无限刑不抵责，是名副其实的大罪人，从而构成大罪人超越法律（极限）原则、大罪人原则。现在，邪恶政权用病毒屠杀全人类超过2000 万人，乌克兰从世界粮仓变成到处是废墟，新冠疫苗行刑毒死毒害全人类，邪恶轴心核武器准备炸毁地球，等等，这些都是资不抵债、刑不抵责的滔天大祸难，就是你把罪犯杀死2000 万次也无法赔偿其所造成的损失，称之为无限死莫赎、无限资不抵债、无限刑不抵责，是名副其实的大罪人，从而构成大罪人超越法律（极限）原则、大罪人原则。病毒屠杀全人类超过2000 万人的问题、各国争夺世界或区域领导权问题、战争问题等，归根结底，就是当年希特勒纳粹问鼎欧洲主权和邪恶轴心刮分世界主权的最终解决在主权层面上的延续，从而构成世界主权最终解决（炸毁地球）原则。依据解决（国家治理、治愈）四海无内外（无分别）原则、解决（国家治理、治愈）排除分裂（力不从心、有心无力、德不配位、有力无心）原则、大罪人超越法律（极限）原则，全球化的前提下，各国千丝万缕的联系无法直接全部切断，今天病毒屠杀全人类超过2000 万人的问题、各国争夺世界或区域领导权问题、战争问题等如果不从全局层面上进行解决，邪恶轴心炸毁地球的结局就无法改变，于是，在排除分裂（力不从心、有心无力、德不配位、有力无心）的前提下，解决这一切问题的前提就是全世界选举出一位世界共主代表全球主权在全局上进行最高解决，才能避免最终解决，即圣心（博爱）国家（民族、国际）化、德才位合（德才位合一），称之为最高（解决）因果、天下共主（万王之王、万主之主）因果，世界共主就是圣经里的万王之王、万主之主，从而构成天下共主（万王之王、万主之主）先于（世界）和平（最高解决、完全解决）原则、天下共主（万王之王、万主之主）先于末日解决原则。病毒屠杀全人类超过2000 万人的问题、各国争夺世界或区域领导权问题、战争问题等，归根结底，就是当年希特勒纳粹问鼎欧洲主权和邪恶轴心刮分世界主权的最终解决在主权层依据一弓3B 解决原则、解决（国家治理、治愈）排除分裂（力不从心、有心无力、德不配位、有力无心）原则、解决（国家治理、治愈）四海无内外（无分别）原则、解决（国家治理、治愈）四海无内外（无分别）原则、博爱（圣心）国家（民族、国际）化先于解决（国家治理、治愈）原则、天位因果三分原则、天子（圣子）定乾坤（安天下）原则，国家要治愈、全世界要解除三步倒，于是，三步弓因果、三线因果、天位三因果（规则因果、造化分布因果、造化安全因果）、天下共主（万王之王、万主之主）因果，构成了全球解决（治理、治愈）3331 原则、解决（治理、治愈）3331 偏置原则。圣心（博爱）国家（民族、国际）化、德才位合（德才位合一），天子剑、天子权杖、天子影响力（天子函数）三位一体于天子在位，解决（治愈）三步走之立弓、开弓、移动分布（移形换位）即平衡、移动平衡、平衡移动，解决（国家治理、治愈）排除分裂（力不从心、有心无力、德不配位、有力无心），解决（国家治理、治愈）四海无内外（无分别），世界才能和平；今天病毒屠杀全人类超过2000 万人的问题、各国争夺世界或区域领导权问题、战争问题等，全世界无一例外祸水滔天，全人类陷入毁灭文明、邪恶轴心炸毁地球的大洪水之中，称之为人祸大洪水、末日大洪水，与史前大洪水相对应。在历前的那场大洪水中，圣经里记载的解决方案是诺亚方舟（Noah's Ark），华夏文明的解决方案是大禹治水立九州，史前大洪水来自自然；今天，毁灭人类文明的人祸大洪水来自邪恶轴心和欧美社会体系的缺陷所导致的腐败，解决方案是什么呢？显然，举世灾难，末日飞机只能让国家元首安全，人们则逃无可逃，全都得死无葬身之地，于是，我们排除了诺亚方舟（Noah's Ark），剩下唯一的方案就是大禹治水保世界、轩辕共主平天下，从而构成末日洪水大禹治水立九州保世界原则，人们才能将中华文明和世界文明一起保存下来，也就是轩辕共主治水（平天下）原则、天下共主（万王之王、万主之主）治水安天下（世界）原则，简称为轩辕共主（万王之王、万主之主）方案、轩辕治水方案即天下共主（万王之王、万主之主）方案，早在公元4700 年前就已经确定。圣心（博爱）国家（民族、国际）化、德才位合（德才位合一），天子剑、天子权杖、天子影响力（天子函数）三位一体于天子在位，解决（治愈）三步走之立弓、开弓、移动分布（移形换位）即平衡、移动平衡、平衡移动，解决（国家治理、治愈）排除分裂（力不从心、有心无力、德不配位、有力无心），解决（国家治理、治愈）四海无内外（无分别），世界才能和平；今天病毒屠杀全人类超过2000 万人的问题、各国争夺世界或区域领导权问题、战争问题等，全世界无一例外祸水滔天，全人类陷入毁灭文明、邪恶轴心炸毁地球的大洪水之中，称之为人祸大洪水、末日大洪水，与史前大洪水相对应。在历前的那场大洪水中，圣经里记载的解决方案是诺亚方舟（Noah's Ark），华夏文明的解决方案是大禹治水立九州，史前大洪水来自自然；今天，毁灭人类文明的人祸大洪水来自邪恶轴心和欧美社会体系的缺陷所导致的腐败，解决方案是什么呢？显然，举世灾难，末日飞机只能让国家元首安全，人们则逃无可逃，全都得死无葬身之地，于是，我们排除了诺亚方舟（Noah's Ark），剩下唯一的方案就是大禹治水保世界、轩辕共主平天下，从而构成末日洪水大禹治水立九州保世界原则，人们才能将中华文明和世界文明一起保存下来，也就是轩辕共主治水（平天下）原则、天下共主（万王之王、万主之主）治水安天下（世界）原则，简称为轩辕共主（万王之王、万主之主）方案、轩辕治水方案即天下共主（万王之王、万主之主）方案，早在公元4700 年前就已经确定。日本内藤湖南说：崖山之后无中华（被游牧民族蒙古灭国但华夏族尚在），明亡之后无华夏（被闭关锁国而被动非法的满夷满清砍毛而灭族），今天，日本保留了大唐的汉家文明，美国保留了近代文明，中共国则连女娲皇帝、轩辕大帝、禹王、孔子、民族英雄岳飞等的坟墓全部被狗刨了，尸骨尽毁，这就是黄俄狗刨政权，此时，其不仅令人想到二战中老首相丘吉尔在《至暗时刻》哭着念的：「于是城门的守将贺拉提斯这样说：对于世上生灵万物，死亡迟早终将降临，为了守护先祖的遗骨与信仰的神殿，直面强敌力战而没，没有别的死亡能如此崇高（Then out spake brave Horatius:The Captain of the Gate. To every man upon this earth. Death come the soon or late. And how can man die better. Than facing fearful odds.）」，为了重建先祖们的遗骨，为了建造和守护全人类的第三圣殿，轩辕的子孙为汉家文明和人类文明立法，置九鼎33 天外天立法治祸水，两木一针天子剑最高解决邪恶轴心的最终解决，从而构成汉家天子立法绝洪水原则，简称汉家天子立法。 15．国家力量防不住原则、俄罗斯合法性立足欧洲原则、情报第零原则、情报第一原则、胜利（女神）第一原则、成功第一原则（1）俄罗斯合法性立足欧洲原则、天灭俄罗斯原则、天灭共党原则 1812 年，拿破仑打如俄罗斯的大纵深如入无人之境，寒冷气候和战斗民族的吃苦韧性并没有发挥任何作用，如果不是瘟疫肆虐法军，如今的俄罗斯早就不存在了；1904 年2 月6 日，日俄战争在中国东北爆发，俄罗斯投入了近两百多万部队进行作战、总动员兵力375 万，日本投入30 万左右兵力，最后，明石元二郎在日内瓦同列宁会面让列宁发动革命，结果大家都看到了，俄罗斯惨败，连日本的30 万部队都打不过，尼古拉二世因此最后全家领了盒饭，共产主义恶魔也由此被释放到人间；1941 年6 月22 日，希特勒闪电战进入苏联势如破竹，之后折腾苏联5 年无人能撼动其江湖地位，最后靠英美援助才赢得战争，综上所述三方面，俄罗斯并非什么战斗民族，气候武器和民族韧性都是一句废话，由此构成俄罗斯三废原则。 15．国家力量防不住原则、俄罗斯合法性立足欧洲原则、情报第零原则、情报第一原则、胜利（女神）第一原则、成功第一原则（1）俄罗斯合法性立足欧洲原则、天灭俄罗斯原则、天灭共党原则 1812 年，拿破仑打如俄罗斯的大纵深如入无人之境，寒冷气候和战斗民族的吃苦韧性并没有发挥任何作用，如果不是瘟疫肆虐法军，如今的俄罗斯早就不存在了；1904 年2 月6 日，日俄战争在中国东北爆发，俄罗斯投入了近两百多万部队进行作战、总动员兵力375 万，日本投入30 万左右兵力，最后，明石元二郎在日内瓦同列宁会面让列宁发动革命，结果大家都看到了，俄罗斯惨败，连日本的30 万部队都打不过，尼古拉二世因此最后全家领了盒饭，共产主义恶魔也由此被释放到人间；1941 年6 月22 日，希特勒闪电战进入苏联势如破竹，之后折腾苏联5 年无人能撼动其江湖地位，最后靠英美援助才赢得战争，综上所述三方面，俄罗斯并非什么战斗民族，气候武器和民族韧性都是一句废话，由此构成俄罗斯三废原则。显然，俄罗斯在亚洲被日本打败，病危改变其命运，其继续在欧洲苟延残喘至于1917 年列宁发动十月革命，俄罗斯随着共产主义政权的到来，俄罗斯欧亚两地无分别地全面改弦易辙，依据全局绝对原则，这就是俄罗斯欧洲改变全局改变绝对原则。如果俄罗斯的合法性不是立足欧洲，那么，其欧洲部分政权的改变不可能是绝对的，记为结论F；然而，依据俄罗斯欧洲改变全局改变绝对原则，俄罗斯的欧洲部分一旦发生改变其全局格局彻底、绝对地改变，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 否定肯定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得俄罗斯合法性非立足欧洲否定（错误）原则和俄罗斯合法性立足欧洲原则，这是俄罗斯历史和政权属性的第一原则，随之又构成俄罗斯（属性、历史、主权）第一原则。如果普京经由XP 军事同盟获取资金、武器征服世界是可能的，那么，依据同一律、前提决定结果原则，普京基于普习军事同盟输送的来自东方亚洲的资金、武器、战略物资等 W 或以之W 为前提发动战争的主权Q（战前只有12 米即GDP 排名世界12）达到H（如 100 米）高度必然是亚洲、东方的，记为结论F；然而，依据罗斯合法性立足欧洲原则，俄罗斯的主权Q 是立足欧洲的而必然是只有战前的12 米高度的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得XP 军事同盟成功可能性否定原则、俄罗斯经由XP 军事同盟征服世界成功否定原则和XP 军事同盟必败（失败）原则、俄罗斯（经由XP 军事同盟）征服世界不可能（成功）原则、俄罗斯（经由XP 军事同盟）绝对归零（分裂、主权分裂）原则、天灭俄罗斯原则。如果普京经由XP 军事同盟获取资金、武器征服世界是可能的，那么，依据同一律、前提决定结果原则，普京基于普习军事同盟输送的来自东方亚洲的资金、武器、战略物资等 W 或以之W 为前提发动战争的主权Q（战前只有12 米即GDP 排名世界12）达到H（如 100 米）高度必然是亚洲、东方的，记为结论F；然而，依据罗斯合法性立足欧洲原则，俄罗斯的主权Q 是立足欧洲的而必然是只有战前的12 米高度的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得XP 军事同盟成功可能性否定原则、俄罗斯经由XP 军事同盟征服世界成功否定原则和XP 军事同盟必败（失败）原则、俄罗斯（经由XP 军事同盟）征服世界不可能（成功）原则、俄罗斯（经由XP 军事同盟）绝对归零（分裂、主权分裂）原则、天灭俄罗斯原则。如果普京兄弟经由XP 军事同盟获取军事力量征服世界是可能的，那么，依据同一律、前提决定结果原则，普京兄弟基于普习军事同盟输送的来自普京的军事力量（核武威慑）W 或以之W 为前提发动战争的主权Q（战前只有1.2 米）达到H（如70 米）高度必然是俄罗斯，记为结论F；然而，中共国的主权Q 是立足亚洲的而必然是只有战前的1.2 米高度的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得XP 军事同盟成功可能性否定原则、共党经由XP 军事同盟征服世界成功否定原则和XP 军事同盟必败（失败）原则、共党（经由 XP 军事同盟）征服世界不可能（成功）原则、共党（经由XP 军事同盟）绝对归零（分裂、主权分裂）原则、天灭共党原则。斯，记为结论F；然而，中共国的主权Q 是立足亚洲的而必然是只有战前的1.2 米高度的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得XP 军事同盟成功可能性否定原则、共党经由XP 军事同盟征服世界成功否定原则和XP 军事同盟必败（失败）原则、共党（经由 XP 军事同盟）征服世界不可能（成功）原则、共党（经由XP 军事同盟）绝对归零（分裂、主权分裂）原则、天灭共党原则。依据俄罗斯（经由XP 军事同盟）征服世界不可能（成功）原则、分裂失败（必败）原则，普京先把自己的主权分裂、腰斩后，然后发神经要去征服世界、统治世界，其必然是失败的、不可能成功的，依此类推至于共党必然是失败的、不可能成功的，由此而来，两个已经被上天的规则腰斩的人，没有必然再作任何的垂死挣扎了，一切已经毫无意义，这是上天的注定也是最终的判决，分裂者、分裂国必败。（2）胜利（女神）第一原则、成功第一原则、温水煮青蛙（高压国、虚假欺骗、共产主义、分裂、神经病、文盲、法盲）从来赢不了原则双方或多方博弈，赢的一方必然是主动的，随之必然是排除被动的，否则即属分裂而被规则归零，从而构成赢者（胜利）主动（排除被动、排除分裂、神经正常、有知识非文盲或法盲）原则、主动（排除被动、排除分裂、神经正常、有知识非文盲或法盲）先于胜利（赢者）原则；进而，任何国家、人、事物存在、发展都必须进行不偏离即失败的主动性维持，从而构成国家（人、失去）维持（维持稳定、维持和平、可持续、存在、发展）主动原则，综上所述两方面即构成胜利（女神）第一原则，即维多利亚（Victoria）第一原则。如果分裂W 能赢得胜利，那么，其必然是主动的，即其为主动是确定的，记为结论F；然而，任何分裂、精神病、神经病都是不确定的，即其是主动的或非主动的都是非（无法）确定的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得分裂赢得胜利否定原则、分裂主动否定原则和分裂失败（必败）原则、分裂非主动原则、分裂（绝对）归零原则、分裂必败（必死）原则。西医治标、中医治本，医生治得好是标本兼治，医不好就治成标本，成为大量尸体工厂的标本后巡展全世界，任何国家U 的政府W 如果将人民Y、社会Q 治理成被动或被动不可排除的却是成功的、能到处打胜仗的，如面向公共领域风险不可排除的电话非实名登记不准购买、使用之被动的，非人脸识别不能进小区回家的，等等，依据同一律，国家U、政府W、高压国的内部全部都是受压迫者，就特权阶级和统治者才是主动的，从而构成高压国（无限）被动（无法排除被动、无法排除分裂）原则。高压国的内部全部都是受压迫者，就特权阶级和统治者才是主动的，从而构成高压国（无限）被动（无法排除被动、无法排除分裂）原则。如果高压国W 是赢得了的，那么，依据赢者主动（排除被动、排除分裂）原则，其必然是排除被动、分裂的，记为结论F；然而，依据高压国（无限）被动（无法排除被动）原则，其是无法排除被动、无法排除分裂的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得高压国（锅）赢得胜利否定原则和高压国从来赢不了原则，依此类推至于如下原则同理成立：温水煮青蛙（虚假欺骗、共产主义、分裂、神经病、文盲、法盲）赢得胜利否定原则和温水煮青蛙（虚假欺骗、共产主义、分裂、神经病、文盲、法盲）从来赢不了原则。（2）胜利（女神）第一原则、成功第一原则、温水煮青蛙（高压国、虚假欺骗、共产主义、分裂、神经病、文盲、法盲）从来赢不了原则西医治标、中医治本，医生治得好是标本兼治，医不好就治成标本，成为大量尸体工厂的标本后巡展全世界，任何国家U 的政府W 如果将人民Y、社会Q 治理成被动或被动不可排除的却是成功的、能到处打胜仗的，如面向公共领域风险不可排除的电话非实名登记不准购买、使用之被动的，非人脸识别不能进小区回家的，等等，依据同一律，国家U、政府W、人民Y 即属无分别之被动的、被动不可排除的，记为结论F；然而，任何人都是主体的而必然是主动的，国家是主权的而必然是主动的，国家U、人民Y 即必然是排除被动的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得治国理政（公共管理、全球治理、国家治理、政府管理）治成标本（被动、中性）否定原则、治成标本否定原则和治国理政（公共管理、全球治理、国家治理、政府管理）治成标本（被动、中性）失败（绝对归零）原则、治国理政（公共管理、全球治理、国家治理、政府管理）主动（排除被动）原则、标本兼治主动（排除被动）原则、治成标本（中性）失败（绝对归零）原则，这就是国家（天子、政府、社会管理）成功第一原则，依此类推至于为人处事被动（失败）否定原则和为人处事主动（排除被动）原则，也就是为人处事成功第一原则。国家（天子、政府、社会管理）成功第一原则和人处事成功第一原则称为成功第一原则。国家（天子、政府、社会管理）成功第一原则和人处事成功第一原则称为成功第一原则。高压国的内部全部都是受压迫者，就特权阶级和统治者才是主动的，从而构成高压国（无限）被动（无法排除被动、无法排除分裂）原则。如果高压国W 是赢得了的，那么，依据赢者主动（排除被动、排除分裂）原则，其必然是排除被动、分裂的，记为结论F；然而，依据高压国（无限）被动（无法排除被动）原则，其是无法排除被动、无法排除分裂的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得高压国（锅）赢得胜利否定原则和高压国从来赢不了原则，依此类推至于如下原则同理成立：温水煮青蛙（虚假欺骗、共产主义、分裂、神经病、文盲、法盲）赢得胜利否定原则和温水煮青蛙（虚假欺骗、共产主义、分裂、神经病、文盲、法盲）从来赢不了原则。（3）问题解决问题三弄（弄巧成拙）原则共产主义和恐怖主义都是从肉体上解决问题，从而构成共产主义（恐怖主义）从肉体上解决问题原则。从肉体上解决问题（非对称解决问题）、战争博弈解决问题、二战后的以金钱和法律（规则）的文斗解决问题、用真理解决问题但不排除武力解决的全解决问题，前三者都是有问题的而不可能真正解决问题，只能是把问题越解决越积恶成习，从而构成了问题解决问题三弄（弄巧成拙）原则和万解之解解决原则（天子剑、天子本征根、天子特征向量、天子权杖解决），问题解决问题三弄（弄巧成拙）比照梅花三弄。从肉体上解决问题（非对称解决问题），肉体是解决了，但制造问题的机制仍在而未解决，如邪恶政权将陈彦霖等17000 名黑衣人全部灭门了，但邪恶政权本身导致反送中运动的腐败、暴政之根源没解决；共党解放后杀害了上海超过50 万名资本家和知识分子，但市场基于区别的根源无法解决而导致大规模饿死人等跟严重的次生后果，等等，这就是从肉体上无法解决问题原则、解决肉体不解决原则、肉体胜负历史轮回原则。共党从肉体上解决问题，不会导致历史轮回，但违背天理规则而必然被天子剑出鞘规则归零，即使规则归零问题没解决，被苍天彻底毁灭。不会导致历史轮回，但违背天理规则而必然被天子剑出鞘规则归零，即使规则归零问题没解决，被苍天彻底毁灭。一战之前，世界上解决问题的方法是用战争进行博弈即武力解决一切，和丛林里的动物武斗是一样的，于是，邪恶的一方和胜利的一方都可能在这种自由搏击中胜出，出问题的筛选机制问题没解决，还会继续制造问题而导致周而复始、循环往复的历史轮回，这就是战争（弱肉强食、自由博弈）无法解决问题原则、（武斗、武力）胜负不解决原则、（武斗、武力）胜负（武力自由竞争）历史轮回原则、丛林历史轮回原则，自然界的繁殖周期脑震荡就这么折腾出来的，何况人类？一战之前，世界上解决问题的方法是用战争进行博弈即武力解决一切，和丛林里的动物武斗是一样的，于是，邪恶的一方和胜利的一方都可能在这种自由搏击中胜出，出问题的筛选机制问题没解决，还会继续制造问题而导致周而复始、循环往复的历史轮回，这就是战争（弱肉强食、自由博弈）无法解决问题原则、（武斗、武力）胜负不解决原则、（武斗、武力）胜负（武力自由竞争）历史轮回原则、丛林历史轮回原则，自然界的繁殖周期脑震荡就这么折腾出来的，何况人类？二战后二战后的雅尔塔建立起以金钱和法律的文斗解决一切问题的舞台剧，让人类文明直立行走的正面筛选机制依然没有建立，人类文明仍然象无头苍蝇乱飞，邪恶的一方和胜利的一方都可能在这种自由搏击中胜出，未被解决的问题积累到一定程度后又会象资本主义危机一样周而复始地暴发，而且还会积恶成习，如同今天美国利益集团协助邪恶轴心打败美国一样，不扭转乾坤最后肯定死无葬身之地，这就是金钱（规则、非七级因果法律）无法解决问题原则、（文斗、规则）输赢不解决原则、（文斗、规则）输赢（文法自由竞争）历史轮回原则、（规则）胜负历史轮回原则。解决肉体不解决原则、战争（弱肉强食、自由博弈）无法解决问题原则、金钱（非七级因果法律）无法解决问题原则，构成了问题解决问题三弄（弄巧成拙）原则、问题解决问题不解决原则、自由竞争历史轮回原则。从肉体上解决问题的共产主义是规则归零问题没解决被直接灰飞烟灭，属于天魔解体的解体体制，绝对多数的共产主义国家都是一夜解体就是这么来的，这就是肉体解决解体原则、解体先于肉体解决（从肉体上解决）原则。解体先于肉体解决（从肉体上解决）原则。（4）民主邪恶联袂不归路原则官僚主义是当官的（包括元首）P 脱离前线，凡事以官僚机构为前提、挡箭牌去解决问题，怕被人从背后捅刀子（象安倍被从背后开枪一样）、或被人找出问题以取而代之，都想和平、不想打仗、不想出事，凡是让别人替自己担当，不冒头、不挑事、不自己担当解决问题，如欧美法系都把判决书写成是陪审团判决嫌犯有罪，美国政客都要政策做成是议会里大家一起通过的，这就是官僚主义圆桌（无战斗部、无方向、无主动、无主）原则，依此类推至于美国的民主宪政一样同理成立，随之构成民主宪政圆桌（无战斗部、无方向、无主动、无主）原则、民主宪政（官僚主义）天下无主原则，违背了绝对归一、无分别归一的天下归一原则、天子制度原则。由此可见，美国的民主宪政实际上就是官僚主义的不担当、推脱责任、六神无主，除了为副不仁的切尼之外，此时，极权的共产主义和民族主义邪恶轴心乘虚而入，不打自招、不言而喻、不战而降，自由竞争导致的周期性危机（覆没）问题无法解决，从而构成圆桌体系（民主宪政、官僚主义）历史轮回（自由竞争、无分别、轮流坐庄）原则；与此同时，依据同一律、前提决定结果原则、结果映射前提原则，圆桌权力机构、机制的存在又让权利无条件运行、无条件服从，依据无条件绝对原则，其即构成圆桌体系（民主宪政、官僚主义）权力运行无条件原则、圆桌体系（民主宪政、官僚主义）无条件服从权力原则、圆桌体系（民主宪政、官僚主义）绝对归一原则。力运行无条件原则、圆桌体系（民主宪政、官僚主义）无条件服从权力原则、圆桌体系（民主宪政、官僚主义）绝对归一原则。力运行无条件原则、圆桌体系（民主宪政、官僚主义）无条件服从权力原则、圆桌体系（民主宪政、官僚主义）绝对归一原则。如丘吉尔(Winston L.S. Churchill)对乔治国王所说首相的位置上上下下是常有的事一样，圆桌体系（民主宪政、官僚主义）的上上下下贻误战机、犹豫不决、拖拖拉拉、徘徊不前是不可避免的，依据圆桌体系（民主宪政、官僚主义）历史轮回（自由竞争）原则、圆桌体系（民主宪政、官僚主义）无条件服从权力原则、圆桌体系（民主宪政、官僚主义）绝对归一原则，圆桌体系（民主宪政、官僚主义）既是绝对归一的又是自由竞争、轮流坐庄而为无分别的、不绝对归一的，显然违反矛盾律而既不可能为真也不可能正确或成立，依据规则绝对归零原则，其即构成圆桌体系（民主宪政、官僚主义）规则归零原则、圆桌体系（民主宪政、官僚主义）规避枪打出头鸟。如丘吉尔(Winston L.S. Churchill)对乔治国王所说首相的位置上上下下是常有的事一样，圆桌体系（民主宪政、官僚主义）的上上下下贻误战机、犹豫不决、拖拖拉拉、徘徊不前是不可避免的，依据圆桌体系（民主宪政、官僚主义）历史轮回（自由竞争）原则、圆桌体系（民主宪政、官僚主义）无条件服从权力原则、圆桌体系（民主宪政、官僚主义）绝对归一原则，圆桌体系（民主宪政、官僚主义）既是绝对归一的又是自由竞争、轮流坐庄而为无分别的、不绝对归一的，显然违反矛盾律而既不可能为真也不可能正确或成立，依据规则绝对归零原则，其即构成圆桌体系（民主宪政、官僚主义）规则归零原则、圆桌体系（民主宪政、官僚主义）规避枪打出头鸟。邪恶轴心直奔主题被规则归零而大解体，民主阵营自带规则归零而走向衰败，这就是末日的世界不归路，从而构成民主邪恶联袂不归路原则。邪恶轴心直奔主题被规则归零而大解体，民主阵营自带规则归零而走向衰败，这就是末日的世界不归路，从而构成民主邪恶联袂不归路原则。（5）国家力量防不住原则、双解体解决原则、情报第零原则、情报第一原则（5）国家力量防不住原则、双解体解决原则、情报第零原则、情报第一原则任何人、国家、民族的防范是立足自身而应变的，即以外来的攻击为前提进行反应性行动而必然是相对的、有限的而不可能是绝对的，从而构成防范（防守、防卫）相对（有限、非绝对）原则。（5）国家力量防不住原则、双解体解决原则、情报第零原则、情报第一原则任何人、国家、民族的防范是立足自身而应变的，即以外来的攻击为前提进行反应性行动而必然是相对的、有限的而不可能是绝对的，从而构成防范（防守、防卫）相对（有限、非绝对）原则。经济建设、民生成绩以获得性的利润为前提或同一性参照基准，不具备输出性的战斗部攻击力，显然不可能是绝对的，从而构成经济非绝对原则、以经济为核心（资本主义）非绝对原则。经济建设、民生成绩以获得性的利润为前提或同一性参照基准，不具备输出性的战斗部攻击力，显然不可能是绝对的，从而构成经济非绝对原则、以经济为核心（资本主义）非绝对原则。依据至上绝对原则，任何国家、民族、地区的主权都是至上的，这就是主权绝对原则；进而，邪恶轴心的俄罗斯民族是只想掠夺土地、共产共妻、工食物链顶端直接掠夺文明成果等，属于基于绝对主权的死性不改之要钱不要命、入土为安不要紧的鬼吹灯盗墓集团，显然是绝对、无限的，从而构成邪恶轴心（侵略、渗透、颠覆、谋杀、斩首、战争、军事行动）绝对原则。依据至上绝对原则，任何国家、民族、地区的主权都是至上的，这就是主权绝对原则；进而，邪恶轴心的俄罗斯民族是只想掠夺土地、共产共妻、工食物链顶端直接掠夺文明成果等，属于基于绝对主权的死性不改之要钱不要命、入土为安不要紧的鬼吹灯盗墓集团，显然是绝对、无限的，从而构成邪恶轴心（侵略、渗透、颠覆、谋杀、斩首、战争、军事行动）绝对原则。安倍晋三（Shinzo Abe）的死、马苏德的死表明，国家力量的谋杀和军事侵略一样是防不住的、无法防范的，因为任何国家、民族的主权P 是至上的，依据至上绝对原则即属无限大的：P→∞，如同打气筒不断地将金钱、人力、人脉资源等输送到战斗部（如秘密组织、军事组织）而制造无限大的以国家主权保持同一的绝对性攻击力量F：P→∞，从而构成国家力量（谋杀、暗杀、侵略、渗透、颠覆）绝对（无限大）原则；而依据防范（防守、防卫）相对（有限、非绝对）原则，任何人、国家、民族、政府的防范（防守、防卫）都是有限的而不可能是无限的国家力量（谋杀、暗杀、侵略、渗透、颠覆）的对手，从而构成国家力量（谋杀、暗杀、侵略、渗透、颠覆、情报）防不住（防不胜防）原则，同时也是任何国家情报系统的情报第零原则、国家力量先于情报原则。所谓没金刚钻不揽瓷器活，没国家力量如何能获取国家主权级别的情报呢？自找死路不仅白白断送了情报人员的生命和门路，更打开了对方反渗透、反向情报的大门，所谓狗洞大开；与此同时，无论你技术多么发达，任何情报都是以情报人员的生命为前提的，如中国在澳大利亚埋下了混血三代的澳大利亚外交部长Penny Wong(黄英贤)，因此，情报人员的生命、合法性身份是情报的第一原则，从而构成（情报人员）生命(合法性身份)先于情报原则、情报第一原则（定律）。澳大利亚埋下了混血三代的澳大利亚外交部长Penny Wong(黄英贤)，因此，情报人员的生命、合法性身份是情报的第一原则，从而构成（情报人员）生命(合法性身份)先于情报原则、情报第一原则（定律）。依据经济（以经济为核心、资本主义）非绝对原则、国家力量（谋杀、暗杀、侵略、渗透、颠覆）绝对（无限大）原则、邪恶轴心（侵略、渗透、颠覆、谋杀、斩首、战争、军事行动）绝对原则，美国、法国、德国等以经济建设为重的资本主义国家肯定不可能与邪恶轴心相抗衡的，也是防不住的，依据不可能绝对原则，其即构成民主阵营不敌（防不住）邪恶轴心（绝对）原则，那么，解决的方案是什么？全人类只有一条路，彻底双解体共党和好吃懒做、只想不劳而获以掠夺为生的民族主义，从而构成双解体解决原则。民匪集中营和土匪集中营都是极权专制的，共党至上、党魁至上，内部是官大一级压死人的无限高压锅系统，从而构成民匪（肉匪）高压锅原则。民匪集中营和土匪集中营都是极权专制的，共党至上、党魁至上，内部是官大一级压死人的无限高压锅系统，从而构成民匪（肉匪）高压锅原则。民匪集中营和土匪集中营都是极权专制的，共党至上、党魁至上，内部是官大一级压死人的无限高压锅系统，从而构成民匪（肉匪）高压锅原则。泽连斯基以总统的一人之力独力支撑着乌克兰的持续，这种情形的国家成为元首（总统）国家、主权国家，依此类推至于其他类似国家同理成立。依据无分别绝对原则，大流士（Darius）有源源不断的外来之希腊雇佣兵Q 做后盾、美国有自己广泛的无分别之民兵R 组织做后盾，等等，这种基于等于是无限的人力做后盾的国家称为动力（民众）国家。泽连斯基以总统的一人之力独力支撑着乌克兰的持续，这种情形的国家成为元首（总统）国家、主权国家，依此类推至于其他类似国家同理成立。依据无分别绝对原则，大流士（Darius）有源源不断的外来之希腊雇佣兵Q 做后盾、美国有自己广泛的无分别之民兵R 组织做后盾，等等，这种基于等于是无限的人力做后盾的国家称为动力（民众）国家。美国是民主圆桌制度，一个人被谋杀了另一个人会顶上去，如肯尼迪被谋杀了约翰逊顶上，这是一种军事化的国家制度，称之为圆桌国家。美国将大量的兵力部署在印太、欧洲等，任何国家W 即便将美国首都华盛顿夷为平地，不出8 个小时（美国兵力是全球8 小时投递制），国家W 就会被美国的海外部队砍瓜切菜，也就是说，象美国这样的国家自带超强复仇功能，任何不自量力的国家进攻美国必然是自取灭亡，明朝是天子守国门，美国是回旋镖的离岸（海外）军队守国门，如同秦始皇坟墓入口的层层机关守护着两千年前的主人一样，称之为（海外、离岸）机关国家。共党从肉体上解决一切问题，不拿人命当事，那同党、人民的死亡当自己发展、赢利的机会（如邪恶政权所设计的疫情经济），其政权以黑箱操作著称、作案以不见天日闻名于时（如杀害香港女儿陈彦霖和灭门其所有相关的亲戚朋友、亲人老师同学），就象一家公司随便找家壳公司借壳上市一样，所惯用武功为杀敌一千自损好几十万（如长津湖的冰雕连）之人盾、人海战术、人民战争等，称之为天魔解体国家（组织、政党）、（大）解体国家（组织、政党）、分裂（精神病、神经病）国家（组织、政党）。元首（总统）国（主权国家）、圆桌国家、动力（民众）国家三国归一于一个国家才算是完成，从而构成国家三分原则、国家主权圆桌动力（支撑）三分原则。美国是民主圆桌制度，一个人被谋杀了另一个人会顶上去，如肯尼迪被谋杀了约翰逊顶上，这是一种军事化的国家制度，称之为圆桌国家。美国是民主圆桌制度，一个人被谋杀了另一个人会顶上去，如肯尼迪被谋杀了约翰逊顶上，这是一种军事化的国家制度，称之为圆桌国家。美国是民主圆桌制度，一个人被谋杀了另一个人会顶上去，如肯尼迪被谋杀了约翰逊顶上，这是一种军事化的国家制度，称之为圆桌国家。上，这是种军事化的国家制度，称之为圆桌国家。美国将大量的兵力部署在印太、欧洲等，任何国家W 即便将美国首都华盛顿夷为平地，不出8 个小时（美国兵力是全球8 小时投递制），国家W 就会被美国的海外部队砍瓜切菜，也就是说，象美国这样的国家自带超强复仇功能，任何不自量力的国家进攻美国必然是自取灭亡，明朝是天子守国门，美国是回旋镖的离岸（海外）军队守国门，如同秦始皇坟墓入口的层层机关守护着两千年前的主人一样，称之为（海外、离岸）机关国家。共党从肉体上解决一切问题，不拿人命当事，那同党、人民的死亡当自己发展、赢利的机会（如邪恶政权所设计的疫情经济），其政权以黑箱操作著称、作案以不见天日闻名于时（如杀害香港女儿陈彦霖和灭门其所有相关的亲戚朋友、亲人老师同学），就象一家公司随便找家壳公司借壳上市一样，所惯用武功为杀敌一千自损好几十万（如长津湖的冰雕连）之人盾、人海战术、人民战争等，称之为天魔解体国家（组织、政党）、（大）解体国家（组织、政党）、分裂（精神病、神经病）国家（组织、政党）。元首（总统）国（主权国家）、圆桌国家、动力（民众）国家三国归一于一个国家才算是完成从而构成国家三分原则国家主权圆桌动力（支撑）三分原则美国将大量的兵力部署在印太、欧洲等，任何国家W 即便将美国首都华盛顿夷为平地，不出8 个小时（美国兵力是全球8 小时投递制），国家W 就会被美国的海外部队砍瓜切菜，也就是说，象美国这样的国家自带超强复仇功能，任何不自量力的国家进攻美国必然是自取灭亡，明朝是天子守国门，美国是回旋镖的离岸（海外）军队守国门，如同秦始皇坟墓入口的层层机关守护着两千年前的主人一样，称之为（海外、离岸）机关国家。美国将大量的兵力部署在印太、欧洲等，任何国家W 即便将美国首都华盛顿夷为平地，不出8 个小时（美国兵力是全球8 小时投递制），国家W 就会被美国的海外部队砍瓜切菜，也就是说，象美国这样的国家自带超强复仇功能，任何不自量力的国家进攻美国必然是自取灭亡，明朝是天子守国门，美国是回旋镖的离岸（海外）军队守国门，如同秦始皇坟墓入口的层层机关守护着两千年前的主人一样，称之为（海外、离岸）机关国家。共党从肉体上解决一切问题，不拿人命当事，那同党、人民的死亡当自己发展、赢利的机会（如邪恶政权所设计的疫情经济），其政权以黑箱操作著称、作案以不见天日闻名于时（如杀害香港女儿陈彦霖和灭门其所有相关的亲戚朋友、亲人老师同学），就象一家公司随便找家壳公司借壳上市一样，所惯用武功为杀敌一千自损好几十万（如长津湖的冰雕连）之人盾、人海战术、人民战争等，称之为天魔解体国家（组织、政党）、（大）解体国家（组织、政党）、分裂（精神病、神经病）国家（组织、政党）。元首（总统）国（主权国家）、圆桌国家、动力（民众）国家三国归一于一个国家才算是完成，从而构成国家三分原则、国家主权圆桌动力（支撑）三分原则。对于天魔解体的共产主义国家，人们只要将其斩首，即处置掉罪魁祸首就能解放人民而解决一切问题，如齐奥塞斯库之于罗马尼亚、戈尔巴乔夫之于前苏联、萨达姆之于伊拉克、卡扎菲之于利比亚、米洛舍维奇之于南斯拉夫，等等，从而构成天魔解体共产主义国家斩首解决（问题、一切）原则。对于象只有主权的主权国家如阿富汗，塔利班就是用「斩首」总统罕默德·阿什拉夫·加尼（Mohammad Ashraf Ghani）来「解决」问题的，从而构成主权国家「斩首」解决（问题、一切）原则。对于圆桌主权国家，如法国、英国等，任何「斩首」、灭国等都很难以至不能一次性解决问题，抵抗力量无法根除而继续存在并蔓延，如希特勒将法国灭亡了，戴高乐继续在英国指挥抵抗力量忙得不亦乐乎，从而构成圆桌国家无法（一次性）「斩首（灭国）」解决（问题、一切）原则。对于动力（民众）国家，如现在的雇佣兵制的北约国家、民兵组织制度的美国，任何「斩首」、灭国等也都很难以至不能一次性解决问题，抵抗力量无法根除而继续存在并蔓延，，从而构成圆桌国家无法（一次性）「斩首（灭国）」解决（问题、一切）原则。对于动力（民众）国家，如现在的雇佣兵制的北约国家、民兵组织制度的美国，任何「斩首」、灭国等也都很难以至不能一次性解决问题，抵抗力量无法根除而继续存在并蔓延，，从而构成圆桌国家无法（一次性）「斩首（灭国）」解决（问题、一切）原则。对于（海外、离岸）机关国家，如专门在海外部署部队的美国，任何「斩首」、攻击本土、灭国等无异于自取灭亡、自找麻烦，从而构成攻击（海外、离岸）机关国家自取灭亡（自找麻烦）原则、攻击（灭国、袭击）机关国家自取灭亡（自找麻烦）原则。对于（海外、离岸）机关国家，如专门在海外部署部队的美国，任何「斩首」、攻击本土、灭国等无异于自取灭亡、自找麻烦，从而构成攻击（海外、离岸）机关国家自取灭亡（自找麻烦）原则、攻击（灭国、袭击）机关国家自取灭亡（自找麻烦）原则。原则自古红颜多薄命，共党是无分别地让上至国家元首、下至黎民百姓的所有人都以最血腥的方式杀人或被杀而无限红颜薄命，这就是共党（共产主义）无限红颜薄命（红颜多薄命）原则、共党（共产主义）自带超长自古红颜多薄命原则，依此类推至于普遍情形同理成立，古今中外真理一样通，所谓近水楼台先得月、向阳花木易为春。动物无法分清粪便，很多动物在自己窝里拉屎撒尿还住得色香味俱全。人要是到了无法分清粪便的地步就恐怖了，自己一文不值大字不认识几个（如齐奥塞斯库、赫鲁晓夫）而在价值上和大便差不多，完全符合安倍晋三的自由价值观，可是，就这种人整天想要永远伟大、光荣、正确地统治全世界而遗臭万年（依据至上绝对原则），不仅祸国殃民、用病毒屠杀全人类超过2000 万还用病毒清零政策和疫苗杀人越货，直接就直奔马粪去了，马克思的大便在此笼罩登场，完全超越了普通的粪便，连畜牲都自叹不如，从而构成共党（共产主义）马粪（马克思大便、超越粪便、马粪光）原则、共党（共产主义）超越粪便（大便）原则、共党（共产主义）畜牲不如（连狗都不如、屎上最强、毒粪便、灭绝人性、丧尽天良、大便不如、粪土当今万户猴）原则。其中，土当今万户猴是指特区阶级通过发行货币、共产共妻等对人民杀人越货、屠村屠城。二战后，日本支持共产党打败蒋介石、帮中共领导人打飞机、发展飞机、细菌病毒（就你小日本干得出）、731 活体解剖的活摘器官，现在安倍付出了生命代价；美国用技术、资金养肥了中共，如今被人用病毒毒死超过100 万人；普京整天兜底他肉匪包皮的政权，俄罗斯举举国之力掠夺中国人民的财富、土地等而穿过大半个中国去睡你：共产共妻，现在，普京成了全世界的头号战犯，综上所述三方，这就是共党（共产主义）无分别（无限、绝对）反噬原则、共党（共产主义）反噬（无分别、无限、绝对）原则。共党（共产主义）无限红颜薄命（红颜多薄命）原则、共党（共产主义）马粪（马克思大便、超越粪便、马粪光）原则、共党（共产主义）超越粪便（大便）原则和共党（共产主义）反噬（无分别、无限、绝对）原则，构成了共党（共产主义）三光（三无限红颜多薄命）原则：自己、人民、大便。金养肥了中共，如今被人用病毒毒死超过100 万人；普京整天兜底他肉匪包皮的政权，俄罗斯举举国之力掠夺中国人民的财富、土地等而穿过大半个中国去睡你：共产共妻，现在，普京成了全世界的头号战犯，综上所述三方，这就是共党（共产主义）无分别（无限、绝对）反噬原则、共党（共产主义）反噬（无分别、无限、绝对）原则。共党（共产主义）无限红颜薄命（红颜多薄命）原则、共党（共产主义）马粪（马克思大便、超越粪便、马粪光）原则、共党（共产主义）超越粪便（大便）原则和共党（共产主义）反噬（无分别、无限、绝对）原则，构成了共党（共产主义）三光（三无限红颜多薄命）原则：自己、人民、大便。金养肥了中共，如今被人用病毒毒死超过100 万人；普京整天兜底他肉匪包皮的政权，俄罗斯举举国之力掠夺中国人民的财富、土地等而穿过大半个中国去睡你：共产共妻，现在，普京成了全世界的头号战犯，综上所述三方，这就是共党（共产主义）无分别（无限、绝对）反噬原则、共党（共产主义）反噬（无分别、无限、绝对）原则。金养肥了中共，如今被人用病毒毒死超过100 万人；普京整天兜底他肉匪包皮的政权，俄罗斯举举国之力掠夺中国人民的财富、土地等而穿过大半个中国去睡你：共产共妻，现在，普京成了全世界的头号战犯，综上所述三方，这就是共党（共产主义）无分别（无限、绝对）反噬原则、共党（共产主义）反噬（无分别、无限、绝对）原则。京成了全世界的头号战犯，综上所述三方，这就是共党（共产主义）无分别（无限、绝对）反噬原则、共党（共产主义）反噬（无分别、无限、绝对）原则。共党（共产主义）无限红颜薄命（红颜多薄命）原则、共党（共产主义）马粪（马克思大便、超越粪便、马粪光）原则、共党（共产主义）超越粪便（大便）原则和共党（共产主义）反噬（无分别、无限、绝对）原则，构成了共党（共产主义）三光（三无限红颜多薄命）原则：自己、人民、大便。共党（共产主义）无限红颜薄命（红颜多薄命）原则、共党（共产主义）马粪（马克思大便、超越粪便、马粪光）原则、共党（共产主义）超越粪便（大便）原则和共党（共产主义）反噬（无分别、无限、绝对）原则，构成了共党（共产主义）三光（三无限红颜多薄命）原则：自己、人民、大便。 16．乌托邦无头原则、情报（系统）无法替代国家（政府、政权、军队）原则、好枪落在神经病（废物、罪犯）手上枪毁人亡（1）乌托邦无头原则、邪恶轴心三战（战争）三惨于阿登战役（突出部之役）原则、分裂（烽火戏诸侯、绑架）规则归零原则如果绑架不是分裂的，那么，任何目标对象W 都是主动的，也就不可能是被动的，记为结论G；然而，事实上，任何绑架的目标对象都是被动的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得绑架非分裂否定原则和绑架分裂原则、绑架规则归零（绝对归零、规则绝对归零、必败、必死）原则。如果分裂（烽火戏诸侯）不是自取灭亡的，那么，任何不灭亡的常态性事物都是正值、正向的，记为结论F；然而，分裂是正负不分的：-\|A\|=\|B\| ⇒ A=B=0，{A}∩{B}=Ø ⇒ A ∈Ø、B∈Ø 而A、B 不存在，其中A、B 为任意实数，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得分裂（烽火戏诸侯）非自取灭亡（绝对归零）否定原则和分裂（烽火戏诸侯）自取灭亡（绝对归零）原则、分裂（烽火戏诸侯）规则归零（绝对归零、规则绝对归零、必败、必死）原则。绑架规则归零（绝对归零、规则绝对归零、必败、必死）原则和分裂（烽火戏诸侯）规则归零（绝对归零、规则绝对归零、必败、必死）原则，构成分裂（烽火戏诸侯、绑架）自取灭亡（绝对归零）原则、分裂（烽火戏诸侯、绑架）规则归零（绝对归零、规则绝对归零原则、必败、必死）原则，称为天子第一剑（原则）。阿登战役（Ardennes Offensive 突出部之役）参战的盟军70 万人、德国20 万人，其一个主要特点是德国兵员一旦损失就几乎无法补充、盟军则一直在迅猛补充，同时存在着燃料必须从盟军手上夺取30~50%的缺口才能逆袭成功，这就是阿登战役（Ardennes Offensive 突出部之役）分裂原则；此时，依据分裂（烽火戏诸侯、绑架）规则归零（绝对归零、规则绝对归零、必败、必死）原则，阿登战役即属天子剑下的规则归零（绝对归零、规则绝对归零、必败、必死）之鬼，从而构成阿登战役（突出部之役）规则归零（绝对归零、规则绝对归零、必败、必死）原则、阿登战役（突出部之役）自取灭亡（绝对归零）原则、阿登战役条件分裂原则。病毒清零政策、封城政策等已经将所有五毛、粪青、官员、公务员、军人、军属、特务、间谍等全部全家捅，而且大部是天天捅、日日捅、夜夜捅，一捅江湖，大量五毛纷纷翻墙出去找真相，由此来看，中共已经是无毛的白虎、纸老虎和临终的病猫，外交部「虎虎虎」的真实写照，就等着开膛剖肚、下锅煎煮、上桌下菜、咀嚼咽下最后一口气，最后的晚餐由此结束，从而构成共党（邪恶轴心）嫌作死不够快（花样作死、病毒清零、疫苗打击彻底五毛无毛）原则、共党（邪恶轴心）白虎（纸老虎、病猫）原则、共党（邪恶轴心）三虎（虎虎虎）投胎（五体投地）原则（林彪林三虎温都尔汗脱毛去汗走火入魔直接挂了）、共党（邪恶轴心）三虎（虎虎虎）成彪原则、共党（邪恶轴心）脱毛去汗（走火入魔）原则、共党（邪恶轴心）薙毛（毛薙、砍毛、鬼剃头）原则。如果严官府出厚贼不成立，那么，苛严法律下人们的选择是不少的，人们至少既可以行善也有可能作恶多端，即是排除别无选择的，记为结论F；然而，苛严法律下的人们几乎是别无选择的，行善是无法排除无法支撑没有任何收入却无限付出之唯一选择之情形的，即是无法排除别无选择的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得严官府出厚贼不成立否定原则和严官府出厚贼（成立、分裂、绝对、绝对归零）原则，因为，象新家坡、日本实行苛刻法律制度的地方都是希望被治理得井井有条的，可真理表明这是分裂的而必然是事与愿违的。毫无信用可言的中共国实行全世界最严厉的征信制度，不配合核酸检测被刑事拘留还影响三代，让无数上海人在清零期间纷纷跳楼、被病死、母婴分离等，从而构成中共国征信刑事犯罪（苛刻制度、影响三代）原则。依据阿登战役条件分裂原则，阿登战役只是条件分裂；现在，邪恶轴心尚未进入状态，几乎全部的国家级情报机密、军事机密、病毒真相等致命信息已经被发到美国向全世界公开，包括83 集团军（勤王军）的无人机等军事机密已经全部被泄露或公开，可见主战力量已经分裂、崩溃，邪恶轴心是至少AI 技术完全不如人的条件分裂加主战力量分裂而构成双分裂；再者，依据分裂（烽火戏诸侯、绑架）规则归零（绝对归零、规则绝对归零原则、必败、必死）原则、严官府出厚贼（成立、分裂、绝对、绝对归零）原则、中共国征信刑事犯罪（苛刻制度、影响三代）原则，邪恶轴心实行不配合核酸检测即刑事犯罪、影响三代的极端征信极度而必然是严官府出厚贼之分裂的，由此而来，严官府出厚贼之分裂、主战力量分裂、技术不如人分裂，邪恶轴心对全人类的战争必然是规则归零（绝对归零、规则绝对归零原则、必败、必死）的，即乌托邦必然是无头的，从而构成乌托邦无头原则、邪恶轴心三战（战争）三惨于阿登战役（Ardennes Offensive 突出部之役）原则、邪恶轴心三战三败（三灭）原则、邪恶轴心三战（战争）规则归零（绝对归零、规则绝对归零原则、必败、必死）原则。术不如人分裂，邪恶轴心对全人类的战争必然是规则归零（绝对归零、规则绝对归零原则、必败、必死）的，即乌托邦必然是无头的，从而构成乌托邦无头原则、邪恶轴心三战（战争）三惨于阿登战役（Ardennes Offensive 突出部之役）原则、邪恶轴心三战三败（三灭）原则、邪恶轴心三战（战争）规则归零（绝对归零、规则绝对归零原则、必败、必死）原则。（2）共党快感政变（床上政变、三代政变、三陪政变）规则归零原则在和平时期，邪恶政权实行绝对的强制管制再拿着人民的外汇血汗钱去各国整人家的黑材料，管生管死管生活，就是对准各国元首、政要、有影响的人进行病毒下的肛拭子服务即人体生物武器，然后用黑材料进行分化瓦解、威胁控制、各种斩首和「斩首」。犹太人几千年来在欧洲用金钱、联姻等方式控制国王、权贵的方法进行高利贷；现在，共党子继父业，用全自动的生理工具、下一代子女（工具红二三代）血缘、黑材料等绑架各国元首、总统、总理、首相、官员、门阀等，肉体和金钱都是中国人民和其他各国人民的，所谓借花杀佛、借花绑佛，进行各种分化瓦解、威胁控制、各种斩首和「斩首」，由此达成各种居心叵测的控制地球的目的，以人为蛊而种蛊、下蛊一条龙、一气呵成，名副其实是老娘夹死一条街，这是乔治奥威尔(George Orwell, Eric Arthur Blair)的《动物庄园（Animal Farm）》、《1984》的现实版、末日版全球无限《动物生理制片场（Animal FuXk）》、世界无限《1984》而绝对统一，智商等于猪的拿破仑（Napoleon）满世界跑控制台，《动物庄园（Animal Farm）》、《1984》就是犯罪指南和作业指导书，依据分裂（绑架、烽火戏诸侯）自取灭亡（绝对归零、规则归零）原则，无限《动物农庄（Animal FuXk）》、《1984》就是一场无限犹太化的无限埋雷政变、雷人政变，军事夺权前的床上夺权、下三路夺权、血缘夺权、红色夺权，从而构成共党快感政变（床上政变、三代政变、雷人政变）床上夺权（下三路夺权、血缘夺权、红色夺权）原则。 1953-1959 年间的美国国务卿约翰·福斯特·杜勒斯（John Foster Dulles）提出了和平演变（peaceful evolution）解放那些被「活摘大脑」和被奴役的人；接着，俄罗斯普京、白俄罗斯等纷纷发动和平政变；然后，中共对德国、英国、法国等进行床上政变、快感政变，不仅没有痛苦、不适还很快了，用一个混血三代者直插澳大利亚等心脏如澳大利亚外交部长 Penny Wong(黄英贤)、用郭亚丽等生三胎直控制英国首相候选人杰里米·亨特（Jeremy Hunt），其中三代包括丈母娘、老婆、孩子等三陪，法国外交部、美国、加拿大等更是直接干三代，完全突破了代沟、年龄、国度和地理限制而为无限纵深的，称之为共党床上（快感、三代）「斩首」（快感政变、床上政变、三代政变、三陪政变、雷人政变），这就是共党快感政变（床上政变、三代政变、雷人政变）三倍和平演变原则、共党快感政变（床上政变、三代政变、三陪政变、雷人政变）超越代沟（年龄、国度、地理限制、因果即人为撮合送货上门）原则、共党快感政变（床上政变、三代政变、三陪政变）雷人（雷人王、排除万难）原则、共党快感政变（床上政变、三代政变、三陪政变、雷人政变）三代（干三代、绑架三代、无限纵深、绝对犹太化、犹太3000 年套路）原则、共党快感政变（床上政变、三代政变、三陪政变、雷人政变）无限纵深（三代纵深）原则、共党床上（快感、三代）「斩首」三倍和平演变原则、共党床上（快感、三代）「斩首」超越代沟（年龄、国度、地理限制、因果即人为撮合送货上门）原则、共党床上（快感、三代）「斩首」人（雷人王、排除万难）原则、共党床上（快感、三代）「斩首」无限纵深（三代纵深）原则。由此可见，邪恶政权是绝对邪恶的太君犹太。被「斩首（精神洗脑、控制）」而奴役、被迫奴役、（获利性）自愿被奴役，从而构成三奴原则、奴役三分原则。人为撮合送货上门）原则、共党床上（快感、三代）「斩首」人（雷人王、排除万难）原则、共党床上（快感、三代）「斩首」无限纵深（三代纵深）原则。由此可见，邪恶政权是绝对邪恶的太君犹太。被「斩首（精神洗脑、控制）」而奴役、被迫奴役、（获利性）自愿被奴役，从而构成三奴原则、奴役三分原则。依据分裂（烽火戏诸侯、绑架）规则归零（绝对归零、规则绝对归零原则、必败、必死）原则、共党三陪政变无限纵深（三代纵深）原则，共党三陪政变必被规则归零（绝对归零、规则绝对归零原则、必败、必死）的，从而构成共党快感政变（床上政变、三代政变、三陪政变）规则归零（绝对归零、规则绝对归零原则、必败、必死）原则、共党床上（快感、三代）「斩首」规则归零（绝对归零、规则绝对归零原则、必败、必死）原则。（2）三枪泛滥成灾杀害耶稣的枪叫朗吉努斯（Longinus）之枪，杀害安倍的枪叫神经病之枪，用国家力量杀人的枪叫万枪之枪。依据至上绝对原则、同一律，国家主权是至上的，握有主权的国家元首、总统等的万枪依据至上绝对原则、同一律，国家主权是至上的，握有主权的国家元首、总统等的万枪之枪必然是至上而为绝对的，从而构成万枪之枪绝对（无限）原则。之枪必然是至上而为绝对的，从而构成万枪之枪绝对（无限）原则。除了北方匈奴民族外，全世界其他民族的人民都都没问题，任何国家、地区的人民都是之枪必然是至上而为绝对的，从而构成万枪之枪绝对（无限）原则。除了北方匈奴民族外，全世界其他民族的人民都都没问题，任何国家、地区的人民都是无分别的而必然是中性的，就象枪支是中性的一样，好人拿它来保家卫国，坏人拿它来杀害安倍，从而人民（枪支）中性（无分别）原则、人民（枪支）没问题原则。如俄罗斯面向北约发起收回1997 年前苏联地盘的入侵乌克兰之战，邪恶政权为颠覆二战后以美国为首的雅尔塔秩序面向世界投毒，XP 军事同盟挑战全世界，等等，这种国际层面上团伙性、战狼性的枪称为万枪之王、神枪。如俄罗斯面向北约发起收回1997 年前苏联地盘的入侵乌克兰之战，邪恶政权为颠覆二战后以美国为首的雅尔塔秩序面向世界投毒，XP 军事同盟挑战全世界，等等，这种国际层面上团伙性、战狼性的枪称为万枪之王、神枪。社会上和军队中的枪支、国家层面的万枪之枪、国际层面上的万枪之王，称为三枪，构成了当今世界的三大枪支问题和今天的灭世之末日危机，随之构成三枪乱世原则。全人类面临的不灭世而首当其冲的问题就是这三枪泛滥成灾的问题。社会上和军队中的枪支、国家层面的万枪之枪、国际层面上的万枪之王，称为三枪，构成了当今世界的三大枪支问题和今天的灭世之末日危机，随之构成三枪乱世原则。全人类面临的不灭世而首当其冲的问题就是这三枪泛滥成灾的问题。俄乌战争、邪恶轴心在东亚的战争、南美洲的擦枪军事演习以至三战，统称为雷人战争。（3）情报（系统）无法替代国家（政府、政权、军队）原则、情报（系统）厉害（胜利、无以匹敌、无敌）不代表国家（政府、政权、军队）厉害（胜利、无以匹敌、无敌）原则、好枪落在神经病（废物、罪犯）手上枪毁人亡、好马配好鞍（成功）原则情报工作是面向对象锁定目标，进而不断地投入力量、资源和技术，直到达成目的，依据至上绝对原则，国家情报工作一般是基于主权的而如同一根打气筒可以将气压升高到绝对之无限大，因此，依据国家力量防不住原则，其必然是令任何人、方都防不胜防、防不住的，从而构成情报（工作）打气筒（聚焦、收敛、获得性）原则。战场上的战争是面向对象无分别的，从而构成战争面向对象（发散、无分别）原则。国家（主权、政权、政府）是情报系统的渊源、电源和支撑，从而构成国家（主权、政权政府）先于情报（系统工作）原则（3）情报（系统）无法替代国家（政府、政权、军队）原则、情报（系统）厉害（胜利、无以匹敌、无敌）不代表国家（政府、政权、军队）厉害（胜利、无以匹敌、无敌）原则、好枪落在神经病（废物、罪犯）手上枪毁人亡、好马配好鞍（成功）原则情报工作是面向对象锁定目标，进而不断地投入力量、资源和技术，直到达成目的，依据至上绝对原则，国家情报工作一般是基于主权的而如同一根打气筒可以将气压升高到绝对之无限大，因此，依据国家力量防不住原则，其必然是令任何人、方都防不胜防、防不住的，（3）情报（系统）无法替代国家（政府、政权、军队）原则、情报（系统）厉害（胜利、无以匹敌、无敌）不代表国家（政府、政权、军队）厉害（胜利、无以匹敌、无敌）利、无以匹敌、无敌）不代表国家（政府、政权、军队）厉害（胜利、无以匹敌、无敌）原则、好枪落在神经病（废物、罪犯）手上枪毁人亡、好马配好鞍（成功）原则情报工作是面向对象锁定目标，进而不断地投入力量、资源和技术，直到达成目的，依据至上绝对原则国家情报工作一般是基于主权的而如同一根打气筒可以将气压升高到绝对利、无以匹敌、无敌）不代表国家（政府、政权、军队）厉害（胜利、无以匹敌、无敌）原则、好枪落在神经病（废物、罪犯）手上枪毁人亡、好马配好鞍（成功）原则情报工作是面向对象锁定目标，进而不断地投入力量、资源和技术，直到达成目的，依据至上绝对原则，国家情报工作一般是基于主权的而如同一根打气筒可以将气压升高到绝对之无限大，因此，依据国家力量防不住原则，其必然是令任何人、方都防不胜防、防不住的，从而构成情报（工作）打气筒（聚焦、收敛、获得性）原则。战场上的战争是面向对象无分别的，从而构成战争面向对象（发散、无分别）原则。国家（主权、政权、政府）是情报系统的渊源、电源和支撑，从而构成国家（主权、政权、政府）先于情报（系统、工作）原则。如果情报系统W 能替代国家军队、政府、政权，或者情报系统的厉害、胜利就代表国家、军队U 一定赢，那么，依据情报（工作）打气筒（聚焦）原则、同一律，军队U 必然是聚焦、收敛的，记为结论F；然而，依据战争面向对象（发散、无分别）原则，与战争保持同一的军队U 必然是面向对象、发散的，与结论F 是完全背道而驰的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得情报（系统）替代军队否定原则和情报（系统）无法（不）替代军队原则；进而，依据分裂（烽火戏诸侯、绑架）规则归零（绝对归零、规则绝对归零原则、必败、必死）原则，情报（系统）替代军队必然是规则归零的，从而构成情报（系统）替代军队规则归零（绝对归零、规则绝对归零原则、必败、必死、灭亡、同归于尽）原则、情报（系统）厉害（胜利、无以匹敌、无敌）军队厉害（胜利、无以匹敌）否定原则和情报（系统）厉害（胜利、无以匹敌、无敌）不代表军队厉害（胜利、无以匹敌、无敌）原则，此时，与情报系统、军队保持同一的国家、政府、政权必然随之被规则归零，从而构成情报利、无以匹敌、无敌）不代表国家（政府、政权、军队）厉害（胜利、无以匹敌、无敌）原则、好枪落在神经病（废物、罪犯）手上枪毁人亡、好马配好鞍（成功）原则情报工作是面向对象锁定目标，进而不断地投入力量、资源和技术，直到达成目的，依据至上绝对原则，国家情报工作一般是基于主权的而如同一根打气筒可以将气压升高到绝对之无限大，因此，依据国家力量防不住原则，其必然是令任何人、方都防不胜防、防不住的，从而构成情报（工作）打气筒（聚焦、收敛、获得性）原则。原则、好枪落在神经病（废物、罪犯）手上枪毁人亡、好马配好鞍（成功）原则情报工作是面向对象锁定目标，进而不断地投入力量、资源和技术，直到达成目的，依据至上绝对原则，国家情报工作一般是基于主权的而如同一根打气筒可以将气压升高到绝对之无限大，因此，依据国家力量防不住原则，其必然是令任何人、方都防不胜防、防不住的，从而构成情报（工作）打气筒（聚焦、收敛、获得性）原则。战场上的战争是面向对象无分别的，从而构成战争面向对象（发散、无分别）原则。国家（主权、政权、政府）是情报系统的渊源、电源和支撑，从而构成国家（主权、政权、政府）先于情报（系统、工作）原则。如果情报系统W 能替代国家军队、政府、政权，或者情报系统的厉害、胜利就代表国家、军队U 一定赢，那么，依据情报（工作）打气筒（聚焦）原则、同一律，军队U 必然是聚焦、收敛的，记为结论F；然而，依据战争面向对象（发散、无分别）原则，与战争保持同一的军队U 必然是面向对象、发散的，与结论F 是完全背道而驰的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得情报（系统）替代军队否定原则和情报（系统）无法（不）替代军队原则；进而，依据分裂（烽火戏诸侯、绑架）规则归零（绝对归零、规则绝对归零原则、必败、必死）原则，情报（系统）替代军队必然是规则归零的，从而构成情报（系统）替代军队规则归零（绝对归零、规则绝对归零原则、必败、必死、灭亡、同归于尽）原则、情报（系统）厉害（胜利、无以匹敌、无敌）军队厉害（胜利、无以匹敌）否定原则和情报（系统）厉害（胜利、无以匹敌、无敌）不代表军队厉害（胜利、无以匹敌、无敌）原则，此时，与情报系统、军队保持同一的国家、政府、政权必然随之被规则归零，从而构成情报（系统）替代军队国家（政府、政权）灭亡（规则归零、绝对归零、规则绝对归零原则、必败、必死、同归于尽）原则。败、必死、同归于尽）原则。如果情报系统W 能替代国家、政府、政权U，或者情报系统的厉害、胜利就代表国家、、政府、政权U 一定赢，那么，情报系统W 必然是先于国家、政府、政权U 的，记为结论F；然而，依据国家（主权、政权、政府）先于情报（系统、工作）原则，国家（主权、政权、政府）是先于情报（系统、工作）的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得情报（系统）替代国家（政府、政权）否定原则和情报（系统）无法（不）替代国家（政府、政权）原则、情报（系统）替代国家（政府、政权）灭亡（规则归零、绝对归零、规则绝对归零原则、必败、必死、同归于尽）原则、情报（系统）厉害（胜利、无以匹敌、无敌）国家（政府、政权）厉害（胜利、无以匹敌）否定原则和情报（系统）厉害（胜利、无以匹敌、无敌）不代表国家（政府、政权）厉害（胜利、无以匹敌、无敌）原则。如李克农象戴笠一样是把好枪W 甚至天下无敌，但却落在了一个神经病、废物、罪犯 U 手上，此时，如果单凭好枪W 就能征服世界、全球治理，那么，好枪W 是先于神经病（废物、罪犯）U 的，枪是指挥党的，记为结论F；然而，好枪W 落在了一个神经病、废物、罪犯U 手上，其意味着神经病、废物、罪犯U 是决定着枪W 的，即神经病（废物、罪犯） U 是先于好枪W 的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得好枪落在神经病（废物、罪犯）手上征服世界（全球治理、有好果子吃）否定原则、好枪一定成功否定原则和好枪落在神经病（废物、罪犯）手上无法（不可能）征服世界（全球治理、有好果子吃）原则、好枪落在神经病（废物、罪犯）手上枪毁人亡（规则归零、绝对归零、规则绝对归零、必败、必死、同归于尽）原则、（单凭）好枪无法（不可能、不一定）成功原则、好马配好鞍（成功）原则。 17．国家元首（总统、君主）禁枪原则（1）心脏天外天原则、圣心透天原则任何人脑死亡或者其他任何器官衰竭，只要心脏还在跳动，就并未真正死绝，植物人都还有苏醒的一天；可是，只要心脏不在跳动，唯有安装叶克膜人工心肺才有可能延续生命，从而构成心脏先于生命原则、心脏第一生命力原则。只要A、B 两个人的HLA 配型是适合的，A 的心脏Q 移植给了B 且成功，这是很普遍的医疗案例，此时，A 的心脏Q 跨越即超越了两个之间的DNA 所代表的质空间P、染色体只要A、B 两个人的HLA 配型是适合的，A 的心脏Q 移植给了B 且成功，这是很普遍的医疗案例，此时，A 的心脏Q 跨越即超越了两个之间的DNA 所代表的质空间P、染色体分裂之间的丝空间Q 和常态性的时空R，如在广西桂林活摘的器官乘坐飞机去了北京再接上去一样没问题而正常工作，也就是说，依据世宇三分原则，A 的心脏Q 实际上处于时空R、质空间P、丝空间Q、生命之外的天外天空间中，从而构成心脏天外天原则、圣心天外天原则、（心脏）圣心原则、圣心透天（玄机）原则，可见，心脏对生命的贡献有多大，依此类推至于普遍情形同理成立。分裂之间的丝空间Q 和常态性的时空R，如在广西桂林活摘的器官乘坐飞机去了北京再接上去一样没问题而正常工作，也就是说，依据世宇三分原则，A 的心脏Q 实际上处于时空R、质空间P、丝空间Q、生命之外的天外天空间中，从而构成心脏天外天原则、圣心天外天原则、（心脏）圣心原则、圣心透天（玄机）原则，可见，心脏对生命的贡献有多大，依此类推至于普遍情形同理成立。（3）国家元首（总统、君主）禁枪原则人民、枪支、核武器、资本本身都没问题，那么，今天全世界的会变成这样，就是枪支、核武器、人民和资本都落到了神经病和恶魔手上，于是，邪恶政权用病毒杀害全世界超过 2000 万人、乌克兰战争已经令全国到处一片废墟、安倍晋三殒命已经表明，世界已经到了自杀性核武战争解决一起的末日时刻。国家元首握有至上而绝对归一的主权，可以源源不断地使用国家力量而让他人不断用枪支发射出子弹屠杀人民和进行战争毁灭世界，此时，为了防止神经病、变态、邪恶获得国家力量的万枪之枪和亲自拿着冲锋枪扫射无辜民众的枪支，全世界在国家元首和政府层面上禁枪已经别无选择并刻不容缓，否则，依据万枪之枪绝对（无限）原则，枪支尤其是国家力量杀人的万枪之枪一旦落入有问题的国家元首、政府官员手上，后果绝对不堪设想，就象现在俄罗斯的万枪之枪落在普京他兄弟手上，中共国的金钱落到普京手上，祸国殃民、毁灭世界、炸毁地球绝对不是虚构而是现实，由此可见，其唯一之解是全世界任何国家都必须无条件禁止神经病、罪犯、邪恶、说谎者等当权，这就是国家元首（总统、君主）禁枪原则。绝对而无限的国家力量的万枪之枪一旦落入神经病、罪犯、邪恶、说谎者等手上，人民、绝对而无限的国家力量的万枪之枪一旦落入神经病、罪犯、邪恶、说谎者等手上，人民、国家、民族以至全世界的后果绝对不堪设想，就象今天世界末日降临一样。（4）刺杀安倍晋自杀（性）核武战争绝对原则（4）刺杀安倍晋自杀（性）核武战争绝对原则（4）刺杀安倍晋自杀（性）核武战争绝对原则决定着万民以至全人类生死的万枪之枪落在一个杀人犯手上，祸越闯越大，其对无辜者释放病毒毒死全世界2000 多万人，接着安倍就倒在地板上，等等，邪恶轴心把全世界都按在地板上摩擦生热，从民众到国家元首、政治世家都难逃其毒手，二哈拆家、二毛拆世界，不可同日而语，杀人犯不伏法而在光天化日之下继续无限杀人，与法律禁止犯罪完全相违背，该死的不死、不该死的却大量死了。俄罗斯是天赋神权四处杀人掠夺，基于不存在的权力而扭曲性地四处无限杀人，这是典型的电动力学模式，是分布消耗型，不思自我表现进取发展只想不劳而获白吃白拿，没有自己的动力来源和基础，产生电动势的内电路不存在，从而构成俄罗斯电动力学（分布性消耗型、无内电路）原则。跨国有组织犯罪集团是用无限杀人、毁灭世界、炸毁地球等自己给自己赋权，就象拿破仑给自己加冕一样，如同发电机，属于摧毁一切的反物质的内电路结型集成窝里斗，其不存在着任何外电路的解决出口，生儿子没屁眼的典型，从而构成共党发电机（结型积累、无外电路）原则、共党无限反物质（枪毁人亡）原则、共党草船借箭绝对原则、共党（共产主义）无限草船借箭发电原则、共党（共产主义）用仇恨发电（用杀戮发电、用死亡发电）原则。共党就象诸葛亮的无限草船借箭系统，人民就是那条船万箭穿心，射箭者是自编自演的共党及其杀手马仔本身、被分化瓦解的群众中的另一派、或如朝鲜战争中的美国军队。日本会为了战争等目的不惜一切代价尤其是切腹自杀，以无限牺牲自我去实现面向对象的对外部的强权建立，如同雷电的等离子云牺牲自我击穿外部界限而产生结型雷电（电闪雷鸣），其解决方案是内电路无限对外扩张之雷击，从而构成日本无限内电路（光速）击穿（无限结型外移放电、无限对外扩张）原则。由此可见，雷电就是一种电源的内电路电极光速无限外移扩张的击穿分布式极限放电，随之构成雷电内电路电极光速无限外移击穿扩张（击穿分布式极限放电、电极分布式光速外移）原则。跨国有组织犯罪集团是用无限杀人、毁灭世界、炸毁地球等自己给自己赋权，就象拿破仑给自己加冕一样，如同发电机，属于摧毁一切的反物质的内电路结型集成窝里斗，其不存在着任何外电路的解决出口，生儿子没屁眼的典型，从而构成共党发电机（结型积累、无外电路）原则、共党无限反物质（枪毁人亡）原则、共党草船借箭绝对原则、共党（共产主义）无限草船借箭发电原则、共党（共产主义）用仇恨发电（用杀戮发电、用死亡发电）原则。共党就象诸葛亮的无限草船借箭系统，人民就是那条船万箭穿心，射箭者是自编自演的共党及其杀手马仔本身、被分化瓦解的群众中的另一派、或如朝鲜战争中的美国军队。日本会为了战争等目的不惜一切代价尤其是切腹自杀，以无限牺牲自我去实现面向对象的对外部的强权建立，如同雷电的等离子云牺牲自我击穿外部界限而产生结型雷电（电闪雷鸣），其解决方案是内电路无限对外扩张之雷击，从而构成日本无限内电路（光速）击穿（无限结型外移放电、无限对外扩张）原则。由此可见，雷电就是一种电源的内电路电极光速无限外移扩张的击穿分布式极限放电，随之构成雷电内电路电极光速无限外移击穿扩张（击穿分布式极限放电、电极分布式光速外移）原则。二战后期，日本用神风特攻队击沉美国包括航空母舰在内的400 多艘军舰（日本方面说击沉73 艘、击伤370 多艘）牺牲两千多架飞机和两千多位飞行员，这就是日本军国主义自杀性袭击传统原则、日本切腹传统原则、日本为国家利益牺牲一切（不惜一切代价、最后不惜一切代价）原则、日本雷霆（雷电、击穿分布式）最终解决原则、日本（大和民族）雷电（雷霆）原则、日本（大和民族）雷电（雷霆）绝对原则，跟今天的本·拉登基地组织是一样的，最后葬送了大日本帝国和部分日本领土，这种基于失败阶段的全民为国群体性玉碎的自杀性行为是不可逆转的，依据不可逆转绝对原则即属绝对的，从而构成日本全民（或无分别）最终（解决）为国切腹自杀（神风特攻队、用命发电、用肉体发电、不可持续、玉碎清盘、最终计划生育）绝对原则、日本（自杀性、同归于尽）死亡国度原则，这是一种用自己的肉体和生命发电的最终雷霆行动。众所周知，罪大恶极的鬼魂下十八层地狱都无法用自杀了终结自己个他人，日本则全民无分别用自杀式终结自己、终结自己同时终结他人作最终了断，奉行最终计划生育解决，至少是十九层地狱，从而构成日本（民族）十九层地狱原则、日本（民族）至少十九层地狱原则。德国纳粹的最终解决是只杀害犹太人而不杀害自己，只是用战争让他人无分别苦难均粘、用集中营让犹太人无分别苦难均粘，等于是十八层地狱的连锁店经营者，从而构成德国（纳粹）十八层地狱原则。粹）十八层地狱原则。可见，日本从一战以至二战的对外扩张战争归根到底为雷电（雷霆）战，比肩德国希特勒的闪电战，而日本就是一个一言不合即雷霆电击他国的雷电国、雷人王，与可持续、电极自守的太阳膏药没什么关系，根本不符合太阳天照精神而是绝对的精神病崩溃之自杀性行为，随之构成日本二战雷电战（雷霆电击、雷电国）原则。显然，日本国必须治愈这种最终解决的自杀性精神病崩溃，才能算是正常国家或真正的脱亚入欧。也就是说，德国和日本一样都具有最终解决的根本特性，德国是以杀害犹太人为己任最终解决，日本是用与对方同归于尽的最终解决，都有问题而必须立刻解决。日本的政治是门阀轮流坐庄的圆桌体系，形式上和美国差不多，依据同一律、至上绝对原则，坐庄的政治门阀与日本的国家主权具有同一性而必然是至上、绝对的，有机会坐庄的政治门阀在圆桌体系中也具有仅次于坐庄门阀的地位，从而构成日本政治门阀圆桌原则、日本政治坐庄门阀主权（至上、绝对）原则。依据日本政治门阀圆桌原则、日本政治坐庄门阀主权（至上、绝对）原则，安倍晋三是日本门阀政治食物链的顶流，也是日本十多年来真正的政策制定者和定海神针，从而构成安倍晋三主权（至上、绝对）原则。倍晋主权（至上、绝对）原则如果代表着日本国家主权尊严的前首相安倍晋三的死不会招致日本发起三战的自杀性战争和对所有参与杀害安倍晋三者的杀身之祸和株连十族，那么，日本的切腹、神风特攻队自杀等传统性玉碎W 就不是绝对的，记为结论F；然而，依据日本（大和民族）雷电（雷霆）绝对原则、日本军国主义自杀性袭击传统原则、日本切腹传统原则、传统常态绝对原则，日本的切腹、神风特攻队自杀等传统性玉碎W 是绝对的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得刺杀安倍晋三不招致自杀性战争（自杀性三战、参与者株连九族、杀身之祸）否定原则和刺杀安倍晋三招致自杀性（核武）战争（自杀性三战、参与者株连九族、杀身之祸）绝对原则、刺杀安倍晋自杀（性）核武战争绝对原则、三战天绝地灭透骨穿心针原则。日本军队、精英在传统上会为了中性的民族生存利益而对外扩张和失败时切腹；现在，依据刺杀安倍晋自杀（性）核武战争绝对原则，俄罗斯基于北方四岛对日本的终极性威胁和共党的渗透，包括安倍晋三遇刺，全人类面对的只能终极的自杀性核武战争。为了不得已解除俄罗斯基于北方四岛的终极性威胁，日本发动先发制人的自杀性核武战争绝对毫无问题。安倍晋太郎在中共国文革后救了中共一命，超过20 亿的中国人的生命因此而命丧邪恶政权之手。今天，任何人没想到的是，日本官员中唯一一个也是第一个承认南京大屠杀的安倍晋三竟然死于共党手上，日本的修宪和因此而回归正常国家之路彻底打开，全世界彻底解决播放病毒杀害全人类超2000 万人的共党和悍然将乌克兰化为到处废墟的俄罗斯民族问题正式进入倒计时，全人类多少人会命丧本次三战抑或是末日大灭绝一铺清地而不只是清袋，（5）安倍（门阀）不站街原则、安倍晋三六层文明（政治爱因斯坦、文明第六支柱）则（5）安倍（门阀）不站街原则、安倍晋三六层文明（政治爱因斯坦、文明第六支柱）原则家族门阀政治体系是延续性的、排他的，从而构成家族门阀体系排他（延续、一脉相承）原则、门阀（家族）排他（延续、一脉相承）原则。街头政治是走平民线路的、无分别的，从而构成街头政治无分别（圆桌、平民路线）原则。象肯尼迪、林肯、甘地等，这些人一旦成为总统、首相等即成为政治明星，随之成为国内外反对派势力唯一确定的众矢之的，依据收敛绝对原则、聚集绝对原则、唯一确定绝对原则，从而构成公众人物（总统、首相、总理）众矢之的（绝对）原则。现代社会有社交媒体、新闻媒体、公关公司等，与人民是无缝对接的，依据无分别（遍及）绝对原则，社交媒体、新闻媒体、公关公司等即属绝对的，从而构成社交媒体（新闻媒体、公关公司）遍及（无分别）绝对原则。日本作为传统的家族门阀政治体系，如果安倍有必要去站街W，那么，街头政治无分别（圆桌、平民路线）原则，门阀站街W 即属无分别的、不排他的，记为结论F；然而，谁能预料？这一切归根结底都是国家层面的万枪之枪和国际层面的万枪之王落入神经病和杀人惯犯手上，所谓恶魔不死国无宁日，害群之枪不禁球无宁地。谁能预料？这一切归根结底都是国家层面的万枪之枪和国际层面的万枪之王落入神经病和杀人惯犯手上，所谓恶魔不死国无宁日，害群之枪不禁球无宁地。本来，邪恶政权放病毒残杀全世界超2000 万人之后，世界上是电动机的土匪、发电机的肉匪和轮流坐庄的海盗之间的高手对决；现在，一家养活中共的安倍晋三之死，雷霆万钧的王匪后来居上，自杀性核武战争随之不可避免，世界变成土匪、肉匪、海盗、王匪之间的四角大战2：2 阵营，海盗曾经丢过原子弹、土匪屠杀了满洲国上亿人、肉匪屠杀中国超20 亿人、王匪干过南京大屠杀，四大灭绝重出江湖，人类不四脚朝天、人仰马翻炸毁地球恐怕是不可能了，全人类不得不执行天门算法而四舍五入应该是唯一之解。全球绝大多数国家降半旗哀悼，表明安倍晋三的以自由价值观为导向的外交、同盟深得人心，英国女王、美国总统、国务卿布林肯、印度莫迪、约翰逊等等纷纷吊唁和几乎所有国家都降半旗致哀，可见安倍晋三的价值非同小可。我们反观回去，此前全世界的领导人过世都没有过高于安倍的殊荣，象列宁、斯大林、胡志名等浪得虚名的魔头也无法与安倍晋三相提并论，显然，人民的眼睛是雪亮的，各国元首的眼睛更是闪闪发光的，那些企图滥竽充数的南郭先生们死上一百次也不可能与安倍相比，要不，请罪魁祸首们立刻死死看？一个瘪三即使统治世界也还是瘪三，安倍即使不统治世界也是百年来的一大伟人，名副其实是生的光荣、死的伟大。悲剧就是把有价值的东西毁给人看，安倍晋三的悲剧表明人类社会处于无辜死刑的人间地狱中，基于规则的雅尔塔体系已经处于土崩瓦解的边沿，联合国需要改革、世界需要改革、日本需要改革，否则，安倍晋三都会被如此残杀，普通人呢？阳光下的种族灭绝、屠村屠城呢？本来，邪恶政权放病毒残杀全世界超2000 万人之后，世界上是电动机的土匪、发电机的肉匪和轮流坐庄的海盗之间的高手对决；现在，一家养活中共的安倍晋三之死，雷霆万钧的王匪后来居上，自杀性核武战争随之不可避免，世界变成土匪、肉匪、海盗、王匪之间的四角大战2：2 阵营，海盗曾经丢过原子弹、土匪屠杀了满洲国上亿人、肉匪屠杀中国超20 亿人、王匪干过南京大屠杀，四大灭绝重出江湖，人类不四脚朝天、人仰马翻炸毁地球恐怕是不可能了，全人类不得不执行天门算法而四舍五入应该是唯一之解。荣、死的伟大。悲剧就是把有价值的东西毁给人看，安倍晋三的悲剧表明人类社会处于无辜死刑的人间地狱中，基于规则的雅尔塔体系已经处于土崩瓦解的边沿，联合国需要改革、世界需要改革、日本需要改革，否则，安倍晋三都会被如此残杀，普通人呢？阳光下的种族灭绝、屠村屠城呢？（5）安倍（门阀）不站街原则、安倍晋三六层文明（政治爱因斯坦、文明第六支柱）原则 18．世宇三分、生命三分原则、九鼎33 因果、因果链（透天乾纲）35721n（33、773333331、 35721、C33）原则（1）质空间（信息空间、法线空间、惯性空间）先于时空原则原则家族门阀政治体系是延续性的、排他的，从而构成家族门阀体系排他（延续、一脉相承原则、门阀（家族）排他（延续、一脉相承）原则。街头政治是走平民线路的、无分别的，从而构成街头政治无分别（圆桌、平民路线）原街头政治是走平民线路的、无分别的，从而构成街头政治无分别（圆桌、平民路线）原则。象肯肯等等政则。象肯尼迪、林肯、甘地等，这些人一旦成为总统、首相等即成为政治明星，随之成为国象肯尼迪、林肯、甘地等，这些人一旦成为总统、首相等即成为政治明星，随之成为国内外反对派势力唯一确定的众矢之的，依据收敛绝对原则、聚集绝对原则、唯一确定绝对原内外反对派势力唯一确定的众矢之的，依据收敛绝对原则、聚集绝对原则、唯一确定绝对原则，从而构成公众人物（总统、首相、总理）众矢之的（绝对）原则。现代社会有社交媒体、新闻媒体、公关公司等，与人民是无缝对接的，依据无分别（遍现代社会有社交媒体新媒体公关公等民无对接依据无分别及）绝对原则，社交媒体、新闻媒体、公关公司等即属绝对的，从而构成社交媒体（新闻媒及）绝对原则，社交媒体、新闻媒体、公关公司等即属绝对的，从而构成社交媒体（新闻媒体、公关公司）遍及（无分别）绝对原则。日本作为传统的家族门阀政治体系，如果安倍有必要去站街W，那么，街头政治无分别（圆桌、平民路线）原则，门阀站街W 即属无分别的、不排他的，记为结论F；然而，依据、门阀（家族）排他（延续、一脉相承）原则，门阀站街W 显然是排他的，记为结论 G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得安倍（门阀）站街否定原则和安倍（门阀）不站街原则。安倍在奈良街头的演讲也就是十几个人，效果不是与民众无缝对接的，却送了不可逆转的生命，可见，日本禁止门阀站街是唯一之举，收入和产出完全不成比例，赔本的生意为什么要继续？安倍晋三的自由价值观是国家以自由价值为基础经由外交发展板级的平等W 之国际合作和同盟，并以此对抗邪恶势力；与此同时，安倍作为首个承认南京大屠杀的日本首相而位列日本头号日奸是基于不争的事实与国际平等，在北方四岛是民族英雄，在钓鱼岛上是维持会会长，综上所述两方面，安倍晋三的自由价值观是将文明第二层面的自由基因常态化并以此构建平等的国际架构，仅次于第六层次（第七因果的天子因果）和最高层次的公义之上帝正果，由此可见，安倍晋三是全人类在社会科学上第一位达到文明之平等框架的圣人，爱因斯坦是科学上第一位以广义相对论的质量等效性（动力学效应）原理达到文明之平等框架的圣人，也就是说，安倍晋三就是1915 年之后百年来社会科学的爱因斯坦，其在政治文明上超越了二战三巨头的罗斯福(Franklin Delano Roosevelt)和邱吉尔(Winston L.S. 原则 Churchill)，是人类5000 年来首个抵达人类七层文明第六层的第二人，前一人是百年前的爱因斯坦，显然，安倍晋三是百年来最靠近上帝、真理的人，从而构成安倍晋三六层文明（政治爱因斯坦、文明第六支柱）原则。安倍晋三的自由价值观（平等）和生命、爱因斯坦的广义相对论之质量等效性（动力学效应）原理，告诉人们文明七基因的代价是什么，依据生命不可再来（不可逆转）绝对原则，安倍晋三的生命即属无价的安倍晋三的生命即属无价的。其中，七层文明、文明七大基因是：①独立、②自由、③民主、④同一、⑤平等、⑥公安倍晋三的生命即属无价的。其中，七层文明、文明七大基因是：①独立、②自由、③民主、④同一、⑤平等、⑥公安倍晋三的生命即属无价的。其中，七层文明、文明七大基因是：①独立、②自由、③民主、④同一、⑤平等、⑥公平 ⑦公义（公正）安倍晋三的生命即属无价的。其中，七层文明、文明七大基因是：①独立、②自由、③民主、④同一、⑤平等、⑥公平、⑦公义（公正）。文明七因果是：①常态因果（植物因果）、相对因果（②获得性因果、③自由因果、④ 民主选择判断因果）、同步因果（⑤本征因果、⑥参数化同步性因果）、⑦天子因果而公义（公正、正义之公义）在天；承受性因果包括本征因果⑤、参数化同步性因果⑥、天子因果 ⑦。安倍晋的命即属无价的其中，七层文明、文明七大基因是：①独立、②自由、③民主、④同一、⑤平等、⑥公平、⑦公义（公正）。平、⑦公义（公正）。文明七因果是：①常态因果（植物因果）、相对因果（②获得性因果、③自由因果、④ 民主选择判断因果）、同步因果（⑤本征因果、⑥参数化同步性因果）、⑦天子因果而公义文明七因果是：①常态因果（植物因果）、相对因果（②获得性因果、③自由因果、④ 民主选择判断因果）、同步因果（⑤本征因果、⑥参数化同步性因果）、⑦天子因果而公义（公正、正义之公义）在天；承受性因果包括本征因果⑤、参数化同步性因果⑥、天子因果 ⑦。民主选择判断因果）、同步因果（⑤本征因果、⑥参数化同步性因果）、⑦天子因果而公义（公正、正义之公义）在天；承受性因果包括本征因果⑤、参数化同步性因果⑥、天子因果 ⑦。（公正、正义之公义）在天；承受性因果包括本征因果⑤、参数化同步性因果⑥、天子因果 ⑦。 18．世宇三分、生命三分原则、九鼎33 因果、因果链（透天乾纲）35721n（33、773333331、 35721、C33）原则（1）质空间（信息空间、法线空间、惯性空间）先于时空原则如果北约、美国在俄罗斯入侵乌克兰时就动手保护乌克兰，在乌克兰变成到处是废墟Y 前拯救乌克兰而投入军事力量和支出W（记为情形1），和等乌克兰变成到处是废墟后再出手相救而投入军事力量和支出W（记为情形2），此时，北约、美国方面在两种情形下投入的军事力量和支出W 都是一样的；可是，英美在战争一开始就出手和乌克兰废墟后再出手，乌克兰的结果就是到处废墟Y 和完整无缺的天壤之别和天然气，英美在开战时就出手和开战后再出手，其结果之间存在着一个时间换空间的代价，一样的原因（投入）出现一个在不同时间上结果之间的差异K，差异K 以不同的时间差G 为前提，从而时间差G（原因）到差异K（结果）之间的因果称为惯性因果、质（空间）因果、法线因果，犹如一艘航空母舰 a 航行在大海b 上，其触礁g 后将震荡冲击力传递到甲板上将其上的舰载机或人员、货物甩出去以至掉进海里的次生因果f 就是航空母舰触礁因果g 的次生因果，此时，次生因果g 和它的上一代因果f 之间存在着法线上的连续性承载h，我们称之为质空间、惯性空间。我们注意到，在一艘航空母舰W 上搭载舰载机U（里面有准备起飞的飞行员Q），W 对U 构成了一个承载性的惯性系统，航空母舰在航行中，停泊在航空母舰W 上的舰载机U 是保持相对运动为零之一致性的，二者之间在质空间上是保持连续性的；进而，当舰载机U 从航空母舰W 上起飞后，舰载机U 里面的飞行员Q 与舰载机U 又是保持一致的而必然是连续性的，由此可见，停泊在航空母舰W 上的舰载机U 里坐着飞行员Q（飞行员肚子里面有食物R），航空母舰W 对舰载机U 的惯性质因果a 和舰载机U 对飞行员Q 的惯性质因果 b 在质空间Y 中是保持连续性的，从而构成质空间的质（惯性）因果连续性定理（原则）、质（惯性）连续性定理（原则），依此类推至于n 阶质因果同理成立。一个胎儿B 在母体A 中怀孕，胎儿B 出生后承载着母亲A（父亲a）的 DNA(m1) 长大成人，母亲B（父亲b）再次将其DNA(m2)遗传给下一代C 的DNA(m3)，此时，DNA(m1) 与DNA(m2)在遗传信息空间中保持着连续性，DNA(m3)与DNA(m1)在遗传信息空间中保持着连续性，等等，依此类推至于无限下一代同理成立，这就是质空间（信息空间、法线空间、惯性空间）的质（惯性、信息）连续性定理（原则），显然，质空间（信息空间、法线空间、惯性空间）的连续性与时空上的连续性无关，但却承载着超越时空的，从而构成质本体、惯性、信息等，随之构成质空间（信息空间、法线空间、惯性空间）先于时空原则、质空间（信息空间、法线空间、惯性空间）脱离时空（与时空无关）原则、质因果（惯性因果、法线因果）先于时空因果原则、质因果（信息因果、法线因果、惯性因果）脱离时空（与时空无关）原则、质因果（信息因果、法线因果、惯性因果）脱离时空因果（与时空无关）原则，也就是说，只要线粒体撤消限制，人是可以长生不老的，人、生物在质空间中的灵魂（人魂）也是存在的，而且直达生物先祖（亚当、夏娃），随之又构成本体（人魂、质空间）灵魂原则，本体（精神、鬼魂、信息、惯性或惯性本体）存在（无限、绝对、守恒）原则、本体（人魂、祖魂、先祖）连续性定理（原则）、质因果（本体因果）连续性定理（原则），依此类推至于n 阶质因果和普遍情形同理成立。（2）（有）丝因果先于时空因果（质因果、惯性因果、法线因果）原则在人类一条精子A 和一个卵子B 的受精过程中，染色体X 和染色体Y 结合，代表着两个质空间中的本体汇合；在生殖细胞的减数分裂过程中，染色体X 和染色体Y 所在的DNA 分别发生带十字中心体的有丝分裂：男性生殖细胞的染色体发生X~Y 的有丝分裂、女性生殖细胞发生X~X 的有丝分裂，由此而来，我们称染色体X 和染色体Y 的结合空间和分裂空间为有丝空间或丝空间，染色体X 和染色体Y 的结合产生男性受精卵R1、染色体X 和染色体X 的结合产生女性受精卵R2，受精卵R1 和R2 的因果称为有丝因果或丝因果。此时，有丝分裂、与有丝分裂相关的逆向受精能无分别地在不同的男女之间发生，黑人与白人都没有任何种族界限、个体界限、时空界限，依据无分别绝对原则、凌驾绝对原则，基于染色体X~Y 的受精所处和进入的有丝空间即属于绝对的、脱离于质空间和时空空间的，从而构成（有）丝空间先于时空（质空间、惯性空间、信息空间）原则、（有）丝空间脱离时空（质空间、惯性空间、信息空间、与时空无关、与质空间无关等）原则、（有）丝因果先于时空因果（质因果、惯性因果、法线因果）原则、（有）丝因果脱离时空（质空间、惯性空间、信息空间、与时空无关、与质空间无关等）原则、（有）丝因果脱离时空因果（质因果、惯性因果、信息因果、与时空因果无关、与质因果无关等）原则，也就是说，基于 DNA 的染色体X/Y 代表着人的本体生命，而在人的本体生命之上还存在着有丝空间的本源生命，超越时空的肉体和质空间的本体，依据绝对无限原则、绝对相等原则、绝对传代原则，直达最原始的先天祖（上帝、造物主），随之构成本源（天魂）先天祖（上帝、造物主）原则、本源（天魂）存在（无限、绝对、守恒）原则、本源（天魂）连续性定理（原则）、（有）丝因果连续性定理（原则），依此类推至于n 阶（有）丝因果和普遍情形同理成立。由此而来，我们称（有）丝空间有上天、天空间、神的空间、造物主空间、本源空间。在人类一条精子A 和一个卵子B 的受精过程中，染色体X 和染色体Y 结合，代表着两个质空间中的本体汇合；在生殖细胞的减数分裂过程中，染色体X 和染色体Y 所在的DNA 分别发生带十字中心体的有丝分裂：男性生殖细胞的染色体发生X~Y 的有丝分裂、女性生殖细胞发生X~X 的有丝分裂，由此而来，我们称染色体X 和染色体Y 的结合空间和分裂空间为有丝空间或丝空间，染色体X 和染色体Y 的结合产生男性受精卵R1、染色体X 和染色体X 的结合产生女性受精卵R2，受精卵R1 和R2 的因果称为有丝因果或丝因果。此时，有丝分裂、与有丝分裂相关的逆向受精能无分别地在不同的男女之间发生，黑人与白人都没有任何种族界限、个体界限、时空界限，依据无分别绝对原则、凌驾绝对原则，基于染色体X~Y 的受精所处和进入的有丝空间即属于绝对的、脱离于质空间和时空空间的，从而构成（有）丝空间先于时空（质空间、惯性空间、信息空间）原则、（有）丝空间脱离时空（质空间、惯性空间、信息空间、与时空无关、与质空间无关等）原则、（有）丝因果先于时空因果（质因果、惯性因果、法线因果）原则、（有）丝因果脱离时空（质空间、惯性空间、信息空间、与时空无关、与质空间无关等）原则、（有）丝因果脱离时空因果（质因果、惯性因果、信息因果、与时空因果无关、与质因果无关等）原则，也就是说，基于 DNA 的染色体X/Y 代表着人的本体生命，而在人的本体生命之上还存在着有丝空间的本源生命，超越时空的肉体和质空间的本体，依据绝对无限原则、绝对相等原则、绝对传代原则，直达最原始的先天祖（上帝、造物主），随之构成本源（天魂）先天祖（上帝、造物主）原则、本源（天魂）存在（无限、绝对、守恒）原则、本源（天魂）连续性定理（原则）、（有）丝因果连续性定理（原则），依此类推至于n 阶（有）丝因果和普遍情形同理成立。由此而来，我们称（有）丝空间有上天、天空间、神的空间、造物主空间、本源空间。由此而来，我们称（有）丝空间有上天、天空间、神的空间、造物主空间、本源空间。（3）人体生命三魂（七魄）原则、生命三分原则当我们将基于DNA 的染色体X/Y 的生殖细胞加热到100 摄氏度过以上，任凭你再怎么样医术高明，染色体X、Y 都不可能再相互或与其他非加热的染色体进行受精，由此可见，人的生命中在DNA 及其X/Y 染色体层面上存在着本体生命、本源生命之下的隐性生命W，称之为动力生命（载命、载魂、地魂、阴魂、生魂），此时，任何生殖细胞只要失去等于主体唯一确定的动力生命即不可能有下一步，依据唯一确定绝对原则，其即构成动力生命（载命、载魂、地魂、阴魂、生魂）唯一确定（绝对、独立、守恒）原则。动力生命、本体生命、本源生命构成人体的三重生命，即地魂、人魂、天魂（主魂、觉魂、生魂，元神、阳神、阴神，或胎光、爽灵、幽精）之三魂，原来都是真的，依此类推，套动力班子七魄随之是真实的：尸狗、伏矢、雀阴、吞贼、非毒、除秽、臭肺，各主精神、气及心、胃、肾、肠，胆、肝、肺，三魂七魄去半便性命危，统称为人体生命的三魂七魄，从而构成（人体）生命三魂（七魄）原则、（人体）生命三分原则，依此类推至于普遍情形同理成立。动力生命、本体生命、本源生命构成人体的三重生命，即地魂、人魂、天魂（主魂、觉魂、生魂，元神、阳神、阴神，或胎光、爽灵、幽精）之三魂，原来都是真的，依此类推，套动力班子七魄随之是真实的：尸狗、伏矢、雀阴、吞贼、非毒、除秽、臭肺，各主精神、气及心、胃、肾、肠，胆、肝、肺，三魂七魄去半便性命危，统称为人体生命的三魂七魄，从而构成（人体）生命三魂（七魄）原则、（人体）生命三分原则，依此类推至于普遍情形同理成立。（4）世宇三分：汉家天子文明16.5 万年（万岁）、弥勒9LC33（九重因果33 天）、天子立法透天原则、7733 天外天因果、7733 天外天秩序、7733 天外天文明、因果链（透天乾纲）35721n（33、773333331、35721、C33）原则、35721n（33、773333331、35721、 C33）阅读框架、汉家天子文明9LC33×CC35721 天外天原则非A 否定肯定原则、非A 肯定否定原则、唯A 否定肯定原则、唯A 肯定否定原则、唯 A 否定否定原则、相反（不同）不相容原则、非A 归属肯定原则、非A 归属失败无关原则，称为七微分因果、微分七因果。七步因果称为时空因果、常态因果、时空七因果、常七态因果；质因果、（有）丝因果七步因果称为时空因果、常态因果、时空七因果、常七态因果；质因果、（有）丝因果称为集成因果、集成三因果。称为集成因果、集成三因果。时空、质空间（惯性空间）、（有）丝空间构成世宇三分原则。实力秩序、规则秩序、效力秩序上升为公正秩序（位元秩序）。从实力出发因果、规则因果、效力性因果构成元（位元、前提、条件）三因果、元（位从实力出发因果、规则因果、效力性因果构成元（位元、前提、条件）三因果、元（位元、前提、条件）因果、以元（位元、前提、条件）达到的公义三因果。波动（波矢）的拟动态分布领域、振动圆频率的动态领域、上述二者卷积后展开后的傅元、前提、条件）因果、以元（位元、前提、条件）达到的公义三因果。波动（波矢）的拟动态分布领域、振动圆频率的动态领域、上述二者卷积后展开后的傅波动（波矢）的拟动态分布领域、振动圆频率的动态领域、上述二者卷积后展开后的傅立叶级数（Fourier series）所处的频域空间，称为三规范空间。波动的波矢是力学在空间的微分导数上平衡的结果，圆频率是力学在时间的微分导数上平衡的结果，频域是非正弦矢量（如电力）以傅立叶级数（Fourier series）展开的分解平衡之结果，上述三因果称为规范三因果。三步弓因果、三线因果、天位三因果（规则因果、造化分布因果、造化安全因果）、天下共主（万王之王、万主之主）因果，构成了全球解决（治理、治愈）3331 原则、解决（治理、治愈）3331 偏置原则。时空七因果、微分七因果、集成三因果、规范三因果构成7×7×3×3 因果，称为7×7 ×3×3（7733、441、C20）因果体系（关系）、7×7×3×3（7733、441、C20）天外天（先天）因果体系（关系）、7×7×3×3（7733、441、C20）天外天（先天）因果。时空七因果、微分七因果、集成三因果、位元三因果、规范三因果、三步弓因果、三线因果、天位三因果、天下共主因果构成7×7×3×3×3×3×3×3×1 因果，称为九重33 因果（9LC33）即九鼎33 天因果、九鼎33 天数学因果、33（773333331、35721、C33）因果体系（关系）、33（773333331、35721、C33）透天因果体系（关系）、33 天（773333331、 35721、C33）透天因果、33 天因果、33 透天（天外天）因果、33 重因果（天）、33 重因果天、33 天文明、33 天（773333331、35721、C33）透天秩序（透天文明秩序）文明。其中，获得性因果即代表一重天，33 因果即代表33 重天，从而构成33 因果33 重天原则，依此类推至于九重因果九鼎（天下）原则同理成立。时空七因果、微分七因果、集成三因果、规范三因果、三步弓因果、三线因果、天位三因果、天下共主因果、公正秩序三位一体构成33（先天、天外天、773333331、35721、C33）文明体系、33（773333331、35721、C33）透天秩序（透天文明秩序）、33（773333331、 35721、C33）透天体系（关系）。时空七因果、微分七因果、集成三因果、规范三因果构成7×7×3×3 因果，称为7×7 ×3×3（7733、441、C20）因果体系（关系）、7×7×3×3（7733、441、C20）天外天（先天）因果体系（关系）、7×7×3×3（7733、441、C20）天外天（先天）因果。时空七因果、微分七因果、集成三因果、位元三因果、规范三因果、三步弓因果、三线因果、天位三因果、天下共主因果构成7×7×3×3×3×3×3×3×1 因果，称为九重33 因果（9LC33）即九鼎33 天因果、九鼎33 天数学因果、33（773333331、35721、C33）因果体系（关系）、33（773333331、35721、C33）透天因果体系（关系）、33 天（773333331、 35721、C33）透天因果、33 天因果、33 透天（天外天）因果、33 重因果（天）、33 重因果天、33 天文明、33 天（773333331、35721、C33）透天秩序（透天文明秩序）文明。其中，获得性因果即代表一重天，33 因果即代表33 重天，从而构成33 因果33 重天原则，依此类推至于九重因果九鼎（天下）原则同理成立。则，依此类推至于九重因果九鼎（天下）原则同理成立。时空七因果、微分七因果、集成三因果、规范三因果、三步弓因果、三线因果、天位三因果、天下共主因果、公正秩序三位一体构成33（先天、天外天、773333331、35721、C33）文明体系、33（773333331、35721、C33）透天秩序（透天文明秩序）、33（773333331、 35721、C33）透天体系（关系）。 7733 因果体系（关系）凌驾于世宇三分和三位一体秩序之外，即属于世外桃源、乾坤之外的先天因果和文明，称之为7733 先天（天外天）体系和先天（天外天）7733 文明，从而构成7733 文明先天（世外桃源、乾坤外先天）原则。微分七因果（Differential）、时空七因果（Normal Spacetime）、集成三因果（Integration）、位元三因果（Exponent）、规范三因果（Rules），一个密码子由碱基ACA AGT 等构成，一条因果链则由上述的五重因果基因构成：DNIER~D(7)N(7)I(3)E(3)R(3)，我们称之为事物的（五道）左函数因果链，也就是说，人世间的事物至少有1323n(n∈N)种的左翼（左函数）因果链，其中1323=7×7×3×3×3（1323、C23），从而构成因果链（透天乾纲）1323n（7 ×7×3×3×3、C23）原则。三步弓因果、三线因果、天位三因果、天下共主因果称为特解函数（四、右函数）因果、右翼（四）因果、保守（四）因果。（五道）左函数因果链和特解函数（四、右函数）因果构成因果链（透天乾纲）35721n （33、773333331、35721、C33）原则、因果链（透天乾纲）35721n（33、773333331、35721、 C33）阅读框架原则、CC35721(causation chain)因果链（乾纲）。 7733 文明先天（世外桃源、乾坤外先天）原则、因果链（透天乾纲）35721n（33、773333331、 35721、C33）阅读框架原则、33（773333331、35721、C33）透天秩序（透天文明秩序）等，称之为涵盖汉家天子立法的汉家天子天外天、透天立法、9LC33×CC35721 计算因果、9LC33 ×CC35721 文明、9LC33×CC35721 佛法、9LC33×CC35721 兵法，从而构成（汉家）天子（天外天）立法透天原则。 7733 因果体系（关系）凌驾于世宇三分和三位一体秩序之外，即属于世外桃源、乾坤之外的先天因果和文明，称之为7733 先天（天外天）体系和先天（天外天）7733 文明，从而构成7733 文明先天（世外桃源、乾坤外先天）原则。微分七因果（Differential）、时空七因果（Normal Spacetime）、集成三因果（Integration）、位元三因果（Exponent）、规范三因果（Rules），一个密码子由碱基ACA AGT 等构成，一条因果链则由上述的五重因果基因构成：DNIER~D(7)N(7)I(3)E(3)R(3)，我们称之为事物的（五道）左函数因果链，也就是说，人世间的事物至少有1323n(n∈N)种的左翼（左函数）因果链，其中1323=7×7×3×3×3（1323、C23），从而构成因果链（透天乾纲）1323n（7 ×7×3×3×3、C23）原则。三步弓因果、三线因果、天位三因果、天下共主因果称为特解函数（四、右函数）因果、右翼（四）因果、保守（四）因果。（五道）左函数因果链和特解函数（四、右函数）因果构成因果链（透天乾纲）35721n （33、773333331、35721、C33）原则、因果链（透天乾纲）35721n（33、773333331、35721、 C33）阅读框架原则、CC35721(causation chain)因果链（乾纲）。 7733 文明先天（世外桃源、乾坤外先天）原则、因果链（透天乾纲）35721n（33、773333331、 35721、C33）阅读框架原则、33（773333331、35721、C33）透天秩序（透天文明秩序）等，称之为涵盖汉家天子立法的汉家天子天外天、透天立法、9LC33×CC35721 计算因果、9LC33 ×CC35721 文明、9LC33×CC35721 佛法、9LC33×CC35721 兵法，从而构成（汉家）天子（天外天）立法透天原则。 7733 因果体系（关系）凌驾于世宇三分和三位一体秩序之外，即属于世外桃源、乾坤之外的先天因果和文明，称之为7733 先天（天外天）体系和先天（天外天）7733 文明，从而构成7733 文明先天（世外桃源、乾坤外先天）原则。微分七因果（Differential）、时空七因果（Normal Spacetime）、集成三因果（Integration）、位元三因果（Exponent）、规范三因果（Rules），一个密码子由碱基ACA AGT 等构成，一条因果链则由上述的五重因果基因构成：DNIER~D(7)N(7)I(3)E(3)R(3)，我们称之为事物的（五道）左函数因果链，也就是说，人世间的事物至少有1323n(n∈N)种的左翼（左函数）因果链，其中1323=7×7×3×3×3（1323、C23），从而构成因果链（透天乾纲）1323n（7 ×7×3×3×3、C23）原则。右翼（四）因果、保守（四）因果。（五道）左函数因果链和特解函数（四、右函数）因果构成因果链（透天乾纲）35721n （33、773333331、35721、C33）原则、因果链（透天乾纲）35721n（33、773333331、35721、 C33）阅读框架原则、CC35721(causation chain)因果链（乾纲）。 7733 文明先天（世外桃源、乾坤外先天）原则、因果链（透天乾纲）35721n（33、773333331、 35721、C33）阅读框架原则、33（773333331、35721、C33）透天秩序（透天文明秩序）等，称之为涵盖汉家天子立法的汉家天子天外天、透天立法、9LC33×CC35721 计算因果、9LC33 ×CC35721 文明、9LC33×CC35721 佛法、9LC33×CC35721 兵法，从而构成（汉家）天子（5）9LC33×CC35721 文明、9LC33×CC35721 佛法（Buddha Dharma）、9LC33 ×CC35721 立法、9LC33×CC35721 兵法、9LC33×CC35721 集成电路（IC、积体电路）时代即十万量子比特时代、安倍晋三超越日本天皇释迦牟尼证得因果，小乘佛法渡己、大乘佛法渡人，亚里斯多德（Aristotle）深入证明了矛盾律和因果律，这就是获得性因果，人类5000 年来的文明一直都基于这个唯一的获得性因果，只是九重C33 因果（9LC33 因果）中的一个本位性因果，9LC33 因果包括如下九重：时空七因果、微分七因果、集成三因果、位元三因果、规范三因果、三步弓因果、三线因果、天位三因果、天下共主因果（主权因果），其中35721 因果链（阅读框架）构成了乾纲，也就是说，一个获得性因果让人类用了5000 年，包括今天的集成块（IC）、量子力学、核武器、飞机、宇航等全部只是基于这么唯一的一个获得性因果，依此类推，九重C33 因果将人类的文明往后推进了：5000×33=165000 年、3000×33=99000 年，CC35721n(causation chain)因果链则在广度上推进到了35721 个领域，因此，汉家天子的因果立法又称为16.5 万年立法、16.5 万岁立法、汉家天子16.5 万年因果立法、汉家天子16.5 万岁因果立法、汉家天子10 万年因果立法、汉家天子10 万岁因果立法，从而构成汉家天子文明16.5 万年（万岁）原则、汉家天子文明10 万年（万岁）原则。自今日起，我将佛门的因果从释迦牟尼的获得性因果延伸到16.5 万年后的9LC33 之九重33 因果即九鼎33 天因果，所有要继续修行释迦牟尼佛教的佛门弟子都必须广修数学和熟悉本文中的33 因果33 重天原则、九重因果九鼎（天下）原则等，做好一切准备我亲自面向全世界的佛门弟子讲解9LC33（九重因果33 天）、CC35721n（CC35721 因果链阅读框架乾纲）。所有佛门弟弟务必相互转告，我就是继承释迦牟尼并且将释迦牟尼的获得性因果佛法推进到16.5 万年后的弥勒，这就是弥勒9LC33（九重因果33 天）、弥勒CC35721n（CC35721 因果链阅读框架乾纲），也是3000 年前佛祖释迦牟尼的预言。全世界迄今为止5000 年，全部的教育、数学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等全部只是处于获得性因果的单因果层面上；现在，9LC33 九层33 因果和 CC35721 因果链阅读框架乾纲将向上再提高到8 层32 因果和CC35721 因果链阅读框架的层面上，9LC33九层33因果数学和CC35721因果链阅读框数学随之建立，依此类推至于，9LC33 九层33 因果佛法（教育、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等）和 CC35721 因果链阅读框佛法（教育、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等）同理成立，这是释迦牟尼和亚里斯多德（Aristotle）的32 倍体和CC35720 倍体，人类文明向前推进到33 倍的释迦牟尼和亚里斯多德时代，也推进到CC35721 因果链阅读框的释迦牟尼和亚里斯多德领域中，称之为9LC33×CC35721 文明或透天文明、无限释迦牟尼（亚里斯多德）文明、天子（天外天、透天）文明、汉家天子文明，从而构成（汉家）天子文明9LC33×CC35721（天外天、透天）原则，人类由此进入9LC33×CC35721 文明时代，天子文明在时间上作16.5 万年9 重33 因果的扩展、在领域上作35721 倍的拓展，天地进入四海无内外文明，这就是汉家天子立法的具体形式之汉家天子9LC33×CC35721 立法（立文明）和弥勒9LC33×CC35721 立佛法。全世界迄今为止5000 年，全部的教育、数学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等全部只是处于获得性因果的单因果层面上；现在，9LC33 九层33 因果和 CC35721 因果链阅读框架乾纲将向上再提高到8 层32 因果和CC35721 因果链阅读框架的层面上，9LC33九层33因果数学和CC35721因果链阅读框数学随之建立，依此类推至于，9LC33 九层33 因果佛法（教育、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等）和 CC35721 因果链阅读框佛法（教育、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等）同理成立，这是释迦牟尼和亚里斯多德（Aristotle）的32 倍体和CC35720 倍体，人类文明向前推进到33 倍的释迦牟尼和亚里斯多德时代，也推进到CC35721 因果链阅读框的释迦牟尼和亚里斯多德领域中，称之为9LC33×CC35721 文明或透天文明、无限释迦牟尼（亚里斯多德）文明、天子（天外天、透天）文明、汉家天子文明，从而构成（汉家）天子文明9LC33×CC35721（天外天、透天）原则，人类由此进入9LC33×CC35721 文明时代，天子文明在时间上作16.5 万年9 重33 因果的扩展、在领域上作35721 倍的拓展，天地进入四海无内外文明，这就是汉家天子立法的具体形式之汉家天子9LC33×CC35721 立法（立文明）和弥勒9LC33×CC35721 立佛法。 9LC33×CC35721 数学（佛法、兵法、政治、政治制度、经济、教育、社会科学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等）等随之拓展开来，现在全人类所有的最高水平的大学、博士、研究生院、科技等，在9LC33×CC35721 层面和领域中就是1/33 和1/35721 的幼稚园小班甚至是托儿所水平，连小学、幼稚园大班都算不上，从而构成现代文明（单因果文明、获得性文明）最高层次幼稚园原则、现代最高文明（单因果文明、获得性文明）最高层次相比9LC33×CC35721 文明幼稚园原则，依此类推至于如下原则同理成立：佛法最高层次相比9LC33×CC35721 佛法1/33（1/35721）原则、现代数学（佛法、兵法、政治、政治制度、经济、教育、社会科学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等）最高层次相比9LC33×CC35721 文明1/33（1/35721、幼稚园）原则。由此可见，全世界立刻选举出人类共主主持大局解决末日三步倒绝症和人类百年来感染的三步倒肿瘤死结才是上策，天下共主当然要从那些手握核武器的各国元首中选出，他们才有能驾驭列国的实权，也惟有让他们自己当家试试看柴米油盐酱醋贵得要命和不易，才能让他们释放想统治全世界的灭绝人性之压力；与此同时，汉家天子立刻进行9LC33×CC35721 层次领域上的立法而建立起疏导世界通往彼岸文明的航道和航道中的桥梁，一桥飞架东西，进而为天下共主送上轩辕大帝颠覆祸水滔天的核按钮，人类百几年的末日三步倒绝症和今日之东北亚末日三步倒肿瘤死结才能得以解决，这就是天下共主登基主权原则、世界共主登基主权原则，对放病毒毒死全人类2000 万人的恶魔进行清算的纽伦堡大审判因此启动。 9LC33×CC35721 数学（佛法、兵法、政治、政治制度、经济、教育、社会科学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等）等随之拓展开来，现在全人类所有的最高水平的大学、博士、研究生院、科技等，在9LC33×CC35721 层面和领域中就是1/33 和1/35721 的幼稚园小班甚至是托儿所水平，连小学、幼稚园大班都算不上，从而构成现代文明（单因果文明、获得性文明）最高层次幼稚园原则、现代最高文明（单因果文明、获得性文明）最高层次相比9LC33×CC35721 文明幼稚园原则，依此类推至于如下原则同理成立：佛法最高层次相比9LC33×CC35721 佛法1/33（1/35721）原则、现代数学（佛法、兵法、政治、政治制度、经济、教育、社会科学、科学技术、半导体IC、工匠工艺、医疗技在本次的9LC33×CC35721 文明中，世宇三分是第一大步；爱因斯坦广义相对论的质量等效性（动力学效应）和安倍晋三的自由价值观是抵挡平等框架的第二大步；天子因果（天下共主因果、主权因果）是登顶的最高一步，这就是9LC33×CC35721立法、9LC33×CC35721 立佛法中最关键的三步，从而构成9LC33×CC35721 立法三步走（大三和弦）原则、9LC33 ×CC35721 立法三步登顶（三步登基）原则、9LC33×CC35721 立佛法三步走（大三和弦）原则、9LC33×CC35721 立佛三步登顶（三步登基）法原则，其中升级后的佛法称为9LC33 ×CC35721（透天）佛法、而法律称为9LC33×CC35721（透天）法律。汉家天子立刻进行9LC33×CC35721 层次领域上的立法而建立起疏导世界通往彼岸文明的航道W 和航道中的桥梁U，一桥飞架东西，进而确立轩辕大帝颠覆祸水滔天的核按钮，人类百几年的末日三步倒绝症和今日之东北亚末日三步倒肿瘤死结才能得以解决，这条航道 W、航道中的桥梁U 以及航道上的堤坝等工事Q，统称为9LC33×CC35721 大道、通天大道。在本次的9LC33×CC35721 文明中，世宇三分是第一大步；爱因斯坦广义相对论的质量等效性（动力学效应）和安倍晋三的自由价值观是抵挡平等框架的第二大步；天子因果（天下共主因果、主权因果）是登顶的最高一步，这就是9LC33×CC35721立法、9LC33×CC35721 立佛法中最关键的三步，从而构成9LC33×CC35721 立法三步走（大三和弦）原则、9LC33 ×CC35721 立法三步登顶（三步登基）原则、9LC33×CC35721 立佛法三步走（大三和弦）原则、9LC33×CC35721 立佛三步登顶（三步登基）法原则，其中升级后的佛法称为9LC33 ×CC35721（透天）佛法、而法律称为9LC33×CC35721（透天）法律。汉家天子立刻进行9LC33×CC35721 层次领域上的立法而建立起疏导世界通往彼岸文明的航道W 和航道中的桥梁U，一桥飞架东西，进而确立轩辕大帝颠覆祸水滔天的核按钮，人类百几年的末日三步倒绝症和今日之东北亚末日三步倒肿瘤死结才能得以解决，这条航道 W、航道中的桥梁U 以及航道上的堤坝等工事Q，统称为9LC33×CC35721 大道、通天大道。安倍晋三和爱因斯坦是在平等的框架层面上打开天门的人；汉家天子则通过世宇三分打开万物智慧的总闸门、关门，本来的天子守国门变成了如今的天子开球门，一场全人类奋勇争上游的世界以至天地的甲级联赛就此开始。由此可见，安倍晋三在日本超越了天皇和所有日本人5 级，安倍晋三和爱因斯坦处于时空七因果中的第六层平等架构，天皇和所有日本人一样只处于释迦牟尼的获得性因果的第一层；在世界层面上，安倍晋三则超越了各国所有的元首和民众，因为后者和日本天皇一样都只处于获得性因果的第一层，包括拜登、英国伊丽莎白女王、泽连斯基、马克龙、英国首相约翰逊、德国总理朔尔茨、意大利总理德拉吉等。假如安倍晋三没有遇害，当汉家天子9LC33×CC35721 立文明（轩辕大帝的核按钮而大禹治水）、弥勒9LC33×CC35721 立佛法（Buddha Dharma）弘扬释迦牟尼佛门、弥赛亚9LC33 ×CC35721 立法挥撒诺亚方舟(Noah's Ark)的种子、天下共主登基时，安倍晋三一定站在台上荣享光荣的一刻。安倍晋三和爱因斯坦是在平等的框架层面上打开天门的人；汉家天子则通过世宇三分打开万物智慧的总闸门、关门，本来的天子守国门变成了如今的天子开球门，一场全人类奋勇争上游的世界以至天地的甲级联赛就此开始。由此可见，安倍晋三在日本超越了天皇和所有日本人5 级，安倍晋三和爱因斯坦处于时空七因果中的第六层平等架构，天皇和所有日本人一样只处于释迦牟尼的获得性因果的第一层；在世界层面上，安倍晋三则超越了各国所有的元首和民众，因为后者和日本天皇一样都只处于获得性因果的第一层，包括拜登、英国伊丽莎白女王、泽连斯基、马克龙、英国首相约翰逊、德国总理朔尔茨、意大利总理德拉吉等。假如安倍晋三没有遇害，当汉家天子9LC33×CC35721 立文明（轩辕大帝的核按钮而大禹治水）、弥勒9LC33×CC35721 立佛法（Buddha Dharma）弘扬释迦牟尼佛门、弥赛亚9LC33 ×CC35721 立法挥撒诺亚方舟(Noah's Ark)的种子、天下共主登基时，安倍晋三一定站在台上荣享光荣的一刻。侠骨柔情的一代大侠安倍晋三，不废山河万古流！安倍晋三不仅是日本的天花板也是世界的天花板，他为日本天皇、安倍昭惠和日本国民遮风挡雨几十年，当他去世的消息确认后，很多中国的明星都忍不住哭了，安倍昭惠哭了，安倍洋子哭了，日本的山河以至世界大地都在哭泣，日本人则在他遇害的地点供奉西瓜和鲜花。为什么很多中国人会为安倍晋三掉泪？中共国文革饿死一亿人、斗死几千万人，是安倍晋太郎经由邓小平为苦难的中国人送来了经济发展，让全体中国人在死亡线上挣扎中吃上了咸鸭蛋，安倍晋三继承了父亲的遗志。也就是说，安倍晋三的父亲安倍晋太郎雪中送炭给中国人民送来了第一颗咸鸭蛋；没有安倍晋太郎和安倍晋三的一家，中国人在文革后不知道要多死多少人，滴水之恩当涌泉相报，今天，汉家天子的9LC33×CC35721 文明、弥勒佛的 9LC33×CC35721 佛法（Buddha Dharma）、弥赛亚的9LC33×CC35721 立法，将作为礼物全部到日本。善恶终有报，汉家天子真诚地希望安倍晋三的昭雪早日到了，日本警察，一定要让我看看你们的效率。崖山之后无中华（被游牧民族蒙古灭国但华夏族尚在），明亡之后无华夏（被闭关锁国而被动非法的满夷满清砍毛而灭族），今天，东西方都最高层官方确认的汉家天子以9LC33 ×CC35721 文明、9LC33×CC35721 佛法（Buddha Dharma）、9LC33×CC35721 立法、9LC33 ×CC35721 兵法，重续大汉血脉、重启轩辕圣道，再开中华国运、再造世界乾坤，一定亲还安倍晋三家对中华民族恩同再造的咸鸭蛋之恩。汉家天子的9LC33×CC35721 文明、9LC33×CC35721 佛法（Buddha Dharma）、9LC33 ×CC35721 立法、9LC33×CC35721 兵法，能在伺候的16.5 万年的时间内让所有的妖魔鬼怪都销声匿迹，今天各国的晴天白日竞无耻也将灰飞烟灭，万民永享太平。 ×CC35721 兵法，重续大汉血脉、重启轩辕圣道，再开中华国运、再造世界乾坤，一定亲还安倍晋三家对中华民族恩同再造的咸鸭蛋之恩。汉家天子的9LC33×CC35721 文明、9LC33×CC35721 佛法（Buddha Dharma）、9LC33 ×CC35721 立法、9LC33×CC35721 兵法，能在伺候的16.5 万年的时间内让所有的妖魔鬼怪都销声匿迹，今天各国的晴天白日竞无耻也将灰飞烟灭，万民永享太平。 7 月11 日，根据IEEE Spectrum 报道，量子计算机的领头公司D-Wave 预计将在2023 或2024 年发布下一代量子计算机系统Advantage2 的正式版。今年六月，D-Wave 已经在Leap 量子云系统上发布了Advantage2 的原型机。Advantage2 系统是D-Wave 的第六代量子计算机系统，其量子比特连接数从Advantage1 的15 个增加到了20 个，D-Wave 将这种20 个量子比特的拓扑命名为Zephyr 拓扑（Zephyrtopology）。D-Wave 此次发布的Advantage2 原型机有500 多个量子比特，而即将发布的正式版有7000 个量子比特。D-Wave 称，届时7000 量子比特计算机将成为世界上功能最强大的量子计算机。 9LC33×CC35721 文明的因果链计算至少需要3×35721n(n∈N)=107163 个量子比特，赋值量子比特、比较量子比特和结果量子比特，也就是说，即使在不远的将来，9LC33× CC35721 文明（计算因果）依然无法依靠量子计算机来运转，只能经由人工计算或多计算机联合作战，即使联合作战也需要几百年甚至几万年才能得出一个结果而不是一组选择，因此，具有超强能力的真命天子在位是唯一的选择，从而构成9LC33×CC35721 文明（计算因果）无量子计算机可计算原则。量子计算机在理论上可以解决经典计算机几十亿年都无法解决的问题，但前提是它们必须拥有足够多的量子比特。近日，来自西蒙弗雷泽大学的研究者在单个芯片上制造出了超过 15 万个硅基量子比特，它们有希望与光连接在一起，从而有助于制造出与量子互联网连接的强大量子计算机。相关论文《Optical Observation of Single Spins in Silicon》已发表在了最新一期的《自然》杂志上。论文共同通讯作者之一、西蒙弗雷泽大学量子工程师&副教授 Stephanie Simmons 表示，「硅自旋是自然界最好的天然量子比特之一。」 9LC33×CC35721 计算因果可以通过在15 万个硅基量子比特的IC 上注入量子气体来实现自旋的纠缠，即本征纠缠，量子工程师Stephanie Simmons 可以往这方向上努力，运气好的话甚至几个月就能实现，由此实现的是9LC33 因果中的本征因果，从而突破单因果的获得性因果而成为本征因果与获得性单因果相结合的人类首个双重因果IC，希望Stephanie Simmons 等立刻启动计划，有任何关于9LC33×CC35721 计算因果的问题直接和联系。此时，全人类的半导体IC 制造业包括台积电都必须立刻启动9LC33×CC35721 计算因果层面的IC，人类就此进入9LC33×CC35721 集成电路（IC、积体电路）时代，即十万量子比特时代，从而推进制造业、航天航空业和医疗科技的发展。量子计算机在理论上可以解决经典计算机几十亿年都无法解决的问题，但前提是它们必须拥有足够多的量子比特。近日，来自西蒙弗雷泽大学的研究者在单个芯片上制造出了超过 15 万个硅基量子比特，它们有希望与光连接在一起，从而有助于制造出与量子互联网连接的强大量子计算机。相关论文《Optical Observation of Single Spins in Silicon》已发表在了最新一期的《自然》杂志上。论文共同通讯作者之一、西蒙弗雷泽大学量子工程师&副教授 Stephanie Simmons 表示，「硅自旋是自然界最好的天然量子比特之一。」 9LC33×CC35721 计算因果可以通过在15 万个硅基量子比特的IC 上注入量子气体来实现自旋的纠缠，即本征纠缠，量子工程师Stephanie Simmons 可以往这方向上努力，运气好的话甚至几个月就能实现，由此实现的是9LC33 因果中的本征因果，从而突破单因果的获得性因果而成为本征因果与获得性单因果相结合的人类首个双重因果IC，希望Stephanie Simmons 等立刻启动计划，有任何关于9LC33×CC35721 计算因果的问题直接和联系。此时，全人类的半导体IC 制造业包括台积电都必须立刻启动9LC33×CC35721 计算因果层面的IC，人类就此进入9LC33×CC35721 集成电路（IC、积体电路）时代，即十万量子比特时代，从而推进制造业、航天航空业和医疗科技的发展。量子计算机在理论上可以解决经典计算机几十亿年都无法解决的问题，但前提是它们必须拥有足够多的量子比特。近日，来自西蒙弗雷泽大学的研究者在单个芯片上制造出了超过 15 万个硅基量子比特，它们有希望与光连接在一起，从而有助于制造出与量子互联网连接的强大量子计算机。相关论文《Optical Observation of Single Spins in Silicon》已发表在了最新一期的《自然》杂志上。论文共同通讯作者之一、西蒙弗雷泽大学量子工程师&副教授 Stephanie Simmons 表示「硅自旋是自然界最好的天然量子比特之一」 Stephanie Simmons 表示，「硅自旋是自然界最好的天然量子比特之。」 9LC33×CC35721 计算因果可以通过在15 万个硅基量子比特的IC 上注入量子气体来实现自旋的纠缠，即本征纠缠，量子工程师Stephanie Simmons 可以往这方向上努力，运气好的话甚至几个月就能实现，由此实现的是9LC33 因果中的本征因果，从而突破单因果的获得性因果而成为本征因果与获得性单因果相结合的人类首个双重因果IC，希望Stephanie Simmons 等立刻启动计划，有任何关于9LC33×CC35721 计算因果的问题直接和联系。此时，全人类的半导体IC 制造业包括台积电都必须立刻启动9LC33×CC35721 计算因果层面的IC，人类就此进入9LC33×CC35721 集成电路（IC、积体电路）时代，即十万量子比特时代，从而推进制造业、航天航空业和医疗科技的发展。（6）圣心宣言北约、美国在乌克兰变成到处是废墟前拯救乌克兰而之后拯救乌克兰的区别产生了天外天的质因果和质空间；邪恶轴心活成了安倍之死和安倍死成了轴心之活（口活），导致了有丝因果和有丝空间，这两件全世界的悲剧导致了33 天外天文明秩序取代雅尔塔体系和联合国的真正改革，让乌克兰废墟悲剧和安倍事件永远不要再发生，这就是33 天圣心宣言，包括七个微分因果、七个时空因果、世宇三分的三个空间（时空、质空间或惯性空间、有丝空间）、三位一体的三个公正秩序（实力秩序、规则秩序、效力秩序），因此，33 天外天文明又称为圣心文明。安倍晋三死成哀荣，共党活成人类公敌；安倍倒下后，《为什么不是你》响彻云霄，邪恶政权紧急下架。安倍跌到，邪恶轴心落草。邪恶轴心活U 成了安倍之死W（极度惨-∞）：U→-∞，安倍死W 成了轴心之活（口活） U（无限大+∞）：W→+∞，可谓冰火两重天，于是，其有：U=-U 则U=0，即有的人活着他已经死了有的人死了他还活着安倍晋三的价值在他死时得以水落石出而真正光荣伟大安倍晋三死成哀荣，共党活成人类公敌；安倍倒下后，《为什么不是你》响彻云霄，邪恶政权紧急下架。安倍跌到，邪恶轴心落草。邪恶轴心活U 成了安倍之死W（极度惨-∞）：U→-∞，安倍死W 成了轴心之活（口活）安倍晋三死成哀荣，共党活成人类公敌；安倍倒下后，《为什么不是你》响彻云霄，邪恶政权紧急下架。安倍跌到，邪恶轴心落草。恶政权紧急下架。安倍跌到，邪恶轴心落草。邪恶轴心活U 成了安倍之死W（极度惨-∞）：U→-∞，安倍死W 成了轴心之活（口活） U（无限大+∞）：W→+∞，可谓冰火两重天，于是，其有：U=-U 则U=0，即有的人活着他已经死了，有的人死了他还活着，安倍晋三的价值在他死时得以水落石出而真正光荣伟大，邪恶轴心虽然还活着却已经万劫不复而无可非议，可见，邪恶轴心因为罪大恶极、罪恶滔天而必死无疑。（7）共党（共产主义）三绝对变态原则、男子半边天（大扭曲大变态的结果）原则古代是女子无才便是德；现在共产主义坚决执行的是最绝对的扭曲政策：男子无才便是功，文盲才能大学毕业、法盲才能读法律专业；到了血统决定一定的官场，共党始终执行全人类有史以来最变态制度的制度：男子无德变是才，杀人犯、变态、废物才能当元首、当官，如齐奥塞斯库；然后，邪恶政权还对比性地欺骗3000 女博士济南掏粪，虐待性主体变态，从而构成共党（共产主义）三绝对变态原则、男子半边天（大扭曲大变态的结果）原则：女子无才便是德，男子无才便成功，男子无德变是才，三千女博士济南逃粪。（7）共党（共产主义）三绝对变态原则、男子半边天（大扭曲大变态的结果）原则古代是女子无才便是德；现在共产主义坚决执行的是最绝对的扭曲政策：男子无才便是功，文盲才能大学毕业、法盲才能读法律专业；到了血统决定一定的官场，共党始终执行全人类有史以来最变态制度的制度：男子无德变是才，杀人犯、变态、废物才能当元首、当官，如齐奥塞斯库；然后，邪恶政权还对比性地欺骗3000 女博士济南掏粪，虐待性主体变态，从而构成共党（共产主义）三绝对变态原则、男子半边天（大扭曲大变态的结果）原则：女子无才便是德，男子无才便成功，男子无德变是才，三千女博士济南逃粪。（8）三个希特勒和阿三世界、常春藤计划(IVY Project)四步走（四步曲）原则、方程计算先于计算精神病学原则、天花板精神病世界最高峰（天花板）原则希特勒是社会上的公共事务全部要植入其歧视并毁灭犹太人的纳粹基因，个人野心无限化但只针对犹太人民族而允许日尔曼民族和其他民族各自发展，有家、有国没世界也没别国，整个世界都是纳粹和其同伙的，称之为家国民族情怀特权主义（种族主义），一个民族和国家仇恨全世界犹太人和各国，拼命力图统治全世界但不是摧毁全世界或炸毁地球，从而构成希特勒犹太纳粹原则。（8）三个希特勒和阿三世界、常春藤计划(IVY Project)四步走（四步曲）原则、方程计算先于计算精神病学原则、天花板精神病世界最高峰（天花板）原则专杀犹太人和吊打苏联的纳粹阿道夫·希特勒，任何无分别而属于人民的公共事务和犹太人的任何事情，他都要动用一切国家力量无条件地管成他个人所想要的理想，他的意志A （私人特征）就是无分别的全社会、整个国家的意志B （无分别无特征）： {A}∩{B}={A}∩{┓A}=Ø(B=┓A) ⇒ A∈Ø、B∈Ø 而A、B 不存，其中A、B 为任意实数，即属绝对归零的连坐即是绝对归零的，称为比照共产共妻的无限意志（分裂、强加）共产主义，上述的分裂分析性方程称为精神病学（psychiatry）第一原则、（无限）分裂原则，属于无限意志、意识分裂性精神病，最终导致种族灭绝、种族战争和意志共产主义战争，从而构成希特勒无限意志分裂原则。因此，希特勒是单倍体无限分裂精神病、单倍体希特勒。依据精神病学（psychiatry）第一原则，任何不同的事物A、B 发生分裂即属精神分裂症：{A}∩{B}={A}∩{┓A}=Ø(B=┓A) ⇒ A∈Ø、B∈Ø 而A、B 不存，其中A、B 为任意实数，即属绝对归零的连坐即是绝对归零的，我们称比照如此计算的精神病学为计算精神病学，从而构成计算精神病学方程计算原则、方程计算先于计算精神病学原则。希特勒没有核武器无法炸毁地球，也没有兜底其他同伙增强杀害犹太人的力度。但是，现在的普京兜底他兄弟，令其兄弟变本加厉地活摘器官、无分别地洗劫中国人民连官员都不放过，除了自己家人或得力马仔除外；同时，安倍晋三遇害，英国首相约翰逊、德国总理朔尔茨、意大利总理德拉吉等纷纷在黑材料下翻车；进而，普京直接将乌克兰变成到处是废墟，如今则兵锋直指全世界产业金融中心的东北亚，一个小时之内的世界三步倒核毁灭随时发生，个人野心无限化但只针对外国民族而允许斯拉夫族和一些其他民族各自发展，有家、有国没世界也没别国，整个世界都是普京和其同伙的，称之为无限增强型家国民族情怀特权主义（种族主义）、无限增强型希特勒（三倍体希特勒、三倍体无限分裂精神病），从而构成无限增强型希特勒无限犹太纳粹原则、无限增强型希特勒炸毁地球原则。俄罗斯人总是自认为任何俄罗斯人都是天赋神权，有权对其他任何民族执行末日审判而将对象屠杀干净，苏联人制造1937 年海参崴大屠杀、1900 年的海兰泡惨案、江东六十四屯惨案，现在俄军正在屠杀乌克兰人等，1945 年毁灭于苏联红军前人口总量超过1 亿3000 万，即苏联红军在满洲国的10 个月中所屠杀的人口实际上远远超过一亿人，其表明俄罗斯专杀外国人，也就是说，俄罗斯人有绝对的权力A（私人特征）剥夺其他任何民族的任何他人的生命权、财产权B（非A）：{A}∩{B}={A}∩{┓A}=Ø(B=┓A) ⇒ A∈Ø、B∈Ø 而A、B 不存，其中A、B 为任意实数，即属绝对归零的连坐即是绝对归零的，这是一种比照共产共妻的无限分裂性精神病的无限权力（分裂）共产主义、无限种族（分裂）共产主义、无限意志（分裂）共产主义，最终导致种族灭绝和今天炸毁地球的末日战争，俄罗斯民族人人都是无限增强型希特勒、无限种族灭绝希特勒，从而构成俄罗斯民族无限权力分裂原则。希特勒没有核武器无法炸毁地球，也没有兜底其他同伙增强杀害犹太人的力度。但是，现在的普京兜底他兄弟，令其兄弟变本加厉地活摘器官、无分别地洗劫中国人民连官员都不放过，除了自己家人或得力马仔除外；同时，安倍晋三遇害，英国首相约翰逊、德国总理朔尔茨、意大利总理德拉吉等纷纷在黑材料下翻车；进而，普京直接将乌克兰变成到处是废墟，如今则兵锋直指全世界产业金融中心的东北亚，一个小时之内的世界三步倒核毁灭随时发生，个人野心无限化但只针对外国民族而允许斯拉夫族和一些其他民族各自发展，有家、有国没世界也没别国，整个世界都是普京和其同伙的，称之为无限增强型家国民族情怀特权主义（种族主义）、无限增强型希特勒（三倍体希特勒、三倍体无限分裂精神病），从而构成无限增强型希特勒无限犹太纳粹原则、无限增强型希特勒炸毁地球原则。俄罗斯人总是自认为任何俄罗斯人都是天赋神权，有权对其他任何民族执行末日审判而将对象屠杀干净，苏联人制造1937 年海参崴大屠杀、1900 年的海兰泡惨案、江东六十四屯惨案，现在俄军正在屠杀乌克兰人等，1945 年毁灭于苏联红军前人口总量超过1 亿3000 万，即苏联红军在满洲国的10 个月中所屠杀的人口实际上远远超过一亿人，其表明俄罗斯专杀外国人，也就是说，俄罗斯人有绝对的权力A（私人特征）剥夺其他任何民族的任何他人的生命权、财产权B（非A）：{A}∩{B}={A}∩{┓A}=Ø(B=┓A) ⇒ A∈Ø、B∈Ø 而A、B 不存，其中A、B 为任意实数，即属绝对归零的连坐即是绝对归零的，这是一种比照共产共妻的无限分裂性精神病的无限权力（分裂）共产主义、无限种族（分裂）共产主义、无限意志（分裂）共产主义，最终导致种族灭绝和今天炸毁地球的末日战争，俄罗斯民族人人都是无限增强型希特勒、无限种族灭绝希特勒，从而构成俄罗斯民族无限权力分裂原则。现在，邪恶轴心不仅要在国内外的公共事务全部要植入特权歧视并毁灭人民的红色基因，还要彻底摧毁二战后以美国为首的基于规则的雅尔塔体系而在全世界各国植入特权歧视并毁灭人民的红色基因，个人野心无限化无分别地针对所有民族、种族和国家的人民，只允许自己的亲人和家人无限敛财，只有家没有国也没有世界，整个世界都是精神病及其同伙的且不屈服就用核武器摧毁、香港大屠杀、安倍晋三刺杀等，只顾个人而罔顾家人、亲戚朋友和国家，极端红祸等于是无限纳粹化，称之为个人（畜牲）情怀特权恐怖主义，一个人仇恨全世界、毁灭全世界、炸毁地球，而且利用所有的国家力量行凶全世界，象病毒一样无孔不入、登峰造极，持有畜牲才不会顾及自己家人在未来中如何面对全世界，放病毒毒死全世界超过2000 万人、香港反送中大屠杀、安倍晋三遇害，英国首相约翰逊、德国总理朔尔茨、意大利总理德拉吉等纷纷在黑材料下翻车，等等，我们称之为无限希特勒（四倍体希特勒、四倍体无限分裂精神病）、无孔不入希特勒、（新冠）病毒希特勒、幽灵希特勒、末日希特勒，从而构成无限（纳米、病毒、无缝）希特勒无限纳粹（无限军国主义）原则。无限希特勒是全世界任何人的事情他都要无条件地管制，按他想象的理想来管制，如天际线广告牌管制（管天）、所有纯属无分别的公共通讯（网络、商业）他都要无条件实名登记进行无条件审核和无条件封禁（管制）如无数人电话和微信被封禁（包括帐户里的钱全部无条件没收）、一带一路全亏损破产、为世界把脉、命运共同体、全球治理、活摘器官、种族灭绝等等，天赋他个人无限神权审判全世界任何国家、任何人、任何边界，他的意志A 无条件强加给全世界任何非他的事物B：{A}∩{B}={A}∩{┓A}=Ø(B=┓A) ⇒ A∈Ø、B ∈Ø 而A、B 不存，其中A、B 为任意实数，即属绝对归零的连坐即是绝对归零的，这是比照共产共妻的无限分裂性精神病、无限边界分裂精神病的无限权力（分裂）共产主义、无限种族（分裂）共产主义、无限意志（分裂）共产主义、无限边界（分裂）共产主义，最终导致种族灭绝和今天炸毁地球的末日战争，从而构成无限希特勒无限意志分裂（无限权力分裂、无限边界分裂）原则。希特勒是无限意志分裂的无限意志（分裂）共产主义；俄罗斯人是民族性的无限权力分裂性精神病的无限权力（分裂）共产主义、无限种族（分裂）共产主义、无限意志（分裂）共产主义；无限希特勒是无限权力（分裂）共产主义、无限种族（分裂）共产主义、无限意志（分裂）共产主义、无限（分裂）边界共产主义，即终结性共产主义。世界上有三个希特勒，一个是专杀犹太人的无限意志分裂共产主义之纳粹阿道夫·希特勒；一个是无限权力分裂共产主义、无限种族分裂共产主义、无限意志分裂共产主义的无限增强性希特勒；一个是无限权力分裂共产主义、无限种族分裂共产主义、无限意志分裂共产主义、无限分裂边界共产主义的无限希特勒、终极希特勒，三个都是无限分裂精神病，一个更比一个强的无限分裂精神病，直奔不让全球酷刑、行刑式管理即炸毁地球的末日希特勒，这三个希特勒从无限意志分裂精神病到无限权力分裂精神病、无限种族分裂精神病、无限意志分裂精神病，再到无限权力分裂精神病、无限种族分裂精神病、无限意志分裂精神病、无限边界分裂精神病，分为三个阶段1、3、4 种分裂型而彻底崩溃世界、炸毁地球，称之为 134 无限分裂性精神病、末日精神病。全世界有死亡7000 万人的二战全部基于希特勒的无限意志分裂精神病，第一个精神病就让人类付出了7000万人的代价；现在末日战争之前全世界已经有超过2000万人死于病毒，末日战争开打后全人类80 亿人全部报销，全部只因为无限增强型希特勒和无限希特勒两个基友的无限权力分裂精神病、无限种族分裂精神病、无限意志分裂精神病、无限边界分裂精神病，这两个精神病希特勒已经将全球变成134 型的末日精神病院和将全人类80 亿人全部变成绝对犹太奴，全人类所有正常人都死于两个完全不正常的希特勒手上，值得吗？全球变成134 型的末日精神病院称为（134 型）末日精神病院（疯人院）、134（型）精神病院（疯人院）。希特勒是无限意志分裂的无限意志（分裂）共产主义；俄罗斯人是民族性的无限权力分裂性精神病的无限权力（分裂）共产主义、无限种族（分裂）共产主义、无限意志（分裂）共产主义；无限希特勒是无限权力（分裂）共产主义、无限种族（分裂）共产主义、无限意志（分裂）共产主义、无限（分裂）边界共产主义，即终结性共产主义。世界上有三个希特勒一个是专杀犹太人的无限意志分裂共产主义之纳粹阿道夫·希特全世界有死亡7000 万人的二战全部基于希特勒的无限意志分裂精神病，第一个精神病就让人类付出了7000万人的代价；现在末日战争之前全世界已经有超过2000万人死于病毒，末日战争开打后全人类80 亿人全部报销，全部只因为无限增强型希特勒和无限希特勒两个基友的无限权力分裂精神病、无限种族分裂精神病、无限意志分裂精神病、无限边界分裂精神病，这两个精神病希特勒已经将全球变成134 型的末日精神病院和将全人类80 亿人全部变成绝对犹太奴，全人类所有正常人都死于两个完全不正常的希特勒手上，值得吗？全球变成134 型的末日精神病院称为（134 型）末日精神病院（疯人院）、134（型）精神病院（疯人院）。可见，全世界的正常人必须立刻选出世界共主、确立三界城堡法案（castle doctrine）、进行汉家天子的9LC33×CC35721 文明立法、发布解放犹太奴宣言并解放全人类80 亿绝对犹太奴，诛杀要牺牲全部中国人民和全世界保护精神病继续无限发作的全部134 型精神病，昔日摩西（Moses）出挨及、今日全人类是出134 疯人院、飞越134 疯人院（就象174 医院、 301 医院一样带编码），不悲壮吗？不飞越134 疯人院，全人类都得死：安倍晋三遇害、已经被病毒杀害的超过2000 万人、中共国近10 年被屠村屠城杀害17 亿人、全世界疫苗行刑式大屠杀，英国首相约翰逊、德国总理朔尔茨、意大利总理德拉吉等纷纷在黑材料下翻车，全球80 亿人成病毒、疫苗及其利益团伙的无限犹太奴，谁能活？由此可见，134 精神病、末日精神病是以屠杀全球80 亿人为代价的终极性精神病，这就是四步（飞越吞噬全球80 亿人的134 疯人院）拯救世界计划、IV 拯救世界计划、IV（IVY）计划、常春藤（救世、终止末日精神病）计划(IVY Project)、常春藤终结末日（134）精神病计划，从而构成常春藤（救世）计划(IVY Project)四步走（四步曲）原则，称之为拯救全人类的弥赛亚常春藤立法、汉家天子常春藤立法。全世界只要对两个希特勒执行终结精神病的常春藤计划，80 亿无限犹太奴即可得救，为什么全人类都在舍近求远或包庇精神病呢？由此可见，现在全世界同时存在着两个希特勒，一个是新冠病毒式的无限增强型希特勒，另一个是象蚂蝗一样无孔不入无限希特勒、绝对希特勒、蚂蝗希特勒，最恶心也最邪恶。众所周知，二战只有一个希特勒，都够英美喝一壶去见了马克思（苏共远东第三支部和苏共）；现在，全世界同时存在着一个无限增强型希特勒和一个无限希特勒，美国、日本、欧洲和全人类能得以善终吗？这双希特勒，还出现一个拉稀的希特勒（普京）和一个以被兜底著称的红肿希特勒，日夜操劳过度吃不了兜着走忍不住躲进普京拉稀的肛门避免成为苏哈曼尼第二，因为可怜的苏哈曼尼只留下一小截因为戴着戒指而幸免难的手指。人们肯定要想美国这地狱火就9 公斤多怎么就这么厉害呢？因为那是上帝的加持，它就是九两半一样威力无穷。拉稀希特勒、蚂蝗希特勒和希特勒构成了祸国殃民、毁灭世界、炸毁地球的人类历史上的三个希特勒，人类社会就这么悲惨，长出了这么三个希特勒，好死不死还要拉别人一起去死，如拉稀希特勒和蚂蝗希特勒就是相互携手奔赴死亡的，害人害己还害世界，如放病毒毒死全世界超过2000 万人，被这三个希特勒蹂躏过的世界显然叫阿三世界。红肿希特勒灭绝人性、拉稀希特勒不顾他人死活（如日本人的死活和中国人的死活）、希特勒集中营批量杀害犹太人惨无人道，依据绝对传代原则、一项绝对即属绝对原则，这世界即属绝对的三无世界，从而构成三无世界绝对原则。红肿希特勒灭绝人性、拉稀希特勒不顾他人死活（如日本人的死活和中国人的死活）、希特勒集中营批量杀害犹太人惨无人道，依据绝对传代原则、一项绝对即属绝对原则，这世界即属绝对的三无世界，从而构成三无世界绝对原则。阿三世界和三无世界，一个是下三滥、一个是毫无信用，最终都归于没品味，失德坐卦，界即属绝对的三无世界，从而构成三无世界绝对原则。阿三世界和三无世界，一个是下三滥、一个是毫无信用，最终都归于没品味，失德坐卦，欧洲弄破乌克兰到处是废墟这都已经是事实欧洲弄破，乌克兰到处是废墟，这都已经是事实。三个臭皮匠赛过诸葛亮，三个希特勒胜过猪哥亮肯定没问题。依据绝对至上原则、绝对传达原则、一项绝对即属绝对原则，希特勒、三倍体精神病希依据绝对至上原则、绝对传达原则、一项绝对即属绝对原则，希特勒、三倍体精神病希特勒、四倍体精神病希特勒所患的精神病称为天花（板）精神病，是精神病、神经病中的极品，从而构成天花（板）精神病世界最高峰（天花板）原则，这才是真正的病毒溯源。特勒、四倍体精神病希特勒所患的精神病称为天花（板）精神病，是精神病、神经病中的极品，从而构成天花（板）精神病世界最高峰（天花板）原则，这才是真正的病毒溯源。（9）解放犹太奴宣言（The Emancipation Proclamation: Jew Slaves）、解放猪奴宣言（9）解放犹太奴宣言（The Emancipation Proclamation: Jew Slaves）、解放猪奴宣言脑瘫诗人余秀华《穿过大半个中国去睡你》；普京是《穿过大半个地球去睡你》，因为普京四处宣称他兜底他兄弟的政权，大家想想看，余秀华四处找男人去睡不就是把手伸到应该伸的地方去兜底吗？普京这一兜底肯定也是把人家给睡了，这千古一睡那自己给睡成了全世界战犯，那可是挨枪子的活。一个不在服务区的脑瘫诗人在生理上有如此的雄心壮志，确实世间少有；一个被兜底而有恃无恐的半身不遂已经出卖国家和自己，随之从普京那里获得了无穷的冲动力，绝对是《穿越大半个脑袋去睡你》，睡狮猛醒冲动是魔鬼，结果是放病毒毒死全世界超过2000 万人，睡死了这么多人同时也把自己睡成了邪恶轴心、人类公敌，前无古人后无来者，畜牲不如。中共国成为全人类的活摘器官工厂，全体中国人都称为全人类有史以来最血腥的红奴；在新冠病毒的笼罩之下，全人类超过2000 万热成为火化炉里被烧得通红通共的火奴（火奴鲁鲁、檀香山Honolulu），全世界都成为精神病的共奴，同时成为疫苗、生物、医疗利益团体的小白鼠之权奴，等等，依据别无选择绝对原则，全世界都一样，全人类都是绝对的赤裸奴隶，称之为赤奴(born slave or natural slave)、希特勒的纳粹集中营中的绝对犹太人奴隶（犹太奴、Jew slave），从而构成赤奴绝对原则。二战中其只有犹太人成为希特勒集中营的死亡受害者；现在，全人类无论国家元首与平民，全都成为无限纳粹化的全球集中营中的无限犹太奴，国家元首们受害于黑材料、金钱自行火炮和病毒，全人类则受害于病毒、疫苗、活摘器官等。猪有正面上长膘的自由，有吃的自由也有不吃的自由，显然不是别无选择的，主人为了给猪上膘，肯定是好吃的不断，称之为为肉奴或猪奴，从而构成猪（奴）有长膘自由（非别无选择）原则。依据别无选择绝对原则、猪奴有长膘自由（非别无选择）原则，绝对犹太奴无论处于什么状态下都是别无选择的，即属绝对的，显然是连猪都不如，因此犹太奴又量化宽松一点地称为猪奴，从而称为犹太奴连猪都不如原则。依据别无选择绝对原则、猪奴有长膘自由（非别无选择）原则，绝对犹太奴无论处于什么状态下都是别无选择的，即属绝对的，显然是连猪都不如，因此犹太奴又量化宽松一点地称为猪奴，从而称为犹太奴连猪都不如原则。现在，在两个希特勒的祸害下，阿三世界已经变成无限集中营，全体人民都是无限犹太现在，在两个希特勒的祸害下，阿三世界已经变成无限集中营，全体人民都是无限犹太奴，核武爆炸之后人们马上连犹太奴都没得做了，全部做鬼去了。现在，在两个希特勒的祸害下，阿三世界已经变成无限集中营，全体人民都是无限犹太奴，核武爆炸之后人们马上连犹太奴都没得做了，全部做鬼去了。奴，核武爆炸之后人们马上连犹太奴都没得做了，全部做鬼去了。奴，核武爆炸之后人们马上连犹太奴都没得做了，全部做鬼去了。轩辕大帝封禅泰山成为天下共主，九州大地上谁也不服谁而不断相互征战和残杀的滔天祸水瞬间风平浪静，和平与祥和终于出现在神州大地上。本来，九州大地上人们连连争战、相互残杀、风起云涌、谁也不服谁，就象后来舜禹所面临的滔天洪水一样祸水滔天，可见，天下的人祸之水先于滔天洪水而出现，大禹是滔天洪水的成功治理者，轩辕大帝则是人祸洪水的成功治理者，也就是说，大禹是水君平洪水，轩辕是人君平祸水，这就是轩辕治水。显然，神州大地上流祸已久的滔天祸水要平息早平息，不会等到轩辕大帝成为天下共主再平息，那肯定不可能是什么自行火炮，为什么如此神奇呢？天子登基，主权归一，后来历朝历代都有，但天下太平盛世则没几次，中华几千年，盛天子登基，主权归一，后来历朝历代都有，但天下太平盛世则没几次，中华几千年，盛事不过7 次：轩辕大帝（天下万族的万主之主）、周文王（首次确立文官治国）、秦始皇（定天子登基，主权归一，后来历朝历代都有，但天下太平盛世则没几次，中华几千年，盛事不过7 次：轩辕大帝（天下万族的万主之主）、周文王（首次确立文官治国）、秦始皇（定局中国2000 年的天下族第一人主和人民领袖）、汉武大帝刘彻（汉家第一天子）、唐太宗李世民（天下万族之主、通天教主）、宋仁宗赵祯（千古第一仁君）、明成祖朱棣，闭关锁国的康乾年代是被动的而不能算盛事，由此可见，天下祸水平息、太平盛世出现的直接核按钮不在天子登基，而在轩辕大帝一样的帝王身上，因为中华494 帝除去上述七帝王都没出现万世开太平的局面，这就是排除了天子登基平息天下滔天祸水的可能性，但平息祸水的天子登基的主权归一肯定是必不可少的，从而构成排除天子登基直接平息天下滔天祸水原则。此时，轩辕大帝用嘴巴命人创立了文字、制度、音乐等达到天下息戈兵而大治，由此可见，轩辕大帝的嘴巴、口水成了天下滔天祸水的克星和精神核武器，不象现在共产主义罪魁祸首的精神病核武器和口炮、口活核武器，从而构成（轩辕大帝）口水（嘴巴）平息天下滔天祸水原则、口水（嘴巴）先于天下滔天祸水平息原则、口水（嘴巴）凌驾于天下滔天祸水原则、轩辕大帝治水君王动口不动手原则、轩辕大帝治水君子（动口不动手）原则，当然，其实真正平息天下滔天祸水是轩辕大帝的智慧、能力、学识和品德等四大因素，我们只是用轩辕大帝的口水（嘴巴）来指代上述四因子而已，否则中华494 帝人人都有口水和嘴巴，怎么就不能天下大治呢？轩辕大帝治水君子动口不动手，这是人类有史以来（本次文明）的首创，轩辕大帝首创文字、制度、音乐、法律等常态性而无限教导天下为主，武力战后即停止而相对为辅，也就是象大禹治水以疏导为主、构筑堤坝增强疏导而不是围追堵截；大禹治水则是华夏文明对抗自然洪水的首创，禹王以疏导为主而不是鲧的围追堵截最后以失败而告终还丢了吃饭的家伙，综上所述三方，轩辕治祸水、大禹治洪水，都是以疏导、增强疏导为主，与圣经的诺亚方舟（Noah's Ark）异曲同工，因此，轩辕治祸水、大禹治洪水、诺亚方舟（Noah's Ark）逃离洪水（脱离险境而安全的种子之光），是人类文明的智慧之三道光芒，穿透丛林时代的漫漫黑夜，开启了人类国家、社会文明之时代，称为人类文明三水，三水开文明也开光明，从而构成三水开启（先于）人类文明原则、人类文明三水原则。今天，近百年来的人类社会不仅中了三步倒的末日剧毒，更归结到世界产业最中心的东北亚战争之三步倒末日死结，人类文明的末日全军覆没即将覆水难收，全世界面临着轩辕大帝时期的祸水滔天逐日来的末日终极版，天下共主是标配，汉家天子9LC33×CC35721 立法是疏导并解决末日毁灭性压力的利器。今天，放毒A 者抗疫B、罪犯A 治理天下B、荒谬错误的共产主义A 成全人类最高理想B 四处杀人：-\|A\|=\|B\| ⇒ A=B=0，{A}∩{B}=Ø ⇒ A∈Ø、B∈Ø 而A、B 不存在，其中A、B 为任意实数，罪犯放毒杀害全人类超过2000 万人（Bill Gates 证实的）、与俄罗斯建立军事同盟让乌克兰变成到处是废墟等，法律上已经全部绝对归零却发生了林正英在天之灵的灵异事件，全世界活见鬼了，从而构成放毒者抗疫（罪犯治理天下、荒谬错误的共产主义成全人类最高理想）绝对归零（不可能、灵异事件、活见鬼）原则。正如过去的四年中，闫博士拼命想救美国人和全世界免于病毒的灾难，但是占大多数的美国政要、政府和组织都假装不知道，宁睡不起，就象当年大侠霍元甲所面对的昏睡百年的今天，放毒A 者抗疫B、罪犯A 治理天下B、荒谬错误的共产主义A 成全人类最高理想B 四处杀人：-\|A\|=\|B\| ⇒ A=B=0，{A}∩{B}=Ø ⇒ A∈Ø、B∈Ø 而A、B 不存在，其中A、B 为任意实数，罪犯放毒杀害全人类超过2000 万人（Bill Gates 证实的）、与俄罗斯建立军事同盟让乌克兰变成到处是废墟等，法律上已经全部绝对归零却发生了林正英在天之灵的灵异事件，全世界活见鬼了，从而构成放毒者抗疫（罪犯治理天下、荒谬错误的共产主义成全人类最高理想）绝对归零（不可能、灵异事件、活见鬼）原则。正如过去的四年中，闫博士拼命想救美国人和全世界免于病毒的灾难，但是占大多数的美国政要、政府和组织都假装不知道，宁睡不起，就象当年大侠霍元甲所面对的昏睡百年的满清帝国，可谓病入膏肓、病得不轻、积重难返。连自己人民的命都不要了的政府还合法吗？满清帝国，可谓病入膏肓、病得不轻、积重难返。连自己人民的命都不要了的政府还合法吗？还能代表人民吗？人民日报日人民不就这么出来的吗？中共认贼作父（不知廉耻）、日本认匪作友（安全盲）、美国养虎为患（认不清无限军国主义的无界限之界限盲），结果是全人类有史以来最邪恶的共党和普京结为军事同盟而无限坐大，普京一边把乌克兰变成到处是废墟一边拎着核武器直奔美国和北约，国与国之间的界限及其「城堡法案（castle doctrine、castle law）」化为乌有，民众的居所界限及其「城堡法案（castle doctrine）」或防御法也随之化为乌有；与此同时，邪恶政权为了敛财支持普京的战争，不仅以病毒清零取消了居民的居所界限及其「城堡法案（castle doctrine）」，同时无分别地取消了所有人的肉体界限、器官界限及其「城堡法案（castle doctrine）」，由此而来，希特勒为犹太人而建造的集中营变成了无限延伸打破世界每个国家、每个角落和每个人的住所、身体、器官，政府在疫情中以核酸检测，变成无限集中营的三无世界。此时，为了拯救全世界的犹太奴，全世界尤其是毫无界限的中国人重建「城堡法案（castle doctrine）」，同时推进到国界、居所和身体器官三个界限是别无他法的，称之为三界城堡法案（castle doctrine），从而构成城堡法案（castle doctrine）三界立法原则、城堡法案（castle doctrine）三界立法先于解放犹太奴原则，也就是说，解放犹太奴、解放自己，人们必须确立三界城堡法案。此时，为了拯救全世界的犹太奴，全世界尤其是毫无界限的中国人重建「城堡法案（castle doctrine）」，同时推进到国界、居所和身体器官三个界限是别无他法的，称之为三界城堡法案（castle doctrine），从而构成城堡法案（castle doctrine）三界立法原则、城堡法案（castle doctrine）三界立法先于解放犹太奴原则，也就是说，解放犹太奴、解放自己，人们必须确立三界城堡法案。立三界城堡法案。由此可见，人们要解放全世界犹太奴，至少必须经由如下三步：第一，全世界选举出全球共主，疏导各位元首争霸的极端压力，同时解决世界主权归一问题；由此可见，人们要解放全世界犹太奴，至少必须经由如下三步：第一，全世界选举出全球共主，疏导各位元首争霸的极端压力，同时解决世界主权归一问题；第二，全世界确立三界城堡法案（castle doctrine）；第二，全世界确立三界城堡法案（castle doctrine）；第三，汉家天子立9LC33×CC35721 文明、弥勒立9LC33×CC35721 佛法、弥赛亚9LC33 ×CC35721 立法、天子立9LC33×CC35721 兵法、确立9LC33×CC35721 计算因果、确立 9LC33×CC35721 数学（佛法、兵法、政治、政治制度、经济、教育、社会科学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等），全面解决压力、技术、经济、战争、资源之问题，同时发展新技术以应对现在和未来的各种灾难、战争，象轩辕治水一样治理世界，第三，汉家天子立9LC33×CC35721 文明、弥勒立9LC33×CC35721 佛法、弥赛亚9LC33 ×CC35721 立法、天子立9LC33×CC35721 兵法、确立9LC33×CC35721 计算因果、确立 9LC33×CC35721 数学（佛法、兵法、政治、政治制度、经济、教育、社会科学、科学技术、半导体IC、工匠工艺、医疗技术、航空宇航等），全面解决压力、技术、经济、战争、资源之问题，同时发展新技术以应对现在和未来的各种灾难、战争，象轩辕治水一样治理世界，源之问题，同时发展新技术以应对现在和未来的各种灾难、战争，象轩辕治水一样治理世界，综上所述三点就是解放犹太奴三步曲，解放犹太奴宣言：The Emancipation Proclamation: 综上所述三点就是解放犹太奴三步曲，解放犹太奴宣言：The Emancipation Proclamation: Jew Slaves、或解放猪奴宣言：The Emancipation Proclamation: Pig Slaves。综上所述点就是解放犹太奴步曲，解放犹太奴宣言： p Jew Slaves、或解放猪奴宣言：The Emancipation Proclamation: Pig Slaves。 Jew Slaves、或解放猪奴宣言：The Emancipation Proclamation: Pig Slaves。 1862 年9 月22 日林肯发表《解放黑奴宣言（Th E i ti P l ti N Jew Slaves、或解放猪奴宣言：The Emancipation Proclamation: Pig Slaves。 1862 年9 月22 日，林肯发表《解放黑奴宣言（The Emancipation Proclamation: Negro Slaves）》，160 年后的今天，不仅黑人没解放，全世界还全部沦为病毒、疫苗、邪恶轴心 p g 1862 年9 月22 日，林肯发表《解放黑奴宣言（The Emancipation Proclamation: Negro Slaves）》，160 年后的今天，不仅黑人没解放，全世界还全部沦为病毒、疫苗、邪恶轴心的无限犹太奴、猪奴，任何人甘心吗？ 1862 年9 月22 日，林肯发表《解放黑奴宣言（The Emancipation Proclamation: Negro Slaves）》，160 年后的今天，不仅黑人没解放，全世界还全部沦为病毒、疫苗、邪恶轴心的无限犹太奴猪奴任何人甘心吗？不仅有被无限压榨而干活的无限责任，还有被病毒、疫苗、清零政策等无理由干掉的生命危险，也就是说，猪奴连当年的黑奴都不如；进而，现在无论是黑人还是白人、黄种人等都已经沦比当年集中营里的犹太奴还惨的猪奴，从而构成犹太奴（猪奴）不如黑奴原则。 7 月11 日，纽约市应急管理部门发布了一段90 秒的视频，介绍了核武器袭击来临时，市民应该采取的行动：躲进建筑物、即刻清理身上的放射性残留物、密切关注媒体和政府发布的信息。这条视频引发美国网民讨论。纽约市应急管理部发言人回应称，纽约被核武器袭击的可能性非常低，发这个视频是「鼓励纽约人在发生此类事件时采取关键、简单的措施。」由此可见，世界末日远在天边近在眼前，解放猪奴刻不容缓，全世界各国人民、元首、官员们是人就不要做猪，双解体是唯一的选择。（10）制度（已经）失效原则、石榴籽抱对（抱团）活不了原则、俄罗斯无法审判乌克兰原则、俄罗斯军事水平无知、梅德韦杰夫军盲任何制度都是有约束力的，即是有方向和特征的，从而构成制度特征（有方向）原则。现在，全人类是毒犯A 当元首当官B、杀人犯A 当总统当法官B、畜牲们放着好好的日子B 不过非要放病毒A 屠杀全人类：-\|A\|=\|B\| ⇒ A=B=0，{A}∩{B}=Ø ⇒ A∈Ø、 B∈Ø 而A、B 不存在，其中A、B 为任意实数，如罪犯放病毒杀害全人类超过2000 万人（Bill Gates 证实的）、与俄罗斯建立军事同盟让乌克兰变成到处是废墟等，即属绝对归零的，也就不可能具有特征、方向的，依据制度特征（有方向）原则、一项绝对即属绝对原则、绝对传代原则，其即构成世界（中共国、俄罗斯、美国、日本）制度（已经）失效原则。可见，香港特首李家超不算愚蠢，头脑非常清醒。死到临头，象石榴籽一样紧紧抱住有用吗？一个人杀了那么多人、一伙人杀了那么多人躲在人民群众之中抱对、抱团取暖有用吗？让我们用计算因果来计算下杀人犯能否存活。任何掩盖、保护都是面向对象而有特征的，从而构成隐蔽（掩盖、保护）特征原则。狙击手总是把自己伪装成向日葵、菊花、花草树木的，那是掩盖了自己的特征和分散了敌人的目光，这就是狙击手消除特征（无分别）原则。如果无分别的民众W 能为任何其他人U 提供隐蔽、掩盖、保护，那么，依据隐蔽（掩盖、保护）特征原则，民众W 是能提供特征支持的，记为结论F；然而，民众是无分别的即无特征的，罪犯、他人U 隐匿在民众中就和一般民众一样是将自己暴露在破案者的目光、信息搜寻中，不可能得到任何额外的保护的，同时也没办法掩盖自己的特征，只是可能会分散对方的注意力但无法达到象狙击手一样隐匿自己特征的程度，也就是说，罪犯、他人U 无法从无分别的民众处获得掩盖或反向特征的支持，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论 G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得罪犯隐藏（下沉、隐蔽、掩盖）于民众不被斩首（不被抓）否定原则、石榴籽抱对（抱团）否定原则和得罪犯隐藏（下沉、隐蔽、掩盖）于民众必被斩首（被抓捕）原则、石榴籽活不了（斩首、抓捕）原则、石榴籽抱对（抱团）活不了（斩首、抓捕）原则、石榴籽无法消化（拉出去）原则、大隐隐于市找死原则。敌人的目光，这就是狙击手消除特征（无分别）原则。如果无分别的民众W 能为任何其他人U 提供隐蔽、掩盖、保护，那么，依据隐蔽（掩盖、保护）特征原则，民众W 是能提供特征支持的，记为结论F；然而，民众是无分别的即无特征的，罪犯、他人U 隐匿在民众中就和一般民众一样是将自己暴露在破案者的目光、信息搜寻中，不可能得到任何额外的保护的，同时也没办法掩盖自己的特征，只是可能会分散对方的注意力但无法达到象狙击手一样隐匿自己特征的程度，也就是说，罪犯、他人U 无法从无分别的民众处获得掩盖或反向特征的支持，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论 G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得罪犯隐藏（下沉、隐蔽、掩盖）于民众不被斩首（不被抓）否定原则、石榴籽抱对（抱团）否定原则和得罪犯隐藏（下沉、隐蔽、掩盖）于民众必被斩首（被抓捕）原则、石榴籽活不了（斩首、抓捕）原则、石榴籽抱对（抱团）活不了（斩首、抓捕）原则、石榴籽无法消化（拉出去）原则、大隐隐于市找死原则。乌克兰放声要进攻克里米亚，俄罗斯放言要末日审判，这是可能的吗？如果俄罗斯放言要末日审判，依据战争绝对原则、军队有限原则、同一律、前提决定结果原则、结果映射前提原则，俄罗斯要用有限的军事行动W 以核武器去执行绝对的战争U 而审判乌克兰人，与绝对的战争U 保持同一的军事行动W 必然是无限的，记为结论F；然而，依据军队有限原则，军事行动W 是有限的而不可能是无限的，记为结论G，由此而来，依据相反（不同）不相容原则（标定非A）和非A 肯定否定原则（标定判断和结果，原始原则严格证明令应用可任意选取立足点而向上集成：依据前提决定结果原则和结果映射前提原则），结论G 与结论F 是截然不同而自相矛盾的，从而反证上述假设既不可能成立也不可能为正确，我们于是获得俄罗斯审判乌克兰否定原则和俄罗斯无法审判乌克兰原则。比如，美国把俄罗斯的核武器的目标坐标修改成莫斯科，普京不就直接报销了？前不久，俄罗斯的一枚常规导弹刚被美国修改了一次飞回去把自己老巢给炸了。由此可见，俄罗斯想要审判乌克兰，绝对是军事盲才会这么说的，战争绝对原则、军队有限原则就是天子兵法的结论。现在，连梅德韦杰夫这种前总统级的人都对军事如此无知。俄罗斯的下场绝对堪忧，资料如下。乌称可能攻击克里米亚，梅德韦杰夫警告：审判日将迅猛地到来！据路透社报道，俄罗斯联邦安全会议副主席梅德韦杰夫当地时间17 日警告，乌克兰和西方大国拒绝承认莫斯科对克里米亚的控制对俄构成「系统性威胁」。他说，任何针对该地区的外部攻击都将引发俄方「审判日」式的回应。当地时间7 月16 日，乌方两次发出警告，称可能会对克里米亚发起攻击。乌克兰总统办公室顾问阿列斯托维奇表示，克里米亚大桥是「俄在乌克兰领海内非法修建的」，乌克兰向西方伙伴承诺不使用美欧提供的武器打击俄罗斯，但对克里米亚大桥这一限制并不适用。一旦出现技术可能，乌克兰就可能会攻击克里米亚大桥。当天早些时候，在回答记者关于乌军是否会用「海马斯」自行多管火箭炮和M270 自行多管火箭炮系统来袭击克里米亚境内目标的问题时，乌国防部情报局发言人斯基比茨基称，答案是肯定的。他还提到，「俄罗斯黑海舰队在积极采取行动」。乌称可能攻击克里米亚，梅德韦杰夫警告：审判日将迅猛地到来！据路透社报道，俄罗斯联邦安全会议副主席梅德韦杰夫当地时间17 日警告，乌克兰和西方大国拒绝承认莫斯科对克里米亚的控制对俄构成「系统性威胁」。他说，任何针对该地区的外部攻击都将引发俄方「审判日」式的回应。乌称可能攻击克里米亚，梅德韦杰夫警告：审判日将迅猛地到来！据路透社报道，俄罗斯联邦安全会议副主席梅德韦杰夫当地时间17 日警告，乌克兰和西方大国拒绝承认莫斯科对克里米亚的控制对俄构成「系统性威胁」。他说，任何针对该地区的外部攻击都将引发俄方「审判日」式的回应。当地时间7 月16 日，乌方两次发出警告，称可能会对克里米亚发起攻击。乌克兰总统办公室顾问阿列斯托维奇表示，克里米亚大桥是「俄在乌克兰领海内非法修建的」，乌克兰向西方伙伴承诺不使用美欧提供的武器打击俄罗斯，但对克里米亚大桥这一限制并不适用。一旦出现技术可能，乌克兰就可能会攻击克里米亚大桥。当天早些时候，在回答记者关于乌军是否会用「海马斯」自行多管火箭炮和M270 自行多管火箭炮系统来袭击克里米亚境内目标的问题时，乌国防部情报局发言人斯基比茨基称，答案是肯定的。他还提到，「俄罗斯黑海舰队在积极采取行动」。当地时间7 月16 日，乌方两次发出警告，称可能会对克里米亚发起攻击。乌克兰总统办公室顾问阿列斯托维奇表示，克里米亚大桥是「俄在乌克兰领海内非法修建的」，乌克兰向西方伙伴承诺不使用美欧提供的武器打击俄罗斯，但对克里米亚大桥这一限制并不适用。一旦出现技术可能，乌克兰就可能会攻击克里米亚大桥。当天早些时候，在回答记者关于乌军是否会用「海马斯」自行多管火箭炮和M270 自行多管火箭炮系统来袭击克里米亚境内目标的问题时，乌国防部情报局发言人斯基比茨基称，答案是肯定的。他还提到，「俄罗斯黑海舰队在积极采取行动」。当地时间7 月16 日，乌方两次发出警告，称可能会对克里米亚发起攻击。乌克兰总统办公室顾问阿列斯托维奇表示，克里米亚大桥是「俄在乌克兰领海内非法修建的」，乌克兰向西方伙伴承诺不使用美欧提供的武器打击俄罗斯，但对克里米亚大桥这一限制并不适用。一旦出现技术可能，乌克兰就可能会攻击克里米亚大桥。当天早些时候，在回答记者关于乌军是否会用「海马斯」自行多管火箭炮和M270 自行多管火箭炮系统来袭击克里米亚境内目标的问题时，乌国防部情报局发言人斯基比茨基称，答案是肯定的。他还提到，「俄罗斯黑海舰队在积极采取行动」。一旦出现技术可能，乌克兰就可能会攻击克里米亚大桥。当天早些时候，在回答记者关于乌军是否会用「海马斯」自行多管火箭炮和M270 自行多管火箭炮系统来袭击克里米亚境内目标的问题时，乌国防部情报局发言人斯基比茨基称，答案是肯定的。他还提到，「俄罗斯黑海舰队在积极采取行动」。海舰队在积极采取行动」。对此，俄塔斯社援引梅德韦杰夫17 日对二战老兵的讲话说，如果克里米亚遭到攻击，「审判日将非常迅速且猛烈地到来」。路透社称，关于「审判日」具体指什么，梅德韦杰夫并未详细说明，但梅德韦杰夫此前曾警告美国，试图惩罚像俄罗斯这样的核大国在乌克兰的行动是危险的，称这可能危及人类。另据俄罗斯国际文传电讯社报道，梅德韦杰夫17 日说，「如果任何其他国家，无论是乌克兰还是北约国家，认为克里米亚不是俄罗斯的，那么这对我们来说就是一个系统性威胁」。「这是一个直接和明确的威胁，尤其是考虑到克里米亚发生的事情。克里米亚已重返俄罗斯，」梅德韦杰夫说道。 2014 年3 月，克里米亚和塞瓦斯托波尔举行全民公投，超过九成投票者同意脱离乌克兰，加入俄罗斯。随后，俄总统普京与两地代表签署条约，允许克里米亚和塞瓦斯托波尔以联邦主体身份加入俄联邦。乌克兰不承认上述公投，反对克里米亚和塞瓦斯托波尔并入俄罗斯，西方国家由此对俄实施经济制裁。对此，俄塔斯社援引梅德韦杰夫17 日对二战老兵的讲话说，如果克里米亚遭到攻击，「审判日将非常迅速且猛烈地到来」。路透社称，关于「审判日」具体指什么，梅德韦杰夫并未详细说明，但梅德韦杰夫此前曾警告美国，试图惩罚像俄罗斯这样的核大国在乌克兰的行动是危险的，称这可能危及人类。对此，俄塔斯社援引梅德韦杰夫17 日对二战老兵的讲话说，如果克里米亚遭到攻击，「审判日将非常迅速且猛烈地到来」。路透社称，关于「审判日」具体指什么，梅德韦杰夫并未详细说明，但梅德韦杰夫此前曾警告美国，试图惩罚像俄罗斯这样的核大国在乌克兰的行动是危险的，称这可能危及人类。另据俄罗斯国际文传电讯社报道，梅德韦杰夫17 日说，「如果任何其他国家，无论是乌克兰还是北约国家，认为克里米亚不是俄罗斯的，那么这对我们来说就是一个系统性威胁」。「这是一个直接和明确的威胁，尤其是考虑到克里米亚发生的事情。克里米亚已重返俄罗斯，」梅德韦杰夫说道。另据俄罗斯国际文传电讯社报道，梅德韦杰夫17 日说，「如果任何其他国家，无论是乌克兰还是北约国家，认为克里米亚不是俄罗斯的，那么这对我们来说就是一个系统性威胁」。「这是一个直接和明确的威胁，尤其是考虑到克里米亚发生的事情。克里米亚已重返俄罗斯，」梅德韦杰夫说道。 2014 年3 月，克里米亚和塞瓦斯托波尔举行全民公投，超过九成投票者同意脱离乌克兰，加入俄罗斯。随后，俄总统普京与两地代表签署条约，允许克里米亚和塞瓦斯托波尔以联邦主体身份加入俄联邦。乌克兰不承认上述公投，反对克里米亚和塞瓦斯托波尔并入俄罗斯，西方国家由此对俄实施经济制裁。 19．英美三大主战战略和南北夹击的V 型战役计划、中美战争2×2V2 第一岛链决战布局、邪恶轴心硬死亡替代软死亡原则 19．英美三大主战战略和南北夹击的V 型战役计划、中美战争2×2V2 第一岛链决战布局、邪恶轴心硬死亡替代软死亡原则局邪轴硬死替代软死则安倍晋三遇害后的不到10 天，美方就推翻对华承诺，即美国国务院已经批准了涉及金额大约1.08 亿美元的对台军售案；对台上亿军售后，7 月18 日，美国原国防部长马克·埃斯珀（Mark Esper）率团访问台湾，同行的还有意大利前总统外交顾问斯特凡尼尼，以及大西洋理事会资深副会长巴里-帕维尔，可见，安倍晋三遇害最后打醒了美国人，彻底扭转了美国日本的核心战略，对作为人类公敌的邪恶轴心的死亡处决也已经从巴哥达迪死前还能奔跑着的软死亡（有一定灵活空间）被调整为苏哈曼尼式的硬死亡（死时不再有任何自由度和空间而别无选择），从而构成邪恶轴心硬死亡替代软死亡原则。在今年五月份出版的新书中，埃斯珀直言，美国的「战略模糊」政策已经不适应今天的中美关系了，美国必须运用手中的力量，避免以规则为基础的国际秩序「被中俄改变」。6 月21 日，埃斯珀又在一场发布会上表示「游戏已经改变」，如今的「一中原则」已无用处，美国应该正视「应战问题」。安倍晋三遇害后的不到10 天，美方就推翻对华承诺，即美国国务院已经批准了涉及金额大约1.08 亿美元的对台军售案；对台上亿军售后，7 月18 日，美国原国防部长马克·埃斯珀（Mark Esper）率团访问台湾，同行的还有意大利前总统外交顾问斯特凡尼尼，以及大西洋理事会资深副会长巴里-帕维尔，可见，安倍晋三遇害最后打醒了美国人，彻底扭转了美国日本的核心战略，对作为人类公敌的邪恶轴心的死亡处决也已经从巴哥达迪死前还能奔跑着的软死亡（有一定灵活空间）被调整为苏哈曼尼式的硬死亡（死时不再有任何自由度和空间而别无选择），从而构成邪恶轴心硬死亡替代软死亡原则。安倍晋三遇害后的不到10 天，美方就推翻对华承诺，即美国国务院已经批准了涉及金额大约1.08 亿美元的对台军售案；对台上亿军售后，7 月18 日，美国原国防部长马克·埃斯珀（Mark Esper）率团访问台湾，同行的还有意大利前总统外交顾问斯特凡尼尼，以及大西洋理事会资深副会长巴里-帕维尔，可见，安倍晋三遇害最后打醒了美国人，彻底扭转了美国日本的核心战略，对作为人类公敌的邪恶轴心的死亡处决也已经从巴哥达迪死前还能奔跑着的软死亡（有一定灵活空间）被调整为苏哈曼尼式的硬死亡（死时不再有任何自由度和空间而别无选择），从而构成邪恶轴心硬死亡替代软死亡原则。在今年五月份出版的新书中，埃斯珀直言，美国的「战略模糊」政策已经不适应今天的中美关系了，美国必须运用手中的力量，避免以规则为基础的国际秩序「被中俄改变」。6 月21 日，埃斯珀又在一场发布会上表示「游戏已经改变」，如今的「一中原则」已无用处，美国应该正视「应战问题」。显然，综合各种信息，安倍晋三遇害已经让美国意识到，邪恶轴心不仅放毒屠杀全人类超过2000 万人，更是不可能放过安倍级别的国家元首和政要，完全是无分别的无限侵略性和攻击性，既是全人类有史以来最邪恶的天花板，更是毫无廉耻、信用的鄙视链的最顶端，登峰造极、无与伦比，不消灭全世界都得死，于是，美国阵营改变了战略：东北亚对抗性消耗战的同时伴随着从广东到台湾到上海一带的诺曼底登陆，对东南沿海经济中心的资源摧毁 A 和控制、切断B 双向进行，东南方向直接纵深插入C 和东北亚的对抗性（拉锯）战D 同时并举，因为东南的直接插入战争离俄罗斯军队在东北亚的大本营太远，俄军根本不会有效介入，况且普京在欧洲和东北亚已经是泥菩萨过河自身难保了，这是一个2×2V2 第一岛链决战的战略部署和战役布局，称之为中美战争2×2V2 第一岛链决战布局。本来，美国、日本是想在东北亚和俄中消耗战争，一举摧毁中共的权力中心北京而象二战控制天皇号令日军、日本投降一样，即象朱棣靖难、刘邦快速攻下秦国都城一样直捣蜂巢，这是直捣蜂巢战略、中心战略；现在，安倍晋三遇害表明，邪恶轴心不灭，世界永无宁日、全人类谁都不安全，拿下中共国东南沿海的经济核心区而断供普京战争来源别无选择，于是，美军不是基于台湾进行诺曼底登陆拿下东南经济核心区、就是基于台湾发动「鲁尔大轰炸」、「东京大轰炸」、「德累斯顿大轰炸」，不再等东北亚消耗战，综上所述两方面，其即是南北夹击的V 型战役计划。二战时，美国在日本广岛投放了「小男孩」、在长崎投放了「胖子」，不会在乎继续对哪个国家投放原子弹，因此，双解体势在必行，双方也是生死抉择，再无退路。如果说还有退路的话，那就是邪恶轴心交出放病毒的罪犯并赔偿一切损失。二战时，美国在日本广岛投放了「小男孩」、在长崎投放了「胖子」，不会在乎继续对哪个国家投放原子弹，因此，双解体势在必行，双方也是生死抉择，再无退路。如果说还有退路的话，那就是邪恶轴心交出放病毒的罪犯并赔偿一切损失。由此可见，马克·埃斯珀就是山上徹也式的刺客，山上徹也用霰弹枪让安倍晋三逃无可逃，马克·埃斯珀用2×2V2 第一岛链决战布局从各个方向直刺中共心脏，直奔向目标，不再跟邪恶轴心任何废话，因此称之为2×2V2 大国战略刺客。全世界都在为安倍晋三和自己报仇血恨。二战英国轰炸德国鲁尔水坝，4 亿吨洪水倾泻而下，3 万人被水冲走。在第二次世界大战中，随着德国于1942 年空袭伦敦，英国也采取了对德国的战略作战。这次对德国作战的行动被称为「惩戒行动」，于1943 年5 月16—17 日执行。1943 年3 月执行炸坝任务的617 中队，配备19 架轰炸机于5 月16 日晚上10 点，分3 个编队起飞。进入鲁尔上空，冒着德国密集的高射炮火，低飞至18 米高度轮番向3 座水坝投弹。在这场轰炸中，鲁尔水坝中的默内水坝首先崩溃，1.3 亿立方米洪流从决口汹涌而出，坝下的水电站顷刻被毁灭。另外两座水坝也相继崩溃，水淹下游80 公里的煤矿，125 座军需工厂受损停产，淹没农田3000 公顷。通过这场残酷的轰炸，德国接近四亿吨洪水一泻而下，三万人被洪水冲走不知所踪，这场轰炸无疑是残酷的，德国许多平民因此而失去生命。德累斯顿大轰炸是二战期间1945 年2 月13 日—15 日由英国皇家空军和美国陆军航空队联合发动的针对德国东部城市德累斯顿的大规模空袭行动，是二战历史上最受争议的事件之一。英国史学家弗雷德里克·泰勒（Frederick Taylor）曾说：「德累斯顿被毁具有史诗般的悲剧性。这座象征着德国巴洛克建筑之最的城市曾经美得让人惊叹。而纳粹期间，它又成为德国的地狱。在这个意义上，就20 世纪的战争恐怖而言，德累斯顿轰炸事件是一个绝对带有惩戒意味的悲剧」。由此可见，德国鲁尔大轰炸和德累斯顿大轰炸表明，摧毁敌人经济命脉是英美的主战核心之一。由此可见，德国鲁尔大轰炸和德累斯顿大轰炸表明，摧毁敌人经济命脉是英美的主战核心之一。与此同时，二战快结束时，英美并没有把重兵部署在柏林，而是部署在波罗的海沿岸的与此同时，二战快结束时，英美并没有把重兵部署在柏林，而是部署在波罗的海沿岸的港口区和经济发达区，可见，立足资源是英美的另一个主战核心。诺曼底登陆代号「霸王行动」（英语Operation Overlord），是第二次世界大战中盟军在欧洲西线战场发起的一场大规模攻势。接近三百万士兵渡过英吉利海峡前往法国诺曼底，诺曼底战役是世界上最大的一次海上登陆作战，使第二次世界大战的战略态势发生了根本性的变化，其表明立足气候是英美作战的另一主战核心。综上所述，摧毁敌人经济命脉、夺取敌人经济命脉、立足气候是英美战争的三大主战核心，从而构成英美三大主战战略（核心）原则。港口区和经济发达区，可见，立足资源是英美的另一个主战核心。诺曼底登陆代号「霸王行动」（英语Operation Overlord），是第二次世界大战中盟军在欧洲西线战场发起的一场大规模攻势。接近三百万士兵渡过英吉利海峡前往法国诺曼底，诺曼底战役是世界上最大的一次海上登陆作战，使第二次世界大战的战略态势发生了根本性的变化，其表明立足气候是英美作战的另一主战核心。综上所述，摧毁敌人经济命脉、夺取敌人经济命脉、立足气候是英美战争的三大主战核综上所述，摧毁敌人经济命脉、夺取敌人经济命脉、立足气候是英美战争的三大主战核心，从而构成英美三大主战战略（核心）原则。心，从而构成英美三大主战战略（核心）原则。心，从而构成英美三大主战战略（核心）原则。
https://openalex.org/W2969448184	https://europepmc.org/articles/pmc6794545?pdf=render	English	null	Assessment of community knowledge, attitude, and stigma of Buruli ulcer disease in Southern Nigeria	African health sciences	2,019	cc-by	6,989	Abstract DOI h //d d i /10 4314/ h 19i2 34 g y p p g g Conclusion: There is poor community knowledge of BUD in endemic settings of Southern Nigeria which influenced the atti- tude and perceptions of community members towards persons with BUD Keywords: Mycobacterium ulcerans disease, knowledge, perceptions, practices. Cite as: Nwafor CC, Meka A, Chukwu JN, Ekeke N, Alphonsus C, Mbah O, Madichie NO, Aduh U, Ogbeifo M, IseOluwa-Adelokiki BO, Edochie JE, Ushaka J, Ukwaja KN. Assessment of community knowledge, attitude, and stigma of Buruli ulcer disease in Southern Nigeria. Afri Health Sci.2019;19(2): 2100-2111. https://dx.doi.org/10.4314/ahs.v19i2.34 Assessment of community knowledge, attitude, and stigma of Buruli ulcer disease in Southern Nigeria Charles Chukwunalu Nwafor1, Anthony Meka1, Joseph Ngozi Chukwu1, Ngozi Ekeke1, Chukwuka Alphonsus1, Obinna Mbah1, Nelson Okechukwu Madichie2, Ufuoma Aduh3, Matthew Ogbeifo3, Bola Olubakin IseOluwa-Adelokiki4, Joseph Ezebunafor Edochie5, Joseph Ushaka6, Kingsley Nnanna Ukwaja7 1. Medical Department, German Leprosy and Tuberculosis Relief Association, Enugu State, Nigeria. 2. St Leo Hospital Enugu, Enugu State, Nigeria. 3. Delta State Tuberculosis, Leprosy and Buruli Ulcer Control Programme, Delta State, Nigeria. 4. Ogun State Tuberculosis, Leprosy and Buruli Ulcer Control Programme, Ogun State, Nigeria. 5. Anambra State Tuberculosis, Leprosy and Buruli Ulcer Control Programme, Anambra State, Nigeria. 6. Cross River State Tuberculosis, Leprosy and Buruli Ulcer Control Programme, Cross River State, Nigeri 7. Department of Medicine, Federal Teaching Hospital Abakaliki, Ebonyi State, Nigeria. 1. Medical Department, German Leprosy and Tuberculosis Relief Association, Enugu State, Nigeria. 2. St Leo Hospital Enugu, Enugu State, Nigeria. Abstract or knowledge can influence timely care-seeking among persons with Buruli ulcer disease (BUD Background: Poor knowledge can influence timely care-seeking among persons with Buruli ulcer disease (BUD). Objectives: To assess community knowledge, attitude and stigma towards persons with BUD in endemic settings of Southern Nigeria. Objectives: To assess community knowledge, attitude and stigma towards persons with BUD in endemic settings of Southern Nigeria. Methods: This was a cross-sectional survey conducted among adult community members in four States of Southern Nigeria. A semi-structured interviewer-administered questionnaire was administered to all participants. Results: Of 491 adults who completed the survey, 315 (64.2%) belonged to the ≤40 years age group, 257 (52.3%) were males and 415 (84.5%) had some formal education. The overall mean (SD) knowledge score was 5.5±2.3 (maximum 10). Only 172 (35.0%) of the participants had a good knowledge of BUD. A total of 327 (66.6%) considered BUD as a very serious illness. Also, there was a high-level of stigma against BUD patients; 372 (75.8%) of the participants felt compassion for and desire to help them, 77 (15.7%) felt compassion but tended to stay away from them, and 53 (10.8%) feared them because they may infect them with the disease. Having a formal education and ethnicity were independent predictors of good knowledge of BUD.l Results: Of 491 adults who completed the survey, 315 (64.2%) belonged to the ≤40 years age group, 257 (52.3%) were males and 415 (84.5%) had some formal education. The overall mean (SD) knowledge score was 5.5±2.3 (maximum 10). Only 172 (35.0%) of the participants had a good knowledge of BUD. A total of 327 (66.6%) considered BUD as a very serious illness. Also, there was a high-level of stigma against BUD patients; 372 (75.8%) of the participants felt compassion for and desire to help them, 77 (15.7%) felt compassion but tended to stay away from them, and 53 (10.8%) feared them because they may infect them with the disease. Having a formal education and ethnicity were independent predictors of good knowledge of BUD. Conclusion: There is poor community knowledge of BUD in endemic settings of Southern Nigeria which influenced the atti- tude and perceptions of community members towards persons with BUD Keywords: Mycobacterium ulcerans disease, knowledge, perceptions, practices. Instruments and data collection The study instrument was an interviewer-administered questionnaire which had been piloted and validated. This questionnaire had four sections namely: demographics; knowledge of BUD; attitude towards the BU disease sufferers; and stigma towards persons with BUD. The knowledge component consisted of 10 questions cover- ing the awareness, aetiology, clinical presentation, trans- mission, treatment and prevention. The questions con- sisted of factual statements that participants responded to with “yes”, “no” or “I don’t know” options. A scoring system was applied to assess the level of knowledge of each respondent and one point was awarded for each cor- rect answer. No point was given for an incorrect answer. In addition, participants were also asked further specif- ic questions regarding their perceptions of the causes, transmission, treatment and prevention of BUD. Five questions each related to attitudes towards the disease, and stigma against persons suffering from the disease were also applied. Background Since then, until recently, there have been scanty reports from different States in Southern Ni- geria4-5. Recently, in 2012 a BUD prevalence of 18.7 per 100,000 population (range: 6.0 to 41.4 per 100,000) was reported in Ogoja territory, Nigeria6. So far, BUD cases have been reported from over 8 States of Nigeria viz. Ad- amawa, Benue, Cross River, Akwa Ibom, Enugu, Anam- bra, Delta, Ogun and Oyo4-8. Recent reports from Nigeria indicate that the number of new BUD cases diagnosed is increasing every year6-8. As a result, there is a growing interest in BUD in the country and case finding strategies for BUD in Nigeria are being scaled-up6-8. Okpe and Ethiope East (Delta State) and Yewa South and Yewa North (Ogun State) having an estimated population of more than one million people constituted the study site. The study participants were heads of households or the next available adult (>18 years) in a selected house- hold in the study communities. Materials & methods Study design This was a community-based cross-sectional survey of BUD-endemic settings in Southern Nigeria between July and August 2016. Sampling Using household census data obtained from each of the selected local government area administrative headquar- ters, 65 households were selected through simple random sampling from each participating local government area (i.e., 130 households per State). In each selected house- hold, the head of the household or the next available adult was interviewed. BUD patients have been found to have substantial delays in seeking appropriate care thereby incurring catastrophic costs as well as acquiring secondary bacterial infections as complications7-8,10. Thus, due to late presentation, they may require extensive surgery and management of the resultant disability9. There is a paucity of research on community knowledge of BUD in endemic settings of West Africa11-14. The WHO recommends evaluation of community-level understanding of BUD in order to de- sign culturally-appropriate and behaviourally-feasible pre- vention and treatment interventions15. This data is crucial in informing the need for changing policies in improv- ing BUD control programme. Therefore, the aim of this study was to assess community knowledge, attitude and stigma of BUD in endemic communities in Southern Ni- geria. Background factors of M. ulcerans infection is contact with swampy areas and swimming in slow-flowing rivers or streams2-3. BUD occurs mainly in tropical and subtropical regions except in Australia, China and Japan1. Most cases of BUD reported from Africa annually come mainly from West and Central Africa, including Benin, Cameroon, Côte d’Ivoire, Democratic Republic of the Congo and Gha- na1. In 2014, 12 of these 15 countries that regularly report data to the World Health Organization (WHO) notified 2200 new cases of BUD2. This figure was more than 50% less than the number of cases they notified in 2009 – in- dicating that except for a few countries, the number of BUD cases has declined since 20102. Buruli ulcer disease (BUD) is a skin disease caused by My- cobacterium ulcerans-a bacterium related to those that cause tuberculosis and leprosy1-3. The exact mode of the disease transmission to humans is still not clear1. The main risk Corresponding author: Kingsley Nnanna Ukwaja, Department of Medicine, Federal Teaching Hospital Abakaliki, Ebonyi State, Nigeria Email: ukwajakingsley@yahoo.co.uk and Central d’Ivoire, Dem na1. In 2014, 1 data to the W 2200 new case less than the n dicating that BUD cases ha African African Health Sciences © 2019 Nwafor et al. Licensee African Health Sciences. This is an Open Access article di License (https://creativecommons.org/licenses/BY/4.0), which permits unrestricted use, di work is properly cited. 2100 Corresponding author: Kingsley Nnanna Ukwaja, Department of Medicine, Federal Teaching Hospital Abakaliki, Ebonyi State, Nigeria Email: ukwajakingsley@yahoo.co.uk and Central Africa, including Benin, Cameroon, Côte d’Ivoire, Democratic Republic of the Congo and Gha- na1. In 2014, 12 of these 15 countries that regularly report data to the World Health Organization (WHO) notified 2200 new cases of BUD2. This figure was more than 50% less than the number of cases they notified in 2009 – in- dicating that except for a few countries, the number of BUD cases has declined since 20102. African Health Sciences Vol 19 Issue 2, June, 2019 African Health Sciences © 2019 Nwafor et al. Licensee African Health Sciences. This is an Open Access article distributed under the terms of the Creative commons Attribution License (https://creativecommons.org/licenses/BY/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2100 African Health Sciences Vol 19 Issue 2, June, 2019 In Nigeria, BUD was first reported from Benue in 1967 in four patients4. Sample size Sample size was calculated using OpenEpi 16. A min- imum sample of 384 persons will be able to detect an estimated 50% prevalence of community members with good knowledge of BUD at 95% confidence level and an absolute sampling error of 0.05. In this study, we in- creased the sample size to 520 households to allow for attrition and further multivariable analysis. Study area and population The study was carried out in four States (Cross River, Anambra, Delta and Ogun) in Southern Nigeria. The States belong to the tropical rain-forest belt characterised by several features including rivers and swamps. In each of the selected States two local government areas (basic management units) notifying the highest number of BUD cases where an active case finding intervention for BUD was ongoing were used as the study sites. Thus, eight local government areas consisting of Ogoja and Yala (Cross River State), Ogbaru and Anambra East (Anambra State), The survey instrument was reviewed by a group of aca- Ethical approval The study was approved by the Ethics and Research Ad- visory Board of the German Leprosy and TB Relief As- sociation, Nigeria. Approval was also obtained from the State TB, Leprosy and Buruli ulcer Control Programme in the study States. All participants gave an oral informed consent to participate in the survey. Two locally recruited research assistants with higher ed- ucation and had participated in a standardised training session were recruited to administer the questionnaire in each State. Demographic characteristics of respondents g p p A total of 491 respondents with complete data were in- cluded in the final analysis. The respondents consisted of 257 (52.3%) male, and 315 (64.2%) were aged ≤40 years (Table 1). Most of the respondents were Christians 453 (92.3%), married 351 (71.5%), farmers 415 (84.5%), and had formal education i.e., completed at least six years of schooling 416 (84.7%). Only 66 (13.5%) of the re- spondent households had a regular income source, 226 (46.0%) had irregular income sources and 119 (40.5%) had no defined income source (Table 1). Data analysis The data were double-entered, and analysed using Epi Info 3.4.1 (CDC, Atlanta, GA USA). A composite score for knowledge of BUD was estimated for each respon- dent. Respondents with a knowledge score of ≥70% were considered to have good knowledge and those with <70% were considered to have poor knowledge of BUD. Continuous variables were summarised as means (± stan- dard deviation SD) and while categorical variables were summarised as proportions. Group comparisons were 2102 African Health Sciences Vol 19 Issue 2, June, 2019 African Health Sciences Vol 19 Issue 2, June, 2019 African Health Sciences Vol 19 Issue 2, June, 2019 2101 demics, infectious disease physicians, epidemiologists and public health physicians within the State Tuberculosis, Leprosy and Buruli ulcer Control Programmes of Cross River, Anambra, Delta and Ogun States who considered it to have face validity. Minor modifications were made to the initial questionnaire following their review. Pre-test- ing was performed among 20 adult community members in Ayamelum local government area, Anambra State not used for the survey which also led to minor changes in the initial questionnaire. made using the χ2 test. A multivariable logistic regression analysis was performed to identify predictors of good knowledge of BUD. A p-value <0.05 was considered sig- nificant. African Health Sciences Vol 19 Issue 2, June, 2019 Table 1: Socio-demographic characteristics of the respondents (N = 491) Variables n (%) Age group (years) ≤40 315 (64.2) >40 176 (35.8) Gender Female 234 (47.7) Male 257 (52.3) Educational status No formal education 75 (15.3) Primary 153 (31.2) Secondary 211 (43.0) Tertiary 52 (10.6) Marital status Single 118 (24.0) Married 351 (71.5) Widowed 22 (4.5) Ethnic group Igbo 128 (26.1) Other 250 (50.9) Yoruba 113 (23.0) Religion Christianity 453 (92.3) Islam 18 (3.7) Traditional religion 20 (4.1) Occupation Civil service 44 (9.0) Farmer 415 (84.5) Other 32 (6.5) Monthly household income sources Irregular 226 (46.0) No defined income 119 (40.5) Regular income 66 (13.5) Table 1: Socio-demographic characteristics of the respondents (N = 491) Monthly household income sources Irregular No defined income Regular income BUD (Table 2). Older individuals (>40years) tended to have better awareness of BUD compared with young- er respondents (86.9% vs. 77.5%; p = 0.01). However, they were less likely to identify a key risk factor of BUD (24.4% vs. 34.6%; p = 0.02), less likely to know that BUD can be prevented (50.6% vs. 57.8%; p = 0.017), less likely to identify one correct preventive measure against BUD (25% vs. 39.1%; p = 0.002). Overall, only 172 (35.0%; 95% C.I. 30.8 to 39.5%) of the respondents had a good knowl- edge of BUD (Table 3).The proportion of respondents with good knowledge of BUD varied across the study States: 46.2% (60/130) in Cross River, 30.2% (38/126) in Anambra, 22.3% (27/121) in Delta, and 41.2% (47/114) in Ogun State (P <0.001). Knowledge of Buruli ulcer disease Knowledge of Buruli ulcer disease Knowledge of Buruli ulcer disease g The respondents were assessed regarding their knowl- edge of BUD (Table 2). The overall mean (SD) knowl- edge score was 5.5±2.3 (maximum 10) suggesting that they generally had a fair knowledge of BUD (Table 2). However, the most knowledge deficit was in identifying a key risk factor of BUD: only 152 (31.0%) of the respon- dents could identify one key risk factor of BUD. Knowledge of Buruli ulcer disease Other major knowledge deficits were: only 240 (48.9%) knew that BUD cannot be transmitted through contact with infected persons, only 271 (55.2%) of the respondents knew that BUD can be prevented and only 167 (34.0%) identified at least one correct preventive measure against 2103 African Health Sciences Vol 19 Issue 2, June, 2019 Table 2: Respondents knowledge of Buruli ulcer disease according to their age groups Variable n (% ) correct Total n (% ) correct (≤40 years) n (% ) correct (>40 years) P-value Overall 491 315 176 Ever heard about the Buruli ulcer disease 397 (80.9) 244 (77.5) 153 (86.9) 0.01 Had an information source of Buruli ulcer disease (N = 397) 397 (100) 244 (100) 153 (100) 0.99 Identified a key risk factor of Buruli ulcer disease 152 (31.0) 109 (34.6) 43 (24.4) 0.02 Identified two or more clinical presentation of Buruli ulcer disease 378 (77.0) 237 (75.2) 141 (80.1) 0.22 Knew that Buruli ulcer cannot be transmitted through contact with infected persons 240 (48.9) 146 (46.3) 94 (53.4) 0.20 Knew that Buruli ulcer disease cannot be transmitted through formites 454 (92.5) 291 (92.4) 163 (92.6) 0.92 Knew that Buruli ucer disease can be prevented 271 (55.2) 182 (57.8) 89 (50.6) 0.017 Identified at least one correct preventative method against Buruli ulcer 167 (34.0) 123 (39.1) 44 (25.0) 0.002 Knew that Buruli ulcer disease could be cured 409 (83.3) 257 (81.6) 152 (86.4) 0.39 Identified that Buruli ulcer disease could be cured though modern medications and surgery 332 (67.6) 211 (67.0) 121 (68.8) 0.69 Table 2: Respondents knowledge of Buruli ulcer disease according to their age groups Table 3: Proportions of respondents’ knowledge of Buruli ulcer according to their age group Variable Total ≤40 years >40years n (%; 95% CI) n (%; 95% CI) n (%; 95% CI) Overall 491 315 176 % of knowledge score ≤49 132 (26.9; 23.1 – 31.1 ) 87 (27.6; 22.8 – 33.0) 45 (25.6; 19.3 – 32.7) 50 – 59 112 (22.8; 19.2 – 26.8 ) 71 (22.5; 18.1 – 27.6) 41 (23.3; 17.3 – 30.2) 60 – 69 75 (15.3; 12.3 – 18.8) 47 (14.9; 11.3 – 19.5) 28 (15.9; 10.8 – 22.2) ≥70 172 (35.0; 30.8 – 39.5) 110 (34.9; 29.7 – 40.5) 62 (35.2; 28.2 – 42.8) Knowledge level Good 172 (35.0; 30.8 – 39.5) 110 (34.9; 29.7 – 40.5) 62 (35.2; 28.2 – 42.8) Poor 319 (65.0; 60.5 – 69.2) 205 (65.1; 59.5 – 70.3) 114 (64.8; 57.2 – 71.8) Table 3: Proportions of respondents’ knowledge of Buruli ulcer according to their age group Some 221 (45.0%) of the respondents indicated that they did not know the aetiology of BUD, 102 (20.8%) per- ceived that the disease was caused by an infective organ- ism, 105 (21.4%) perceived it to be caused by witchcraft, 95 (19.3%) perceived the disease to be caused by contact with swamps, and 63 (12.8%) perceived it was caused by swimming in rivers/streams (Figure 1). Knowledge of Buruli ulcer disease African Health Sciences Vol 19 Issue 2, June, 2019 Knowledge of Buruli ulcer disease Furthermore, the majority 372 (75.8%) of the respondents indicated that they did not know the mode of transmission of BUD. However, a few of the respondents believed it could be transmitted through aerosol 36 (7.3%); while others be- lieved it could be transmitted through fomites in public places 37 (7.5%); sharing of cups for drinking water 24 (4.9%), or eating from the same plates with affected per- sons 20 (4.1%). In addition, the respondents had a var- ied perception on the preventability of BUD (Figure 1). African Health Sciences Vol 19 Issue 2, June, 2019 2104 Some of the respondents perceived that the disease could be prevented by: avoiding swimming in rivers and streams 89 (18.1%), wearing protective clothing in swamps 78 (15.9%), or drinking clean/portable water 68 (13.8%). Some of the respondents perceived that the disease could be prevented by: avoiding swimming in rivers and streams 89 (18.1%), wearing protective clothing in swamps 78 (15.9%), or drinking clean/portable water 68 (13.8%). Furthermore, 332 (67.6%) of the respondents perceived that BUD can be cured using modern medications and surgery, 173 (35.2%) perceived it could be cured by herbal remedies only, and 69 (14.1%) perceived it could be cured through prayers (Figure 1) Figure 1: Respondents knowledge of the aetiology, transmission, treatment and prevention of Buruli ulcer in Southern Nigeria Figure 1: Respondents knowledge of the aetiology, transmission, treatment and prevention of Buruli ulcer in Southern Nigeria Figure 1: Respondents knowledge of the aetiology, transmission, treatment and prevention of Buruli ulcer in Southern Nigeria Attitudes to Buruli ulcer disease A total of 327 (66.6%) and 55 (11.2%) considered BUD as a very serious or a somewhat serious illness, respec- tively (Table 4). Also, 189 (38.5%) and 72 (14.7%) con- sidered BUD to be a very serious or a somewhat serious illness in their community, respectively. The respondents’ attitude to being diagnosed with BUD mainly includes; fear 120 (24.4%), sadness or hopelessness 116 (23.6%), Attitudes to Buruli ulcer disease surprise 112 (22.8%) and shame 50 (10.2%). Although only 130 (26.5%) of the respondents had a positive at- titude towards the curability of BUD, most indicated that their first source of advice/help following being diagnosed with the disease is a healthcare worker, 257 (52.3%), others indicated that they would seek help from their spouse 74 (15.1%), parents 55 (11.2%) or a close friend 25 (5.1%). African Health Sciences Vol 19 Issue 2, June, 2019 2105 Table 4: Attitude to Buruli ulcer disease among the study respondents (N = 491) Table 4: Attitude to Buruli ulcer disease among the study respondents (N = 491) Variable n (%) Attitude to seriousness of Buruli ulcer disease as an illness Very serious 327 (66.6) Somewhat serious 55 (11.2) Not very serious 60 (12.2) Don’t know 49 (10.0) Attitude to seriousness of Buruli ulcer disease in respondent’s community Very serious 189 (38.5) Somewhat serious 72 (14.7) Not very serious 165 (33.6) Don’t know 65 (13.2) Attitude to the curability of Buruli ulcer disease Yes 130 (26.5) No 283 (57.6) Don’t know 78 (15.9) Respondent’s reaction to being diagnosed with Buruli ulcer disease Fear 120 (24.4) Surprise 112 (22.8) Shame 50 (10.2) Sadness or hopelessness 116 (23.6) Others 93 (18.9) Respondents’ first source of advice/help following being diagnosed with Buruli ulcer disease Healthcare worker / doctor 257 (52.3) Spouse 74 (15.1) Parent 55 (11.2) Close friend 25 (5.1) No one 19 (3.9) Others 61 (12.4) (56.4%) of them indicated that they are allowed to freely interact with persons with the disease, 388 (79.0%) indi- cated that they don’t allow their children to freely interact with persons with the disease, 403 (82.1%) reported that they don’t allow persons with BUD to work as teachers in the community, and 385 (78.4%) indicated that they do not welcome persons with BUD into their homes. Only 202 (41.1%) of the respondents perceived that some in- dividuals are more likely to develop BUD than others. However, when asked to identify such individuals, their answers ranged from men only 31 (6.3%) or women only 31 (6.3%), to children 107 (21.8%), and only adults 150 (30.5%). (56.4%) of them indicated that they are allowed to freely interact with persons with the disease, 388 (79.0%) indi- cated that they don’t allow their children to freely interact with persons with the disease, 403 (82.1%) reported that they don’t allow persons with BUD to work as teachers in the community, and 385 (78.4%) indicated that they do not welcome persons with BUD into their homes. Only 202 (41.1%) of the respondents perceived that some in- dividuals are more likely to develop BUD than others. However, when asked to identify such individuals, their answers ranged from men only 31 (6.3%) or women only 31 (6.3%), to children 107 (21.8%), and only adults 150 (30.5%). Community stigma towards persons with Buruli ul- cer disease A total of 344 (70.1%) of the respondents knew a person who have or have had BUD in their community (Table 5). Also, respondents were asked what they felt for per- sons with BUD (Table 5): 372 (75.8%) felt compassion and desire to help them, 77 (15.7%) felt compassion but tended to stay away from them, 53 (10.8%) feared them because they may infect them with the disease, and 30 (6.1%) indicated that they have no particular feelings to- wards them. The respondents were further asked how persons with BUD are treated in their community: 277 2106 African Health Sciences Vol 19 Issue 2, June, 2019 African Health Sciences Vol 19 Issue 2, June, 2019 African Health Sciences Vol 19 Issue 2, June, 2019 Table 5: Community perceptions and stigma towards persons with Buruli ulcer disease (N = 491) Variable n (%) Knew a person/persons who have or have had Buruli ulcer disease Yes 344 (70.1) No 114 (23.2) Don’t know 33 (66.7) What the respondents feels for persons with Buruli ulcer disease Feels compassion and desire to help 372 (75.8) Feels compassion but tended to stay away from them 77 (15.7) Fears them because they may infect him/her 53 (10.8) Have no particular feelings towards them 30 (6.1) Others 40 (8.1) How persons with Buruli ulcer disease are treated in the community We are allowed to freely interact with persons with Buruli ulcer 277 (56.4) We don’t allow our children freely interact with a Buruli ulcer patient 388 (79.0) We don’t allow persons with Buruli ulcer to work as teachers in the community 403 (82.1) We don’t We don’t allow persons with Buruli ulcer to work as teachers in the community 385 (78.4) Most people are friendly to them, but they generally try to avoid him or her 90 (18.3) The community mostly do not support and/or help them 355 (72.3) Other Respondents perception on some persons being more likely to develop Buruli ulcer disease than others Yes 202 (41.1) No 185 (37.7) Not sure 104 (21.2) In your opinion, which persons are more likely to develop Buruli ulcer disease Men 31 (6.3) Women 31 (6.3) Adult male and females 150 (30.5) Children 107 (21.8) Others 42 (8.6) I don’t know 81 (16.5) who are from the Igbo and Yoruba ethnic groups were more likely to have good knowledge of BUD compared to those from “other” ethnic groups (p = 0.004). Bivari- ate and multivariable logistic regression analysis was per- formed to determine socio-demographic predictors of good knowledge of BUD among the respondents (Table S2). The independent predictors for good knowledge of BUD among the respondents were: completing a prima- ry adjusted odds ratio (aOR) 2.2, 95% CI 1.04 –5.5 or secondary (aOR 3.0, 95% CI 1.4 – 6.1) or a tertiary edu- cation (aOR 4.5, 95% CI 1.9 – 10.5). Further more, be- longing to the “other ethnic group” was an independent predictor of lower knowledge of BUD (aOR 0.6, 95% CI 0.4 – 0.9). African Health Sciences Vol 19 Issue 2, June, 2019 Factors associated with knowledge of Buruli ulcer disease The relationship between the socio-demographic charac- teristics of the respondents and knowledge of BUD are as shown in (Supplementary Tables S1, and S2; Appendix 1). Good knowledge of BUD did not differ according to age (p = 0.95) or gender (p = 0.70) categories. Also, good knowledge of BUD did not differ according to marital status (p = 0.56), religion (p = 0.61), occupation (p = 0.16) or household income sources (p = 0.09). Howev- er, respondents with a formal education were more like- ly to have good knowledge of BUD compared to those with no formal education (p = 0.009), and respondents African Health Sciences Vol 19 Issue 2, June, 2019 2107 Table S1: Relationship between respondents profile and knowledge of Buruli ulcer disease in Nigeria Variables Poor knowledge n (%) Good knowledge n (%) Chi-square P-value Age (years) 0.005 0.95 ≤40 205 (65.1) 110 (34.9) >40 114 (64.8) 62 (35.2) Gender 0.15 0.70 Female 150 (64.1) 84 (35.9) Male 169 (65.8) 88 (34.2) Educational status 11.5 0.009 No formal education 59 (78.7) 16 (21.3) Primary 97 (63.4) 56 (36.6) Secondary 137 (64.9) 74 (35.1) Tertiary 26 (50.0) 26 (50.0) Marital status 1.1 0.56 Single 81 (68.6) 37 (31.4) Married 225 (64.1) 126 (35.9) Widowed 13 (59.1) 9 (40.9) Ethnic group 11.2 0.004 Igbo 75 (58.6) 53 (41.4) Other 180 (72.0) 70 (28.0) Yoruba 64 (56.6) 49 (43.4) Religion 0.99 0.61 Christianity 297 (65.6) 156 (34.4) Islam 10 (55.6) 8 (44.4) Traditional religion 12 (60.0) 8 (40.0) Occupation 3.6 0.16 Civil service 23 (52.3) 21 (47.7) Farmer 276 (66.5) 139 (33.5) Other 20 (62.5) 12 (37.5) Income sources 4.8 0.09 Irregular 150 (66.4) 76 (33.6) No defined source 134 (67.3) 65 (32.7) Regular 35 (53.0) 31 (47.0) ween respondents profile and knowledge of Buruli ulcer disease in Nigeria able S1: Relationship between respondents profile and knowledge of Buruli ulcer disease in Nigeria 2108 African Health Sciences Vol 19 Issue 2, June, 2019 African Health Sciences Vol 19 Issue 2, June, 2019 African Health Sciences Vol 19 Issue 2, June, 2019 Discussion propriately perceived that wearing a mask to cover their nose and mouth as well as drinking potable water could prevent BUD. Thus, despite a high awareness of BU in the community, respondents generally showed a poor knowledge of its prevention. In addition, we observed some variation in the proportion of respondents with good knowledge of BUD in the study States which fol- lowed the pattern of the integration of the States with the BUD Control Programme. Some of the observations in this study disagree with those of Akoachere et al.,14 in Cameroun who found that almost half (49.4%) of their respondents thought that BUD could be transmitted from one person to another. This study has shown that majority of individuals living in endemic settings in Nigeria had poor knowledge of BUD. Only about a fifth of them knew that the disease is caused by an infective organism; and similar proportions reported that it may be caused by contact with swamps or witchcraft. Our finding was consistent with reports from Cameroon and Ghana which showed a high variation in community knowledge of the aetiology of BUD with a substantial proportion attributing the disease to witch- craft11-14. This wrong knowledge of the cause of BUD led affected persons to consult traditional medical practi- tioners and faith healers for help7,9,11. Although majority of the respondents indicated not knowing the mode of transmission of BUD, only very few (<10% each) inap- propriately perceived that the disease could be transmit- ted through fomites, aerosol droplet, handshake or shar- ing of household items like cups and plates. It is crucial that in undertaking further health education programme in populations with BUD there is a need to highlight that there is no evidence of BUD transmission through these means. Also, we found that a relatively high proportion of the respondents believed in the preventability of BUD. This suggests that the ongoing sensitisation and outreach pro- grammes in BUD-endemic settings of Southern Nigeria may have contributed to an improved knowledge of the preventability of the disease. This is unlike a similar study in Cameroon which found that more than half of the re- spondents believed BUD cannot be prevented suggesting that the community sensitization on BUD in the study area is insufficient and needs to be reinforced14. African Health Sciences Vol 19 Issue 2, June, 2019 Table S2: Logistic regression analysis of factors associated with good knowledge of Buruli ulcer disease in Nigeria Variables Good knowledge Crude OR Adjusted OR Adjusted n (%) (95% CI) (95% CI) P-value Age (years) ≤40 110 (34.9) 1 1 >40 62 (35.2) 1.01 (0.7 – 1.5) 1.2 (0.7 – 1.8) 0.55 Gender Female 84 (35.9) 1.1 (0.7 – 1.6) 1.1 (0.7 – 1.6) 0.79 Male 88 (34.2) 1 1 Educational status No formal education 16 (21.3) 1 1 Primary 56 (36.6) 2.2 (1.2 – 4.2) 2.7 (1.4 – 5.5) 0.005 Secondary 74 (35.1) 2.0 (1.1 – 3.7) 3.0 (1.4 – 6.1) 0.004 Tertiary 26 (50.0) 3.4 (1.6 – 7.4) 4.5 (1.9 – 10.5) <0.001 Marital status Single 37 (31.4) 0.8 (0.5 – 1.3) 0.8 (0.5 – 1.4) 0.44 Married 126 (35.9) 1 1 Widowed 9 (40.9) 1.2 (0.5 – 3.0) 2.6 (0.9 – 7.0) 0.07 Ethnic group Igbo 53 (41.4) 1 1 Other 70 (28.0) 0.6 (0.4 – 0.9) 0.6 (0.4 – 0.9) 0.02 Yoruba 49 (43.4) 1.1 (0.7 – 1.8) 1.4 (0.8 – 2.4) 0.27 Income sources Irregular 76 (33.6) 1 1 No defined source 65 (32.7) 1.02 (0.7 – 1.5) 1.1 (0.7 – 1.6) 0.76 Regular 31 (47.0) 1.7 (1.0 – 2.9) 1.6 (0.7 – 1.6) 0.10 OR; odds ratio, 95% CI = 95% confidence interval Table S2: Logistic regression analysis of factors associated with good knowledge of Buruli ulcer disease in Nigeria African Health Sciences Vol 19 Issue 2, June, 2019 Discussion Some of the preventive measures mentioned by the respondents who perceived BUD to be preventable in this study have been reported in previous studies17-19. Furthermore, less than one-sixth of the respondents correctly knew that wearing of protective clothing and avoiding swimming in rivers and swamps could help in the prevention of BUD. Some of the respondents inap- African Health Sciences Vol 19 Issue 2, June, 2019 2109 This study revealed some positive attitudes towards BUD with the majority of respondents perceiving the illness to be a serious problem in their communities. Only few of the respondents had a positive attitude towards the cur- ability of the disease. As a result, the majority of respon- dents will react to a diagnosis of BUD by being afraid, ashamed, surprised or depressed. However, majority of them indicated a positive attitude towards appropriate care-seeking by identifying a health worker / doctor as their choice for advice if they develop BUD. This study has some strengths and limitations. A key strength of this study was it was carriedout in communi- ties in Nigeria where little or no literature is available. In addition, it was performed in four States with the highest burden of BUD in Nigeria. Therefore, the findings of this study can help stakeholders and other health poli- cymakers to plan culturally-appropriate and behavioural- ly-feasible community education and prevention inter- ventions against BUD in the country. However, as this was a descriptive cross-sectional study we cannot make any causal inferences. Moreover, there may be other con- founders and predictors of knowledge of BUD not ex- plored. A qualitative study would strengthen the findings of this study and improve upon these limitations. This study revealed some positive attitudes towards BUD with the majority of respondents perceiving the illness to be a serious problem in their communities. Only few of the respondents had a positive attitude towards the cur- ability of the disease. As a result, the majority of respon- dents will react to a diagnosis of BUD by being afraid, ashamed, surprised or depressed. However, majority of them indicated a positive attitude towards appropriate care-seeking by identifying a health worker / doctor as their choice for advice if they develop BUD. y p Although majority of the respondents indicated a willing- ness to help BUD sufferers, we found a high level of so- cial stigma towards persons with BUD. Discussion This is consistent with other published studies which reported on stigma and discrimination faced by patients with BUD11,20,21. This high level of social stigma e.g., not allowing persons with BUD to work as teachers, suggests that the stigma demon- strated may be due to fear of contacting the disease11,21. However, in Ghana there was a high level of acceptance of BUD-affected persons with persistent community ed- ucation and exposure to these persons. Stienstra et al.,11 hypothesised that there may be a link between reduced stigma and burden of BUD in a given area i.e., a greater level of acceptance of BUD because of familiarity with the disease in an area leads to increased case detection and high prevalence rates. Conclusion We found that there is poor knowledge of BUD in en- demic settings of Nigeria which influenced the attitude of community members and increased stigma towards persons with BUD. We recommend that the National Tu- berculosis, Leprosy and Buruli Ulcer Control Programme of Nigeria should strengthen community education pro- grammes on the presentation, known risk factors of the disease and its preventive measures in order to improve community attitudes and reduce stigma towards persons with BUD. Further emphasis of such educational pro- grammes should be on early recognition of symptoms of BUD and prompt referral for appropriate care. Only educational status and ethnicity were found to be independent predictors of good knowledge of BUD. We found that all levels of formal education were predictors of good knowledge of BUD. Improved education of community members could contribute to easy dissemina- tion of information. The role of education in improved community knowledge of BUD has been shown by stud- ies in Ghana and Cameroun12,14,22. Education remains a key instrument in driving social change and helps in changing ones perception about a disease e.g., acceptance of BUD patients by community members. We also found that ethnicity was an independent predictor of knowledge of BUD with patients belonging to the “other” smaller ethnic groups (besides the major Igbo and Yoruba eth- nic groups) in the study setting had poorer knowledge of BUD. These communities and the health care workers serving them will benefit from further targeted educa- tional programmes in order to improve their knowledge of the disease23. References 1. Sizaire V, Nackers F, Comte E, Portaels F. Mycobacte- rium ulcerans infection: control, diagnosis, and treatment. Lancet Infect Dis. 2006; 6 (5): 288-296. 1. Sizaire V, Nackers F, Comte E, Portaels F. Mycobacte- rium ulcerans infection: control, diagnosis, and treatment. Lancet Infect Dis. 2006; 6 (5): 288-296. 2. WHO. Buruli ulcer disease factsheet. Geneva: World Health Organization; 2017. http://www.who.int/media- centre/factsheets/fs199/en/ 2. WHO. Buruli ulcer disease factsheet. Geneva: World Health Organization; 2017. http://www.who.int/media- centre/factsheets/fs199/en/ 3. Wansbrough-Jones M, Phillips R. Buruli ulcer: emerg- ing from obscurity. Lancet 2006; 367(9525): 1849-1858. 4. Gray HH, Kingma S, Kok SH. Mycobacterial skin ul- cers in Nigeria. Trans R Soc Trop Med Hyg. 1967; 61 (5): 712–714. 3. Wansbrough-Jones M, Phillips R. Buruli ulcer: emerg- ing from obscurity. Lancet 2006; 367(9525): 1849-1858. 4. Gray HH, Kingma S, Kok SH. Mycobacterial skin ul- cers in Nigeria. Trans R Soc Trop Med Hyg. 1967; 61 (5): 712–714. 5. Oluwasanmi JO, Solankee TF, Olurin EO, Itayemi SO, Alabi GO, Lucas AO. Mycobacterium ulcerans (Buruli) skin ulceration in Nigeria. Am J Trop Med Hyg. 1976; 25(1): 122–128. African Health Sciences Vol 19 Issue 2, June, 2019 2110 15. World Health Organization. Report of the 5th WHO Advisory Group Meeting on Buruli Ulcer: March 11-14 2002. Geneva, Switzerland 2003. 6. Ukwaja KN, Meka AO, Chukwuka A, Asiedu KB, Hu- ber KL, Eddyani M, et al. Buruli ulcer in Nigeria: Results of a pilot case study in three rural districts. Infect Dis Pov- erty. 2016;5:39 15. World Health Organization. Report of the 5th WHO Advisory Group Meeting on Buruli Ulcer: March 11-14 2002. Geneva, Switzerland 2003. 16. Dean AG, Sullivan KM, Soe MM. OpenEpi: Open Source Epidemiologic Statistics for Public Health, Ver- sion. www.OpenEpi.com, updated 2014/09/22. (ac- cessed 2016/05/24). 16. Dean AG, Sullivan KM, Soe MM. OpenEpi: Open Source Epidemiologic Statistics for Public Health, Ver- sion. www.OpenEpi.com, updated 2014/09/22. (ac- cessed 2016/05/24). 7. Meka AO, Chukwu JN, Nwafor CC, Oshi DC, Madi- chie NO, Ekeke N, et al. Diagnosis delay and duration of hospitalisation of patients with Buruli ulcer in Nigeria. Trans R Soc Trop Med Hyg. 2016; 110 (9): 502-509 17. Raghunathan PL, Whitney EA, Asamoa K, Stienstra Y, Taylor TH Jr, Amofah GK, et al. Risk factors for Bu- ruli ulcer disease (Mycobacterium ulcerans infection): results from a case-control study in Ghana. Clin Infect Dis. 2005; 40 (10):1445–1453. 8. References Chukwu JN, Meka AO, Nwafor CC, Oshi DC, Madi- chie NO, Ekeke N, et al. Financial burden of health care for Buruli ulcer patients in Nigeria: the patients' perspec- tive. Int Health. 2017; 9 (1): 36-43. for Buruli ulcer patients in Nigeria: the patients' perspec- tive. Int Health. 2017; 9 (1): 36-43. 18. Pouillot R, Matias G, Wondje CM, Portaels F, Valin N, Ngos F, et al. Risk factors for Buruli ulcer: a case control study in Cameroon. PLoS Negl Trop Dis. 2007; 1(3) :e101. 9. Huang GKL, Johnson PD. Epidemiology and manage- ment of Buruli ulcer. Expert Rev Anti Infect Ther. 2014; 12(7): 855–865. 19. Kenu E, Nyarko KM, Seefeld L, Ganu V, Käser M, Lartey M, et al. Risk factors for Buruli ulcer in Ghana-a case control study in the Suhum-Kraboa-Coaltar and Akuapem South Districts of the Eastern region. PLoS Negl Trop Dis. 2014; 8(11) :e3279. 10. Anyim MC, Meka AO, Chukwu JN, Nwafor CC, Oshi DC, Madichie NO, et al. Secondary bacterial isolates from previously untreated Buruli ulcer lesions and their antibi- otic susceptibility patterns in Southern Nigeria. Rev Soc Bras Med Trop. 2016; 49 (6): 746-751 11. Stienstra Y, van der Graaf WT, Asamoa K, van der Werf TS. Beliefs and attitudes toward Buruli ulcer in Ghana. Am J Trop Med Hyg. 2002; 67 (2): 207–213. 20. Tawiah PE, Adongo PB, Aikins M. Mental Health-Re- lated Stigma and Discrimination in Ghana: Experience of Patients and Their Caregivers. Ghana Med J. 2015; 49(1):30-6. Werf TS. Beliefs and attitudes toward Buruli ulcer in Ghana. Am J Trop Med Hyg. 2002; 67 (2): 207–213. 12. Renzaho AM, Woods PV, Ackumey MM, Harvey SK, Kotin J. Community-based study on knowledge, attitude and practice on the mode of transmission, prevention and treatment of the Buruli ulcer in Ga West District, Ghana. Trop Med Int Health. 2007; 12 (3): 445-458. 13. Kamga LF, Nsagha DS, Assob JCN, Njunda AL, Ndefon P, Palle JN, Ngowe NM et al. Buruli ulcer in Cameroon: an assessment of the community knowledge pattern. African Journal of Integrated Health. 2013; 02:36-39 14. Akoachere JF, Nsai FS, Ndip RN. A Community Based Study on the Mode of Transmission, Prevention and Treatment of Buruli Ulcers in Southwest Cameroon: Knowledge, Attitude and Practices. PLoS One. 2016; 11(5) :e0156463. 12. Renzaho AM, Woods PV, Ackumey MM, Harvey SK, Kotin J. References Community-based study on knowledge, attitude and practice on the mode of transmission, prevention and treatment of the Buruli ulcer in Ga West District, Ghana. Trop Med Int Health. 2007; 12 (3): 445-458. 21. Ocaya A, Kironde F, Odongo-Aginya FI. Knowledge and attitude towards Buruli ulcer disease in Adjumani dis- trict, Northwestern Uganda. East Afr Med J. 2005; 92 (11): 537-541. 13. Kamga LF, Nsagha DS, Assob JCN, Njunda AL, Ndefon P, Palle JN, Ngowe NM et al. Buruli ulcer in Cameroon: an assessment of the community knowledge pattern. African Journal of Integrated Health. 2013; 02:36-39 14. Akoachere JF, Nsai FS, Ndip RN. A Community Based Study on the Mode of Transmission, Prevention and Treatment of Buruli Ulcers in Southwest Cameroon: Knowledge, Attitude and Practices. PLoS One. 2016; 11(5) :e0156463. 22. Owusu-Sekyere E, Kwame OA, Nkuah JK. Percep- tions and attitudes: the challenge of managing Buruli ul- cer morbidity in Ghana. Int J Sci. 2013; 2(3):16–24 23. Ekeke N, Meka AO, Chukwu JN, Nwafor NC, Al- phonsus C, Mbah OK, et al. Assessment of health care workers’ knowledge, attitude and risk perception of Bu- ruli ulcer disease in Southern Nigeria. Trans R Soc Trop Med Hyg. 2017; 111(5):226-232 2111 African Health Sciences Vol 19 Issue 2, June, 2019 African Health Sciences Vol 19 Issue 2, June, 2019
https://openalex.org/W2981372472	https://europepmc.org/articles/pmc6854102?pdf=render	English	null	Expression of alternative developmental pathways in the cabbage butterfly, <i>Pieris melete</i> and their differences in life history traits	Ecology and evolution	2,019	cc-by	8,775	O R I G I N A L R E S E A R C H O R I G I N A L R E S E A R C H Expression of alternative developmental pathways in the cabbage butterfly, Pieris melete and their differences in life history traits Jian‐Jun Tang1 \| Hai‐Min He2 \| Shao‐Hui Wu3 \| Cao Zou2 \| Fang‐Sen Xue2 \| Lan Xiao4,5 Jian‐Jun Tang1 \| Hai‐Min He2 \| Shao‐Hui Wu3 \| Cao Zou2 \| Fang‐Sen Xue2 \| Lan Xiao4,5 \| Hai‐Min He2 \| Shao‐Hui Wu3 \| Cao Zou2 \| Fang‐Sen Xue2 \| 1College of Computer and Information Engineering, Jiangxi Agricultural University, Nanchang, China 2Institute of Entomology, Jiangxi Agricultural University, Nanchang, China 3Department of Entomology, University of Georgia, Tifton, GA, USA 4School of Education, Huazhong University of Science and Technology, Wuhan, China 5Foreign Language School, Jiangxi Agricultural University, Nanchang, China 1College of Computer and Information Engineering, Jiangxi Agricultural University, Nanchang, China 2Institute of Entomology, Jiangxi Agricultural University, Nanchang, China 3Department of Entomology, University of Georgia, Tifton, GA, USA 4School of Education, Huazhong University of Science and Technology, Wuhan, China 5Foreign Language School, Jiangxi Agricultural University, Nanchang, China Abstract Abstract The seasonal life cycle of the cabbage butterfly, Pieris melete is complicated because there are three options for pupal development: summer diapause, winter diapause, and nondiapause. In the present study, we tested the influence of temperature, day length, and seasonality on the expression of alternative developmental pathways and compared the differences in life history traits between diapausing and directly developing individuals under laboratory and field conditions. The expression of de- velopmental pathway strongly depended on temperature, day length, and seasonal- ity. Low temperatures induced almost all individuals to enter diapause regardless of day length; relatively high temperatures combined with intermediate and longer day lengths resulted in most individuals developing without diapause in the laboratory. The field data revealed that the degree of phenotypic plasticity in relation to devel- opmental pathway was much higher in autumn than in spring. Directly developing individuals showed shorter development times and higher growth rates than did dia- pausing individuals. The pupal and adult weights for both diapausing and directly de- veloping individuals gradually decreased as rearing temperature increased, with the diapausing individuals being slightly heavier than the directly developing individuals at each temperature. Female body weight was slightly lower than male body weight. The proportional weight losses from pupa to adult were almost the same in diapaus- ing individuals and in directly developing individuals, suggesting that diapause did not affect weight loss at metamorphosis. Our results highlight the importance of the expression of alternative developmental pathways, which not only synchronizes this butterfly's development and reproduction with the growth seasons of the host plants but also exhibits the bet‐hedging tactic against unpredictable risks due to a dynamic environment. Correspondence Lan Xiao, School of Education, Huazhong University of Science and Technology, Wuhan 430074, China and Foreign Language School, Jiangxi Agricultural University, Nanchang 330045, China. Email: 185859707@qq.com This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Received: 8 July 2019 \| Revised: 6 September 2019 \| Accepted: 11 September 2019 Received: 8 July 2019 \| Revised: 6 September 2019 \| Accepted: 11 September 2019 Received: 8 July 2019 \| Revised: 6 September 2019 \| Accepted: 11 September 2019 DOI: 10.1002/ece3.5731 K E Y W O R D S bet‐hedging, diapause, direct development, life history trait, Pieris melete, temperature \| 12311 www.ecolevol.org 2 \| MATERIALS AND METHODS The cabbage butterfly, Pieris melete Ménétriés is a serious pest of crucifers in the mountain areas of the Jiangxi Province, PR China and has a multivoltine life cycle with both summer and winter dia- pause in the pupal stage. The effects of temperature and photope- riod on diapause induction and termination have been evaluated in detail in this butterfly species under laboratory and field condi- tions (Xiao, Li, Wei, & Xue, 2008; Xiao, Wu, He, Chen, & Xue, 2012; Xiao, Wu, Wang, Zhu, & Xue, 2009; Xue, Kallenborn, & Wei, 1997). 12312 \| 1 \| INTRODUCTION Understanding the expression of two alternative developmental pathways may be crucial for studying life history evolution and developing successful pest management programs (Nylin, 2001). The cabbage butterfly, Pieris melete Ménétriés is a serious pest of crucifers in the mountain areas of the Jiangxi Province, PR China and has a multivoltine life cycle with both summer and winter dia Therefore, this insect species may serve as an excellent experi- mental model to test the differences in life history traits between the diapausing and directly developing individuals. In the present study, we tested the influence of temperature, day length, and seasonality on the expression of alternative developmental pathways in P. melete under laboratory and field conditions and their differences in larval and pupal development time, pupal weight and growth rate, and adult weight and weight loss, aiming to understand how temperature, day length, and seasonality affect the evolution of their life‐history traits. 12312 \| 1 \| INTRODUCTION However, diapause has another important role that is often ignored—it permits the insects to breed dispersively at different periods, because their chances of survival are greatly enhanced (Danks, 1987; Masaki, 1980; Tauber, Tauber, & Masaki, 1986; Xue & Kallenborn, 1993). Portions of many insect populations are known to enter into diapause while the remaining insects continue to develop and reproduce. For example, 6 years of field observations of summer diapause in the zygaenid moth, Pseudopidorus fasciata have shown that only 20%–28% of individuals of the overwintering generation and 49%–60% of the first genera- tion entered summer diapause as prepupae, while the rest contin- ued to develop and produce the next generation (Xue & Kallenborn, 1998). In the fly, Pegomyia bicolor, 6 years of field observations in- dicated that 41%–70% of individuals that pupated during April 5–7 entered pupal diapause, while the rest continued to emerge and ovi- posit and produced the second generation (Xue, Zhu, & Shao, 2001). In the cabbage beetle, Colaphellus bowringi, there were always some individuals (17%–67%) hatched between August 5 and October 5 entering diapause as adults, while the rest mated and produced the next generation (Xue, Spieth, Li, & Hua, 2002). That part of the pop- ulation entering dormancy (summer or winter diapause) and the rest the population whose development continues have been regarded to constitute a bet‐hedging or risk‐spreading reproductive strat- egy that avoids the situation in which individuals “put all their eggs into one basket” (Hopper, 1999; Masaki, 1980; Tauber et al., 1986; Waldbauer, 1978; Wise, 1980; Xue & Kallenborn, 1993) and sub- jects them all simultaneously to the possibility of meeting unfavor- able environmental conditions. Individual insects that follow these two alternative pathways of diapause and direct development are typically described as presenting alternative developmental path- ways (Aalberg Haugen & Gotthard, 2015; Fischer & Fiedler, 2000; Gotthard & Berger, 2010; Kivelä, Svensson, Tiwe, & Gotthard, 2015; Kivelä, Välimäki, & Gotthard, 2016; Kivelä, Välimäki, & Mäenpää, 2012). However, there have been a few studies showing that the expression of life‐history traits may differ between these path- ways (Aalberg Haugen, Berger, & Gotthard, 2012; Aalberg Haugen & Gotthard, 2015; Chen, Xia, Xiao, Xiao, & Xue, 2014; Gotthard & Berger, 2010; Kivelä, Välimäki, & Gotthard, 2013). Understanding the expression of two alternative developmental pathways may be crucial for studying life history evolution and developing successful pest management programs (Nylin, 2001). 12312 \| 1 \| INTRODUCTION It has been clearly recognized that diapause is an important mech- anisms for synchronizing seasonal development and activity in sub- tropical and temperate zone insects. However, diapause has another important role that is often ignored—it permits the insects to breed dispersively at different periods, because their chances of survival are greatly enhanced (Danks, 1987; Masaki, 1980; Tauber, Tauber, & Masaki, 1986; Xue & Kallenborn, 1993). Portions of many insect populations are known to enter into diapause while the remaining insects continue to develop and reproduce. For example, 6 years of field observations of summer diapause in the zygaenid moth, Pseudopidorus fasciata have shown that only 20%–28% of individuals of the overwintering generation and 49%–60% of the first genera- tion entered summer diapause as prepupae, while the rest contin- ued to develop and produce the next generation (Xue & Kallenborn, 1998). In the fly, Pegomyia bicolor, 6 years of field observations in- dicated that 41%–70% of individuals that pupated during April 5–7 entered pupal diapause, while the rest continued to emerge and ovi- posit and produced the second generation (Xue, Zhu, & Shao, 2001). In the cabbage beetle, Colaphellus bowringi, there were always some individuals (17%–67%) hatched between August 5 and October 5 entering diapause as adults, while the rest mated and produced the next generation (Xue, Spieth, Li, & Hua, 2002). That part of the pop- ulation entering dormancy (summer or winter diapause) and the rest the population whose development continues have been regarded to constitute a bet‐hedging or risk‐spreading reproductive strat- egy that avoids the situation in which individuals “put all their eggs into one basket” (Hopper, 1999; Masaki, 1980; Tauber et al., 1986; Waldbauer, 1978; Wise, 1980; Xue & Kallenborn, 1993) and sub- jects them all simultaneously to the possibility of meeting unfavor- able environmental conditions. Individual insects that follow these two alternative pathways of diapause and direct development are typically described as presenting alternative developmental path- ways (Aalberg Haugen & Gotthard, 2015; Fischer & Fiedler, 2000; Gotthard & Berger, 2010; Kivelä, Svensson, Tiwe, & Gotthard, 2015; Kivelä, Välimäki, & Gotthard, 2016; Kivelä, Välimäki, & Mäenpää, 2012). However, there have been a few studies showing that the expression of life‐history traits may differ between these path- ways (Aalberg Haugen, Berger, & Gotthard, 2012; Aalberg Haugen & Gotthard, 2015; Chen, Xia, Xiao, Xiao, & Xue, 2014; Gotthard & Berger, 2010; Kivelä, Välimäki, & Gotthard, 2013). 12312 \| 1 \| INTRODUCTION 12312 TANG et al. TANG et al. Under laboratory conditions, the photoperiodic response curves in P. melete showed an intermediate response type: that is, short day lengths (8–11 hr) induced winter diapause; the intermediate day lengths (12–13 hr) permitted some pupae to develop without dia- pause, and the long day lengths (14–16 hr) induced summer diapause (Xiao et al., 2009). These studies also revealed that high tempera- tures strongly weakened the diapause‐inducing effects of long day length and significantly reduced the incidence of summer diapause; whereas winter diapause can be induced under short day‐length at relatively high temperatures (Xiao et al., 2009; Xue et al., 1997). In the field, there are two distinct infestation peaks per year, one in the spring and a second in autumn. According to our field observa- tions for 9 years (1988, 1989, 1994, 1995, 2003, 2004, 2005, 2006, and 2007), if the overwintered pupae eclosed into adults between mid‐March and early April (1988, 1989, 1994, 1995, 2003, 2005, and 2007), almost all their progenies would have entered summer diapause and produced one generation. However, if adults emerged between late February and late March, some progenies produced by the early emerged adults would have developed without diapauses (33.33% in 2004; 34.04% in 2006; Xiao et al., 2012), these proge- nies emerged as adults in late April and produced the second gen- eration. In autumn, aestivating individuals emerge between the end of August and early November. Early‐emerging individuals can pro- duce three generations in autumn under conditions of relatively high temperatures and intermediate day lengths. However, late‐emerging individuals produce only one generation because of the relatively low temperature and short day lengths occurring in late autumn. Thus, there are one to three generations in autumn (Xue, Zhu, & Wei, 1996). Furthermore, there are always some individuals entering winter diapause regardless of temperature, as indicated by the fact that 3.85% of individuals in 2003, 4.65% in 2004, and 6.78% of indi- viduals in 2005 that hatched in August entered winter diapause even under high temperatures—from 26.4 to 31.2°C (Xiao et al., 2012). It has been clearly recognized that diapause is an important mech- anisms for synchronizing seasonal development and activity in sub- tropical and temperate zone insects. 2.2 \| Experimental arrangement The data of the mean daily temperature experienced by larvae for each generation were collected from the weather station of Jiangxi Agricultural University. (30 × 30 × 35 cm) for pupation and hibernation and estivation under natural conditions. Adults from the overwintering or aestivating pupae were released to an outdoor web‐screened insectary with cultivated flowering Chinese cabbage, Brassica chinensis for mating and oviposition in the spring or autumn, respectively. Eggs laid on leaves were collected in Petri dishes (height 2 cm; diameter 9.0 cm) lined with moistened filter paper every day and were used to con- duct the experiments. terminate pupal diapause and observe the adult emergence. The number of females and males was recorded daily. terminate pupal diapause and observe the adult emergence. The number of females and males was recorded daily. In the autumn generations, diapausing and nondiapausing pupae were obtained using the same method as that used for the spring generations. Nondiapause pupae generally emerged within 7–21 days in the autumn generations. Thus, each pupa that did not emerge within 25 days was assumed to be in diapause. The diapause pupae were treated under the same conditions as were the aestivat- ing pupae to observe the adult emergence. The diapause pupae of the second autumn generation were maintained under natural con- ditions until adult eclosion the following spring. 2.4 \| Statistical analyses Statistical analyses were conducted using the SPSS 17.0 statistical software package (IBM, www.ibm.com). Life history traits were ana- lyzed in relation to temperature, development pathway, and sex with the general linear model. The nonsignificant three‐way term (tem- perature‐by‐development pathway‐by‐sex) was dropped from the final model in the analysis. One‐way analysis of variance (ANOVA) was used to determine whether there were significant differences in life history traits in different development pathways at each tem- perature. One‐way analysis of variance and Duncan's test were used to compare the differences in life history traits between sexes in each development pathway and at each temperature. Throughout the text, all means are given with ±1 SEs. 2.3 \| Measurement methods For each diapausing and directly developing individual obtained from both laboratory and field conditions, we measured the larval and pupal development time from hatching to pupation and adult eclosion, pupal and adult weight, growth rate, and proportional weight loss at metamorphosis. We calculated the pupal weight on the 2nd day after pupation and adult weight after the release of the meconium by using an electronic balance (AUY120; Shimadzu). The individual growth rate of each larva used in the experiments was calculated according to the methods of Gotthard, Nylin, and Wiklund (1994): Growth rate = ln (pupal weight)/larval time × 100. This formula gives a relative growth rate representing the mean weight gain per day. Weight loss between pupation and adult eclosion was calculated using the following for- mula: proportion weight loss = 1 − (adult weight/pupal weight). 2.2.1 \| Laboratory experiments After hatching, young larvae from the spring generation were reared in Petri dishes (height 2 cm; diameter 9.0 cm) containing moistened filter paper and fresh leaves of B. chinensis with four larvae in a Petri dish. The Petri dishes were randomly divided into four groups and were placed in four illuminated incubators (LRH‐250‐GS, Guangdong Medical Appliances Plant) with constant temperatures of 16, 19, 22, and 25°C. The photoperiod was identical in all treatments (24‐hr L/D cycle, L:D 12.5:11.5 hr). At least 30 Petri dishes were used for each temperature treatment. The Petri dishes were checked daily and supplied with new fresh leaves when needed. After pupation, pupae were placed individually in a transparent plastic box (3.5 cm in diame- ter and 6 cm in height) lined with filter paper and the box was covered with gauze. The pupae were monitored for eclosion to determine the developmental pathway for each individual. Based on the current ex- periment, if they did not emerge within 35 days at 16°C, 15 days at 19°C, 12 days at 22°C, and 9 days at 25°C, they were assumed to be in diapause. Diapause pupae were placed at 8°C for 30 days in continuous darkness and then transferred to L:D 15:9 hr and 18°C conditions to terminate the pupal diapause and observe the adult emergence. The number of females and males was recorded daily. 2.2.2 \| Field experiments Newly hatched larvae from the spring generation were transferred to B. chinensis plants grown in an outdoor web‐screened insec- tary. When the larvae matured they were placed individually in a transparent plastic box (3.5 cm in diameter and 6 cm in height) lined with filter paper and fresh leaves for pupation. After pupa- tion, the pupae were transferred individually to a clean transpar- ent plastic box for eclosion. Adults emerging from nondiapause pupae were released into another outdoor web‐screened insec- tary to mate and produce the second generation using the same protocol. Nondiapause pupae generally emerged within 8–13 days in the spring generations. Thus, each pupa that did not emerge within 15 days in spring generations was assumed to be in sum- mer diapause. Similar to the actions during laboratory experiment, aestivating pupae were placed at 8°C (a low temperature that can accelerate the development of diapaus and shortened diapause duration) for 30 days in continuous darkness (Xiao, Wu, Chen, & Xue, 2013), and then transferred to LD 15:9 hr, 18°C conditions to 2.1 \| Experimental insects The cabbage butterflies, P. melete used in the experiments originated from a wild population in the Tonggu County (28.5°N, 114.4°E; at an altitude of approximately 240 m above sea level), Jiangxi Province, PR China. Mature larvae prior to pupation were collected from crucifers in the vegetable gardens in mid‐November, 2015 and late April, 2016 and then were transferred to wooden insectaries TANG et al. 12313 TA B LE 2 Results from a linear model analysis of fixed effects on larval time, pupal weight, growth rate, adult weight, and weight loss of Pieris melete at different constant temperatures in relation to temperature, development pathway (develop. path), and sex TA B LE 2 Results from a linear model analysis of fixed effects on larval time, pupal weight, growth rate, adult weight, and weight loss of Pieris melete at different constant temperatures in relation to temperature, development pathway (develop. path), and sex regardless of the favorable day length (Table 1). However, when lar- vae were reared at relatively high temperatures, some individuals un- derwent direct development. The percentage of directly developing pupae reached 44.0% at 19°C, 77.0% at 22°C, and 63.1% at 25°C. The intermediate day length of 12.5 hr combined with the intermediate temperature of 22°C promoted direct development most effectively. regardless of the favorable day length (Table 1). However, when lar- vae were reared at relatively high temperatures, some individuals un- derwent direct development. The percentage of directly developing pupae reached 44.0% at 19°C, 77.0% at 22°C, and 63.1% at 25°C. The intermediate day length of 12.5 hr combined with the intermediate temperature of 22°C promoted direct development most effectively. p , p p y ( p p ), Traits Fixed effects df F p Larval time Temp 3 2,222.199 <.001 Develop. path. 1 72.072 <.001 Sex 1 0.217 .641 Temp × develop. path. 3 6.245 .02 Temp × sex 3 0.295 .829 Develop. path. × sex 1 0.106 .745 Pupal weight Temp 3 59.346 <0.001 Develop. path. 1 2.946 .087 Sex 1 17.740 <.001 Temp × develop. path. 3 0.109 .897 Temp × sex 3 0.343 .795 Develop. path. × sex 1 0.002 .960 Growth rate Temp 3 1,028.836 <.001 Develop. path. 1 60.015 <.001 Sex 1 1.095 .296 Temp × develop. path. 3 0.838 .433 Temp × sex 3 0.077 .972 Develop. path. × sex 1 0.064 .800 Adult weight Temp 3 41.559 <.001 Develop. path. 1 8.639 .003 Sex 1 3.465 .063 Temp × develop. path. 3 0.231 .794 Temp × sex 3 0.165 .920 Develop. path. × sex 1 0.116 .734 Weight loss Temp 3 2.688 .046 Develop. path. 1 3.357 .068 Sex 1 3.837 .051 Temp × develop. path. 2 1.576 .208 Temp × sex 3 1.199 .310 Develop. path. × sex 1 0.683 .409 Note: The significant effects are highlighted in bold. TA B LE 1 Incidence of developmental pathways taken by Pieris melete pupae at different constant temperatures TA B LE 1 Incidence of developmental pathways taken by Pieris melete pupae at different constant temperatures Temperature significantly affected weight loss from pupa to adult (Table 1). The proportional weight losses of diapausing pupae were similar to those of directly developing individuals at all temperatures (55%–59%; see Table S1). There were no significant differences in weight loss between males and females at any temperature (Table S1). T (°C) N Developmental pathway Direct Diapause Direct (%) 16 112 2 110 1.8 19 91 40 51 44.0 22 87 67 20 77.0 25 195 123 72 63.1 Note: The photoperiod (24‐hr L:D cycle, L:D 12.5:11.5) was identical in all treatments. 3.2 \| Comparisons of life‐history traits between diapausing and directly developing individuals at constant temperatures Temperature, developmental pathway, and their interactions (tem- perature × developmental pathway) significantly affected larval development time (Table 2). Larval development time significantly decreased as rearing temperature increased, with the larval develop- ment time of directly developing individuals being shorter than that of diapausing individuals and with significant differences at 19 and 22°C (Figure 1, Table S1, p < .05). Pupal weight was significantly affected by temperature and sex (Table 2). Pupal weight gradually decreased as rearing temperature increased from 16 to 25°C (Figure 1), which is consistent with the general pattern in ectothermic animals (Atkinson, 1994). Males were slightly larger than females at each temperature, but this difference was not significant (see Table S1, p > .05). Individuals that developed directly into adults attained relatively lower pupal weights than did individuals entering diapause (Figure 1), although significant differ- ences were not found at each temperature (Table S1, p > .05). The temperature and developmental pathway significantly af- fected larval growth rate (Table 2). The growth rate increased sig- nificantly as the rearing temperature increased. Individuals that developed directly into adults had significantly higher growth rates than individuals entering diapause (Figure 1, Table S1). Adult weight was significantly affected by temperature and de- velopmental pathway (Table 1). Adult weight gradually decreased as the rearing temperature increased (Figure 1). Although Table 1 shows a significant effect on adult weight induced by developmental pathway, there was no significant difference in adult weight between diapausing and di- rectly developing individuals at any temperature, with diapausing individuals being slightly larger than directly developing individuals (Figure 2, Table S1). Male adults were slightly larger than female adults (Figure 2, Table S1). 3.1 \| Developmental pathway at constant temperatures When larvae were reared under an intermediate day length of 12.5 hr, 98.2% of the pupae entered diapause at the low temperature of 16°C 12314 TANG et al. TANG et al. 3.3 \| Developmental pathways under field conditions In the spring generations, almost all individuals entered summer dia- pause (97.1% in the first spring generation, 93.5% in the second spring Note: The photoperiod (24‐hr L:D cycle, L:D 12.5:11.5) was identical in all treatments. \| 12315 \| 12315 TANG et al. 12315 \| 12315 TANG et al. FI G U R E 1 Comparisons of larval time, pupal weight, and growth rate between diapausing and directly developing individuals of Pieris melete at constant temperatures of 16, 19, 22, and 25°C. The symbols of ▫, □, and represent mean values, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausing and directly developing individuals at a significant level of 0.05 FI G U R E 1 Comparisons of larval time, pupal weight, and growth rate between diapausing and directly developing individuals of Pieris melete at constant temperatures of 16, 19, 22, and 25°C. The symbols of ▫, □, and represent mean values, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausing and directly developing individuals at a significant level of 0.05 in directly developing individuals than in diapausing individuals, with significant differences at 16.8°C for females and at 17.6 and 25.6°C for males (Figure 3, Table S2, p < .05). generation), only a few individuals underwent direct development (Table 2). This is because they experienced relatively low mean daily temperatures and gradually prolonging day lengths. In the autumn gen- erations, most individuals developed without diapause (90% in the first autumn generation, 78.5% in the second autumn generation) when they experienced gradually shortening day lengths (close to the intermediate day lengths) and relatively high mean daily temperatures (Table 3). Pupal weight was significantly affected by temperature, sex, de- velopmental pathway, and a significant interaction (developmental pathway × sex; Table 4). Pupal weight gradually decreased as the mean daily temperature increased, with diapausing individuals being slightly larger than directly developing individuals (Table S2). Male pupae were generally slightly larger than female pupae, with a signif- icant difference at 20.8°C (Figure 3, Table S2, p < .05). 3.4 \| Comparisons of life‐history traits between diapausing and directly developing individuals under field conditions In nature, such response patterns for diapause induction play an important role in regulating the life cycle of P. melete. The low‐temperature control of diapause not only ensures almost all larvae that grow in the spring enter sum- di th idi d ti d i d FI G U R E 2 Comparisons of weight loss from pupae to adults between diapausing and directly developing individuals of Pieris melete at constant temperatures of 16, 19, 22, and 25°C. The symbols of ▫, □, and represent the mean value, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausing and directly developing individuals at a significant level of 0.05 12316 \| TANG et al. FI G U R E 2 Comparisons of weight loss from pupae to adults between diapausing and directly developing individuals of Pieris melete at constant temperatures of 16, 19, 22, and 25°C. The symbols of ▫, □, and represent the mean value, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausing and directly developing individuals at a significant level of 0.05 FI G U R E 2 Comparisons of weight loss from pupae to adults between diapausing and directly developing individuals of Pieris melete at constant temperatures of 16, 19, 22, and 25°C. The symbols of ▫, □, and represent the mean value, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausing and directly developing individuals at a significant level of 0.05 significantly different between diapausing and directly developing individuals at a significant level of 0.05 Temperature and developmental pathway significantly affected adult weight (Table 4). Adult weight gradually decreased as the mean daily temperature increased (Figure 4), with diapausing individuals being slightly larger than directly developing individuals (Table S2). Male adults were larger than female adults, with a significant differ- ence at 20.8°C (Figure 4, Table S2, p < .05). the developmental pathway is highly plastic, depending on tempera- ture, day length, and their interaction (see Tables 1 and 3). Constant low temperatures of 16°C and the mean daily spring temperatures of 16.8 and 20.8°C combined with gradually increasing day lengths resulted in almost all individuals entering pupal diapause (98.2% and 97.1%). 3.4 \| Comparisons of life‐history traits between diapausing and directly developing individuals under field conditions Higher constant temperatures (19, 22, and 25°C) and mean daily autumn temperatures (17.6 and 25.6°C) combined with gradu- ally shortened autumn day lengths induced most individuals to de- velop without diapause (90% and 78.5%). In nature, such response patterns for diapause induction play an important role in regulating the life cycle of P. melete. The low‐temperature control of diapause not only ensures almost all larvae that grow in the spring enter sum- mer diapause, thus avoiding reproduction during adverse summer conditions (e.g., drought and food shortage) but also ensures that that the species synchronize its development and reproduction with the growth seasons of the host plants (Xue et al., 1997). In the field, all cruciferous vegetables are harvested in May and are generally not cultivated until autumn. The relatively high autumn temperature in- duction of direct development enables the butterfly to maximize the Proportional weight loss from pupa to adult was significantly af- fected by temperature (Table 4). However, the proportional weight loss did not show significant differences between diapausing in- dividuals and directly developing individuals at any temperature, although diapausing individuals experienced significantly longer du- rations of pupae than did directly developing individuals (67–97 vs. 7–13 days). 3.4 \| Comparisons of life‐history traits between diapausing and directly developing individuals under field conditions Growth rate was significantly affected by temperature and developmental pathway; developmental pathway and sex had a significant interaction (Table 4). The growth rate increased sig- nificantly as the mean daily temperature increased. Moreover, the growth rate was higher in directly developing individuals than diapausing individuals, showing significant differences for males at mean daily temperatures of 17.6, 20.8 and 35.6°C (Figure 3, Table S2, p < .05). Larval development time was significantly influenced by tempera- ture and developmental pathway (Table 4). There were significant interactions (temperature × developmental pathway, tempera- ture × sex and temperature × developmental pathway × sex). The larval development time significantly decreased with increasing the mean daily temperature. The larval development times were shorter 12316 \| 12316 \| TANG et a FI G U R E 2 Comparisons of weight los from pupae to adults between diapausing and directly developing individuals of Pieris melete at constant temperatures of 16, 19, 22, and 25°C. The symbols of ▫, □, and represent the mean value, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausin and directly developing individuals at a significant level of 0.05 TANG et al. 12316 12316 \| TANG et al. Temperature and developmental pathway significantly affected adult weight (Table 4). Adult weight gradually decreased as the mean daily temperature increased (Figure 4), with diapausing individuals being slightly larger than directly developing individuals (Table S2). Male adults were larger than female adults, with a significant differ- ence at 20.8°C (Figure 4, Table S2, p < .05). Proportional weight loss from pupa to adult was significantly af- fected by temperature (Table 4). However, the proportional weight loss did not show significant differences between diapausing in- dividuals and directly developing individuals at any temperature, although diapausing individuals experienced significantly longer du- rations of pupae than did directly developing individuals (67–97 vs. 7 13 d ) the developmental pathway is highly plastic, depending on tempera- ture, day length, and their interaction (see Tables 1 and 3). Constant low temperatures of 16°C and the mean daily spring temperatures of 16.8 and 20.8°C combined with gradually increasing day lengths resulted in almost all individuals entering pupal diapause (98.2% and 97.1%). Higher constant temperatures (19, 22, and 25°C) and mean daily autumn temperatures (17.6 and 25.6°C) combined with gradu- ally shortened autumn day lengths induced most individuals to de- velop without diapause (90% and 78.5%). TA B LE 4 Results from a linear model analysis of fixed effects on larval time, pupal weight, growth rate, adult weight, and weight loss of Pieris melete under field conditions in relation to temperature, development pathway (develop. path), and sex allows the butterfly to escape from various unpredictable physical or biotic factors, such as farming practices (insecticide applications, thinning of the seedlings and harvesting), interspecific competition (aphids and the beetle Phaedon brassicae), and autumn drought, thus avoiding the catastrophic elimination of an entire population. ( ) p p y p p Traits Fixed effects df F P Larval time Temp 3 985.126 <.001 Sex 1 3.695 .055 Develop. path. 1 49.794 <.001 Temp × sex 3 3.064 .028 Temp × develop. path 3 2.105 .099 Develop. path. × sex 1 2.712 .100 Temp × develop. path. × sex 3 4.123 .007 Pupal weight Temp 3 15.796 <.001 Sex 1 7.603 .006 Develop. path. 1 5.659 .018 Temp × sex 3 0.053 .984 Temp × develop. path 3 0.249 .862 Develop. path. × sex 1 4.098 .044 Growth rate Temp 3 827.492 <.001 Sex 1 0.153 .696 Develop. path. 1 37.210 <.001 Temp × sex 3 2.542 .56 Temp × develop. path 3 0.942 .420 Develop. path. × sex 1 6.829 .009 Adult weight Temp 3 15.255 <.001 Sex 1 2.563 .197 Develop. path. 1 5.414 .02 Temp × sex 3 0.284 .837 Temp × develop. path 3 0.012 .998 Develop. path. × sex 1 2.243 .135 Weight loss Temp 3 5.951 .001 Sex 1 0.863 .353 Develop. path. 1 1.719 .190 Temp × sex 3 0.844 .470 Temp × develop. path 3 0.576 .631 Develop. path. × sex 1 0.000 .982 Note: The significant effects are highlighted in bold. Given the physiological responses that control the propensity to enter diapause, the expression of an alternative development path- way in P. melete may be dependent upon a threshold. Those individu- als above a certain threshold value may enter diapause; those below it may avert diapause. Alternatively, alternative developmental path- ways may entail that conditionally expressed genes, although carried by all individuals within a population, are expressed and exposed to selection only a fraction of these individuals at any given time (Van Dyken & Wade, 2010). Individual insects that follow the two alternative pathways of diapause and direct development often display substantial phe- notypic differences in life history traits. Directly developing in- dividuals generally have shorter development times and higher growth rates than do diapausing individuals (Aalberg Haugen et al., 2012; Blanckenhorn & Fairbairn, 1995; Kivelä et al., 2012; Nylin, 1992; Pöykkö & Hyvärinen, 2012; Wiklund & Friberg, 2011; Wiklund, Nylin, & Forsberg, 1991). Compared to that of diapausing individuals, the body size of directly developing individuals var- ies with species and may be relatively larger (Kivelä et al., 2012; Wiklund et al., 1991), relatively smaller (Aalberg Haugen et al., 2012; Mousseau & Roff, 1989; Pöykkö & Hyvärinen, 2012; Teder, Esperk, Remmel, Sang, & Tammaru, 2010), or equal (Blanckenhorn, 1994; Blanckenhorn & Fairbairn, 1995; see also Välimäki, Kivelä, Mäenpää, & Tammaru, 2013). In the present study, we found that compared with the diapause pathway, the direct development pathway in P. melete is generally associated with shorter larval de- velopment times, higher larval growth rates and relatively lower pupal, and adult weights. Under both laboratory and field conditions, pupal and adult weights for both diapausing and directly developing individuals of P. melete gradually decreased with increasing temperature, showing a typical thermal reaction norm for ectotherm body size, denoted as the temperature‐size rule (TSR; Atkinson, 1994). However, increasing evidence has shown that the reverse TSR in insects is also common. For example, reversals of the TSR have been found in four species of mayfly (Atkinson, 1995); four species of British grasshoppers (Willott & Hassall, 1998); the tropical butterfly, Bicyclus anynana (Fischer, Bot, & Brakefield, 2004); the small cabbage white, butterfly, Pieris rapae (Kingsolver, Massie, Ragland, & Smith, 2007); the Asian corn borer, Ostrinia furnacalis (He, Tang, Huang, Gao, & Xue, 2019; Xiao et al., 2016); and the rice stem borer, Chilo suppressalis (Fu, He, Zhou, Xiao, & Xue, 2016; Huang, Xiao, He, & Xue, 2018). As such, why do some insect species follow the TSR and some exhibit the reverse TSR? We speculate whether an insect species follows the TSR or not may be related to its diapause characteristic. Those species with summer dia- pause may exhibit the TSR, as indicated by the cabbage beetle, C. bow‐ ringi (Tang, He, Chen, Fu, & Xue, 2017) and this butterfly, P. melete because their reproductive periods occur in the spring and autumn Note: The significant effects are highlighted in bold. use of the available food resources (approximately 3.5 months) for building up the population. 4 \| DISCUSSION The experimental results in P. melete under both laboratory and field conditions showed similar patterns in that the expression of TA B LE 3 Incidence of developmental pathways taken by Pieris melete pupae at different mean daily temperatures and altered day lengths in the outdoor screened insectary Generation The mean daily temperature (°C) and day length (hr) experienced by larvae Date from hatching to pupation N Developmental pathway Direct Diapause Direct (%) Spring 16.8°C 12.6–13.5 hr Mar 21–Apr 18 547 16 531 2.9 Spring 20.8°C 13.5–14.4 hr Apr 22–May 23 307 20 287 6.5 Autumn 25.6°C 13.3–12.1 hr Sept 3–Sept 23 50 45 5 90 Autumn 17.6°C 12.1–11.3 hr Oct 16–Nov 15 121 95 26 78.5 opmental pathways taken by Pieris melete pupae at different mean daily temperatures and altered day lengths TA B LE 3 Incidence of developmental pathways taken by Pieris melete pupae at different mean daily temperatures and altered day lengths in the outdoor screened insectary TA B LE 3 Incidence of developmental pathways taken by Pieris melete pupae at different mean daily tempe in the outdoor screened insectary 12317 TANG et al. The higher autumn temperature induc- tion of diapause in some individuals reflects a bet‐hedging tactic that 12318 \| TANG et FI G U R E 3 Comparisons of larval tim pupal weight, and growth rate between diapausing and directly developing individuals of Pieris melete at different mean daily temperatures in the field. The symbols of ▫, □, and represent the mean value, ± SEs and ± SDs, respective The values with different lowercase letters are significantly different betwe diapausing and directly developing individuals at a significant level of 0.05. The mean daily temperatures of 16.8, 17.6, 20.8, and 25.6°C represent the firs spring generation, the second autumn generation, the second spring generatio and the first autumn generation, respectively TANG et al. 12318 12318 \| TANG et al. FI G U R E 3 Comparisons of larval time, pupal weight, and growth rate between diapausing and directly developing individuals of Pieris melete at different mean daily temperatures in the field. The symbols of ▫, □, and represent the mean value, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausing and directly developing individuals at a significant level of 0.05. The mean daily temperatures of 16.8, 17.6, 20.8, and 25.6°C represent the first spring generation, the second autumn generation, the second spring generation, and the first autumn generation, respectively FI G U R E 3 Comparisons of larval time, pupal weight, and growth rate between diapausing and directly developing individuals of Pieris melete at different mean daily temperatures in the field. The symbols of ▫, □, and represent the mean value, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausing and directly developing individuals at a significant level of 0.05. The mean daily temperatures of 16.8, 17.6, 20.8, and 25.6°C represent the first spring generation, the second autumn generation, the second spring generation, and the first autumn generation, respectively and because these insects have experienced strong selection for body size under relatively low environmental temperatures during the pro- cess of evolution. Those species with winter diapause triggered by shortening day lengths combined with high autumn temperatures may exhibit the reverse TSR, as indicated by the Asian corn borer, O. fur‐ nacalis (He et al., 2019; Xiao et al., 2016), and the rice stem borer, C. suppressalis (Fu et al., 2016; Huang et al., 2018). These two spe- cies enter winter diapause in response to high autumn temperatures and experience strong selection for body size under warm conditions. Additional insect species with similar diapause characteristics will be investigated to confirm this speculation. conditions, despite those diapausing individuals exhibiting much lon- ger pupal durations than did the directly developing individuals (Tables S1 and S2). Such a case indicates that the process of diapause did not affect body weight change during metamorphosis. Furthermore, the weights of both female and male adults were still slightly higher in dia- pausing individuals than in the directly developing individuals. Thus, newly emerged female adults from diapause development should have relatively high fecundity than those from direct development because female fecundity is generally positively correlated with adult body weight when the number of eggs is assessed as lifetime fecundity under standard conditions or by dissection (Honek, 1993). Therefore, relatively large body sizes in diapausing individuals are generally con- sidered to be adaptive because of their greater reserves (Hahn & Denlinger, 2007) and may ameliorate the negative cost of diapause. To date, few studies have tested the differences in weight loss between the diapausing and directly developing individuals. In the cotton bollworm, Helicoverpa armigera, proportional weight losses were slightly lower in diapausing individuals than in directly develop- ing individuals at 20 and 22°C, but slightly higher at 25°C (Chen et al., 2014). In the present study, the proportional weight losses were similar between the diapausing and directly developing individuals under both laboratory and field conditions at the same temperature CONFLICT OF INTEREST The authors declare that they have no conflict of interest. The authors declare that they have no conflict of interest. Atkinson, D. (1994). Temperature and organism size‐a biological law for ectotherms? Advance in Ecological Research, 25, 1–58. Atkinson, D. (1995). Effects of temperature on the size of aquatic ecto- therms: Exceptions to the general rule. Journal of Thermal Biology, 20, 61–74. https://doi.org/10.1016/0306-4565(94)00028-H ACKNOWLEDGMENTS We thank all in the laboratory for their technical assistance with the experiment. This research was supported by a grant from the \| 12319 TANG et al. 12319 \| 12319 TANG et al. National Natural Science Foundation of the People's Republic of Chi (31260430) life‐history traits in a butterfly. Journal of Evolutionary Biology, 25, 1377 1388 https://doi org/10 1111/j 1420-9101 2012 02525 x FI G U R E 4 Comparisons of weight loss from pupae to adults between diapausing and directly developing individuals of Pieris melete at different mean daily temperatures in the field. The symbols of ▫, □, and represent the mean values, ± SEs and ± SDs, respectively. The values with different lowercase letters are significantly different between diapausing and directly developing individuals at a significant level of 0.05. The mean daily temperatures of 16.8, 17.6, 20.8, and 25.6°C represent the first spring generation, the second autumn generation, the second spring generation, and the first autumn generation, respectively National Natural Science Foundation of the People's Republic of China (31260430). life‐history traits in a butterfly. Journal of Evolutionary Biology, 25, 1377–1388. https://doi.org/10.1111/j.1420-9101.2012.02525.x Aalberg Haugen, I. M., & Gotthard, K. (2015). Diapause induction and relaxed selection on alternative developmental pathways in a butterfly. Journal of Animal Ecology, 84, 464–472. https://doi. org/10.1111/1365-2656.12291 ORCID Jian‐Jun Tang https://orcid.org/0000-0003-0756-4135 Fang‐Sen Xue https://orcid.org/0000-0002-7609-7414 Lan Xiao https://orcid.org/0000-0003-4665-4440 Fischer, K., & Fiedler, K. (2000). Sex‐related differences in reaction norms in the butterfly Lycaena tityrus (Lepidoptera: Lycaenidae). Oikos, 90, 372–380. https://doi.org/10.1034/j.1600-0706.2000.900218.x Fu, D. M., He, H. M., Zhou, C., Xiao, H. J., & Xue, F. S. (2016). Life‐his- tory responses of the rice stem borer Chilo suppressalis to temperature change: Breaking the temperature–size rule. Journal of Thermal Biology, 61, 115–118. https://doi.org/10.1016/j.jtherbio.2016.09.006 AUTHOR CONTRIBUTIONS FS‐X conceived and designed the research. JJ‐T, HM‐H, and C‐Z conducted experiments and analyzed the data. L‐X and SH‐ Wu wrote the manuscript. All authors read and approved the manuscript. Blanckenhorn, W. U. (1994). Fitness consequences of alternative life histories in water striders, Aquarius remigis (Heteroptera: Gerridae). Oecologia, 97, 354–365. https://doi.org/10.1007/BF00317325 Blanckenhorn, W. U., & Fairbairn, D. J. (1995). Life history adapta- tion along a latitudinal cline in the water strider Aquarius remigis (Heteroptera: Gerridae). Journal of Evolutionary Biology, 8, 21–41. Chen, C., Xia, Q. W., Xiao, H. J., Xiao, L., & Xue, F. S. (2014). A comparison of the life‐history traits between diapause and direct development individuals in the cotton bollworm, Helicoverpa armigera. Journal of Insect Science, 14, 19. DATA AVAILABILITY STATEMENT Empirical data have been archived in DataDryad: https://doi. org/10.5061/dryad.s5n7t4d Danks, H. V. (1987). Insect dormancy: An ecological perspective. Ottawa, ON: Entomological Society of Canada. Fischer, K., Bot, A. N. M., Zwaan, B. J., & Brakefield, P. M. (2004). Genetic and environmental sources of egg size variation in the butterfly Bicyclus anynana. Heredity, 92, 163–169. https://doi.org/10.1038/ sj.hdy.6800382 butterfly. Journal of Evolutionary Biology, 23, 1129–1137. https://doi. org/10.1111/j.1420-9101.2010.01994.x Tauber, M. J., Tauber, C. A., & Masaki, S. (1986). Seasonal adaptations of insects. New York, NY: Oxford University Press. Gotthard, K., Nylin, S., & Wiklund, C. (1994). Adaptive variation in growth rate: Life history costs and consequences in the speckled wood butterfly, Pararge aegeria. Oecologia, 99, 281–289. https://doi. org/10.1007/BF00627740 Teder, T., Esperk, T., Remmel, T., Sang, A., & Tammaru, T. (2010). Counterintuitive size patterns in bivoltine moths: Late‐season lar- vae grow larger despite lower food quality. Oecologia, 162, 117–125. https://doi.org/10.1007/s00442-009-1439-1 Hahn, D. A., & Denlinger, D. L. (2007). Meeting the energetic de- mands of insect diapause: Nutrient storage and utilization. Journal of Insect Physiology, 53, 760–773. https://doi.org/10.1016/j.jinsp hys.2007.03.018 Välimäki, P., Kivelä, S. M., Mäenpää, M. I., & Tammaru, T. (2013). Latitudinal clines in alternative life histories in a geometrid moth. Journal of Evolutionary Biology, 26, 118–129. https://doi.org/10.1111/ jeb.12033 He, H. M., Tang, J. J., Huang, L. L., Gao, Y. L., & Xue, F. S. (2019). Inter‐geo- graphic hybridization in the corn borer Ostrinia furnacalis reduced the fitness of hybrids. Journal of Pest Science, 92, 1027–1037. https://doi. org/10.1007/s10340-019-01119-3 Van Dyken, J. D., & Wade, M. J. (2010). The genetic signature of condi- tional expression. Genetics, 184, 557–570. https://doi.org/10.1534/ genetics.109.110163 Waldbauer, G. P. (1978). Phenological adaptation and the polymodal emergence patterns of insecte. In H. Dingle (Ed.), Evolution of insect migration and diapause (pp. 127–144). New York, NY and Heidelberg, Berlin, Germany: Springer. Honěk, A., & Honek, A. (1993). Intraspecific variation in body size and fecundity in insects—A general relationship. Oikos, 66, 483–492. https://doi.org/10.2307/3544943 Wiklund, C., & Friberg, M. (2011). Seasonal development and vari- ation in abundance among four annual flight periods in a but- terfly: A 20‐year study of the speckled wood (Pararge aegeria). Biological Journal of the Linnean Society, 102, 635–649. https://doi. org/10.1111/j.1095-8312.2010.01581.x Hopper, K. R. (1999). Risk‐spreading and bet‐hedging in insect popula- tion biology. Annual Review of Entomology, 44, 535–560. https://doi. org/10.1146/annurev.ento.44.1.535 Huang, X. L., Xiao, L., He, H. M., & Xue, F. S. (2018). Effect of rearing conditions on the correlation between larval development time and pupal weight of the rice stem borer, Chilo suppressalis. Ecology and Evolution, 8, 12694–12701. Wiklund, C., Nylin, S., Forsberg, J., & Nylin, S. (1991). Sex‐related varia- tion in growth rate as a result of selection for large size and protandry in a bivoltine butterfly, Pieris napi. Oikos, 60, 241–250. https://doi. org/10.2307/3544871 Kingsolver, J. G., Massie, K. R., Ragland, G. J., & Smith, M. H. (2007). Rapid population divergence in thermal reaction norms for an invading species: Breaking the temperature–size rule. Journal of Evolutionary Biology, 20, 892–900. https://doi. org/10.1111/j.1420-9101.2007.01318.x Willott, J., & Hassall, M. (1998). Life‐history responses of British grasshop- pers (Orthoptera: Acrididae) to temperature change. Functional Ecology, 12, 232–241. https://doi.org/10.1046/j.1365-2435.1998.00180.x Kivelä, S. M., Svensson, B., Tiwe, A., & Gotthard, K. (2015). Thermal plas- ticity of growth and development varies adaptively among alterna- tive deveolopmental pathways. Evolution, 69, 2339–2413. Wise, E. J. (1980). Seasonality distribution and life histories of Ephemeroptera in a Northumbrian river. Freshwater Biology, 10, 101–111. Kivelä, S. M., Välimäki, P., & Gotthard, K. (2013). Seasonality main- tains alternative life history phenotypes. Evolution, 67, 3145–3160. https://doi.org/10.1111/evo.12181 Xiao, H. J., Li, F., Wei, X. T., & Xue, F. S. (2008). A comparison of photo- periodic control of diapause between aestivation and hibernation in the cabbage butterfly Pieris melete. Journal of Insect Physiology, 54, 755–764. https://doi.org/10.1016/j.jinsphys.2008.01.009 Kivelä, S. M., Välimäki, P., & Gotthard, K. (2016). Evolution of alternative insect life histories in stochastic seasonal environments. Ecology and Evolution, 6, 5596–5613. https://doi.org/10.1002/ece3.2310 Xiao, H., Wu, S., Chen, C., & Xue, F. (2013). Optimal low temperature and chilling period for both summer and winter diapause development in Pieris melete: Based on a similar mechanism. PLoS ONE, 8(2), e56404. https://doi.org/10.1371/journal.pone.0056404 Kivelä, S. M., Välimäki, P., & Mäenpää, M. I. (2012). Genetic and phe- notypic variation in juvenile development in relation to tem- perature and developmental pathway in a geometrid moth. Journal of Evolutionary Biology, 25, 881–891. https://doi. org/10.1111/j.1420-9101.2012.02478.x Xiao, H. J., Wu, S. H., He, H. M., Chen, C., & Xue, F. S. (2012). Role of natural day‐length and temperature in determination of summer and winter diapause in Pieris melete (Lepidoptera: Pieridae). Bulletin of Entomological Research, 102, 267–273. Masaki, S. (1980). Summer diapause. Annual Review of Entomology, 25, 1–25. https://doi.org/10.1146/annurev.en.25.010180.000245 Xiao, H. J., Wu, X. F., Wang, Y., Zhu, X. F., & Xue, F. S. (2009). Diapause induction and clock mechanism in the cabbage butterfly Pieris me‐ lete Ménétriés. Journal of Insect Physiology, 55, 488–493. https://doi. org/10.1016/j.jinsphys.2008.12.012 Mousseau, T. A., & Roff, D. A. (1989). Adaptation to seasonality in a cricket: Patterns of phenotypic and genotypic variation in body size and diapause expression along a cline in season length. Evolution, 43, 1483–1496. https://doi.org/10.1111/j.1558-5646.1989.tb02598.x Xiao, L., He, H. M., Huang, L. REFERENCES Aalberg Haugen, I. M., Berger, D., & Gotthard, K. (2012). The evolution of alternative developmental pathways: Footprints of selection on Gotthard, K., & Berger, D. (2010). The diapause decision as a cascade switch for adaptive developmental plasticity in body mass in a 12320 TANG et al. TANG et al. \| 12321 How to cite this article: Tang J‐J, He H‐M, Wu S‐H, Zou C, Xue F‐S, Xiao L. Expression of alternative developmental pathways in the cabbage butterfly, Pieris melete and their differences in life history traits. Ecol Evol. 2019;9:12311– 12321. https://doi.org/10.1002/ece3.5731 Chrysomelidae). Journal of Insect Physiology, 48, 279–286. https://doi. org/10.1016/S0022-1910(01)00172-X How to cite this article: Tang J‐J, He H‐M, Wu S‐H, Zou C, Xue F‐S, Xiao L. Expression of alternative developmental pathways in the cabbage butterfly, Pieris melete and their differences in life history traits. Ecol Evol. 2019;9:12311– 12321. https://doi.org/10.1002/ece3.5731 How to cite this article: Tang J‐J, He H‐M, Wu S‐H, Zou C, Xue F‐S, Xiao L. Expression of alternative developmental pathways in the cabbage butterfly, Pieris melete and their differences in life history traits. Ecol Evol. 2019;9:12311– 12321. https://doi.org/10.1002/ece3.5731 How to cite this article: Tang J‐J, He H‐M, Wu S‐H, Zou C, Xue F‐S, Xiao L. Expression of alternative developmental pathways in the cabbage butterfly, Pieris melete and their differences in life history traits. Ecol Evol. 2019;9:12311– 12321. https://doi.org/10.1002/ece3.5731 Xue, F. S., Zhu, X. F., & Shao, Z. Y. (2001). Control of summer and winter diapause in the leaf‐mining fly Pegomyia bicolor (Diptera: Antomylidae). Journal of Applied Entomology, 125, 181–187. Xue, F. S., Zhu, X. F., & Wei, H. Y. (1996). Biology of Pieris melete Pieris melete Ménétriés (in Chinese). Jiangxi Plant Protection, 19, 1–4. L., Geng, T., Fu, S., & Xue, F. S. (2016). Variation of life‐history traits of the Asian corn borer, Ostrinia fur‐ nacalis in relation to temperature and geographical latitude. Ecology and Evolution, 6, 5129–5143. https://doi.org/10.1002/ece3.2275 Nylin, S. (1992). Seasonal plasticity in life history traits: Growth and development in Polygonia c‐album (Lepidoptera: Nymphalidae). Biological Journal of Linnean Society, 47, 301–323. https://doi. org/10.1111/j.1095-8312.1992.tb00672.x Xue, F. S., & Kallenborn, H. G. (1993). Dispersive breeding in agricultural pest insects and its adaptive significance. Journal of Applied Entomology, 116, 170–177. https://doi.org/10.1111/j.1439-0418.1993.tb01185.x Nylin, S. (2001). Life history perspectives on pest insects: What's the use? Australian Journal of Ecology, 26, 507–517. https://doi. org/10.1046/j.1442-9993.2001.01134.x Xue, F. S., & Kallenborn, H. G. (1998). Control of summer and winter dia- pause in Pidorus euchromioides (Lepidoptera; Zygaenidae) on Chinese sweetleaf Symplocs chinensis. Bulletin of Entomological Research, 88, 207–211. Pöykkö, H., & Hyvärinen, M. (2012). To grow fast or to grow big? Time limited larvae of Eilema depressum speed up their growth and reduce the number of instars. Entomolgia Experimentalis et Applicata, 142, 145–152. Xue, F. S., Kallenborn, H. G., & Wei, H. Y. (1997). Summer and winter diapause in pupae of the cabbage butterfly, Pieris melete Menetries. Journal of Insect Physiology, 143, 701–707. Tang, J. J., He, H. M., Chen, C., Fu, S., & Xue, F. S. (2017). Latitudinal cogradient variation of development time and growth rate and a neg- ative latitudinal body weight cline in a widely distributed cabbage beetle. PLoS ONE, 12(7), e0181030. https://doi.org/10.1371/journ al.pone.0181030 Xue, F. S., Spieth, H. R., Li, A. Q., & Hua, A. (2002). The role of photo- period and temperature in determination of summer and winter diapause in the cabbage beetle, Colaphellus bowringi (Coleoptera: TANG et al. 12321 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of the article. Additional supporting information may be found online in the Supporting Information section at the end of the article.
https://openalex.org/W4317204819	https://journal.spera.asn.au/index.php/AIJRE/article/download/408/494	English	null	A Teaching Program in Rural Education	Australian & international journal of rural education	1,996	cc-by	4,147	ABSTRACT This paper describes an innovative rural education project undertaken at Edith Cowan University through funding from the Committee for the Advancement of University Teaching (CAUT). The project sought to create and implement an instructional course based on contemporary learning theories and to enhance learning outcomes in a pre-service teacher education course dealing with rural education. It sought to do so through the use of telematics in teaching and learning. The project involved the development of a module of work that enabled students to experience the technology and to construct their own perceptions of the use of telecommunications as a delivery medium for rural education. It was planned that the students would learn about teaching and learning with telematics by themselves being taught with that technology. The outcomes and initial findings from an implementation of these ideas provided very positive results. The project has demonstrated a powerful alternative to the conventional teaching format, one which is both efficient as a delivery medium and effective in the outcomes that are achieved. A TEACmNG PROGRAM IN RURAL EDUCATION: LEARNING THROUGH EXPERIENTIAL ACTIVITIES RonOliver MurrayLake Edith Cowan University INTRODUCTION One of the most consistent themes evident in the literature dealing with rural education is that of rural disadvantage. Of the matrix of factors leading to that disadvantage, geographical isolation and the extent to wltich it restricts access, is a major concern. If access is a matter of fairness and equity, it is fair and equitable that all children, regardless of location, have access to the broadest range of quality educational experience. Many rural children, however, are denied that access. Traditional educational delivery systems will not suffice. Telecommunication technology offers great promise and, in some cases, provides the only viable means of providing access to the educational services required. Furthermore, there is clear evidence that the very best level of service can be brought to all rural schools regardless of size or geographical location or the specificity of the requirement. Teacher education institutions have a role to play in this process. They are aware that the majority of their graduates will be appointed to rural schools or will be transferred to such schools early in their careers. They are aware also that a significant and increasing number of rural schools utilise telecommunication technology in their instructional program. Accordingly, as noted by Tomlinson (1994), their role is essential in ensuring that the promise and potential of instructional technology is fully realised. The project described in this paper, was designed to develop a curriculum to instruct teacher- education students more effectively in the use of telecommunications technologies and teaching and learning in rural settings. In past years the rural education course at Edith Cowan University has used conventional teaching methods in its program delivery. Students were exposed to the procedures and practices within rural education settings through such activities as formal lectures, site visits, observation of classroom activities as well as reading and reviewing relevant literature. This form of delivery provided the students with a broad view of the nature of teaching and learning in rural education and with elementary practical classroom skills. The Education in Rural Australia, V 01. 6 (2) ... 1 Copyright Agency J;~lniied (CAL) l'icensed cop~'.fl1rthef.'co~pytng and Communication pFohibitcd except on payment of fee per <Sop)' or Commuication And otherwise in ac~6rd:1nce with the licer.<:'e from CAI:.'to ACER.For more Infnrmation-contact'"@-J\b-tm-({)2t939+--76e(Torinfu@CopjTIghtconr.au- Copyright Agency J;~lniied (CAL) l'icensed cop~'.fl1rthef.'co~pytng and Communication pFohibitcd except on payment of fee per <Sop)' or Commuication And otherwise in ac~6rd:1nce with the licer.<:'e from CAI:.'to ACER.For more Infnrmation-contact'"@-J\b-tm-({)2t939+--76e(Torinfu@CopjTIghtconr.au- knowledge and skills, however, ran the risk of being general and peripheral to actual applications, a factor that has been found to limit the development of a full understanding of the technology and its applications (Robinson, 1993; Downes, 1993; Byrnm & Cashman, 1993). knowledge and skills, however, ran the risk of being general and peripheral to actual applications, a factor that has been found to limit the development of a full understanding of the technology and its applications (Robinson, 1993; Downes, 1993; Byrnm & Cashman, 1993). Theoretical Framework Theoretical Framework The design of the curriculum developed iu this project was based on contemporary learning theories that espoused the value of knowledge construction and contextual learning rather than knowledge transmission. The relationship between teaching and learning was based very mucb on the types and levels of activity engendered in the learners. Cognitive psychologists bave promoted the notion that learners receive knowledge from environmental stimuli and that learning depends on how that knowledge is used. Piaget (1954 ) argued that information is coded and learned accordiug to the meaniug ascribed by the learner. Bruner (1960) showed that developing and establishing relationships between items of knowledge is critical to learning. He used the term 'meaniugfullearning' to describe that which accompanied the construction of liuks to existing knowledge. Ausubel (1960) went further to describe the need for iustruction to build networks and frameworks into which new knowledge could be placed with meaningful links and connections. The view is also promoted by some educators that learning is enhanced by active environments in which students have cause to be engaged and reflective in the learning process. Similarly, there is support the notion that students learn through a process of constructing knowledge. This constructivist view maintains that knowledge is gained by learners through a process of knowledge building. When a learner is confronted with new knowledge, the learner's intentions, previous experiences and metacoguitive strategies are all essential elements in determiuing what becomes of the knowledge. Education in Rural Australia, Vol. 6 (2) ... 2 Education in Rural Australia, V 01. 6 (2) ... 1 In this process, the learner is regarded not as an empty vessel into which knowledge is poured, but more as an individual replete with pre-existing knowledge, aptitudes and motivations (Reeves, 1993). The project sought to create a curriculum that would support a constructivist learning environment. Students were to become active learners who would process lesson content through generative' activities.' Wittrock (1974) describes generative processing as deep processing that emerges from activities that cause students to interpret and assimilate new information into existing mental structures and to reorganise existing structures in the light of newly interpreted information. The project further sought to create an environment that facilitated high levels of learner control. Learner control has been a heavily researched dimension of learning environments in recent years (Steinberg, 1989). In rural settings, both students and teachers are often required to assume control and take responsibility of their own learning programs. New teachers in rural schools need to have the skills and confidence to seek information from their own resources. They need to be able to create and support learning environments that use and build on the students' capacities for self-regulation. These forms of teachiug and learning are quite foreign to many student-teachers. Through their own schooling and tertiary studies they are likely to have met only conventional forms of instruction. Traditionally the role of the teacher in such environments is didactic and transmissive. But in settings where the teacher assumes more a facilitative and coaching role, students become more active in the learning process. Rather than attending to a teacher-student dialogue, students can be working in a more autonomous role, either individually or among themselves. When teachers facilitate learning, the level of student control is raised. Student control and personal autonomy in their learning was also seen to enhance knowledge development and enable them to experience, fIrst-hand, the learning environments to which their own students would be exposed. This, in turn, was seen as a means of developing a realistic set of expectations and impressions of the instructional settings which they would be required to develop and create in their future classrooms. Education in Rural Australia, Vol. 6 (2) ... 2 Education in Rural Australia, Vol. 6 (2) ... 2 Perhaps the aspect of the project with the strongest potential for enhanced learning outcomes was its practical context. TELEMATICS To provide students with a learning environment that involved all the elements of constructive learning, it was proposed to integrate the content of the teaching and the delivery medium. A course was proposed that incorporated a medium that had particular application to rural education, telematics, to teach about rural education. Telematics is the technology that enables simultaneous interactive commnnication between teacher and students using telecommunications (Oliver & Reeves, 1994). It is used widely in some Australian states for the delivery of educational programs to rural schools. The application of this technology is seen to provide a learning environment that is meaningful and effective (Haywood & Nomtan, 1988; Diem, 1989: Novak & Knowles, 1991). The full range of issues and subject content relating to rural education could be treated in a manner that was likely to increase the likelihood of transfer and generalisibility of the skills and knowledge (Brown, 1985) . Education in Rural Australia, V 01. 6 (2) ... 1 There is significant support in the literature for the benefits of contextual learning where students are taught and learn in environments that closely match reality. The earliest type of systematic learning activity probably involved some sort of apprenticeship whereby a novice worked side by side with a master. Apprenticeships have high concrete and experiential value. More abstract learning activities, such as classroom lectures, were developed much later in history. A major criticism of much of the current dominant pedagogical schemes is that they are too abstract, removed as they are from 'real world' experience (Brown, Collins, & Dugnid, 1989). An important concern for educators and trainers alike is the degree to which classroom learning transfers to external situations in which the application of knowledge, skills, and attitudes is appropriate. The cognitive theories of Newell and Simon (1972), Brown (1985), and others support the fundamental principle that the way in which knowledge, skills, and attitudes are initially learned plays an important role in the degree to which these abilities can be used in other contexts. To put it simply, if knowledge, skills, and anitudes are learned in a context of use, they will be used in that and similar contexts. Education in Rural Australia, Vol. 6 (2) ... 3 THE TELEMATICS COURSE A curriculum was developed to expose the students to the advantages, difficulties and disadvantages associated with teaching and learning with telematics this medium. The curriculum was developed around a series of modules with support materials for independent student use. It was planned to be implemented in a virtual classroom among groups of students using telematics in two locations across campuses. The students would receive instruction and direction through attendance in telematics lessons and would then iudependently complete a series of prescribed tasks and activities. Through this form of delivery, it was intended that students would learn: Through this form of delivery, it was intended that students would learn: • how to operate and apply the hardware associated with telecommunications, the mechanisms and processes by which the telecommunications technology operated, • how to operate and apply the hardware associated with telecommunications, the mechanisms and processes by which the telecommunications technology operated, • the skills reqttired to independently use computers, telecommunications and the appropriate software, • the instructional skills and relevant pedagogy associated with teaching and learning with telecommunications in rural schools, and Education in Rural Australia, Vol. 6 (2) ... 3 • through the integrated nature of the instruction, develop transferable and generalisable skills and knowledge applicable in future settings (Hollingsworth, 1989; Prawat, 1992; Rovegno, 1993). COURSE MATERIALS A range of learning materials was developed to form the basis of the independent student activity. While it was planned that students would attend the telematics sessions to establish a learning program, the bulk of the learning was to be undertaken independently and collaboratively in the students' own time. The materials developed were as follows: a course guide, a booklet!letailing the course, the modules and instructional episodes and the activities and tasks to be completed, a set of readings, relevant materials describing the technology, its application and implementations, video materials showing telematics teaching and learning, instruction manuals and guides for the telematics equipment and resources, g q p ROM with appropriate materials to guide the planuing and creation of telematics lessons, g q p a CD-ROM with appropriate materials to guide the planuing and creation of telematics l pp p g p g instruction manuals and activities for the Macintosh computer system, p y self-paced laboratory activities describing the use of the telematics software, and self-paced laboratory activities describing the use of the telematics software, and an electronic document on the World Wide Web describing distance and rural education technologies and their applications. an electronic document on the World Wide Web describing distance and rural education technologies and their applications. The selection of the materials involved the broadest possible range of instructional technologies and media to provide students with exposure and experience in use of the many forms of technology that could support them in their rural education teaching. Education in Rural Australia, Vol. 6 (2) ... 4 OUTCOMES Student responses provided strong support for the claim that the course was both effective and efficient in achieving its objectives. Of the 23 students who participated in the project, most had minimal computing' experience. That experience ranged from very limited use of computer- assisted learning packages to having used a word processor for assignment preparation. The technology emphasis in this course required students to significantly extend their practical computing skills and knowledge. Course Strengths: The aspect of the course that most students regarded as its major strength was its practical and hands-on format. Most students commented on the value they saw being gained from the practical activities. Other strengths included the collaborative nature of the learning environment, the capacity to work in groups and the high levels of participation. The students enjoyed working with the technology and felt that the independent nature of the learning led to confidence building in the use of the various pieces of hardware and software. It was of interest to note that many of the attributes of distance learning that often are seen as impediments to successful teaching and learning, for example, independent learning, were seen by these students to be a strength of the course. Course Weaknesses: Students noted a number of weaknesses in the way in which the course was delivered. Those weaknesses were all brought about by the remote learning environment and the incapacity of the technology to fully replicate face-to-face teaching. In a more traditional course students would have been told of the weaknesses of teaching with telematics. In this course, however, they experienced those weaknesses. The students indicated that they would have preferred to have met the off-site instructor. They felt that this limited the knowledge they could draw from his experience. It could be argued, however, that the knowledge they drew from being distant probably significantly outweighed the benefits that which would have been gained from direct contact. The students further felt that the course itself was of too short a duration and would have preferred more time to work with the technology and prepare their teaching sequences. This can be quite easily remedied in future implementations by breaking this component of the course into non-contiguous blocks. Comparison With Conventional Instruction: All students saw strong comparisons between the new format and conventional modes of teaching. Students were asked to comment on what aspects they saw as superior and inferior. COURSE DELIVERY This course, delivered to students in 1995, was based on a series of short telematics lessons supported by independent learning activities. The telematics lessons were delivered by the lecturer from a separate campus. Another lecturer acted as a support for the students in much the same way as the classroom teacher coordinates school-based telematics programs. Students were expected to complete all the assigned tasks across a five week period leading to the final planuing and delivery of an actual telematics lesson. Throughout the course, communication with the support lecturer was limited to the telematics technology and telephone, a restriction that was used to make students fully aware of the conditions and limitations of learning through such technologies. Records were kept of the students' progress in the different activities and, three months after the course, they were asked to complete a questionnaire to gather more specific details of their anitudes to telematics teaching and learning. The questionnaire sought responses to the following questions: What did students perceive to be the strengths of the course? What were the perceived weaknesses? How did the course compare to conventional teaching formats? How much was learned and retained? What parts of the course were the most effective and provided the best learning opportunities? What did students perceive to be the strengths of the course? What were the perceived weaknesses? How did the course compare to conventional teaching formats? How much was learned and retained? What parts of the course were the most effective and provided the best learning opportunities? What did students perceive to be the strengths of the course? What were the perceived weaknesses? How did the course compare to conventional teaching formats? How much was learned and retained? What parts of the course were the most effective and provided the best learning opportunities? Education in Rural Australia, Vol. 6 (2) ... 4 Education in Rural Australia, Vo!. 6 (2) ... 5 SUMMARY The purpose of this project was to create and implement a learning environment based on contemporary learning theories to enhance learning outcomes in a pre-service teacher education course. The module that was created provided a setting where students were able to construct their own meaning and understanding of issues and outcomes arising from the use of telecommunications technologies as a delivery medium for distance and rural education. The outcomes and initial findings from an implementation of these ideas has provided very positive results. The project has demonstrated a powerful alternative to the conventional teaching format, one which is both efficient as a delivery medium and effective in the outcomes that are achieved. Implementation is planned for 1996 and will involve a more rigorous examination of learning outcomes to more fully ascertain strengths and weaknesses of the alternative teaching approach. The further practical implications arising from this project are clear. Pre-service teacher education institutions are in a position to provide their students with the necessary understandings and skills in the use of telecommunication technology. This, however, calls for a concerted effort. Unless telecommunication technology is afforded priority status, its full potential will not be realised, opportunities will be lost, the present disparity between educational services available in rural and urban schools will continue to widen and teacher educators will again have failed to provide those experiences that help to ensure that rural children are provided with the extended educational access that is their right. OUTCOMES There were many more examples of superiority given. The more frequently mentioned superior elements tended to be those associated with active and constructive learning. The students recognised and valued the approach that placed importance on learning through experience. Those aspects listed as inferior tended to relate to restrictions caused by teaching with telematics and as such were strong and contributory components of the teaching program. Learning: The students were very positive in their descriptions of what they learned and retained. The questionnaire, completed some time after the end of the course, provided some time for the course content to wane. The majority of students considered that they retained a strong level of knowledge of the principles and practices covered in the course. Many considered that they needed more experience with the technology but at the same time knew the areas where their weaknesses lay and had the means to overcome these in the form of self-paced instructional materials. Most Effective Components: There was little agreement in student responses about those components of the course that each judged to be most effective. Some considered the planning and delivery of their own telematics lesson to be the most useful component while others considered the class telematics sessions to be the most effective. It was expected that the students would rate the practical and technology-based activities as those through which most was learned. Many students rated the reading and video viewing as activities of high educational Education in Rural Australia, Vo!. 6 (2) ... 5 value. It was apparent that among the group there was a range of preferred learning styles and the availabili,ty of a wide range of activities and learning opportunities provided an environment that appeared to cater well for all students. value. It was apparent that among the group there was a range of preferred learning styles and the availabili,ty of a wide range of activities and learning opportunities provided an environment that appeared to cater well for all students. Education in Rural Australia, Vol. 6 (2) ... 6 REFERENCES Ausubel, D. (1960). The use of advance organisers in the learning and retention of meaningful verbal material. Journal of Educational Psychology, 51, 267-272. Brown, J. (1985). Process versus product: A perspective on tools for communal and informal electronic learning. Journal of Educational Computing Research, 1, 179-201. Brown, J., Collins, A., & Duguid, P. (1989). Situated cognition and the culture of learning. Educational Researcher, 18(1),32-41. ( ) Bmner, J. (1960). The Process of Education. Cambridge, Mass: Harvard University Press B D & C h C (1993) P i h i i i d i l Byrum, D., & Cashman, C. (1993). Pre-service teacher training in educational computing: Problems, perceptions and preparation. Journal of Technology and Teacher Education, 1(3), 259-274. Diem, R. (1989) Preservice teachers and computer ntilisation: A case study. Educational Technology, 29(12), 34-36. wnes, T. (1993) Student-teachers' experiences in using computers during teaching practice Journal of Computer Assisted Learning, 9(1), 17-33. wood, G. & Norman, P. (1988) Problems of educational innovation: The primary teacher response to using the microcomputer. Journal of Computer Assisted Learning, 4(1), 34-43. y p y response to using the microcomputer. Journal of Computer Assisted Learning, 4(1), 34-43. Hollingsworth, S. (1989). Prior beliefs and cognitive change in learning to teach. American Educational Research Journal, 26(2),160-189. Hollingsworth, S. (1989). Prior beliefs and cognitive change in learning to teach. American Educational Research Journal, 26(2),160-189. ( ) Newell, A., & Simon, H. (1972). Human problem-solving. Englewood Cliffs, NJ: Prentice- Hall. Novak, D. & Knowles, J. (1991) Beginning elementary teachers' use of computers in classroom instruction. Action in Teacher Education, 8(2), 43-51. Oliver, R. & Reeves, T. (1994). Telematics in Rural Education. An investigation of the use of telematics for the delivery of specialist programs for students in rural schools. Mt Lawley Western Australia: InTech Research, Edith Cowan University. y Piaget, J. (1954). The construction of reality in the child. New York: Basic Books. R Prawat, R. (1992). Teachers' beliefs about teaching and learning: A constructivist perspective. American Journal of Education, 100(3), 354-395. Robinson, B. (1993). The English national curriculum and the information technology curriculum for teacher education. Journal of Technology and Teacher Education, (I), 1,73- 80. Reeves, T. (1993). Interactive learning systems as mind tools. in Viewpoints 2 (Ed. P. Newhouse). Perth: Educational Computing Association ofWA. R I (1993) C k l d i i i d i d d h i i Rovegno, I. (1993). ABOUT THE AUTHORS Ron Oliver is a Senior Lecturer specialising in multimedia and information technologies at Edith Cowan University. He has extensive experience in implementing and researching applications of computer technologies in school curricula and has a particular interest in technologies that support open-learning and distance education. He leads a number of school-based research projects involving use of the Internet and telecommunications for instructional purposes. Murray Lake is Director of the Australian Centre for Education and Training at Edith Cowan University and Senior Lecturer in Education. He has been involved in rural education for more than 15 years both as a teacher and researcher, has carried out rural education projects for the State Education Department, Commonwealth Schools Commission and was a member of the Ministerial Review of Rural Education in Western Australia. Murray's work has involved him in projects in Mauritius and the Seychelles and, most recently, in a study of small rural schools in Malaysia. Education in Rural Australia, Vol. 6 (2) ... 6 Education in Rural Australia, Vol. 6 (2) ... 6 REFERENCES Education in Rural Australia, Vol. 6 (2) ... 7 REFERENCES Content-knowledge acquisition during undergraduate teacher training: Overcoming cultural templates and learning through practice. American Educational Research Journal, 30(3),611-642. ( ) Steinberg, E. (1989). Cognition and learner control: A literature review, 1977-88. Journal of Computer-Based Instruction, 16(4), 117-121. Tomlinson, D. (1994). Schooling in Rural Western Australia: Report of the Ministerial Review of Schooling in Rural Western Australia. Perth: Education Department of Western Australia. Wittrock, M. (1974). Learning as a generative activity. Educational Psychologist, 11, 87-95. Education in Rural Australia, Vol. 6 (2) ... 7 Education in Rural Australia, Vol. 6 (2) ... 7
https://openalex.org/W3207274240	https://ojs.unimal.ac.id/mjml/article/download/2737/2822	English	null	Performance Analysis of Students Through Critical Thinking Ability Based on Mathematic Ability	Malikussaleh journal of mathematics learning	2,021	cc-by-sa	4,451	1. INTRODUCTION In terms of education, it is all aspects of activities that have a positive impact on all subjects and objects in education . Activities in education are not just the delivery of subject matter such as providing explanations to students, but there are many basic things that need to be considered such as guidance and direction, as well as instructions for each student (Fattah, 2003; Tilaar, 2012; Alwasilah, 2012; Sauri,2006). Activities like this are things that can affect aspects of knowledge and motivation of students in carrying out learning activities. (Sardiman, 2007; Wardati & Jauhar, 2011; Sukardi & Kusmawati, 2008; Sumarwiyah, 2009). With the development of an era, of course education will also continue to develop over time, this can be exemplified and found in terms of science or technology. Technology that has developed drastically has certainly entered our lives. This needs to be considered by all education observers as the development of a technology that has a role in increasing capabilities in all fields in general. such as the abilities contained in three indicators, namely intellectual, psychomotor and affective abilities. In developing these abilities, of course there are many factors that influence it (Yusuf, 2005; Siswanto, 2016). Therefore educators need to prepare what activities will be carried out by students and the need to prepare as early as possible by paying attention to the many factors that influence it. The statement above is in accordance with the opinion of Sakerebau (2018) which clearly explains that many factors influence a person in learning, where these factors are classified into internal factors, which come from within a person consisting of psychological, fatigue, and physical items, while other factors, namely external factors are those that come from outside the person such as parents, educators, and the surrounding community. In learning mathematics, which is part of a scientific discipline, it has a major role in the development of science and also in the field of technology, this is because mathematics is said to be one of the sciences that underlie other sciences (Shadiq, 2014; Sumarmo, 2007; Mandur, 2013; Jihad, 2008). In studying mathematics, we can learn to develop meticulous and accurate aspects, we can also improve Learning in terms is known as two-way communication activities by involving support for each student in order to achieve learning goals and change behavior in a positive direction (Mulyadi, 2010; Mulyasa, 2007). Malikussaleh Journal of Mathematics Learning (MJML) Vol. 4, No. 1, May 2021, pp. 23-27 ISSN 2620-6315 (print), 2620-6323 (online) Malikussaleh Journal of Mathematics Learning (MJML) Vol. 4, No. 1, May 2021, pp. 23-27 ISSN 2620-6315 (print), 2620-6323 (online) Malikussaleh Journal of Mathematics Learning (MJML) Vol. 4, No. 1, May 2021, pp. 23-27 ISSN 2620-6315 (print), 2620-6323 (online) DOI: https://doi.org/10.29103/mjml.v4i1.2737 Available online at http://ojs.unimal.ac.id/index.php/mjml Performance Analysis of Students Through Critical Thinking Ability Based on Mathematic Ability Qurrota A’yun1, Rosita Yanuarti1, Dimas Anditha Cahyo Sujiwo2 1 Department of Informatics Engineering, University of Muhammadiyah Jember, Jember, Indonesia, 68121 2 Department of Mathematics Education, IKIP PGRI Jember, Jember, Indonesia, 68121 Corresponding author: qurrota.ayun@unmuhjember.ac.id \| Phone Number: +6282231308136 Qurrota A’yun1, Rosita Yanuarti1, Dimas Anditha Cahyo Sujiwo2 1 Department of Informatics Engineering, University of Muhammadiyah Jember, Jember, Indonesia, 68121 2 Department of Mathematics Education, IKIP PGRI Jember, Jember, Indonesia, 68121 Corresponding author: qurrota.ayun@unmuhjember.ac.id \| Phone Number: +6282231308136 ARTICLE HISTORY ARTICLE HISTORY The standard of critical thinking in this study, namely: first, interpretation is on a problem can showing/ writing what they know and what is asked about the problem correctly; second, analysis is the activity of identifying the relationship between statements, questions and concepts in a problem through making mathematical models and accompanied by the right reasons; third, evaluation is the right strategy in solving problems and fourth, conclusion is accuracy in drawing conclusions from what is asked. While the type of research used is descriptive qualitative type. The instruments used were interview guidelines and test questions. The interview used is a structured interview and the test used in the form of a math problem test with derived application material consisting of 3 problem descriptions with material that has been received by students. Student performance levels in this study are at the Apprentice level and the Novice level. The acquisition of data at the Apprentice level the average value is different, this is because the acquisition of data on each indicator is different overall. This is because the subject is not accustomed to or accustomed to working on open questions or also problem stories/problems. Student performance levels in this study are at the Apprentice level and the Novice level. The acquisition of data at the Apprentice level the average value is different, this is because the acquisition of data on each indicator is different overall. This is because the subject is not accustomed to or accustomed to working on open questions or also problem stories/problems. Received : 11 August 2020 Revised : 11 March 2021 Accepted : 10 April 2021 KEYWORDS Received : 11 August 2020 Revised : 11 March 2021 Accepted : 10 April 2021 KEYWORDS Critical Thinking Skills; Level of Performance; Received : 11 August 2020 Revised : 11 March 2021 Accepted : 10 April 2021 This is an open access article under the CC–BY-SA license. 1. INTRODUCTION 1. INTRODUCTION 1. INTRODUCTION In their duties, educators need to develop the basic abilities of their students, 23 Malikussaleh Journal of Mathematics Learning (MJML) Vol. 4, No. 1, May 2021, pp. 23-27 A’yun, Yanuarti & Sujiwo critical and logical thinking skills, reasoning and can be positive, creative, responsible, and the ability to work with peers (Ismaimuza, 2010; Riyanto, 2010; Trianto, 2010; Isjoni, 2009; Dwitagama and Wijaya, 2012; Haryani, 2012; Julita). This is in accordance with Suherman et al (2003: 56) which states that there are three functions in learning mathematics, namely patterns of thought, tools, and knowledge. Furthermore, Vincent (Dahlan, 2017) explains that thinking is any mental activity that can help formulate or solve a problem, make decisions or fulfill the desire to understand something. Not a few students argue that it is difficult to learn and understand mathematics subjects because the object of study studied in mathematics is abstract and moreover learning is only from educators. Memory limitations are also the cause of students to memorize mathematical formulas that are considered meaningful. This kind of thing can have an impact on mathematics which is still seen as a difficult subject for students and society in general (Muijs & Reynolds, 2005). The thinking ability of each individual is certainly not the same, so each individual needs to practice and develop their abilities in studying mathematics subjects. This way of learning that is carried out by students certainly needs a change, namely by increasing their thinking skills, one of which is critical thinking. they have with the new knowledge they get while studying the lessons they follow, and are able to connect mathematical concepts but are still part of the process; 3) Practitioner, students have begun to be able to have the correct strategy to solve problems, reasons and the process of proof have started to be logical, able to partially communicate ideas, able to connect old knowledge with new ones, and able to construct mathematical and scientific concepts but still part of the process construction; 4) Expert, this level shows that students have started to be able to have the right strategy to solve problems, the reasons and the proof process have started to be logical, able to communicate complete ideas, able to connect old knowledge with new ones, and able to construct mathematical and scientific concepts. This research is based on the level set by Exemplars. 2. METHODS This type of descriptive qualitative research is used in this study Researchers describe the performance level of Information Technology students. In other words, it can be said that the researcher describes the level of performance of this Information Technology student based on the student's critical thinking ability in solving math problems given during research activities. The researcher also described the level of student performance in critical thinking in solving problems in the form of problems. The researcher checks and reports the results of the student's problem solving by matching the results of the student's answers with the criterion of critical thinking. The data in the study were interview guidelines that would be applied to students and test questions that would be carried out by six selected students majoring in Information Technology class A Muhammadiyah University of Jember which were categorized as high, medium, and low. Data obtained in the form of test results and interviews. The test for performance levels is based on students' critical thinking skills. The research subjects were Muhammadiyah Jember Information Technology students. Subject taking was obtained from previous students' scores to group students with high, medium and low abilities and continued by selecting two students from each category. From the test activities given, then conducting interviews with each student as the research subject. Taking two students of Information Technology class A Muhammadiyah University of Jember in each of these categories aims to determine whether students in the same category are at the same level or not. So that in this study the researcher took as many as 6 subjects with two high category students, two medium category students, and two low category students who would be analyzed for their performance levels based on their critical thinking skills in solving math problems that had been prepared before the activity. Dahlan (2017) explained that the four levels of performance appraisal for students were in full according to the NCTM standard. 1. INTRODUCTION So that researchers will observe student performance levels, namely: Novice, Apprentice, Practitioner and Expert. To find out the level of students in this study is to connect each level with the predetermined standard indicators of thinking. Performance level category based on NCTM stipulations adopted by Dahlan, (2017). Tabel 2. Performance level category Tabel 2. Performance level category Score Performance level Expert Practitioner Apprentice Novice g Choy & Cheah (2009) define critical thinking as complex which requires a high level of cognitive processing information. Furthermore, Ennis (2011) explains that critical thinking is the ability to think reflective and reasoned which is focused on what is believed or done. Students can think critically with a skill through the results of activities to analyze and prove the truth. According to Fisher (2009), it explains that critical thinking is a skill in thinking about everything in an appropriate way in interpreting and evaluating activities such as observation, interaction with various other sources of information. Critical thinking carried out by students in the form of questioning questions, answering logically, finding information in an efficient time. A person's ability can be improved with regular practice in solving math problems. The maximum effort that must be done is to give students experience knowledge so that students' performance abilities will increase. To find out this, a method is needed that can inform learning outcomes, namely the achievement of student competencies, which is called an assessment. The appropriate form of assessment is student performance assessment. While Arifin (2012) performance appraisal is a way of assessing the level of mastery of students' skills through performance tests or demonstrations or real work practices. The level of student performance consists of four levels with reference to the level set by Exemplars. The four levels are Novice, Apprentice, Practitioner, and Expert. 3. RESULTS AND DISCUSSION 3. RESULTS AND DISCUSSION In the implementation of this research, the research instruments that will be used are validated first by the experts in their fields. There are three validators who will provide an assessment of the instruments to be used. By carrying out this validation activity, it is hoped that the level of validity of this research instrument can be measured so that it can be used properly. The instruments that will be used are the instrument test questions and interview instruments. The following are the values of the three validators. The results of validator 1 indicate that the test questions instrument is in the range of 2.5833333333 or Va = 2.5833333333. These results indicate that the value of validator 1 for the question instrument is in the valid criteria. The result of validator 2 shows that the result of the instrument validation test is 2.6666666667 or Va = 2.6666666667. While validator 3 shows the results of the instrument validation test are 2.6666666667 or Va = 2.6666666667. So that the average validation result of the three validators for the test instrument is 2.638888889. According to the instrument validity criteria, then the test instrument is valid in the 2.5 < Va < 3 category range. Furthermore, in the interview instrument, the average value of the three validators was obtained by 2.5833333333. The details of each validator for the validation of the guideline instrument are as follows. validator 1 gives a score of 2.5 for the interview instrument, the value of the validator 2 is 2.75, and the value of the validator 3 is 2.5. So that the average obtained in the validation of the interview instrument for the three validators is 2.5833333333. This value is in the valid category and falls into the 2.5 < Va < 3 category. research can be used for research activities. For the two low-category students, one of the students was not clear or did not understand the problem. This means that the student is not precise or incomplete in writing what he knows and the questions asked. This was reinforced during interviews, students were not yet precise or clear in terms of describing a problem. One of the other students was able to say that he understood enough about the problem. 2. METHODS The following is the explanation: 1) Novice, students have a strategy in solving problems that contain problems, participants do not provide logical explanations in mathematical concepts related to the process of proof, are unable to communicate ideas in their thinking, are unable to connect old knowledge with new ones so that little experience, and unable to construct mathematical concepts; 2) Apprentice, at this level, students can be said to have been able to have or carry out a correct strategy to solve a problem, can provide reasons and the process of proof can be said to be logical even though it is not yet orderly, able to communicate ideas partially, can associate the knowledge 24 Malikussaleh Journal of Mathematics Learning (MJML) Vol. 4, No. 1, May 2021, pp. 23-27 A’yun, Yanuarti & Sujiwo The test used is in the form of a math problem test with derivative application material consisting of 3 description questions with material that has been received by students. Each question will test the criteria or indicators of student performance assessment based on critical thinking skills. The purpose of working on these questions is to make sure that the student can work on the questions given with the same type. The interview activity was carried out after giving the final test questions. To avoid the same answer, interviews were carried out alternately for each ability group and carried out individually. The type of interview used is a structured interview because the guidelines were prepared from the start. The analysis was carried out in critical thinking skills, namely the analysis of the test results obtained from the answer sheet. Student answers are analyzed with an assessment sheet based on standard critical thinking indicators, which will then be categorized into performance levels in accordance with the scoring guidelines for the performance levels that have been made, scoring on student work results on test questions is adjusted to indicator achievement. After giving scores on the students 'work on the test questions, an interview was conducted which aimed to see whether the students' answers were consistent between the answers on the test questions and during the interviews. students are dominant at the apprentice level, through this research it is hoped that students can hone their critical thinking skills in solving problems. Critical thinking can be said as thinking that processes and results by analyzing and evaluating them. 2. METHODS This certainly shows that students can think critically with their own skills by analyzing and proving the truth. The following is the result of the percentage of students' critical thinking on each predetermined indicator. Tabel 2. The average gain from the performance level indicator Tabel 2. The average gain from the performance level indicator Item Name Average Category 1 BE 2,111111111 Apprentice 2 AL 2,111111111 Apprentice 3 AA 2,851851852 Apprentice 4 AF 2,518518519 Apprentice 5 AX 1,740740741 Novice 6 AU 1,666666667 Novice Based on the table above, it shows that the results of low-ability critical thinking skills appear to be at the novice level stage. Medium abilities are at the apprentice level, as well as high abilities are at the apprentice level. Based on this data, it shows that students have different ability to think. Students need to be accustomed to being given problems that require working on questions that have a creative thinking level. In research activities, it is at the interpretation stage where students are asked to understand the questions related to the problem. This stage requires students to understand the problem, students have the ability to think in different ways. 3. RESULTS AND DISCUSSION Overall students are in the high category both in solving problems on the questions and in delivering them in interview activities. This is because in the data validity activity, the triangulation of the test and interview method produces appropriate results, meaning that each student in the high category is so consistent between the answer sheet and the delivery that was raised by the student during the interview activity. With the results of data validity through this triangulation, it can be said that the results of the data obtained from this method are "valid". In high category students, both students are at the Apprentice level, but the average obtained from the two students is different, this is because the data acquisition of the two students on each indicator of critical thinking is different as a whole. Dahlan, A. (2017). Leveling of Student Performance Based on Critical Thinking Ability of Class VII Students of MTs Negeri Jember 1 Filial in Solving Mathematical Problems Related to the Coffee Theme. Jurnal Matematika. Jember : Universitas Jember. Dwitagama, D andWijaya, K. (2012). Get to know Classroom Action Research. Jakarta: Indeks. Fattah, N. (2003). Concept of School Based Management and School Board. Bandung : Pustaka Bani . Fisher, A. (2009). Critical Thinking: An Introduction. Jakarta: Erlangga. Haryani, Desti. (2012). Shaping Critical Thinking Students through Mathematics Learning. Proceedings of the National Seminar on Mathematics and Mathematics Education, Nov:10. Yogyakarta. Isjoni.(2009).“Cooperative Learning Increases Communication Intelligence among Students”. Yogyakarta: Pustaka Pelajar. Ismaimuza, D. (2010). The Effect of Problem Based Learning with Cognitive Conflict Strategies on Mathematical Critical Thinking Ability and Attitudes of Junior High School Students. Jurnal Pendidikan Matematika UNSRI, (Online), Vol. 4 No.1 Jihad, A. (2008). Mathematics Curriculum Development. Yogyakarta: Multi Pressindo. Julita. (2014). Developing Mathematical Critical Thinking Skills through Concept Achievement Learning. Proceedings of the National Seminar on Mathematics Education for the Postgraduate Program of STKIP Siliwangi, Nov:27. Bandung. 4. CONCLUSION Mandur, K. (2013). Contribution of Connection Ability, Percentage Ability, and Mathematical Disposition on Mathematics Learning Achievement of Private High School Students in Manggarai Regency. E-Journal PPs Universitas Pendidikan Ganesha. Vol. 2. Thn. 2013. Halaman: 4. Based on the discussion and analysis results, it can be concluded that the level of student performance in the critical thinking ability of Information Technology students shows at the Apprentice level and the Novice level. Of the six existing subjects, 2 subjects are at the Novice level. 3. RESULTS AND DISCUSSION Therefore, this student is expected to be careful in solving all the questions given in the future. Overall, this medium category student is quite good at working on questions and interview activities. This is shown in the triangulation activity of the test and interview method which shows that each student with the moderate category is very consistent between the answer sheet and the student's answer during the interview. The validity of the data through this triangulation shows the validity of the data results. In the middle category students indicate that the two students are at the Apprentice level, but the average obtained from the two students is different, this is because the data acquisition of the two students on each indicator of critical thinking is different as a whole. A.M, Sardiman. (2007). Teaching and Learning Interaction and Motivation. Jakarta: PT Raja Grafindo Persada. Alwasilah, A,c. (2012). Anyway Engineering Literacy. Bandung: PT Kiblat Buku Utama. Arifin, Zainal. (2012). Learning Evaluation. Bandung : PT. Remaja Rosdakarya. Choy, S. C., & Cheah, P. K. (2009). Teacher Perception of Critical Thinking Among Students and Its Influence on Higher Education. International Journal of Teaching and Learning in Higher Education, 20(2), 198—206. Retrieved from: http://www.isetl.org/ijtlhe/pdf/IJTLHE336.pdf. http://www.isetl.org/ijtlhe/pdf/IJTLHE336.pdf. Ennis, R.H. (2011). The Nature of Critical Thinking:An Outline of Critical Thinking Disposition and Abilities. Last Revised. Emeritus Proffessor: University of Illinois Students with high abilities at this critical thinking stage, it can be said that students with high categories already understand the questions well. Students are also able to write down the problem questions by writing down what is written on the questions clearly. Students are also able to plan the problem solving properly and also in accordance with the answers to solving the questions. However, in the indicators of writing another alternative way, students have not been able to write well. So that in this indicator students get a score of 1. While in making conclusions about solving the questions, the two students are very clear and can be said to be good at making conclusions, the reasons given in the conclusions also reflect correct problem solving. Then discussing interview activities with the two students with this high category. The explanation of the things conveyed in the interview activities by the two students was very consistent with the answer sheets they worked on. 3. RESULTS AND DISCUSSION This is evidenced in the answer sheet, which shows that the student can write down what is known and what is being asked about the question. In the interview activity too, the student's statement can be said to be consistent with the answer sheet. With consistency between student answer sheets in the low category and what was conveyed during the interview, AU students are at the novice level because most of them do not fulfill all critical thinking indicators, while AX students are also at the novice level, but the average results of the scores critical thinking indicator is different from AU. AU obtained an average value of 1.6666666667 in the novice level category and AX got a score of 1.740740741 which is also at the novice level. In other words, there is a difference in the results of the average acquisition of critical thinking indicators for students with the low category, this is because the data acquisition for each indicator is different as a whole. Students with moderate ability at this critical thinking stage, it can be said that they have understood the problem well, this is known when they can do or write things that are known, overall there are still some errors and inaccuracies. another thing in the process, students in the medium category are good enough in presenting a problem that is in the problem in clear language. In the interview activity with students in the medium category, as a whole the students explained what was asked in the interview according to what they wrote in their respective answer sheets. In terms of writing down the student's understanding of the questions, the student can rewrite the answers to what is known and Of the 6 students who were the subject, the level of performance of each student was obtained by different indicators of achievement. This student performance level is based on their critical thinking skills which are classified into two levels, namely the apprentice level and the novice level. This leveling grouping is based on an analysis of student answers in solving a problem. In the results of this leveling, 25 Malikussaleh Journal of Mathematics Learning (MJML) Vol. 4, No. 1, May 2021, pp. 23-27 A’yun, Yanuarti & Sujiwo REFERENCES what is asked in the questions. Although in writing a strategy plan for solving and solving problems, there are still things that are unclear and inaccurate. 3. RESULTS AND DISCUSSION This subject is a subject with low ability. While at the Apprentice level there are four subjects. The four subjects have different abilities, namely two subjects with moderate ability and the other two subjects are subjects with high abilities. However, in this category with the same Apprentice level the average obtained by the four subjects is different, this is because the data obtained by these four subjects on each indicator are different as a whole. In this study, none of the six subjects were at the critical thinking level category with the Practitioner level and the expert level. This is because the subject is not accustomed or accustomed to working on open questions or story questions/problems. Muijs, D., & Reynolds, D. (2005). Effective teaching evidence and practice. London: SAGE Publications.p.212. Mulyadi. (2010). Diagnosis of Learning Difficulties and Guidance on Specific Learning Difficulties. Yogyakarta: Nuha Litera. Mulyasa, E. (2007). Competency Standards and Teacher Certification. Bandung: Remaja Rosdakarya. Riyanto, Y. (2010). New Paradigm of Learning. Jakarta: Kencana Prenada Media Group. Sakerebau, J. (2018). Memahami Peran Psikologi Pendidikan Bagi Pembelajaran. BIA: Jurnal Teologi dan Pendidikan Kristen Kontekstual 1.1, hal 96-111. 26 Malikussaleh Journal of Mathematics Learning (MJML) Vol. 4, No. 1, May 2021, pp. 23-27 A’yun, Yanuarti & Sujiwo Sauri, S. (2006). Building Communication in the Family. Bandung: PT Genesindo. Shadiq, F. (2014). Mathematics Learning (How to Improve Students' Thinking Ability). Yogyakarta: Graha Ilmu. Siswanto, & B. Tri. (2016). Factors Influencing Student Learning Outcomes On Teaching Practical Automotive High School Schools in Yogyakarta City. Jurnal pendidikan vokasi. 6 (1): 2476- 9401. Suherman.E, et al. (2003). Strategi Pembelajaran Matematika Kontemporer (Edisi Revisi). Bandung: JICA-Universitas Pendidikan Indonesia (UPI). Sukardi, Ketut, D dan Kusmawati, N. (2008). Guidance and Counseling Process in Schools. Jakarta: PT Rineka Cipta. Sumarmo, U & Permana Y. (2007). Develop High School Students' Reasoning Ability and Mathematical Connection Through Problem Based Learning. Jurnal Educationist. Vol. I. No 2. page: 117. Sumarwiyah. (2009). The Effect of the Application of Tutoring Services on Study Habits and Learning Achievement. Jurnal Sosial and Budaya. Vol:2 No 2. Tilaar, H. A. R. (2012). National Education Kaleidoscope. Jakarta: Gramedia. Trianto. (2010). Designing Innovative-Progressive Learning Models. Jakarta: Kencana Prenada Media Group. Wardati & Jauhar, M. (2011). Guidance and Counseling Implementation in Schools. Jakarta: Prestasi Pustakaraya. Yusuf, S. (2005). Guidance and Counseling Foundation. Bandung: PT. Remaja Rosda Karya. 27
https://openalex.org/W3049104711	http://ruor.uottawa.ca/bitstream/10393/40849/1/13024_2020_Article_397.pdf	English	null	The role of TDP-43 mislocalization in amyotrophic lateral sclerosis	Molecular neurodegeneration	2,020	cc-by	17,666	© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. The role of TDP-43 mislocalization in amyotrophic lateral sclerosis Terry R. Suk1,2 and Maxime W. C. Rousseaux1,2,3,4* Terry R. Suk1,2 and Maxime W. C. Rousseaux1,2,3,4* Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 https://doi.org/10.1186/s13024-020-00397-1 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 https://doi.org/10.1186/s13024-020-00397-1 Background TDP-43, a central player in amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) is a fatal neurode- generative disease characterized by the selective loss of motor neurons resulting in mortality within an average of 2-5 years [1]. Though most cases of ALS are sporadic (sALS), approximately 10% are familial (fALS) in origin. The identification of these familial cases, now spanning TDP-43 bridges the divide between sporadic and familial ALS and remains a dominant protein of interest to understand disease pathogenesis. TDP-43 was identified as a primary component of ubiquiti- nated and hyper-phosphorylated cytosolic aggregates * Correspondence: max.rousseaux@uottawa.ca 1University of Ottawa Brain and Mind Research Institute, Ottawa, Canada 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada Full list of author information is available at the end of the article tawa, Canada l list of author information is available at the end of the article Abstract Since its discovery as a primary component in cytoplasmic aggregates in post-mortem tissue of patients with Amyotrophic Lateral Sclerosis (ALS), TAR DNA Binding Protein 43 kDa (TDP-43) has remained a central focus to understand the disease. TDP-43 links both familial and sporadic forms of ALS as mutations are causative for disease and cytoplasmic aggregates are a hallmark of nearly all cases, regardless of TDP-43 mutational status. Research has focused on the formation and consequences of cytosolic protein aggregates as drivers of ALS pathology through both gain- and loss-of-function mechanisms. Not only does aggregation sequester the normal function of TDP-43, but these aggregates also actively block normal cellular processes inevitably leading to cellular demise in a short time span. Although there may be some benefit to therapeutically targeting TDP-43 aggregation, this step may be too late in disease development to have substantial therapeutic benefit. However, TDP-43 pathology appears to be tightly linked with its mislocalization from the nucleus to the cytoplasm, making it difficult to decouple the consequences of nuclear- to-cytoplasmic mislocalization from protein aggregation. Studies focusing on the effects of TDP-43 mislocalization have demonstrated both gain- and loss-of-function consequences including altered splicing regulation, over responsiveness to cellular stressors, increases in DNA damage, and transcriptome-wide changes. Additionally, mutations in TARDBP confer a baseline increase in cytoplasmic TDP-43 thus suggesting that small changes in the subcellular localization of TDP-43 could in fact drive early pathology. In this review, we bring forth the theme of protein mislocalization as a key mechanism underlying ALS, by highlighting the importance of maintaining subcellular proteostasis along with the gain- and loss-of-functional consequences when TDP-43 localization is dysregulated. Additional research, focusing on early events in TDP-43 pathogenesis (i.e. to the protein mislocalization stage) will provide insight into disease mechanisms, therapeutic targets, and novel biomarkers for ALS. Keywords: ALS, TDP-43, Mislocalization, Pathology, Nucleocytoplasmic shuttling over 20 genes (reviewed by Nguyen et al. [2]) has highlighted the importance of various cellular processes in the pathogenesis of ALS [3]. Indeed, some rare genetic cases – such as the identification of mutations in TAR DNA Binding Protein 43 kDa (TARDBP, encoding TDP-43) have provided crucial insight into common pathogenic themes in ALS [4–7]. * Correspondence: max.rousseaux@uottawa.ca 1University of Ottawa Brain and Mind Research Institute, Ottawa, Canada 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada Full list of author information is available at the end of the article Page 2 of 16 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 nearly all ALS-causing mutations on TDP-43 cluster within this domain [4, 6, 7]. observed from post-mortem tissue of patients with ALS [8, 9]. This pathological phenomena is considered a hallmark of ALS as it is observed in approximately 97% of all ALS patients regardless of the mechanisms of disease onset, with the notable exceptions of familial ALS (fALS) caused by mutations in Zn/Cu Superoxide Dismutase 1 (SOD1) and Fused in Sarcoma (FUS) [1, 10–15]. Further- more, since the first report in 2008, over 50 mutations in TARDBP have been linked to ALS, further supporting TDP-43 dysfunction as a critical component in ALS [4–6, 16–18]. Therefore, TDP-43 dysfunction provides common ground in an otherwise convoluted disease, thus gaining notoriety and attention from researchers aiming to un- cover the mechanisms causing TDP-43 aggregation. It is also important to note that mutations in TARDBP can also cause frontotemporal lobar dementia (FTLD), which itself shares some clinical parallels with ALS and displays TDP-43 pathology in ~ 45% of cases [8, 9, 19–21]. Here, however, we will focus on TDP-43 dysfunction as a central mechanism connecting multiple pathways in the context of ALS. observed from post-mortem tissue of patients with ALS [8, 9]. This pathological phenomena is considered a hallmark of ALS as it is observed in approximately 97% of all ALS patients regardless of the mechanisms of disease onset, with the notable exceptions of familial ALS (fALS) caused by mutations in Zn/Cu Superoxide Dismutase 1 (SOD1) and Fused in Sarcoma (FUS) [1, 10–15]. Further- more, since the first report in 2008, over 50 mutations in TARDBP have been linked to ALS, further supporting TDP-43 dysfunction as a critical component in ALS [4–6, 16–18]. Therefore, TDP-43 dysfunction provides common ground in an otherwise convoluted disease, thus gaining notoriety and attention from researchers aiming to un- cover the mechanisms causing TDP-43 aggregation. It is also important to note that mutations in TARDBP can also cause frontotemporal lobar dementia (FTLD), which itself shares some clinical parallels with ALS and displays TDP-43 pathology in ~ 45% of cases [8, 9, 19–21]. Here, however, we will focus on TDP-43 dysfunction as a central mechanism connecting multiple pathways in the context of ALS. In ALS, truncated forms of TDP-43 are found in ALS aggregates, more predominantly in the cortex but also to a lesser extent in the spinal cord [55–59]. The N- terminally truncated, C-terminal fragments 35 kDa (CTF35) and 25 kDa (CTF25) are the most notable “species” of TDP-43 [8, 60–62]. Several species of TDP- 43 exist and are produced through translation of alterna- tively spliced isoforms or through proteolytic cleavage at the post-translational level (Fig. 1). CTF35 and CTF25 can be generated through proteolytic cleavage via Caspases 3 and 7 after asparagine-89, and Caspase 4 after asparagine-174, respectively, and caspase activity is also modulated by the ALS-linked protein Progranulin (PGRN) [63–69]. Alternative splicing also contributes to short forms of TDP-43 where a second splice isoform was identified through cDNA sequencing encoding an N-Terminally truncated, ~ 32 kDa isoform of TDP-43 [70]. Additionally, CTF35 fragment can also be gener- ated through non-canonical splicing in exon 2 and alternative translational initiation at methionine-85 [59]. C-terminally truncated species can also be generated through proteolytic cleavage. δ-secretase cleaves TDP-43 after asparagine-291 and -306 to generate a ~ 32 kDa and ~ 35 kDa species, respectively [71]. The calcium- dependant cysteine proteases, calpains, also play a role in TDP-43 cleavage generating ~ 35 kDa and ~ 25 kDa species associated with cell toxicity [72, 73]. As many of the truncated species of TDP-43 are of similar molecular weights many studies simply nest them as “CTF35” or “CTF25” based on molecular weight without investiga- tion to the exact species observed which may limit the understanding of TDP-43 species contribution to ALS as different species display distinctive properties [59, 63, 74, 75]. The exact functions of these truncated species remain unclear and are generally thought to be toxic, but have also been proposed to serve a protective role in the cell to promote TDP-43 clearance [59, 63, 73, 75–80]. It is important to recognize that other species of TDP-43 CTFs have been identified at 15-16 kDa, 22-25 kDa, and 33-37 kDa in ALS/ALS-FTLD, however due to low levels of reporting their prevalence in disease remains elusive [56, 74, 75, 81–85]. Main text M1,M3,M5 (Red): Mitochondria Localization Sequences; NLS (Turquoise): Bipartite Nuclear Localization Sequence; RRM1,RRM2 (Blue): RNA Recognition Motif; NES (Light Purple): Controversial Nuclear Export Signal; NES (Dark Purple): Nuclear Export Signal; CTD (Grey): C-Terminal Domain; Yellow Box: Alternate Amino Acid Sequence (N-Terminus of “Isoform 2” and C-Terminus of “Short TDP-43”); Dashed Lines: Cleavage Sites Fig. 1 Structure of TDP-43 including functional domains and identified short-species. M1,M3,M5 (Red): Mitochondria Localization Sequences; NLS (Turquoise): Bipartite Nuclear Localization Sequence; RRM1,RRM2 (Blue): RNA Recognition Motif; NES (Light Purple): Controversial Nuclear Export Signal; NES (Dark Purple): Nuclear Export Signal; CTD (Grey): C-Terminal Domain; Yellow Box: Alternate Amino Acid Sequence (N-Terminus of “Isoform 2” and C-Terminus of “Short TDP-43”); Dashed Lines: Cleavage Sites linked ubiquitin-binding autophagic adaptor Sequesto- some 1 (SQSTM1, also known as p62) [8, 55–60, 82, 86–89]. in the field as to whether cytoplasmic TDP-43 structures indeed colocalize as a component of SGs or are mostly dis- tinct from these bodies [90, 91, 93, 94, 103, 104, 106–109]. Under prolonged stress conditions, phase-separated TDP- 43 transitions from a liquid-like droplet to form gel-like inclusions inhibiting their ability to dissociate [110–112]. These gel-like inclusions eventually accumulate several hallmarks the TDP-43 inclusions seen in ALS [61, 108, 109, 111]. The exact mechanisms mediating the formation of TDP-43 aggregates remain elusive. In ALS aggregates, TDP-43 was found to colocalize with important markers of stress granules (SGs) [90–95]. SGs are membraneless organelles that form in the cytoplasm comprised primarily of ribonuclear proteins and mRNA stalled in translation (Reviewed by Wolozin & Ivanov [96]). The formation of SGs occurs through a process called liquid-liquid phase separation (LLPS) where SG proteins and associated mRNA will de-mix into a liquid phase distinct from the cytosol [97, 98]. Two prominent proteins that are indica- tive of a SG are Ras GTPase-activating protein-binding protein 1 (G3BP1) and TIA1 cytotoxic granule-associated RNA binding protein (TIA1, [99–102]). Interestingly, mutations in the LCD of TIA1 – a domain that plays a key role in LLPS – cause ALS, further supporting the in- volvement of the cellular stress response in disease [103]. Clearance of TDP-43 remains an important biological process tightly coupled with cytotoxicity. The ubiquitin- proteasome system is disrupted by ALS-linked mutations in Ubiquilin-2 (UBQLN2), and is important for degrading full-length TDP-43 in addition to CTF-35 and CTF-25 [113–120]. Main text TDP-43 function, dysfunction, and aggregation TDP-43 is a highly conserved and essential DNA/RNA binding protein belonging to the heterogenous ribonucleo- protein family that preferentially recognizes UG-rich and TG-rich motifs of RNA and DNA, respectively [22–26]. TDP-43 is ubiquitously expressed in all cell types and is predominantly localized to the nucleus, but is also present in the cytoplasm and mitochondria [27–29]. Importantly, TDP-43 is highly regulated, particularly by autoregulation through cryptic exon repression within the 3’UTR of TARDBP mRNA [30–32]. Deletion of TDP-43 results in embryonically lethality in mice, and its depletion or overex- pression causes toxicity or cell death in cell and animal models [33–48]. Structurally, TDP-43 has a bipartite NLS sequence in the N-terminal domain upstream of the first RNA recognition motif (RRM), a nuclear export signal (NES) within the second RRM, and 5 putative mitochondria localization signals (M1-M5) of which 3 (M1, M3, and M5) are functionally characterized [14, 24, 28, 29]. The NLS and NES are important for shuttling TDP-43 between the nucleus and cytoplasm, however the involvement of the NES remains controversial as some studies suggest the NES is non-functional [27, 49–51]. These motifs reside within the N-terminal portion of TDP-43 forming a globular tertiary structure [22, 52, 53]. The C-terminal domain (CTD) – sometimes referred to as the low-complexity domain (LCD), glycine-rich region, intrinsically disordered region (IDR), or prion-like domain (PrLD) – remains rela- tively unstructured and is thought to be critically important for TDP-43 toxicity in disease [4, 53, 54]. Not only is the unstructured nature of the CTD aggregation-prone, but There are several features that commonly define aggre- gates of TDP-43 in ALS. These include the accumulation of post translational modifications such as ubiquitination, poly-ubiquitination, and aberrant phosphorylation (some- times referred to as hyperphosphorylation) of full length TDP-43; specifically phosphorylation of TDP-43 at serine 409 and 410 (S409/410) is widely used as an indicator of aggregated TDP-43 [8, 9, 56, 58, 61]. TDP-43 aggregates in ALS also accumulate full length and lower molecular weight species of TDP-43 and stain positive for the ALS- Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 Page 3 of 16 Fig. 1 Structure of TDP-43 including functional domains and identified short-species. Additional avenues of TDP-43 toxicity Studies have suggested that not all aggregates are equal in their ability to cause toxicity. Similar to other neuro- degenerative diseases, large protein aggregates such as amyloid-like structures may not be as toxic as smaller ones that preceded them such as oligomers [143–147]. However, describing aggregates simply as “large” or “small” is a gross oversimplification as there are thought to be multiple species of aggregates based on the proper- ties of protein misfolding which may mediate altered toxicity at different stages [53, 147–150]. Although TDP-43 aggregation is apparent in various modes of cellular dysfunction, critics argue that TDP-43 aggre- gates may simply be an artifact of neuronal degeneration observed at the time of post-mortem analyses [14, 151]. In cell and animal models, TDP-43 aggregation is not ne- cessarily essential to cause cellular toxicity [14, 152–157]. This would suggest that TDP-43 aggregates may act as a bystander alongside a cell death pathway or work in parallel with an alternate mechanism to promote toxicity. Further- more, TDP-43 aggregates are not exclusive to motor neurons, they can also be observed in glia and muscle tissue of ALS patients and are observed to spread in a prion-like manner throughout the brain [8, 9, 15, 57, 158–162]. Yet in ALS, motor neurons selectively degenerate suggesting that the presence of TDP-43 aggregates may not necessarily drive cell-death. Clearly, TDP-43 aggregation is not the only feature at play. p The cellular stress model, particularly oxidative stress (e.g. via sodium arsenite treatment), to induce SGs and TDP-43 inclusions is a widely used model to study TDP- 43 mislocalization and aggregation [96, 164]. Yet systemic stress makes it difficult to differentiate phenotypes associ- ated with TDP-43 mislocalization and accumulation to general cellular stress responses. To overcome this limita- tion, a novel model expressed TDP-43 fused with an Arabidopsis thaliana-derived Cryptochrome 2 (CRY2) protein to allow for optogenetic instigation of LLPS [108, 109, 111, 165]. In contrast to prolonged sodium arsenite treatment, prolonged LLPS through optogenetic stimula- tion in wild-type conditions results in TDP-43 inclusions within the nucleus absent of ALS hallmarks including hyperphosphorylation and SQSTM1 sequestration [108]. However, prolonged induction of LLPS on TDP-43ΔNLS or mildly stressed cells inducing a mild mislocalization resulted in cytoplasmic inclusions positive for ALS-like hallmarks. Main text Inhibiting this mode of clearance in primary neurons results in a greater accumulation of cytoplasmic TDP-43 aggregates compared to other cell stressors [113, 121, 122]. Recently a gain-of-function mutation in CYLD Lysine 63 Deubiquitinase (CYLD) was identified to cause ALS and FTLD [123]. The authors demonstrated in mouse primary neurons that this mutation increased deu- biquitinase activity, decreased autophagy function and caused TDP-43 mislocalization, along with TDP-43 aggre- gation in the human brain. Autophagy also plays a role in clearing aggregated forms of TDP-43 and is linked to ALS through mutations in autophagy-related proteins SQST M1, TANK Binding Kinase 1 (TBK1) and Optineurin (OPTN) [87, 113, 124–128]. Of particular importance, the TDP-43 plays an important role in regulating the dynamics of SG formation and disassembly where loss of TDP-43 reduces SG formation [104, 105]. Treatment of cells with cell stressors used to study the formation of SGs, such as oxidative stressors (e.g. Sodium Arsenite), osmotic stressors (e.g. D-Sorbitol) or heat shock, results in the formation of phase-separated TDP-43 structures in the cytoplasm. Nevertheless, there remains a debate Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 Page 4 of 16 Page 4 of 16 cellular phenotypes may be caused by mislocalization or the mutation itself. Studies have exploited the NLS sequence on TDP-43 through genetic manipulation to shed light on the consequences of mislocalization inde- pendent of mutations or aggregate formation as in the cell stress models. In cellular models, expression of TDP- 43ΔNLS resulted in depletion of endogenous TDP-43WT from the nucleus and promoted the formation of insoluble inclusions in the cytoplasm [29]. In a transgenic mouse model expressing human TDP-43ΔNLS under a neurofila- ment heavy chain promoter for brain and spinal cord expression, mice displayed a rapidly progressive motor phenotype, loss of body mass, neuromuscular denervation, and spinal motor neuron loss [155]. These mice also exhib- ited high levels of phosphorylated TDP-43 aggregates throughout the brain and spinal cord. Interestingly, the authors of this study describe progressive endogenous nuclear TDP-43 depletion followed by aggregate formation in the brain and to a lesser extent the spinal cord. This infers that TDP-43 mislocalization can promote nuclear depletion and is likely upstream of aggregation. Much of the toxicity, however, was attributed to the high level of transgene expression in the animal model which can function to exacerbate the effect of induced TDP-43 mislocalization by inducing cellular stress from TDP-43 overexpression. Main text sequestration of SQTSM1 into TDP-43 aggregates, one of the aforementioned hallmarks of ALS aggregates, leads to the inhibition of proteasome function in addition to autophagy, further promoting the accumulation of toxic, misfolded proteins in cells [129, 130]. The reduced clearance of aggregates can lead to another toxic gain-of-function mechanism: blocking intracellular transport. Aggregates are observed through- out the cytoplasm, often in the soma, but are also observed in the axons and dendrites [131, 132]. Inhibit- ing axonal transport is a common feature in ALS and particularly relevant as mutations in genes involved in cellular transport, namely KIF5A or DCTN1, cause ALS [133–139]. This may provide some insight into selective neuron vulnerability in ALS as motor neuron axons are particularly long and susceptible to changes in trafficking dynamics [139]. Additionally, TDP-43 plays an important role in axonal trafficking of mRNA granules, a function lost when it is mutated or aggregated [131, 132, 140–142]. The contribution of TDP-43 mislocalization to cellular toxicity in ALS abilities of TDP-43 have been linked to TDP-43 toxicity, though some studies suggest that RNA binding is a protective mechanism [111, 184–189]. An important finding suggests that TDP-43 RNA binding regulates its solubility and lack of RNA promotes aberrant inclusions in the cytoplasm [111, 189]. Mislocalization of TDP-43 may inhibit proper RNA trafficking to the cytoplasm and subsequently promote an environment where TDP- 43 is less soluble. Increasing evidence suggests that nuclear-to-cytoplasmic mislocalization of TDP-43 induces toxicity through both loss- and gain-of-function mechanisms. Classic roles for TDP-43 pertain to mRNA maturation in the nucleus, specifically acting as a repressor of alternate splicing, cryptic exon splicing, and alternate polyadenylation [25, 166–173]. Loss of these functions through mislocaliza- tion or depletion have widespread deleterious effects on the cell [170, 173]. For example, recently it was discov- ered that loss-of-TDP-43 decreases microtubule out- growth specifically in motor neurons through premature polyadenylation of the Stathmin2 (STMN2) transcript [167, 174]. TDP-43 is also involved in mRNA transport, a mechanism that is dysregulated within ALS, as well as local translational regulation [131, 175]. Disruption of ei- ther of these mechanisms may effectively trap TDP-43 in the cytoplasm, inhibiting its normal functions. This hypothesis is substantiated by transcriptomic evidence showing that diseased neurons and mouse models of ALS demonstrate increases in alternative splicing events, cryptic exon inclusion and alternate polyadenylated sequences [168, 176–178]. Recently, the CTD of TDP-43 was found to mediate its recognition of G-quadruplex structures on RNA, facilitating subcellular transport to neurites for local translation and nucleocytoplasmic trafficking [179, 180]. Interestingly, C9ORF72 hexanu- cleotide repeat expansion results in G-quadruplex formation, however the relationship between TDP-43 and C9ORF72 in the context of these structures has not yet been explored [181–183]. Additionally, RNA binding The nuclear functions of TDP-43 are not limited to its RNA binding functions; TDP-43 also binds DNA at TG- rich regions to regulate gene expression and exon skipping [25, 190]. For example, TDP-43 normally binds to the promoter of Vacuolar Protein Sorting 4B (VPS4B) to repress its transcription [191]. Loss of function due to mislocalization results in a loss of VPS4B repression leading to an increased interaction with the ALS-linked protein Charged Multivesicular Body Protein 2B (CHMP2B) thereby disrupting dendritic recycling-endosome trafficking and reducing ALS-linked ERB-B2 Receptor Tyrosine Kinase 4 (ERBB4) surface expression [191–193]. Additional avenues of TDP-43 toxicity Additionally this model demonstrated in vitro that there is a relatively short time course between initial induction of LLPS to aggregate formation (within hours) and inevitable cell death in less than 6 weeks [108, 111]. This suggests a slippery slope between aggregate forma- tion and neurodegeneration, thereby inferring that thera- peutically targeting the aggregation step may be too late to have substantial impact on disease progression (Fig. 2). Increasingly, the field is focusing on mechanisms outside of TDP-43 aggregation to identify early drivers of disease. In the presence of ALS-causing mutations, TDP-43 often demonstrates an altered nucleocytoplasmic distribution (increased cytosolic, decreased nuclear) in comparison to its wild-type counterpart [153, 163]. This may suggest that TDP-43 dysfunction can promote cytoplasmic accumula- tion. However, it remains difficult to differentiate whether Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 Page 5 of 16 Fig. 2 TDP-43 (Red) mislocalizes (partially or completely) from the nucleus to the cytoplasm due to genetic and/or environmental factors causing deleterious effects to the cell. Prolonged mislocalization promotes aggregation. Under physiological conditions the cell can clear small TDP-43 aggregates through proteasomal, endosomal, or autophagic degradation. Prolonged The accumulation of TDP-43 aggregates disrupts physiological functioning (e.g. sequestration of SQSTM1) thereby exacerbating pathology and promoting neuronal degeneration. Early interventions normalizing TDP-43 localization hold the potential to prevent cellular demise Fig. 2 TDP-43 (Red) mislocalizes (partially or completely) from the nucleus to the cytoplasm due to genetic and/or environmental factors causing deleterious effects to the cell. Prolonged mislocalization promotes aggregation. Under physiological conditions the cell can clear small TDP-43 aggregates through proteasomal, endosomal, or autophagic degradation. Prolonged The accumulation of TDP-43 aggregates disrupts physiological functioning (e.g. sequestration of SQSTM1) thereby exacerbating pathology and promoting neuronal degeneration. Early interventions normalizing TDP-43 localization hold the potential to prevent cellular demise The contribution of TDP-43 mislocalization to cellular toxicity in ALS The contribution of TDP-43 mislocalization to cellular toxicity in ALS These mechanisms can cover a range of biological aspects intrinsic to TDP-43 function as well as systemic cellular function [202]. [ ] As previously described, TDP-43 contains several subcellular-regulatory sequences including an NLS, the controversial NES, and mitochondrial localization sequences M1, M3, and M5. ALS-causing mutations how- ever rarely reside within these motifs (with the exceptions of A90V in the NLS, and mutations between amino acids 294-300 in M5), and TDP-43 mislocalization exists outside of TARDBP mutations, suggesting extrinsic factors from TDP-43 govern its subcellular localization [4, 6, 28, 151, 211–222]. Understanding the contribution of these domains to TDP-43 biology remains an important step to understand disease. To this end, targeted mutagenesis of TDP-43 NLS sequence suggests that TDP-43 is actively transported into the nucleus [49]. Furthermore, knock- down of nuclear import machinery (e.g. Importin-β) impair TDP-43 nuclear localization, increasing the cytoplasmic abundance of TDP-43 [223]. Mutagenesis of the NES does not alter TDP-43 localization suggesting the NES is non- functional, however manipulation of export machinery (e.g. Exportin 1) yields conflicting results; thus there may be overlapping mechanisms of TDP-43 export [49–51]. Nevertheless, nuclear pore trafficking is important to some extent for normal TDP-43 localization. Perhaps unsurpris- ingly, in ALS, nuclear pore trafficking is disrupted, espe- cially in cases of patients bearing mutations in TARDBP or C9ORF72 [223–228]. Aggregates of TDP-43 sequester nuclear pore proteins which would likely exacerbate TDP- 43 mislocalization and accumulation into the protein aggregates [224]. In cases of C9ORF72 repeat expansion, dipeptide repeats (DPRs) generated through repeat- associated non-AUG (RAN) translation of the expanded hexanucleotide (CCCCGG) repeat blocks and disrupts the nuclear pores leading to TDP-43 pathology [229–235]. TDP-43 mislocalization was also shown to exacerbate RAN translation of C9ORF72 DPRs and could contribute to nuclear pore defects in conjunction or independently from DPRs [235]. This study suggests that C9ORF72 neurotoxicity may be mediated by TDP-43, and that TDP- 43 mislocalization independent of C9ORF72 DPRs can dis- rupt nuclear pore function. Thus, nuclear pore complex disruption is an important part of TDP-43 pathogenesis, however, this mechanism may not always precede TDP-43 Although we have focused strictly on nuclear and cytoplasmic TDP-43, it is important to highlight a role for TDP-43 at mitochondria. TDP-43 misregulation through genetic manipulation of the NLS, presence of an ALS-causing mutation, or overexpression result in an increased localization to mitochondria [28]. The contribution of TDP-43 mislocalization to cellular toxicity in ALS Another nuclear role for TDP-43 is in its response to genomic double stranded breaks (DSBs) which accumulate in ALS patients [163, 194–200]. Mislocalization of TDP-43 through an ALS-causing mutation impair the nuclear localization of DSB-repair proteins and result in the accumulation of DNA damage promoting cell death [163, 194, 201, 202]. Loss of nuclear TDP-43 can also affect chromatin accessibility leading to altered gene expression [203, 204]. Not all consequences of TDP-43 mislocalization are attributed to nuclear loss-of-function as TDP-43 has defined roles in the cytoplasm including stress granule Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 Page 6 of 16 Page 6 of 16 regulation, mRNA stability, translational regulation, local synaptic RNA regulation, mRNA trafficking, microRNA regulation, and regulation of autophagy (extensively reviewed by Birsa et al. [205]). The exact consequences of increased cytoplasmic TDP-43 on these cellular func- tions remains largely unknown as most studies focus on protein aggregation resulting in an effective loss-of- TDP-43 function. TDP-43 is cleared through both the ubiquitin-proteasome system and lysosomal degradation pathways (highlighted above) [206–208]. Interestingly, TDP-43 mislocalization through overexpression or pathogenic mutations causes vacuole fragmentation, causing cellular disruption in addition to altering its own clearance [206]. Additionally, mislocalization may prime cells to respond abnormally in certain circumstances. As TDP-43 can readily undergo phase separation, the increase in cytoplasmic density biophysically promotes LLPS to occur (reviewed by Boeynaems et al. [98]). This is apparent in models of cellular stress when TDP-43 is mislocalized as there is a significant increase in the cellular stress response including rapid formation of stress granules and TDP-43 granules [109, 111, 165]. Therefore, mislocalization may sensitize the cell to re- spond disproportionately to a cellular stress than it may normally be less responsive to. This was recently exem- plified in a study where induced pluripotent stem cell (iPSC)-derived motor neurons, but not astrocytes, with mislocalized TDP-43 showed an increase level of cell death when seeded with TDP-43 aggregates from patient tissue [209]. Potential mechanisms driving TDP-43 Mislocalization g It is apparent that TDP-43 mislocalization on its own is toxic and can contribute to many of the cellular charac- teristics observed in ALS. However, the mechanisms governing TDP-43 localization remain largely elusive. Identifying the mechanisms driving mislocalization will be crucial to identify key mechanisms that are misregu- lated early in disease and can be therapeutically targeted to prevent TDP-43 pathology all together (Fig. 2). Approaches to study TDP-43 Mislocalization to better understand ALS understand ALS As with TDP-43 aggregates, interpreting the extent of TDP-43 mislocalization in patient tissue remains challenging as observations are made post-mortem at the late stage of disease. It may be implied that mislocali- zation of TDP-43 has occurred where there are cytoplas- mic TDP-43 aggregates, however the mechanism(s) of biogenesis of this pathogenic hallmark remain(s) elusive. Future studies should systematically analyze the extent of TDP-43 mislocalization in addition to aggregation in hu- man tissue to gain a better understanding of TDP-43 pathogenesis. Relying on key late-stage hallmarks such as phosphorylation of TDP-43 or SQSTM1 sequestration may limit the understanding of early pathogenesis and may lead to the dismissal of models that do not recapitu- late late-stage pathology. Though studies have not system- atically analyzed the extent of TDP-43 mislocalization throughout the central nervous system, data suggest that TDP-43 mislocalization correlates with aging in the vul- nerable motor neurons of mouse spinal cord tissue [256]. Further understanding the basic biology and general extent of TDP-43 mislocalization throughout the central nervous system will help gain insight into the cell-type specific vulnerabilities to key stages of TDP-43 pathology. There is a need for developing better models that re- capitulate important aspects of ALS, including behav- ioural, pathological, and molecular phenotypes to further understanding of disease. Although most cases of ALS display TDP-43 proteinopathy, only some transgenic mouse models, and fewer endogenous mouse models, recapitulate TDP-43 pathology and ALS-like phenotypes (ALS mouse models recently reviewed by De Giorgio et al. [257]). An interesting exception is in mice bearing mutations in Senataxin (SETX), a poorly understood protein thought to act as a DNA/RNA helicase which cause a rare juvenile-onset form of fALS and sALS [258–260]. Both mouse models bearing transgenic and endogenous mutations in SETX recapitulate TDP-43 mislocalization, aggregation, and ALS-like phenotypes observed in patients [258]. Within TARDBP models, many models that display TDP-43 proteinopathy rely on overexpression approaches. With the advent of genome engineering (e.g. via CRISPR/Cas9) in animals and iPSCs, models have pushed towards studying endogen- ous TDP-43, moving away from the heavy reliance on As with TDP-43 aggregates, interpreting the extent of TDP-43 mislocalization in patient tissue remains challenging as observations are made post-mortem at the late stage of disease. It may be implied that mislocali- zation of TDP-43 has occurred where there are cytoplas- mic TDP-43 aggregates, however the mechanism(s) of biogenesis of this pathogenic hallmark remain(s) elusive. The contribution of TDP-43 mislocalization to cellular toxicity in ALS Within the mitochondrion, mutant TDP-43 also preferentially binds mitochondria-resident mRNA, presumably causing Complex 1 disassembly through altered expression of its components [28]. Nevertheless, this finding has been debated, as studies in cell and animal models of ALS suggest that mitochondrial energetics and metabolism are unaltered [210]. The important contribution of mitochondria in ALS however remains a focus due to mutations in the mitochondrial protein SOD1 as a primary genetic cause of fALS [11]. However, it is inter- esting to consider that fALS caused by mutations in SOD1 rarely present with TDP-43 pathology [15]. Together, these data suggest that both loss- and gain- of-TDP-43 function mediated by nuclear-to-cytoplasmic mislocalization cause systemic cellular dysfunction in ALS. This recognition calls for a better understanding of the native subcellular functions of TDP-43 and the con- sequences of mislocalization independent of aggregation. Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 Page 7 of 16 Page 7 of 16 from ALS patients’ spinal cord and tissue samples. Clearly, focusing exclusively on full length TDP-43 is not encom- passing to understand its contribution to ALS. Further understanding the biological roles and consequences of cleaved and alternately spliced forms of TDP-43 will provide novel insight into ALS pathogenesis and aid our interpretations of TDP-43 contributions to disease. mislocalization. These studies warrant further investigation into mechanisms that may hinder TDP-43 translocation into the nucleus as potential aggravators of disease. p gg It is clear that regulation of TDP-43 is crucial for proper function, yet relatively little is known about how TDP-43 is regulated. Post translational modifications play an important role in regulating protein function (Reviewed by Buratti [236]). Along with phosphorylation at S409/410, toxic TDP-43 generally displays an over- abundance of phosphate modifications leading to the general consensus that phosphorylation of TDP-43 is toxic [58, 61, 237–245]. However, phosphorylation may play a protective role and promote normal function within the cell [246]. For example, phosphorylation of TDP-43 at T153 and T155 by Mitogen Activated Protein Kinase Kinase (MEK) regulates TDP-43 localization to the nucleolus after heat shock, suggesting a normal maintenance role for phosphorylation in TDP-43 biology [247]. Roles for other post translational modifications such as acetylation, poly-ADP ribosylation (PARylation), oxidation, and ubiquitination have been described sug- gesting that post translational modifications are likely important for normal TDP-43 function and may have unappreciated roles regulating subcellular localization [112, 236, 248–250]. Approaches to study TDP-43 Mislocalization to better understand ALS Future studies should systematically analyze the extent of TDP-43 mislocalization in addition to aggregation in hu- man tissue to gain a better understanding of TDP-43 pathogenesis. Relying on key late-stage hallmarks such as phosphorylation of TDP-43 or SQSTM1 sequestration may limit the understanding of early pathogenesis and may lead to the dismissal of models that do not recapitu- late late-stage pathology. Though studies have not system- atically analyzed the extent of TDP-43 mislocalization throughout the central nervous system, data suggest that TDP-43 mislocalization correlates with aging in the vul- nerable motor neurons of mouse spinal cord tissue [256]. Further understanding the basic biology and general extent of TDP-43 mislocalization throughout the central nervous system will help gain insight into the cell-type specific vulnerabilities to key stages of TDP-43 pathology. [ ] The role of TDP-43 cleavage into CTF35 and CTF25 is gaining traction as potential contributors of normal and toxic TDP-43 function. CTF35 for example assembles into SGs and plays roles in RNA processing, however, CTF25 does not localize to SGs and remains diffuse throughout the cell [251]. Cytoplasmic localization of CTF35 and CTF25 may be due to the partial and full loss of the bipartite NLS upon cleavage, respectively [59, 63, 252]. Additionally, mutations in TDP-43 and CTF35 also in- crease mitochondrial localization to the mitochondrial matrix and intermembrane space, respectively [252]. This study highlights that full length TDP-43, not CTF35, may cause oxidative stress, in turn increasing TDP-43 cleavage and promoting mislocalization and aggregation. As these fragments are observed in TDP-43 aggregates, increased in disease, and may induce neuronal toxicity, they remain an interesting mechanism that may provide insight into TDP-43 biology and ALS [64, 77, 251–253]. Several stud- ies have identified alternative spliced isoforms of TDP-43, yet few have been functionally characterized [22, 30, 254]. Recently, a C-terminally truncated alternatively spliced isoform of TDP-43 (“short TDP-43” or sTDP-43) was characterized and found to encode a functional NES within the alternative C-terminus (Fig. 1) resulting in a more cytoplasmic localization compared to TDP-43 [255]. Interestingly, sTDP-43 was upregulated in response to increased neuronal activity, induced mislocalization of en- dogenous TDP-43, and caused neurotoxicity. Additionally, the sTDP-43 isoform was abundant in TDP-43 aggregates p y g p gy There is a need for developing better models that re- capitulate important aspects of ALS, including behav- ioural, pathological, and molecular phenotypes to further understanding of disease. Approaches to study TDP-43 Mislocalization to better understand ALS For example, the TDP-43Q331K knock-in mouse model does not display significant motor phenotype whereas the TDP-43Q331K transgenic mouse display some, but not robust, motor deficits [31, 263]. This difference may be due to synergistic effects of the TDP-43 mutation and overexpression in the transgenic model. There are sev- eral iPSC models for ALS (recently reviewed by Hawrot et al. [264]) however many of them do not recapitulate key hallmarks of ALS pathology. Neurons derived from TDP-43A90V patient iPSCs display TDP-43 mislocaliza- tion (likely due to disruption of the NLS), TDP-43M337V results in slight cytoplasmic granular staining of TDP-43 in iPSC-derived motor neurons, and mislocalization of TDP-43 is observed in TDP-43M337V patient iPSC-derived astrocytes [265–267]. The difficulty in recapitulating TDP-43 pathology may suggest that there are additional mechanisms contributing to ALS in conjunction with TDP-43 mutations, such as changes associated with human aging or chronic stress on the cell. Attempts to exacerbate ALS pathology or behaviour deficits in physio- logically relevant models may help to elucidate more complex mechanisms driving disease. generally limited to germline mutations and does not account for potential mosaicism which may arise during an individual’s lifetime which could lead to ALS [268]. For example, an ALS patient was reported to express TDP-43 bearing the ALS-causing Q331K mutation spe- cifically in spinal cord neurons, but not in the occipital lobe suggesting this ALS-causing mutation is somatic and thus not likely identified through germline genome sequencing [163]. Biomarkers serve as a potential diag- nostic feature in addition to providing insight into dis- ease progression. Current biomarker candidates such as Neurofilament Light Chain are increased in ALS patients and may provide insight into disease progression, but is non-specific to ALS and only provides foresight by about 12 months before symptoms occur [269–273]. As TDP- 43 mislocalization is a central feature in ALS and biomarkers based on phenotypes associated with mislo- calization may provide the specificity and foreshadowing required for early diagnosis. q y g Integrative “omic” approaches will be important to identify robust biomarkers capable of diagnosis and pro- viding early insight into disease progression. Determin- ing the direct consequences of TDP-43 mislocalization as they pertain to ALS remains a challenge due to the incomplete understanding of TDP-43 function and general dysfunction associated with disease. Therefore unbiased, systems-based approaches will be important to understand TDP-43 biology surrounding mislocalization. Approaches to study TDP-43 Mislocalization to better understand ALS Bulk RNA sequencing has provided great insight into transcriptomic changes in ALS mediated by TDP-43. This technology, however, has limited abilities to detect subtle biologically significant changes that may be cell- type specific. Incorporating single cell RNA sequencing or methods of enriching populations of interest (i.e. flow cytometry, spatial transcriptomics) will help target cell- type specific changes affected by TDP-43 mislocalization in animal and cell models that can translate to human disease [274, 275]. As TDP-43 plays important roles in regulating alternative splicing, alternate polyadenylation, and cryptic exon inclusion, deep RNA sequencing will help identify rare toxic species of RNA that can give insight into ALS progression or lead to biomarker for early disease [276, 277]. Interestingly, mislocalization of FUS was recently identified in iPSCs derived from pa- tients bearing mutations in Vasolin Containing Protein (VCP) in addition to spinal cords from sALS patients [278]. FUS mislocalization was suggested to occur due to binding of aberrantly retained introns, namely in the SFPQ gene. The authors further suggest FUS mislocaliza- tion may be a more common hallmark of ALS than previ- ously recognized however more evidence is required. Proteomic approaches such as immunoprecipitation-mass spectrometry or proximity-labeling mass spectrometry (e.g. APEX Proteomics or BioID) comparing wild-type to Although protein aggregation has gained the most attention to understand ALS and identify novel thera- peutic targets, studying the earlier components of TDP- 43 mislocalization may provide an important level of insight into the disease onset and progression. However, studying subcellular localization comes with its own challenges ranging from limitations in technology to convoluted interpretations. TDP-43 mislocalization is primarily studied using cellular fractionation methods or microscopy-based methods. Developing and optimizing more reliable methods to quantitatively analyze TDP-43 subcellular localization will enhance our understanding of critical regulators of TDP-43. For example, identifying nuclear-, mitochondrial-, and cytosol-specific post trans- lational modifications will allow for the generation of antibodies facilitating more rapid and quantifiable detec- tion of mislocalized TDP-43. These would parallel the antibodies raised against phospho-S409/410 TDP-43 which function as a gold-standard for detection of TDP- 43 aggregates through microscopy [61]. The generation of reliable tools will help to resolve potential issues with subjectivity and enhance reproducibility to understand and characterize the consequences of TDP-43 mislocali- zation. Although much weight is placed on DNA se- quencing for modern diagnosis, this technique offers relatively little diagnostic ability in the cases of complex diseases such as ALS. Approaches to study TDP-43 Mislocalization to better understand ALS Although most cases of ALS display TDP-43 proteinopathy, only some transgenic mouse models, and fewer endogenous mouse models, recapitulate TDP-43 pathology and ALS-like phenotypes (ALS mouse models recently reviewed by De Giorgio et al. [257]). An interesting exception is in mice bearing mutations in Senataxin (SETX), a poorly understood protein thought to act as a DNA/RNA helicase which cause a rare juvenile-onset form of fALS and sALS [258–260]. Both mouse models bearing transgenic and endogenous mutations in SETX recapitulate TDP-43 mislocalization, aggregation, and ALS-like phenotypes observed in patients [258]. Within TARDBP models, many models that display TDP-43 proteinopathy rely on overexpression approaches. With the advent of genome engineering (e.g. via CRISPR/Cas9) in animals and iPSCs, models have pushed towards studying endogen- ous TDP-43, moving away from the heavy reliance on Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 Page 8 of 16 Page 8 of 16 transgenic and toxic overexpression models. Several knock-in mouse models expressing mutant TDP-43 dis- play a range of phenotypes depending on the mutation, but most behavioural phenotypes are quite subtle and occur at a very-late stage [254, 257, 261, 262]. For example, the TDP-43Q331K knock-in mouse model does not display significant motor phenotype whereas the TDP-43Q331K transgenic mouse display some, but not robust, motor deficits [31, 263]. This difference may be due to synergistic effects of the TDP-43 mutation and overexpression in the transgenic model. There are sev- eral iPSC models for ALS (recently reviewed by Hawrot et al. [264]) however many of them do not recapitulate key hallmarks of ALS pathology. Neurons derived from TDP-43A90V patient iPSCs display TDP-43 mislocaliza- tion (likely due to disruption of the NLS), TDP-43M337V results in slight cytoplasmic granular staining of TDP-43 in iPSC-derived motor neurons, and mislocalization of TDP-43 is observed in TDP-43M337V patient iPSC-derived astrocytes [265–267]. The difficulty in recapitulating TDP-43 pathology may suggest that there are additional mechanisms contributing to ALS in conjunction with TDP-43 mutations, such as changes associated with human aging or chronic stress on the cell. Attempts to exacerbate ALS pathology or behaviour deficits in physio- logically relevant models may help to elucidate more complex mechanisms driving disease. transgenic and toxic overexpression models. Several knock-in mouse models expressing mutant TDP-43 dis- play a range of phenotypes depending on the mutation, but most behavioural phenotypes are quite subtle and occur at a very-late stage [254, 257, 261, 262]. Abbreviations ALS: Amyotrophic Lateral Sclerosis; C9orf72: Chromosome 9 Open Reading Frame 72; CHMP2B: Charged Multivesicular Body Protein 2B; CNS: Central Nervous System; CRY2: Cryptochrome 2; CTD: C-Terminal Domain; CTF25: [TDP-43] C-Terminal Fragment 25 kDa; CTF35: [TDP-43] C-Terminal Fragment 35 kDa; CYLD: CYLD Lysine 63 Deubiquitinase; DCTN1: Dynactin 1; DSB: Double Stranded Breaks; ERBB4: RB-B2 Receptor Tyrosine Kinase 4 (ERBB4); fALS: Familial ALS; FTLD: Frontotemporal Lobar Dementia/ Frontotemporal Dementia; FUS: Fused in Sarcoma; G3BP1: Ras-GTPase Activating Protein-Binding Protein 1; iPSC: Induced Pluripotent Stem Cell; KIF5A: Kinesin Heavy Chain Isoform A; LCD: Low-Complexity Domain; LLPS: Liquid-Liquid Phase Separation; M1-M5: [TDP-43] Mitochondrial Localization Sequence 1-5; MEK: Mitogen Activated Protein Kinase Kinase; NES: Nuclear Export Signal; NLS: Nuclear Localization Signal; OPTN: Optineurin; PARylation: Poly-ADP Ribosylation; PGRN: Progranulin; PrLD: Prion-Like Domain; RAN: Repeat-Associated Non-AUG (translation); RRM: RNA Recognition Motif; sALS: Sporadic ALSSGStress Granule; SOD1: Zn/ Funding T.R.S. acknowledges the ALS Society of Canada in partnership with the Brain Canada Foundation through the Brain Canada Research Fund, with the financial support of Health Canada, in addition to the Éric Poulin Center for Neuromuscular Disease for financial support through the ALS Trainee Award Program 2019 and Scholarship in Translational Research awards, respectively. M.W.C.R. acknowledges the Parkinson’s Foundation (PF-JFA-1762), Parkinson’s Canada (New Investigator Award), the New Frontiers in Research Fund (NFRFE-2018-00264), Brain Canada-Azrieli Foundation (Early-Career Capacity Building Grant) and Partners Invested in Parkinson’s Research (PIPR). None of these funding sources played a role in writing or editing of this manuscript and the comments in this manuscript solely represent those of the authors. The views expressed herein do not necessarily represent the views of the Minister of Health or the Government of Canada. Authors’ contributions Authors’ contributions T.R.S. and M.W.C.R. wrote and edited the manuscript. The author(s) read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. References 1. Hardiman O, Al-Chalabi A, Chio A, Corr EM, Logroscino G, Robberecht W, et al. Amyotrophic lateral sclerosis. Nat Rev Dis Prim. Nature Publishing Group. 2017;3:17071. 1. Hardiman O, Al-Chalabi A, Chio A, Corr EM, Logroscino G, Robberecht W, et al. Amyotrophic lateral sclerosis. Nat Rev Dis Prim. Nature Publishing Group. 2017;3:17071. Consent for publication Not applicable. Consent for publication Not applicable. Author details 1U f O Author details 1University of Ottawa Brain and Mind Research Institute, Ottawa, Canada. 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada. 3Eric Poulin Center for Neuromuscular Diseases, Ottawa, Canada. 4Ottawa Institute of Systems Biology, Ottawa, Canada. Received: 1 April 2020 Accepted: 7 August 2020 Approaches to study TDP-43 Mislocalization to better understand ALS Additionally, DNA sequencing is Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 Page 9 of 16 Page 9 of 16 mislocalized TDP-43 will provide insight into locale- specific interactors [224, 279, 280]. Additionally, cross analysis with proteomic data from ALS tissue may provide insight into potential toxic protein-protein interactions as a result of TDP-43 mislocalization serving as early therapeutic targets. New technologies are also focusing on the subcellular localization of RNA such as APEXseq [281–283]. Enhancing this technology with datasets that monitor Protein-RNA binding (e.g. CLIP-seq), could greatly enhance our understanding of which transcripts are bound by TDP-43 in various subcellular compart- ments by comparing nuclear, cytoplasmic, and mitochon- dria TDP-43-RNA interactions [284]. Integrating these systems-based approaches will help to uncover novel markers of TDP-43 mislocalization and elucidate path- ways leading to cellular demise in ALS. Cu Superoxide Dismutase 1; SFPQ: Splicing Factor Proline-, and Glutamine- Rich; SQSTM1: Sequestosome-1; sTDP-43: Short (Alternatively-Spliced) TDP-43; STXN: Senataxin; TARDBP: TAR DNA-Binding Protein 43 kDa (Gene); TBC1D1: TBC1 Domain Family Member 1; TBK1: TANK Binding Kinase-1; TDP- 43: TAR DNA-Binding Protein 43 kDa (Protein); TIA1: TIA1 Cytotoxic Granule Associated RNA Binding Protein; UBQLN2: Ubiquilin-2; VCP: Vasolin- Containing Protein; VPS4B: Vacuolar Protein Sorting 4B; WT: Wild Type Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Conclusions Increasing evidence suggests that TDP-43 aggregation is not a single driver of pathology in ALS. TDP-43 mislo- calization plays significant roles in cellular dysfunction independently and in parallel to aggregation. Increas- ingly the field has begun to focus on understanding the regulatory mechanisms of TDP-43 mislocalization. To this end, as protein mislocalization is likely more readily reversible than protein aggregation, understanding the mechanisms regulating TDP-43 subcellular localization will be critical for therapy development. Specifically, a better understanding of TDP-43 localization regulators will surely shed light on novel therapeutics that have the potential to be more effective earlier in disease, more generalizable to most ALS cases, and more informative biomarkers for diagnosis and analysis of progression for ALS. Lastly, given that TDP-43 pathology can also coexist with other aggregate-prone proteins, such as C9ORF72 DPRs, Tau, α-Synuclein, and poly-Q expanded Hunting- tin, insight into the role of TDP-43 mislocalization in its pathogenic function will serve to better understand path- ology and modes of degeneration across a spectrum of neurodegenerative diseases [184, 285–288]. Acknowledgments Acknowledgments Figures were created with Biorender.com Acknowledgments Figures were created with Biorender.com Figures were created with Biorender.com Abbreviations ALS A t h White MA, Kim E, Duffy A, Adalbert R, Phillips BU, Peters OM, et al. TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD. Nat Neurosci. Nature Publishing Group. 2018;21:1138. 10. Kiernan MC, Vucic S, Cheah BC, Turner MR, Eisen A, Hardiman O, et al. Amyotrophic lateral sclerosis. Lancet (London, England). Elsevier. 2011;377:942–55. 32. Sugai A, Kato T, Koyama A, Koike Y, Konno T, Ishihara T, et al. Non- genetically modified models exhibit TARDBP mRNA increase due to perturbed TDP-43 autoregulation. Neurobiol Dis. Academic Press. 2019;130: 104534. 11. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, et al. Mutations in cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. Nat Publ Group. 1993;362:59–62. 12. Kwiatkowski TJ, Bosco DA, LeClerc AL, Tamrazian E, Vanderburg CR, Russ C, et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science. American Association for the Advancement of Science. 2009;323:1205–8. 33. Yang C, Wang H, Qiao T, Yang B, Aliaga L, Qiu L, et al. Partial loss of TDP-43 function causes phenotypes of amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. National Academy of Sciences. 2014;111:E1121–9. 13. Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009;323:1208–11. 34. Schmid B, Hruscha A, Hogl S, Banzhaf-Strathmann J, Strecker K, Van Der Zee J, et al. Loss of ALS-associated TDP-43 in zebrafish causes muscle degeneration, vascular dysfunction, and reduced motor neuron axon outgrowth. Proc Natl Acad Sci U S A. National Academy of Sciences. 2013; 110:4986–91. 14. Hergesheimer RC, Chami AA, De Assis DR, Vourc’h P, Andres CR, Corcia P, et al. The debated toxic role of aggregated TDP-43 in amyotrophic lateral sclerosis: A resolution in sight? Brain. Oxford University Press. 2019;142: 1176–94. 35. Iguchi Y, Katsuno M, Niwa JI, Takagi S, Ishigaki S, Ikenaka K, et al. Loss of TDP-43 causes age-dependent progressive motor neuron degeneration. Brain. 2013;136:1371–82. 15. Mackenzie IRA, Bigio EH, Ince PG, Geser F, Neumann M, Cairns NJ, et al. Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann Neurol. Wiley- Blackwell. 2007;61:427–34. 36. Wu LS, Cheng WC, Shen CKJ. Abbreviations ALS A t h 2. Nguyen HP, Van Broeckhoven C, van der Zee J. ALS genes in the genomic era and their implications for FTD. Trends Genet. Elsevier. 2018;34:404–23. 2. Nguyen HP, Van Broeckhoven C, van der Zee J. ALS genes in the genomic era and their implications for FTD. Trends Genet. Elsevier. 2018;34:404–23. 3. Chia R, Chiò A, Traynor BJ. Novel genes associated with amyotrophic latera sclerosis: diagnostic and clinical implications. Lancet Neurol. Lancet Publishing Group. 2018;17:94–102. 3. Chia R, Chiò A, Traynor BJ. Novel genes associated with amyotrophic lateral sclerosis: diagnostic and clinical implications. Lancet Neurol. Lancet Publishing Group. 2018;17:94–102. 4. Van Deerlin VM, Leverenz JB, Bekris LM, Bird TD, Yuan W, Elman LB, et al. TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis. Lancet Neurol. NIH Public Access. 2008;7:409–16. 5. Pesiridis GS, Lee VMY, Trojanowski JQ. Mutations in TDP-43 link glycine-rich domain functions to amyotrophic lateral sclerosis. Hum Mol Genet. 2009;18: R156–62. 6. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. American Association for the Advancement of Science. 2008;319:1668–72. Page 10 of 16 Page 10 of 16 Page 10 of 16 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 7. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, Vande VC, et al. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. Nature Publishing Group. 2008;40:572–4. 29. Winton MJ, Igaz LM, Wong MM, Kwong LK, Trojanowski JQ, Lee VMY. Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2008; 283:13302–9. 8. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. American Association for the Advancement of Science. 2006;314:130–3. 30. Avendaño-Vázquez SE, Dhir A, Bembich S, Buratti E, Proudfoot N, Baralle FE. Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection. Genes Dev. Cold Spring Harbor Laboratory Press. 2012;26:1679–84. 9. Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, Mori H, et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun. 2006;351:602–11. 31. Abbreviations ALS A t h Targeted depletion of TDP-43 expression in the spinal cord motor neurons leads to the development of amyotrophic lateral sclerosis-like phenotypes in mice. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2012;287:27335–44. 16. Gitcho MA, Baloh RH, Chakraverty S, Mayo K, Norton JB, Levitch D, et al. TDP-43 A315T mutation in familial motor neuron disease. Ann Neurol. John Wiley & Sons, Ltd. 2008;63:535–8. 37. Wils H, Kleinberger G, Janssens J, Pereson S, Joris G, Cuijt I, et al. TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A. National Academy of Sciences. 2010;107:3858–63. 17. Buratti E. Functional significance of TDP-43 mutations in disease. Adv Genet. Academic Press. 2015;91:1–53. 18. Xu ZS. Does a loss of TDP-43 function cause neurodegeneration? Mol Neurodegener. BioMed Central. 2012;7:27. 38. Herzog JJ, Deshpande M, Shapiro L, Rodal AA, Paradis S. TDP-43 misexpression causes defects in dendritic growth. Sci Rep. Nature Publishing Group. 2017;7:1–13. 19. Karch CM, Wen N, Fan CC, Yokoyama JS, Kouri N, Ross OA, et al. Selective genetic overlap between amyotrophic lateral sclerosis and diseases of the frontotemporal dementia Spectrum. JAMA Neurol. 2018;75(7):860–75. 39. Wegorzewska I, Bell S, Cairns NJ, Miller TM, Baloh RH. TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A. National Academy of Sciences. 2009;106:18809–14. 20. Borroni B, Bonvicini C, Alberici A, Buratti E, Agosti C, Archetti S, et al. Mutation within TARDBP leads to frontotemporal dementia without motor neuron disease. Hum Mutat. John Wiley & Sons, Ltd. 2009;30:E974–83. 40. Xu Y-FF, Gendron TF, Zhang Y-JJ, Lin W-LL, D’Alton S, Sheng H, et al. Wild- type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits, and early mortality in transgenic mice. J Neurosci. Society for Neuroscience. 2010;30:10851–9. 21. Kwong LK, Neumann M, Sampathu DM, Lee VMY, Trojanowski JQ. TDP-43 proteinopathy: The neuropathology underlying major forms of sporadic and familial frontotemporal lobar degeneration and motor neuron disease. Acta Neuropathol. Springer. 2007;114:63–70. 41. Fiesel FC, Schurr C, Weber SS, Kahle PJ. TDP-43 knockdown impairs neurite outgrowth dependent on its target histone deacetylase 6. Mol Neurodegener. BioMed Central. 2011;6:64. 22. Wang HY, Wang IF, Bose J, Shen CKJ. Structural diversity and functional implications of the eukaryotic TDP gene family. Genomics. Academic Press Inc. 2004;83:130–9. 42. Abbreviations ALS A t h Iguchi Y, Katsuno M, Niwa JI, Yamada SI, Sone J, Waza M, et al. TDP-43 depletion induces neuronal cell damage through dysregulation of rho family GTPases. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2009;284:22059–66. 23. Dreyfuss G, Matunis MJ, Pinol-Roma S, Burd CG. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. Annual Reviews. 1993;62:289–321. 24. Buratti E, Baralle FE. Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2001;276:36337–43. 43. Kabashi E, Lin L, Tradewell ML, Dion PA, Bercier V, Bourgouin P, et al. Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo. Hum Mol Genet. 2009;19:671–83. 44. Vanden Broeck L, Naval-Sánchez M, Adachi Y, Diaper D, Dourlen P, Chapuis J, et al. TDP-43 loss-of-function causes neuronal loss due to defective steroid receptor-mediated gene program switching in Drosophila. Cell Rep. Elsevier. 2013;3:160–72. 25. Buratti E, Brindisi A, Pagani F, Baralle FE. Nuclear factor TDP-43 binds to the polymorphic TG repeats in CFTR intron 8 and causes skipping of exon 9: a functional link with disease penetrance [2]. Am J Hum Genet. University of Chicago Press. 2004;74:1322–5. 45. Diaper DC, Adachi Y, Sutcliffe B, Humphrey DM, Elliott CJH, Stepto A, et al. Loss and gain of Drosophila TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration by loss-of- function phenotypes. Hum Mol Genet. 2013;22:1539–57. 26. Kuo PH, Chiang CH, Wang YT, Doudeva LG, Yuan HS. The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids. Nucleic Acids Res. 2014:4712–22 Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985631/. Cited 2020 Jun 9. 46. Kraemer BC, Schuck T, Wheeler JM, Robinson LC, Trojanowski JQ, Lee VMY, et al. Loss of murine TDP-43 disrupts motor function and plays an essential role in embryogenesis. Acta Neuropathol. Springer. 2010;119: 409–19. 27. Ayala YM, Zago P, D’Ambrogio A, Xu Y-FFY-F, Petrucelli L, Buratti E, et al. Structural determinants of the cellular localization and shuttling of TDP-43. J Cell Sci. The Company of Biologists Ltd. 2008;121:3778–85. 28. Wang W, Wang L, Lu J, Siedlak SL, Fujioka H, Liang J, et al. The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity. Nat Med. Nature Publishing Group. 2016;22:869–78. 47. Abbreviations ALS A t h Springer Verlag. 2015;130:49–61. 80. Huang CC, Bose JK, Majumder P, Lee KH, Huang JTJ, Huang JK, et al. Metabolism and mis-metabolism of the neuropathological signature protein TDP-43. J Cell Sci. Company of Biologists Ltd. 2014;127:3024–38. 60. Arai T, Hasegawa M, Nonoka T, Kametani F, Yamashita M, Hosokawa M, et al. Phosphorylated and cleaved TDP-43 in ALS, FTLD and other neurodegenerative disorders and in cellular models of TDP-43 proteinopathy. Neuropathology. 2010;30:170–81. 81. De Marco G, Lomartire A, Mandili G, Lupino E, Buccinnà B, Ramondetti C, et al. Reduced cellular Ca2+ availability enhances TDP-43 cleavage by apoptotic caspases. Biochim Biophys Acta. Elsevier. 2014;1843:725–34. 61. Neumann M, Kwong LK, Lee EB, Kremmer E, Flatley A, Xu Y, et al. Phosphorylation of S409/410 of TDP-43 is a consistent feature in all sporadic and familial forms of TDP-43 proteinopathies. Acta Neuropathol. NIH Public Access. 2009;117:137–49. 82. Shenoy J, El Mammeri N, Dutour A, Berbon M, Saad A, Lends A, et al. Structural dissection of amyloid aggregates of TDP-43 and its C-terminal fragments TDP-35 and TDP-16. FEBS J. 2019;287:2449–67. 62. Jeon GS, Shim YM, Lee DY, Kim JS, Kang MJ, Ahn SH, et al. Pathological modification of TDP-43 in amyotrophic lateral sclerosis with SOD1 mutations. Mol Neurobiol. Springer US. 2018;56:1–15. 83. Osuru HP, Pramoonjago P, Abhyankar MM, Swanson E, Roker LTA, Cathro H, et al. Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium. Mol Reprod Dev. John Wiley and Sons Inc. 2017;84:675–85. 63. Li Q, Yokoshi M, Okada H, Kawahara Y. The cleavage pattern of TDP- 43 determines its rate of clearance and cytotoxicity. Nat Commun. 2015;6:1–12. 84. YAT K, Alemu S, Lamari A, Loew N, Brower CS. The N termini of TAR DNA- binding protein 43 (TDP43) C-Terminal fragments influence degradation, aggregation propensity, and morphology. Mol Cell Biol. American Society for Microbiology. 2018;38:e00243–18. 64. Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, et al. Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43. J Neurosci. Society for Neuroscience. 2007;27:10530–4. 85. Nonaka T, Kametani F, Arai T, Akiyama H, Hasegawa M. Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43. Hum Mol Genet. 2009;18:3353–64. 65. Dormann D, Capell A, Carlson AM, Shankaran SS, Rodde R, Neumann M, et al. Proteolytic processing of TAR DNA binding protein-43 by caspases produces C-terminal fragments with disease defining properties independent of progranulin. J Neurochem. Abbreviations ALS A t h Afroz T, Hock EM, Ernst P, Foglieni C, Jambeau M, Gilhespy LAB, et al. Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation. Nat Commun. Nature Publishing Group. 2017;8:45. 74. Brower CS, Piatkov KI, Varshavsky A. Neurodegeneration-associated protein fragments as short-lived substrates of the N-end rule pathway. Mol Cell. Elsevier. 2013;50:161–71. 54. Guo L, Kim HJ, Wang H, Monaghan J, Freyermuth F, Sung JC, et al. Nuclear- import receptors reverse aberrant phase transitions of rna-binding proteins with prion-like domains. Cell. Cell Press. 2018;173:677–692.e20. 75. Igaz LM, Kwong LK, Chen-Plotkin A, Winton MJ, Unger TL, Xu Y, et al. Expression of TDP-43 C-terminal fragments in vitro recapitulates pathological features of TDP-43 proteinopathies. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2009;284:8516–24. 55. Berning BA, Walker AK. The pathobiology of TDP-43 C-terminal fragments in ALS and FTLD. Front Neurosci. Frontiers Media S.A. 2019;13:335. 76. Suzuki H, Lee K, Matsuoka M. TDP-43-induced death is associated with altered regulation of BIM and Bcl-xL and attenuated by caspase-mediated TDP-43 cleavage. J Biol Chem. 2011;286:13171–83. 56. Tsuji H, Arai T, Kametani F, Nonaka T, Yamashita M, Suzukake M, et al. Molecular analysis and biochemical classification of TDP-43 proteinopathy. Brain. Narnia. 2012;135:3380–91. 77. Zhang YJY-J, Xu YFY-F, Cook C, Gendron TF, Roettges P, Link CD, et al. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci. National Academy of Sciences. 2009;106:7607–12. 57. Smethurst P, Sidle KCL, Hardy J. Review: prion-like mechanisms of transactive response DNA binding protein of 43 kDa (TDP-43) in amyotrophic lateral sclerosis (ALS). Neuropathol Appl Neurobiol. Blackwell Publishing Ltd. 2015;41:578–97. 78. Wang X, Fan H, Ying Z, Li B, Wang H, Wang G. Degradation of TDP-43 and its pathogenic form by autophagy and the ubiquitin-proteasome system. Neurosci Lett. Elsevier. 2010;469:112–6. 58. Hasegawa M, Arai T, Nonaka T, Kametani F, Yoshida M, Hashizume Y, et al. Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Ann Neurol. Wiley-Blackwell. 2008;64:60–70. 79. Walker AK, Tripathy K, Restrepo CR, Ge G, Xu Y, Kwong LK, et al. An insoluble frontotemporal lobar degeneration-associated TDP-43 C-terminal fragment causes neurodegeneration and hippocampus pathology in transgenic mice. Hum Mol Genet. 2015;24:7241–54. 59. Xiao S, Sanelli T, Chiang H, Sun Y, Chakrabartty A, Keith J, et al. Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death. Acta Neuropathol. Abbreviations ALS A t h Sephton CF, Good SK, Atkin S, Dewey CM, Mayer P, Herz J, et al. TDP-43 is a developmentally regulated protein essential for early embryonic Page 11 of 16 Page 11 of 16 Page 11 of 16 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 development. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2010;285:6826–34. 68. Schymick JC, Yang Y, Andersen PM, Vonsattel JP, Greenway M, Momeni P, et al. Progranulin mutations and amyotrophic lateral sclerosis or amyotrophic lateral sclerosis-frontotemporal dementia phenotypes. J Neurol Neurosurg Psychiatry. 2007;78:754–6. 48. Wu LS, Cheng W, Hou SC, Yan YT, Jiang ST, Shen CKJ. TDP-43, a neuro- pathosignature factor, is essential for early mouse embryogenesis. Genesis. Genesis. 2010;48:56–62. 69. Yin P, Guo X, Yang W, Yan S, Yang S, Zhao T, et al. Caspase-4 mediates cytoplasmic accumulation of TDP-43 in the primate brains. Acta Neuropathol. Springer Verlag. 2019;137:919–37. 49. Pinarbasi ES, Caǧatay T, Fung HYJ, Li YC, Chook YM, Thomas PJ. Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization. Sci Rep. Nature Publishing Group. 2018;8:7083. 70. Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, et al. Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat Genet. Nature Publishing Group. 2004;36:40–5. 50. Archbold HC, Jackson KL, Arora A, Weskamp K, Tank EMH, Li X, et al. TDP43 nuclear export and neurodegeneration in models of amyotrophic lateral sclerosis and frontotemporal dementia. Sci Rep. Nature Publishing Group. 2018;8:1–18. 71. Herskowitz JH, Gozal YM, Duong DM, Dammer EB, Gearing M, Ye K, et al. Asparaginyl endopeptidase cleaves TDP-43 in brain. Proteomics. 2012;12: 2455–63. 51. Ederle H, Funk C, Abou-Ajram C, Hutten S, Funk EBE, Kehlenbach RH, et al. Nuclear egress of TDP-43 and FUS occurs independently of Exportin-1/ CRM1. Sci Rep. Nature Publishing Group. 2018;8:1–18. 72. Yamashita T, Hideyama T, Hachiga K, Teramoto S, Takano J, Iwata N, et al. A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology. Nat Commun. Nature Publishing Group. 2012;3:1–13. 52. Kuo PH, Doudeva LG, Wang YT, Shen CKJ, Yuan HS. Structural insights into TDP-43 in nucleic-acid binding and domain interactions. Nucleic Acids Res. Oxford University Press. 2009;37:1799–808. 73. Rao MV, Campbell J, Palaniappan A, Kumar A, Nixon RA. Calpastatin inhibits motor neuron death and increases survival of hSOD1 G93A mice. J Neurochem. Blackwell Publishing Ltd. 2016;137:253–65. 53. Abbreviations ALS A t h Stress granules and neurodegeneration. Nat Rev Neurosci. Nature Publishing Group. 2019;20:649–66. 97. Protter DSW, Parker R. Principles and properties of stress granules. Trends Cell Biol. Elsevier. 2016;26:668–79. 118. Millecamps S, Corcia P, Cazeneuve C, Boillée S, Seilhean D, Danel-Brunaud V, et al. Mutations in UBQLN2 are rare in French amyotrophic lateral sclerosis. Neurobiol Aging. Elsevier Inc. 2012;33:839.e1–3. 98. Boeynaems S, Alberti S, Fawzi NL, Mittag T, Polymenidou M, Rousseau F, et al. Protein phase separation: a new phase in cell biology. Trends Cell Biol. Elsevier Ltd. 2018;28:420–35. 119. van Doormaal PTC, van Rheenen W, van Blitterswijk M, Schellevis RD, Schelhaas HJ, de Visser M, et al. UBQLN2 in familial amyotrophic lateral sclerosis in the Netherlands. Neurobiol Aging. Elsevier Inc. 2012;33:2233.e7–8. 99. Kedersha NL, Gupta M, Li W, Miller I, Anderson P. RNA-binding proteins TIA- 1 and TIAR link the phosphorylation of eIF-2α to the assembly of mammalian stress granules. J Cell Biol. The Rockefeller University Press. 1999;147:1431–41. 120. Daoud H, Suhail H, Szuto A, Camu W, Salachas F, Meininger V, et al. UBQLN2 mutations are rare in French and French-Canadian amyotrophic lateral sclerosis. Neurobiol Aging. Elsevier Inc. 2012;33:2230.e1–5. 100. Kedersha N, Cho MR, Li W, Yacono PW, Chen S, Gilks N, et al. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. J Cell Biol. The Rockefeller University Press. 2000;151:1257–68. 121. van Eersel J, Ke YD, Gladbach A, Bi M, Götz J, Kril JJ, et al. Cytoplasmic accumulation and aggregation of TDP-43 upon proteasome inhibition in cultured neurons. Iijima KM, editor. PLoS One. Public Library of Science. 2011;6:e22850. 101. Tourrière H, Chebli K, Zekri L, Courselaud B, Blanchard JM, Bertrand E, et al. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol. The Rockefeller University Press. 2003;160:823–31. 122. Ishii T, Kawakami E, Endo K, Misawa H, Watabe K. Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging. PLoS One. Public Library of Science. 2017;12:e0179375. 102. Matsuki H, Takahashi M, Higuchi M, Makokha GN, Oie M, Fujii M. Both G3BP1 and G3BP2 contribute to stress granule formation. Genes Cells. John Wiley & Sons, Ltd. 2013;18:135–46. 123. Dobson-Stone C, Hallupp M, Shahheydari H, Ragagnin AMG, Chatterton Z, Carew-Jones F, et al. CYLD is a causative gene for frontotemporal dementia- amyotrophic lateral sclerosis. Brain. 2020;143:783–99. 103. Mackenzie IR, Nicholson AM, Sarkar M, Messing J, Purice MD, Pottier C, et al. Abbreviations ALS A t h John Wiley & Sons, Ltd. 2009; 110:1082–94. 86. Mizuno Y, Amari M, Takatama M, Aizawa H, Mihara B, Okamoto K. Immunoreactivities of p62, an ubiqutin-binding protein, in the spinal anterior horn cells of patients with amyotrophic lateral sclerosis. J Neurol Sci. Elsevier. 2006;249:13–8. 66. Beel S, Herdewyn S, Fazal R, De Decker M, Moisse M, Robberecht W, et al. Progranulin reduces insoluble TDP-43 levels, slows down axonal degeneration and prolongs survival in mutant TDP-43 mice 11 medical and health sciences 1109 neurosciences. Mol Neurodegener. BioMed Central Ltd. 2018;13:55. 87. Fecto F, Yan J, Vemula SP, Liu E, Yang Y, Chen W, et al. SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis. Arch Neurol. American Medical Association. 2011;68:1440–6. 88. Tanji K, Zhang H-X, Mori F, Kakita A, Takahashi H, Wakabayashi K. p62/ sequestosome 1 binds to TDP-43 in brains with frontotemporal lobar degeneration with TDP-43 inclusions. J Neurosci Res. John Wiley & Sons, Ltd. 2012;90:2034–42. 67. Sleegers K, Brouwers N, Maurer-Stroh S, Van Es MA, Van Damme P, Van Vught PWJ, et al. Progranulin genetic variability contributes to amyotrophic lateral sclerosis. Neurology. Lippincott Williams and Wilkins. 2008;71:253–9. Page 12 of 16 Page 12 of 16 Page 12 of 16 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 89. Nakano T, Nakaso K, Nakashima K, Ohama E. Expression of ubiquitin-binding protein p62 in ubiquitin-immunoreactive intraneuronal inclusions in amyotrophic lateral sclerosis with dementia: analysis of five autopsy cases with broad clinicopathological spectrum. Acta Neuropathol. Springer. 2004; 107:359–64. 109. Gasset-Rosa F, Lu S, Yu H, Chen C, Melamed Z, Guo L, et al. Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death. Neuron. Elsevier. 2019;102:339–357.e7. 110. Babinchak WM, Haider R, Dumm BK, Sarkar P, Surewicz K, Choi JK, et al. The role of liquid-liquid phase separation in aggregation of the TDP-43 low- complexity domain. J Biol Chem. 2019;294:6306–17. 90. Colombrita C, Zennaro E, Fallini C, Weber M, Sommacal A, Buratti E, et al. TDP-43 is recruited to stress granules in conditions of oxidative insult. J Neurochem. John Wiley & Sons, Ltd (10.1111). 2009;111:1051–61. 111. Mann JR, Gleixner AM, Mauna JC, Gomes E, DeChellis-Marks MR, Needham PG, et al. RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43. Neuron. Elsevier. 2019;102:321–338.e8. 91. Liu-Yesucevitz L, Bilgutay A, Zhang YJ, Vanderwyde T, Citro A, Mehta T, et al. Abbreviations ALS A t h Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue. PLoS One. 2010;5: e13250. 112. McGurk L, Gomes E, Guo L, Mojsilovic-Petrovic J, Tran V, Kalb RG, et al. Poly(ADP-ribose) prevents pathological phase separation of TDP-43 by promoting liquid Demixing and stress granule localization. Mol Cell. 2018; 71:703–717.e9. 92. Volkening K, Leystra-Lantz C, Yang W, Jaffee H, Strong MJ. Tar DNA binding protein of 43 kDa (TDP-43), 14-3-3 proteins and copper/zinc superoxide dismutase (SOD1) interact to modulate NFL mRNA stability. Implications for altered RNA processing in amyotrophic lateral sclerosis (ALS). Brain Res. Elsevier B.V. 2009;1305:168–82. 113. Scotter EL, Vance C, Nishimura AL, Lee Y-BYB, Chen HJH-J, Urwin H, et al. Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species. J Cell Sci. Company of Biologists. 2014;127:1263–78. 93. Dormann D, Rodde R, Edbauer D, Bentmann E, Fischer I, Hruscha A, et al. ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin- mediated nuclear import. EMBO J. John Wiley & Sons, Ltd. 2010;29:2841–57. 114. Deng HX, Chen W, Hong ST, Boycott KM, Gorrie GH, Siddique N, et al. Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Nature. Nature Publishing Group. 2011;477:211–5. 94. McGurk L, Lee VM, Trojanowksi JQ, Van Deerlin VM, Lee EB, Bonini NM. Poly- a binding Protein-1 localization to a subset of TDP-43 inclusions in amyotrophic lateral sclerosis occurs more frequently in patients harboring an expansion in C9orf72. J Neuropathol Exp Neurol. Lippincott Williams and Wilkins. 2014;73:837–45. 115. Picher-Martel V, Renaud L, Bareil C, Julien JP. Neuronal expression of UBQLN2P497H exacerbates TDP-43 pathology in TDP-43G348C mice through interaction with ubiquitin. Mol Neurobiol. 2018;56:1–17. 116. Le NTT, Chang L, Kovlyagina I, Georgiou P, Safren N, Braunstein KE, et al. Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS-FTD-linked UBQLN2 mutations. Proc Natl Acad Sci U S A. National Academy of Sciences. 2016;113:E7580–9. 95. Bentmann E, Neumann M, Tahirovic S, Rodde R, Dormann D, Haass C. Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43). J Biol Chem. American Society for Biochemistry and Molecular Biology. 2012;287:23079–94. 117. Wu Q, Liu M, Huang C, Liu X, Huang B, Li N, et al. Pathogenic Ubqln2 gains toxic properties to induce neuron death. Acta Neuropathol. Springer Verlag. 2015;129:417–28. 96. Wolozin B, Ivanov P. Abbreviations ALS A t h Page 13 of 16 Page 13 of 16 Page 13 of 16 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 130. Seibenhener ML, Babu JR, Geetha T, Wong HC, Krishna NR, Wooten MW. Sequestosome 1/p62 is a Polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol Cell Biol. American Society for Microbiology. 2004;24:8055–68. 151. Geser F, Robinson JL, Malunda JA, Xie SX, Clark CM, Kwong LK, et al. Pathological 43-kDa transactivation response DNA-binding protein in older adults with and without severe mental illness. Arch Neurol. NIH Public Access. 2010;67:1238–50. 152. Liu R, Yang G, Nonaka T, Arai T, Jia W, Cynader MS. Reducing TDP-43 aggregation does not prevent its cytotoxicity. Acta Neuropathol Commun. BioMed Central. 2014;2:49. 131. Fallini C, Bassell GJ, Rossoll W. The ALS disease protein TDP-43 is actively transported in motor neuron axons and regulates axon outgrowth. Hum Mol Genet. 2012;21:3703–18. 132. Alami NH, Smith RB, Carrasco MA, Williams LA, Winborn CS, Han SSW, et al. Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations. Neuron. 2014;81:536–43. 153. Barmada SJ, Skibinski G, Korb E, Rao EJ, Wu JY, Finkbeiner S. Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J Neurosci. 2010;30: 639–49. 133. Nicolas A, Kenna KP, Renton AE, Shaw CE, Traynor BJ, Landers Correspondence JE, et al. Genome-wide Analyses Identify KIF5A as a Novel ALS Gene. Neuron. 2018;97:1268–1282.e6. 154. Igaz LM, Kwong LK, Lee EB, Chen-Plotkin A, Swanson E, Unger T, et al. Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J Clin Invest. American Society for Clinical Investigation. 2011;121:726–38. 134. Brenner D, Yilmaz R, Müller K, Grehl T, Petri S, Meyer T, et al. Hot-spot KIF5A mutations cause familial ALS. Brain. 2018;141:688–97. 135. Puls I, Jonnakuty C, LaMonte BH, Holzbaur ELF, Tokito M, Mann E, et al. Mutant dynactin in motor neuron disease. Nat Genet. Nature Publishing Group. 2003;33:455–6. 155. Walker AK, Spiller KJ, Ge G, Zheng A, Xu Y, Zhou M, et al. Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta Neuropathol. Springer Berlin Heidelberg. 2015;130: 643–60. 136. Münch C, Sedlmeier R, Meyer T, Homberg V, Sperfeld AD, Kurt A, et al. Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS. Neurology. 2004;63:724–6. 156. Abbreviations ALS A t h Co-regulation of mRNA translation by TDP-43 and fragile X syndrome protein FMRP. Acta Neuropathol. Springer Verlag. 2016;132:721–38. 162. Cykowski MD, Powell SZ, Appel JW, Arumanayagam AS, Rivera AL, Appel SH. Phosphorylated TDP-43 (pTDP-43) aggregates in the axial skeletal muscle of patients with sporadic and familial amyotrophic lateral sclerosis. Acta Neuropathol Commun. NLM (Medline). 2018;6:28. 143. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature. Nature Publishing Group. 2004;431:805–10. 144. Parone PA, Da Cruz S, Han JS, McAlonis-Downes M, Vetto AP, Lee SK, et al. Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis. J Neurosci. Society for Neuroscience. 2013;33:4657–71. 163. Guerrero EN, Mitra J, Wang H, Rangaswamy S, Hegde PM, Basu P, et al. Amyotrophic lateral sclerosis (ALS)-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis. Hum Mol Genet. 2019;28(5):2459–76. 145. Treusch S, Cyr DM, Lindquist S. Amyloid deposits: Protection against toxic protein species? Cell Cycle. Taylor and Francis Inc. 2009;8:1668–74. 164. D’Amico E, Factor-Litvak P, Santella RM, Mitsumoto H. Clinical perspective on oxidative stress in sporadic amyotrophic lateral sclerosis. Free Radic Biol Med. NIH Public Access. 2013;65:509–27. 146. Kitamura A, Yuno S, Muto H, Kinjo M. Different aggregation states of a nuclear localization signal-tagged 25-kDa C-terminal fragment of TAR RNA/ DNA-binding protein 43 kDa. Genes Cells. Blackwell Publishing Ltd. 2017;22: 521–34. 165. Asakawa K, Handa H, Kawakami K. Optogenetic modulation of TDP-43 oligomerization accelerates ALS-related pathologies in the spinal motor neurons. Nat Commun. Nature Research. 2020;11:1–16. 147. French RL, Grese ZR, Aligireddy H, Dhavale DD, Reeb AN, Kedia N, et al. Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2019;294:6696–709. 166. Modic M, Rot G, Grosch M, Lepko T, Shaposhnikov D, Cacchiarelli D, et al. Cross-regulation between TDP-43 and Paraspeckles promotes pluripotency- differentiation transition. SSRN Electron J. 2018;74:951–65. 167. Melamed Z, López-Erauskin J, Baughn MW, Zhang O, Drenner K, Sun Y, et al. Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci. 2019; 22:180–90. 148. Fang YS, Tsai KJ, Chang YJ, Kao P, Woods R, Kuo PH, et al. Abbreviations ALS A t h Sasaguri H, Chew J, Xu YF, Gendron TF, Garrett A, Lee CW, et al. The extreme N-terminus of TDP-43 mediates the cytoplasmic aggregation of TDP-43 and associated toxicity in vivo. Brain Res. Elsevier BV. 2016; 1647:57–64. 137. Kieran D, Hafezparast M, Bohnert S, Dick JRT, Martin J, Schiavo G, et al. A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice. J Cell Biol. The Rockefeller University Press. 2005; 169:561–7. 157. Arnold ES, Ling S-C, Huelga SC, Lagier-Tourenne C, Polymenidou M, Ditsworth D, et al. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43. Proc Natl Acad Sci U S A. National Academy of Sciences. 2013;110:E736–45. 138. Bilsland LG, Sahai E, Kelly G, Golding M, Greensmith L, Schiavo G. Deficits in axonal transport precede ALS symptoms in vivo. Proc Natl Acad Sci U S A. National Academy of Sciences. 2010;107:20523–8. 158. Maniecka Z, Polymenidou M. From nucleation to widespread propagation: a prion-like concept for ALS. Virus Res. Elsevier. 2015;207:94–105. 139. De Vos KJ, Hafezparast M. Neurobiology of axonal transport defects in motor neuron diseases: Opportunities for translational research? Neurobiol Dis. Academic Press Inc. 2017;105:283–99. 159. Porta S, Xu Y, Restrepo CR, Kwong LK, Zhang B, Brown HJ, et al. Patient- derived frontotemporal lobar degeneration brain extracts induce formation and spreading of TDP-43 pathology in vivo. Nat Commun. Nature Publishing Group. 2018;9:4220. 140. Sleigh JN, Tosolini AP, Gordon D, Devoy A, Fratta P, Fisher EMC, et al. ALS mice carrying pathological mutant TDP-43, but not mutant FUS, display axonal transport defects in vivo. Cell Rep. 2020;31:3655–62. 160. Mori F, Tada M, Kon T, Miki Y, Tanji K, Kurotaki H, et al. Phosphorylated TDP- 43 aggregates in skeletal and cardiac muscle are a marker of myogenic degeneration in amyotrophic lateral sclerosis and various conditions. Acta Neuropathol Commun. BioMed Central Ltd. 2019;7:165. 141. Coyne AN, Siddegowda BB, Estes PS, Johannesmeyer J, Kovalik T, Daniel SG, et al. FUTSCH/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of amyotrophic lateral sclerosis. J Neurosci. Society for Neuroscience. 2014;34:15962–74. 161. Vogler TO, Wheeler JR, Nguyen ED, Hughes MP, Britson KA, Lester E, et al. TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle. Nature. Nature Publishing Group. 2018;563:508–13. 142. Majumder P, Chu JF, Chatterjee B, Swamy KBS, Shen CKJ. Abbreviations ALS A t h TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics. Neuron. Cell Press. 2017;95:808–816.e9. 124. Maruyama H, Morino H, Ito H, Izumi Y, Kato H, Watanabe Y, et al. Mutations of optineurin in amyotrophic lateral sclerosis. Nature. Nature Publishing Group. 2010;465:223–6. 104. McDonald KK, Aulas A, Destroismaisons L, Pickles S, Beleac E, Camu W, et al. TAR DNA-binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1. Hum Mol Genet. Oxford University Press. 2011;20:1400–10. 125. Freischmidt A, Wieland T, Richter B, Ruf W, Schaeffer V, Müller K, et al. Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia. Nat Neurosci. Nature Publishing Group. 2015;18:631–6. 105. Khalfallah Y, Kuta R, Grasmuck C, Prat A, Durham HD, Vande Velde C. TDP-43 regulation of stress granule dynamics in neurodegenerative disease-relevant cell types. Sci Rep. Nature Publishing Group. 2018;8:7551. 126. de Majo M, Topp SD, Smith BN, Nishimura AL, Chen HJ, Gkazi AS, et al. ALS- associated missense and nonsense TBK1 mutations can both cause loss of kinase function. Neurobiol Aging. Elsevier Inc. 2018;71:266.e1–266.e10. 106. Dewey CM, Cenik B, Sephton CF, Dries DR, Mayer P, Good SK, et al. TDP-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor. Mol Cell Biol. American Society for Microbiology. 2011;31: 1098–108. 127. Weinreich M, Shepheard SR, Verber N, Wyles M, Heath PR, Highley JR, et al. Neuropathological characterization of a novel TANK binding kinase (TBK1) gene loss of function mutation associated with amyotrophic lateral sclerosis. Neuropathol Appl Neurobiol. 2019;46:279–91. 107. Chen Y, Cohen TJ. Aggregation of the nucleic acid– binding protein TDP-43 occurs via distinct routes that are coordinated with stress granule formation. J Biol Chem. American Society for Biochemistry and Molecular Biology Inc. 2019;294:jbc.RA118.006351. 128. Brady OA, Meng P, Zheng Y, Mao Y, Hu F. Regulation of TDP-43 aggregation by phosphorylation andp62/SQSTM1. J Neurochem. 2011;116: 248–59. 129. Gal J, Ström AL, Kilty R, Zhang F, Zhu H. p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2007;282:11068–77. 108. Zhang P, Fan B, Yang P, Temirov J, Messing J, Kim HJ, et al. Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology. Elife. 2019;8:e39578. Abbreviations ALS A t h Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients. Nat Commun. Nature Publishing Group. 2014;5:1–13. 168. Ling JP, Pletnikova O, Troncoso JC, Wong PC. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. American Association for the Advancement of Science. 2015;349:650–5. 149. Cao Q, Boyer DR, Sawaya MR, Ge P, Eisenberg DS. Cryo-EM structures of four polymorphic TDP-43 amyloid cores. Nat Struct Mol Biol. Nature Publishing Group. 2019;26:619–27. 169. Buratti E. Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease. Front Biosci. 2008;13:867–78. 150. Laferrière F, Maniecka Z, Pérez-Berlanga M, Hruska-Plochan M, Gilhespy L, Hock E-M, et al. TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates. Nat Neurosci. Nature Publishing Group. 2019;22:65–77. 170. Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, et al. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci. 2011;14:452–8. Page 14 of 16 Page 14 of 16 Page 14 of 16 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 171. Buratti E, Dörk T, Zuccato E, Pagani F, Romano M, Baralle FE. Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. EMBO J. 2001;20:1774–84. and alters trophic signaling in neurons. EMBO J. John Wiley & Sons, Ltd. 2016;35:2350–70. and alters trophic signaling in neurons. EMBO J. John Wiley & Sons, Ltd. 2016;35:2350–70. 192. Cox LE, Ferraiuolo L, Goodall EF, Heath PR, Higginbottom A, Mortiboys H, et al. Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS). PLoS One. 2010;5:e9872. 172. Rot G, Wang Z, Huppertz I, Modic M, Lenče T, Hallegger M, et al. High- resolution RNA maps suggest common principles of splicing and polyadenylation regulation by TDP-43. Cell Rep. Elsevier B.V. 2017;19: 1056–67. amyotrophic lateral sclerosis (ALS). PLoS One. 2010;5:e9872. 193. Takahashi Y, Fukuda Y, Yoshimura J, Toyoda A, Kurppa K, Moritoyo H, et al. Erbb4 mutations that disrupt the neuregulin-erbb4 pathway cause amyotrophic lateral sclerosis type 19. Am J Hum Genet. Cell Press. 2013;93: 900–5. 173. Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY, et al. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat Neurosci. Nat Publ Group. 2011;14:459–68. 194. Kim BW, Jeong YE, Wong M, Martin LJ. Abbreviations ALS A t h Kawaguchi T, Rollins MG, Moinpour M, Morera AA, Ebmeier CC, Old WM, et al. Changes to the TDP-43 and FUS Interactomes induced by DNA damage. J Proteome Res. 2020;19:360–70. 180. Ishiguro A, Kimura N, Watanabe Y, Watanabe S, Ishihama A. TDP-43 binds and transports G-quadruplex-containing mRNAs into neurites for local translation. Genes to Cells. Blackwell Publishing Ltd. 2016;21:466–81. 201. Roos WP, Kaina B. DNA damage-induced cell death by apoptosis. Trends Mol Med. 2006;12:440–50. 202. Wu C, Jin L, Wang I, Wei W, Ho P, Liu Y, et al. HDAC1 dysregulation induces aberrant cell cycle and DNA damage in progress of TDP-43 proteinopathies. EMBO Mol Med. John Wiley & Sons, Ltd. 2020;12(6):e10622. 181. Zhou B, Liu C, Geng Y, Zhu G. Topology of a G-quadruplex DNA formed by C9orf72 hexanucleotide repeats associated with ALS and FTD. Sci Rep. Nature Publishing Group. 2015;5:1–7. 203. Tibshirani M, Zhao B, Gentil BJ, Minotti S, Marques C, Keith J, et al. Dysregulation of chromatin remodelling complexes in amyotrophic lateral sclerosis. Hum Mol Genet. 2017;26:4142–52. 182. Conlon EG, Lu L, Sharma A, Yamazaki T, Tang T, Shneider NA, et al. The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains. Elife. eLife Sciences Publications Ltd. 2016;5:e17820. 204. Liu EY, Russ J, Cali CP, Phan JM, Amlie-Wolf A, Lee Correspondence EB. Loss of nuclear TDP-43 is associated with Decondensation of LINE retrotransposons. Cell Rep. 2019;27:1409–1421.e6. 183. Fay MM, Anderson PJ, Ivanov P. ALS/FTD-associated C9ORF72 repeat RNA promotes phase transitions in vitro and in cells. Cell Rep. 2017;21:3573–84. 205. Birsa N, Bentham MP, Fratta P. Cytoplasmic functions of TDP-43 and FUS and their role in ALS. Semin Cell Dev Biol. Academic Press. 2019;99:193–201. 184. Elden AC, Kim HJ, Hart MP, Chen-Plotkin AS, Johnson BS, Fang X, et al. Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. Nature. Nature Publishing Group. 2010;466:1069–75. 206. Leibiger C, Deisel J, Aufschnaiter A, Ambros S, Tereshchenko M, Verheijen BM, et al. TDP-43 controls lysosomal pathways thereby determining its own clearance and cytotoxicity. Hum Mol Genet. 2018;27:1593–607. 185. Flores BN, Li X, Martinez J, Beg AA, Barmada SJ. An Intramolecular Salt Bridge Linking TDP43’s RNA Recognition Motifs Dictates RNA Binding and TDP43-Dependent Neurodegeneration. Cell Rep. Cell Press. 2019;27:1133– 1150.e8. 207. Liu G, Coyne AN, Pei F, Vaughan S, Chaung M, Zarnescu DC, et al. Endocytosis regulates TDP-43 toxicity and turnover. Nat Commun. Abbreviations ALS A t h DNA damage accumulates and responses are engaged in human ALS brain and spinal motor neurons and DNA repair is activatable in iPSC-derived motor neurons with SOD1 mutations. Acta Neuropathol Commun. BioMed Central Ltd. 2020;8:1–26. 174. Klim JR, Williams LA, Limone F, Guerra San Juan I, Davis-Dusenbery BN, Mordes DA, et al. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat Neurosci. Nature Publishing Group. 2019;22:167–79. 195. Mitra J, Guerrero EN, Hegde PM, Liachko NF, Wang H, Vasquez V, et al. Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects. Proc Natl Acad Sci. National Academy of Sciences. 2019;116:4696–705. 175. Chu JF, Majumder P, Chatterjee B, Huang SL, Shen CKJ. TDP-43 Regulates Coupled Dendritic mRNA Transport-Translation Processes in Co-operation with FMRP and Staufen1. Cell Rep. Elsevier B.V. 2019;29:3118–3133.e6. 196. Shaikh AY, Martin LJ. DNA base-excision repair enzyme apurinic/apyrimidinic endonuclease/redox factor-1 is increased and competent in the brain and spinal cord of individuals with amyotrophic lateral sclerosis. NeuroMolecular Med. Springer Science and Business Media LLC. 2002;2:47–60. 176. Amlie-Wolf A, Ryvkin P, Tong R, Dragomir I, Suh E, Xu Y, et al. Transcriptomic changes due to cytoplasmic TDP-43 expression reveal dysregulation of histone transcripts and nuclear chromatin. Buratti E, editor. PLoS One. 2015;10:e0141836. 197. Kim SH, Engelhardt JI, Henkel JS, Siklós L, Soós J, Goodman C, et al. Widespread increased expression of the DNA repair enzyme PARP in brain in ALS. Neurology. 2004;62:319–22. 177. Wu LS, Cheng WC, Chen CY, Wu MC, Wang YC, Tseng YH, et al. Transcriptomopathies of pre- and post-symptomatic frontotemporal dementia-like mice with TDP-43 depletion in forebrain neurons. Acta Neuropathol Commun. NLM (Medline). 2019;7:50. 198. Olkowski ZL. Mutant AP endonuclease in patients with amyotrophic lateral sclerosis. Neuroreport. Lippincott Williams and Wilkins. 1998;9:239–42. 178. Jeong YH, Ling JP, Lin SZ, Donde AN, Braunstein KE, Majounie E, et al. Tdp- 43 cryptic exons are highly variable between cell types. Mol Neurodegener. 2017;12:13. 199. Higelin J, Catanese A, Semelink-Sedlacek LL, Oeztuerk S, Lutz AK, Bausinger J, et al. NEK1 loss-of-function mutation induces DNA damage accumulation in ALS patient-derived motoneurons. Stem Cell Res. Elsevier B.V. 2018;30: 150–62. 179. Ishiguro A, Kimura N, Noma T, Shimo-Kon R, Ishihama A, Kon T. Molecular dissection of ALS-linked TDP-43: involvement of the Gly-rich domain in interaction with G-quadruplex mRNA. FEBS Lett. 2020. https://doi.org/10. 1002/1873-3468.13800. 200. Abbreviations ALS A t h Ticozzi N, LeClerc AL, van Blitterswijk M, Keagle P, McKenna-Yasek DM, Sapp PC, et al. Mutational analysis of TARDBP in neurodegenerative diseases. Neurobiol Aging. Elsevier. 2011;32:2096–9. 234. Mori K, Arzberger T, Grässer FA, Gijselinck I, May S, Rentzsch K, et al. Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins. Acta Neuropathol. 2013;126:881–93. 215. Van Blitterswijk M, Van Es MA, Hennekam EAM, Dooijes D, Van Rheenen W, Medic J, et al. Evidence for an oligogenic basis of amyotrophic lateral sclerosis. Hum Mol Genet. 2012;21:3776–84. 235. Solomon DA, Stepto A, Au WH, Adachi Y, Diaper DC, Hall R, et al. A feedback loop between dipeptide-repeat protein, TDP-43 and karyopherin-α mediates C9orf72-related neurodegeneration. Brain. 2018;141:2908–24. 216. Nozaki I, Arai M, Takahashi K, Hamaguchi T, Yoshikawa H, Muroishi T, et al. Familial ALS with G298S mutation in TARDBP: a comparison of CSF tau protein levels with those in sporadic ALS. Intern Med. The Japanese Society of Internal Medicine. 2010;49:1209–12. 236. Buratti E. TDP-43 post-translational modifications in health and disease. Expert Opin Ther Targets. 2018;22(3):279–93. 237. Liachko NF, McMillan PJ, Guthrie CR, Bird TD, Leverenz JB, Kraemer BC. CDC7 inhibition blocks pathological TDP-43 phosphorylation and neurodegeneration. Ann Neurol. John Wiley & Sons, Ltd. 2013;74:39–52. 217. Winton MJ, Van Deerlin VM, Kwong LK, Yuan W, Wood EMC, Yu CE, et al. A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro. FEBS Lett. NIH Public Access. 2008s;582:2252–6. 238. Alquezar C, Salado IG, De La Encarnación A, Pérez DI, Moreno F, Gil C, et al. Targeting TDP-43 phosphorylation by casein kinase-1δ inhibitors: a novel strategy for the treatment of frontotemporal dementia. Mol Neurodegener. 2016;11:36. 218. Conforti FL, Sproviero W, Simone IL, Mazzei R, Valentino P, Ungaro C, et al. TARDBP gene mutations in south Italian patients with amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry. BMJ Publishing Group Ltd. 2011;82: 587–8. 239. Kametani F, Nonaka T, Suzuki T, Arai T, Dohmae N, Akiyama H, et al. Identification of casein kinase-1 phosphorylation sites on TDP-43. Biochem Biophys Res Commun. Academic Press. 2009;382:405–9. 219. Corrado L, Ratti A, Gellera C, Buratti E, Castellotti B, Carlomagno Y, et al. High frequency of TARDBP gene mutations in italian patients with amyotrophic lateral sclerosis. Hum Mutat. Wiley-Liss Inc. 2009;30:688–94. 240. Nonaka T, Suzuki G, Tanaka Y, Kametani F, Hirai S, Okado H, et al. Abbreviations ALS A t h Li HY, Yeh PA, Chiu HC, Tang CY, Tu BP. Hyperphosphorylation as a defense mechanism to reduce TDP-43 aggregation. Ginsberg SD, editor. PLoS One. Public Library of Science. 2011;6:e23075. 226. Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011;72:257–68. 247. Li W, Reeb AN, Lin B, Subramanian P, Fey EE, Knoverek CR, et al. Heat shock- induced phosphorylation of TAR DNA-binding protein 43 (TDP-43) by MAPK/ERK kinase regulates TDP-43 function. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2017;292:5089–100. 227. Majounie E, Renton AE, Mok K, Dopper EGP, Waite A, Rollinson S, et al. Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol. Lancet Publishing Group. 2012;11:323–30. 248. Ikeda K, Tsuchiya K. Motor neuron disease group accompanied by inclusions of unidentified protein signaled by ubiquitin. Neuropathology. 2004;24:117–24. 228. Ditsworth D, Maldonado M, McAlonis-Downes M, Sun S, Seelman A, Drenner K, et al. Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis. Acta Neuropathol. Springer Verlag. 2017;133:907–22. 249. Cohen TJ, Hwang AW, Unger T, Trojanowski JQ, Lee VMY. Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. EMBO J. 2012;31:1241–52. 229. Boeynaems S, Bogaert E, Michiels E, Gijselinck I, Sieben A, Jovičić A, et al. Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD. Sci Rep. Nature Publishing Group. 2016;6:20877. 250. Cohen TJ, Hwang AW, Restrepo CR, Yuan CX, Trojanowski JQ, Lee VMY. An acetylation switch controls TDP-43 function and aggregation propensity. Nat Commun. Nature Publishing Group. 2015;6:5845. 230. Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee KH, et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. Nature Publishing Group. 2015;525:129–33. 251. Nishimoto Y, Ito D, Yagi T, Nihei Y, Tsunodo Y, Suzuki N. Characterization of alternative isoforms and inclusion body of the TAR DNA-binding protein-43. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2010;285:608–19. 231. Jovičič A, Mertens J, Boeynaems S, Bogaert E, Chai N, Yamada SB, et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci. Nature Publishing Group. 2015; 18:1226–9. 252. Salvatori I, Ferri A, Scaricamazza S, Giovannelli I, Serrano A, Rossi S, et al. Abbreviations ALS A t h Phosphorylation of TAR DNA-binding protein of 43 kDa (TDP-43) by truncated casein kinase 1δ triggers mislocalization and accumulation of TDP-43. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2016;291:5473–83. 220. Del Bo R, Ghezzi S, Corti S, Pandolfo M, Ranieri M, Santoro D, et al. TARDBP (TDP-43) sequence analysis in patients with familial and sporadic ALS: identification of two novel mutations. Eur J Neurol. John Wiley & Sons, Ltd. 2009;16:727–32. 221. Williams KL, Durnall JC, Thoeng AD, Warraich ST, Nicholson GA, Blair IP. A novel TARDBP mutation in an Australian amyotrophic lateral sclerosis kindred. J Neurol Neurosurg Psychiatry. BMJ Publishing Group. 2009;80: 1286–8. 241. Krach F, Batra R, Wheeler EC, Vu AQ, Wang R, Hutt K, et al. Transcriptome– pathology correlation identifies interplay between TDP-43 and the expression of its kinase CK1E in sporadic ALS. Acta Neuropathol. Springer Berlin Heidelberg. 2018;136:405–23. 242. Choksi DK, Roy B, Chatterjee S, Yusuff T, Bakhoum MF, Sengupta U, et al. TDP-43 phosphorylation by casein kinase iε promotes oligomerization and enhances toxicity in vivo. Hum Mol Genet. 2014;23:1025–35. 222. Pamphlett R, Luquin N, McLean C, Jew SK, Adams L. TDP-43 neuropathology is similar in sporadic amyotrophic lateral sclerosis with or without TDP-43 mutations. Neuropathol Appl Neurobiol. John Wiley & Sons, Ltd. 2009;35:222–5. 243. Liachko NF, Guthrie CR, Kraemer BC. Phosphorylation promotes neurotoxicity in a Caenorhabditis elegans model of TDP-43 proteinopathy. J Neurosci. Society for Neuroscience. 2010;30:16208–19. 223. Aizawa H, Yamashita T, Kato H, Kimura T, Kwak S. Impaired nucleoporins are present in sporadic amyotrophic lateral sclerosis motor neurons that exhibit mislocalization of the 43-kDa TAR DNA-binding protein. J Clin Neurol. Korean Neurological Association. 2019;15:62–7. 244. Liachko NF, McMillan PJ, Strovas TJ, Loomis E, Greenup L, Murrell JR, et al. The tau tubulin kinases TTBK1/2 promote accumulation of pathological TDP-43. PLoS Genet. Public Library of Science. 2014;10:e1004803. 224. Chou CC, Zhang YJY, Umoh ME, Vaughan SW, Lorenzini I, Liu F, et al. TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nat Neurosci. Nature Publishing Group. 2018;21:228–39. 245. Taylor LM, McMillan PJ, Liachko NF, Strovas TJ, Ghetti B, Bird TD, et al. Pathological phosphorylation of tau and TDP-43 by TTBK1 and TTBK2 drives neurodegeneration. Mol Neurodegener. BioMed Central Ltd. 2018;13:7. 225. Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P, et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. Nature Publishing Group. 2015;525:56–61. 246. Abbreviations ALS A t h Nature Publishing Group. 2017;8:2092. 186. Ihara R, Matsukawa K, Nagata Y, Kunugi H, Tsuji S, Chihara T, et al. RNA binding mediates neurotoxicity in the transgenic Drosophila model of TDP- 43 proteinopathy. Hum Mol Genet. 2013;22:4474–84. 208. Liu G, Byrd A, Warner AN, Pei F, Basha E, Buchanan A, et al. Cdc48/VCP and Endocytosis Regulate TDP-43 and FUS Toxicity and Turnover. Mol Cell Biol. American Society for Microbiology. 2019;40:e00256–19. 187. Voigt A, Herholz D, Fiesel FC, Kaur K, Müller D, Karsten P, et al. TDP-43- mediated neuron loss in vivo requires RNA-binding activity. Feany MB, editor. PLoS One. Public Library of Science. 2010;5:e12247. 209. Smethurst P, Risse E, Tyzack GE, Mitchell JS, Taha DM, Chen YR, et al. Distinct responses of neurons and astrocytes to TDP-43 proteinopathy in amyotrophic lateral sclerosis. Brain. 2020;143:430–40. 188. Chen H-J, Topp SD, Hui HS, Zacco E, Katarya M, McLoughlin C, et al. RRM adjacent TARDBP mutations disrupt RNA binding and enhance TDP-43 proteinopathy. Brain. 2019;142:3753–70. 210. Kawamata H, Peixoto P, Konrad C, Palomo G, Bredvik K, Gerges M, et al. Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in vivo. Mol Neurodegener. 2017;12:37. 211. Benajiba L, Le BI, Camuzat A, Lacoste M, Thomas-Anterion C, Couratier P, et al. TARDBP mutations in motoneuron disease with frontotemporal lobar degeneration. Ann Neurol. John Wiley and Sons Inc. 2009;65:470–3. 189. Maharana S, Wang J, Papadopoulos DK, Richter D, Pozniakovsky A, Poser I, et al. RNA buffers the phase separation behavior of prion-like RNA binding proteins. Science. American Association for the Advancement of Science. 2018;360:918–21. 212. Borghero G, Pugliatti M, Marrosu F, Marrosu MG, Murru MR, Floris G, et al. Genetic architecture of ALS in Sardinia. Neurobiol Aging. Elsevier Inc. 2014; 35:2882.e7–2882.e12. 190. Lalmansingh AS, Urekar CJ, Reddi PP. TDP-43 is a transcriptional repressor: the testis-specific mouse acrv1 gene is a TDP-43 target in vivo. J Biol Chem. American Society for Biochemistry and Molecular Biology. 2011;286:10970–82. 213. Millecamps S, Salachas F, Cazeneuve C, Gordon P, Bricka B, Camuzat A, et al. SOD1, ANG, VAPB, TARDBP, and FUS mutations in familial amyotrophic lateral sclerosis: genotype-phenotype correlations. J Med Genet. BMJ Publishing Group Ltd. 2010;47:554–60. 191. Schwenk BM, Hartmann H, Serdaroglu A, Schludi MH, Hornburg D, Meissner F, et al. TDP-43 loss of function inhibits endosomal trafficking Page 15 of 16 Page 15 of 16 Page 15 of 16 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 214. Abbreviations ALS A t h Differential toxicity of TAR DNA-binding protein 43 isoforms depends on their submitochondrial localization in neuronal cells. J Neurochem. Blackwell Publishing Ltd. 2018;146:585–97. 232. Shi KY, Mori E, Nizami ZF, Lin Y, Kato M, Xiang S, et al. Toxic PRn poly- dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export. Proc Natl Acad Sci U S A. National Academy of Sciences. 2017; 114:E1111–7. 253. Fung G, Shi J, Deng H, Hou J, Wang C, Hong A, et al. Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis. Cell Death Differ. 2015;22:2087–97. 233. Zu T, Liu Y, Bañez-Coronel M, Reid T, Pletnikova O, Lewis J, et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc Natl Acad Sci U S A. 2013;110:E4968–77. 254. Fratta P, Sivakumar P, Humphrey J, Lo K, Ricketts T, Oliveira H, et al. Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis. EMBO J. 2018;37:e98684. Page 16 of 16 Page 16 of 16 Suk and Rousseaux Molecular Neurodegeneration (2020) 15:45 GRM3 and USP47 in amyotrophic lateral sclerosis. Neuropathol Appl Neurobiol. Blackwell Publishing Ltd. 2020. https://doi.org/10.1111/nan.12597. GRM3 and USP47 in amyotrophic lateral sclerosis. Neuropathol Appl Neurobiol. Blackwell Publishing Ltd. 2020. https://doi.org/10.1111/nan.12597. 255. Weskamp K, Tank EM, Miguez R, McBride JP, Gómez NB, White M, et al. Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS. J Clin Invest. 2020;130:1139–55. 275. Maniatis S, Äijö T, Vickovic S, Braine C, Kang K, Mollbrink A, et al. Spatiotemporal dynamics of molecular pathology in amyotrophic lateral sclerosis. Science. American Association for the Advancement of Science. 2019;364:89–93. 256. Hideyama T, Teramoto S, Hachiga K, Yamashita T, Kwak S. Co-occurrence of TDP-43 Mislocalization with reduced activity of an RNA editing enzyme, ADAR2, in aged mouse motor neurons. Kano MR, editor. PLoS One. Public Library of Science. 2012;7:e43469. 276. Otake K, Kamiguchi H, Hirozane Y. Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid. BMC Med Genomics. BioMed Central Ltd. 2019;12:7. 257. De Giorgio F, Maduro C, EMC F, Acevedo-Arozena A. Transgenic and physiological mouse models give insights into different aspects of amyotrophic lateral sclerosis. Dis Model Mech. Company of Biologists Ltd. 2019;12(1):dmm037424. 277. Zucca S, Gagliardi S, Pandini C, Diamanti L, Bordoni M, Sproviero D, et al. Abbreviations ALS A t h RNA-seq profiling in peripheral blood mononuclear cells of amyotrophic lateral sclerosis patients and controls. Sci Data. Nature Publishing Groups. 2019;6:1–8. 258. Bennett CL, Dastidar SG, Ling SC, Malik B, Ashe T, Wadhwa M, et al. Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients. Acta Neuropathol. Springer Verlag. 2018;136:425–43. 278. Tyzack GE, Luisier R, Taha DM, Neeves J, Modic M, Mitchell JS, et al. Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis. Brain. 2019;142(9):2572–80. 259. Grunseich C, Patankar A, Amaya J, Watts JA, Li D, Ramirez P, et al. Clinical and molecular aspects of Senataxin mutations in amyotrophic lateral sclerosis 4. Ann Neurol. John Wiley and Sons Inc. 2020;87:547–55. 279. Lobingier BT, Hüttenhain R, Eichel K, Miller KB, Ting AY, von Zastrow M, et al. An approach to spatiotemporally resolve protein interaction networks in living cells. Cell. 2017;169:350–360.e12. 260. Chen YZ, Bennett CL, Huynh HM, Blair IP, Puls I, Irobi J, et al. DNA/ RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am J Hum Genet. University of Chicago Press. 2004;74:1128–35. 280. Roux KJ, Kim DI, Burke B. BioID: A screen for protein-protein interactions. Curr Protoc Protein Sci. Blackwell Publishing Inc. 2013;2013:19.23.1–19.23.14. 281. Fazal FM, Han S, Parker KR, Kaewsapsak P, Xu J, Boettiger AN, et al. Atlas of subcellular RNA localization revealed by APEX-Seq. Cell. Elsevier. 2019;178:1–18. 261. Huang SL, Wu LS, Lee M, Chang CW, Cheng WC, Fang YS, et al. A robust TDP-43 knock-in mouse model of ALS. Acta Neuropathol Commun. BioMed Central Ltd. 2020;8:3. 282. Padrón A, Iwasaki S, Ingolia NT. Proximity RNA Labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules. Mol Cell. Cell Press. 2019;75:875–887.e5. 262. Ebstein SY, Yagudayeva I, Shneider NA. Mutant TDP-43 causes early-stage dose-dependent motor neuron degeneration in a TARDBP Knockin mouse model of ALS. Cell Rep. Elsevier. 2019;26:364–373.e4. 283. Markmiller S, Soltanieh S, Server KL, Bennett EJ, Lé E, Yeo Correspondence GW. Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules. Cell. 2018;172:590–598.e13. 263. Mitchell JC, Constable R, So E, Vance C, Scotter E, Glover L, et al. Wild type human TDP-43 potentiates ALS-linked mutant TDP-43 driven progressive motor and cortical neuron degeneration with pathological features of ALS. Acta Neuropathol Commun. BioMed Central. 2015;3:36. 284. Ule J, Jensen KB, Ruggiu M, Mele A, Ule A, Darnell RB. Abbreviations ALS A t h CLIP Identifies Nova- Regulated RNA Networks in the brain. Science. American Association for the Advancement of Science. 2003;302:1212–5. 264. Hawrot J, Imhof S, Wainger BJ. Modeling cell-autonomous motor neuron phenotypes in ALS using iPSCs. Neurobiol Dis. Academic Press Inc. 2020;134: 104680. 285. Schwab C, Arai T, Hasegawa M, Yu S, McGeer PL. Colocalization of transactivation-responsive DNA-binding protein 43 and huntingtin in inclusions of Huntington disease. J Neuropathol Exp Neurol. Oxford Academic. 2008;67:1159–65. 265. Bilican B, Serio A, Barmada SJ, Nishimura AL, Sullivan GJ, Carrasco M, et al. Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability. Proc Natl Acad Sci U S A. National Academy of Sciences. 2012;109:5803–8. 286. Nakashima-Yasuda H, Uryu K, Robinson J, Xie SX, Hurtig H, Duda JE, et al. Co-morbidity of TDP-43 proteinopathy in Lewy body related diseases. Acta Neuropathol. 2007;114:221–9. 266. Serio A, Bilican B, Barmada SJ, Ando DM, Zhao C, Siller R, et al. Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy. Proc Natl Acad Sci U S A. National Academy of Sciences. 2013;110:4697–702. 287. Chornenkyy Y, Fardo DW, Nelson PT. Tau and TDP-43 proteinopathies: kindred pathologic cascades and genetic pleiotropy. Lab Investig. Nature Publishing Group. 2019;99:993–1007. 288. Nonaka T, Masuda-Suzukake M, Hosokawa M, Shimozawa A, Hirai S, Okado H, et al. C9ORF72 dipeptide repeat poly-GA inclusions promote intracellular aggregation of phosphorylated TDP-43. Hum Mol Genet. 2018;27:2658–70. 288. Nonaka T, Masuda-Suzukake M, Hosokawa M, Shimozawa A, Hirai S, Okado H, et al. C9ORF72 dipeptide repeat poly-GA inclusions promote intracellular aggregation of phosphorylated TDP-43. Hum Mol Genet. 2018;27:2658–70. 267. Zhang Z, Almeida S, Lu Y, Nishimura AL, Peng L, Sun D, et al. Downregulation of MicroRNA-9 in iPSC-derived neurons of FTD/ALS patients with TDP-43 mutations. Pandey U, editor. PLoS One. Public Library of Science. 2013;8:e76055. Publisher’s Note 268. Frank SA. Somatic evolutionary genomics: mutations during development cause highly variable genetic mosaicism with risk of cancer and neurodegeneration. Proc Natl Acad Sci U S A. National Academy of Sciences. 2010;107:1725–30. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 269. Benatar M, Wuu J, Andersen PM, Lombardi V, Malaspina A. Neurofilament light: a candidate biomarker of presymptomatic amyotrophic lateral sclerosis and phenoconversion. Ann Neurol. John Wiley and Sons Inc. 2018; 84:130–9. 270. Gisslén M, Price RW, Andreasson U, Norgren N, Nilsson S, Hagberg L, et al. Plasma concentration of the Neurofilament light protein (NFL) is a biomarker of CNS injury in HIV infection: a cross-sectional study. EBioMedicine. Elsevier B.V. 2016;3:135–40. 271. Li QF, Dong Y, Yang L, Xie JJ, Ma Y, Du YC, et al. Neurofilament light chain is a promising serum biomarker in spinocerebellar ataxia type 3. Mol Neurodegener. BioMed Central Ltd. 2019;14:39. 272. Preische O, Schultz SA, Apel A, Kuhle J, Kaeser SA, Barro C, et al. Serum neurofilament dynamics predicts neurodegeneration and clinical progression in presymptomatic Alzheimer’s disease. Nat Med. Nature Publishing Group. 2019;25:277–83. 273. Cai L, Huang J. Neurofilament light chain as a biological marker for multiple sclerosis: a meta-analysis study. Neuropsychiatr Dis Treat. Dove Medical Press Ltd. 2018;14:2241–54. 273. Cai L, Huang J. Neurofilament light chain as a biological marker for multiple sclerosis: a meta-analysis study. Neuropsychiatr Dis Treat. Dove Medical Press Ltd. 2018;14:2241–54. 274. Gregory JM, McDade K, Livesey MR, Croy I, Marion de Proce S, Aitman T, et al. Spatial transcriptomics identifies spatially dysregulated expression of 274. Gregory JM, McDade K, Livesey MR, Croy I, Marion de Proce S, Aitman T, et al. Spatial transcriptomics identifies spatially dysregulated expression of
https://openalex.org/W2089989500	https://europepmc.org/articles/pmc3940913?pdf=render	English	null	Two Kinds of Ferritin Protect Ixodid Ticks from Iron Overload and Consequent Oxidative Stress	PloS one	2,014	cc-by	10,512	Abstract Ticks are obligate hematophagous parasites that have successfully developed counteractive means against their hosts’ immune and hemostatic mechanisms, but their ability to cope with potentially toxic molecules in the blood remains unclear. Iron is important in various physiological processes but can be toxic to living cells when in excess. We previously reported that the hard tick Haemaphysalis longicornis has an intracellular (HlFER1) and a secretory (HlFER2) ferritin, and both are crucial in successful blood feeding and reproduction. Ferritin gene silencing by RNA interference caused reduced feeding capacity, low body weight and high mortality after blood meal, decreased fecundity and morphological abnormalities in the midgut cells. Similar findings were also previously reported after silencing of ferritin genes in another hard tick, Ixodes ricinus. Here we demonstrated the role of ferritin in protecting the hard ticks from oxidative stress. Evaluation of oxidative stress in Hlfer-silenced ticks was performed after blood feeding or injection of ferric ammonium citrate (FAC) through detection of the lipid peroxidation product, malondialdehyde (MDA) and protein oxidation product, protein carbonyl. FAC injection in Hlfer-silenced ticks resulted in high mortality. Higher levels of MDA and protein carbonyl were detected in Hlfer-silenced ticks compared to Luciferase-injected (control) ticks both after blood feeding and FAC injection. Ferric iron accumulation demonstrated by increased staining on native HlFER was observed from 72 h after iron injection in both the whole tick and the midgut. Furthermore, weak iron staining was observed after Hlfer knockdown. Taken together, these results show that tick ferritins are crucial antioxidant molecules that protect the hard tick from iron- mediated oxidative stress during blood feeding. Citation: Galay RL, Umemiya-Shirafuji R, Bacolod ET, Maeda H, Kusakisako K, et al. (2014) Two Kinds of Ferritin Protect Ixodid Ticks from Iron Overload and Consequent Oxidative Stress. PLoS ONE 9(3): e90661. doi:10.1371/journal.pone.0090661 Editor: Ben J. Mans, Onderstepoort Veterinary Institute, South Africa Received October 24, 2013; Accepted February 4, 2014; Published March 3, 2014 Received October 24, 2013; Accepted February 4, 2014; Published March 3, 2014 ay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Galay et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This study was supported by a joint research grant (25-joint-2) of the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Morinaga Foundation, and grants-in-aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS). RLG and ETB are supported by the Japanese Government Ministry of Education, Culture, Sports, Science, and Technology Scholarship (Monbukagakusho: MEXT) for doctoral fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tetsuya@ms.kagoshima-u.ac.jp Remil Linggatong Galay1,2, Rika Umemiya-Shirafuji3, Eugene T. Bacolod4,5, Hiroki Maeda1,2, Kodai Kusakisako2, Jiro Koyama6, Naotoshi Tsuji7, Masami Mochizuki1,2, Kozo Fujisaki7, Tetsuya Tanaka1,2* Remil Linggatong Galay1,2, Rika Umemiya-Shirafuji3, Eugene T. Bacolod4,5, Hiroki Maeda1,2, Kodai Kusakisako2, Jiro Koyama6, Naotoshi Tsuji7, Masami Mochizuki1,2, Kozo Fujisaki7, Tetsuya Tanaka1,2* 1 Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi, Japan, 2 Laboratory of Emerging Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima, Japan, 3 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan, 4 The United Graduate School of Agricultural Sciences, Kagoshima University, Shimoarata, Kagoshima, Japan, 5 Department of Chemistry, College of Arts and Sciences, University of San Carlos, Cebu City, Philippines, 6 Education and Research Center for Marine Resources and Environment, Faculty of Fisheries, Kagoshima University, Shimoarata, Kagoshima, Japan, 7 National Agricultural and Food Research Organization, Kannondai, Tsukuba, Ibaraki, Japan Introduction Iron-binding proteins, such as transferrin and ferritin, are present in most living organisms that function to regulate iron levels and prevent iron toxicity. Most ferritins consist of 24 subunits folded in a helical bundle, forming an almost spherical protein shell with a large cavity that can hold up to 4,000 iron atoms [4]. Mammalian ferritins serve mainly as intracellular iron storage proteins, while insect ferritins also function in iron transport [5]. Aside from iron transport and storage functions, ferritin was also implicated in immune response [6] and oxidative stress [7]. Iron is an essential element required for various physiological processes in most living organisms. Iron metabolism involves a continuous redox cycling between the ferrous (Fe2+) and ferric (Fe3+) states. Fe2+ is potentially toxic due to its ability to catalyze the formation of reactive oxygen species (ROS) through Fenton reaction [1]. High levels of ROS can lead to cellular damage and death, resulting from damage to biomolecules including lipid peroxidation, DNA and protein oxidation, which is collectively known as oxidative stress [2]. Oxidative stress occurs when the level of ROS overwhelms the antioxidant defense mechanisms, accompanied by the accumulation of oxidative stress products. These products of oxidative damage to biomolecules can be used as indicators in evaluating oxidative stress, termed biomarkers [3]. Ticks are important blood-feeding parasites of wild and domestic animals and humans, primarily because they serve as vectors of different pathogens. Aside from dealing with the host’s hemostatic and immune mechanism [8], ticks must also cope with the potentially toxic molecules in their large blood meal, including iron. However, many aspects of iron metabolism of ticks remain March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 1 March 2014 \| Volume 9 \| Issue 3 \| e90661 Tick Ferritins against Oxidative Stress unclear. Heme transport [9,10] and detoxification [11] have already been investigated. An intracellular and a secretory ferritin in two species of hard ticks, Ixodes ricinus [12] and Haemaphysalis longicornis [13] have been reported to be crucial in blood feeding and reproduction. The other functions of ferritin, particularly its role in tick survival, have not yet been fully elucidated. Existing knowledge on the antioxidant defense of ticks, especially during blood feeding, is limited. without dsRNA injection. Ticks and experimental animals Parthenogenetic (Okayama strain) adult female H. longicornis ticks were used throughout this study. Ticks have been maintained by feeding on the ears of Japanese white rabbits (Kyudo, Kumamoto, Japan) for several generations at the Laboratory of Emerging Infectious Diseases, Joint Faculty of Veterinary Med- icine, Kagoshima University, Kagoshima, Japan [14]. Rabbits were kept in a temperature- and humidity-controlled room, with a constant supply of water and commercial rabbit pellets. Rabbit care and use in this study has been approved by the Animal Care and Use Committee of Kagoshima University (Approval number VM13007). Protein extraction Blood-fed or FAC-injected whole ticks were homogenized in phosphate-buffered saline (PBS). Midguts and salivary glands were also collected and homogenized in Tris-buffered saline (TBS) with a protease inhibitor (Complete Mini EDTA-free, Roche, Man- heim, Germany). Hemolymph was collected from the amputated legs of immobilized ticks. Hemocytes were separated by centrifu- gation. Protein from whole ticks and organs was extracted as previously described [15]. Protein samples were kept at 280uC until use. Introduction To check the survival rate of Hlfer- silenced ticks after exposure to iron, 100 mM FAC was injected in the same manner as dsRNA injection. Likewise, sterilized high- purity water was injected to dsRNA-injected ticks for additional control. Thirty ticks for each group were used for this experiment. After injection of FAC, ticks were held as mentioned above and monitored for mortality every 12 h for 11 days. The survival experiment was repeated three times to confirm the reproducibility of results. Otherwise, unfed adult ticks not injected with dsRNA were injected with 50 or 100 mM FAC or sterilized high-purity water for control to evaluate mRNA and protein expression and iron staining in response to iron treatment. Here we showed that RNA interference (RNAi)-mediated silencing of H. longicornis ferritin genes predisposed the ticks to oxidative stress by detecting the levels of a product of lipid peroxidation and a product of protein oxidation after blood feeding or iron injection. Our results show that the two ferritins of H. longicornis are essential antioxidant molecules that prevent iron- mediated oxidative stress during blood feeding and are crucial to its survival. Electrophoresis, Western blot analysis and gel iron staining To investigate the protein expression, protein samples were separated in 12% SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis as described previously [13]. Specific mouse anti-ferritin sera [13] or anti-b-tubulin serum for control [16] were used as primary antibodies. Protein signals were detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and images were taken using the FluorChem FC2 Imaging System (Protein Simple, Santa Clara, CA, USA). Western blotting was performed at least three times. To accurately determine differ- ences in the protein expression, band densitometry analysis was performed using Alpha View Software (Alpha Innotech, Protein Simple). The band densitometry analysis results shown in this study represent the mean of three trials of Western blot analysis. injection An indirect immunofluorescent antibody test (IFAT) was performed as previously described [13]. Briefly, midguts and salivary glands were dissected from unfed adult ticks then fixed overnight in 4% paraformaldehyde in PBS with 0.1% glutaralde- hyde and washed with a sucrose series before being embedded in Tissue-Tek O.C.T Compound (Sakura Finetek Japan, Tokyo, Japan). After cutting, tissue sections were air-dried and then blocked overnight with 5% skim milk in PBS at 4uC. Sections were RNA interference and tick infestation The silencing of Hlfer in unfed adult female ticks was induced by injection of double-stranded RNA (dsRNA) prepared as previously described [13]. Briefly, ticks were attached to glass slides and then injected with 1 mg per 0.5 ml of Hlfer1 or Hlfer2 dsRNA through the fourth coxae using an IM 300 Microinjector (Narishige, Tokyo, Japan). Control ticks were injected with the same amount of firefly Luciferase (Luc) dsRNA. To confirm silencing, total RNA was extracted from whole ticks 4 days post-injection of dsRNA for RT-PCR analysis. Ticks injected with dsRNA were held in a humidity chamber kept in a 25uC incubator for 18 h before infestation to rabbits or for 4 days before injection with ferric ammonium citrate (FAC). To stain native HlFER for ferric iron, protein extracts were separated in 6% native PAGE. Protein concentration was adjusted after determination of protein concentration using a Micro BCA Assay kit (Thermo Scientific, Rockford, IL, USA) or based on control immunoblotting with b-tubulin as described above [16]. The gel was stained in a freshly prepared Prussian blue staining solution (equal volume of 10% K4[Fe(CN)6] and 10% HCl) at room temperature for 48 h as previously reported [17]. The high molecular weight marker (GE Healthcare), which contains ferritin from equine spleen for the 440 kDa band, as well as the commercially-prepared horse holoferritin (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. For rabbit infestation, a total of 50 ticks per dsRNA injected group were attached in separate ears of rabbits, individually covered with an ear bag. Attached ticks were allowed to feed until they naturally dropped off. From the total number of engorged ticks, 30 ticks from each group were used for the thiobarbituric acid reactive species (TBARS) assay in the whole ticks. Five pooled midgut samples, comprising of three ticks each for Hlfer1and Hlfer2-silenced ticks and two ticks each for Luc-injected group, were also prepared for the TBARS assay. The remaining ticks were used for immunoblot detection of oxidative stress biomarkers, described in the succeeding sections. All ticks were stored in 2 80uC until use. Measurement of total ferrous iron The ferrozine assay for measuring non-heme iron was adapted to determine the amount of ferrous iron in whole ticks injected with FAC after Hlfer knockdown [19,20]. Ten whole ticks from each group were collected 72 h after the injection of FAC and homogenized in lysis buffer (20 mM Tris, 137 mM NaCl, 1% Triton X-100, 1% glycerol). Protein concentration was measured using a Micro BCA Assay Kit (Thermo Scientific). Concentrated HCl was added and then heated to 95uC. After cooling to room temperature, the mixture was centrifuged and the supernatant was obtained, to which 10 mM ferrozine was added. Color develop- ment was accomplished by the addition of saturated ammonium acetate. Absorbance was measured at 550 nm and iron concen- tration was calculated based on a molar extinction coefficient of the iron-ferrozine complex of 27 900 M21 cm21 and based on protein concentration. Assessment of oxidative stress Oxidative stress was evaluated by detecting oxidative stress biomarkers including malondialdehyde (MDA) and protein carbonyl (PC). MDA was demonstrated through immunoblotting using the Oxiselect Malondialdehyde Immunoblot Kit (Cell Biolabs, San Diego, CA, USA) following the manufacturer’s recommendation. Engorged whole ticks were homogenized individually, whereas midguts and unfed adult ticks injected FAC were pooled. Protein was adjusted based on tubulin profile. Bands were viewed using Clarity Western ECL Substrate (Bio-rad Laboratories, Hercules, CA, USA) and the MDA level relative to tubulin was calculated after band densitometry analysis. TBARS assay was also performed to quantify MDA [18]. For the TBARS assay, tick homogenates were mixed with TBARS reagent (0.37% (w/v) thiobarbituric acid, 15% (w/v) trichloroacetic acid in 0.25 M HCl) and then placed in boiling water bath for 15 min and allowed to cool. Absorbance was measured at 532 nm and MDA content was calculated using the molecular extinction coefficient for MDA. PC was also demonstrated following the immunoblot assay using the Oxiselect Protein Carbonyl Immu- noblot Kit (Cell Biolabs) according to the manufacturer’s instruction and analyzed similar to MDA. FAC injection has no effect on transcription but stimulates protein expression of ferritins p p We evaluated whether injection of FAC as exogenous iron source can affect Hlfer transcript level and HlFER protein expression. Artificial feeding or in vitro exposure of cells to iron in different organisms induced up-regulation of ferritin mRNA [21,22,23,24,25]. Different concentrations of FAC were injected to the hemocoel of normal unfed adult female ticks or sterilized high- purity water for the control group. The transcript level in whole ticks was then checked at 24 h and 72 h after FAC injection, whereas protein expression was examined from 24 h to 96 h after FAC injection. RT-PCR analysis showed no difference among the groups at any time point (Fig. S2). However, increased protein expression particularly of HlFER1 was observed in both concen- trations of FAC from 24 h to 96 h post-injection (Fig. 2). Band densitometry analysis was performed to accurately determine the differences in protein expressions. We also examined the HlFER expression in organs at 24 h and 72 h post-injection and we found that both HlFER1 and HlFER2 levels were higher in the midguts (Fig. 3A) of FAC injected ticks but not in the salivary glands (Fig. S3). In the hemolymph where only HlFER2 is present, its expression is also higher after FAC injection compared to the control (Fig. 3B). These findings suggest that iron injection can stimulate HlFER expression in unfed ticks. Injection of ferric ammonium citrate (FAC) We previously found that the silencing of either Hlfer1 or Hlfer2 had a negative effect on tick survival after blood feeding [13] and we concluded that this was caused by iron overload. Thus, to further investigate the effect of high levels of iron on ticks, different concentrations of FAC were injected into unfed adult ticks, with or March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 2 Tick Ferritins against Oxidative Stress incubated with a 1:50 dilution of anti-ferritin sera or normal mouse serum as a control for the primary antibody and a 1:1,000 dilution of Alexa Fluor 594-conjugated goat anti-mouse IgG (Invitrogen, Eugene, OR, USA) for the secondary antibody for an hour each at room temperature. Following washes with PBS, sections were mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA) and then viewed on a fluorescence microscope mounted with a DP71 camera (Olympus, Tokyo, Japan). survival rate was related to iron overload in the absence of ferritin, here we exposed unfed Hlfer-silenced adult female ticks to iron by injecting 100 mM FAC into the hemocoel. Silencing was confirmed by RT-PCR analysis (data not shown). After FAC injection, ticks were kept at 25uC and survival was monitored every 12 h. No mortality was observed in the control group injected with Luc dsRNA (Fig. 1). In contrast, both Hlfer1- and Hlfer2-silenced groups had a continuously decreasing survival rate (P,0.0001). Eleven days after FAC injection, the Hlfer2 dsRNA- injected group showed the lowest survival rate. As an additional negative control, high-purity sterilized water was similarly injected after RNAi but this did not result in high mortality as in the case of FAC injection (Fig. S1). This result supports our previous conclusion that the mortality after blood feeding in Hlfer-silenced ticks was due to iron toxicity. FAC injection led to ferric iron accumulation in HlFER in the whole tick and the midgut After observing that FAC can stimulate HlFER expression in the whole tick and in the midgut, we determined whether there was a corresponding accumulation of ferric iron on native HlFER. After separating the tick protein in native PAGE, HlFER was stained using Prussian blue staining to indicate ferric iron. Both the high molecular weight marker containing ferritin from equine spleen as the 440 kDa band, and the commercial horse holoferritin strongly stained for ferric iron (Fig. 4A). In whole ticks, increased staining was observed at 72 h and 96 h after injection of any concentration of FAC (Fig. 4A). Ferric iron staining also increased in the midgut and hemolymph at 72 h post- injection of FAC but not in the salivary glands (Fig. 4B) or ovary (data not shown). In all experiments, only one band was stained with Prussian blue, with an estimated molecular weight of around 440 kDa. We confirmed that the bands stained for ferric iron were HlFERs through Western blot analysis after native PAGE (Fig. S4 and 4B). HlFER1 and HlFER2 had almost the same molecular weight on native PAGE. Statistical analyses For band densitometry analysis, Student’s t-test or the Mann- Whitney U test was performed, depending on data distribution. For the survival experiment, the Mantel-Cox log-rank test was performed using GraphPad Prism software. In all statistical analyses, significant difference between groups is defined by P, 0.05. Hlfer-silenced ticks had low survival rate after FAC injection High mortality was previously observed in Hlfer-silenced ticks after blood feeding [13]. To further demonstrate that the low March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 3 Tick Ferritins against Oxidative Stress Figure 1. Survival rate of Hlfer-silenced ticks after injection of FAC. Unfed adult female ticks were injected with H. longicornis fer1 (Hlfer1), H. longicornis fer2 (Hlfer2) or Luciferase (Luc) dsRNA for the control to induce RNAi. Silencing was confirmed through RT-PCR. After 4 days, 100 mM FAC was injected, and mortality was monitored. Both Hlfer1- and Hlfer2-dsRNA-injected groups had a lower survival rate compared to Luc. n = 30 ticks per group. The graph here represents the result of a single independent trial. Bars represent standard error. Significant difference was determined using the log-rank Mantel-Cox test (P,0.0001, Luc vs. Hlfer1 or Hlfer2). doi:10.1371/journal.pone.0090661.g001 Figure 1. Survival rate of Hlfer-silenced ticks after injection of FAC. Unfed adult female ticks were injected with H. longicornis fer1 (Hlfer1), H. longicornis fer2 (Hlfer2) or Luciferase (Luc) dsRNA for the control to induce RNAi. Silencing was confirmed through RT-PCR. After 4 days, 100 mM FAC was injected, and mortality was monitored. Both Hlfer1- and Hlfer2-dsRNA-injected groups had a lower survival rate compared to Luc. n = 30 ticks per group. The graph here represents the result of a single independent trial. Bars represent standard error. Significant difference was determined using the log-rank Mantel-Cox test (P,0.0001, Luc vs. Hlfer1 or Hlfer2). doi:10.1371/journal.pone.0090661.g001 Figure 2. Protein expression of H. longicornis ferritins in whole, unfed ticks at different hours after injection of different concentrations of FAC. Sterilized high-purity water was injected into the control group (0 mM). (A) Western blot analysis after incubation with specific anti-sera against H. longicornis FER1 (HlFER1) or H. longicornis FER2 (HlFER2). Tubulin was used as an internal control. (B) Band densitometry analysis for HlFER1 and HlFER2. The relative expression was calculated based on tubulin. Significant increase in expression was particularly found in HlFER1. Data represent the means of three independent trials 6 SE. Statistical significance (P,0.05) was determined using the Mann-Whitney test. doi:10.1371/journal.pone.0090661.g002 Figure 2. Protein expression of H. longicornis ferritins in whole, unfed ticks at different hours after injection of different concentrations of FAC. Sterilized high-purity water was injected into the control group (0 mM). (A) Western blot analysis after incubation with specific anti-sera against H. longicornis FER1 (HlFER1) or H. longicornis FER2 (HlFER2). Hlfer-silenced ticks had low survival rate after FAC injection Tubulin was used as an internal control. (B) Band densitometry analysis for HlFER1 and HlFER2. The relative expression was calculated based on tubulin. Significant increase in expression was particularly found in HlFER1. Data represent the means of three independent trials 6 SE. Statistical significance (P,0.05) was determined using the Mann-Whitney test. doi:10.1371/journal.pone.0090661.g002 March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 4 Tick Ferritins against Oxidative Stress Figure 3. Protein expression of H. longicornis ferritins in midguts (A) and hemolymph (B) of unfed ticks injected with different concentrations of FAC. Midguts and hemolymph were collected at 24 h and 72 h after injection of 50 mM or 100 mM FAC or sterilized high purity water for the control group (0 mM). Hemocyte was separated from the hemolymph by centrifugation. Western blot analysis was performed using specific anti-sera against H. longicornis FER1 (HlFER1) or H. longicornis FER2 (HlFER2). Tubulin was used as an internal control. The relative expression of HlFER1 and HlFER2 was calculated based on tubulin after band densitometry analysis. Significant increase in expression was particularly found in HlFER1. Data represent the means of three independent trials 6 SE. Statistical significance (P,0.05) was determined using the Mann-Whitney test. doi:10.1371/journal.pone.0090661.g003 Tick Ferritins against Oxidative Stress Figure 3. Protein expression of H. longicornis ferritins in midguts (A) and hemolymph (B concentrations of FAC. Midguts and hemolymph were collected at 24 h and 72 h after injection of 5 water for the control group (0 mM). Hemocyte was separated from the hemolymph by centrifugation specific anti-sera against H. longicornis FER1 (HlFER1) or H. longicornis FER2 (HlFER2). Tubulin was used of HlFER1 and HlFER2 was calculated based on tubulin after band densitometry analysis. Significant in HlFER1. Data represent the means of three independent trials 6 SE. Statistical significance (P,0.05) w doi:10.1371/journal.pone.0090661.g003 Figure 3 Protein expression of H longicornis ferritins in midguts (A) and hemolymph (B) of unfed ticks injected with Figure 3. Protein expression of H. longicornis ferritins in midguts (A) and hemolymph (B) of unfed ticks injected with different concentrations of FAC. Midguts and hemolymph were collected at 24 h and 72 h after injection of 50 mM or 100 mM FAC or sterilized high purity water for the control group (0 mM). Hemocyte was separated from the hemolymph by centrifugation. Western blot analysis was performed using specific anti-sera against H. longicornis FER1 (HlFER1) or H. Hlfer-silenced ticks had low survival rate after FAC injection longicornis FER2 (HlFER2). Tubulin was used as an internal control. The relative expression of HlFER1 and HlFER2 was calculated based on tubulin after band densitometry analysis. Significant increase in expression was particularly found in HlFER1. Data represent the means of three independent trials 6 SE. Statistical significance (P,0.05) was determined using the Mann-Whitney test. doi:10.1371/journal.pone.0090661.g003 FAC injected into the hemocoel stimulated HlFER expression of digestive cells in the midgut and salivary glands were collected from normal unfed adult female ticks 72 h after injection of FAC or sterile high-purity water. Increased fluorescence was observed in digestive cells 72 h after injection of 50 mM and 100 mM FAC (Fig. 5). For HlFER1, extensive fluorescence was observed throughout the midgut, from the basal lamina up to the inner digestive cells lining the lumen. For HlFER2, much of the fluorescence was observed along the basal lamina. In contrast, very weak fluorescence for both HlFERs was observed in the salivary glands (Fig. S5). It is interesting that injection of FAC to the hemocoel stimulated HlFER expression in the midgut, as shown by Western blot analysis, and that the midgut can also store the iron from the hemolymph, as demonstrated by ferric iron staining on native PAGE. We wanted to know the extent of the effect of FAC on HlFER expression of digestive cells, therefore we performed IFAT. The salivary gland was also examined for comparison. Midguts March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 5 Tick Ferritins against Oxidative Stress Figure 4. Staining of HlFER for ferric iron on native PAGE gel after FAC injection. Ticks were injected with 50 or 100 mM FAC or sterilized high-purity water (0 mM) for the control group. Whole ticks were collected from 24 h to 96 h and total protein was extracted (A). Each lane of native PAGE gel was loaded with 20 mg of total protein and then the gel was stained with an equal volume of 10% K4[Fe(CN)6] and 10% HCl for up to 48 h. A single band stained for ferric iron. The high molecular weight marker (M), which contains ferritin from equine spleen, also stained for ferric iron, as well as the horse holoferritin (HF), used as positive control. Midguts, salivary glands, and the hemolymph (B) were also collected from ticks 72 h after injection of FAC or sterilized high-purity water. Each lane was loaded with 10 mg of protein. Western blot analysis using specific anti-HlFER sera was also performed to confirm that the band stained for ferric iron is HlFER. Arrows indicate approximately 440 kDa, which is the theoretical molecular weight of native ferritin. doi:10.1371/journal.pone.0090661.g004 Fi S i i f HlFER f f i i i PAGE l f FAC i j i Ti k Figure 4. Hlfer1-silenced ticks did not accumulate ferric iron after blood feeding or injection of FAC Iron is stored in ferritin as ferric iron. We hypothesized that ferric iron accumulation should be reduced in the Hlfer-silenced ticks after blood feeding or FAC injection. To evaluate this hypothesis, staining of ferric iron after native PAGE was performed. Protein concentration was adjusted based on the tubulin level. In whole ticks and midguts after blood feeding, as well as in whole ticks injected with FAC, Hlfer1-silenced ticks weakly stained for ferric iron (Fig. 9A). Interestingly, the Hlfer2- silenced group still showed strong staining. Immunodetection using specific antibodies against MDA (Fig. 6) and PC (Fig. 7) showed that Hlfer-silenced ticks have significantly higher (P,0.05) levels of these oxidative stress biomarkers than the control group after blood feeding or FAC injection. Band densitometry analysis was performed to calculate the relative MDA or PC content of the samples based on tubulin. Hlfer1- silenced ticks showed the highest levels of MDA and PC, including engorged whole ticks and midguts and unfed ticks injected with FAC. Hlfer-silenced ticks had higher levels of oxidative stress biomarkers after blood feeding or FAC injection Hlfer-silenced ticks had higher levels of oxidative stress biomarkers after blood feeding or FAC injection damage [3]. The results showed that lipid peroxidation was higher in both Hlfer-silenced groups as compared to the Luc-injected control group, either in whole ticks or in midguts (Fig. 8). The highest level of MDA was observed in Hlfer1- and Hlfer2-silenced groups in whole ticks and midguts, respectively. Iron is known to catalyze the formation of ROS in living cells, thus promoting oxidative stress. We previously found that Hlfer- silenced ticks had abnormal midgut morphology and high mortality after blood feeding and we hypothesized that this was caused by oxidative stress. Thus, we evaluated the oxidative status of Hlfer-silenced ticks after blood feeding or exposure to exogenous iron through demonstration of known oxidative stress biomarkers. We detected malondialdehyde (MDA), a known product of lipid peroxidation [3], and protein carbonyl (PC) resulting from the oxidation of proteins [26]. FAC injected into the hemocoel stimulated HlFER expression of digestive cells in the midgut Staining of HlFER for ferric iron on native PAGE gel after FAC injection. Ticks were injected with 50 or 100 mM FAC or sterilized high-purity water (0 mM) for the control group. Whole ticks were collected from 24 h to 96 h and total protein was extracted (A). Each lane of native PAGE gel was loaded with 20 mg of total protein and then the gel was stained with an equal volume of 10% K4[Fe(CN)6] and 10% HCl for up to 48 h. A single band stained for ferric iron. The high molecular weight marker (M), which contains ferritin from equine spleen, also stained for ferric iron, as well as the horse holoferritin (HF), used as positive control. Midguts, salivary glands, and the hemolymph (B) were also collected from ticks 72 h after injection of FAC or sterilized high-purity water. Each lane was loaded with 10 mg of protein. Western blot analysis using specific anti-HlFER sera was also performed to confirm that the band stained for ferric iron is HlFER. Arrows indicate approximately 440 kDa, which is the theoretical molecular weight of native ferritin. doi:10 1371/journal pone 0090661 g004 doi:10.1371/journal.pone.0090661.g004 Higher level of ferrous iron was detected in Hlfer2- silenced ticks injected with FAC In performing the ferrozine assay, the addition of ascorbic acid was omitted to avoid the reduction of ferric to ferrous iron. The ferrozine assay showed that Hlfer1-silenced ticks had only a slightly higher ferrous iron level, while Hlfer2-silenced ticks had a significantly higher (P,0.05) ferrous iron level 72 h after FAC injection than the control group (Fig. 9B). HlFER2 being abundant in the hemolymph, this result suggests that knockdown of Hlfer2 may have caused the accumulation of ferrous iron in the hemolymph of ticks. more rapid increase in mortality and a lower survival rate at the end of the observation period compared to both Hlfer1-silenced and Luc-injected control groups. The injection of FAC introduced high levels of free iron in the hemocoel. Only the secretory HlFER2 is present in the tick’s hemolymph. After the knockdown of Hlfer2, excessive ferrous iron, as we have demonstrated through the ferrozine assay, could have caused oxidative damage in the ticks that eventually lead to mortality. Conversely, the absence of HlFER1 after its knockdown could have led to high levels of intracellular ferrous iron. To confirm that the mortality after FAC injection in Hlfer- silenced ticks is related to ferritin function, we performed additional experiments after injecting FAC into normal unfed adult ticks, including RT-PCR, Western blotting and IFAT. In contrast to reports of ferritin up-regulation on mosquitoes following artificial feeding and in vitro exposure of cells to iron [21,22,23], and after injection of iron in Macrobrachium rosenbergii [24] and Bombus ignitus [25], the transcript level of either Hlfer did not change in response to iron injection. Meanwhile, Western blot analyses showed an increasing protein level in whole ticks, particularly of HlFER1, in a time-dependent manner after FAC injection. In agreement with our previous conclusion, these results demonstrated the translational regulation of HlFER1 through the binding of the iron-responsive element (IRE) to the iron-regulatory protein (IRP) [28]. Higher level of ferrous iron was detected in Hlfer2- silenced ticks injected with FAC We also hypothesized that in the absence of HlFER, ferrous iron cannot be stored as ferric iron and should accumulate. Thus, the ferrozine assay for measuring non-heme iron was performed to determine the amount of ferrous iron in whole ticks injected with The level of MDA in Hlfer-silenced ticks after blood feeding was further evaluated using the TBARS assay, the most common technique employed in studying lipid peroxidation and oxidative March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 6 Tick Ferritins against Oxidative Stress Figure 5. An indirect immunofluorescent antibody test (IFAT) in the midgut after injection of FAC. Different concentrations of FAC or sterilized high-purity water (0 mM) were injected to unfed adult ticks and midguts were collected after 72 h. To visualize the extent of the stimulation of HlFER expression caused by FAC injection, frozen sections of the midgut were incubated with specific mouse anti-HlFER1 or anti-HlFER2 sera. Mouse normal serum was used as a negative control. Anti-mouse IgG conjugated with Alexa 594 was used as secondary antibody and nuclei were visualized using DAPI. Arrowheads point to areas with increased fluorescence. (Bars = 20 mm). doi:10.1371/journal.pone.0090661.g005 Figure 5. An indirect immunofluorescent antibody test (IFAT) in the midgut after injection of FAC. Different concentrations of FAC or sterilized high-purity water (0 mM) were injected to unfed adult ticks and midguts were collected after 72 h. To visualize the extent of the stimulation of HlFER expression caused by FAC injection, frozen sections of the midgut were incubated with specific mouse anti-HlFER1 or anti-HlFER2 sera. Mouse normal serum was used as a negative control. Anti-mouse IgG conjugated with Alexa 594 was used as secondary antibody and nuclei were visualized using DAPI. Arrowheads point to areas with increased fluorescence. (Bars = 20 mm). doi:10.1371/journal.pone.0090661.g005 FAC after Hlfer knockdown [19,20]. In performing the ferrozine assay, the addition of ascorbic acid was omitted to avoid the reduction of ferric to ferrous iron. The ferrozine assay showed that Hlfer1-silenced ticks had only a slightly higher ferrous iron level, while Hlfer2-silenced ticks had a significantly higher (P,0.05) ferrous iron level 72 h after FAC injection than the control group (Fig. 9B). HlFER2 being abundant in the hemolymph, this result suggests that knockdown of Hlfer2 may have caused the accumulation of ferrous iron in the hemolymph of ticks. FAC after Hlfer knockdown [19,20]. Discussion Ticks are known for their ability to ingest large amounts of blood from their host, reaching more than a hundred times their unfed body weight. The numerous bioactive molecules in their saliva allow them to evade the host’s immune and hemostatic mechanisms, which is important for successful attachment and feeding [27]. However, they also must cope with potentially toxic molecules in the host blood, including iron. Ferritin is an iron- storage protein involved in iron homeostasis in most living organisms. The physiological importance of ferritin in blood feeding and reproduction of the hard ticks I. ricinus [12] and H. longicornis [13] has been demonstrated through RNAi; however, the specific role of tick ferritins has not been demonstrated. In this study, we showed that H. longicornis ferritins act as antioxidant molecules that minimize oxidative stress. Interestingly, Western blot analysis of different organs showed that FAC injection stimulated the expression of both HlFER1 and HlFER2 in the midgut but not in the salivary glands or ovary. IFAT also showed the extensive fluorescence of digestive cells for HlFER1 after FAC injection, extending from the basal lamina to the cells close to the lumen, whereas HlFER2 was strong particularly along the basal lamina in the midgut. In contrast, no fluorescence was found in the salivary glands. These results suggest that the iron in the hemolymph may cross the basal lamina of the midgut for storage in HlFER1 of digestive cells. In Aside from the effects of Hlfer silencing on blood feeding and reproduction, we also previously reported that Hlfer-silenced ticks had high mortality after blood feeding. We showed here that this mortality is related to the iron-storage function of ferritin. For the first time, we exposed the ticks to exogenous iron by injecting FAC into the hemocoel. The silencing of Hlfer alone (data not shown) or with injection of water in unfed adult female ticks did not result in any mortality; however, mortality increased with each day after FAC injection. The group injected with Hlfer2 dsRNA showed a March 2014 \| Volume 9 \| Issue 3 \| e90661 March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 7 Tick Ferritins against Oxidative Stress Figure 6. Detection of malondialdehyde (MDA) from Hlfer-silenced ticks after blood feeding or injection of FAC. Discussion Total protein was extracted from whole ticks (A) and midguts (B) after blood feeding and whole ticks 72 h after injection of 100 mM FAC (C). Western blot analysis was performed and the membrane was incubated with a specific anti-MDA antibody. Tubulin was used as internal control. The relative content of MDA (clearest band) to tubulin was calculated after band densitometry analysis. Both Hlfer1- and Hlfer2-silenced ticks had significantly higher MDA compared to the control (Luc) group. Data represent the means of three independent trials 6 SE. P,0.05, significantly different vs. Luc, Student’s t- test. doi:10.1371/journal.pone.0090661.g006 Figure 6. Detection of malondialdehyde (MDA) from Hlfer-silenced ticks after blood feeding or injection of FAC. Total protein was extracted from whole ticks (A) and midguts (B) after blood feeding and whole ticks 72 h after injection of 100 mM FAC (C). Western blot analysis was performed and the membrane was incubated with a specific anti-MDA antibody. Tubulin was used as internal control. The relative content of MDA (clearest band) to tubulin was calculated after band densitometry analysis. Both Hlfer1- and Hlfer2-silenced ticks had significantly higher MDA compared to the control (Luc) group. Data represent the means of three independent trials 6 SE. P,0.05, significantly different vs. Luc, Student’s t- test. Figure 6. Detection of malondialdehyde (MDA) from Hlfer-silenced ticks after blood feeding or injection of FAC. Total protein was extracted from whole ticks (A) and midguts (B) after blood feeding and whole ticks 72 h after injection of 100 mM FAC (C). Western blot analysis was performed and the membrane was incubated with a specific anti-MDA antibody. Tubulin was used as internal control. The relative content of MDA (clearest band) to tubulin was calculated after band densitometry analysis. Both Hlfer1- and Hlfer2-silenced ticks had significantly higher MDA compared to the control (Luc) group. Data represent the means of three independent trials 6 SE. P,0.05, significantly different vs. Luc, Student’s t- test. d i 10 1371/j l 0090661 006 doi:10.1371/journal.pone.0090661.g006 doi:10.1371/journal.pone.0090661.g006 mammals, circulating iron bound to transferrin can enter the basolateral membrane of enterocytes through transferrin receptor 1 [1]. However in the ticks, the function of transferrin in iron metabolism remains to be elucidated. Discussion Meanwhile, the increased fluorescence of HlFER2 along the basal lamina of the midgut after FAC injection may imply that iron in the hemolymph stimulated its expression with subsequent secretion, since the HlFER2 level in the hemolymph also increased after FAC injection. In mosquitoes, iron treatment resulted in an increase in the secretion of ferritin [22]. Here, since a high level of iron was present in the hemolymph, HlFER2 could have been secreted to sequester iron. Moreover, we previously concluded that HlFER2 is secreted from the midgut to remove iron and distribute it to other organs of the tick, in agreement with the model of iron metabolism in ticks proposed by Hajdusek et al. [12]. Presently, the other components of iron metabolism in ticks, as well as the regulatory signals in iron distribution, remain to be elucidated. Iron traffic during blood feeding in ticks must be systemically regulated, involving complex signal pathways. Whereas a series of signal pathways are known to be involved in iron traffic aside from the iron-binding proteins in mammals [29] and several proteins have already been identified in arthropods such as Drosophila melanogaster [30] and Anopheles gambiae [31], to function in iron absorption, these aspects require further investigations in the ticks. mammals, circulating iron bound to transferrin can enter the basolateral membrane of enterocytes through transferrin receptor 1 [1]. However in the ticks, the function of transferrin in iron metabolism remains to be elucidated. Meanwhile, the increased fluorescence of HlFER2 along the basal lamina of the midgut after FAC injection may imply that iron in the hemolymph stimulated its expression with subsequent secretion, since the HlFER2 level in the hemolymph also increased after FAC injection. In mosquitoes, iron treatment resulted in an increase in the secretion of ferritin [22]. Here, since a high level of iron was present in the hemolymph, HlFER2 could have been secreted to sequester iron. Moreover, we previously concluded that HlFER2 is secreted from the midgut to remove iron and distribute it to other organs of the tick, in agreement with the model of iron metabolism in ticks proposed by Hajdusek et al. [12]. Presently, the other components of iron metabolism in ticks, as well as the regulatory signals in iron distribution, remain to be elucidated. Iron traffic during blood feeding in ticks must be systemically regulated, involving complex signal pathways. Discussion Whereas a series of signal pathways are known to be involved in iron traffic aside from the iron-binding proteins in The Prussian blue staining for ferric iron in native HlFER after native PAGE was useful in the assessment of ferric iron accumulation. We found increased staining after FAC injection, which may reflect the increased HlFER level and iron uptake of HlFER molecules at these time points. The increased ferric iron accumulation, together with the increased levels of both HlFERs in the midgut we mentioned earlier, supports our previous conclusion that the midgut is the primary organ for iron metabolism, most likely being the first organ exposed to large amounts of iron during blood feeding. Interestingly, ferric iron staining was weakened after Hlfer1-silencing but not after the silencing of Hlfer2. We previously found that HlFER1 was still expressed after Hlfer2 silencing, particularly in the midgut [13]. Thus, the present result on ferric iron staining in Hlfer2-silenced ticks implicates HlFER1. March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 Tick Ferritins against Oxidative Stress Figure 7. Detection of protein carbonyl groups (PC) from Hlfer-silenced ticks after blood feeding or injection of FAC. Total protein was extracted from whole ticks (A) and midguts (B) after blood feeding and whole ticks 72 h after injection of 100 mM FAC (C). After the transfer of proteins to a membrane through Western blot, the membrane was incubated first with dinitrophenylhydrazine (DNPH) for pre-derivitazation of the carbonyl group, before incubation with a specific anti-DNP antibody. Tubulin was used as internal control. The relative content of PC (strongest band) to tubulin was calculated after band densitometry analysis. Both Hlfer1- and Hlfer2-silenced ticks had significantly higher PC levels compared to the control (Luc) group. Data represent the means of three independent trials 6 SE. P,0.05, significantly different vs. Luc, Student’s t-test. doi:10.1371/journal.pone.0090661.g007 Figure 7. Detection of protein carbonyl groups (PC) from Hlfer-silenced ticks after blood feeding or injection of FAC. Total protein was extracted from whole ticks (A) and midguts (B) after blood feeding and whole ticks 72 h after injection of 100 mM FAC (C). After the transfer of proteins to a membrane through Western blot, the membrane was incubated first with dinitrophenylhydrazine (DNPH) for pre-derivitazation of the carbonyl group, before incubation with a specific anti-DNP antibody. Tubulin was used as internal control. Tick Ferritins against Oxidative Stress Tick Ferritins against Oxidative Stress Figure 8. Thiobarbituric acid reactive species (TBARS) assay for Hlfer-silenced ticks after blood feeding. Whole bodies or midguts of ticks injected with Hlfer1, Hlfer2, or Luc dsRNA were collected after dropping from the host. Individual whole ticks or pooled midguts were weighed before being homogenized. After ultrasonication, the super- natants were obtained and boiled with TBARS reagent. Upon cooling and centrifugation, the absorbance of the supernatants was measured at OD532. The relative amount of MDA was calculated based on the sample weight and expressed as nmol/g. Both Hlfer1- and Hlfer2- silenced ticks had higher MDA levels in both whole ticks or midguts than the control (Luc) ticks. Values are means of 30 samples for each group 6 SE. P,0.05, significantly different vs. control, Student’s t-test. doi:10.1371/journal.pone.0090661.g008 transition metals including iron, ROS, products of lipid peroxi- dation including MDA, and glycoxidation. Protein carbonyls cannot be repaired and accumulation may lead to cell death [26]. Our present results also show that the midguts from Hlfer1-silenced ticks had the highest level of either oxidative stress biomarker, which corresponds to our previous observation of more severe abnormalities in the digestive cells of Hlfer1-silenced ticks. Moreover, we also previously reported a decrease in hematin production after Hlfer1 silencing, indicative of impaired digestive activity. Oxidative stress can also alter physiological processes including heme detoxification in the midgut [37]. Figure 8. Thiobarbituric acid reactive species (TBARS) assay for Hlfer-silenced ticks after blood feeding. Whole bodies or midguts of ticks injected with Hlfer1, Hlfer2, or Luc dsRNA were collected after dropping from the host. Individual whole ticks or pooled midguts were weighed before being homogenized. After ultrasonication, the super- natants were obtained and boiled with TBARS reagent. Upon cooling and centrifugation, the absorbance of the supernatants was measured at OD532. The relative amount of MDA was calculated based on the sample weight and expressed as nmol/g. Both Hlfer1- and Hlfer2- silenced ticks had higher MDA levels in both whole ticks or midguts than the control (Luc) ticks. Values are means of 30 samples for each group 6 SE. P,0.05, significantly different vs. control, Student’s t-test. doi:10.1371/journal.pone.0090661.g008 Several antioxidant enzymes that counteract reactive oxygen species, such as superoxide dismutase, glutathione S-transferase and thioredoxin, have been identified in hard ticks [38,39]. These enzymes prevent oxidative stress by keeping the level of free radicals to a minimum. Tick Ferritins against Oxidative Stress In the hard tick Dermacentor variabilis, these antioxidant enzymes were found in the midgut at day 6 of blood feeding, corresponding to the rapid feeding stage [38]. In this study, we demonstrated that, by sequestering ferrous iron and keeping it in the oxidized ferric form, ferritin is also an important antioxidant molecule in the hard tick because it prevents oxidative stress. In summary, the silencing of two ferritin genes in the hard tick H. longicornis resulted to increased levels of oxidative stress biomarkers after a blood meal or injection of iron. Our results provide evidence for the first time that two kinds of ferritin act as antioxidant molecules in a hard tick that prevent oxidative stress during blood feeding, thus ensuring tick survival. This paper provides a clearer explanation on the crucial importance of ferritin in the ticks that we reported in our previous paper on H. longicornis [13], and also the other work in another hard tick, I. ricinus [12]. Moreover, our iron-injection experiment, which to our knowledge is employed for the first time in ticks, demonstrated that iron in the hemocoel can stimulate HlFER expression of the midgut and that carbonyl. Lipid peroxidation leads to alterations of biological membranes and gives rise to several products that are known to induce diverse biological effects [35]. MDA, which is one of the most known and most studied products of lipid peroxidation, is highly toxic and can interact with DNA and proteins and thus can impair physiological functions [36]. Aside from the direct injury caused by reactive oxygen species, products of lipid peroxidation including MDA can promote further injury. Protein carbonylation is another hallmark of oxidative stress resulting from irreversible oxidative modification of proteins that can be induced by carbonyl. Lipid peroxidation leads to alterations of biological membranes and gives rise to several products that are known to induce diverse biological effects [35]. MDA, which is one of the most known and most studied products of lipid peroxidation, is highly toxic and can interact with DNA and proteins and thus can impair physiological functions [36]. Aside from the direct injury caused by reactive oxygen species, products of lipid peroxidation including MDA can promote further injury. Protein carbonylation is another hallmark of oxidative stress resulting from irreversible oxidative modification of proteins that can be induced by Figure 9. Evaluation of iron accumulation in Hlfer-silenced ticks. Discussion The relative content of PC (strongest band) to tubulin was calculated after band densitometry analysis. Both Hlfer1- and Hlfer2-silenced ticks had significantly higher PC levels compared to the control (Luc) group. Data represent the means of three independent trials 6 SE. P,0.05, significantly different vs. Luc, Student’s t-test. doi:10.1371/journal.pone.0090661.g007 Iron is known to promote the formation of reactive oxygen species that can result in damage to macromolecules, including DNA, proteins and lipids—the condition collectively termed oxidative stress [2]. Iron was particularly reported to induce lipid peroxidation [32,33] and oxidation of several amino acid residues in proteins [26]. Thus, the function of ferritin as a repository for excess iron is crucial to preventing oxidative damage. Here we showed that the knockdown of either Hlfer resulted in oxidative stress in ticks exposed to high levels of iron, either from blood meal or FAC injection. Similar to our previous study, Hlfer-silenced ticks infested on rabbits failed to engorge, weighing less than half of the Luc-injected ticks’ engorged body weight, meaning they ingested a lower amount of blood. Oxidative stress was confirmed by the detection of malondialdehyde and protein carbonyl, which are products of lipid peroxidation and protein oxidation, respectively, and observation of higher levels in Hlfer1- and Hlfer2-silenced ticks than in Luc-injected ticks after blood feeding or injection of FAC. The TBARS assay was also employed to assess lipid peroxidation after blood feeding and similarly, it showed that Hlfer-silenced ticks had a higher degree of lipid peroxidation compared to the control. We also attempted to perform TBARS assay on unfed Hlfer- silenced ticks injected with FAC but due to the low sensitivity of this test [34], we were unable to detect the presence of MDA on the samples. Taken together, these results imply that without HlFER1 or HlFER2, free iron predisposed the ticks to oxidative stress that led to death. We previously found abnormalities in the digestive cell morphology in Hlfer-silenced ticks during blood feeding, including altered shape, disrupted microvilli and cell membrane and vacuolated cytoplasm [13]. We hypothesized that these abnor- malities resulted from oxidative damage. Here we show that the midgut of Hlfer-silenced ticks had high levels of MDA and protein March 2014 \| Volume 9 \| Issue 3 \| e90661 March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 9 Tick Ferritins against Oxidative Stress Ferric iron accumulation was evaluated by staining HlFER on native PAGE (A). Total protein was extracted from whole bodies and midguts after blood feeding and whole bodies 72 h after FAC injection. The amount of protein was adjusted based on the tubulin profile after Western blotting. Weak staining was observed in Hlfer1-silenced ticks. Ferrozine assay for ferrous iron 72 h after injection of 100 mM FAC to unfed Hlfer-silenced ticks. (B). Ten ticks from each group were homogenized and total protein concentration was measured. Ferrous iron was extracted with concentrated HCl and then detected using ferrozine. Absorbance was measured at OD550. Relative ferrous iron content was calculated based on protein concentration. Hlfer2-silenced ticks had significantly higher ferrous iron content than the control (Luc) group. P,0.05, significantly different vs. Luc, Student’s t-test. doi:10.1371/journal.pone.0090661.g009 Figure 9. Evaluation of iron accumulation in Hlfer-silenced ticks. Ferric iron accumulation was evaluated by staining HlFER on native PAGE (A). Total protein was extracted from whole bodies and midguts after blood feeding and whole bodies 72 h after FAC injection. The amount of protein was adjusted based on the tubulin profile after Western blotting. Weak staining was observed in Hlfer1-silenced ticks. Ferrozine assay for ferrous iron 72 h after injection of 100 mM FAC to unfed Hlfer-silenced ticks. (B). Ten ticks from each group were homogenized and total protein concentration was measured. Ferrous iron was extracted with concentrated HCl and then detected using ferrozine. Absorbance was measured at OD550. Relative ferrous iron content was calculated based on protein concentration. Hlfer2-silenced ticks had significantly higher ferrous iron content than the control (Luc) group. P,0.05, significantly different vs. Luc, Student’s t-test. doi:10.1371/journal.pone.0090661.g009 March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 10 Tick Ferritins against Oxidative Stress primary antibodies against H. longicornis FER1 (HlFER1) or H. longicornis FER2 (HlFER2). Tubulin was used as an internal control. No significant difference was observed among groups. (TIF) iron molecules can be apparently transported from the hemo- lymph to digestive cells. However, further experiments are needed to elucidate this aspect of iron transport mechanism in ticks. Moreover, the iron-sequestration function of ferritin is implicated in immune response in many organisms [6]; thus, we are interested in the possible role of HlFERs in the tick immunity. References 1. Wang J, Pantopoulos K (2011) Regulation of cellular iron metabolism. Biochem J 434: 365–281. 14. Fujisaki K (1978) Development of acquired resistance and precipitating antibody in rabbits experimentally infested with females of Haemaphysalis longicornis (Ixodoidea: Ixodidae). Natl Inst Anim Health Q 18: 27–38. 2. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M (2006) Free radicals, metals and antioxidants in oxidative-stress induced cancer. Chem Biol Interact 160: 1–40. Q 15. Aung KM, Boldbaatar D, Liao M, Umemiya-Shirafuji R, Nakao S, et al. (2011) Identification and characterization of class B scavenger receptor CD36 from the hard tick, Haemaphysalis longicornis. Parasitol Res 108: 273–285. 3. Niki E (2013) Biomarkers of lipid peroxidation in clinical material. Biochim Biophys Acta. 16. Umemiya-Shirafuji R, Tanaka T, Boldbaatar D, Tanaka T, Fujisaki K (2012) Akt is an essential player in regulating cell/organ growth at the adult stage in the hard tick Haemaphysalis longicornis. Insect Biochem Mol Biol 42: 164–173. 4. Arosio P, Ingrassia R, Cavadini P (2009) Ferritins: A family of molecules for iron storage, antioxidation and more. Biochim Biophys Acta 1790: 589–599. p y g 17. Tang X, Zhou B (2013) Ferritin is the key to dietary iron absor iron detoxification in Drosophila melanogaster. FASEB J 27: 1–11. 17. Tang X, Zhou B (2013) Ferritin is the key to dietary iron absorp 5. Pham DQD, Winzerling JJ (2010) Insect ferritins: typical or atypical. Biochim Biophys Acta 1800: 824–833. iron detoxification in Drosophila melanogaster. FASEB J 27: 1–11 18. Chauhan A, Chauhan V, Brown WT, Cohen I (2004) Oxidative stress in autism: Increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin - the antioxidant proteins. Life Sciences 75. 6. Ong ST, Ho JZS, Ho B, Ding JL (2006) Iron-witholding strategy in innate immunity. Immunobiology 211: 295–314. 7. Orino K, Lehman L, Tsuji Y, Ayaki H, Torti SV, et al. (2001) Ferritin and the response to oxidative stress. Biochem J 357: 241–247. 19. Missirlis F, Holmberg S, Georgieva T, Dunkov BC, Rouault TA, et al. (2006) Characterization of mitochondrial ferritin in Drosophila. Proc Natl Acad Sci U S A 103: 5893–5898. 8. Mans BJ, Neitz AWH (2004) Adaptations of ticks to a blood-feeding environment: evolution from a functional perspective. Insect Biochem Mol Biol 34: 1–17. 20. Lang M, Braun CL, Kanost MR, Gorman MJ (2012) Multicopper oxidase-1 is a ferroxidase essential for iron homeostasis in Drosophila melanogaster. Supporting Information Figure S1 Survival rate of Hlfer-silenced ticks after injection of sterilized high-purity water. Four days after injection of Hlfer1, Hlfer2, or Luciferase dsRNA, sterilized high- purity was injected, and mortality was monitored. Low mortality was observed from all the three groups. n = 25 ticks per group. Bars represent standard error. Figure S1 Survival rate of Hlfer-silenced ticks after injection of sterilized high-purity water. Four days after injection of Hlfer1, Hlfer2, or Luciferase dsRNA, sterilized high- purity was injected, and mortality was monitored. Low mortality was observed from all the three groups. n = 25 ticks per group. Bars represent standard error. (TIF) Figure S5 An IFAT examination of salivary glands 72 h after injection of different concentrations of FAC compared to control group injected with sterilized high-purity water. Frozen sections of the salivary glands were incubated with specific mouse anti-HlFER1 or anti-HlFER2 sera. Normal mouse serum was used as negative control. Anti-mouse IgG conjugated with Alexa 594 was used as secondary antibody and nuclei were visualized using DAPI. No fluorescence was observed among groups. (Bars = 20 mm). (TIF) Figure S2 Transcription profile of H. longicornis adult ticks injected with FAC. Unfed adult ticks were injected with 50 mM or 100 mM FAC. Sterilized high-purity water was injected into the control group (0 mM). Total RNA was extracted from whole ticks at 24 h and 72 h after injection and RT-PCR analysis was performed using specific primers for Hlfer1 and Hlfer2. cDNA was adjusted based on control amplification for Hlactin. No significant difference was observed among groups. (TIF) Figure S2 Transcription profile of H. longicornis adult ticks injected with FAC. Unfed adult ticks were injected with 50 mM or 100 mM FAC. Sterilized high-purity water was injected into the control group (0 mM). Total RNA was extracted from whole ticks at 24 h and 72 h after injection and RT-PCR analysis was performed using specific primers for Hlfer1 and Hlfer2. cDNA was adjusted based on control amplification for Hlactin. No significant difference was observed among groups. (TIF) Figure S3 Protein expression of H. longicornis ferritins in salivary glands of unfed ticks injected with different concentrations of FAC. Salivary glands were collected from ticks at 24 h and 72 h after injection of 50 mM or 100 mM FAC. Sterilized high-purity water was injected into the control group (0 mM). Western blot analysis was performed using specific Author Contributions Conceived and designed the experiments: RLG TT. Performed the experiments: RLG ETB. Analyzed the data: RLG RUS ETB HM KK JK NT MM KF TT. Contributed reagents/materials/analysis tools: RUS ETB JK. Wrote the paper: RLG. Conceived and designed the experiments: RLG TT. Performed the experiments: RLG ETB. Analyzed the data: RLG RUS ETB HM KK JK NT MM KF TT. Contributed reagents/materials/analysis tools: RUS ETB JK. Wrote the paper: RLG. Tick Ferritins against Oxidative Stress Together with our previous results, our present study shows that ferritin is an important protective antigen of ticks that can be utilized to design a control strategy. Figure S4 Coomassie blue staining and Western blot analysis after native PAGE. To further confirm that the bands stained for ferric iron from tick protein samples were HlFER, we performed Coomassie blue staining and Western blot analysis using specific anti-HlFER sera. (A) Coomassie blue staining showed all the bands of high molecular weight marker (M), the commercially prepared horse holoferritin (HF) and the tick protein (T). The weak band of approximately 440 kDa in the tick protein sample was presumed to be ferritin. Western blot analyses for HlFER1 (B) and HlFER2 (C) showed a single band of approximately 440 kDa. Arrow indicates the 440 kDa band in the high molecular marker while arrowheads point to tick ferritin. (TIF) References Proc Natl Acad Sci U S A 109: 13337–13342. 9. Maya-Monteiro CM, Daffre S, Logullo C, Lara FA, Alves EW, et al. (2000) HeLp, a heme lipoprotein from the hemolymph of the cattle tick, Boophilus microplus. J Biol Chem 275: 36584–36589. 21. Dunkov BC, Georgieva T, Yoshiga T, Hall M, Law JH (2002) Aedes aegypti ferritin heavy chain homologue: feeding of iron or blood influences message levels, lengths and subunit abundance. J Insect Sci 2: 7. 10. Lara FA, Lins U, Bechara GH, Oliveira PL (2005) Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus. J Exp Biol 208: 3093–3101. 22. Geiser DL, Zhang D, Winzerling JJ (2006) Secreted ferritin: Mosquito defense against iron overload? Insect Biochem Mol Biol 36: 177–187. 11. Lara FA, Lins U, Paiva-Silva G, Almeida IC, Braga CM, et al. (2003) A new intracellular pathway of haem detoxification in the midgut of the cattle tick Boophilus microplus: aggregation inside a specialized organelle, the hemosome. J Exp Biol 206: 1707–1715. 23. Pham DQD, Winzerling JJ, Dodson MS, Law JH (1999) Transcriptional control is relevant in the modulation of mosquito ferritin synthesis by iron. Eur J Biochem 266: 236–240. 24. Qiu GF, Zheng L, Liu P (2008) Transcriptional regulation of ferritin mRNA levels by iron in the freshwater giant prawn, Macrobrachium rosenbergii. Comp Biochem Physiol B 150: 320–325. 12. Hajdusek O, Sojka D, Kopacek P, Buresova V, Franta Z, et al. (2009) Knockdown of proteins involved in iron metabolism limits tick reproduction and development. Proc Natl Acad Sci U S A 106: 1033–1038. development. Proc Natl Acad Sci U S A 106: 1033–1038. 25. Wang D, Kim BY, Lee KS, Yoon HJ, Cui Z, et al. (2009) Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignitus. Comp Biochem Physiol B 2009: 20– 27. 13. Galay RL, Aung KM, Umemiya-Shirafuji R, Maeda H, Matsuo T, et al. (2013) Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis. J Exp Biol 216: 1905–1915. March 2014 \| Volume 9 \| Issue 3 \| e90661 March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org 11 Tick Ferritins against Oxidative Stress Tick Ferritins against Oxidative Stress Tick Ferritins against Oxidative Stress 26. Fedorova M, Bollineni RC, Hoffman R (2013) Protein carbonylation as a major hallmark of oxidative damage: Update of analytical strategies. Mass Spectrom Rev. 34. Grotto D, Santa Maria L, Valetini J, Paniz C, Schmitt G, et al. (2009) Importance of the lipid peroxidation biomarkers and methodological aspects for malondialdehyde quantification. Quim Nova 32: 169–174. 27. Francischetti IMB, Sa-Nunes A, Mans BJ, Santos IM, Ribeiro JMC (2010) The role of saliva in tick feeding. Front Biosci 14: 2051–2088. 35. Niki E (2009) Lipid peroxidation: physiological levels and dual biological effects. Free Radic Biol Med 47: 469–484. g 28. Anderson CP, Shen M, Eisenstein RS, Leibold EA (2012) Mammalian iron metabolism and its control by iron regulatory proteins. Biochim Biophys Acta 1823: 1468–1483. 36. Del Rio D, Stewart AJ, Pellegrini N (2005) A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis 15: 316–328. 29. Gkouvatsos K, Papanikolaou G, Pantopoulos K (2012) Regulation of iron transport and the role of transferrin. Biochim Biophys Acta 1820: 188–202. 37. Citelli M, Lara FA, Vaz IdJ, Oliveira PL (2007) Oxidative stress impairs heme detoxification in the midgut of the cattle tick, Rhipicephalus (Boophilus) microplus. Mol Biochem Parasitol 151: 81–88. 30. Mandilaras K, Pathmanathan T, Missirlis F (2013) Iron absorption in Drosophila melanogaster. Nutrients 5: 1622–1647. 38. Anderson JM, Sonenshine DE, Valenzuela JG (2008) Exploring the mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae). BMC Genomics 9: 552. 31. Winzerling J, Pham DQD (2006) Iron metabolism in insect disease vectors: Mining the Anopheles gambiae translated protein database. Insect Biochem Mol Biol 36: 310–321. 39. Ibrahim MA, Mohamed MM, Ghazy AM, Masoud HMM (2013) Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii. Comp Biochem Physiol B 164: 221–228. 32. Braughler JM, Duncan LA, Chase RL (1986) The involvement of iron in lipid peroxidation. J Biol Chem 261: 10282–10289. 32. Braughler JM, Duncan LA, Chase RL (1986) T 33. Fuhrman B, Oiknine J, Aviram M (1994) Iron induces lipid peroxidation in cultured macrophages, increases their ability to oxidatively modify LDL, and affects their secretory properties. Atherosclerosis 111: 65–78. March 2014 \| Volume 9 \| Issue 3 \| e90661 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 12
https://openalex.org/W2884358097	https://europepmc.org/articles/pmc6145240?pdf=render	English	null	Scavenging effect of pasipay (<i>passiflora incarnate</i> L.) on singlet oxygen generation and fatty acid photooxygenation	Food science & nutrition	2,018	cc-by	4,370	Received: 23 March 2018 \| Revised: 3 June 2018 \| Accepted: 13 June 2018 Received: 23 March 2018 \| Revised: 3 June 2018 \| Accepted: 13 June 2018 Received: 23 March 2018 \| Revised: 3 June 2018 \| Accepted: 13 June 2018 DOI: 10.1002/fsn3.731 DOI: 10.1002/fsn3.731 K E Y W O R D S antioxidant, fatty acid, Pasipay incarnate L, reactive oxygen species, singlet oxygen antioxidant, fatty acid, Pasipay incarnate L, reactive oxygen species, singlet oxygen © 2018 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1670 \| www.foodscience-nutrition.com the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, y cited Abstract Correspondence Mahdi Hajimohammadi, Faculty of Chemistry, Kharazmi University, G. C, Mofateh, Tehran, 14911-15719, Iran. Email: hajimohammadi@khu.ac.ir Funding information Kharazmi University Scavenging effect of pasipay (passiflora incarnate L.) on singlet oxygen generation and fatty acid photooxygenation Mahdi Hajimohammadi \| Parisa Nosrati Mahdi Hajimohammadi \| Parisa Nosrati Faculty of Chemistry, Kharazmi University, Tehran, Iran Abstract Anthracene as a chemical probe is usually used to trap the singlet oxygen and then detection and quantification can be based on absorbance. In this study, oxidation of anthracene declared that rate of singlet oxygen quenching in the presence of pasipay (passiflora incarnate L.) as a natural antioxidant, 1,4 Diazabicyclo [2.2.2] octane (DABCO) as a well-known singlet oxygen scavenger and highly effective synthetic antioxidants in food industry such as Butylated hydroxytoluene (BHT), Butylated hydroxyanisole (BHA), tert-Butylhydroquinone (TBHQ) decreased in the order of DABCO >pasi- pay > TBHQ > BHT > BHA. On the other hand, lipid photooxidation is the undesirable chemical process in which singlet oxygen result in the peroxidation of fatty acids. The results of this study also showed that oleic acid oxidation with singlet oxygen in the presence of pasipay (contains 0.4576 mg flavonoid compounds) diminished about 11% which shows pasipay has an effective role to inhibit lipid peroxidation. inhabitants rely on traditional medicine for their primary health care needs and most of this therapy involves the use of plant ex- tracts or their active phenolic components (Bruneton, 1995) which have an efficient antioxidant capacity. Pasipay is the largest and most important genus of the family Pasipayceae, comprising about 500 species, distributed mostly in warm temperate and tropical regions (Dhawan, Dhawan, & Sharma, 2004) Previous studies have described the presence of flavonoids as the major constituents of pasipay (Dhawan, Dhawan, & Sharma, 2004). There are few stud- ies on the efficacy of natural antioxidant as O2 (1Δg) quenchers and their roles in the prevention of lipid oxidation (Niki, 2015; Terao, Minami, & Bando, 2010) because scavenging of DPPH free radical is the basis of a common antioxidant assay and most often an overall antioxidant effect was measured. However, singlet oxy- gen has not radical nature. (Ruiz-González et al., 2017) This project was designed to characterize the antioxidant potential of passiflora incarnate L. as a natural antioxidant in compare with well-known singlet oxygen scavenger such as DABCO and the highly effective antioxidants such as BHA, BHT, and TBHQ. flavonoid) separately, were added to anthracene (4 × 10−4 M) and MB (1 × 10−4 M). Continuous irradiation of samples was carried out using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) for 5 min at room temperature under 1 atm of bubbling of air in the solution at room temperature. Determination of products was recorded on a Shimadzu 2100 spectrophotometer at 375 nm. 2.1 \| Material In all analyses, three replicates were applied and analysis of the re- sults was achieved using SAS software, version 3.9 and then average the results were compared using Duncan test. Also, with Excel soft- ware diagrams were drawn. Hydroalcoholic extract of passiflora incarnate L. was obtained from Iran darouk Co. Anthracene, oleic acid, acetonitrile, methylene blue (MB), DABCO, BHT, BHA, TBHQ were purchased from Fluka and Merck and used without further purification. Tetraphenyl porphyrin (H2TPP), ZnTPP, and MgTPP were synthesized according to the lit- eratures (Lindsey & Wagner, 1989). 2.2.2 \| Sample preparation for photooxygenation of fatty acid with singlet oxygen 0.0016 mmol antioxidant 7 ml acetonitrile, (DABCO, BHT, BHA, TBHQ and pasipay (contains 0.4576 mg flavonoid) separately, were added to oleic acid (4.6 × 10−3 M) and H2TPP (1 × 10−3 M). Continuous irradiation of samples was carried out using solar sim- ulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) for 120 min at room temperature under 1 atm of bubbling of air in the solution. Determination of products and were analyzed on a Bruker AMX 300 MHz spectrometer using TMS as internal standard and iodometric titration method. Peroxide value (PV) (meq O2/kg) of the samples was determined according to the literature (Barthel & Grosch, 1974). 1 \| INTRODUCTION and induces the synthesis of matrix metalloproteinases (Kligman, 1989). The use of antioxidants to protect human skin from the harmful effects of UV radiation is a topic that has attracted grow- ing interest in recent years within the world of photoprotection research (Martinez et al., 2017). DABCO recognized as a very effi- cient quencher of singlet oxygen in the organic media (Lengfelder, Cadenas, & Sies, 1983) and synthetic antioxidants such as TBHQ, BHA, and BHT have been found to have a strong singlet oxygen quenching ability (Lee & Jung, 2010). Recent research has focused on isolation and characterization of effective natural antioxidants (Fang et al., 2017; Nimse & Pal, 2015). Natural antioxidants act (a) as reducing agents, (b) as free radical scavengers, and (c) as quenchers of the formation of singlet oxygen. They can be used in the food industry and there is evidence that they may exert their antioxidant effects within the human body (Hamid, Aiyelaagbe, Usman, Ameen, & Lawal, 2010; Koski et al., 2002). People receive antioxidant supplements directly from fresh fruits and vegetables. The World Health Organization estimated that <80% of the earth’s 1 Oxygen molecule in its ground state has two unpaired electrons and when oxygen molecule has excess energy, these unpaired elec- trons in the external orbital can be pair and generate singlet oxy- gen (Min & Boff, 2002). One of the physical methods for producing singlet oxygen is photosensitization reaction. Great photosensitiz- ers have received attention, due in part to their direct relevance to many biological systems. Electrophilic tendency of singlet ox- ygen causes lipids, amino acids, nucleic acids, and electron rich molecular can be its target (Korytowski, Schmitt, & Girotti, 2010). Singlet oxygen can be easily produced in food systems under light illumination, especially in the presence of photosensitizers such as riboflavin and chlorophylls (Greer, 2006). Lipids also can be a target of singlet oxygen due to the electrophilic inherent, this re- active species attacked to unsaturated fatty acid and produce lipid hydroperoxides as primary products (Girotti, 1998). On the other hand UV radiation causes DNA damage and protein oxidation Food Sci Nutr. 2018;6:1670–1675. HAJIMOHAMMADI and NOSRATI 1671 3.3 \| Effect of pasipay on fatty acid photooxgenation Singlet oxygen generation (Type II) and its reaction with the substrates is the foremost mechanism that occurs in our cir- cumstances, since conversions of oleic acid obey the order of H2TPP > ZnTPPCl > MgTPPCl (Table 1 entry 3, 4, and 5). Paramagnetic metals are claimed to quench singlet oxygen by en- ergy transfer mechanism from oxygen to the low-lying d orbital electron levels and have very short triplet lifetimes (Table 1, entry 4) (Bonnett & Martınez, 2001) also diamagnetic metals quench sin- glet oxygen by a charge transfer mechanism (Bonnett & Martınez, 2001) (Table 1, entry 5). In addition, in the presence of DABCO, which is a well-known singlet oxygen scavenger (Lengfelder et al., 1983) (Table 1, entry 6) photooxidation of oleic acid was inhibited. According to the literature, singlet oxygen lifetime in dimethyl sulf- oxide (DMSO) solvent is 19 μs, in acetonitrile (CH3CN) solvent is 65 μs, and in ethanol solvent is 38 μs which was corresponded with the results in Table 1 (entry 3,7, and 8) (Bressan & Morvillo, 1989; Chen et al., 2001; Toffoli, Gomes, Junior, & Courrol, 2008). Table 1 entry 3, 7, and 8 indicates that conversion of oleic acid in acetonitrile as solvent is higher than ethanol and DMSO that correlated with sin- glet oxygen lifetimes in this solvents. For investigation of the type I mechanism, we performed our reaction in the presence superoxide The photosensitized production of singlet oxygen has signifi- cance in the areas of the photooxidation of organic compounds (Hajimohammadi, Schwarzinger, & Knör, 2012) and food chem- istry [1, 2, 4]. Photooxygenation of oleic acid as a one of the targets of singlet oxygen was investigated to evaluate the an- tioxidant effect of pasipay. Figure 5 shows the conversion of oleic acid to the peroxide products in an oxygenated solution of acetonitrile and H2TPP photosensenisitizer under visible light in the presence of pasipay, well-known singlet oxygen (DABCO) and highly effective antioxidants such as BHT, BHA and TBHQ. After 120 min irradiation, the rate of oleic acid oxidation by 1O2 as a very reactive ROS reduced 11.6% in the presence of pasipay (contains 0.4576 mg flavonoid compounds) that shows pasipay can be used as an effective additive to fatty acids for the pres- ervation of them. TA B LE 1 PV of oleic acid oxidation with singlet oxygen in different conditiona anion radical (O2 •–). In the presence of O2 •–, the rates of oxidation reaction significantly decreased (Table 1 entry 9). TA B LE 1 PV of oleic acid oxidation with singlet oxygen in different conditiona B LE 1 PV of oleic acid oxidation with singlet oxygen Entry Condition PV 1 Oleic acid + CH3CN + light + air Trace 2 Oleic acid + CH3CN + air + H2TPP Trace 3 Oleic acid + CH3CN + air + H2TPP + ight 101.67 4 Oleic acid + CH3CN + light + air + ZnTPP 79.33 5 Oleic acid + CH3CN + light + air + MgTPP 70.39 6 Oleic acid + CH3CN + H2TPP + light + DABC O Trace 7 Oleic acid + H2TPP + light + air + DMSO 27.93 8b Oleic acid + H2TPP + light + air + Ethanol 89.38 9 Oleic acid + O− 2 Trace a4.6⨯10−3 mol oleic acid, 5 ml solvent, 1 ml (0.001 M) sensitizer, air (1 atm) and 288 power LED lamps, 1 W, 2.3 V (59660 LUX). bO− 2 was pre- pared by dissolving K2O in dried DMSO. 3.2 \| Effect of pasipay on Anthracene photooxygenation Spectrophotometry is a more convenient option for detection of excited oxygen molecules. A chemical probe is usually used to trap the singlet oxygen and then detection and quantification can be based on absorbance. A very characteristic reaction of singlet oxygen is the [4 + 2] cycloaddition to conjugated cyclic dienes and polycyclic aromatic hydrocarbons such as anthracene (Aubry, Pierlot, Rigaudy, & Schmidt, 2003). Anthracene traps re- versibly singlet oxygen. Singlet oxygen generation by MB is evi- denced by chemical trapping of 1O2 with anthracene. The UV–Vis spectra of anthracene as the function of time irradiation by using of MB as a photosensitizer are displayed in Figure 4a. A reduc- tion of the emission intensity absorption band of anthracene (λmax = 375 nm) was observed with the increase in irradiation time. This response is a consequence of the anthracene-9,10- endoperoxide formation (see Figure 4). During the reaction, the addition of well-known singlet oxygen scavenger such as DABCO and highly effective synthetic antioxidants in food in- dustry in foods such as BHT, BHA, TBHQ, and pasipay inhibited the oxidation of anthracene in the order of DABCO > pasi- pay > TBHQ > BHT > BHA (Figure 4a,b). Moreover, the oxidation reaction did not occur under dark conditions. This confirms that the anthracene oxidation occurs by singlet oxygen under visible irradiation. These results show pasipay act as a very effective deterrent on singlet oxygen generation. a4.6⨯10−3 mol oleic acid, 5 ml solvent, 1 ml (0.001 M) sensitizer, air (1 atm) and 288 power LED lamps, 1 W, 2.3 V (59660 LUX). bO− 2 was pre- pared by dissolving K2O in dried DMSO. which has worked few studies on it (Terao, Minami, & Bando, 2010). Photooxygenation of oleic acid with H2TPP as a photosensitizers was investigated as a typical standard sample to evaluate singlet oxygen production (Figure 1). It is important to note that 1H NMR spectroscopy and iodo- metric method revealed oxidation of oleic acid to peroxide product stopped in the absence of photosensitizers (Figure 2 and Table 1 entry 1) or when the irradiation was interrupted (Table 1 entry 2). Accordingly, the presence of a H2TPP, light and O2 are essential for the conversion oleic acid to corresponding products (Table 1 entry 3). According to the literature, there are two major pathways for photooxygenation reactions in the presence of nonmetal photosen- sitizers, Type I and Type II (Figure 3a) (Laing, 1989). 2.2.1 \| Sample preparation for anthracene oxidation with singlet oxygen In this work, the oxidative alterations of oleic acid as a result of oxi- dation with singlet oxygen were analyzed in the presence and ab- sence of pasipay as a source of natural phenolic compounds. Our target was fatty acid oxidation with singlet oxygen as a noble species In a typical experiment, 15 ml acetonitrile, 0.002 mmol antioxi- dant (DABCO, BHT, BHA, TBHQ, and pasipay (contains 0.5731 mg FI G U R E 1 Oleic acid photooxygenation in the presence and absence of Pasipay (a). Structure of different applied photosensitizers (b) FI G U R E 1 Oleic acid photooxygenation in the presence and absence of Pasipay (a). Structure of different applied photosensitizers (b) HAJIMOHAMMADI and NOSRATI 1672 3.3 \| Effect of pasipay on fatty acid photooxgenation Also, the rate of oleic acid preservation in the presence of these types of antioxidants decreased in the order of TBHQ > DABCO > BHT > BHA > pasipay. Herbal and natural source of flavonoid and polyphenol com- pounds have been reported to act as scavengers of various HAJIMOHAMMADI and NOSRATI 1673 oxidizing species (Dulf, Vodnar, & Socaciu, 2016). On the other hand according to the literature flavonoid compounds trap sin- glet oxygen and produce FLA-O2 compound (Majer, Neugart, Krumbein, Schreiner, & Hideg, 2014) (Figure 3b). Pasipay because of its flavonoid compounds can use as a highly efficient singlet oxygen scavenger. FI G U R E 2 1H NMR spectra of oleic acid (3.1 × 10−3 mol) after photooxidation in the absence (a) and in the presence (b) of H2TPP (0.001M) FI G U R E 2 1H NMR spectra of oleic acid (3.1 × 10−3 mol) after photooxidation in the absence (a) and in the presence (b) of H2TPP FI G U R E 2 1H NMR spectra of oleic acid (3.1 × 10−3 mol) after photooxidation in the absence (a) and in the presence (b) of H2TPP (0.001M) oxidizing species (Dulf, Vodnar, & Socaciu, 2016). On the other hand according to the literature flavonoid compounds trap sin- glet oxygen and produce FLA-O2 compound (Majer, Neugart, Krumbein, Schreiner, & Hideg, 2014) (Figure 3b). Pasipay because of its flavonoid compounds can use as a highly efficient singlet oxygen scavenger. FI G U R E 2 1H NMR spectra of oleic acid (3.1 × 10−3 mol) after photooxidation in the absence (a) and in the presence (b) of H2TPP (0.001M) FI G U R E 2 1H NMR spectra of oleic acid (3.1 × 10−3 mol) after photooxidation in the absence (a) and in the presence (b) of H2TPP (0.001M) oxidizing species (Dulf, Vodnar, & Socaciu, 2016). On the other hand according to the literature flavonoid compounds trap sin- glet oxygen and produce FLA-O2 compound (Majer, Neugart, oxidizing species (Dulf, Vodnar, & Socaciu, 2016). On the other hand according to the literature flavonoid compounds trap sin- glet oxygen and produce FLA-O2 compound (Majer, Neugart, oxidizing species (Dulf, Vodnar, & Socaciu, 2016). On the other hand according to the literature flavonoid compounds trap sin- glet oxygen and produce FLA-O2 compound (Majer, Neugart, Krumbein, Schreiner, & Hideg, 2014) (Figure 3b). 3.3 \| Effect of pasipay on fatty acid photooxgenation Pasipay because of its flavonoid compounds can use as a highly efficient singlet oxygen scavenger. 1674 \| HAJIMOHAMMADI and NOSRAT FI G U R E 3 The mechanisms for producing reactive oxygen species in the presence of photosensitizers (a) The mechanism of flavonoids (FLA) barricade of Pasipay against singlet oxygen (b) HAJIMOHAMMADI and NOSRATI 1674 FI G U R E 3 The mechanisms for producing reactive oxygen species in the presence of photosensitizers (a) The mechanism of flavonoids (FLA) barricade of Pasipay against singlet oxygen (b) FI G U R E 3 The mechanisms for producing reactive oxygen species in the presence of photosensitizers (a) The mechanism of flavonoids (FLA) barricade of Pasipay against singlet oxygen (b) FI G U R E 4 UV–visible spectra of anthracene photooxygenation with singlet oxygen in the presence of different kind of singlet oxygen scavengers (λmax = 375 nm) after 5 min using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) under 1 atm of bubbling of air in the acetonitryl (a) The scavenging capacity of different kind of singlet oxygen scavengers after 5 min using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) under 1 atm of bubbling of air in the acetonitrile oxygen was comprehensively assessed by anthracene oxida assay and evaluation of fatty acid oxidation. It was showed pasi has an effective role in restricting of singlet oxygen generation limitation of fatty acid photooxidation. ACKNOWLEDGMENT We gratefully acknowledge support from the Khara University. CONFLICTS OF INTEREST The authors declare that they have no conflict of interest. The authors declare that they have no conflict of interest. The authors declare that they have no conflict of interest. 4 \| CONCLUSION This article does not contain any studies with human participants or animals performed by any of the authors. Due to the increase of diseases such as cancer, Alzheimer’s disease, skin disorders, etc. with ROS especially singlet oxygen and light, finding efficient antioxidant is very important. In this study, antioxi- dant capacity for pasipay and synthetic polyphenolics against singlet 3.3 \| Effect of pasipay on fatty acid photooxgenation ETHICAL STATEMENT FI G U R E 4 UV–visible spectra of anthracene photooxygenation with singlet oxygen in the presence of different kind of singlet oxygen scavengers (λmax = 375 nm) after 5 min using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) under 1 atm of bubbling of air in the acetonitryl (a) The scavenging capacity of different kind of singlet oxygen scavengers after 5 min using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) under 1 atm of bubbling of air in the acetonitrile FI G U R E 5 Diagram of oleic acid preservation in the presence of pasipay and well-known fatty acid antioxidants (BHA, BHT an TBHQ) and chemical singlet oxygen scavenger (DABCO) FI G U R E 5 Diagram of oleic acid preservation in the presence of pasipay and well-known fatty acid antioxidants (BHA, BHT and TBHQ) and chemical singlet oxygen scavenger (DABCO) FI G U R E 5 Diagram of oleic acid preservation in the presence of pasipay and well-known fatty acid antioxidants (BHA, BHT and TBHQ) and chemical singlet oxygen scavenger (DABCO) oxygen was comprehensively assessed by anthracene oxidation assay and evaluation of fatty acid oxidation. It was showed pasipay has an effective role in restricting of singlet oxygen generation and limitation of fatty acid photooxidation. ACKNOWLEDGMENT We gratefully acknowledge support from the Kharazmi University. UV–visible spectra of anthracene photooxygenation FI G U R E 4 UV–visible spectra of anthracene photooxygenation with singlet oxygen in the presence of different kind of singlet oxygen scavengers (λmax = 375 nm) after 5 min using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) under 1 atm of bubbling of air in the acetonitryl (a) The scavenging capacity of different kind of singlet oxygen scavengers after 5 min using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) under 1 atm of bubbling of air in the acetonitrile p p yg with singlet oxygen in the presence of different kind of singlet oxygen scavengers (λmax = 375 nm) after 5 min using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) under 1 atm of bubbling of air in the acetonitryl (a) The scavenging capacity of different kind of singlet oxygen scavengers after 5 min using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) under 1 atm of bubbling of air in the acetonitrile CONFLICTS OF INTEREST The authors declare that they have no conflict of interest. REFERENCES Research and Technology, 214, 294–298. https://doi.org/10.1007/ s00217-001-0479-5 Research and Technology, 214, 294–298. https://doi.org/10.1007/ s00217-001-0479-5 Aubry, J. M., Pierlot, C., Rigaudy, J., & Schmidt, R. (2003). Reversible binding of oxygen to aromatic compounds. Accounts of chemical re- search, 36, 668–675. https://doi.org/10.1021/ar010086g Laing, M. (1989). The three forms of molecular oxygen. Journal of Chemical Education, 66, 453. https://doi.org/10.1021/ed066p453 Lee, J. H., & Jung, M. Y. (2010). Direct spectroscopic observation of sin- glet oxygen quenching and kinetic studies of physical and chemical singlet oxygen quenching rate constants of synthetic antioxidants (BHA, BHT, and TBHQ) in methanol. Journal of food science, 75, https://doi.org/10.1111/j.1750-3841.2010.01669.x Barthel, G., & Grosch, W. (1974). Peroxide value determination compari- son of some methods. Journal of the American Oil Chemists Society, 51, 540–544. https://doi.org/10.1007/BF02636025 Bonnett, R., & Martınez, G. (2001). Photobleaching of sensitisers used in photodynamic therapy. Tetrahedron, 57, 9513–9547. https://doi. org/10.1016/S0040-4020(01)00952-8 Lengfelder, E., Cadenas, E., & Sies, H. (1983). Effect of DABCO (1, 4-diazabicyclo [2, 2, 2]-octane) on singlet oxygen monomol (1270 nm) and dimol (634 and 703 nm) emission. FEBS letters, 164, 366–370. https://doi.org/10.1016/0014-5793(83)80318-4 Bressan, M., & Morvillo, A. (1989). Alkene epoxidation by ruthenium (II) phosphine complexes A kinetic investigation. Inorganic Chemistry, 28, 950–953. https://doi.org/10.1021/ic00304a028 Lindsey, J. S., & Wagner, R. W. (1989). Investigation of the synthe- sis of ortho-substituted tetraphenylporphyrins. Journal of Organic Chemistry, 54, 828–836. https://doi.org/10.1021/jo00265a021 Bruneton, J. (1995). Pharmacognosy, phytochemistry, medicinal plants. Paris: Lavoisier publishing. Chen, Y., Xu, S., Li, L., Zhang, M., Shen, J., & Shen, T. (2001). Active oxygen generation and photo-oxygenation involving temporfin (m-THPC). Dyes and pigments, 51, 63–69. https://doi.org/10.1016/ S0143-7208(01)00071-7 Majer, P., Neugart, S., Krumbein, A., Schreiner, M., & Hideg, É. (2014). Singlet oxygen scavenging by leaf flavonoids contributes to sun- light acclimation in Tilia platyphyllos. Environmental and Experimental Botany, 100, 1–9. https://doi.org/10.1016/j.envexpbot.2013.12.001 Dhawan, K., Dhawan, S., & Sharma, A. (2004). Passiflora: A review update. Journal of ethnopharmacology, 94, 1–23. https://doi.org/10.1016/j. jep.2004.02.023 Martinez, R. M., Pinho-Ribeiro, F. A., Steffen, V. S., Caviglione, C. V., Fattori, V., Bussmann, A. J. C., … Baracat, M. M. (2017). Trans-Chalcone, a Flavonoid Precursor, Inhibits UV-Induced Skin Inflammation and Oxidative Stress in Mice by Targeting NADPH Oxidase and Cytokine Production. Photochemical & Photobiological Sciences, 16, 1162–1173. https://doi.org/10.1039/c6 pp00442c Dulf, F. V., Vodnar, D. C., & Socaciu, C. (2016). Effects of solid-state fermentation with two filamentous fungi on the total phenolic con- tents, flavonoids, antioxidant activities and lipid fractions of plum fruit (Prunus domestica L.) by-products. ORCID Mahdi Hajimohammadi http://orcid.org/0000-0002-5979-0196 Mahdi Hajimohammadi http://orcid.org/0000-0002-5979-0196 HAJIMOHAMMADI and NOSRATI 1675 REFERENCES Food chemistry, 209, 27–36. https://doi.org/10.1016/j.foodchem.2016.04.016 Min, D. B., & Boff, J. M. (2002). Chemistry and reaction of singlet oxygen in foods. Comprehensive Reviews in Food Science and Food Safety, 1, 58–72. https://doi.org/10.1111/j.1541-4337.2002.tb00007.x Fang, T., Wu, X., Cao, W., Jia, G., Zhao, H., Chen, X., … Liu, G. (2017). Effects of dietary fiber on the antioxidant capacity, immune status, and antioxidant-relative signaling molecular gene expression in rat organs. Royal Science Chemistry Advances, 7(7), 19611–19620. https:// doi.org/10.1039/C7RA02464A Niki, E. (2015). Lipid oxidation in the skin. Free radical research, 49, 827– 834. https://doi.org/10.3109/10715762.2014.976213 Nimse, S. B., & Pal, D. (2015). Free radicals, natural antioxidants, and their reaction mechanisms. Royal Science Chemistry Advances, 5, 27986– 28006. https://doi.org/10.1039/C4RA13315C Girotti, A. W. (1998). Lipid hydroperoxide generation, turnover, and effec- tor action in biological systems. Journal of lipid research, 39, 1529–1542. Ruiz-González, R., Bresolí-Obach, R., Gulías, Ò., Agut, M., Savoie, H., Boyle, R. W., … Giuntini, F. (2017). NanoSOSG: A nanostructured fluorescent probe for the detection of intracellular singlet oxygen. Angewandte Chemie International Edition, 56, 2885–2888. https://doi. org/10.1002/anie.201609050 Greer, A. (2006). Christopher Foote’s discovery of the role of singlet ox- ygen [1O2 (1Δg)] in photosensitized oxidation reactions. Accounts of chemical research, 39, 797–804. https://doi.org/10.1021/ar050191g Hajimohammadi, M., Schwarzinger, C., & Knör, G. (2012). Controlled multistep oxidation of alcohols and aldehydes to carboxylic acids using air, sunlight and a robust metalloporphyrin sensitizer with a pH-switchable photoreactivity. Royal Science Chemistry Advances, 2, 3257–3260. https://doi.org/10.1039/C2RA01076C Terao, J., Minami, Y., & Bando, N. (2010). Singlet molecular oxygen- quenching activity of carotenoids: Relevance to protection of the skin from photoaging. Journal of Clinical Biochemistry and Nutrition, 48, 57–62. https://doi.org/10.1002/anie.201609050 Hamid, A. A., Aiyelaagbe, O. O., Usman, L. A., Ameen, O. M., & Lawal, A. (2010). Antioxidants: its medicinal and pharmacological applications. African Journal of pure and applied Chemistry, 4, 142–151. Toffoli, D. J., Gomes, L., Junior, N. D. V., & Courrol, L. C. (2008). Enhancement on the hypocrellin B singlet oxygen gener- ation quantum yield in the presence of rare earth ions. AIP Conference Proceedings, 992, 1207–1212. https://doi. org/10.1063/1.2926819 Kligman, L. H. (1989). Prevention and repair of photoaging: Sunscreens and retinoids. Cutis, 43, 458–465. Korytowski, W., Schmitt, J. C., & Girotti, A. W. (2010). Surprising Inability of Singlet Oxygen-generated 6-Hydroperoxycholesterol to Induce Damaging Free Radical Lipid Peroxidation in Cell Membranes. Photochemistry and photobiology, 86, 747–751. https://doi. org/10.1111/j.1751-1097.2010.00722.x How to cite this article: Hajimohammadi M, Nosrati P. How to cite this article: Hajimohammadi M, Nosrati P. Scavenging effect of pasipay (passiflora incarnate L.) on singlet oxygen generation and fatty acid photooxygenation. Food Sci Nutr. 2018;6:1670–1675. https://doi.org/10.1002/fsn3.731 REFERENCES Scavenging effect of pasipay (passiflora incarnate L.) on singlet oxygen generation and fatty acid photooxygenation. Food Sci Nutr. 2018;6:1670–1675. https://doi.org/10.1002/fsn3.731 Koski, A., Psomiadou, E., Tsimidou, M., Hopia, A., Kefalas, P., Wähälä, K., & Heinonen, M. (2002). Oxidative stability and minor constitu- ents of virgin olive oil and cold-pressed rapeseed oil. European Food
https://openalex.org/W4362569812	https://pubs.rsc.org/en/content/articlepdf/2023/ra/d3ra01408h	English	null	Effects of throat sizing and gasification agents in a biomass downdraft gasifier: towards CO<sub>2</sub>-free syngas production	RSC advances	2,023	cc-by	12,827	1. Introduction and also time consuming. Consequently, researchers are using modelling to simulate and predict gasiers behaviour. Diﬀerent modelling tools are used in the gasication process varying from equilibrium12,13 to kinetic,11,14–17 and Computational Fluid Dynamics (CFD).10,18–20 1. The gradual use of fossil fuels for energy production is esca- lating the negative impacts on the environment and climate change due to CO2 production.1–3 The increased rate of deple- tion of fossil fuels and the worlds' increased energy demands are all leading to the focus on renewable energy sources. Biomass is a renewable and sustainable resource for energy and has CO2 neutrality. Energy recovery from biomass could be done through combustion, pyrolysis, and gasication.4–6 One of the most promising ways for energy production from biomass is gasication. It is estimated that 10% of energy production around the world is met from biomass.7,8 y ( ) Equilibrium12,13,21,22 and kineti15–17,23,24 models are widely used in pyrolysis and gasication of biomass. However, there are some limitations which restrict the applicability of both the kinetic and equilibrium models. For example, gasier design is a complex process aﬀecting the production of syngas and tar content. Kinetic models can only address chemical reactions and rates which do not depend on the gasier geometry. A robust modelling tool should also consider multiphase uid dynamics, heat and mass transfer, and chemical transport. The solid and gas phase reactions and their interactions cannot be covered through kinetic models.9,25 To address all these factors, CFD modelling techniques are strongly recommended.9,18 Designing a gasier requires complicated steps and considers diﬀerent aspects e.g., required thermal power, as well as biomass type, size, moisture, and ash content. As a result, it requires a time consuming experiment or a detailed numerical modelling which proves its ability in the gasication process simulation and design.9,11 Although experiments are eﬀective and reliable in designing a gasier, it is a costly, sometime risky CFD models are widely used in the process of gasication inuenced with diﬀerent chemical kinetics, and rates of reac- tions. The approaches of variations are based on the gasier geometry, design, feedstock, operating parameters, and gasi- fying agent. RSC Advances 1 Introduction Cite this: RSC Adv., 2023, 13, 10221 Received 2nd March 2023 Accepted 22nd March 2023 DOI: 10.1039/d3ra01408h rsc.li/rsc-advances PAPER Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. his article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Eﬀects of throat sizing and gasiﬁcation agents in a biomass downdraft gasiﬁer: towards CO2-free syngas production Cite this: RSC Adv., 2023, 13, 10221 Ahmed M. Salem *ac and Manosh C. Paulb The gasiﬁcation process in a downdraft biomass gasiﬁer is investigated using Computational Fluid Dynamics (CFD). The aim is to develop a novel approach to reduce CO2 emissions from producer syngas while increasing the higher heating value (HHV). To this end, the eﬀects of varying the throat diameter of the gasiﬁer and gasifying media (air and oxygen) on the performance of gasiﬁcation are investigated. The results reveal that as the throat ratio decreases for oxy-gasiﬁcation, more CO, H2, and CH4 are produced, thus resulting in a HHV of 12.1 MJ Nm−3. For the same working conditions (ER, MC, and feedstock), the suggested design/optimum throat ratio of 0.14 is found to reduce CO2 by ∼55% compared to any other higher throat ratios, while simultaneously increasing HHV by ∼20% for both air and oxy-gasiﬁcation cases. Additionally, the suggested throat ratio increases the gasiﬁcation eﬃciency, carbon conversion and producer gas yield by 19%, 33%, and 22% respectively. Therefore, it shows a signiﬁcant potential for CO2-free syngas production in the gasiﬁcation process, demonstrating a promising technique that does not require any solvents, catalysts, absorbers, or additional CO2 removal. Lower throat ratios further favour the higher yield of syngas, HHV, gasiﬁcation and conversion eﬃciencies, with better gasiﬁer performance. Received 2nd March 2023 Accepted 22nd March 2023 DOI: 10.1039/d3ra01408h rsc.li/rsc-advances bSystems, Power & Energy Research Division, James Watt School of Engineering, University of Glasgow, Glasgow, G12 8QQ, UK. E-mail: Manosh.Paul@glasgow.ac.uk cMechanical Power Department, Faculty of Engineering, Tanta University, Tanta, 31521, Egypt. E-mail: Ahmed_salem@f-eng.tanta.edu.eg aSchool of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK aSchool of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK bSystems, Power & Energy Research Division, James Watt School of Engineering, University of Glasgow, Glasgow, G12 8QQ, UK. E-mail: Manosh.Paul@glasgow.ac.uk cMechanical Power Department, Faculty of Engineering, Tanta University, Tanta, 31521, Egypt. E-mail: Ahmed_salem@f-eng.tanta.edu.eg Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The model built by Kumar and Paul,10 for a downdra biomass gasier used ANSYS Fluent soware, and simulated a 2D, 20 kW downdragasier. The four main gasication zones were included in the model by the Euler–Lagrangian discrete phase approach. The model was validated against the experimental data and kinetic model of ref. 31. Additionally, diﬀerent feedstocks were used with diﬀerent air equivalence ratio (ER) to study the model sensitivity on the gasication process. Although the model showed stable and reliable results, it could not perform better under ERs below 0.35. Furthermore, the model was converted to a 3D model using rubber wood as a feedstock.18 The 3D model found a good agreement with the previous experimental data at same working conditions. On the other hand, an experimental study was carried out by Montuori et al.42 They studied the eﬀect of the throat diameter sizing on gasier performance, and the whole gasication plant stability was coupled with an internal combustion engine. The xed bed gasier performance was examined in conjunction with syngas production and electricity generation. Air was used as a gasifying medium with two throat diameters 7 and 10 cm. They reported that 10 cm throat diameter is the most conve- nient for syngas production (31% increment), with the plant electricity generation reaching 40%. While Gunarathne et al.43 experimentally examined the eﬀect of changing three throat diameters (125 mm, 150 mm and 175 mm) on downdra gasier output. Gasier performance was reported by studying the specic syngas production, conversion eﬃciency, and heating value. They concluded that changing throat diameter has no signicant eﬀect on the producer gas composition. The highest rate of gas production was observed at a throat diameter of 175 mm, with ER being 0.425. Although previous studies included the eﬀect of throat ratio and nozzle's diameter/height e.g. ref. 44, 41 and 45, the eﬀect of changing gasifying medium and throat ratio on gasier performance and CO2 emissions has yet to be investigated. Additionally, all studies used air as gasifying medium, and the main eﬀect was on enrich hydrogen production. Furthermore, throat ratios below 0.25 was not examined in any of the mentioned studies. More details about CFD modelling within diﬀerent gasiers could be found in ref. 32, 33, 34, 35 and 36. 1. Introduction Using the appropriate modelling techniques, CFD models are expected to reduce the time to design a gasier and predict gasication output of each experiment based on a specic feedstock and working parameters.26 As a result, CFD models are emerging as an eﬀective method in the gasication process simulation for diﬀerent gasier types.26–28 RSC Adv., 2023, 13, 10221–10238 \| 10221 © 2023 The Author(s). Published by the Royal Society of Chemistry Paper View Article Online Paper View Article Online View Article Online © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Advances Furthermore, one of the key parameters during the design of a gasier is the throat diameter. It has a great eﬀect on the producer gas composition, gasier power, and tar formation, as shown in the kinetic model study of.31 Some CFD studies focused on the eﬀect of throat angle,37,38 while others studied the eﬀects of number and angle of nozzles e.g. (ref. 39 and 40). However, few numerical and experimental studies mentioned the throat diameter eﬀect on the gasication process. Pra- sertcharoensuk et al.41 numerically studied the optimization process of a 20 cm throat of a downdragasier using ANSYS CFD. Producer gas composition and temperature distribution were examined for diﬀerent throat diameters. The modelling results were validated against experimental results and found to have a good agreement. Maximum value of H2 was found to be 31.2 vol%, and H2/Co ratio was found to be 1.25 at a throat diameter of 0.4. They used the throat to gasier diameter ratio varying from 0.25 to 0.5. However, the eﬀect of reducing the throat/gasier diameter below 0.25 was not examined. L. Yu et al.29 introduced a numerical model for coal gasi- cation inside a uidized bed gasier. They combined Arrhenius rate reactions for coal gasication with a kinetic theory of granular ow (KTGF). Aer the validation of model against experimental data, it was then used to study the eﬀect of changing gasier height on the syngas composition, velocity, and temperature along the gasier bed. Whereas a detailed model was built by Fletcher et al.30 using CFX4 package, for the gasication of biomass in an entrained ow gasier. They used Lagrangian approach in modelling the particles entering the gasier, followed by volatiles release and gasication. The concentrations of gas species are obtained by solving the transport equations and heterogeneous reactions. Producer gas composition with gasication temperature was presented at the gasier outlet and found in a good agreement with experi- mental results. RSC Advances View Article Online Paper production of CO2 during the gasication process as possible and this research addresses it. combustion zone. The nozzles (D = 1.6 cm each) are specied at xed height (7.8 cm) above the throat diameter based on the previous recommendations described in ref. 31. The feedstock is fed from top while producer gas is derived from bottom as showed in the gure. All the gasier dimensions are illustrated in the gure based on the kinetic model predictions.31 The model assumes all the char is consumed during the reduction/ gasication – the same assumption was made in the kinetic model.31 In addition, the model is considering the following assumptions: Steady-state simulations. Uniform spherical particle size. Tar and other higher hydrocarbons are neglected in the Table 2 Oxidation zone reactions Reactions A (1/s) E (kJ mol−1) Ref. 2C + O2 / 2CO 1.47 × 105 112.99 58 2H2 + O2 / 2H2O 2.2 × 109 109 59 CO + 0.5O2 / CO2 1.0 × 1010 126 59 CH4 + 2O2 / CO2 + 2H2O 4.4 × 1011 126 60 Table 3 Reduction zone reactions Reactions A (1/s) E (kJ mol−1) Ref. C + CO2 / 2CO 8.268 188.2 58 0.5C + H2 / 0.5CH4 8.8894 × 10−6 67.16 58 C + H2O / CO + H2 42.5 142 58 CO + H2O / CO2 + H2 2.35 × 1010 288 61 CH4 + H2O / CO + 3H2 3 × 108 125 59 CO2 + H2 / CO + H2O 1.785 × 1012 326 61 Table 1 Feedstocks data used in validation and testing the model18,57 Ultimate analysis db% Proximate analysis db% C H O N S Vol. FC Ash MC Wood chips 54 6.0 40 0 0 70.0 20.0 0.338 7.36 Rubber wood 50.6 6.5 42 0.2 0.7 81.1 19.1 0.7 18.5 Table 1 Feedstocks data used in validation and testing the model18,57 To the best of authors' knowledge, previous studies, as per the literature review presented above, do not adequately cover throat sizing and its relationship with gasication processes when combining with diﬀerent gasifying mediums. Addition- ally, the impact of varying agents, particularly oxy and oxy–air, on the producer gas quality, yield, carbon conversion, and gasication eﬃciency, and the subsequent heating value is not fully explored. RSC Advances View Article Online Furthermore, one of the major goals of this paper, which addresses a crucial knowledge gap in the eld, is to investigate the eﬀect of modifying throat ratio and gasifying agent on minimising carbon dioxide emissions while simulta- neously boosting hydrogen yield. Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Table 2 Oxidation zone reactions 2. CFD model description All the gasier dimensions are illustrated in the gure based on the kinetic model predictions.31 The model assumes all the char is consumed during the reduction/ gasication – the same assumption was made in the kinetic model.31 In addition, the model is considering the following assumptions: Fig. 1 2D schematic of the proposed gasiﬁer design. Steady-state simulations. Steady-state simulations. Uniform spherical particle size. Tar and other higher hydrocarbons are neglected in the current model, for their complex formation and reaction rates. All reactions take place under atmospheric pressure. Turbulence intensity and hydraulic diameter where speci- ed for all inlets/exits for uniform distribution of ow inside the gasier. Two equations k-epsilon model is specied for turbulence. Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. However, most of the previous works do not include oxy-gasication eﬀect in CFD modelling, and its eﬀect on the gasier design and output. Hence, the main goal of the current research is to put more focus on the eﬀect of air and oxy-gasication towards improving the yield of hydrogen enrich bio-syngas and how the gasica- tion agent alternation further inuences the key design parameter of a downdragasier i.e., the throat ratio (e.g., throat/gasier diameter). Consequently, their combined eﬀects on the overall gasier performance will be further examined and explained. Couto et al.35 presented a 2D numerical model based on CFD framework along with experiments to study the eﬀect of using oxygen enriched air on the process of biomass gasication. Eulerian–Eulerian approach was used in exchanging mass, energy, and momentum. The model was validated against their experimental data and found a good agreement. The inuence of oxygen on steam to biomass ratio, syngas composition, and temperature along gasier was examined. They found that N2 and H2 concentrations decrease as a function of oxygen content, while CO2 concentrations were found to increase. They used KTGF, DPM, and k-epsilon turbulent model in the simulation process. However, they did not argue over the use of pure oxygen on gasication performance and producer gas quality. Addi- tionally, the study does not include any eﬀect of gasier design and geometry, as well as the corresponding impacts of using diﬀerent oxidizers. y A gasication process produces gases (CO, CO2, CH4, H2, N2, H2O), tar, and solid residues. The amount of CO2 produced depends on the gasier type, feedstock, working conditions, and gasifying medium. Depending on the gasifying medium, the CO2 mol% of producer gas from steam, air, oxygen, and CO2 gasication produce (12–30)%, (15–38)%, (10–48)%, and (5– 15)% respectively.46–48 The US dep. Of Energy reported in 2018 that 64 commercial plants for CO2 removal/capture is associ- ated with syngas production plants. The most widely used technologies for removal are absorption-based (∼60%), fol- lowed by cryogenics (18%), adsorbers (10%), and other tech- nologies.49 However, such technologies are still developing and cost intensive. Hence, it is better to focus on eliminating the © 2023 The Author(s). Published by the Royal Society of Chemistry 10222 \| RSC Adv., 2023, 13, 10221–10238 RSC Advances View Article Online 2. CFD model description Table 3 Reduction zone reactions Table 3 Reduction zone reactions Reactions A (1/s) E (kJ mol−1) Ref. C + CO2 / 2CO 8.268 188.2 58 0.5C + H2 / 0.5CH4 8.8894 × 10−6 67.16 58 C + H2O / CO + H2 42.5 142 58 CO + H2O / CO2 + H2 2.35 × 1010 288 61 CH4 + H2O / CO + 3H2 3 × 108 125 59 CO2 + H2 / CO + H2O 1.785 × 1012 326 61 The gasier design is based on the kinetic model developed by the current authors31 in which a 20 kW downdrabiomass gasier is modelled. The integrated model considers three zones – drying and pyrolysis, combustion, followed by gasication/reduction as illustrated in Fig. 1. Each zone is controlled by a set of detailed kinetic rate reactions used in ANSYS 19.0 (Tables 2 and 3). Further details for the gasier schematic diagram in Fig. 1, and its dimensions are fully covered in ref. 9 and 31, and for brevity they are not repeated here. A zoomed in section from the top right-hand side of the gasier is also presented in Fig. 1 to illustrate the structural mesh distribution created inside the gasier. Air or oxygen is injected through the two nozzles at the gasier sides within the combustion zone. The nozzles (D = 1.6 cm each) are specied at xed height (7.8 cm) above the throat diameter based on the previous recommendations described in ref. 31. The feedstock is fed from top while producer gas is derived from bottom as showed in the gure. All the gasier dimensions are illustrated in the gure based on the kinetic model predictions.31 The model assumes all the char is consumed during the reduction/ gasication – the same assumption was made in the kinetic model.31 In addition, the model is considering the following assumptions: combustion zone. The nozzles (D = 1.6 cm each) are specied at xed height (7.8 cm) above the throat diameter based on the previous recommendations described in ref. 31. The feedstock is fed from top while producer gas is derived from bottom as showed in the gure. RSC Advances Paper Volatiles / x1CO + x2CO2 + x3CH4 + x4H2 (8) (8) a continuous phase of uid in which an interaction between the particles takes place considering the mass and heat transfer equations. The conservation equations of mass, momentum, energy, and species transport are numerically solved under the turbulent ow steady-state condition with a set of nite rate kinetic reactions. These equations are presented as follows:50,51 Volatiles / x1CO + x2CO2 + x3CH4 + x4H2 Volatiles / x1CO + x2CO2 + x3CH4 + x4H2 The volatiles are composed of gases (CO, CO2, H2, and CH4) and other HC components. The process of pyrolysis and biomass devolatilization starts aer the drying process. Depending on its composition, biomass is decomposed into volatiles, char, tar, and ash. The model carries out an elemental mass balance for the volatiles to estimate its products. However, Mass conservation: Mass conservation: V$(r~v) = Sm (1) V$(r~v) = Sm (1) (1) V$(r~v) = Sm the CO concentrations are rst calculated using the model proposed by56 which calculates the mass fraction of every species based on the pyrolysis temperature. Momentum conservation: Momentum conservation: Eqn (8) describes the volatiles break-up based on the model proposed by.56 The model is further implemented inside the ANSYS directory to describe the species release during the pyrolysis process (eqn (7), and (8)) based on the ultimate anal- ysis of the feedstock. V$(r~v~v) = −V (s ̿) + r~g + F⃑ (2) Energy conservation: Energy conservation: V$ v!ðrE þ pÞ ¼ V$ leffVT X hjJj þ seff !$~v þ Sh (3) 2.5. Convergence criteria (6) The set of models and solution methods, and residuals control used are all concluded in Table 4. where i refers to diﬀerent species in the simulation, Sct is the turbulent Schmidt number and is represented by the ratio of turbulent viscosity to eddy diﬀusivity, and Ri is the net rate of the production of diﬀerent species (i) by the chemical reactions. Two phase equations are solved numerically by an implicit nite volume method in ANSYS. A pressure–velocity coupling algorithm is used which solves the combined momentum and pressure-based equations.51 A spatial discretization for pressure is solved by PREssure STaggering Option (PRESTO) method which gives better accuracy and conversion for volume of uids 2.4. Char and gas phase reactions v vxi ðr3uiÞ ¼ v vxj m þ m sk þ ve vxj þ C13 3 k ðGk þ G33GbÞ C23r 32 k þ S3 Tables 2 and 3 describe the diﬀerent reactions used in the current model based on the recommendations of ref. 13 and 20, where A is the pre-exponential factor (1/s), and E is the activa- tion energy in (kJ mol−1). The reactions represent the kinetic rate reaction data which take place in the oxidation and reduction zones. All the reactions are implemented inside the ANSYS code, including the volatiles decomposition reactions illustrated earlier. (5) where the parameters C13 = 1.44, C23 = 1.92, Sk = Se = 1, and Ym = 0.09. Sm is the mass added to the phase (kg), hj is the enthalpy of species (j), s̿ the stress tensor (pa), leﬀis the eﬀective conductivity, and 3 is the turbulent dissipation rate (m2 s−3). The species transport equation:52 The species transport equation:52 V$ðr~vYiÞ ¼ V$ rDi;m þ mt Sct VYi þ Ri (6) The turbulence k-epsilon RNG model is represented by The turbulence k-epsilon RNG model is represented by Two feedstocks are used in the current model for validation and studying the eﬀect of varying the throat diameter on the gasier performance and species behaviour. v vxi ðr k uiÞ ¼ v vxj m þ m sk þ vk vxj þ Gk þ Gb r3 Ym þ Sk (4) v vxi ðr3uiÞ ¼ v vxj m þ m sk þ ve vxj þ C13 3 k ðGk þ G33GbÞ C23r 32 k þ S3 (5) v vxi ðr k uiÞ ¼ v vxj m þ m sk þ vk vxj þ Gk þ Gb r3 Ym þ Sk (4) (4) (4) Biomass / volatiles + moisture + tar + char + ash 2.1. Governing equations Species transport model is used along with the discrete ordi- nates (DO) radiation and k-epsilon turbulence models. Air and biomass are fed at 600 K, and 300 K respectively. The feedstock particles are modelled using a Lagrangian approach – discrete phase model (DPM). DPM considers the particles trajectories as Fig. 1 2D schematic of the proposed gasiﬁer design. RSC Adv., 2023, 13, 10221–10238 \| 10223 © 2023 The Author(s). Published by the Royal Society of Chemistry © 2023 The Author(s). Published by the Royal Society of Chemistry Paper View Article Online View Article Online 3.1. Mesh independency test The mesh independency test is carried out using ve diﬀerent mesh sizes with cell numbers of 225 267, 201 593, 161 554, 74 360, and 57 456 respectively. The mole fraction of producer gas composition and its heating value are illustrated in Fig. 2, where air is used as a gasifying agent for wood chips gasication at ER of 0.3, and at a throat diameter of 8.8 cm. The results of producer gas composition (mol%) and heating value (MJ Nm−3) for wood gasication showed slight variations in all the grid sizes used. The heating value of producer gas exhibits similar results with variances of less than 0.5%, demonstrating the consistency of the results throughout the ve mesh sizes used. The mesh sizes higher than 74 360 cell numbers, show no variations in gas composition and heating 3. Results and discussions Besides the mesh independency test, which proves the model's stability, validation against experimental results57 is performed. The validation is carried out with the same feedstock (wood chips), ER (0.35), gasifying agent (air), and gasier design (Tables 1 and 6). Additionally, rubber wood gasication is used as second feedstock and the results are compared with experi- mental data,15 and kinetic model results.31 Besides the mesh independency test, which proves the model's stability, validation against experimental results57 is performed. Following the mesh resolution study, the model is validated using data from a downdragasier with the same design and working conditions. The eﬀect of the throat/gasier ratio on the producer gas heating value will be discussed, as well as process optimization. The results will be divided into two main cate- gories; air gasication followed by oxy-gasication eﬀects. The validation is carried out with the same feedstock (wood chips), ER (0.35), gasifying agent (air), and gasier design (Tables 1 and 6). Additionally, rubber wood gasication is used as second feedstock and the results are compared with experi- mental data,15 and kinetic model results.31 The set of results illustrated by Fig. 3 shows the dry gas composition at the gasier outlet for (A) wood chips, and (B) rubber wood gasication. The results are validated under the same working conditions (i.e., MC 7.36%, ER 0.35, and gasier design) for wood pellets. On the other hand, rubber wood gasication simulations are run under (MC 18.5%, and ER 0.326). The HHV variations for wood pellets and rubber wood are (<3%, and <7%) respectively, while other gas species are showing smaller variations. The model's ability to replicate the process of gasication in downdragasiers is demonstrated by a satisfactory agreement between the current model, kinetic model, and the experimental data. Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. value, implying stability of the results predicted. However, the higher grid size is a time intensive process and that requires higher computational cost. As a result, the mesh size of 74 360 is selected for the rest of the simulations carried out in this study. (VOF), and multi-phase modelling. Upwind scheme is used for solving the energy, momentum, and gas species discretization. Other boundary conditions are specied in Table 5. 2.2. Devolatilization and biomass decomposition Default drying model within the ANSYS directory51 is the Lee model53 which predicts the moisture evaporation and drying model for mixtures. It is applicable and shows good stability for the VOF multi-phase, and Euler–Lagrangian models. Conse- quently, it will be used in the current simulation. Table 4 Solution methods followed in the CFD modelling Euler–Lagrangian Turbulence: k-epsilon 2 equations Species transport for nite rate/Eddy transport kinetic model Radiation: discrete ordinates Intensity and hydraulic diameter specication Pressure–velocity coupling, coupled Pressure discretization scheme, PRESTO Momentum and energy; 2nd order upwind discretization scheme 10−3 for all variables, for energy and radiation 10−6 Phases Models included The process of gasication is composed of four main steps. Drying, followed by pyrolysis and volatiles break-up, combus- tion, and gasication/reduction. The heat released during the combustion process drives the biomass drying and decompo- sition in the pyrolysis zone. Aer drying, the biomass rst decomposes into volatiles and char, followed by further decomposition to form char and volatiles as illustrated by eqn (7) and (8).54,55 (7) 10224 \| RSC Adv., 2023, 13, 10221–10238 © 2023 The Author(s). Published by the Royal Society of Chemistry Paper Table 5 Boundary conditions used in the model Inlet Mass ow inlets for air nozzles and biomass feed Always supposed as normal to boundary Outlet Two exits for syngas zero-gauge pressure Back ow temperature was assumed 1000 K Walls Stationary walls Turbulence For assuring fully developed ows for air and biomass feeding, the turbulence is identied by the intensity and hydraulic diameter Table 5 Boundary conditions used in the model Inlet Mass ow inlets for air nozzles and biomass feed Always supposed as normal to boundary Outlet Two exits for syngas zero-gauge pressure Back ow temperature was assumed 1000 K Walls Stationary walls Turbulence For assuring fully developed ows for air and biomass feeding, the turbulence is identied by the intensity and hydraulic diameter Table 6 Gasiﬁer design for current model and experimental data for validation Table 5 Boundary conditions used in the model Table 6 Gasiﬁer design for current model and experimental data for validation Gasier design Current model Experiment57 Height, cm 90 91.7 External diameter, cm 21.8 21.9 Throat diameter, cm 8.8 8.8 Throat/gasier D ratio, r 0.4 0.4 3.3. Air gasication The eﬀect of changing the throat ratio when using air as a gasication medium is investigated. The production of Fig. 2 Producer gas composition at diﬀerent cell numbers. © 2023 The Author(s) Published by the Royal Society of Chemistry RSC Adv 2023 13 10221–10238 \| 10225 Fig. 2 Producer gas composition at diﬀerent cell numbers. © 2023 The Author(s). Published by the Royal Society of Chemistry © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 10221–10238 \| 10225 Paper View Article Online Paper View Article Online RSC Advances Fig. 3 Current model validation for (A) wood pellets,57 and (B) rubber wood.15 Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 Current model validation for (A) wood pellets,57 and (B) rubber wood.15 a good agreement with55,62,63 as well as the results derived from the kinetic model.31 syngas, its heating value, velocity, and temperature distribu- tions, as well as the composition of H2, CO, and CO2 at the producer is further illustrated. Decreasing the throat diameter results in a gradual increase in the temperature inside the gasier. This is clearly because of more throttling at the end of combustion zone which results in a longer residence time and higher turbulence (Fig. 4b), which in turn increasing the temperature. The volume of combustion zone has changed slightly because of the throttling eﬀect. However, the model considers xed owrate of biomass and gasifying medium, which ensures the same owrate inside the gasier in all cases of changing throat size. As a result, when throat diameter is decreased, this led to an increase in turbu- lence, and residence time, and consequently, favours the oxidation reactions. Higher residence time and turbulence also encourage the combustion reactions (exothermic), leading to an increase in temperature and consumption of H2 which will be explored in more detail in the next sections. Also, as discussed that decreasing throat ratio leads to more turbulence inside the gasier and within the combustion zone, which causes higher temperatures and velocity (Fig. 4b). Maximum velocity within the range of 1–1.2 m s−1 is achieved around the exit nozzles and at the throat area. 3.3.1. © 2023 The Author(s). Published by the Royal Society of Chemistry 3.3. Air gasication Throat diameter eﬀect on air gasication process. Gasier throat diameter is expected to aﬀect the reactions and residence time inside the gasier. As a result, it needs a careful consideration when designing a gasier. A new dimensionless parameter, so called a throat ratio r is generated to simplify the procedure, where r is the ratio between the throat diameter and the gasier diameter (also known as the re box/pyrolysis diameter). Four diﬀerent values for r will be used in the current study (0.4, 0.28, 0.23, and 0.14) to evaluate the eﬀect of throat on the gasier performance and syngas production. 3.3.2. Temperature and velocity distributions. Fig. 4 illus- trates the eﬀect of changing throat ratio on the distribution of temperature (a), velocity (b), and turbulent kinetic energy (c) along the gasier. Rubber wood is used with an ER of 0.3 and air as the gasifying medium. The default throat diameter based on the kinetic model31 predictions is 6.2 cm, and the gasier diameter is 21.8 cm. Maximum temperatures around the nozzles (ignition temperature) are ∼2300 K, while at the centreline/centre zone of the gasier ∼1650 K at the smallest throat ratio of 0.14 examined. For the design case, the maximum temperature along centreline is ∼1300 K which is in The set of results illustrated in Fig. 4c depicts the turbulence kinetic energy associated with air gasication at diﬀerent throat © 2023 The Author(s). Published by the Royal Society of Chemistry 10226 \| RSC Adv., 2023, 13, 10221–10238 RSC Advances View Article Online RSC Advances View Article Online Paper an turbulent kinetic energy per unit mass mass starts at the pyrolysis then decrease of static temperature (a), velocity (b), and turbulent kinetic energy (c) along gasiﬁer for air gasiﬁcatio Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 Contours of static temperature (a), velocity (b), and turbulent kinetic energy (c) along gasiﬁer for air gasiﬁcation at diﬀerent throat diameters rs of static temperature (a), velocity (b), and turbulent kinetic energy (c) along gasiﬁer for air gasiﬁcation Fig. 4 Contours of static temperature (a), velocity (b), and turbulent kinetic energy (c) along gasiﬁer for air gasiﬁcation at diﬀerent throat diameters. ratios. The mean turbulent kinetic energy per unit mass generated during the gasication process shows higher values for the smallest throat diameters. 3.3. Air gasication More turbulence per unit mass starts at the pyrolysis then decrease along the gasier height. As shown previously in Fig. 4c, higher velocities are formed around the air nozzles and the syngas exits. © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 10221–10238 \| 10227 Paper View Article Online View Article Online View Article Online Paper rticle Online RSC Advances Paper Additionally, for smaller throat ratios, higher turbulence and velocity are found. This is because of the higher residence time due to throttling and more ability for reactions to place. On the other hand, throttling generates higher velocities, and hence, higher turbulence. reactions take place between the gasier heights of 40–60 cm. These reactions are exothermic, generating heat for the whole gasication process consisting of drying, pyrolysis decomposi- tion, and gasication reactions. As a result, the combustion zone inside the gasier has higher temperatures (Fig. 4). Higher reaction rates are found for CO, followed by H2, and CH4 respectively. This is because of increased activity of CO and H2, and thus larger amounts are produced during pyrolysis compared to CH4. reactions take place between the gasier heights of 40–60 cm. These reactions are exothermic, generating heat for the whole gasication process consisting of drying, pyrolysis decomposi- tion, and gasication reactions. As a result, the combustion zone inside the gasier has higher temperatures (Fig. 4). Higher reaction rates are found for CO, followed by H2, and CH4 respectively. This is because of increased activity of CO and H2, and thus larger amounts are produced during pyrolysis compared to CH4. 3.3.3. Producer gas composition and heating value. As illustrated in Fig. 5, the volatile break-up process starts slightly below the top of the gasier, i.e., the pyrolysis zone. While at a height of 45 cm of the gasier, all the volatiles tend to be fully decomposed and converted to other compounds in the combustion and gasication zones. The volatiles are converted into tar, char, and gases. The combustion rate of diﬀerent gases is taking place at the combustion zone where it meets the oxidant (air) as illustrated clearly in the gure. The reaction rates in (kmol m−3 s−1) for CO, H2, and CH4 combustion for wood gasication at ER 0.3 is discussed. The combustion The results shown in Fig. 6 depict the volumetric gas composition of the producer gas at diﬀerent throat ratios. 10228 \| RSC Adv., 2023, 13, 10221–10238 3.3. Air gasication The throat ratio is set to r = 0.28 by default; however, increasing the throat does not signicantly aﬀect the producer syngas composition or heating value. In contrast, decreasing the throat diameter leads to an increase in the producer gas heating value. This is because a smaller throat diameter induces more Fig. 5 Volatiles decomposition and combustion reactions rate along gasiﬁer. Fig. 6 Producer gas compositions at diﬀerent throat ratios (r) for air gasiﬁcation. 10228 \| RSC Adv., 2023, 13, 10221–10238 © 2023 The Author(s). Published by the Royal Society of Chemistry Open Access Article. Published on 04 April 2023. Downloaded on This article is licensed under a Creative Commons A Open Access Article. Published on 04 April 2023. Downloaded This article is licensed under a Creative Commons Fig. 5 Volatiles decomposition and combustion reactions rate along gasiﬁer. Fig. 6 Producer gas compositions at diﬀerent throat ratios (r) for air gasiﬁcation. Fig. 6 Producer gas compositions at diﬀerent throat ratios (r) for air gasiﬁcation. Fig. 6 Producer gas compositions at diﬀerent throat ratios (r) for air gasiﬁcation. 10228 \| RSC Adv., 2023, 13, 10221–10238 10228 \| RSC Adv., 2023, 13, 10221–10238 © 2023 The Author(s). Published by the Royal Society of Chemistry © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Advances View Article Online RSC Advances View Article Online Paper throttling in the combustion area and increases residence time, which encourages heterogeneous combustion reactions (Fig. 5). This subsequently led to enhanced gasication process, resulting in an increase in CO, CH4. The boudouard, metha- nation and other reduction zone reactions are more likely to occur due to the rising temperature, resulting to consumption of CO2, and consequently, an increase in CO, and CH4, as shown in Fig. 6. Furthermore, the nitrogen concentration drops slightly, while the heating value tends to increase while reducing the throat ratio, again due to increase in the syngas composition. Optimum throat diameter is observed with high- est values of CO, CH4, and H2, and low CO2 concentrations (i.e., the r = 0.14). As previously illustrated in Fig. 4, the smaller throat ratios lead to high residence time, and turbulence inside the gasier. Consequently, more consumption for hydrogen as seen in Fig. 6. However, the decrease in H2 is ∼13% when using r = 0.14. © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper (0.28). As a result, optimum throat diameters (r = 0.14) produce heating values ∼15% higher than other cases. (0.28). As a result, optimum throat diameters (r = 0.14) produce heating values ∼15% higher than other cases. achieves higher temperatures because of the absence of nitrogen. As a result, fuel consumption is reduced, and higher ame temperature is achieved. On the other hand, the velocity distribution inside the gasier with oxy-gasication reaches a maximum of 0.4 m s−1, compared to 1.2 m s−1 with air gasication. As discussed earlier, for the same ER, a lower amount of oxygen is required to gasify the same amount of biomass. As a result, with the same throat diameter, smaller ow rates are achieved, resulting in lower velocities inside the gasier. 3.3. Air gasication On the other hand, there is increase in CO production by ∼43% when using r = 0.14 rather than default throat ratio of CO2, and consequently, an increase in CO, and CH4, as shown in Fig. 6. Furthermore, the nitrogen concentration drops slightly, while the heating value tends to increase while seen in Fig. 6. However, the decrease in H2 is ∼13% r = 0.14. On the other hand, there is increase in CO by ∼43% when using r = 0.14 rather than default Fig. 7 Contours of static temperature (top) and velocity along gasiﬁer, (K) for oxygen at diﬀerent throat diameters. © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 10221–1 Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 A This article is licensed under a Creative Commons Attribution 3.0 Unporte Fig. 7 Contours of static temperature (top) and velocity along gasiﬁer, (K) for oxygen at diﬀerent throat diameters. Contours of static temperature (top) and velocity along gasiﬁer, (K) for oxygen at diﬀerent throat diameters. RSC Adv., 2023, 13, 10221–10238 \| 10229 © 2023 The Author(s). Published by the Royal Society of Chemistry View Article Online View Article Online RSC Advances © 2023 The Author(s). Published by the Royal Society of Chemistry 3.4. Oxy-gasication 3.4.1. Temperature and velocity distributions. Fig. 7 depicts the temperature and velocity distribution along the gasier when oxygen is used instead of air as the gasifying medium. Rubber wood is used at ER of 0.3, and an MC of 18.5%. All simulations are run under the same conditions for easier comparisons and optimum results. The temperature reached their highest level at 2400–3700 K near the oxygen injection points (nozzles). Temperature inside the gasier rises while the throat diameter decreases, as expected, and already discussed with air gasication. It also exhibits temperature variations along the gasier centreline from (1300–1700) K, and around 1050 K at the gasier exit, which is consistent with experimental data in ref. 35. Furthermore, as previously discussed with air gasication, reducing throat leads to higher residence time, turbulence, and oxidation inside the gasier, resulting in a temperature increase. Compared to air, oxy-gasication 3.4.2. Producer gas composition. Fig. 8a illustrates the volumetric concentration of syngas species on a dry basis at the gasier exit. In the absence of nitrogen, higher concentrations of syngas species are found, and hence resulting in a higher heating value for the producer gas. At the same working conditions of biomass, ER, and MC, the heating value is ex- pected to be two times higher than that of air-gasication, which is in strong agreement with the results derived from previous research.64–66 Reduction in the throat ratio leads to an increase in the producer gas heating value. This is because of throttling, Fig. 8 Producer gas volumetric composition (a: dry, and b: wet basis) at diﬀerent throat ratios for oxy-gasiﬁcation. Producer gas volumetric composition (a: dry, and b: wet basis) at diﬀerent throat ratios for oxy-gasiﬁcation. Fig. 8 Producer gas volumetric composition (a: dry, and b: wet basis) at diﬀerent throat ratios for oxy-gasiﬁcation. 10230 \| RSC Adv., 2023, 13, 10221–10238 © 2023 The Author(s). Published by the Royal Society of Chemistry © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Advances View Article Online RSC Advances View Article Online Paper causing turbulence and higher temperature and residence time inside the gasier, further leading to an increase in the gasi- cation reaction rates with higher CO and lower CO2 concen- trations. Higher concentrations of CO are due to increased rates of Boudouard reaction which consumes CO2 as noticed in the results. 3.4. Oxy-gasication Slight diﬀerences in heating value were found while changing the throat ratio. The ndings are matching with the same results from air gasication. Optimum throat ratio of (r = 0.14) leads to the higher production of CO, leading to increase the values of HHV to the maximum of 12.1 MJ Nm−3. eﬀects are found during air and oxy-gasication. Additionally, the continuous consumption of H2, CH4 is also leading to H2O formation as illustrated by Fig. 8b referring to the above- mentioned discussions and also as seen from the reactions at (Tables 2 and 3). © 2023 The Author(s). Published by the Royal Society of Chemistry 3.5. Towards CO2 free gasication Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 10221–10238 \| 10231 View Article Online RSC Advances Paper increase in CO leads to a higher increase in the heating value of the produced gas. This is due to the fact that the ratio of CO increase is higher than H2 reduction, since it relies on CO2 consumption as previously shown in Fig. 9. the heating value of producer gas. For a throat ratio of 0.14, the heating value was found to increase by ∼(6–14%) than default throat ratio. Hence, throat sizing seems to be a very promising option for eliminating CO2 emissions within the process gasi- cation. Although the study was aiming to produce CO2-free syngas, the massive reduction in the produced values (i.e., ∼52% reduction) without the further use of solvents, catalysts, or another means of CO2 capture is encouraging and oﬀers a major improvement in the gasication process. Fig. 10 illustrates the producer gas composition at diﬀerent ER for the air, and oxy gasication at the same working condi- tions. Rubber wood is used as feedstock at ER of 0.2, 0.25, 0.3, and 0.35, for the same throat ratio (0.14). One of main aims of the current study is reducing/eliminating the production of N2, and CO2. As shown in the gure, air gasication produces higher amounts of N2 (40–45) mol% because of its higher nitrogen content. On the other hand, oxy-gasication shows zero content of N2. This is clearly because it does not have any content of N2. Throat ratio change has no eﬀect on N2 production because it only changes with the amount of air injected (i.e., the equivalence ratio) as seen in Fig. 6. While reducing the throat ratio, the results in both the cases (air and oxygen) follow the same behaviour of increasing CO, and a decrease of H2. An increase of CO production was found to be up to 43% for both air and oxy-gasication, while for the same throat decrease, the H2 values are found to drop by (15– 19%). As previously discussed, one of the main aims of the current study is the decrease of CO2. As a result, an increase in CO was found, because of the continuous use of CO2 in the boudouard and the methanation reactions. 3.5. Towards CO2 free gasication Sensitivity analysis is carried out to further study the eﬀects of changing ER on both the syngas production (HHV) and CO2 emissions. Air and oxygen are used as gasifying medium while rubber wood is the feedstock. A xed (the smallest) throat ratio (r = 0.14) is used because it proves to give higher heating values with lower CO2 production e.g., see Fig. 6 and 8. On the other hand, reversed steam reforming (CO2 + H2 CO + H2O) which has the highest activation energy, and pre- exponential factor (Table 3) leads to more consumption of H2 due to the higher temperatures (for lower throat ratios). As a result, lower H2 concentrations are found with low throat ratios. On the other hand, although higher temperature favours higher formation of CH4 through methanation and reforming reactions, CH4 concentration drops because of lower throat ratios (Fig. 6 and 8). This is further inuenced by the higher reaction rates of reversed steam reforming and methane reforming reactions resulted in more CO with consumption of CH4. Additionally, this favours the formation of CO2. However, in the presence of char and higher temper- atures, CO is formed through the boudouard reaction. Same Fig. 9 illustrates the eﬀect of throat sizing on the H2, CO, CO2 produced during the gasication process, and the correspond- ing heating value, where the default value of throat ratio r = 0.28. For air, and oxy-gasication, throat ratio of (r = 0.14) leads to (∼52%) reduction in CO2 production. The reduction in CO2 amount is because of the previous discussions showing that small throat leads higher temperatures, higher residence time, and hence encourage the heterogenous reactions to take place (Fig. 4, 5, and 7). As a result, the methanation, and boudouard reactions are taking place and consuming more CO2. Thus, higher CO production is also achieved resulting in increasing Fig. 9 Eﬀect of throat sizing on CO2, HHV, H2, and CO for air, and oxy-gasiﬁcation. Fig. 9 Eﬀect of throat sizing on CO2, HHV, H2, and CO for air, and oxy-gasiﬁcation. Fig. 9 Eﬀect of throat sizing on CO2, HHV, H2, and CO for air, and oxy-gasiﬁcation. © 2023 The Author(s). Published by the Royal Society of Chemistry © 2023 The Author(s). 3.5. Towards CO2 free gasication The aforementioned factors are all aﬀecting the yield of produced gas as illustrated by Fig. 11. Air has higher yield than oxy-gasication – although same ER – nitrogen content in the air tends to feed higher amounts of air than oxygen as a feeding medium for the same working condi- tions. As a result, this tends to increase the gasication eﬃciency for the same feedstock (eqn (11)). Lower throat ratios tend to produce higher velocities, temperatures, and heating values for produced gas as previously illustrated. As a result, this eﬀect leads to higher velocities near the exit of the gasier, and volume owrate for the producer gas, and correspondingly higher yield. On the other hand, lower throat ratios are found to produce higher syngas composition, which in turn favours higher heating values resulting in higher gasication eﬃciencies. As previously suggested in Fig. 6 and 8, and in the current gure, the optimum throat ratio is r = 0.14. At r = 0.14, the gasication eﬃciency increased that the base design case (r = 0.28) by 32, 37% for oxy, and air gasication respectively. While the producer gas yield is found to increase at the optimum throat ratio than the base case by 22, 19% for oxy, and air gasication respectively. Air and oxy-gasication producer gas yield are ranging between (1.9–2.4), and (0.88– 1.1) Nm3 kg−1 of biomass respectively. Additionally, the gasi- cation eﬃciency ranges between (54–79)%, and (45–68)% for air and oxy-gasication respectively. The results meet fair agree- ment with literature data of ref. 68, 69, and 70. The research also aims to increase the amounts of H2 and CH4 which in turn increase the heating value as shown in the gure. Lower heating values with lower syngas composition is noted for air compared to oxygen gasication. This is because of the N2 dilution in air gasication (∼50%). On the other hand, oxygen tends to increase the production of CO, H2, and CH4 as shown in the gure. The smallest throat ratio, with lower ER of 0.2, leads to the highest amounts produced from CO, H2, and CH4 which increase the heating value to the maximum 12.7 MJ Nm−3. As discussed earlier, decreasing the throat ratio, leads to higher residence time, higher temperature, better mixing, and turbulence. All the previous mentioned factors lead to higher production of CO, H2, and CH4 which further increases the heating value. 3.5. Towards CO2 free gasication Also, H2 is consumed because of higher residence time and in the presence of CO2 to be further converted into CO (CO2 + H2 / CO + H2O). Consequently, this aﬀects the concentration of other species leading to decrease of H2. Although H2 is decreasing, the While varying the throat ratio, the amounts of CO2 produc- tion show similar amounts for both air and oxygen. However, it shows small amounts of CO2 during air gasication (CO2 ∼5.7– 6 mol%). This is mainly due to the throttling which tends to increase the residence time inside the gasier, temperature Fig. 10 Eﬀect of changing ER on syngas production for (a) air, and (b) oxy-gasiﬁcation. This article is licensed under a Fig. 10 Eﬀect of changing ER on syngas production for (a) air, and (b) oxy-gasiﬁcation. Fig. 10 Eﬀect of changing ER on syngas production for (a) air, and (b) oxy-gasiﬁcation. 10232 \| RSC Adv., 2023, 13, 10221–10238 © 2023 The Author(s). Published by the Royal Society of Chemistry © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper (Fig. 7) and gives the opportunity to boudouard reaction to take place, and more CO2 consumption. the corresponding heating value for air and oxy-gasication. However, a full understanding of the process should include the yield of produced gas and the gasication eﬃciency for full understanding of the whole process. Gasication eﬃciency is calculated as follows:38 the corresponding heating value for air and oxy-gasication. However, a full understanding of the process should include the yield of produced gas and the gasication eﬃciency for full understanding of the whole process. Gasication eﬃciency is calculated as follows:38 p p Fig. 10b also shows the same eﬀect of CO2 reduction while reducing the ER and using smaller throat ratio. However, oxygen tends to produce more CO2 than air gasication for the same working parameters (ER, Feedstock, and throat ratio). Nitrogen free gasifying mediums (oxygen) tends to produce higher concentrations of other components. As a result, higher CO2 production than air gasication. Additionally, slight changes in all gas composition and the corresponding heating value were reported in this case (r = 0.14), irrespective to the change of ER. For the same ER, the change of r from 0.28 to 0.14 results in increase in CO and HHV by 41% and 8% respectively, while reducing CO2 and H2 concentrations by 53% and 16% respectively. 3.5. Towards CO2 free gasication This in general tends to increase HHV, though H2 concentration is decreasing. As a result, the throat change has an eﬀect on increasing syngas heating value and reducing CO2 emissions. Lower ER tends to produce more CO, H2, CH4, resulting in higher HHV. However, particular to note for the lower throat ratio of 0.14 that ER eﬀect is found to be small (Fig. 10b). This is because of the throttling eﬀect which consumes higher amounts of CO2, H2, and CH4 resulting in higher production of CO as previously illustrated in Fig. 6 and 8. Nevertheless, this eﬀect was not clear in air gasication because of the nitrogen dilution in the gasifying medium. However, in oxy-gasication, since the optimal condition was achieved at r = 0.14, the maximum production of CO with lowest amounts of CO2 was achieved (regardless of ER change). Moreover, lower throat ratio is associated with higher combustion and gasi- cation temperatures, and reaction rates (Fig. 7) even at lower ER, which favours the CO formation and results in HHV increase as ER increases from 0.2 to 0.35 and results in decrease of CO,H2, and HHV by 3.5%, 7.5%, and 7.3% respectively. Simultaneously, this results in CO2 reduction by 11%. hth ¼ GpQg Qb ; (9) (9) where Qg is the syngas LHV in (MJ Nm−3), Gp is the produced gas yield in Nm3 kg−1, and Qb is the biomass LHV in MJ kg−1 and estimated as following.68 Qb = 0.339 C + 1.029 H + 0.109 S −0.112 O −0.025 W (10) Qg = 0.126 CO + 0.108 H2 + 0.358 CH4 (11) (11) where C, H, O, S are the elemental composition of the feedstock, and W is the moisture content. While CO, H2, and CH4 are the volume fraction of diﬀerent species in the producer gas. The results illustrated by Fig. 11 depict the eﬀect of changing throat ratio on the producer gas yield, and the gasication eﬃ- ciency for rubber wood at xed ER = 0.3, and MC 18.5%. Under a certain ER, the model uses xed owrate of biomass and gasifying medium no matter the throat ratio changes, resulting in the same owrate for all cases. However, the throat ratio changes lead to a change in temperature, velocity, and diﬀerent gas species concentrations, and the corresponding heating value of the produced gas (Fig. 4, 6, 7, and 8). 3.5. Towards CO2 free gasication Furthermore, the highest heating value in the current work is obviously higher than previous works using oxy- gasication e.g. ref. 67 (10.1 MJ Nm−3), ref. 41 (10.12 MJ Nm−3) and ref. 61 (11 MJ Nm−3). This is because of the eﬀect of throat ratio on the gasication process. 3.7. Carbon conversion The throat diameter change has a great impact on the producer gas quality (Fig. 6, 8, and 10) including gas composition, and Carbon is the main component during the process gasication. As a result, the carbon conversion from the biomass to the © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 10221–10238 \| 10233 RSC Advances Fig. 11 Producer gas yield (a), and gasiﬁcation eﬃciency (b) for air and oxy-gasiﬁcation. n Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. ucer gas yield (a), and gasiﬁcation eﬃciency (b) for air and oxy-gasiﬁcation. Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 11 Producer gas yield (a), and gasiﬁcation eﬃciency (b) for air and oxy-gasiﬁcation. product gas is represented by carbon conversion eﬃciency hcc. Carbon conversion eﬃciency is the proportion of converted carbon into gases (in producer gas) to the total amount of carbon in the feedstock and is estimated from ref. 71 and 72 as following. this tends to increase the conversion of carbon during air gasication. Lower throat ratios are associated with higher amount of carbon fraction in producer gas (Fig. 6 and 8) and higher yield of syngas, resulting in higher carbon conversion than higher throat ratios. The carbon conversion during air and oxy- gasication is ranging between (71–98), and (55–82)% respec- tively. For the optimum throat ratio, carbon conversion is higher than the design/base case by 28.8, and 33% for air, and oxy-gasication respectively. This nds a strong agreement with previous works of ref. 71, 72, 73. hcc ¼ 12 ðCO þ CO2 þ CH4Þ Gp 22:4 C 100 (12) (12) where CO, CO2, CH4 are the volume concentrations of diﬀerent species in the producer gas, C is the carbon concentration in the feedstock, and Gp is the yield of producer gas. Fig. 12 represents the carbon conversion eﬃciency during rubber wood gasication. Air and oxygen are used as gasifying mediums under the same working conditions of ER = 0.3, and MC = 18.5%. Fixed working parameters are used for easier comparison between air and oxy-gasication, and during throat ratio change. Air yields higher conversion eﬃciencies than oxygen under all cases. © 2023 The Author(s). Published by the Royal Society of Chemistry Nomenclature Upper case letters A Pre-exponential factor, (units vary) D Diameter (m) Di,m Mass diﬀusion coeﬃcient for species i in the mixture DT,i Thermal diﬀusion coeﬃcient for species i Dt Turbulent diﬀusivity E Energy, (kJ mol−1) Fi External body forces, (N) Gb Turbulence kinetic energy due to buoyancy Gk Turbulence kinetic energy due to the mean velocity gradients H Enthalpy, (kJ mol−1) I Unit tensor Ji Diﬀusion ux of species i K Kinetic constant, (s−1) M Molecular mass, (kg mol−1) P Pressure, (Pa) R Net rate of formation, (mol m−3 s−1) Re Reynolds number Ri Net rate of production of species i by chemical reaction Sk Source terms for the kinetic energy St Mass added to the continuous phase from the dispersed phase S3 Source terms for rate of dissipation Sct Schmidt number for turbulent ow T Temperature, (K) TR Temperature of radiation (K) V Volume (m3) Yi Mass fraction of species i YM Contribution of the uctuating dilatation in compressible turbulence to the overall dissipation rate Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:4 This article is licensed under a Creative Commons Attribution 3.0 U Fig. 12 Carbon conversion eﬃciency for air and oxy-gasiﬁcation. sizing is applicable in any downdraor updrasystem and independent on gasier size/capacity. downdrabiomass gasier (small industrial scale). However, the results derived from the model are applicable in both small and large industrial scales. The dry gas composition results are based on specic working conditions (ER, MC, feedstock) regardless of gasier scale (Fig. 6 and 8). Additionally, the results represented in (Fig. 11, and 12) for gasier performance are independent of the gasier capacity since gas yield (Nm3 per kg of biomass), and the eﬃciencies in %. As a result, the nd- ings represented by the current research could be applied in diﬀerent scales of gasiers and for multiple applications. © 2023 The Author(s). Published by the Royal Society of Chemistry 3.7. Carbon conversion Although carbon fraction in producer gas (CO + CO2 + CH4) is higher during oxy-gasication, but the yield of producer gas during air gasication is more than double oxy-gasication during same conditions (Fig. 11). As a result, The unit cost of natural gas was reported to be around 1–3 US$ per GJ.74,75 On the other hand, for the syngas produced by oxy-gasication, the unit cost is estimated to be 2.0 US$ per GJ. However, this requires a detailed economic study to evaluate the exact cost of the syngas based on feedstock, gasifying agent, technology, and maintenance. As a result, lower throat ratios are eﬀective in reducing CO2 emissions, boosting gasier performance, increasing syngas yield, HHV, gasication eﬃ- ciency, and achieves higher carbon conversion during the process gasication. The gasier model is based on a 20 kW © 2023 The Author(s). Published by the Royal Society of Chemistry 10234 \| RSC Adv., 2023, 13, 10221–10238 RSC Advances View Article Online Paper Fig. 12 Carbon conversion eﬃciency for air and oxy-gasiﬁcation. Fi 12 C b i ﬃi f i d iﬁ ti Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 4. Conclusions A CFD model was developed to investigate the eﬀects of varying gasifying mediums and throat ratios on the gasication process performance. Producer gas composition, heating value, CO2, N2, temperature, and velocity distributions were presented and discussed. The model is validated through mesh independency test, and then against results derived from experiment for the same gasier type, dimensions, feedstock, and working conditions. The results revealed higher heating value for oxy-gasication than air gasication. Additionally, 4 throat ratios were exam- ined in the current study (0.14, 0.23, 0.28, and 0.4) and lower throat ratios tend to increase the producer gas heating value, and temperature along the gasier. Lower throat ratios are also preferred when it comes to reducing CO2 amounts for air gasication. Furthermore, the lowest throat ratio resulted in a CO2 reduction of more than 55% and a 20% increase in HHV, as compared to the default cases used in previous designs. Furthermore, lowest throat ratio yields higher production of producer gas, gasication, and carbon conversion eﬃciency by 22, 37, and 33% respectively. As a result, the current study gives promising outcomes in reducing CO2 and N2 emissions in the gasication process without the need of using any lters, removal, or catalysts. Additionally, the change in design/throat Schmidt number for turbulent ow Contribution of the uctuating dilatation in compressible turbulence to the overall dissipation rate © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 10221–10238 \| 10235 View Article Online View Article Online Lower case letters gi Gravitational body forces h Convective heat transfer coeﬃcient (W m−2 K) hfg Latent heat (J kg−1) mp Mass of the particle (kg) xi Number of mole species Greek letters r Density P Summation D Change in state si,j Stress tensor m Molecular viscosity sk Turbulent Prandtl numbers for k s3 Turbulent Prandtl numbers for 3 mt Turbulent viscosity rp Density of the particle 3p Particle emissivity s Stefan Boltzmann constant, 5:67 108 kg s3K4 List of acronyms VOF Volume of uid MC Moisture content, (%) A/F Air to fuel ratio ER Equivalence ratio HHV Higher heating value (MJ Nm−3) Nm3 Normal cubic meter CFD Computational Fluid Dynamics DPM Discrete phase model PRESTO PREssure Staggering Option RANS Reynolds Averaged Navier–Stokes RSC Advances Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. Conﬂicts of interest 14 A. M. Salem and M. C. Paul, Syngas Production and Combined Heat and Power from Scottish Agricultural Waste Gasication – A Computational Study, Sustainability, 2022, 14(7), 3745. There are no conicts to declare. 4. Conclusions This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Paper 4 A. M. Sepe, J. Li and M. C. Paul, Assessing biomass steam gasication technologies using a multi-purpose model, Energy Convers. Manage., 2016, 126, 216–226. 5 J. Li, M. C. Paul and K. M. Czajka, Studies of ignition behaviour of biomass particles in a down-re reactor for improving co-ring performance, Energy Fuels, 2016, 30(7), 5870–5877. 6 J. Li, M. C. Paul, P. L. Younger, I. Watson, M. Hossain and S. Welch, Prediction of high-temperature rapid combustion behaviour of woody biomass particles, Fuel, 2016, 165, 205–2014. 7 S. Safarian, R. Unnp´orsson and C. Richter, A review of biomass gasication modelling, Renewable Sustainable Energy Rev., 2019, 110, 378–391. 8 X. Chen, S. Song, H. Li and G. Gözaydın, Expanding the Boundary of Biorenery: Organonitrogen Chemicals from Biomass, Acc. Chem. Res., 2021, 54(7), 1711–1722. 9 A. M. Salem, I. N. Zaini, M. C. Paul and W. Yang, The evolution and formation of tar species in a downdra gasier: Numerical modelling and experimental validation, Biomass Bioenergy, 2019, 130, 105377. 10 U. Kumar and M. C. Paul, CFD modelling of biomass gasication with a volatile break-up approach, Chem. Eng. Sci., 2019, 195, 413–422. 11 A. M. Salem, U. Kumar, A. N. Izaharuddin, H. Dhami, T. Sutardi, and M. C. Paul, Advanced numerical methods for the assessment of integrated gasication and CHP generation technologies, in Coal and Biomass Gasication, Energy, Environment, and Sustainability, Springer, 2018, pp. 307–330. 12 R. Budhathoki, Three zone modeling of Downdrabiomass Gasication: Equilibrium and nite Kinetic Approach, Msc Thesis, University of Jyv¨askyl¨a, 2003. 13 Z. A. Zainal, R. Ali, C. H. Lean and K. N. Seetharamu, Prediction of performance of a downdragasier using equilibrium modeling for diﬀerent biomass materials, Energy Convers. Manage., 2001, 42(12), 1499–1515. References 15 P. N. Sheth and B. V. Babu, Modeling and simulation of reduction zone of downdrabiomass gasier: Eﬀect of char reactivity factor, Energy Convers. Manage., 2006, 47, 2602–2611. 1 S. Safarian, S. Sattari, R. Unnthorsson and Z. Hamidzadeh, Prioritization of bioethanol production systems from agricultural and waste agricultural biomass using multi- criteria decision making, Biophys. Econ. Resour. Qual., 2019, 4, 4. 1 S. Safarian, S. Sattari, R. Unnthorsson and Z. Hamidzadeh, Prioritization of bioethanol production systems from agricultural and waste agricultural biomass using multi- criteria decision making, Biophys. Econ. Resour. Qual., 2019, 4, 4. 16 N. Bianco, M. C. Paul, G. B. E. Brownbridge, D. Nurkowski, A. M. Salem, U. Kumar, A. N. Bhave and M. Kra, Automated advanced calibration and optimization of thermochemical models applied to biomass gasication and pyrolysis, Energy Fuels, 2018, 32(10), 10144–10153. 2 C. Mondelli, G. Gözaydın, N. Yan and J. P´erez-Ram´ırez, Biomass valorisation over metal-based solid catalysts from nanoparticles to single atoms, Chem. Soc. Rev., 2020, 49, 3764–3782. 17 A. K. Sharma, Modeling and simulation of a downdra biomass gasier 1. Model development and validation, Energy Convers. Manage., 2011, 52, 1386–1396. 3 M. Mehrpooya, M. Khalili and M. M. Sharifzadeh, Model development and energy and exergy analysis of the biomass gasication process (Based on the various biomass sources), Renewable Sustainable Energy Rev., 2018, 91, 869–887. 18 U. Kumar and M. C. Paul, Sensitivity analysis of homogeneous reactions for thermochemical conversion of biomass in a downdragasier, Renewable Energy, 2020, 151, 332–341. 10236 \| RSC Adv., 2023, 13, 10221–10238 © 2023 The Author(s). Published by the Royal Society of Chemistry RSC Advances View Article Online RSC Advances View Article Online Paper Paper 19 A. M. Salem and M. C. Paul, CFD modelling of spatiotemporal evolution of detailed tar species in a downdragasier, Biomass Bioenergy, 2023, 168, 106656. 36 E. Monteiro, et al., Experimental and modeling studies of Portuguese peach stone gasication on an autothermal bubbling uidized bed pilot plant, Energy, 2018, 142, 862– 877. 20 R. Gupta, P. Jain and S. Vyas, CFD Modeling and Simulation of 10KWE Biomass DowndraGasier, Int. J. Curr. Eng. Technol., 2017, 7(4), 1446–1452. 37 S. Sivakumar, K. Pitchandi and E. Natarajan, Modelling and simulation of down drawood gasier, J. Appl. Sci., 2008, 8(2), 271–279. 21 M. Formica, S. Frigo and R. Gabbrielli, Development of a new steady state zero-dimensional simulation model for woody biomass gasication in a full scale plant, Energy Convers. ccess Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 38 D. Dzulfansyah, L. O. Nelwan and D. Wulandani, Computational uid dynamic analysis for desiging downdra-rice hush gasier, J. Keteknikan Pertanian, 2014, 2, 133–140. 22 N. Deng, A. Zhang, Q. Zhang, G. He, W. Cui, G. Chen, et al., Simulation analysis and ternary diagram of municipal solid waste pyrolysis and gasication based on the equilibrium model, Bioresour. Technol., 2017, 235, 371–379. 39 R. Kumar, N. Baba, S. Kumar and V. Vishnu, CFD simulation of down drabiomass gasier, Int. J. Appl. Eng., 2016, 3(1), 58–64. 40 S. Zhao, Y. Su, W. Wu, Y. Zhange, Y. Wang and Y. Luo, Numerical simulation of partial combustion for biomass tar elimination in two-stage gasier, J. Sustainable Bioenergy Syst., 2013, 3, 86–92. 23 S. Hameed, N. Ramzan, Z.-u Rahman, M. Zafar and S. Riaz, Kinetic modeling of reduction zone in biomass gasication, Energy Convers. Manage., 2014, 78, 367–373. 24 P. C. Roy, A. Datta and N. Chakraborty, Modelling of a downdrabiomass gasier with nite rate kinetics in the reduction zone, Int. J. Energy Res., 2009, 33, 833–851. 41 P. Prasertcharoensuk, D. A. Hernandez, S. J. Bull and A. Phan, Optimisation of a throat downdragasier for hydrogen production, Biomass Bioenergy, 2018, 116, 216–226. 25 T. K. Patra and P. N. Sheth, Biomass gasication models for downdragasier: A state-of-the-art review, Renewable Sustainable Energy Rev., 2015, 50, 583–593. 42 L. Montuori, C. Salgado and M. Ortega, Impact of the throat sizing on the operating parameters in an experimental xed bed gasier: Analysis, evaluation and testing, Renewable Energy, 2015, 83, 615–625. 26 A. Ramos, E. Monteiro and A. Rouboa, Numerical approaches and comprehensive models for gasication process: A review, Renewable Sustainable Energy Rev., 2019, 110, 188–206. 43 D. Gunarathne, S. S. Jatunarachchi, N. S. Senanayake and B. Wei, The Eﬀect of Throat Diameter on the Performance a DowndraBiomass Gasier, Int. J. Energy Eng., 2013, 3(3), 171–175. 27 R. I. Singh, A. Brink and M. Hupa, CFD modeling to study uidized bed combustion and gasication, Appl. Therm. Eng., 2013, 52(2), 585–614. 44 A. N. Azlan, et al., Three Dimensional CFD Simulation of Air- Blown Gasication in a DowndraReactor: Eﬀect of Throat Diameter and Air Inlet Position, Fuel Mixture Form. Combust. Process, 2021, 3(1), 1–8. 28 A. Ramos, et al., Co-gasication and recent developments on waste-to-energy conversion: a review, Renewable Sustainable Energy Rev., 2018, 81, 380–398. 29 L. Yu, J. Lu, X. Zhang and S. References Manage., 2016, 120, 358–369. ccess Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 10221–10238 \| 10237 View Article Online View Article Online RSC Advances Paper Paper Paper critical review on reactors, catalysts, catalytic mechanisms and mathematical models, Renewable Sustainable Energy Rev., 2019, 116, 109426. 52 N. Ngamsidhiphongsa, P. Ponpesh, A. Shotipruk and A. Arpornwichanop, Analysis of the Imbert downdra gasier using a species-transport CFD model including tar- cracking reactions, Energy Convers. Manage., 2020, 213, 112808. 65 R. C. Saxena, D. Seal, S. Kumar and H. B. Goyal, Thermo- chemical routes for hydrogen rich gas from biomass: a review, Renewable Sustainable Energy Rev., 2008, 12, 1909–1927. 53 W. H. Lee, A Pressure Iteration Scheme for Two-Phase Modeling, Technical Report LA-UR 79-975, Los Alamos Scientic Laboratory, Los Alamos, New Mexico, 1979. Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 66 Y. Kalincia, A. Hepbasli and I. Dincer, Biomass-based hydrogen production: A review and analysis, Int. J. Hydrogen Energy, 2009, 34, 8799–8817. 54 C. A. Koufopanosi, G. Maschio and A. Lucchesit, Kinetic Modelling of the Pyrolysis of Biomass and Biomass Components, Can. J. Chem. Eng., 1989, 67, 75–84. 67 L. Hongtao, C. Feng, P. Xia, Y. Kai and L. Shuqin, Method of oxygen-enriched two-stage underground coal gasication, Min. Sci. Technol., 2011, 21, 191–196. 55 T. H. Jayah, L. Aye, R. J. Fuller and D. F. Stewart, Computer simulation of a downdrawood gasier for tea drying, Biomass Bioenergy, 2003, 25, 459–469. Open Access Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42 This article is licensed under a Creative Commons Attribution 3.0 Unp 68 C. Gai and Y. Dong, Experimental study on non-woody biomass gasication in a downdra gasier, Int. J. Hydrogen Energy, 2012, 37(6), 4935–4944. 56 A. Dufour, E. Masson, P. Girods, Y. Rogaume and A. Zoulalian, Synthesis gas production by biomass pyrolysis: Eﬀect of reactor temperature on product distribution, Int. J. Hydrogen Energy, 2009, 43, 1726–1734. 69 A. Anukam, S. Mamphweli, P. Reddy, E. Meyer and O. Okoh, Pre-processing of sugarcane bagasse for gasication in a downdrabiomass gasier system: A comprehensive review, Renewable Sustainable Energy Rev., 2016, 36, 775–801. 57 M. A. Chawdhury and K. Mahkamov, Development of a Small Downdra Biomass Gasier for Developing Countries, J. Sci. Res., 2011, 3(1), 51–64. 70 M. ccess Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Zhang, Numerical simulation of the bubbling uidized bed coal gasication by the kinetic theory of granular ow (KTGF), Fuel, 2007, 86, 722–734. 45 M. Simone, F. Barontini, C. Nicolella and L. Tognotti, Gasication of pelletized biomass in a pilot scale downdragasier, Bioresour. Technol., 2012, 116, 403–412. 30 D. F. Fletcher, B. S. Haynes, F. C. Christo and S. D. Joseph, A CFD based combustion model of an entrained ow biomass gasier, Appl. Math. Models, 2000, 24, 165–182. 46 Z. Zhang and S. Pang, Experimental investigation of tar formation and producer gas composition in biomass steam gasication in a 100 kW dual uidised bed gasier, Renewable Energy, 2019, 132, 416–424. 31 A. M. Salem and M. C. Paul, An integrated kinetic model for downdragasier based on a novel approach that optimises the reduction zone of gasier, Biomass Bioenergy, 2018, 109, 172–181. 47 T. Hanaoka, S. Hiasa and Y. Edashige, Syngas production by CO2/O2 gasication of aquatic biomass, Fuel Process. Technol., 2013, 116, 9–15. 48 H. Wibowo, et al., Recent developments of deep eutectic solvent as absorbent for CO2 removal from syngas produced from gasication: Current status, challenges, and further research, J. Environ. Chem. Eng., August 2021, 9(4), 105439. 32 M. Vascellari, et al., From laboratory-scale experiments to industrial-scale CFD simulations of entrained ow coal gasication, Fuel, 2015, 152, 58–73. 33 B. Dou and Y. Song, A CFD approach on simulation of hydrogen production from steam reforming of glycerol in a uidized bed reactor, Int. J. Hydrogen Energy, 2010, 35(19), 10271–10284. 49 K. Rahimi, S. Riahi, M. Abbasi and Z. Fakhroueian, Modication of multi-walled carbon nanotubes by 1,3- diaminopropane to increase CO2 adsorption capacity, J. Environ. Manage., 2019, 242, 81–89. 34 A. Klimanek, et al., Towards a hybrid Eulerian-Lagrangian CFD modeling of coal gasication in a circulating uidized bed reactor, Fuel, 2015, 152, 131–137. 50 P. C. Murugan and S. J. Sekhar, Species–Transport CFD model for the gasication of rice husk (Oryza Sativa) using downdragasier, Comput Electron Agric., 2017, 139, 33–40. 35 N. Couto, et al., Using an Eulerian-granular 2-D multiphase CFD model to simulate oxygen air enriched gasication of agroindustrial residues, Renewable Energy, 2015, 77, 174– 181. 51 ANSYS 15 Fluent Theory Guide, Canonsburg, PA 15317, 2013. © 2023 The Author(s). © 2023 The Author(s). Published by the Royal Society of Chemistry ccess Article. Published on 04 April 2023. Downloaded on 10/24/2024 5:42:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Asadullah, Barriers of commercial power generation using biomass gasication gas: a review, Renewable Sustainable Energy Rev., 2014, 29, 201–215. 58 J. Xie, W. Zhong, B. Jin, Y. Shao and H. Liu, Simulation on gasication of forestry residues in uidized beds by Eulerian Lagrangian approach, Bioresour. Technol., 2010, 121, 36–46. 71 Z. Wang, et al., Gasication of biomass with oxygen-enriched air in a pilot scale two-stage gasier, Fuel, 2015, 150, 386– 393. 59 B. L. Gomez-Barea, Modeling of biomass gasication in uidized bed, Prog. Energy Combust. Sci., 2010, 36, 444–509. 72 F. Guo, Y. Dong, L. Dong and C. Guo, Eﬀect of design and operating parameters on the gasication process of biomass in a downdraxed bed: An experimental study, Int. J. Hydrogen Energy, 2014, 39(11), 5625–5633. 60 J. Tomeczek, Z. Jastrzçab and B. Grado´n, Lateral diﬀusion of solids in a uidized bed with submerged vertical tubes, Powder Technol., 1992, 72(1), 17–22. 73 F. M. Guangul, S. A. Sulaiman and A. Ramli, Gasier selection, design and gasication of oil palm fronds with preheated and unheated gasifying air, Bioresour. Technol., 2012, 126, 224–232. 61 P. Nakod, Modeling and validation of oxy-red and air-red entrained ow gasiers, Int. J. Chem. Phys. Sci., 2013, 2(6), 2074–2091. 62 M. Barrio and M. Fossum, Operational characteristics of a small-scale stratied downdragasier, in Technologies and Combustion for a Clean Environment Sixth International Conference, 2001. 74 T. Blumberg, G. Tsatsaronis and T. Morosuk, On the economics of methanol production from natural gas, Fuel, 2019, 256, 115824. 63 T. H. Jayah, L. Aye, R. J. Fuller and D. F. Stewart, Computer simulation of a downdrawood gasier for tea drying, Biomass Bioenergy, 2003, 25, 459–469. 75 T. Nakyai and D. Saebea, Exergoeconomic comparison of syngas production from biomass, coal, and natural gas for dimethyl ether synthesis in single-step and two-step processes, J. Cleaner Prod., 2019, 241, 118334. 64 J. Ren, J. Cao, X. Zhao, F. Yang and X. Wei, Recent advances in syngas production from biomass catalytic gasication: A 10238 \| RSC Adv., 2023, 13, 10221–10238 © 2023 The Author(s). Published by the Royal Society of Chemistry
https://openalex.org/W2594791763	https://amt.copernicus.org/articles/11/6589/2018/amt-11-6589-2018.pdf	English	null	Joint retrieval of surface reflectance and aerosol properties with continuous variation of the state variables in the solution space – Part 1: theoretical concept	Atmospheric measurement techniques	2,018	cc-by	11,919	Joint retrieval of surface reﬂectance and aerosol properties with continuous variation of the state variables in the solution space – Part 1: theoretical concept Yves Govaerts and Marta Luffarelli Rayference, 1030 Brussels, Belgium Correspondence: Yves Govaerts (yves.govaerts@rayference.eu) Received: 29 January 2017 – Discussion started: 7 March 2017 Revised: 26 November 2018 – Accepted: 30 November 2018 – Published: 14 December 2018 Correspondence: Yves Govaerts (yves.govaerts@rayference.eu) Received: 29 January 2017 – Discussion started: 7 March 2017 Revised: 26 November 2018 – Accepted: 30 November 2018 – Published: 14 December 2018 Received: 29 January 2017 – Discussion started: 7 March 2017 Revised: 26 November 2018 – Accepted: 30 November 2018 – Published: 14 December 2018 to solve a radiative system composed of at least two sets of layers, where the upper layers include aerosols and the bot- tom ones represent the soil–vegetation strata. The problem can be further complicated by the intrinsic anisotropic radia- tive behaviour of natural surfaces due to the mutual shadow- ing of the scattering elements, which is also affected by the amount of incident radiation (Govaerts et al., 2010b, 2016). In most cases, an increase in aerosol concentration is respon- sible for an increase in the fraction of diffuse sky radiation which, in turn, smooths the effects of surface reﬂectance anisotropy. Though multispectral information is critical for the retrieval of aerosol properties, the spectral dimension alone does not allow full characterisation of the underlying surface reﬂectance, which often offers a signiﬁcant contribu- tion to the total signal observed at the satellite level. In this regard, the additional information contained in multispectral and multi-angular observations has proven essential to char- acterising aerosol properties over land surfaces. Abstract. This paper presents a new algorithm for the joint retrieval of surface reﬂectance and aerosol properties with continuous variations of the state variables in the solution space. This algorithm, named CISAR (Combined Inversion of Surface and AeRosol), relies on a simple atmospheric ver- tical structure composed of two layers and an underlying sur- face. Surface anisotropic reﬂectance effects are taken into ac- count and radiatively coupled with atmospheric scattering. For this purpose, a fast radiative transfer model has been ex- plicitly developed, which includes acceleration techniques to solve the radiative transfer equation and to calculate the Ja- cobians. The inversion is performed within an optimal esti- mation framework including prior information on the state variable magnitude and regularisation constraints on their spectral and temporal variability. Joint retrieval of surface reﬂectance and aerosol properties with continuous variation of the state variables in the solution space – Part 1: theoretical concept In each processed wave- length, the algorithm retrieves the parameters of the surface reﬂectance model, the aerosol total column optical thickness and single-scattering properties. The CISAR algorithm func- tioning is illustrated with a series of simple experiments. Pinty et al. (2000a) pioneered the development of a re- trieval method dedicated to the joint retrieval of surface re- ﬂectance and aerosol properties based on the inversion of a physically based radiative model. This method has been subsequently improved to permit the processing of any geo- stationary satellites accounting for their actual radiometric performance (Govaerts and Lattanzio, 2007). This new ver- satile version of Pinty’s algorithm has permitted the gener- ation of a global surface albedo product from archived data acquired by operational geostationary satellites around the globe (Govaerts et al., 2008). These data included obser- vations acquired by an old generation of radiometers with only one broad solar channel on board the European Me- teosat First Generation satellite, the US Geosynchronous Op- Atmos. Meas. Tech., 11, 6589–6603, 2018 https://doi.org/10.5194/amt-11-6589-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 3.0 License. Atmos. Meas. Tech., 11, 6589–6603, 2018 https://doi.org/10.5194/amt-11-6589-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 3.0 License. 1 Introduction Radiative coupling between atmospheric scattering and sur- face reﬂectance processes prevents the use of linear relation- ships for the retrieval of aerosol properties over land surfaces. The discrimination between the contribution of the signal re- ﬂected by the surface and that scattered by aerosols repre- sents one of the major issues when retrieving aerosol prop- erties using space-borne passive optical observations over land surfaces. Conceptually, this problem can be modelled 2 Lessons learned from previous approaches Even considering a large number of aerosol models, LUT-based approaches underperform com- pared to methods with multivariate continuity in the solution space (Kokhanovsky et al., 2010). The GSA algorithm has been further improved for the pro- cessing of SEVIRI data on board MSG for the retrieval of the total column AOT from observations acquired in three solar bands centred at 0.6, 0.8 and 1.6 µm (Govaerts et al., 2010b; Wagner et al., 2010). The method developed by these authors relies on an OE approach wherein surface reﬂectance and daily aerosol load are simultaneously retrieved. The in- version is performed independently for each aerosol model and the one with the smallest cost function is selected. A physically based radiative transfer model accounting for non- Lambertian surface reﬂectance and its radiative coupling with atmospheric scattering is inverted against daily accumu- lated SEVIRI observations. However, this land daily aerosol (LDA) algorithm suffers from two major limitations: (i) the use of predeﬁned aerosol models, and (ii) the algorithm de- livers only one mean aerosol value per day when applied to MSG SEVIRI data (Govaerts et al., 2010a). This latter issue has been addressed by Luffarelli et al. (2016), who retrieve an aerosol optical thickness value for each SEVIRI obser- vation. The former issue prevents a continuous variation of the state variables characterising the aerosol single-scattering properties as required to ﬁnd the minimum of the cost func- tion. A consistent implementation of such an approach is not straightforward since aerosol models are deﬁned as prior knowledge of the observed medium but uncertainties cannot A new joint surface reﬂectance–aerosol properties re- trieval approach is presented here that overcomes the limi- tations resulting from precomputed RTE solutions stored in LUTs. The advantages of a continuous variation of the aerosol properties in the solution space against a LUT-based ap- proach is discussed in Sect. 3. The proposed method ex- presses the single-scattering albedo and phase function val- ues as a linear mixture of basic aerosol models. The forward radiative transfer model that includes the Jacobians, i.e. the partial derivative, is described in Sect. 4. With the excep- tion of gaseous transmittance, this model no longer relies on LUTs, and the RTE is explicitly solved. The inversion method is described in Sect. 5. Finally, the ability to express aerosol single-scattering properties as a linear combination is in illustrated Sect. 2 Lessons learned from previous approaches Pinty et al. (2000a) proposed an algorithm for the joint retrieval of surface reﬂectance and aerosol properties to demonstrate the possibility of generating essential climate variables (ECVs) from data acquired by operational weather geostationary satellites. Due to limited operational compu- tational resources available at that time in the EUMETSAT ground segment, where the data were processed, the devel- opment of this algorithm was subject to strong constraints. The RTE solutions were precomputed and stored in LUTs with a very coarse resolution, limiting the maximum aerosol optical thickness (AOT) to 1, which represented a severe lim- itation over the Sahara region where AOT values can eas- ily exceed such a limit. Furthermore, the radiative coupling between aerosol scattering and gaseous absorption was not taken into account. This algorithm, referred to as geostation- ary surface albedo (GSA), has been subsequently modiﬁed by Govaerts and Lattanzio (2007) to include an estimation of the retrieval uncertainty. This updated version has permit- ted the generation of a global aerosol product derived from observations acquired by operational weather geostationary satellites (Govaerts et al., 2008). Since then, it has been rou- tinely applied in the framework of the SCOPE-CM initiative to generate a climate data record (CDR) of surface albedo (Lattanzio et al., 2013). The strengths and weaknesses of the algorithm proposed by Govaerts et al. (2010b) are discussed in Sect. 2. In their approach, the solutions of the radiative transfer equation (RTE) are pre-calculated and stored in look-up tables (LUTs) for a limited number of state variable values. Aerosol prop- erties are limited to six different models dominated either by ﬁne or coarse particles. Two major drawbacks result from the use of predeﬁned aerosol models stored in precomputed LUTs. Firstly, only a limited region of the solution space is sampled as a result of the reduced range of variability for state variables stored in the LUTs. For instance, in order to reduce the size of the LUTs, Pinty et al. (2000b) limit the maximum aerosol optical thickness to 1. Secondly, the use of predeﬁned aerosol models constitutes a major drawback since the solution space is not continuously sampled. Dubovik et al. (2011) and Diner et al. (2012), among others, demonstrated the advantages of a retrieval approach based on continuous variations of the aerosol properties as opposed to a LUT-based approach relying on a set of pre- deﬁned aerosol models. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol satellite data are addressed in Luffarelli and Govaerts (2018) (hereafter referred to as Part 2). erational Environmental Satellite (GOES) and the Japanese Geostationary Meteorological Satellite (GMS). It is now rou- tinely applied in the framework of the Sustained and COor- dinated Processing of Environmental satellite data for Cli- mate Monitoring (SCOPE-CM) initiative for the generation of essential climate variables (Lattanzio et al., 2013). An im- proved version of this algorithm has been proposed by Go- vaerts et al. (2010b) to take advantage of the multispectral capabilities of Meteosat Second Generation Spinning En- hanced Visible and Infrared Imager (MSG SEVIRI) operated by EUMETSAT and includes an optimal estimation (OE) in- version scheme using a minimisation approach based on the Marquardt–Levenberg method (Marquardt, 1963). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 6590 Atmos. Meas. Tech., 11, 6589–6603, 2018 3 Continuous variation of aerosol properties in the solution space be easily assigned to this decision. Consequently, the esti- mated retrieval uncertainty is inconsistent as it does not ac- count for the use of prior information and its associated un- certainties. Aerosol single-scattering properties include the single- scattering albedo ω0 and the phase function 8 in RTE. Gov- aerts et al. (2010b) explained the beneﬁts of representing pre- deﬁned aerosol models in a two-dimensional solution space composed of these aerosol single-scattering properties. For the sake of clarity, they limited the phase function in that 2- D space to the ﬁrst term of the Legendre coefﬁcients, i.e. the asymmetry parameter g. However, one should keep in mind that the reasoning applied in this section should be applied to the entire phase function 8. These aerosol single-scattering properties are themselves determined by aerosol microphys- ical properties, such as the particle size distribution, shape and their complex index of refraction. The objective of re- trievals that assume aerosol models is to provide a reasonable sampling of the {g,ω0} space. Omitting areas of that space may produce biased retrievals, as discussed in Govaerts et al. (2010b). The inversion process proposed by in this paper re- lies on a set of six models which have been deﬁned from AErosol RObotic NETwork (AERONET) data aggregation (Dubovik et al., 2006). That choice of models was intended to provide a sampling of solution space representative of real- world conditions. The inversion is repeated for each aerosol class and the result with the best ﬁt is reported, rather than having continuously varying aerosol properties, as would be preferable. Diner et al. (2012) demonstrated the advantages of a re- trieval method based on continuous variations of aerosol single-scattering properties in the solution space as opposed to a LUT-based approach derived for a limited number of predeﬁned aerosol models. Dubovik et al. (2011) proposed an original method for the retrieval of aerosol microphys- ical properties, which also does not necessitate the use of predeﬁned aerosol models. This method retrieved more than 100 state variables, therefore requiring a considerable num- ber of observations, such as those provided by multi-angular and -polarisation radiometers like Polarisation et Anisotropie des Réﬂectances Au SOmmet de l’Atmosphère (PARASOL) (Serene and Corcoral, 2006) or the future Multi-viewing Multi-channel Multi-polarization Imaging (3MI) instrument on board EUMETSAT’s Polar System Second Generation (Manolis et al., 2013). 2 Lessons learned from previous approaches 6 with simulated data representing var- ious scenarios, including small and large particles. Practical aspects of the application of the CISAR algorithm for the retrieval of both surface and aerosol properties from actual www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 6591 Figure 1. Aerosol dual-mode models based on Govaerts et al. (2010b) in the {g,ω0} space derived from the aggregation of aerosol single-scattering properties retrieved from AERONET observations (Dubovik et al., 2006). Classes 1 to 3 are dominated by the ﬁne mode and 4 to 6 by the coarse one. Figure 2. Example of sensitivity of aerosol single-scattering prop- erties to particle median radius (green arrows) and imaginary part of the refractive index (red arrows) at 0.44 and 0.87 µm for ﬁne-mode, F (rmf = 0.1 µm), and coarse mode, C (rmc = 2.0 µm). The length of the arrows reﬂects the magnitude of the change. Figure 2. Example of sensitivity of aerosol single-scattering prop- erties to particle median radius (green arrows) and imaginary part of the refractive index (red arrows) at 0.44 and 0.87 µm for ﬁne-mode, F (rmf = 0.1 µm), and coarse mode, C (rmc = 2.0 µm). The length of the arrows reﬂects the magnitude of the change. Aerosol dual-mode models based on Govaerts et a Figure 1. Aerosol dual-mode models based on Govaerts et al. (2010b) in the {g,ω0} space derived from the aggregation of aerosol single-scattering properties retrieved from AERONET observations (Dubovik et al., 2006). Classes 1 to 3 are dominated by the ﬁne mode and 4 to 6 by the coarse one. www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRos The isolines show the probability that the aerosol single- scattering properties derived from AERONET observations with the method of Dubovik et al. (2006) fall within the delineated spaces. scattering albedo and phase function values derived from AERONET observations acquired in a speciﬁc region of in- terest for a given period (Dubovik et al., 2006). The red iso- line in Fig. 3 delineates the area [g,ω0] of the solution space where 99.7 % of the aerosol single-scattering properties de- rived by Dubovik et al. (2006) from AERONET observations are located. The green and blue lines respectively show the 95 % and 68 % probability regions. These values have been derived using all available level 2 AERONET observations since 1993. Finally, the model proposed by Schuster et al. (2005) can be used to determine the spectral variations of the single-scattering properties outside the spectral bands mea- sured by AERONET. This work relies on simulated data and the aerosol vertices have been positioned to sample the solu- tion space in a realistic way. When processing actual satellite data over a speciﬁc region or period, it is advised to cal- culate the isolines corresponding to that region of interest from AERONET observations and to adjust the position of the aerosol vertices accordingly as performed in Part 2. scattering albedo and phase function values derived from AERONET observations acquired in a speciﬁc region of in- terest for a given period (Dubovik et al., 2006). The red iso- line in Fig. 3 delineates the area [g,ω0] of the solution space where 99.7 % of the aerosol single-scattering properties de- rived by Dubovik et al. (2006) from AERONET observations are located. The green and blue lines respectively show the 95 % and 68 % probability regions. These values have been derived using all available level 2 AERONET observations since 1993. Finally, the model proposed by Schuster et al. (2005) can be used to determine the spectral variations of the single-scattering properties outside the spectral bands mea- sured by AERONET. This work relies on simulated data and the aerosol vertices have been positioned to sample the solu- tion space in a realistic way. When processing actual satellite data over a speciﬁc region or period, it is advised to cal- culate the isolines corresponding to that region of interest from AERONET observations and to adjust the position of the aerosol vertices accordingly as performed in Part 2. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRos 6592 gions in the {g,ω0} space according to the dominant parti- cle size distribution, i.e. ﬁne or coarse. Within that space, an aerosol model is deﬁned by the spectral behaviour of the {g(λ),ω0(λ)} pairs, where λ indicates the wavelength. The proposed ﬁne-mode models vary mostly as a function of ω0, which is largely determined by the imaginary part of the re- fractive index ni. Conversely, aerosol models dominated by coarse particles show little dependency on g and are there- fore organised parallel to the ordinate axis. The main param- eter discriminating these latter models is the median radius rm, which essentially determines the asymmetry parameter value at a given wavelength. To illustrate the dependence of g and ω0 on the median radius rm (http://eodg.atm.ox.ac.uk/user/grainger/research/ aerosols.pdf, last access: (9 December 2018) and imaginary part of the refractive index ni, ﬁne- and coarse-mono-mode aerosol models were generated with rm = 0.15 and 2.0 µm respectively. The other microphysical values have been ﬁxed to σr = 0.5 µm, nr = 1.42 and ni = 0.008, where σr is the ra- dius standard deviation and nr the real part of the refractive index. These values were selected to ease the explanation of the organisation of the aerosol models in Fig. 1. Black dots in Fig. 2 show the corresponding location of {g,ω0} at 0.44 and 0.87 µm. The magnitude of the red arrows illustrate the sensitivity to a ni change of ±0.0025 and the green ones to a rm change of ±25 %. For the ﬁne mono-mode (F), changes in ni essentially translate in displacement along the ω0 axis, while changes in rm result in changes almost parallel to the g axis. There is also a clear relationship between the particle size and g for that mode. A change in the particle size results in a change in g, while ω0 remains relatively unchanged. The situation is quite different for the coarse mono-mode, where changes in both ni and rm induce displacement parallel to the ω0 axis with limited impact on g values. Figure 3. Example of a region (light-blue area) in the {g,ω0} solu- tion space at 0.44 µm deﬁned by four aerosol vertices: single ﬁne- mode non-absorbing (FN), single ﬁne-mode absorbing (FA), coarse mode with small radius (CS) and coarse mode with large radius (CL). 3 Continuous variation of aerosol properties in the solution space Instruments delivering such a large number of observations are rather scarce, as most of the cur- rent passive optical sensors do not offer instantaneous multi- angular observation capabilities nor information on polariza- tion. The primary objective of this paper is to address the limitations resulting from conventional approaches based on LUTs and/or a limited number of predeﬁned aerosol models by proposing a method that can be applied to observations acquired by single or multi-view instruments. A visual inspection of Fig. 1 based on Govaerts et al. (2010b) reveals that aerosol models occupy different re- www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 M. Luffarelli: Combined Inversion of Surface and AeRosol Figure 3. Example of a region (light-blue area) in the {g,ω0} solu- tion space at 0.44 µm deﬁned by four aerosol vertices: single ﬁne- mode non-absorbing (FN), single ﬁne-mode absorbing (FA), coarse mode with small radius (CS) and coarse mode with large radius (CL). The isolines show the probability that the aerosol single- scattering properties derived from AERONET observations with the method of Dubovik et al. (2006) fall within the delineated spaces. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 4.1 Overview ym(x,b;m) = TLg(b;m)I ↑ s (x,b;m) + I ↑ m(x,b;m) E↓ 0 (m)µ0 , (1) where The forward model, named FASTRE, simulates the TOA bidirectional reﬂectance factor (BRF) ym(x,b;m) as a func- tion of the independent parameters m deﬁning the observa- tion conditions and a series of state variables x describing the state of the atmosphere and underlying surface. Model parameters b represent variables such as total column water vapour that inﬂuence the value of ym(x,b;m) but cannot be retrieved from the processed space-based observations due to the lack of information. The independent parameters m in- clude the illumination and viewing geometries (0,v) and the wavelength dependence. The RTE is solved with the ma- trix operator method (Fischer and Grassl, 1984) optimised by Liu and Ruprecht (1996) for a limited number of quadrature points. (1) where where – I ↑ s (x,b;m) is the upward radiance ﬁeld at level Za due to the single scattering, – I ↑ m(x,b;m) is the upward radiance ﬁeld at level Za due to the multiple scattering, – TLg(b;m) denotes the total transmission factor in the Lg layer, – E↓ 0 (m) denotes the solar irradiance at level Zs corrected for the Sun–Earth distance variations. The model simulates observations acquired within spec- tral bandseλ characterised by their spectral response. Gaseous transmittances in these bands are precomputed and stored in LUTs. The model computes the contributions from sin- gle and multiple scattering separately, the latter being solved in Fourier space. In order to reduce the computation time, the forward model relies on the same atmospheric vertical struc- ture as in Govaerts et al. (2010b), i.e. a three-level system containing two layers (Fig. 4). The lowest level, Z0, repre- sents the surface. The lower layer, La, ranging from levels Z0 to Za, contains the aerosol particles. Molecular scattering and absorption also take place in that layer, which is radia- tively coupled with the surface for both single and the mul- tiple scattering. The upper layer, Lg, ranging from Za to Zs, is only subject to molecular absorption. The single-scattering contribution is written The single-scattering contribution is written I ↑ s (x,b;m) = E↓ 0 (m)µ0 π exp −τ La µ0 rs(xs,b;m)exp −τ La µv , (2) (2) where τ La is the total optical thickness of layer La. µ0 and µv are the cosine of the illumination and viewing zenith an- gles respectively. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRos The actual extent of solutions in the {g,ω0} space for a given spectral band can be outlined by a series of vertices de- ﬁned by aerosol single-scattering properties (Fig. 3). Follow- ing Fig. 2, these vertices are deﬁned by absorbing and non- absorbing ﬁne mono-mode models with small radii of about 0.1 µm, labelled respectively FA and FN, and by two coarse mono-modes with different radii, i.e. large (1 µm) and small (0.3 µm), labelled respectively CL and CS. In Sect. 4, we will see how any pair of single-scattering albedo and phase func- tion values can be expressed as a linear combination of the vertex properties. The position of these vertices is critical as they should encompass the most likely aerosol single-scattering prop- erties that could be observed at a given time and location. Different approaches could be used to deﬁne the position of these vertices. The positions can be derived from the analysis of typical aerosol single-scattering properties avail- able in databases such as the Optical Properties of Aerosols and Clouds (OPAC) (Hess et al., 1998). Alternatively, it is also possible to follow a similar approach to the one pro- posed in Govaerts et al. (2010b), who analysed the single- www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 Y. Govaerts and M. Luffarelli: Combined Inversion of Sur Figure 4. Atmospheric vertical structure of the FASTRE model. The surface is at level Z0 and radiatively coupled with the lower layer La extending from level Z0 to Za. This layer includes scatter- ing and absorption processes. The upper layer, Lg, runs from level Za to Zs and only accounts for gas absorption processes. 6593 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol model characterised by four parameters, xs = {ρ0,k,2,ρc}, that are all wavelength dependent (Rahman et al., 1993). The ρ0 parameter, included in the [0,1] interval, controls the mean amplitude of the BRF and strongly varies with wavelengths. The k parameter is the modiﬁed Minnaert’s contribution that determines the bowl or bell shape of the BRF and typically varies between 0 and 2. The asymmetry parameter 2 of the Henyey–Greenstein phase function varies between −1 and 1. The ρc parameter controls the amplitude of the hotspot due to the “porosity” of the medium. This parameter varies between −1 and 1. 4 Forward radiative transfer model The FASTRE model expresses the TOA BRF in a given spectral band eλ as a sum of the single I ↑ s and multiple I ↑ m scattering contributions as in Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRos For the simulations over the ocean, the Cox–Munk model (Cox and Munk, 1954) is used as implemented in Ver- mote et al. (1997). Figure 4. Atmospheric vertical structure of the FASTRE model. The surface is at level Z0 and radiatively coupled with the lower layer La extending from level Z0 to Za. This layer includes scatter- ing and absorption processes. The upper layer, Lg, runs from level Za to Zs and only accounts for gas absorption processes. Figure 4. Atmospheric vertical structure of the FASTRE model. The surface is at level Z0 and radiatively coupled with the lower layer La extending from level Z0 to Za. This layer includes scatter- ing and absorption processes. The upper layer, Lg, runs from level Za to Zs and only accounts for gas absorption processes. Aerosol single-scattering properties in the layer La are represented by an external mixture of a series of predeﬁned aerosol vertices as explained in Sect. 4.2. The Lg layer only contains absorbing gas not included in the scattering layer, such as high-altitude ozone, the part of the total column wa- ter vapour not included in layer La and a few well-mixed gases. www.atmos-meas-tech.net/11/6589/2018/ 4.4 FASTRE model accuracy τa(eλ) = X v τv(eλ). (3) τa(eλ) = X v τv(eλ). (3) The simple atmospheric vertical structure composed of two layers is the principal assumption of the FASTRE model. In order to evaluate the accuracy of FASTRE, a similar proce- dure to that in Govaerts et al. (2010b) has been applied. The outcome of FASTRE has been evaluated against a more elab- orated 1-D radiative transfer model (RTM) (Govaerts, 2006) for sun and viewing angles varying from 0 to 70◦, for var- ious types of aerosols, surface reﬂectance and total column water vapour values. This reference RTM represents the ver- tical structure of the atmosphere with 50 layers. The mean relative bias and relative root mean square error (RMSE) be- tween the reference model and FASTRE have been estimated in the main spectral bands used for aerosol retrievals. The rel- ative RMSE, Rr, is estimated as The phase function 8v(eλ,g) is characterised by a limited number Nκ of Legendre coefﬁcients equal to 2Nθ −1. The decision to use this number results from a trade-off between accuracy and computational time. When Nκ is too small, the last Legendre moment is often not equal to zero and the delta- M approximation is applied (Wiscombe, 1977). In this case, the αd coefﬁcient of the delta-M approximation is equal to 8v(Nκ). The Legendre coefﬁcients κj become cj = κj −αd 1 −αd , (4) (4) and the truncated phase function is denoted by 8′ v. The cor- rected optical thickness τ ′ v(eλ) and single-scattering albedo ω′ 0,v(eλ) of the corresponding aerosol model become Rr = v u u t 1 N X N ym(x,b;m) −yr(x,b;r) yr(x,b;r) 2 , (10) (10) τ ′ v(eλ) = (1 −ω0,vαd)τv(eλ) (5) τ ′ v(eλ) = (1 −ω0,vαd)τv(eλ) (5) and τ ′ v(eλ) = (1 −ω0,vαd)τv(eλ) (5) and ′ (eλ) 1 −αd (eλ) (6) (5) and and where yr(x,b;m) is the TOA BRF calculated with the ref- erence model. In this paper, the FASTRE model solves the RTE using 16 quadrature points Nθ, which provides a good compromise between speed and accuracy. Results are shown in Table 1. The relative RMSE between FASTRE and the reference model is typically in the range of 1 %–3 %. An- other comparison of FASTRE has been made against actual Project for On-Board Autonomy-Vegetation (PROBA-V) ob- servations (Luffarelli et al., 2017). These comparisons show an RMSE in the range [0.024–0.038]. 4.2 Scattering layer La properties The layer La contains a set of mono-mode aerosol mod- els v characterised by their single-scattering properties, i.e. the single-scattering albedo ω0,v(eλ) and phase function 8v(eλ,g) where g represents the scattering angle. The dif- ferent vertices are combined into this layer according to their respective optical thickness τv(eλ) with the total aerosol opti- cal thickness τa(eλ) of the layer being equal to Spectral bands (µm) 0.44 0.55 0.67 0.87 Relative bias (%) −1.1 −0.3 0.0 +0.3 Relative RMSE (%) 2.8 1.8 1.3 1.2 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Table 1. Relative bias and root mean square error in percentage between FASTRE and the reference RTM in various spectral bands. 5.1 Overview 8′(eλ,g) = P c8′ v(eλ,g)τ ′ v(eλ) τ ′a(eλ) . (9) (9) Surface reﬂectance characterisation requires multi-angular observations ye3, the acquisition of which can take between several minutes, as is the case for the Multi-angle Imaging SpectroRadiometer (MISR) instrument, and several days, as is the case for the Ocean and Land Colour Instrument (OLCI) on board Sentinel-3 or the Moderate Resolution Imaging Spectroradiometer (MODIS). In the former case, data are of- ten assumed to be acquired almost instantaneously, i.e. with the atmospheric properties remaining unchanged during the acquisition time. Such a situation considerably reduces the calculation time required to solve the RTE, as the multiple 5 Inversion process and the layer average phase function and the layer average phase function 4.4 FASTRE model accuracy ω′ 0,v(eλ) = 1 −αd 1 −ω0,vαd ω0,v(eλ). (6) (6) The layer total optical thickness, τLa, is the sum of the gaseous, τg, the aerosol, τ ′ a and the Rayleigh, τr, optical depth τLa(eλ) = τg(eλ) + τ ′ a(eλ) + τr(eλ), (7) (7) with τ ′ a(eλ) = P vτ ′ v(eλ). The single-scattering albedo of the scattering layer is equal to with τ ′ a(eλ) = P vτ ′ v(eλ). The single-scattering albedo of the scattering layer is equal to ω′ 0(eλ) = P cω′ 0,v(eλ)τ ′ v(eλ) τ ′a(eλ) (8) (8) 4.1 Overview ↑ The multiple-scattering contribution, I ↑ m(x,b;m), is solved in the Fourier space for all illumination and viewing directions of the quadrature directions Nθ for 2Nθ −1 az- imuthal directions. The contribution I ↑ m(x,b;m) in the direc- tion (0,v) is interpolated from the surrounding quadra- ture directions. Finally, the Jacobian kxi = ∂ym(xi,b;m) ∂xi of ym(x,b;m) for parameter xi are calculated as ﬁnite differ- ences. The surface reﬂectance rs(xs,b;m) over land is repre- sented by the so-called RPV (Rahman–Pinty–Verstraete) www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 6594 5.2 Cost function The fundamental principle of OE is to maximise the proba- bility P = P (x\|ye3,xb,b) with respect to the values of the state vector x, conditional to the value of the measurements and any prior information. The conditional probability takes on the quadratic form (Rodgers, 2000): τeλl,v τeλl+1,v = eeλl eeλl+1 , (15) (15) P (x) ∝exp h − ym(x,b;m) −ye3 T S−1 y ym(x,b;m) −ye3 i + exp h − x −xb T S−1 x x −xb i + exp h −xT HT a S−1 a Hax i + exp h −xT HT l S−1 l Hlx i , (11) where eeλl is the extinction coefﬁcient in bandeλl. where eeλl is the extinction coefﬁcient in bandeλl. λl Maximising the probability function in Eq. (11) is equiva- lent to minimising the negative logarithm J(x) = Jy(x) + Jx(x) + Ja(x) + Jl(x), (16) (16) with (11) Jy(x) = ym(x,b,) −ye3 S−1 y ym(x,b,) −ye3 T (17) Jx(x) = x −xb S−1 x x −xb T (18) Ja(x) = xT HT a S−1 a Hax (19) Jl(x) = xT HT l S−1 l Hlx. (20) where the ﬁrst two terms represent weighted deviations from measurements and the prior state parameters respectively, the third represents the AOT temporal smoothness constraints and the fourth represents the AOT spectral constraint, with respective uncertainty matrices Sa and Sl. The algorithm pro- posed by Dubovik et al. (2011) implements similar temporal and spectral smoothness constraints. The two matrices Ha and Hl, representing respectively the temporal and spectral constraints, can be written as block diagonal matrices: where the ﬁrst two terms represent weighted deviations from measurements and the prior state parameters respectively, the third represents the AOT temporal smoothness constraints and the fourth represents the AOT spectral constraint, with respective uncertainty matrices Sa and Sl. The algorithm pro- posed by Dubovik et al. (2011) implements similar temporal and spectral smoothness constraints. The two matrices Ha and Hl, representing respectively the temporal and spectral constraints, can be written as block diagonal matrices: (20) Notice that the cost function J is minimised with respect to the state variable x, so that the derivative of J is independent of the model parameters b. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol where the four blocks Hρ0, Hk, Hθ and Hρc express the spec- tral constraints between the surface parameters. Their values are set to zero when these constraints are not active. The sub- matrix Hτ a can also be written using blocks Hτ a;eλ,v along the diagonal. For a given spectral band eλ and aerosol vertex v, the block Hτ a;eλ,v is deﬁned as follows: scattering term I ↑ m(x,b;m) needs to be estimated only once per spectral band. In the latter case, atmospheric properties cannot be assumed to be invariant and the multiple scattering contribution needs to be solved for each observation. When geostationary observations are processed, the accumulation period is often reduced to 1 day, and the assumption that the atmosphere does not change can be converted into an equiva- lent radiometric uncertainty (Govaerts et al., 2010b). Strictly speaking, it should be assumed that atmospheric properties have changed when the accumulation time exceeds several minutes (Luffarelli et al., 2016). Hτ a;eλ,v τeλ,v =   1 −1 0 ... ... 0 1 −1 0 ... ... ... ... ... ... ... ... ... 1 −1 ... ... ... ... 0     τeλ,v,1 τeλ,v,2 ... τeλ,v,Nt−1 τeλ,v,1,Nt   . (13) The retrieved state variables in each spectral band eλ are composed of the xs parameters characterising the state of the surface and the set of aerosol optical thicknesses τv for the aerosol vertices that are mixed in layer La. Prior informa- tion consists of the expected values xb of the state variables x characterising the surface and the atmosphere on one side, and regularisation of the spectral and/or temporal variability of τv on the other side. Uncertainty matrices Sx are assigned to this prior information. Finally, uncertainties in the mea- surements Sy are assumed to be normally distributed with zero mean. The inversion process of the FASTRE model will be herein referred to as the Combined Inversion of Surface and AeRosol (CISAR) algorithm. (13) In the same way, the submatrix Hτ l can be written using blocks Hτ l;v,t. For a given aerosol vertex v and time t, the block Hτ l;v,t is deﬁned as follows: Hτ l;v,t τ v,t =   0 0 0 ... 0 −ϵ2 ϵ1 1 0 ... 0 0 −ϵ3 ϵ2 1 ... 0 ... ... ... ... 0 ... ... ... Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol − ϵNλ ϵNλ−1 1     τ1,v,t τ2,v,t τ3,v,t ... τNeλ,v,t   , (14) where the ϵl represents the uncertainties associated with the AOT spectral constraints of the individual vertex v, bound- ing the solution space. The spectral variations of τv between bandeλl andeλl+1 are assumed to vary, as 4.3 Gaseous layer properties It is assumed that only molecular absorption takes place in layer Lg. The height of level Za is used to partition the total column water vapour and ozone concentration in each layer assuming a US76 standard atmosphere vertical proﬁle. This height is not retrieved and is therefore a model parameter of FASTRE, which should be derived from some climatological values. TLg denotes the total transmission of that layer. www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 6595 www.atmos-meas-tech.net/11/6589/2018/ 6.2 Results (23) 5.4 Acceleration methods The minimisation of Eq. (16) relies on an iterative approach with ym(x,b;m) and the associated Jacobians Kx being es- timated at each iteration. In order to reduce the calculation time dedicated to the estimation of ym(x,b;m) and Kx, a se- ries of methods have been implemented. All quantities that do not explicitly depend on the state variables, such as the observation conditions m, model parameters b, quadrature point weights, etc. are computed only once prior to the opti- misation. When solving the RTE, the estimation of the multiple scat- tering term is by far the most time-consuming step. Hence, during the iterative optimisation process, when the change 1τa(eλ) between iteration j and j + 1 is small, the multiple scattering contribution at iteration j + 1 is estimated with Values used for the RPV parameters in the four selected bands are indicated in Table 5. They correspond to typical BRF values that would be observed over a vegetated surface with a leaf area index value of 3 and a bright underlying soil. I ↑ m(τa(j + 1,eλ),b;m) = I ↑ m(τa(j,eλ),b;m) + ∂I ↑ m(τa(j,eλ),b;m) ∂τa 1τa(eλ). (23) 6.1 Experimental set-up A simple experimental set-up based on simulated data has been deﬁned to illustrate the performance of the CISAR al- gorithm as a function of the chosen solution space. More speciﬁcally, the capability of CISAR to continuously sample the {g,ω0} solution space is examined in detail. For the sake of simplicity, a noise-free multi-angular observation vector, ye3, where expresses the illumination and viewing ge- ometries, is assumed to be acquired instantaneously in the principal plane and in the spectral bands listed in Table 1. A radiometric uncertainty of 3 % is assumed to compose Sy. In this ideal conﬁguration, the solar zenith angle (SZA) is set to 30◦. In these experiments, to concentrate on the retrieval of aerosol properties, the surface parameters prior values are set to the true values used in the simulation (Table 5) with an ascribed uncertainty of 0.03. The ﬁrst guess values are ran- domly chosen within this uncertainty interval. Part 2 explains how prior information on the surface parameters can be de- rived. No prior information is assumed for the aerosol optical thickness; i.e. the prior uncertainty is set to very large values. Only regularisation of the spectral variations of τa is applied. 5.3 Retrieval uncertainty estimation The retrieval uncertainty is based on the OE theory, assuming linear behaviour of ym(x,b;m) in the vicinity of the solution ˆx. Under this condition, the retrieval uncertainty σˆx is deter- mined by the shape of J(x) at ˆx σ 2 ˆx = ∂2Js(x) ∂x2 −1 = KT x S−1 y Kx + S−1 x + HT a S−1 a Ha + HT l S−1 l Hl −1 , (21) (21) where Kx is the Jacobian matrix of ym(x,b;m) calculated in ˆx. Combining Eqs. (21) and (8), the uncertainty in the re- trieval of ω0 in bandeλ is written σ 2 ˆω0(eλ) = X v ω0,v(eλ) −ω0(eλ) τa(eλ) 2 σ 2 ˆτv(eλ). (22) (22) The CISAR algorithm performance evaluation is based on a series of experiments corresponding to different selec- tions of aerosol properties, both for the forward simulation of the observations and their inversion. Three different aerosol models are used in the forward simulations: F0, which only contains ﬁne-mode; F1, which contains a dual-mode particle size distribution dominated by small particles; and F2, com- posed of a dual-mode distribution dominated by the coarse particles. Table 2 contains the values of the size distribution and refractive indices of these aerosol models. Values for the four vertices (FA, FN, CL and CS) enclosing the solution space as illustrated in Fig. 3 are given in Table 3. When the observations simulated with aerosol types F0, F1 or F2 are inverted, the list of vertices actually used depends on the type of experiment as indicated in Table 4. For all these scenarios, an AOT of 0.4 at 0.55 µm is assumed. A similar equation can be derived for the estimation of σ 2 g . Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 6 Algorithm performance evaluation the variance should decrease. Accordingly, the magnitude of the elements of the covariance matrix should decrease as 1/√ny. Thus, repeating similar observations results in some enhancements of retrieval accuracy, which is proportional to the ratio ny/nx. Hence, the cost function which is actually minimised is Js(x) = Jy(x)+ny/nx(Jx(x)+Ja(x)+Jl(x)). 5.2 Cost function The need for angular sampling to document the surface anisotropy leads to an unbalanced dimension of nx and ny with ny > nx, where ny and nx rep- resent the number of observations and state variables respec- tively. According to Dubovik et al. (2006), these additional observations should improve the retrieval as, from a statisti- cal point of view, repeating similar observations implies that H =   Hρ0 0 0 0 0 0 Hk 0 0 0 0 0 Hθ 0 0 0 0 0 Hρc 0 0 0 0 0 Hτ   , (12) (12) Atmos. Meas. Tech., 11, 6589–6603, 2018 6596 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol The last column indicates the name of the aerosol model used to simulate the observations. the F0 aerosol model. The retrieved AOT is also in very good agreement with the true values as can be seen on the right panel. The retrieval error ϵτ is deﬁned as the difference be- tween the retrieved and the true AOT values. Results are sum- marised in Table 6. This ﬁrst experiment demonstrates that it is possible to retrieve the properties of the aerosol model F0 as a linear combination of the vertices FA and FN when only the absorption varies, the particle median radius being con- stant. Experiment Active vertices Forward type FA FN CS CL F00 × × F0 F10 × × F1 F11 × × × F1 F12 × × × F1 F13 × × × × F1 F21 × × × F2 F22 × × × F2 F23 × × × × F2 A comparison between the true and retrieved values in Ta- ble 5 shows that the surface parameters are very accurately retrieved. As stated in Sect. 6.1, prior information on the magnitude of the RPV parameter is assumed to be unbiased with an uncertainty of 0.03. The corresponding posterior un- certainties exhibit a signiﬁcant decrease for the ρ0 parame- ter at all wavelengths. Similar behaviour is not observed for the other parameters. As explained in Wagner et al. (2010), the k and 2 parameters, controlling the surface reﬂectance anisotropy, are strongly correlated with the amount of atmo- spheric scattering. Consequently, the retrieved uncertainties decrease with increasing wavelengths, i.e. as a function of the actual AOT. Despite the observations taking place in the principal plane, the posterior uncertainty on the hot spot pa- rameter remains equal to the prior one as a result of atmo- spheric scattering. This fact is attributed to the relatively high value of the true AOT, and the consequent amount of scatter- ing attenuating the hot spot. Results for the surface parameter as for the FN and FA vertices used for the inversion. Hence, the retrieval is limited to the imaginary part of the index of refraction, the real part being set to 1.4. With a retrieval con- ﬁguration restricted to the use of only two vertices, the so- lution space for each wavelength is limited to a straight line between the two vertices. Results are shown in Fig. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 6597 Table 2. List of aerosol properties used for the simulations. The parameters rmf and rmc are the median ﬁne- and coarse-mode radii expressed in µm. Their respective standard deviations are σrmf and σrmc. The parameters nr and ni are the real and imaginary part of the refractive index in the indicated bands. Nf and Nc are the ﬁne- and coarse-mode particle concentration in number of particles per cm3. Table 2. List of aerosol properties used for the simulations. The parameters rmf and rmc are the median ﬁne- and coarse-mode radii expressed in µm. Their respective standard deviations are σrmf and σrmc. The parameters nr and ni are the real and imaginary part of the refractive index in the indicated bands. Nf and Nc are the ﬁne- and coarse-mode particle concentration in number of particles per cm3. Centre band in µm 0.44 0.55 0.67 0.87 Type rmf rmc ni nr nr nr Nf Nc F0 0.08 – 1.396 1.393 1.391 1.388 – – F1 0.10 0.93 1.419 1.427 1.436 1.442 9.587 0.002 F2 0.08 0.77 1.498 1.520 1.544 1.542 8.975 0.024 σrmf σrmc ni ni ni ni F0 0.45 – 0.012 0.012 0.012 0.012 – – F1 0.43 0.62 0.006 0.005 0.005 0.005 F2 0.50 0.62 0.005 0.005 0.004 0.004 Table 3. Microphysical parameter values for the four vertices (FA, FN, CS and CL) in the selected spectral bands. Radius are given in µm. Table 3. Microphysical parameter values for the four vertices (FA, FN, CS and CL) in the selected spectral b ter values for the four vertices (FA, FN, CS and CL) in the selected spectral bands. Radius are given in µm. Table 3. Microphysical parameter values for the four vertices (FA, FN, CS and CL) in the selected spectral bands. Radius are given in µm Centre band in µm 0.44 0.55 0.67 0.87 0.44 0.55 0.67 0.87 Type rm σrm nr nr nr nr ni ni ni ni FN 0.08 0.45 1.3958 1.3932 1.3909 1.3879 0.0006 0.0006 0.0006 0.0006 FA 0.08 0.45 1.3958 1.3932 1.3909 1.3879 0.0207 0.0207 0.0207 0.0205 CS 0.30 0.55 1.4889 1.4878 1.4845 1.4763 0.0029 0.0029 0.0029 0.0029 CL 1.00 0.55 1.4889 1.4878 1.4845 1.4763 0.0029 0.0029 0.0029 0.0029 Table 4. List of experiments: the names are provided in the ﬁrst column. The active vertices in each experiment are indicated with the × symbol. 6.2.1 Experiment F00 This approximation is not used in two successive iterations to avoid inaccurate results, and the single-scattering contri- bution is always explicitly estimated. This approximation is not used in two successive iterations to avoid inaccurate results, and the single-scattering contri- bution is always explicitly estimated. The purpose of the ﬁrst experiment (F00) is to demonstrate that the CISAR algorithm can accurately retrieve aerosol properties in a simple situation, showing therefore that the in- version process works correctly. The F0 aerosol model used to simulate the observations is only composed of ﬁne par- ticles with a median radius of 0.08 µm, i.e. the same value Atmos. Meas. Tech., 11, 6589–6603, 2018 www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Luffarelli: Combined Inversion of Surface and AeRosol 6598 Table 5. Values of the true and retrieved surface RPV parameters for experiment F00. Wavelengths are given in µm. Values of the true and retrieved surface RPV parameters for experiment F00. Wavelengths are given in µm. Table 5. Values of the true and retrieved surface RPV parameters for experiment F00. Wavelengths are given in µm. Table 6. Retrieved AOT error and uncertainties for the six experiments. The error ϵτ is calculated as the difference between the retrieved and the true values, δτ is the relative error in percent and στ is the retrieval uncertainty estimated with Eq. (21). Wavelengths are given in µm. Band 0.44 0.55 0.67 0.87 Experiment ϵτ δτ στ ϵτ δτ στ ϵτ δτ στ ϵτ δτ στ Units – (%) – – (%) – – (%) – – (%) – F00 0.001 −0.1 0.203 −0.002 0.6 0.133 −0.000 0.0 0.095 −0.004 3.3 0.079 F10 0.062 −11.0 0.199 0.042 −10.5 0.130 0.022 −7.8 0.094 0.026 −15.6 0.078 F11 0.005 −0.9 0.239 −0.021 5.3 0.164 −0.037 13.2 0.125 −0.047 27.8 0.095 F12 0.041 −7.3 0.228 0.013 −3.3 0.152 −0.004 1.5 0.113 −0.015 8.6 0.089 F13 −0.001 0.1 0.295 −0.028 6.9 0.199 −0.041 14.5 0.145 −0.051 30.5 0.103 F21 0.018 −3.9 0.252 0.037 −9.2 0.172 0.042 −11.9 0.129 0.071 −22.9 0.096 F22 −0.018 3.9 0.236 −0.007 1.8 0.158 −0.004 1.1 0.116 0.008 −2.6 0.090 F23 −0.041 8.8 0.296 −0.031 7.8 0.200 −0.027 7.5 0.145 −0.018 6.0 0.103 retrieval exhibits very similar behaviour for the other experi- ments and will not be shown. is particularly large at 0.87 µm, but rather underestimated at 0.44 µm. The improvement in the AOT retrieval accuracy is noticeable in the 0.44 and 0.55 µm bands where the magni- tude of ϵr is reduced from 0.062 to 0.005 and from 0.042 to −0.021 respectively (Table 6). At larger wavelengths, the beneﬁt of adding the CS vertex is less noticeable though the magnitude of ϵr remains below 0.05. Finally, the retrieval un- certainty slightly increases from 0.199 up to 0.239 for the 0.44 µm band because of the use of additional state variables τv associated with the inclusion of an additional vertex. Sim- ilar behaviour is observed in the other bands. 6.2.2 Experiment F10 Let us now examine the case in which both rm and ni differ from those of the vertices used for the inversion. The aerosol type F1 is used for the forward simulation with rmf = 0.1 µm for the predominant ﬁne mode and rmc = 0.93 µm for the coarse mode. The same aerosol vertices as in experiments F00 are used for the inversion. The results in Fig. 6 show that ω0 is reasonably well re- trieved unlike the g parameter, which is systematically under- estimated. At any given wavelengths, it is not possible to re- trieve g values outside the bounds deﬁned by the FA and FN vertices. Consequently, the retrieved AOT values are under- estimated by about 10 % (Table 6). This example illustrates the retrieval failure when the actual solution lays outside the {g,ω0} space deﬁned by the active vertices. For experiment F12, the CS vertex is substituted by vertex CL which has a median radius of 1.0 µm. The use of this ver- tex instead of CS considerably improves the retrieval of g and of ω0 at large wavelengths (Fig. 8). As can be seen in Fig. 2, the sensitivity of aerosol single-scattering properties to parti- cle median radius and imaginary part of the refractive index depends on the wavelength. Hence, a similar performance of the algorithm at all wavelengths should not be expected. The errors ϵτ in this experiment F12 are further reduced com- pared to experiment F11 with the exception of the 0.44 µm band. The CISAR algorithm retrieves total AODs consistent with the truth. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 5 for the atmosphere and Table 5 for the surface. The asymmetry factor g and single-scattering albedo ω0 are almost exactly retrieved. There is practically no uncertainty in the retrieval of g because of the constraints imposed by the fact that the particle radius is the same as for www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 6598 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Table 5. Values of the true and retrieved surface RPV parameters for experiment F00. Wavelengths are given in µm. True Retrieved Band ρ0 k 2 ρc ρ0 k 2 ρc 0.44 0.025 0.666 −0.150 0.125 0.025 ± 0.006 0.666 ± 0.030 −0.150 ± 0.030 0.125 ± 0.030 0.55 0.047 0.657 −0.114 0.023 0.047 ± 0.004 0.657 ± 0.029 −0.116 ± 0.028 0.023 ± 0.030 0.67 0.056 0.710 −0.096 0.025 0.056 ± 0.004 0.711 ± 0.028 −0.096 ± 0.026 0.025 ± 0.030 0.87 0.238 0.706 −0.019 0.030 0.238 ± 0.011 0.705 ± 0.025 −0.020 ± 0.017 0.029 ± 0.030 Table 6. Retrieved AOT error and uncertainties for the six experiments. The error ϵτ is calculated as the difference between the retrieved and the true values, δτ is the relative error in percent and στ is the retrieval uncertainty estimated with Eq. (21). Wavelengths are given in µm. Band 0.44 0.55 0.67 0.87 Experiment ϵτ δτ στ ϵτ δτ στ ϵτ δτ στ ϵτ δτ στ Units – (%) – – (%) – – (%) – – (%) – F00 0.001 −0.1 0.203 −0.002 0.6 0.133 −0.000 0.0 0.095 −0.004 3.3 0.079 F10 0.062 −11.0 0.199 0.042 −10.5 0.130 0.022 −7.8 0.094 0.026 −15.6 0.078 F11 0.005 −0.9 0.239 −0.021 5.3 0.164 −0.037 13.2 0.125 −0.047 27.8 0.095 F12 0.041 −7.3 0.228 0.013 −3.3 0.152 −0.004 1.5 0.113 −0.015 8.6 0.089 F13 −0.001 0.1 0.295 −0.028 6.9 0.199 −0.041 14.5 0.145 −0.051 30.5 0.103 F21 0.018 −3.9 0.252 0.037 −9.2 0.172 0.042 −11.9 0.129 0.071 −22.9 0.096 F22 −0.018 3.9 0.236 −0.007 1.8 0.158 −0.004 1.1 0.116 0.008 −2.6 0.090 F23 −0.041 8.8 0.296 −0.031 7.8 0.200 −0.027 7.5 0.145 −0.018 6.0 0.103 retrieval exhibits very similar behaviour for the other experi- ments and will not be shown is particularly large at 0.87 µm, but rather underestimated at 0 44 µm The improvement in the AOT retrieval accuracy is Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Y. Govaerts and M. 6.2.3 Experiments F11–F13 In order to improve the retrieval of the F1 aerosol model properties, the additional aerosol CS vertex with rm = 0.3 µm has been added for the inversion process. Results of exper- iment F11 are displayed in Fig. 7. Retrieved g values are no longer underestimated. The single-scattering albedo is slightly underestimated. It should be noted that the estimated uncertainty associated with g increases with wavelength and Finally, in experiment F13 the inversion was performed using all four vertices (Fig. 9). This additional “degree of freedom” translates into an increase of the estimated uncer- tainty σˆτ as a result of the large number of possible way to combine these four vertices to retrieve the properties of the www.atmos-meas-tech.net/11/6589/2018/ www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 6599 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 6599 Figure 5. (a) Results of experiment F00 in the {g,ω0} space. The aerosol vertices used for the inversion are FN (blue) and FA (green). The forward aerosol properties are shown in black and the retrieved ones in red. Vertical and horizontal red bars indicate the uncertainty of the retrieved values. (b) Retrieved AOT (red circles). The retrieval uncertainty is shown with the vertical red lines. True values are indicated with black crosses. True and retrieved values are slightly staggered to ease the reading. Figure 5. (a) Results of experiment F00 in the {g,ω0} space. The aerosol vertices used for the inversion are FN (blue) and FA (green). The forward aerosol properties are shown in black and the retrieved ones in red. Vertical and horizontal red bars indicate the uncertainty of the retrieved values. (b) Retrieved AOT (red circles). The retrieval uncertainty is shown with the vertical red lines. True values are indicated with black crosses. True and retrieved values are slightly staggered to ease the reading. Figure 6. Same as Fig. 5 but for experiment F10. Figure 6. Same as Fig. 5 but for experiment F10. F23 (Fig. 11) and FN, FA, CS and CL for experiment F22 (Fig. 11). Essentially the same conclusions hold as for the retrieval of aerosol model F1. The retrieval of the F2 model properties expressed as a linear combination of the FN, FA and CL vertices provides the best solution, with both g and ω0 being retrieved at all wavelengths. aerosol model F1. 6.2.3 Experiments F11–F13 In other words, using these two coarse mode vertices does not improve the characterisation of F1. The actual beneﬁt of adding this fourth vertex, i.e. expand- ing the solution space, is therefore not straightforward, and it should be noted that increasing the number of vertices im- pacts the computational time. This series of experiments has shown that, of the four considered, the use of the FN, FA and CL vertices provides the best combination for the retrieval of aerosol model F1. With this combination, the FN and FA vertices allow for control of the amount of radiation absorbed by the aerosols, while the effects of the particle size are con- trolled by the CL vertex. 7 Discussion and conclusion This paper describes the CISAR algorithm designed for the joint retrieval of surface reﬂectance and aerosol properties. Previous attempts to perform a joint retrieval have been re- viewed, discussing their advantages and weaknesses. The limitations due to a combined used of discrete and continu- ous state variables in retrieval methods based on OE as in Go- vaerts et al. (2010b) are discussed in Sect. 2. The new method presented in this paper speciﬁcally addresses these limita- tions, allowing continuous variations of the aerosol single- scattering properties in the solution space without the aerosol microphysical properties explicitly appearing as state vari- ables. Atmos. Meas. Tech., 11, 6589–6603, 2018 6.2.4 Experiments F21–F23 The retrieval of aerosol model F2, a dual-mode particle size distribution dominated by coarse particles, is now exam- ined. This model is composed of a ﬁne mode with radius rmf = 0.08 µm and a coarse mode with radius rmc = 0.77 µm. As for the retrieval of the F1 aerosol model, three combina- tions of vertices have been explored, i.e. FN, FA and CS for experiment F21 (Fig. 10), FN, FA and CL for experiment www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 6600 Y. Govaerts and M. Luffarelli: Combined Inversion Figure 7. Same as Fig. 5 but for experiment F11. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 6600 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Figure 7. Same as Fig. 5 but for experiment F11. Figure 8. Same as Fig. 5 but for experiment F12. 6600 Figure 7. Same as Fig. 5 but for experiment F11. Figure 7. Same as Fig. 5 but for experiment F11. Figure 8. Same as Fig. 5 but for experiment F12. Figure 9. Same as Fig. 5 but for experiment F13. Atmos. Meas. Tech., 11, 6589–6603, 2018 www.atmos-meas-tech.net/11/6 Figure 7. Same as Fig. 5 but for experiment F11. Figure 7. Same as Fig. 5 but for experiment F11. Figure 8. Same as Fig. 5 but for experiment F12. Figure 8. Same as Fig. 5 but for experiment F12. Figure 8. Same as Fig. 5 but for experiment F12. Figure 9. Same as Fig. 5 but for experiment F13. Atmos. Meas. Tech., 11, 6589–6603, 2018 www.atmos-meas-tech.net/11/6589/2018/ Figure 8. Same as Fig. 5 but for experiment F12. Figure 9. Same as Fig. 5 but for experiment F13. Figure 9. Same as Fig. 5 but for experiment F13. Figure 9. Same as Fig. 5 but for experiment F13. Figure 9. Same as Fig. 5 but for experiment F13. Atmos. Meas. Tech., 11, 6589–6603, 2018 www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 erts and M. Luffarelli: Combined Inversion of Surface and AeRosol Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 6601 Figure 10. Same as Fig. 5 but for experiment F21. Figure 11. Same as Fig. 5 but for experiment F22. Figure 10. Same as Fig. 5 but for experiment F21. Figure 10. Same as Fig. 5 but for experiment F21. Figure 11. Same as Fig. 5 but for experiment F22. Figure 12. Same as Fig. 5 but for experiment F23. www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech. Figure 10. Same as Fig. 5 but for experiment F21. Figure 10. Same as Fig. 5 but for experiment F21. Figure 11. Same as Fig. 5 but for experiment F22. Figure 12. Same as Fig. 5 but for experiment F23. www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 Figure 11. Same as Fig. 5 but for experiment F22. Figure 11. Same as Fig. 5 but for experiment F22. Figure 12. Same as Fig. 5 but for experiment F23. www.atmos-meas-tech.net/11/6589/2018/ Atmos. Meas. Tech., 11, 6589–6603, 2018 Figure 12. Same as Fig. 5 but for experiment F23. Figure 12. Same as Fig. 5 but for experiment F23. Atmos. Meas. Tech., 11, 6589–6603, 2018 Atmos. Meas. Tech., 11, 6589–6603, 2018 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol place far from the principal plane, where most of the angular variations occur. Part 2 addresses the performance of CISAR when applied to actual satellite data. A fast-forward radiative transfer model has been designed which solves the radiative transfer equation without relying on precomputed look-up tables. This model considers two at- mospheric layers. The upper layer only hosts molecular ab- sorption. The lower layer accounts for both absorption and scattering processes due to aerosols and molecules and is ra- diatively coupled with the surface represented with the RPV BRF model. Single-scattering aerosol properties in this layer are expressed as a linear combination of the properties of ver- tices enclosing part of the solution space. Data availability. Results presented in Sect. 6 are available from the authors upon request. Supplement. It includes the plots of case F22, adding a 3 % Gaussian noise to the simulated TOA BRF for AOT = 0.05 (Fig. S1), AOT = 0.2 (Fig. S2), AOT = 0.4 (Fig. S3) and AOT = 0.8 (Fig. S4). Figure S5 shows the merged results in the {g,ω0} space of experiments F11, F12 and F13. Figure S6 shows the merged results in the {g,ω0} space of experiments F21, F22 and F23. The supplement related to this article is available online at: https://doi.org/10.5194/amt-11-6589-2018-supplement. A series of different experiments has been devised to anal- yse the behaviour of the CISAR algorithm and its capability to retrieve aerosol single-scattering properties as well as the optical thickness. This discussion focuses on the retrieval of aerosol models dominated by the ﬁne mode or coarse mode. These two models have pretty different spectral behaviour in the {g,ω0} space and yet the CISAR algorithm is capable of retrieving the corresponding single-scattering properties in both cases with estimated uncertainties of about 15 %. Author contributions. YG developed the FASTRE model, con- ceived the experimental set-up and wrote the paper. ML contributed to the development of the inversion method. These experiments illustrate the possibility of using Eqs. (8) and (9) for the continuous retrieval of the aerosol single-scattering albedo and phase function. These equations assume linear behaviour of ω0 and g in the solution space. Such assumptions have proven to be valid for the case ad- dressed in experiment F00. This assumption is not exactly true for the retrieval aerosol models of a ﬁne- and a coarse- particle size modes. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol However, the retrieved aerosol single- scattering properties are derived much more accurately than with a method based on a limited number of predeﬁned aerosol models as in Govaerts et al. (2010b), where the single-scattering properties of only predeﬁned models are re- trieved. It thus represents a major improvement with respect to these types of retrieval approaches without requiring the use of a large number of state variables as in the method pro- posed by Dubovik et al. (2011), where aerosol microphysical properties are explicitly included in the set of retrieved state variables. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. The authors would like to thank the reviewers for their fruitful suggestions. Edited by: Andrew Sayer Reviewed by: three anonymous referees www.atmos-meas-tech.net/11/6589/2018/ www.atmos-meas-tech.net/11/6589/2018/ 6602 Atmos. Meas. Tech., 11, 6589–6603, 2018 Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol 6603 Luffarelli, M., Govaerts, Y., and Damman, A.: Assessing hourly aerosol properties retrieval from MSG/SEVIRI observations in the framework of aeroosl-cci2, in: Living Planet Symposium 2016, 9–13 May, Prague, Czech Republic, 2016. Fischer, J. and Grassl, H.: Radiative transfer in an atmosphere- ocean system: an azimuthally dependent matrix-operator ap- proach, Appl. Optics, 23, 1032–1039, 1984. Govaerts, Y. and Lattanzio, A.: Retrieval Error Esti- mation of Surface Albedo Derived from Geostation- ary Large Band Satellite Observations: Application to Meteosat-2 and -7 Data, J. Geophys. Res., 112, D05102, https://doi.org/10.1029/2006JD007313, 2007. Luffarelli, M., Govaerts, Y., Goossens, C., Wolters, E., and Swin- nen, E.: Joint retrieval of surface reﬂectance and aerosol prop- erties from PROBA-V observations, part I: algorithm perfor- mance evaluation, in: Proceedings of MultiTemp 2017, 27–29 June, Bruges, Belgium, 2017. Govaerts, Y., Luffarelli, M., and Damman, A.: Effects of Sky Ra- diation on Surface Reﬂectance: Implications on The Derivation of LER from BRF for the Processing of Sentinel-4 Observations, in: Living Planet Symposium 2016, 9–13 May, Prague, Czech Republic, 2016. Manolis, I., Grabarnik, S., Caron, J., Bézy, J.-L., Loiselet, M., Betto, M., Barré, H., Mason, G., and Meynart, R.: The MetOp second generation 3MI instrument, Proc. SPIE Remote Sens., 88890J, https://doi.org/10.1117/12.2028662, 2013. Govaerts, Y. M.: RTMOM V0B.10 User’s Manual, 2006. Marquardt, D.: An Algorithm for Least-Squares Estimation of Non- linear Parameters, SIAM J. Appl. Math., 11, 431–441, 1963. Govaerts, Y. M., Lattanzio, A., Taberner, M., and Pinty, B.: Generating global surface albedo products from multiple geo- stationary satellites, Remote Sens. Environ., 112, 2804–2816, https://doi.org/10.1016/j.rse.2008.01.012, 2008. Pinty, B., Roveda, F., Verstraete, M. M., Gobron, N., Govaerts, Y., Martonchik, J. V., Diner, D. J., and Kahn, R. A.: Surface albedo retrieval from Meteosat: Part 1: Theory, J. Geophys. Res., 105, 18099–18112, 2000a. Govaerts, Y. M., Wagner, S., and Dubovik, O.: Enhanced retrieval of loading and detailed micro-physics of atmospheric aerosol from MTG/FCI observations, in: EUMETSAT Meteorological User Conference, 20–24 September, Córdoba, Spain, 2010a. Pinty, B., Roveda, F., Verstraete, M. M., Gobron, N., Govaerts, Y., Martonchik, J. V., Diner, D. J., and Kahn, R. A.: Surface albedo retrieval from Meteosat: Part 2: Applications, J. Geophys. Res., 105, 18113–18134, 2000b. Govaerts, Y. M., Wagner, S., Lattanzio, A., and Watts, P.: Joint retrieval of surface reﬂectance and aerosol optical depth from MSG/SEVIRI observations with an optimal estimation approach: 1. Theory, J. Geophys. Res., 115, 10.1029/2009JD011779, https://doi.org/10.1029/2009JD011779, 2010b. Rahman, H., Pinty, B., and Verstraete, M. Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol M.: Coupled surface- atmosphere reﬂectance (CSAR) model, 2. Semiempirical surface model usable with NOAA Advanced Very High Resolution Ra- diometer Data, J. Geophys. Res., 98, 20791–20801, 1993. Hess, M., Koepke, P., and Schult, I.: Optical properties of aerosols and clouds: The software package OPAC, B. Am. Meteorol. Soc., 79, 831–844, 1998. Rodgers, C. D.: Inverse methods for atmospheric sounding, Series on Atmospheric Oceanic and Planetary Physics, World Scien- tiﬁc, Oxford, 2000. Kokhanovsky, A. A., Deuzé, J. L., Diner, D. J., Dubovik, O., Ducos, F., Emde, C., Garay, M. J., Grainger, R. G., Heckel, A., Herman, M., Katsev, I. L., Keller, J., Levy, R., North, P. R. J., Prikhach, A. S., Rozanov, V. V., Sayer, A. M., Ota, Y., Tanré, D., Thomas, G. E., and Zege, E. P.: The inter-comparison of major satellite aerosol retrieval algorithms using simulated intensity and polar- ization characteristics of reﬂected light, Atmos. Meas. Tech., 3, 909–932, https://doi.org/10.5194/amt-3-909-2010, 2010. Schuster, G. L., Dubovik, O., Holben, B. N., and Clothiaux, E. E.: Inferring black carbon content and speciﬁc absorption from Aerosol Robotic Network (AERONET) aerosol retrievals, J. Geophys. Res., 110, 1017–1017, 2005. Rozanov, V. V., Sayer, A. M., Ota, Y., Tanré, D., Thom Serene, F. and Corcoral, N.: PARASOL and CALIPSO: Experi- ence Feedback on Operations of Micro and Small Satellites, in: SpaceOps 2006 Conference, 21 June, American Institute of Aeronautics and Astronautics, https://doi.org/10.2514/6.2006- 5919, 2006. Lattanzio, A., Schulz, J., Matthews, J., Okuyama, A., Theodore, B., Bates, J. J., Knapp, K. R., Kosaka, Y., and Schüller, L.: Land Sur- face Albedo from Geostationary Satelites: A Multiagency Col- laboration within SCOPE-CM, B. Am. Meteorol. Soc., 94, 205– 214, https://doi.org/10.1175/BAMS-D-11-00230.1, 2013. Vermote, E. F., Tanré, D., Deuzé, J. L., Herman, M., and Morcrette, J. J.: Second simulation of the satellite signal in the solar spec- trum, 6S: An overview, IEEE T. Geosci. Remote, 35, 675–686, 1997. Liu, Q. and Ruprecht, E.: Radiative transfer model: matrix operator method, Appl. Optics, 35, 4229–4237, 1996. Wagner, S. C., Govaerts, Y. M., and Lattanzio, A.: Joint retrieval of surface reﬂectance and aerosol optical depth from MSG/SEVIRI observations with an optimal estimation approach: 2. Imple- mentation and evaluation, J. Geophys. Res., 115, D02204, https://doi.org/10.1029/2009JD011780, 2010. Luffarelli, M. and Govaerts, Y.: Joint retrieval of surface reﬂectance and aerosol properties with continuous variation of the state variables in the solution space: Part 2: Application to geosta- tionary and polar orbiting satellite observations, Atmos. Meas. Tech. References Cox, C. and Munk, W.: Measurement of the Roughness of the Sea Surface from Photographs of the Sun’s Glitter, J. Opt. Soc. Am., 44, 838–850, https://doi.org/10.1364/JOSA.44.000838, 1954. The choice of vertices outlining the {g,ω0} solution space is critical. In these experiments, the best retrieval is ob- tained using three vertices, i.e. one vertex composed of small weakly absorbing particles (FN), one vertex composed of small absorbing particles (FA) and one vertex composed of large particles (CL). The use of a fourth vertex (CS) does not improve the retrieval and increases the estimated retrieval un- certainty. Diner, D. J., Hodos, R. A., Davis, A. B., Garay, M. J., Mar- tonchik, J. V., Sanghavi, S. V., von Allmen, P., Kokhanovsky, A. A., and Zhai, P.: An optimization approach for aerosol re- trievals using simulated MISR radiances, Atmos. Res., 116, 1– 14, https://doi.org/10.1016/j.atmosres.2011.05.020, 2012. Dubovik, O., Sinyuk, A., Lapyonok, T., Holben, B. N., Mishchenko, M., Yang, P., Eck, T. F., Volten, H., Munoz, O., Veihelmann, B., van der Zande, W. J., Leon, J. F., Sorokin, M., and Slutsker, I.: Application of spheroid models to account for aerosol particle nonsphericity in remote sensing of desert dust, J. Geophys. Res.- Atmos., 111, 11208–11208, 2006. This set of experiments represents ideal conditions, i.e. noise-free observations in the principal plane with no bias on the surface prior. This choice is motivated by the need to keep the result interpretation simple to illustrate how the new retrieval concept developed in this paper works. These experiments show the possibility of retrieving aerosol single- scattering properties within the solution space provided it is correctly bounded by the vertices. It is clear that adding noise to the observations will degrade the quality of the retrieval. Similar conclusions can hold if the observations are taking Dubovik, O., Herman, M., Holdak, A., Lapyonok, T., Tanré, D., Deuzé, J. L., Ducos, F., Sinyuk, A., and Lopatin, A.: Statistically optimized inversion algorithm for enhanced re- trieval of aerosol properties from spectral multi-angle polari- metric satellite observations, Atmos. Meas. Tech., 4, 975–1018, https://doi.org/10.5194/amt-4-975-2011, 2011. Atmos. Meas. Tech., 11, 6589–6603, 2018 www.atmos-meas-tech.net/11/6589/2018/ Y. Govaerts and M. Luffarelli: Combined Inversion of Surface and AeRosol Discuss., https://doi.org/10.5194/amt-2018-265, in review, 2018. Wiscombe, W. J.: The Delta-M Method: Rapid Yet Accurate Radia- tive Flux Calculations for Strongly Asymmetric Phase Functions, J. Atmos. Sci., 34, 1408–1422, 1977. Atmos. Meas. Tech., 11, 6589–6603, 2018 www.atmos-meas-tech.net/11/6589/2018/

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